Book of Abstracts V International Conference on Antimicrobial Research - ICAR2018 Torremolinos, Málaga (Spain), May 2018

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1 Book of Abstracts V International Conference on Antimicrobial Research - ICAR2018

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3 Introduction Plenary Lecture A new look into an old natural biopolyester - the antimicrobial activity and chemistry of suberin 1 A novel Pseudomonas strain capable to produce antifungal substances 2 Advancing a new class of methionyl-trna synthetase (MetRS) antibiotics 3 Aeromonas production of antimicrobial substance active against clinical resistant strains 4 Ag Bi nanoalloys: Synthesis by femtosecond laser irradiation and antibacterial activity against MRSA and MSSA 5 Allyl isothiocyanate and cinnamaldehyde against mycotoxigenic fungi in vitro and their combined use in stored corn 6 An innovative, electrochemical in situ ozone generator 7 Analogous derived from C-terminal salmonids IL-8 as potential antimicrobial agents for infectious diseases 8 Antibacterial activity of α-ag 2-2xMxWO 4 (M = Cu 2+ ) Solid Solutions Obtained by Coprecipitation Method 9 Antibiotic potential of defense peptides from wild cereals 10 Antibiotic resistance ABCF proteins reset the peptidyl transferase center of the ribosome to counter translational arrest 11 Antibiotic resistance mediated by integrons in Pseudomonas spp. genus 12 Antifungal properties of silver tungstate (Ag 2 WO 4 ) irradiated by electron and femtosecond laser 13 Antimicrobial activity assay of Pseudomonas luteola VFB Antimicrobial activity of a methanolic extract of Pinguicula moranensis Kunth 15 Antimicrobial effect of cinnamaldehyde, carvacrol and thymol and their combined effect with penicillin and trimethoprim-sulfamethoxazole against resistant Streptococcus suis strains 16 Antimicrobial effect of lysozyme in serum in patients with ventilation-associated pneumonia 17 Antimicrobial Functionalization of Copper Surfaces by Ultrashort Pulsed Direct Laser Interference Patterning 18 Antimicrobial resistance for bacterial pathogens isolated from bovine mastitis 19 Antimicrobial resistance in rare mycobacteria from hospital environment and diabetic foot ulcers 20 Antiviral, antimicrobial and biocompatible polymer-based coatings with natural bioactive substances Arthrobacter globiformis as a source of bioactive compounds for pharmaceutical applications 23 Assessment of the lytic ability of bacteriophages specific for Staphylococcus aureus, associated with bovine mastitis in South Africa 24 Bacteria-triggered antimicrobial coatings to maintain drinking water quality 25 Bactericidal activity of AP-CECT7121 combined with colistin against biofilmproducers Escherichia coli and Pseudomonas aeruginosa from catheters 26 Bacteriocin produced by Lactobacilli from Algerian goat's milk against pathogenic bacteria 27 Bark beetles, a novel source of strains capable to inhibit Candida sp. 28 Bark beetles, a source of Pseudomonas strains with antimicrobial activity 29 Biofilm formation in clinical isolates in ICU 30 Bioinformatics study on multi-resistant bacteria that express β-lactamases 31 Biological and molecular characterization of South African bacteriophages infective against Streptococcus uberis, a predominant causal agent of bovine mastitis 32 Coating of titanium surface with Ag 2 WO 4 inhibits methicillin-resistant Staphylococcus aureus adhesion 33 Comparison of antimycotic efficacy of biosynthesized selenium and tellurium nanoparticles against grapevine (Vitis vinifera) pathogenic fungi 34 V VII I

4 Comparison of sporulation and germination between clinical and hospital environment strains of Clostridium difficile and Clostridium perfringens 35 Copper alloys door handles: weapons against biofilms formation? 36 Designer polymeric biocides 37 Detection of marker proteins for bacteria antimicrobial resistance by mass spectrometry 38 Determining the rate and cost of in vitro resistance development in Methicillin Resistant Staphylococcus aureus against moenomycin A 39 Development of novel disinfectants against pathogens occur in the hospital environment 40 Effect of an organic acids based feed additive and enrofloxacin on the prevalence of antibioticresistant E. coli in broilers 41 Effect of Isothiocyanates on cholera toxin s gene expression 42 Effects of cerium oxide nanoparticles on bacterial outer membrane permeability and their potential application as antibiotic adjuvant in MDR pathogenic bacteria Effects on cell viability, growth and morphology of C. albicans SC5314 biofilms after kinpen 09 plasma treatment 45 Engineering of Next-Generation Antimicrobial Peptides (AMPs) I: mechanisms behind how AMPs act and how bacteria develop resistance against them 46 Engineering of Next-Generation Antimicrobial Peptides (AMPs) II: next generation AMPs with high bactericidal activity and low susceptibility to bacterial 47 resistance Essential oils of wild Algerian endemic Lamiaceae as antibacterial against opportunistic bacteria involved in nosocomial infections 48 Estimation of Non-Specific Immunity of Patients with Patients with Inflammatory Diseases of Large Salivary Glands Evaluation of antibacterial properties of copper alloys surfaces in long-term geriatric care facilities 51 Extensive drug resistant (XDR) Acinetobacter baumannii in coronary care units of a hospital in Kuwait 52 Fmoc Chemical Synthesis of a Cyclotide with Antibacterial Activity against Aquaculture Pathogens 53 Garlic essential oil as an antimicrobial agent towards mycotoxigenic fungi 54 Graphene-based Oleo-polyurethane Nanocomposites with Biocidal Function as Active Food Packaging Materials: Synthesis and Mechanism of Antimicrobial Action 55 High prevalence of extended-spectrum β-lactamase producing Enterobacteriaceae among clinical isolates from cats and dogs in Switzerland 56 How different sterols contribute to cytotoxic action of sea cucumber saponins 57 Identification of compound with microbial activity extracted from the cyanobacteria Arthrospira maxima 58 In vitro Efficacy of Ocimum Basilicum Essential oil extracted by Supercritical Carbon Dioxide on Lactococcus garviae. Preliminary Results 59 Insects novel source for antimicrobial natural products 60 Insight into the metabolomics inventory and exploring metabolic adaptation of Streptococcus pneumoniae to antibiotic stresses 61 Investigating levels of evolved Arg 10 -teixobactin resistance in methicillin-resistant Staphylococcus aureus 62 Involvement of Oligopeptidase B in Bacterial Resistance against the Antimicrobial Peptide Bac7 63 Low-molecular amphiphilic compounds regulation of pore-forming activity of cecropins 64 Methicillin resistant Staphylococcus aureus (MRSA) in raw buffalo milk from Italy: preliminary results 65 Microorganisms in nurses hands, clinical uniforms, and pocket medical devices 66 Mo-W-O system: A study on the mechanisms of antibacterial action via protons release 67 MRSA in horses and humans working in contact with them 68 II

5 New antibiotic leads targeting bacterial RNA polymerase holoenzyme assembly identified with a BRET drug discovery platform 69 New antimicrobial compounds from microorganisms 70 Photo-inactivation of Gram-positive and Gram-negative bacteria by an octahedral molybdenum cluster complex 71 Potential uses of genetically engineered bacteriocins 72 Quorum sensing and Quorum quenching in Pseudomonas spp.: role of antibiotics as quorum quenchers at subclinical concentrations 73 Remineralization effect of hydroxyapatite and its potential for substituting fluoride in dentifrice 74 Study of the chemical component and evaluation of essential oils of Daucus carota (Apiaceae) and Chenopodium ambrosioïdes (Chenopodiaceae) 75 Surface-Active, Nonleaching Antimicrobial Boron-Modified Hyperbranched Polyurethane 76 Synergistic antibacterial activity of phenolic compounds with commercial antibiotics 77 Synthesis and in-vitro biological activity of some novel 2-pyazinecarboxylic acid derivatives 78 The effects of adequate education through the implementation of antibiotic stewardship programs in long-term care facilities on successful reduction of antibiotic usage, antibiotic resistance, and 79 Clostridium difficile infections Typing Burkholderia cepacia complex isolated from non-cystic fibrosis patients in Kuwait 80 Ureolitic and arginolitic activity of dental plaque and saliva and the influence over the bacteria abundance in samples taken from children with or without caries 81 Use of marine and freshwater cyanobacteria as biological control agents in agriculture 82 Vancomycin loaded superparamagnetic MnFe 2 O 4 nanoparticles coated with PEGylated chitosan to enhance antibacterial activity 83 Winery waste as an antimicrobial and antioxidant additive in packaging 84 Φ of Streptococcus pyogenes is a Functional Bacteriophage carrying the macrolide efflux gene pair mef(a)/msr(d) 85 III

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7 INTRODUCTION This book contains a selection of the abstracts that were accepted for presentation at the V International Conference on Antimicrobial Research (ICAR2018), which was held in Torremolinos, Málaga, Spain, from 24 th to 25 th May, The ICAR conference series aims at establishing as a key forum in Europe for the presentation, exchange and dissemination of information and experiences on anti-microbe strategies. "Anti" is here taken in the broadest sense as "against cell cycle, adhesion, or communication", when harmful for the human health, industry or economy (infectious diseases, chemotherapy, food, biomedicine, agriculture, livestock, biotechnology, water systems...). It also covers topics on antimicrobial resistance, (early) microbial and resistance detection, enhancement of innate defenses against pathogens, as well as methods and techniques. The organization called for research papers dealing with the following topics (related on either bacteria, fungi, microbial parasites or viruses): Antimicrobial natural products I - Peptides: Antimicrobial peptides. cyclic lipopeptides Antimicrobial natural products II - Terrestrial and marine organisms: Antimicrobial substances from terrestrial and marine organisms. Essential oils. Bioactive phytochemicals. Plant/Herbal extracts Biocontrol. Biosynthesis of antimicrobials: Microbial-derived toxins. Bacteriocins (colicins, microcins, lantibiotics ). Archaeocins. Biocontrol approach to microbial invasions (probiotics, lactic acid bacteria ). Biosynthesis of antibiotics. Genetic and metabolic engineering. Gene regulation Bacteriophages: Phage therapy and biocontrol in humans, animals (agriculture-farm animals, aquaculture), plants, food industry Materials functionalization with bacteriophages. Using bacteriophages for microbiological detection... Biofilms: Biofilm formation, control and eradication. Microbial adhesion to surfaces. Novel characterization techniques. Biofouling. Biofilms susceptibility to antimicrobials. Antibiotic resistance of microorganisms in biofilms. Genomics and Proteomics... Antimicrobial materials science and surface chemistry. Antimicrobials in consumer products: Antimicrobial, anti-adhesive surfaces & coatings. Microbial adhesion to surfaces. Physical and chemical (inorganic (e.g. silver, copper compounds) and organic) surface modification. Cationic surfaces. Functionalization strategies for polymers, metals, metal oxides, ceramics. Drug-eluting concepts... Antimicrobial chemistry: Synthesis and screening of novel chemical compounds for antimicrobial action. Natural, synthetic and semi-synthetic antibiotics. Analogs. Structural determination. In-silico/ab-initio/de-novo antimicrobials discovery. New targets for antimicrobials. Rational design of antimicrobials. Bioinformatics and comparative genomics for the identification of antimicrobial targets V

8 Non-antibiotic biocides: Disinfectants, antiseptics, preservatives Mechanism of action. Resistance to non-antibiotic biocides. Combination of physical and chemical treatments. Hygiene and Sterilizing. Sanitizers. Regulatory issues. Good practices Antimicrobial physics: Exploitation of physical properties for killing/inactivating microbes: surface tension (nanoemulsions), radiation, ultrasounds, temperature, specific properties of nano-materials (nano-particles, nanotubes/wires, nano-crystals, nano-grained materials ). Resistance to physical agents... Clinical and medical microbiology, infectious diseases and antimicrobials. Public health Strengthening of innate immune system as antimicrobial strategy: Immunotherapy, immunomodulating agents, cytokines (interleukins, colony-stimulating factors, interferons ), hormones Novel vaccines for preventing or treating disease Antimicrobial resistance. Mechanisms of action of antimicrobial agents: Microbial resistance to antibiotics and biocides. Resistance genes. Prevention of resistance. Surveillance & statistics. Genetics and Proteomics Inhibitors of bacterial cell wall biosynthesis. Inhibition of protein biosynthesis. Inhibition of nucleic acid biosynthesis. Alteration of cell membrane function. Inhibition of cell metabolism (antimetabolites). Superbugs. Multi-resistant strains. Emerging and re-emerging bacteria and fungi in humans, animals, and plants. Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin Intermediate/Resistant Staphylococcus aureus (VISA/VRSA), Clostridium difficile, Mycobacterium tuberculosis, Vancomycinresistant Enterococcus (VRE), Cryptosporidium, Plasmodium parasites, Plasmodium falciparum, Leishmania species, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, Cryptococcus, Escherichia coli O157:H7, Helicobacter spp., Enterobacter sakazakii, Serratia spp., Pseudomonas aeruginosa, Fluoroquinolone-Resistant Pseudomonas aeruginosa (FQRP)... Attenuation of virulence as antimicrobial strategy: Interfering microbe-microbe communication (quorum sensing). New strategies... Techniques and Methods: Susceptibility Testing. Rapid microbial and resistance detection. Detection of antibiotics in environmental samples. Microscopy, microanalysis & spectroscopy, single-cell studies, highthroughput studies, nanomechanical studies, microfluidics, lab-on-a-chip concepts, miniaturized science, analysis of microbial surfaces, heterogeneity, statistics. Interaction of antimicrobial drugs with model membranes. Analytical techniques We hope readers will find the content of this fourth edition of the conference inspiring and stimulating and look forward to seeing another fruitful edition in A. Méndez-Vilas ICAR2018 General Coordinator Formatex Research Center C/Zurbarán 1, Planta 2, Oficina Badajoz Spain VI

9 Medical devices decorated with stimuli-responsive polymers for contact-killing surfaces and antimicrobial competitive release Carmen Álvarez-Lorenzo* Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, R+DPharma Group (GI-1645), Facultad de Farmacia and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain * Corresponding author: carmen.alvarez.lorenzo@usc.es The use of medical devices is associated to an inherent risk of infection. Prosthetic heart valves, catheters, hip prosthesis, vascular grafts, sutures, and contact lenses, among many other common implantable or insertable devices, are particularly prone to adhesion of certain host proteins soon after implantation, which in turn favors the subsequent bacteria adhesion and proliferation until biofilm formation by single or mixed species. These medical devices are indeed considered as a heaven for opportunistic bacteria and are responsible of almost 50% of nosocomial infections, which in turn have an impact on patients morbidity and mortality. Decoration of medical devices with polymers that modify their features as a function of the level and nature of the surrounding microorganisms can endow medical devices with improved performance against biofilms. Strong efforts are being made to design stimuli-responsive polymers that expose contact-killing groups when microorganisms try to adhere, and bioinspired nanostructures that recognize microorganisms for triggered (competitive/affinity-driven) drug release [1]. Relevant examples of both approaches will be presented. Prophylaxis and treatment of infections may benefit from polymers that are responsive to the unique changes that microbial growth causes in the surrounding environment or that even recognize the microorganism itself or its Quorum Sensing signals may offer novel tools prophylaxis and treatment of health-care related infections. [1] C. Alvarez-Lorenzo, C.A. Garcia-Gonzalez, E. Bucio, A. Concheiro. Stimuli-responsive polymers for antimicrobial therapy: drug targeting, contact-killing surfaces and competitive release. Expert Opinion on Drug Delivery 13, (2016). VII

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11 A new look into an old natural biopolyester - the antimicrobial activity and chemistry of suberin Rubén Rodrigues, Vanessa G. Correia, Artur Bento, Joana Pais, Alistair W.T. King and Cristina Silva Pereira 1 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, , Oeiras, Portugal 2 Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, A.I. Virtasen aukio 1 (Chemicum), PL 55, University of Helsinki, Finland * Corresponding author: spereira@itqb.unl.pt Suberin, a complex biopolyester built essentially from poly-functional long-chain fatty acids and glycerol, can be found in specialized plant cell walls [1-3]. Our team has established a unique system that allows recovery of suberin near its native state. This system uses the ionic liquid cholinium hexanoate in the dual roles of solvent and catalyst [4-7]. The catalyst mediates specifically a mild hydrolysis of acylglycerol ester bonds in the polymer, whilst linear aliphatic ester bonds are preserved [8]. Suberin-moieties can spontaneously self-assemble recreating the polyester-defensive barrier in plant, hence a water-proof, bactericidal and anti-biofouling material [9]. Inspired by the above, we are giving a new look into the bioactivity and the chemistry of this old polymer, essentially as to establish knowledge grounds to push for the development of functional suberin-based materials. Sequential centrifugation allowed the recovery of suberin moieties with low polydispersity. As opening studies our focus was to characterize the near-native suberin antimicrobial activity by MIC determination against both S. aureus and E.coli bacterial cells as well as by morphological evaluation of Aspergillus sp. after contact with the polymer. Linking the biological activity with the suberin chemistry functionalities is a critical challenge with interesting results - a suite of spectroscopic methods (liquid-state 1D- and 2D-NMR) were applied successfully in this polymer for the first time. Collectively our observations are revealing secrets that the old polymer suberin still conceals. Keywords: antimicrobial natural polymers; bacteria; fungi; suberin Acknowledgement We acknowledge funding from the European Research Council through grants ERC-2014-CoG and FLIPT , and to Fundação para a Ciência e Tecnologia (FCT) through grants UID/Multi/04551/2013 (Research unit GREEN-it "Bioresources for Sustainability") and PTDC/AGR-TEC/1191/2014. RR is grateful to FCT through the grant SFRH/BD/110467/2015. [1] Graça. Suberin: the biopolyester at the frontier of plants. Frontiers in Chemistry : [2] Kolattukudy. Bio-polyester membranes of plants - cutin and suberin. Science. 1980; 208: [3] Schreiber. Transport barriers made of cutin, suberin and associated waxes. Trends in Plant Science. 2010;15: [4] Petkovic, Ferguson, Gunaratne, Ferreira, Leitão, Seddon, Rebelo & Silva Pereira. Novel biocompatible cholinium-based ionic liquids-toxicity and biodegradability. Green Chemistry. 2010; 12: [5] Ferreira, Garcia, Sousa, Petkovic, Lamosa, Freire, Silvestre, Rebelo & Silva Pereira. Suberin isolation from cork using ionic liquids: characterisation of ensuing products. New Journal of Chemistry. 2012; 36: [6] Garcia, Ferreira, Petkovic, Ferguson, Leitão, Gunaratne, Seddon, Rebelo & Silva Pereira. Dissolution of cork biopolymers in biocompatible ionic liquids. Green Chemistry. 2010; 12: [7] Ferreira, Garcia, Sousa, Freire, Silvestre, Rebelo & Silva Pereira. Isolation of suberin from birch outer bark and cork using ionic liquids: A new source of macromonomers. Industrial Crops and Products. 2013; 44: [8] Ferreira, Garcia, Sousa, Guerreiro, Duarte, Freire, Calhorda, Silvestre, Rebelo & Silva Pereira. Unveiling the dual role of the cholinium hexanoate ionic liquid as solvent and catalyst in suberin depolymerisation. RSC Advances. 2014; 4: [9] Ferreira, Garcia, Martins, Sousa, Freire, Silvestre, Kunz, Rebelo & Silva Pereira. Ex-situ reconstitution of the plant biopolyester suberin as a film. Biomacromolecules. 2014; 15:

12 A novel Pseudomonas strain capable to produce antifungal substances Zaki Saati-Santamaría 1,2, Miroslav Kolarik 3, Raúl Rivas 1,2 and Paula García-Fraile 1,2,3 1 Microbiology and Genetics Department. University of Salamanca. Doctores de la Reina SN, Salamanca, Spain 2 Spanish-Portuguese Institute for Agricultural Research (CIALE), Salamanca, Spain 3 Institute of Microbiology of the CAS, v. v. i., Průmyslová 595, Vestec, Czech Republic Since the beginning of the use of antifungal drugs to treat human illnesses, two problems have been shared its development. First of all, its intrinsic adverse reactions, as well as nefrotoxicity, among others, of this group of drugs that cause the failure of many treatments(1). On the other hand, it is increasing the role of resistances acquired by fungi due to its incorrect use. Taking into account these circumstances, it is crucial to find new antifungal substances which could act as a reservoir to use when resistances appear, as well as they could have less important adverse reactions. In this work we isolated bacterial strains from an unexplored niche, bark beetles, which could be a source of novel bacteria or compounds with antifungal activities. Indeed, we found an undescribed Pseudomonas strain (named IA19) which can inhibit the growth of filamentous fungi (Aspergilus sp., Fusarium sp.), and not filamentous Candida sp., and Pichia sp. by cross streak method. Once known the potential of this strain, we mined its genome. The draft genome was obtained using Illumina MiSeq platform and assembled by Velvet Gene calling and annotation was performed using RAST(2). The study of the genome was carried out by The SEED viewer framework(3) and antismash 3.0(4). This showed up the potential of this strain to produce some substances similar to already known antifungal compounds, as well as bromopyrroles, caryoynencin, and PM PM100118, besides other antimicrobial and antiviral compounds. Moreover, we found some clusters of genes related with the production of non-described secondary metabolites, which could be the responsible of the observed activity against fungus. Keywords: Genome mining; secondary metabolites [1] Girois SB, Chapuis F, Decullier E, Revol BGP. Adverse effects of antifungal therapies in invasive fungal infections: review and meta-analysis. Eur J Clin Microbiol Infect Dis. 2006; 25(2):138. [2] Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008;9:75. [3] Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014;42(D1):D [4] Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, et al. antismash 3.0 a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015;43(W1):W

13 Advancing a new class of methionyl-trna synthetase (MetRS) antibiotics Mansour Bassiri 1, Michael Goodson 2 and Richard Gless 1 1 Bioxiness Pharmaceuticals, Inc. 750 Alfred Nobel Drive, Suite 106, Hercules, CA 94547, USA 2 One Shields Avenue, University of California, Davis, CA 95616, USA Bioxiness Pharmaceuticals, Inc. (BXN) has synthesized a library of small molecule inhibitors of methionyltrna synthetase (MetRS). The Bioxiness technology uses a novel tri-partite scaffold that combines a (1) head group, a warhead that substitutes hydroxamic acid for the methionine carboxyl group, (2) modification(s) to methionine sidechain, and (3) an amino acid tail attached to the alpha-amino group of methionine, to facilitate membrane transport. Although methionine hydroxamate without the amino acid tail was shown previously to inhibit MetRS (Lee et al, 1998), the unmodified warhead did not enter cells efficiently. Bioxiness compounds are designed to enter the cell and inhibit aminoacylation step by MetRS, thereby disrupting the first critical discriminatory step in initiation of protein synthesis and also the elongation of protein synthesis, where methionine is also required. Using molecular docking, and a structure activity relationship (SAR) approach, we synthesized ~ 80 firstgeneration compounds and obtained broad spectrum antimicrobial activity (measured as MIC) against gramnegative strains Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and a drug-resistant strain of Streptococcus pneumoniae (ATCC 49619), and gram-positive strains Staphylococcus aureus, S. epidermis and Bacillus subtilis. Of the pathogens considered serious by the CDC-Threat Report, we have shown activity against drug resistant E. coli and N. gonorrhea. Our initial screening panel includes gram-negative bacteria that represent a public health risk, and drug-resistant strains that must be addressed per FDA guidance as well as strains of interest to the US military. Other in vitro studies showed that Bioxiness prototype compounds: (i) are active in whole cells assays at about 2-64 µg/ml when all three components - warhead, sidechain and peptide transporters are integrated; (ii) engage target MetRS with a binding affinity of approximately 300 μm (IC50), cf. natural methionine which binds at μm (Km value); (iii) are selective for the bacterial MetRS enzyme; (iv) show low cytotoxicity in human cells; (v) have no detectable metal-chelating effects on Fe and Zn; and (vi) perhaps most importantly, are not subject to efflux, and (vii) most recently in E. coli infected mice skin infection model, demonstrated statistically significant reduction of bacteria as compared to infected, but untreated animals (controls). Bioxiness compounds are synthetic, water soluble, and relatively easy to produce. Bioxiness Pharmaceuticals, Inc. recently closed on $700K of a $1M financing co-led by Life Science Angels (Palo Alto, CA) and Launch Capital (New Haven, CT) and syndicated with additional Angel Network members. The company s Series A milestones are to (1) optimize its library (ICAGEN, Tucson, AZ) by screening compounds in vitro for potency, effectiveness and low cytotoxicity (Micromyx, Kalamazoo, MI), then (2) identify 2-4 lead compounds in a series of in vivo studies (University of North Texas, Fort Worth, TX). The company s pro forma therapeutic product profile is an intravenous formulation to address in-hospital gramnegative infections. Keywords: Gram-negative bacteria; Antibiotic resistant; Methionyl-tRNA Synthetase [1] MA Deniziak, J Barciszewski, Methionyl-tRNA synthetase. Acta Biochemica (2001) [2] Ref: Lwis, K, New approaches to antimicrobial discovery. Biochemical Pharmacology (2017) 3

14 Aeromonas production of antimicrobial substance active against clinical resistant strains S. Silva, F. Guedes, S. Mederos, F.S. Diniz, J.R. Ribeiro, F. Thompsom, A. Macrae and S.S. Oliveira Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, CCS, UFRJ, Rio de Janeiro, Brasil Aeromonas are Gram-negative rod shaped facultative anaerobe bacteria that can be found in aquatic environments, human intestinal tract and in some cold blooded animals. They are considered opportunistic pathogens for humans and their infections can be severe. They are generally pathogenic to fish and have economic implications. The occurrence of bacterial antibiotic resistance in strains isolated from the environment and from clinical samples has been reported in the literature and also by our group. This is cause for concern and highlights the need to prospect for new antimicrobial substances. In this study, we analyzed 78 Aeromonas strains, isolated from water and fish, sampled from two large bays (Guanabara and Sepetiba) and an urban lagoon (Rodrigo de Freitas) from Rio de Janeiro. We evaluated the production of antimicrobial substances by seeding the bacterial isolates in spots on Petri dishes. After incubation, the cells were killed with chloroform and the indicator strain pour plated. Incubations were all done at 30oC for 18 hours. Strains of different genera, representing the Gram-negative, Gram-positive and clinical strains were used as indicators. Evidence of antimicrobial substances was found in 39 strains. Antibiotic activity against important pathogens highly resistant to antibiotics including Klebsiella pneumoniae KPC, Escherichia coli ESBL, Acinetobacter baumannii and Pseudomonas aeruginosa SPM was detected. The chemical nature of the substances that caused these activities is being investigated. Preliminary analyses have shown that inhibition zones detected were resistant to neutralization with NaOH and to pronase treatment. One strain that presented a broad inhibition spectrum was chosen for DNA purification and genome sequencing. Among the putative proteins, 13 sequences linked to bacteriocin metabolism, were found. The use 16S comparisons and in silico DNA-DNA hybridization identified the producer strain as Aeromonas allosaccharophila. Keywords: Aeromonas; bacteriocins; nosocomial strains; antimicrobial substances 4

15 Ag Bi nanoalloys: Synthesis by femtosecond laser irradiation and antibacterial activity against MRSA and MSSA J. Andrés ; C.C. De Foggi ; T. R. Machado ;M. Assis ; M. Oliva ; G. Minguez-Vega # ; E. Condoncillo $ ; H. Beltran-Mir $ ; C. Doñate-Buendia # ; N.G. Macedo ; M.M. Teixeira and E. Longo CDMF, LIEC, Chemistry Department of the Federal University of São Carlos (UFSCar), P.O. Box 676, São Carlos, SP, Brazil Department of Analytical and Physical Chemistry, University Jaume I (UJI), Castelló 12071, Spain # GROC UJI, Institut de Noves Tecnologies de la Imatge (INIT, University Jaume I (UJI), Castelló 12071, Spain. $ Department of Inorganic and Organic Chemistry, University Jaume I (UJI), Castelló 12071, Spain. Ag Bi nanoalloys is an emerging material which attracted interest due to its superior photocatalytic H 2 production from water splitting, degradation of organic pollutants in wastewater and nitric oxide (NO) [1;2]. It is well-known that femtosecond (Fs) laser irradiation elicit physical-chemical interactions which may result in the emergence of changes in the macroscopic behavior of materials due to internal modifications. The aim of this study is to evaluate the potential use of Fs laser irradiation on Ag Bi nanoalloys (Ag 2 WO 4 pure; NaBiO 3 pure and Ag 2 WO 4 /NaBiO 3 ) and its antibacterial activity against methicilin susceptible (MSSA) and resistant (MRSA) Staphylococcus aureus. The microorganisms were grown in TSB culture medium until reaching the mid-log phase of growth. Then, the concentration of the inoculum was standardized spectrophotometrically in 10 8 cells per ml. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined according described by Clinical and Laboratory Standards Institute. Confocal laser scanning microscopy (CLSM) was used to observe the living and dead cells, after marking with the Live and Dead BacLight, according manufacturer s instructions. The MIC found against MRSA was the same for Ag 2 WO 4 and Ag 2 WO 4 /NaBiO 3 (3.91 g /ml). For the susceptible strain, Ag 2 WO 4 (1.95 g /ml) was more efficient than Ag 2 WO 4 /NaBiO 3 (3.91. g /ml). Although NaBiO 3 showed no antibacterial activity against any of the strains tested, when associated to Ag 2 WO 4, it presented MIC similar to pure Ag 2 WO 4. CLSM micrographs corroborate to MIC/MBC results. Although NaBiO 3 did not present antibacterial activity, its association to Ag 2 WO 4 promoted similar results compared to pure Ag 2 WO 4. Significant reduction of live cells was observed for both bacteria when treated with Ag 2 WO 4 and Ag 2 WO 4 /NaBiO 3. 1 Z. Jiao, Y. Zhang, S. Ouyang, H. Yu, G. Lu, J. Ye and Y. Bi, ACS Appl. Mater. Interfaces, 2014, 6, J. Gong, C. Lee, Y. Chang and Y. Chang, Chem. Commun., 2014, 50,

16 Allyl isothiocyanate and cinnamaldehyde against mycotoxigenic fungi in vitro and their combined use in stored corn K.C.P. Bocate 1, A.G. Evangelista 1, G. Meca 2 and F.B. Luciano 1 1 Graduate Program in Animal Science, Pontifical Catholic University of Paraná, R. Imaculada Conceição, 1155, , Curitiba, Brazil 2 Laboratory of Food Toxicology, University of Valencia, Av. Vicent Andrés Estelles, s/n, 46100, Valencia, Spain It is estimated that 25% of worldwide production of grains is contaminated with mycotoxins. According to the International Agency for Research on Cancer (IARC), mycotoxins can be classified as carcinogenic, nephrotoxic, hepatotoxic, and teratogenic. Alternative measures have been proposed to reduce the growth of mycotoxigenic fungi and the production of toxins, especially natural compounds. Allyl isothiocyanate (AITC) and cinnamaldehyde, which are the active ingredients of mustard and cinnamon essential oils, respectively, have shown antifungal activity in previous studies. As natural compounds, their use can attend the commercial demand as substitutes of traditional preservatives that are chemically synthesized. Thus, the focus of this study was to evaluate the efficacy of AITC and cinnamaldehyde against the growth of Aspergillus parasiticus CECT 2681, Fusarium verticillioides CECT 2983, and Giberella zeae CECT 2150 producers of aflatoxins (AFs), fumonisins and zearalenona, respectively in culture media and also by fumigation of contaminated corn kernels. The minimum inhibitory concentration (MIC) of each compound was determined by microdilution for the three mycotoxigenic species, with subsequent determination of the interaction between the natural compounds. Evaluation of growth inhibition (halo diameter in petri dishes containing potato dextrose agar) was also performed for these compounds after volatilization in hermetic glass jars, which were kept at 25 o C for 7 d. The MICs were used as the basis for the application of AITC and cinnamaldehyde in corn kernels contaminated by either of the fungal species and maintained in hermetic glass jars at 25 o C. Fungal population and aflatoxin production were evaluated after 30 d. In culture media, concentrations of 7.81, 31.25, and μl/l of AITC, and 31.25, 250, and 62.5 μl/l of cinnamaldehyde were found as the MIC for A. parasiticus, F. verticillioides, and G. zeae, respectively. When used together, 3.91 μl/l of AITC and 7.81 μl/l of cinnamaldehyde were required to inhibit A. parasiticus; 3.91 μl/l of AITC and 250 μl/l of cinnamaldehyde for F. verticillioides; and 7.81 and μl/l to inhibit G. zeae. Combinations of 0.62, 0.47, and 0.23 μl/l (ratio 1 AITC : 4 cinnamaldehyde) were able to significantly reduce growth of A. parasiticus, G. zeae, and F. verticillioides respectively, with total inhibition at 1.25, 0.94, and 0.94 μl/l. The same combinatory ratio was used to evaluate the effect of the oils in corn kernels against the mycotoxigenic fungi. The combined doses used were 30 and 300 μl/l. Significant reduction was found at 30 μl/l for all fungal species. Moreover, 300 μl/l reduced the population of all species below the detection limit (DL = 1.66 Log UFC/g). Aflatoxin production was also inhibited by the natural formulation, where the control group presented ppb of total AFs, 30 μl/l of AITC/cinnamaldehyde had ppb and 300 μl/l of AITC/cinnamaldehyde ppb. These results confirm the antifungal action of both natural bioactive compounds analysed, demonstrating their effectiveness against mycotoxigenic fungi growth. Therefore, AITC and cinnamaldehyde may be a viable alternative to the traditional chemicals used for the application in stored corn to avoid fungal contamination and mycotoxin production. Keywords: Natural antifungals; Bioactive natural compounds; Gaseous antimicrobials; Stored grains 6

17 An innovative, electrochemical in situ ozone generator B. Behrendt-Fryda, D. Brackemeyer, A. Hampel, L. Heesch, C. Li, D. Mathiak, O. Schumacher and M. Fryda CONDIAS GmbH, Fraunhoferstrasse 1b, Itzehoe, Germany The need to use clean water for everyday use will play an increasingly important role in the future. In general, there are many possibilities for water extraction and water recycling. The production of disinfectants by electrolysis is also established in some industrial applications, especially the chlorine electrolysis is used, which now has the disadvantage of resistance in biofilms. We would like to present a new concept of ozone production. The ozone is produced by an in situ EAOP process (electrochemical advanced oxidation process). The boron doped diamond electrodes (BDD) can be used in different water matrices, but it is wrong to think that simple electrolysis with anode and cathode alone is successful. Central know how here is the interaction of cell design and the understanding of the function of the associated technology. Since the process can be adapted to the environment, a precise adjustment of the ozone concentration for the disinfection of contaminated water is important. In this context we would like to introduce a tool that is in a prototype phase. By simulation and specification of the water matrix to be treated, a prototype could be constructed which is capable of direct disinfection considering the limit values of the German drinking water and WHO regulation. Fig. 1 Reduction factor of microorganism vs in situ dissolved ozone concentration. Figure 1 shows high efficiency of reduction factor for several microorganism depending on in situ ozone concentration. Furthermore we show MIKROZON ozone generator, specific process parameters as well as data of by-product risk [1]. Keywords: in situ ozone; EAOP ; simulation; disinfection; electrolysis-by-products [1] Electrochemical disinfection with boron doped diamond electrodes and reduction of electrolysis by-products; October 2015; Disinfection By-products in Drinking Water; Chapter: 16; Publisher: The Royal Society of Chemistry 7

18 Analogous derived from C-terminal salmonids IL-8 as potential antimicrobial agents for infectious diseases P.A. Santana 1 and F. Guzmán Quimbayo 2 1 Instituto de Ciencias Químicas Aplicadas. Facultad de Ingeniería. Universidad Autónoma de Chile. El Llano Subercaseaux 2801, Santiago. Chile 2 Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso (PUCV), Avenida Universidad #330, Valparaíso. Chile Atlantic salmon and rainbow trout are species of national commercial importance that develop diseases produced mainly by Gram-negative bacteria, which result in mayor economic losses. Vaccines and antibiotics are used to control and prevent these diseases, which have not been effective since the over use of antibiotics has lead to the generation of resistant bacteria strains and vaccines only provide partial degrees of protection and more research is needed on its mode of action [1,2]. Therefore, studies have focused on finding new forms of treatments compatible with the environment and in reducing the concentration of antibiotics. The antimicrobial peptides (AMPs) are excellent alternative as therapeutic agents. In salmonids, piscidins, cathelicidins, β-defensins and hepcidins have been described as AMPs [3]. Currently, our research group has identified AMPs derived from the carboxyl-terminal end of the salmonids chemokine IL-8, which have antibacterial properties against Gramnegative bacteria and have attractive structural features that can be used as drug delivery vectors. These vectors are known as Cell-Penetrating Peptides (CPPs). Some AMPs or peptides analogous to these have been described, which share structural features to CPPs and have the ability to translocate across cellular membranes as well as carry drugs [4]. To date, there are no studies on AMPs or peptides analogous to those of salmonids used as potential CPPs. Thus, in this work we identifing by scan-ala the key residues in antimicrobial activity of trout IL-8 derived peptide. Subsequently both the IL-8-derived peptide and its analogues were characterized by circular dichroism spectroscopy. This analysis showed that the peptides has different alpha helix propensity. The antimicrobial activity against Aeromonas salmonicida was determined at 10 to 30 µm peptide concentration. By Scanning Electron Microscope the reference peptide showed the ability to disrupt the bacterial membrane of A. salmonicida. Finally, no cytotoxicity was observed for IL-8-derived peptide. These bioactive molecules are a basis for the design of new drug and their used in the treatment of salmonids infectious disease. Keywords: salmonids; IL-8 chemokine; antimicrobial activity [1] Fortt A. and Buschmann A. (2007). Oceana [2] Maisey K., Montero R. and Christodoulides M., (2017).Expert Review of Vaccines. 16 (3): [3] Masso-Silva J.A. and Diamond G. (2014).Pharmaceuticals. 7 (3): [4] Milletti F. (2012). Drug Discovery Today. 17 (15 16):

19 Antibacterial activity of α-ag 2-2xMxWO 4 (M = Cu 2+ ) Solid Solutions Obtained by Coprecipitation Method E. Longo ; C.C. De Foggi ; P.F.S. Pereira, M. Assis, C.E. Vergani*, and J. Andrés CDMF, LIEC, Chemistry Department of the Federal University of São Carlos (UFSCar), P.O. Box 676, São Carlos, SP, Brazil Department of Analytical and Physical Chemistry, University Jaume I (UJI), Castelló 12071, Spain * São Paulo State University (Unesp), School of Dentistry, Araraquara, SP, Brazil Metal nanoparticles made of one or more elements has attract great interest due to their advantageous properties, as in the case of antimicrobial activity promoted by silver (Ag) and copper (Cu). Ag 2 WO 4 associated with Cu was synthesized by coprecipitation method, as previously described [1]. The minimum inhibitory (MIC) and bactericidal (MBC) concentration were determined against methicillin-resistant Staphylococcos aureus (MRSA, ATCC 33591), accordingly to Clinical and Laboratory Standards Institute (CLSI M7-A7). The samples tested had different Cu 2+ proportions in their structure. DRX patterns data indicated that all the samples obtained experimentally presented orthorhombic structures. MR and FT-IR spectroscopies confirmed that all vibrational modes are characteristic of the orthorhombic structure. FE-SEM images revealed that the incorporation of Cu 2+ in the α-ag 2 WO 4 structure affected the shape and size of α-ag 2 WO 4 microcrystals and shifted the electronic and optical properties. The incorporation of Cu 2+ to α-ag 2 WO 4 increased the antibacterial activity in a dosedependent manner, suggesting a synergistic effect of Ag and Cu against MRSA cells. The MIC found were: 500 g/ml for Ag 2 WO 4, Ag 2 WO 4 with 0.5% and 1%Cu; 250 g/ml for Ag 2 WO 4 with 2% and 4% Cu; 125 g/ml for Ag 2 WO 4 with 8% and 10%Cu; and g/ml for Ag 2 WO 4 with 16%Cu. Even at sub-inhibitory concentrations, all materials reduced the viability of MRSA cells. We conclude that incorporation of Cu to Ag 2 WO 4 microcrystals has potential to enhance the antibacterial properties of Ag 2 WO 4. 1 P. F. S. Pereira, C. C. Santos, A. F. Gouveia, M. M. Ferrer, I. M. Pinatti, G. Botelho, J. R. Sambrano, I. L. V. Rosa, J. Andrés, and E. Longo. Inorg. Chem., 2017, 56,

20 Antibiotic potential of defense peptides from wild cereals E.A. Rogozhin 1,2 and A.N. Smirnov 3 1 Department of Molecular Neurobiology, M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya Str., Moscow, Russian Federation 2 Department of Chemical Study of Biologically Active Compounds of Microbial Origin, G.F. Gause Institute of New Antibiotics, 11 Bol shaya Pirogovskaya Str, , Moscow, Russian Federation 3 Departmant of Plant Protection, K.A. Timiryazev Russian State Agrarian University, 49 Timiryazevskaya Str., , Moscow, Russian Federation The inhibitory action of peptide complexes from barnyard grass (Echinochloa crusgalli), lyme grass (Leymus arenarius) and couch-grass (Agropyron repens) seeds belonging to some RP-proteins classes (defensins, alphahairpinins, lipid-transfer proteins, protease inhibitors from Bowman-Birk and bifunctional inhibitors of trypsin/alpha-amylase from cereals) was evaluated against a set of plant pathogenic (from Fusarium genus) and animal pathogenic (from Aspergillus genus) filamentous fungi. The alveolar method was used and the bactericidal effect against Gram-Positive bacterium Staphylococcus aureus using flow cytometry and photometry was measured. A preliminary antifungal effect against collection cultures of Aspergillus spp. (A. oryzae, A. niger, A. terreus, A. nutans) and Fusarium spp. (F. solani and F. graminearum) was demonstrated, as expressed by quantitative suppression of hyphal growth and spore germination. Statistically reliable bactericidal action was demonstrated by the EcLTP peptide from E. crusgalli and a complex of high homologous defensins from L. arenarius. The data indicate that the wild cereal studied could be a potential donor of high-active polypeptides for protection of cultivated monocotyledonous plants from diseases caused by specific fungal pathogens. On the other hand, the cereal could also be a source of next-generation natural peptide antibiotics. Acknowledgement This study was supported by Russian Foundation for Basic Research (project mol_a_dk). Keywords: plant antibiotics; wild cereals; antimicrobial activity; peptides 10

21 Antibiotic resistance ABCF proteins reset the peptidyl transferase center of the ribosome to counter translational arrest V. Murina 1,2, M. Kasari 1, V. Hauryliuk 1,2,3,* and G.C. Atkinson 1 1 Department of Molecular Biology, Umeå University, Umeå, Sweden 2 Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden 3 University of Tartu, Institute of Technology, Tartu, Estonia Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we have dissected the molecular mechanism of Staphylococcus haemolyticus VgaA LC and Enterococcus faecalis LsaA on the ribosome [1]. We demonstrate that VgaA LC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ 2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ 2 mutant, is the extended inter-abc domain linker region. Deletion of the linker region renders wild type VgaA LC inactive in antibiotic protection and the EQ 2 mutant inactive in PTC inhibition. Keywords: ribosome; ABCF; Staphylococcus; Enterococcus [1] V. Murina, M. Kasari, V. Hauryliuk, G. C. Atkinson, Nucleic Acids Research, 2018, accepted. 11

22 Antibiotic resistance mediated by integrons in Pseudomonas spp. genus A. do Brito, M. Robas Mora 1, A. Probanza 2 and P.A. Jiménez Gómez 1 1 Section of Microbiology, Facultad de Farmacia; Departamento de Ciencias, Farmaceúticas y de la Salud, Universidad San Pablo CEU, Campus de Montepríncipe, Ctra. Boadilla del Monte, Km 5.300, Boadilla del Monte, Madrid, Spain 2 Section of Plant Biology, Facultad de Farmacia; Departamento de Ciencias, Farmaceúticas y de la Salud, Universidad San Pablo CEU, Campus de Montepríncipe, Ctra. Boadilla del Monte, Km 5.300, Boadilla del Monte, Madrid, Spain Gram negative bacteria, where Pseudomonas genus are included, constitute, nowadays, an increasing hazard to human and animal health, due to their ability to accumulate Pan-resistance (PDR) or Extensive-resistance (XDR). This means that resistance against all (or substantially all) of the today available antibiotics, respectively [1]. It s estimated that, in the European Union more than annual deaths are caused by the inefficacy of traditionally used antibiotics against multi-resistant bacteria. This fact reveals the magnitude of the risk that human and animal Medicine assumes, compromising goals achieved thanks to modern Medicine in the past few decades. Considering all this, as well as the great impact of antibiotic resistance (AR), it is a priority to make a rational use of them. Thus, several international organisms have published numerous documents intended to promote a general consensus on AR, with the main aim of establishing programs at a national and supranational scale with a holistic approach. However, it s not yet been implemented as generalized and coordinated measures likely to increase the chances of success [2]. Genetic recombination plays an essential role in modulating microbial genome plasticity, as it enables genetic exchange, as well as allowing the viability of the non-replicative DNA acquired by bacteria. Pseudomonas aeruginosa bacterial species is a natural porter of the vast majority of the enzymatic and mutational resistance mechanisms described up to date [3]. The acquisition of resistance mechanisms mediated by mobile genetic elements, as integrons, increase significantly the difficulty to treat infectious diseases caused by this species. Due to this fact, that this work reviews the current situation of the most important resistance mechanisms mediated by integrons to antipseudomonic antibiotics, frequently employed in antimicrobial therapy against Pseudomonas aeruginosa, particularly β-lactams and aminoglycosides [4]. Keywords: Pseudomonas aeruginosa; antibiotic resistance; integrons [1] Magiorakos, A.P. et al. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18(3): [2] AEMPS. (2015). Reunión de coordinación del Plan Nacional de Resistencia a los Antibióticos. Nota informativa. Available in: reunionplan-antibioticos-2015.pdf Last visit: 16/04/2018. [3] McGowan, J.E. (2006). Resistance in nonfermenting gram-negative bacteria: multidrug resistance to the maximum. Am. J. Med. 119(6 Suppl 1): S29-36; discussion S [4] OECD. (2013). Health at a Glance: OECD Indicators, OECD Publishing. Available in: Last visit: 16/04/

23 Antifungal properties of silver tungstate (Ag 2 WO 4 ) irradiated by electron and femtosecond laser E. Longo ; C.C. De Foggi ; M. Assis ; A. Kubo ; P.A. Barbugli*; C. E. Vergani* and J. Andrés ; M. Oliva ; G. Minguez-Vega # ; E. Condoncillo, E. $ ; H. Beltran-Mir $ ; R. Torres-Mendieta % CDMF, LIEC, Chemistry Department of the Federal University of São Carlos (UFSCar), P.O. Box 676, São Carlos, SP, Brazil Department of Analytical and Physical Chemistry, University Jaume I (UJI), Castelló 12071, Spain * São Paulo State University (Unesp), School of Dentistry, Araraquara, SP, Brazil # GROC UJI, Institut de Noves Tecnologies de la Imatge (INIT, University Jaume I (UJI), Castelló 12071, Spain. $ Department of Inorganic and Organic Chemistry, University Jaume I (UJI), Castelló 12071, Spain. % Institute for Nanomaterials, Advanced Technologies and Innovation Technical University of Liberec, Studentská 1402/2, Liberec, Czech Republic. Silver tungstate (Ag 2 WO 4 ) is widely used in several areas due to its optical and photoluminescent characteristics, catalytic and photocatalytic properties, apart from its antimicrobial activity [1;2]. The development of technologies that favors modifications of materials properties, such as morphological characteristics, broadly increases its capacity of use, including therapeutics. Our group previously reported the structural modifications of Ag 2 WO 4 through electron beam irradiation and femtosecond laser; in which both methods increased Ag 2 WO 4 antimicrobial activity [3;4]. Electron beam irradiation of α-ag 2 WO 4 provokes the formation and growth of silver (Ag) nanofilaments onto the particle surface [3]. In turn, when α-ag 2 WO 4 is irradiated with femtosecond laser, spherical Ag nanoparticles are formed onto materials surface, removing α-ag 2 WO 4 equilibrium system [4]. Based on the well-known activity of Ag against a broad spectrum of microorganisms, this research focused on studying the antimicrobial potential of Ag 2 WO 4 materials. α-ag 2 WO 4 was synthesized by coprecipitation method and irradiated by field emission gun scanning electron microscope (Ag 2 WO 4 : Electron - 15kV for 5 minutes) and femtosecond laser (Ag 2 WO 4 :NonFocused and Ag 2 WO 4 :Focused - Ti:sapphire laser; 30 fs; 800nm; 1kHz). A laser beam of 6 mm in diameter, at the 1/e2 point, and mean power of 200 mw was focused onto the surface of a pellet target of α-ag 2 WO 4 with a 75 mm lens. In order to obtain the focused sample, an 8mm lens was used. The antifungal effect was determined against Candida albicans ATCC biofilm, and evaluated by colony forming units per ml and confocal laser and scanning electron microscopy. All forms of Ag 2 WO 4 used (Ag 2 WO 4; Ag 2 WO 4 : E; Ag 2 WO 4 :NF and Ag 2 WO 4 :F) showed antibiofilm activity against C. albicans, and the most efficient were Ag 2 WO 4 :NF and Ag 2 WO 4 :F. The samples presented antifungal activity and have potential to be used in different biomaterials. [1] Roca, Roman A., et al. "Formation of ag nanoparticles on β-ag 2 WO 4 through electron beam irradiation: a synergetic computational and experimental study." Inorganic chemistry (2016): [2] de Foggi, Camila C., et al. "Tuning the Morphological, Optical, and Antimicrobial Properties of α-ag2wo4 Microcrystals Using Different Solvents." Crystal Growth & Design (2017): [3] Longo, Valéria M., et al. "Potentiated electron transference in α-ag2wo4 microcrystals with Ag nanofilaments as microbial agent." The Journal of Physical Chemistry A (2014): [4] Assis, Marcelo, et al. "Towards the scale-up of the formation of nanoparticles on α-ag 2 WO 4 with bactericidal properties by femtosecond laser irradiation." Scientific reports 8.1 (2018):

24 Antimicrobial activity assay of Pseudomonas luteola VFB122 MªÁngeles Calvo Torras and Brian Morales Segovia Department of Animal healt and Anatomy. Autonomous University of Barcelona. Travesera dels Turons Cerdanyola del Vallès, Barcelona, Spain From an environmental sampling done through the SAS system of a university classroom and after the development of the colonies, was observed the growth of a colony with inhibitory potential against of the remaining. Through isolation, Gram stain and biochemical tests this strain was identified as Pseudomonas luteola and was deposited in the cultures collection of the Veterinary Faculty of the Autonomous University of Barcelona (VFB) After the identification of the strain, a study was carry out to determine the possible antagonic effect of Pseudomonas luteola VFB 122 against of the following microorganisms: Kocuria rhizophila, Streptococcus sp, Proteus mirabilis, Staphylococcus aureus, Escherichia coli, Salmonella tiphymurium, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, Listeria innocua, Bacillus subtilis and Bacillus cereus. To carry out the study, the following techniques were used to evaluate the antimicrobial activity: two variables of the Kirby-Bauer method and a modification of the Wickerham technique. In all tests two types of culture medium were used: TSA agar and agar to favor the production of antimicrobial metabolites. In the first variable of the Kirby-Bauer method 0.5 McFarland dilutions of P. luteola were made to impregnate the sensidiscs and apply them on the surface of the culture media previously seeded with the problem microorganisms. In the second variable of the Kirby-Bauer method, P. luteola was seeded in a TSA plate and incubated for 24h ± 2h at 37ºC ± 1ºC. Once the incubation time had elapsed, agar discs were made in the same plate by a punch. The agar disks were applied to the surface of the culture media by the growth face with P. luteola. The modification of the Wickerham technique consisted in previously making a streak in each of the culture plates with P.luteola and incubating them during 24h ± 2h at 37ºC ± 1ºC. After the incubation time, 0,5McFarland dilutions of the problem microorganisms were carry out and through swabs three parallel streaks were made perpendicular to the P. luteola in their respective plates. Among the results obtained we can highlight: - In the first variable of the Kirby-Bauer method were observed inhibition halos against Bacillus cereus and Bacillus subtilis. - In the case of the second variable of the Kirby-Bauer method, the halos were of greater diameter and affected a greater number of microorganisms: Kocuria rhizophila, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Streptococcus sp, Bacillus cereus, Listeria monocytogenes and Listeria innocua - In the modification of the Wickerham test, relevant inhibitions were observed against Kocuria rhizophila, Escherichia coli, Staphylococcus aureus and Streptococcus sp. - As to agar medium was observed that the agar medium to favor the production of antimicrobial metabolites showed inhibition halos of a larger diameter than TSA. Keywords: Pseudomonas luteola; Antimicrobial activity; Kirby-Bauer; Wickerham 14

25 Antimicrobial activity of a methanolic extract of Pinguicula moranensis Kunth. R. Olvera-Ramírez, N.B. Medina-Jaritz and E. Rojas Iniestra Plant Physiology Lab., Department of Botany, National School of Biological Sciences,National Politechnic Institute, Prol. Carpio s/n, Mexico City, Mexico The emergence of bacterial strains resistant to current antibiotics is a global concern, which makes it necessary that the search for new antimicrobial compounds be constant. Plants are a potential source of these compounds [1]. The genus Pinguicula is very represented in the Mexican territory, 44 species have been described, most of it distributed in the Sierra Madre Oriental; belongs to the family Lentibulariaceae (Lamiales), the most abundant species and with a very wide distribution is Pinguicula moranensis Kunth, its common name is canyon violet, is a carnivorous plant that lives in forests of oak, pine-oak and mountain mesophyll, in an altitude range of 800 to 3200 m a.s.l.[2]. P. moranensis presents a seasonal dimorphism, during the winter it forms a compact basal rosette with fleshy leaves, small (10-30 X 2-6 mm), tightly pressed; while during the summer it forms a lax basal rosette with leaves extended and reclined on the ground, obovate, elliptical to almost circular, up to 55 to 130 mm long by 23 to 75 mm wide, these leaves are covered with numerous pedunculated glands that secrete mucilage [2]. The mucilage allows the plant to trap and digest small arthropods and prevents the attack of saprobic microorganisms on the prey or phytopathogens [3]. Some authors claim that the most important secondary metabolite of plants of this genus is cinnamic acid [4], a compound with fungicidal properties and necrosant activity and, although it has been shown that some species of Pinguicula have antibacterial properties against Staphylococcus aureus and S. epidermidis [5], no studies have been conducted on the possible antibacterial activity of Pinguicula moranensis. The aim of this study was to evaluate the antibacterial activity of a Pinguicula moranensis methanolic extract. The antimicrobial activity was performed against Staphylococcus aureus (ATCC 15920) and Salmonella typhi (ATCC 6584); for which the Kirby-Bauer diffusion method was used on Müeller Hinton agar plates with paper discs impregnated with the test extract, or methanol as a negative control and chloramphenicol as a positive control. The antibacterial activity of the tested extract was stronger against the Gram positive bacteria. Keywords: antibacterial; antimicrobial assays; carnivorous; methanolic extracts; Pinguicula [1] Santos Pereira V, da Silva JW, Pereira de Lima RD Antimicrobial activity of Myracrodruon urundeuva. In: Antimicrobial Research: Novel bioknowledge and educational programs (Microbiology Book Series #6). Edited by A. Méndez-Vilas, Formatex Research Center, Badajoz, Spain [2] Zamudio RS Lentibulariaceae. In: Flora del Bajío y de Regiones Adyacentes (Fascículo 136). Instituto de Ecología, A. C. Pátzcuaro, Michoacán, México. 2005: 32pp. [3] Jensen SR, Franzyk H & Wallander E Chemotaxonomy of the Oleaceae: iridoids as taxonomic markers. Phytochemistry 60: [4] Von Christen K, Gordonoff T Das wirksame Prinzip der Pinguicula vulgaris L. Arzneimittelforschung 10: [5] Grevenstuk T Phytochemical Studies and biological activity of carnivorous plants from the mediterranean region. Ph. D. Dissertation. Tesis doctoral. Algarve University. Faro, Portugal. 250pp. 15

26 Antimicrobial effect of cinnamaldehyde, carvacrol and thymol and their combined effect with penicillin and trimethoprim-sulfamethoxazole against resistant Streptococcus suis strains F. de Aguiar, A.L. Solarte, C. Tarradas, I. Luque, A. Maldonado, R. Astorga, B. Barrero-Domínguez, A. Galán-Relaño and B. Huerta Animal Health Department, Faculty of Veterinary Medicine. Campus Universitario de Rabanales, 14075, Córdoba, Spain. Agrifood Campus of International Excellence ceia3, CeiA3. University of Córdoba Streptococcus suis is a Gram positive bacteria causing different clinical conditions in pigs, such as meningitis, septicaemia, arthritis, endocarditis, pneumonia and septic shock. In addition, this pathogen can affect to humans, being individuals in contact with swine or pork production the main groups at risk. The control of this disease is based on antimicrobial therapy and biosecurity measures. However, nowadays an important pressure to reduce the use of antimicrobials in pig farms is advised; one of the strategies proposed is the use of natural products alone or in combination with conventional antimicrobials. The susceptibility of Streptococcus suis strains against cinnamaldehyde, carvacrol and thymol and the combined effect of those products with penicillin (P) and trimethoprim-sulfamethoxazole (SXT) against resistant strains are the objectives of this work. The broth microdilution method was carried out to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 60 S. suis strains against two-fold serial dilutions ( µg/ml) of these natural products, according to the guidelines [1]. The MIC 50,90, MBC 50, 90 were calculated. The bactericidal power (rate MBC 50 and MBC 90 / MIC 50 and MIC 90 ) was determined, considering a rate 4 as bactericide [2]. Five resistant S. suis strains to P or SXT drugs were used to study the combined effect by the Checkerboard method. The fractional inhibitory concentration index (FIC index ) was determined for the best combination that inhibited the bacterial growth. A synergistic effect was considered when FIC index 0.5; additive >0.5-1; indifferent >1 <2; and antagonist when FIC index 2 [3]. Carvacrol and thymol showed important antimicrobial activity, with MIC 50, 90 and MBC 50, 90 values of µg/ml. The MIC 50, 90 and MBC 50 values of cinnamaldehyde were of µg/ml, whereas the MBC 90 reached the value of 625 µg/ml. The MBC/MIC rates were 2, showing their strong bactericide character and their potential effectiveness as an alternative to traditional antibiotics to control S. suis infections. Additive effect (FIC index = 0.75) was obtained for some strains when combined with SXT and cinnamaldehyde or thymol, with a considerable reduction of the effective concentration of the antimicrobials (4-fold MIC). Combination of these natural products with trimethoprim-sulfamethoxazole or penicillin generally showed an indifferent effect (FIC index = 1.25). No antagonistic and synergic effect was detected for any S. suis strain. This experiment represents the first study on the distribution of the susceptibility of S. suis against cinnamaldehyde, carvacrol and thymol, the results obtained highlight the bactericidal potential of the natural products and support their possible use for the control of S. suis infections. Our results are of interest for future options of combined treatment of conventional antimicrobials with natural products against this swine disease. Keywords: Streptococcus suis; antimicrobial activity; natural products; combined effect [1] Clinical and Laboratory Standards Institute (CLSI), Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals. Approved Standard VET01.A4, Fourth Edition. Wayne, PA. [2] Montravers P. & Dupont H Monitorage d'une antibiothérapie. Conférences d'actualisation, Elsevier, Paris, et SFAR, p [3] European Society of Clinical Microbiology and Infectious Diseases (EUCAST), Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. European Committee for Antimicrobial Susceptibility Testing of the definitive document E. Def 1.2, , CMI, Vol

27 Antimicrobial effect of lysozyme in serum in patients with ventilation-associated pneumonia V.Y. Ziamko Vitebsk State Medical University, Vitebsk, Belarus Introduction: In modern concepts of pathogenesis of inflammatory diseases, much attention is paid to nonspecific humoral factors of immune system, including lysozyme. The aim of the study was to evaluate level of lysozyme activity in serum in patients with ventilation-associated pneumonia. Materials and methods: A complex examination of 43 patients with ventilation-associated pneumonia and 43 practically healthy people was performed. Blood was taken from the ulnar vein before meals into sterile test tubes. Results: We created a simpler and a cheaper way of evaluating lysozyme activity in biological fluids, which demands little time and has clear evaluation criteria. The invention relates to medicine, namely to laboratory diagnosis. From cell walls of Micrococcus lysodeikticus culture ATCC 4698 peptidoglycan substrate was prepared, and the activity of lysozyme was calculated by the formula obtained after constructing calibration curve for lysozyme, which showed the dependence of the concentration of lysozyme and the optical density of the solution of Congo red. The positive effect of the proposed method is that the substrate for the production of reaction is prepared once and can be used for a long time. The way is easy and simple in play. The sensitivity of this method to the human lysozyme produced by Sigma (Lysozyme human recombinant expressed in rice 100,000 Unit / mg) was 0.06 mg / ml. This is enough to determine immunodeficiency states, because the content of lysozyme in biological fluids varies according to various data in a wide range from 0.2 to 28 mg / ml in normal. It was found that the lysozyme activity in the serum was lower in patients with ventilation-associated pneumonia (246.7; µl / ml) than that in donors (445.5; µl / ml). After inactivation of the complement both in patients with ventilation-associated pneumonia (116.0; мкг/ µl) and in donors (246,0; 183,6-305,7 µl /ml) a statistically significant decrease in lysozyme activity occurred. There is a statistically significant decrease of nonspecific humoral resistance in patients with bacterial infection, namely, lysozyme, which is a risk factor for the development of ventilation-associated pneumonia. 17

28 Antimicrobial Functionalization of Copper Surfaces by Ultrashort Pulsed Direct Laser Interference Patterning Daniel Wyn Müller, Philipp Grützmacher, Jiaqi Luo and Frank Mücklich Department of Material Science and Engineering, Chair of Functional Materials, Saarland University, Saarbrücken, Germany The augmentation of antibiotic resistance in a growing number of microbial strains is giving rise to a higher risk of infection, as well as a more critical course of disease in many public facilities, where a transfer takes place due to a deposition and receiving of germs via contact areas. To reduce the number of infections triggered by the transportation and spreading especially of multiresistant germs (MRGs), the potential route of transfer by contact areas has to be eliminated, which can be done by the use of actively antimicrobial metallic materials like copper and its alloys. A promising technique to additionally modify such metallic surfaces topographically and chemically is laser structuring using ultrashort pulse lengths. Copper is among the most suitable metallic materials for this type of processing due to its specific properties. An additional enhancement of the potential antimicrobial properties was made by the modification of surface topography and wettability by ultrashort pulsed laser structuring. Line patterns with tailored structural features were applied on copper surfaces adapting the chemical as well as the topographical surface properties. By this, both structural features in the size of single bacteria, as well as a significant hydrophobic wetting behavior were achieved. This modification leads to a lower transfer of germs to the metallic surface first-hand, while the topography itself enhances the contact area of adherent germs to the substrate leading to a higher transfer of ions and thus a highly efficient contact-killing of the microbes. Keywords: antimicrobial; copper; laser structuring; superhydrophobic 18

29 Antimicrobial resistance for bacterial pathogens isolated from bovine mastitis Khaled M Al-Qudah* Faculty of Health Science, Veterinary Science Division, Higher Colleges of Technology, P.O.Box: 17155, Al Ain, United Arab Emirates * Corresponding author: kalqudah@hct.ac.ae Antibiotics are fundamental for the control of infectious diseases in man and animals. In recent years, however, there has been considerable concern about the emergence and diffusion of antibiotic resistance resulting from haphazard antibiotic use in the veterinary sector, with possible risks for the human population. A pilot study, on emergence of antibiotics resistance among bovine mastitis pathogens, was carried out in the Veterinary Health Center (VHC) in Jordan. Records reviews were carried out on 205 mastitis cases. Culture and sensitivity tests were done. The most prevalent pathogens isolated were Staphylococcus aureus (S. aureus) (53.4%), E.coli (16 %), Streptococcus non-agalactiea (Strep non-ag) (5.9%), and coagulase negative Staphylococci (CNS)(5.9%). The antibiotics for which, most of the isolated pathogens were sensitive to were enrofloxacin (61.2%) and ciprofloxacin (58.7%). While the ones that the pathogens were mostly resistant against were sulfa/trimethoprim (87.4%), and penicillin (84.5%). Results shows the risk of an emergence of an antimicrobial resistance among mastitis bacterial pathogens in Jordan. 19

30 Antimicrobial resistance in rare mycobacteria from hospital environment and diabetic foot ulcers S. Alarico 1, S.G. Pereira 1, A. Maranha 1, I. Tiago 2, D. Reis 1, D. Nunes-Costa 1, O. Cardoso 3,4 and N. Empadinhas 1,5 1 CNC Centre for Neuroscience and Cell Biology, University of Coimbra, Portugal 2 CFE - Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Portugal 3 FFUC - Faculty of Pharmacy, University of Coimbra, Portugal 4 CIEPQPF Chemical Process Engineering and Forest Products Center, University of Coimbra, Portugal 5 IIIUC - Interdisciplinary Research Institute, University of Coimbra, Portugal Nontuberculous mycobacteria (NTM) are ubiquitous in nature and lately increasingly recognized as opportunistic agents, particularly, of human pulmonary and skin atypical infections. Evaluation of their prevalence in anthropogenic environments and associated antimicrobial resistance phenotypes have been rarely reported. In this work, minimal inhibitory concentrations of 24 antimicrobials were determined for rapidly growing Mycobacterium mucogenicum and M. obuense isolates, as well as for the rare slowly growing M. paragordonae isolates, recovered from 102 strains isolated from amenities of a tertiary-care hospital in central Portugal. Mycobacterium mucogenicum isolate showed a resistance profile at least to four different classes of antimicrobials (sulfonamides, aminoglycosides, tetracyclines and fluoroquinolones). Mycobacterium obuense isolate, also exhibited a resistance profile, namely to ciprofloxacin, clarithromycin, imipenem and tobramycin. In contrast to other strains isolated elsewhere [1,2], M. obuense and M. mucogenicum strains examined in this study were resistant to different Clinical Laboratory Standards Institute (CLSI) recommended drugs, suggestive of multidrug resistant (MDR) profiles. To our knowledge, no M. paragordonae strains have been tested yet for antimicrobial susceptibility prior to this work. The isolates tested here were only susceptible to four of the CLSI antimicrobials recommended for slowly growing mycobacteria, which also suggests MDR profiles. Susceptibilities of other clinically relevant actinobacterial isolates collected from the same hospital surfaces were also determined. Corynebacterium sp. isolates were highly resistant to some of the CLSI-recommended drugs, namely imipenem and linezolid. Separately, selective microbial isolation from swabs retrieved from 102 diabetic patients foot ulcers (DFU) and skin, recruited from the ambulatory Diabetic foot consults from the same hospital, allowed the identification of 3 NTM isolates, which to our knowledge were never reported in this patients. The isolates were provisionally assigned as M. frederiksbergens, M. murale and M. pulveris species, after partial 16S sequencing. Antimicrobial susceptibility studies are underway. At a time when infections by NTM and related bacteria are increasing and poor epidemiological surveillance thwarts estimation of their real prevalence in healthcare settings are neglected, this study highlights the importance of implementing effective measures to screen and control these opportunists, whose antimicrobial resistance profiles represent a serious threat to vulnerable individuals. Keywords: Nontuberculous mycobacteria; diabetic foot ulcers; opportunistic agents; nosocomial environment Acknowledgement Fundação para a Ciência e a Tecnologia and COMPETE 2020, Operational Programme for Competitiveness and Internationalization (POCI) project UID/NEU/04539/2013 and SFRH/BPD/108299/2015 to SA and FRH/BD/117777/2016 to DN-C. European Regional Development Fund, Centro 2020 Regional Operational Programme (CENTRO FEDER HealthyAging2020). Infarmed, Fundo para a Investigação em Saúde, FIS-FIS _DIA_ [1] Li G, Pang H, Guo Q, Huang M, Tan Y, Li C, Wei J, Xia Y, Jiang Y, Zhao X et al. Antimicrobial susceptibility and MIC distribution of 41 drugs against clinical isolates from China and reference strains of nontuberculous mycobacteria. Int J Antimicrob Agents. 2017; 49(3): [2] van Ingen J, van der Laan T, Dekhuijzen R, Boeree M, van Soolingen D. In vitro drug susceptibility of 2275 clinical non-tuberculous Mycobacterium isolates of 49 species in The Netherlands. Int J Antimicrob Agents. 2010; 35(2):

31 Antiviral, antimicrobial and biocompatible polymer-based coatings with natural bioactive substances C. Gneupel and S. Reinemann Research Team, Biological Active Systems, Department of Plastics Research, Thuringian Institute of Textile and Plastics Research, Breitscheidstr. 97, Rudolstadt, Germany The motivation for the development and use of such virucidal and antimicrobial polymer coatings lies in the fact that, especially in areas with high population density and in developing countries, infectious diseases are emerging more and more frequently due to contaminated drinking water and poor hygiene. Especially in times of globalization, in the worst case, this can lead to a pandemic. For example, from 2009 to 2010, H1N1 pandemic, a variant of the influenza A virus, occurred, which is also popularly known as "swine flu". During the pandemic phase, cases of infection with H1N1 had been confirmed in laboratories from a total of 214 countries and overseas territories deaths are believed to be related to viral infection. [1] For current reasons, the Ebola Fever epidemic 2014 is to be mentioned in Central and West Africa. The Ebola fever is according to current knowledge in 50 to 90 percent of all cases deadly. Thus, according to official figures from the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) of , 1752 confirmed cases and 897 deaths have been identified in this epidemic [2] [3]. Another reason for the development and use of virucidal and antimicrobial polymers is that, according to a WHO estimate, 90% of all known pathogens of infectious diseases are viruses; 50% of all patients who have an infectious disease are infected with a virus. As a result, it is mainly viruses that appear as pathogens of diseases with a, even today often difficult to calculate risk potential [4]. The challenge is that contact surfaces such as door handles, light switches or handrails are touched very often and in a short time by several people, which inevitably leads to germ transmission. In addition to already established hygiene measures, such as hand disinfection, new solutions have to be found to minimize the risk potential. Virus-inactivating and antimicrobial materials can significantly support the fight against infections as an additional barrier. For these reasons, the equipment of polymer-based coatings with virucidal and antimicrobial additives is quite desirable. Such coated materials can be used in medical, food processing, public and high-traffic buildings and facilities, drinking water-conducting systems, plumbing, air conditioning and swimming pool technology, crisis areas and developing countries where poor sanitary conditions are prevailed and no clean drinking water is available. At TITK different types of polymers (PET-G, PC and PA) were tested as matrix materials for antiviral and antimicrobial coating systems, whereby the polyamide (PA) turned out to be the best matrix material. This PA is a polyamide specifically designed for coatings which has a maximum moisture content of more than 10%, which facilitates the drug delivery process. In this matrix material different antimicrobial and / or antiviral agents were incorporated in different concentrations and examined for their biological properties. Thus, different humic extracts were used as a natural antimicrobial and antiviral agent and compared with the established biocides PHMB, Dextrane Sulfate, Foscarnet and Azidocarboxamide. The aim was to develop a coating system with maximum antimicrobial and antiviral efficacy and simultaneously existing biocompatibility against mammalian cells. Thus, it has been demonstrated that the natural antimicrobial and antiviral agent Humin 3 achieves approximately the same antibacterial activity against S. aureus (Figure a) and a better activity against the virus strain MVA (Figure b) with simultaneously existing biocompatibility than with the established antimicrobial and antiviral active substances. Keywords: antiviral humin; natural antiviral agents [1] World Health Organization. Global Alert and Response (GAR): Pandemic (H1N1) update 112. [Online] [Cited: September 04, 2014.] [2] Ebola virus disease update - west Africa. Disease outbreak news: 28 August [Online] August 28, [Cited: August 28, 2014.] [3] Centers for Disease Control and Prevention Ebola Outbreak in West Africa. [Online] August 28, [Cited: August 28, 2014.] [4] von Rheinhaben, F. and Wolff, M.H. Handbuch der viruswirksamen Desinfektionen. s.l. : Springer-Verlag Berlin Heidelberg, 2002, p. Vorwort. 21

32 Acknowledgements The authors are grateful to the Federal Ministry of Economics and Technology (BMWi) for the financial support of the research project INNO-KOM-Ost VF a) b) Fig. 1 a) Results of the measurement of the antibacterial activity R of the polymer-based coatings with natural bioactive substances, the polymer-based coatings, aided by conventional biocides and their reference materials based on ISO 22196; b) Results of the measurement of virus inactivating effectiveness of the polymer coatings against MVA of the polymer-based coatings with natural bioactive substances, the polymer-based coatings, aided by conventional biocides and their reference materials according to the Guideline of the Robert Koch Institute for the testing the virucidal effect of the chemistry of surface disinfectants and instrument disinfectants included in the list according to 10c of the Federal Epidemic Law. Federal Health Report, Health Research, Health Protection 1995; 6:

33 Arthrobacter globiformis as a source of bioactive compounds for pharmaceutical applications Zaki Saati-Santamaría 1,2, Miroslav Kolarik 2, Raúl Rivas 1,2 and Paula García-Fraile 1,2,3 1 Microbiology and Genetics Department. University of Salamanca. Doctores de la Reina SN, Salamanca, Spain 2 Spanish-Portuguese Institute for Agricultural Research (CIALE), Salamanca, Spain 3 Institute of Microbiology of the CAS, v. v. i., Průmyslová 595, Vestec, Czech Republic During thousands of years, natural molecules obtained from plants, animals or microorganisms have been the main source of drugs to treat human illnesses. However, the field of chemistry has greatly influenced the availability of drugs by using combinatorial chemistry to produce new molecules. Currently, natural sources are receiving more attention, as the production of new drugs chemically is becoming more restricted because they can t achieve the complex characteristics of natural molecules(1),and it is becoming more difficult to solve the problems that arise in association with Public Health. In particular, Actinobacteria is a group of Gram-positive bacteria that known to produce two-thirds of all naturally derived antibiotics in current clinical use, as well as many anticancer, anthelmintic, and antifungal compounds(2). In this study, several strains were isolated from the bark beetle Ips acuminatus and screened against different microorganisms, finding that one of the most promising strains was strain IA7, an actinobacterium belonging to the species Arthrobacter globiformis. Its genome was sequenced using Illumina MiSeq platform and assembled by Velvet Gene calling and annotation was performed using RAST(3). The mining of the genome was carried out by using The SEED viewer framework and the program antismash 3.0(4). These analyses allowed us the finding of several clusters of genes that are related to the production of antibiotics, such as streptomycin, kanamycin, sisomicin, as well as antifungal (amphotericin) and antiparasitics (meilingmycin), among other bioactive substances. Nevertheless, in many of these gene clusters we found that the percentage of similitude to already described gene clusters it is not high, so the substances that they encode could be a promising way to find new drug derivatives. Moreover, there is a chance to find new bioactive molecules, taking into account that there are several clusters of genes that are predicted to encode secondary metabolites which have not been described yet. These facts make this strain as a promising one to further explore its capability to produce compounds for pharmaceutical applications. Keywords: Actinobacteria; Genome mining [1] Harvey AL, Edrada-Ebel R, Quinn RJ. The re-emergence of natural products for drug discovery in the genomics era. Nat Rev Drug Discov. 2015;14(2): [2] Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Klenk H-P, et al. Taxonomy, Physiology, and Natural Products of Actinobacteria. Microbiol Mol Biol Rev MMBR ;80(1):1-43. [3] Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008;9:75. [4] Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, et al. antismash 3.0 a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res ;43(W1):W

34 Assessment of the lytic ability of bacteriophages specific for Staphylococcus aureus, associated with bovine mastitis in South Africa I.H. Basdew and M.D. Laing Discipline of Plant Pathology, University of KwaZulu-Natal, Private Bag X01, Scottsville, Pietermaritzburg, 3209, Republic of South Africa South Africa is the third largest producer of fresh milk in Africa, after Sudan and Kenya. The South African dairy industry is also an important generator of foreign exchange with major export destinations for milk-based products being Mozambique, Zimbabwe and Angola. However, the physical and biochemical effects of diseases, specifically Staphylococcus aureus-induced bovine mastitis, can severely affect both the export and local market commerciability of milk. A holistic control strategy comprising the hosts natural defence system, cultural measures and antibiotic therapy are routinely applied in an attempt to curb disease onset and spread. However, S. aureus has the ability to quickly develop resistance towards such measures, particularly the inhibitory effects imposed by antibiotics. Interest has accordingly shifted from antibiotic therapies towards the field of biological control of the disease. One such option involves the use of lytic bacterial viruses called bacteriophages that are specific to S. aureus. Phages boost the capacity to control microbes over multi-disciplinary fields ranging from agriculture, to human and animal medicine. Phages demonstrate a unique system of infection and replication within their target bacteria. This intricate network of events is so specific that all other microbes in that environment (pathogenic or commensal) remain unaffected. This, coupled with their ability to multiply between fold within a host bacterium, makes phages an ideal candidate for bacterial disease therapy. However, while much research has been conducted on the use of phages to control bovine mastitis, a viable phage-based product has yet to be commercialized for this purpose. The research conducted in the following study was initiated with this long-term objective in mind. Significant strides have been made over the duration of the project. A total of 38 S. aureus strains were isolated from raw bovine milk located in three of the major milk producing regions of KwaZulu-Natal. Kirby-Bauer disc-diffusion assays showed that all 38 bacterial strains expressed resistance to the major antibiotics used, with the exception of tetracycline. Of the 38 bacterial strains, 3 were carried forward into phage studies. After phage isolations and initial in vitro screening, 3 of the 112 phages isolated, were carried forward for in-depth in vitro screening. These phages demonstrated a wide host range (lytic against 26 of the 38 S. aureus isolates), stability under stress conditions (temperature fluctuations, formulation variations), high titers, and exceptional lethal effects as they were able to reduce live bacterial cell counts by up to 95%. Morphological and genomic classification of the phages revealed that they were of the order Caudovirales, family Myoviridae. In vivo trials then followed. Phages were screened for mastitis control in clinically infected dairy cows. In Trial 1, cows were specifically pre-screened for S. aureus-induced bovine mastitis. Six animals were selected for the experiment; three animals were treated with a phage cocktail comprising of a mixture of the three different phages, and three animals were treated with a control preparation of 40% glycerin, for 5 days. Sampling commenced for 15 days; milk samples were assayed for somatic cell counts (SCC), and S. aureus and phage counts. Results from Trial 1 indicated a significant drop in S. aureus counts in the treated cows. This however, was correlated with a corresponding increase in SCC of the same treated animals. The Control animals showed a more or less constant population of S. aureus. In Trial 2, the number of experimental animals was increased from 6 to 12, with 6 treated and 6 untreated animals, and milk was analyzed over 21 days. Again, a decrease in S. aureus cell counts was noted, that corresponded with an increase in SCC, in treated versus untreated animals. Phage replication appeared stable across all the treated animals versus the untreated animals for both Trials 1 and 2, with a significant increase in phage counts over time noted in milk drawn from treated animals. Initial results were promising as the typical patterns expected of a lytic phage:bacterium interaction were noted (sharp drop in bacterial cell counts, exponential phage counts). However, experimental revisions need to be implemented in trials going forward, particularly when taking into account the SCC. The long-term vision for the study is to establish phage therapy for bovine mastitis as a part of standard veterinary practise. Keywords: bacteriophages; bovine mastitis; biological control; Staphylococcus aureus 24

35 Bacteria-triggered antimicrobial coatings to maintain drinking water quality M.K.L.N. Sikosana, A. Ruland, L.D. Renner, R. Helbig and C. Werner Leibniz-Institut für Polymerforschung e.v., Max Bergmann Center of Biomaterials, Dresden, Germany Biofilm-related infections attributable to unsafe water, sanitation and poor hygiene, remain a global public health concern [1]. On the one hand, the necessary transition from disinfectants to more biofilm preventative measures such as antimicrobial and structured surfaces in water treatment has begun. However, to date, these strategies are unilateral and focus on drug conjugation and diffusion-limited systems, which could continue to support bacterial resistance. While on the other hand, the full ecology of bacterial behaviour and biocidal resistance in aqueous conditions are not fully understood nor adequately addressed on a lab-scale. The ability to tackle both imperatives simultaneously, whilst mitigating cost and environmental harm, has become something of a grand challenge for concerned researchers. With this backdrop, we highlight our novel approach to introduce bioresponsive antimicrobial coatings that could rise to the occasion, by maintaining drinking water quality (even) under realistic conditions. These antimicrobial coatings are based on a surface-adhesive anchor polymer backbone with a functional group that can be variably conjugated according to the required application, e.g. with antiseptics, or antibiotics. This polymer toolbox can be extended with a responsive core, i.e. an enzymatically cleavable peptide-linker between the polymer and functional group. The functionalized hydrophobic anchor polymers afford the coating its critical and unique ability to strongly adsorb to various surfaces from aqueous media. If equipped with an enzymatic cleavable peptide linker, common extracellular proteases secreted by bacteria cleave the surface s peptide-linkers and ultimately release the biocide. Upon microbial death, protease secretion seizes as well, the coating degradation comes to halt hence preserving its protective capacity to fight potential infections subsequently. Also, this precision could mitigate the potential for excessive biocide concentrations in solution deterring breeding opportunities for bacterial resistance. We demonstrate the biocidal release, stability and the antibacterial performance of this bio-responsive coating when tested on materials found in typical water distribution systems, Polystyrene (PS) and Polyethylene (PE). Specifically, antimicrobial tests reveal that the coating functionalized with hexetidine effectively reduces the bacterial viability of typical lab grown Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Moreover, the outcomes of how well these antifouling claims stand when tested against the Growth Advantage in Stationary Phase (GASPx; x=regeneration cycles) bacteria, are presented. These fitter bacteria, cultured under stressed conditions express alternative metabolic pathways that are more indicative of natural water environments [2]. The reported results suggest that auto-regulative coating systems allow for the adaptive administration of a broad spectrum of antimicrobial drugs; maximizing efficacy while mitigating both cost and environmental harm. When robust enough under realistic conditions, self-cleaning water storage and filter equipment could help address some of 2 million water related deaths reported annually [1]. Keywords: antimicrobial; polyphenols; bio-responsive coating; water treatment; GASP bacteria [1] World Health Organization (WHO). Water, Sanitation and Hygiene Links to Health. WHO; Geneva, Switzerland: [2] Finkel SE (2006) Long-term survival during stationary phase: evolution and the GASP phenotype. Nat Rev Microbiol 4:

36 Bactericidal activity of AP-CECT7121 combined with colistin against biofilmproducers Escherichia coli and Pseudomonas aeruginosa from catheters G. Delpech 1, M. Ceci 2, M. Bistoletti 2, S. Lissarrague 1 and M. Sparo 1, * 1 Microbiología Clínica, Medicina, Universidad Nacional del Centro de la Provincia de Buenos Aires, Av. Pringles 4375, 7400, Olavarría, Argentina 2 Centro de Estudios Bioquímicos, Av. Colón 1388, 7000, Tandil, Argentina * Corresponding author: monicasparo@gmail.com Escherichia coli and Pseudomonas aeruginosa are prevalent etiologic agents of catheter-related infections. They adhere to abiotic surfaces (e.g. catheters) leading to biofilm formation, which contribute with antimicrobial resistance. In addition, an increase of resistance to colistin, one of the last therapeutical options for Gram negative`s catheter-related infections, has been reported. Strategies for control of biofilm formation include the identification of new biocides effective against biofilm producers, such as bacteriocins. AP-CECT7121 (AP) is an enterocin produced by the probiotic strain Enterococcus faecalis CECT7121. The aim of the study was to investigate the bactericidal activity of AP-CECT7121combined with colistin (C) against biofilm-producer E. coli and P. aeruginosa isolated from catheters. E. coli HRS338 and P. aeruginosa HRS457 were isolated and characterized from catheters of patients at the Intensive Care Unit in the Hospital Ramón Santamarina (Tandil City, Buenos Aires Province, Argentina). Strains MIC AP and MIC C were determined by broth microdilution (CLSI, 2017). Time-kill curves were carried out against biofilm E. coli and P. aeruginosa cells in order to assess in vitro bactericidal activity of AP-CECT7121combined with colistin. Curves were performed three times, in duplicate. Briefly, suspensions of brain-heart infusion broth with glucose (1%) were added to 96-wells plates. Then, it was incubated at 35 ± 2 ºC, for 24 h. Formed biofilms were incubated with AP-CECT7121 and colistin for 1 h and 24 h. Viable cell counts were done. In addition, scanning electron microscopy (SEM) was carried out with an inert surface pre-treated with AP-CECT7121. Strains were added and biofilm formation was observed. AP-CECT7121 (MIC > 128 mg/l) alone not showed an inhibitory effect against E. coli; colistin presented anti- E. coli activity (MIC 0.3 mg/l). AP-CECT7121 combined with colistin showed bactericidal activity against E. coli (MIC AP/C 16/0.02 mg/l). After 24 h of incubation, AP-CECT7121 (MICx4) showed bactericidal activity against biofilm cells of E. coli (-3.1 ΔLog 10 FCU/mL) and P. aeruginosa (-3.0 ΔLog 10 FCU/mL). The peptide together with colistin showed bactericidal activity against E. coli and P. aeruginosa. Through SEM, prevention of biofilm formation with the enterocin was observed. AP-CECT7121 constitutes an attractive candidate for its use as a natural tool, in combination with conventional antimicrobial agents such as colistin, against biofilm cells of E. coli and P. aeruginosa. Keywords: E. coli; P. aeruginosa; biofilm; colistin; AP-CECT

37 Bacteriocin produced by Lactobacilli from Algerian goat's milk against pathogenic bacteria A. Marroki 1, L. Bousmaha-Marroki 1,2 and G. Pérez-Martínez 3 1 Department of Biology, Faculty of Natural and Life Sciences, University Djillali Liabes, Sidi Bel Abbès, 22000, Algeria 2 Microbiology and Natural Product Group, Laboratory of Vegetal Biodiversity, Faculty of Natural and Life Sciences, University Djillali Liabes, Sidi Bel Abbès, 22000, Algeria 3 Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (C.S.I.C), polígono de la Coma s/n, Burjassot (Valencia), Spain Lactobacilli are rods belonging to the Lactic Acid Bacteria (LAB), and include: Gram-positive, catalase negative, non-motile, non-sporulating, facultative anaerobes, growing under microaerophilic to strictly anaerobic conditions. Recently, we focused our research for select strains from goat s milk capable to producing antibacterial substances, such as bacteriocin and with safety aspect [1, 2, 3]. Strains intended for the use in food systems as starters or probiotics adjunct culture should therefore be carefully examined following important criteria [4]. The objective of this research was to study the antimicrobial activity of seventy one (71) strains of Lactobacillus isolated from Algerian goat's milk: L. plantrum, L. rhamnosus, L. pentosus and L. fermentum. Lactobacilli strains were tested against the indicator strains Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The agar spot assays allows that: a) 19.70% strains show antibacterial activity against L. monocytogenes CECT 932 T ; b) 25,35% inhibit Sta. aureus CECT 86 T or Sta.aureus UT 206; c) 19.70% produce an inhibition zone on agar against B. cereus AVZ 421. Well-diffusion test, shows that: a) 14% of strains produce antibacterial substance against L. monocytogenes CECT 932 T ; b)15.50% against Sta. aureus CECT 86 T and 12.70% displayed inhibitory activity against Sta. aureus UT 206; c)11.30% exhibit an antibacterial activity against B.cereus AVZ 421. The antimicrobial activity inactivation of the supernatants by the proteolytic enzyme proteinase K, confirms the proteinaceous nature of the antibacterial produced substance. In addition, these antibacterial showed a wide range of thermostability (80 and 100 C for 10 min) and conserve their antibacterial activity over a wide range of ph (4.5 and 6.5). These results suggest that the antibacterial substance produced by some Lactobacilli tested strains is bacteriocin type inhibiting pathogenic organisms. These strains from raw goat's milk might be useful as a biopreservative in different dairy industry in Algeria. Keywords: Lactobacillus; raw goat's milk; bacteriocin; antibacterial activity; pathogenic bacteria [1] Ahmed Marroki, Manuel Zúñiga, Mabrouk Kihal and Gaspar Pérez-Martínez (2011). Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rdna sequencing and their technological properties. Braz. J. Microbiol. 42: [2] Ahmed Marroki, and Leila Bousmaha-Marroki (2014). Lactobacilli Isolated from Algerian Goat s Milk as Adjunct Culture in Dairy Products. Brazilian Archives of Biology and Technology. 57: [3] Leila Bousmaha-Marroki and Ahmed Marroki. Antibiotic susceptibility and heterogeneity in technological traits of lactobacilli isolated from Algerian goat s milk (2015). J Food Sci Technol. 52(8): [4] Frédéric Leroy, Luc de Vuyst (2004). Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol. 15:

38 Bark beetles, a novel source of strains capable to inhibit Candida sp. Zaki Saati-Santamaría 1,2, Alejandro Jiménez-Gómez 1,2, Miroslav Kolarik 2, Raúl Rivas 1,2 and Paula García- Fraile 1,2,3 1 Microbiology and Genetics Department. University of Salamanca. Doctores de la Reina SN, Salamanca, Spain 2 Spanish-Portuguese Institute for Agricultural Research (CIALE), Salamanca, Spain 3 Institute of Microbiology of the CAS, v. v. i., Průmyslová 595, Vestec, Czech Republic Candidiasis is a fungal infection that represents the most common opportunistic oral infection in humans, as well as means the cause of a lot of genitalia, respiratory tract, esophageal and topic infections. Moreover, it cause dangerous sepsis in immunodeficient patients(1). In addition, the antimicrobial resistance that the rest of microorganisms are acquiring, and which threats Public Health affects also to these infections. For all this, research studies focused in the discovery of new compounds that could tackle this problem are of vital importance. In this work, different strains have been isolated from different bark beetles life stages (Ips acuminatus, Pityogenes bidentatus, Pityophthorus pityographus, Cryphalus piceae) taken from trees (Pinus sylvestris and Abies alba). These strains have been screened against different type of microorganisms and the results showed that the 70% of them are capable to inhibit Candida sp. Moreover, 59% of these strains were also able to inhibit another yeast-like, Pichia fermentans. Taking this into account, we can conclude that bark beetles represent a niche with a great potential for finding interesting bacteria capable to produce some substance that could fight the worldwide problem that candidiasis implies. Keywords: Antifungal activity; New antibiotic sources [1] Wingard JR. Candida tropicalis: A Major Pathogen in Immunocompromised Patients. Ann Intern Med. 1979;91(4):

39 Bark beetles, a source of Pseudomonas strains with antimicrobial activity Zaki Saati-Santamaría 1,2, Alejandro Jiménez-Gómez 1,2, Miroslav Kolarik 3, Raúl Rivas 1,2 and Paula García- Fraile 1,2,3 1 Microbiology and Genetics Department. University of Salamanca. Doctores de la Reina SN, Salamanca, Spain 2 Spanish-Portuguese Institute for Agricultural Research (CIALE), Salamanca, Spain 3 Institute of Microbiology of the CAS, v. v. i., Průmyslová 595, Vestec, Czech Republic Antimicrobial resistance is one of the major risks to public health, threating the effectiveness of treatments against microbial infections(1). Therefore, the study of unexplored ecological niches that can serve as novel sources for potentially novel antimicrobial substances is of utmost importance to the development of new drugs to treat those microbial infections produced by multi-resistance microbial strains(2). In this study, several bacterial strains have been isolated from bark beetles and they have been screened against different kinds of microorganisms. Out of these isolates, the most promising strains, those with the capability to inhibit more of the reference microorganisms, belonged to the genus Pseudomonas. Some of the best Pseudomonas strains were then selected to obtain their genome sequence, in order to further study their genetic potential to produce antimicrobial substances. Their genome sequence was mined to find clusters of genes that are related with the production of different compounds with antimicrobial activity, among other secondary metabolites with potential bioactive functions. Many of the predicted gene clusters of these genomes implicated in bioactive substances synthesis are not 100% similar to those previously described or, moreover, they are not related with the synthesis of any known substances. That is why we conclude that these strains are promising bacteria as producers of potentially novel substances that could be implicated in the development of new drugs. Keywords: Genome; New drugs [1] Hoffman SJ, Outterson K, Røttingen J-A, Cars O, Clift C, Rizvi Z, et al. An international legal framework to address antimicrobial resistance. Bull World Health Organ. 2015;93(2): [2] Cragg GM, Newman DJ. Biodiversity: A continuing source of novel drug leads. Pure Appl Chem. 2009;77(1):

40 Biofilm formation in clinical isolates in ICU V.Y. Ziamko and V.K. Okulich Vitebsk State Medical University, Vitebsk, Belarus Background. Microorganisms are capable of forming a kind of community - a biofilm, which increases their resistance to the adverse effects of environment. The aim of the study was to study biofilm formation of clinical isolates in ICU. Materials and methods. To study the ability to form microbial biofilms in a polystyrene plate 173 clinical isolates from ICU were studied. S. aureus comprised 20 isolates (11.5%), Acinetobacter spp isolates (32.9%), K. pneumoniae - 56 isolates (32.3%), P. aeruginosa - 26 isolates (15%), P. mirabilis - 4 isolates (2.3%), E. faecium - 5 isolates (2.8%), E. coli - 3 isolates (1.7%), S. epidermidis and S. haemolyticus -1 isolate (0.57%). In 24 hours after incubation optical density was measured. Results. When studying the results of measuring the mass of the formed microbial biofilm, it was found that the greatest value of this parameter was found in biofilms formed by isolates of P. mirabilis (56.4 ± μg / well), S. aureus (39.74 ± 23.4 μg / well) and K. pneumoniae (39.70 ± 20.1 μg / well). The study found that 45% isolates of S. aureus had a high ability to form a biofilm (n = 9). 69% of isolates of K. pneumoniae had a high (n = 39) and 30.36% moderate capacity to form biofilms (n = 17). Isolates of P. aeruginosa and Acinetobacter spp formed biofilm a bit worse (37.46 ± μg / well and ± 24.2 μg / well, respectively). Between the ability to form a biofilm and the increase in the number of groups of antibacterial drugs to which microorganisms were resistant, a correlation of average strength was revealed, r = 0.63; p <0.05. Conclusion. A large amount of intercellular matrix and, correspondingly, a significant mass of biofilm can significantly impede the penetration of antiseptics and antibiotics to bacteria in the biofilm, which leads to a decrease in the sensitivity of the microorganism to antibacterial drugs. Keywords: Biofilm; ICU; antibiotic resistance [1] O'Toole, G.; Kaplan, H.B.; Kolter, R., 2000: Biofilm formation as microbial development. Annual Review of Microbiology 54:

41 Bioinformatics study on multi-resistant bacteria that express β-lactamases C. Polanco Departamento de Matemáticas, Facultad de Ciencias, Universidad Nacional Autónoma de México, D.F., Mexico Bacteria become multi-resistant to antibiotics such as cephalosporins, monobactams, carbapenems, and penicillins when they express β-lactamase proteins. This paper introduces the main results of a bioinformatics study about protein mutation extracted from UniProt database (Septembe 10, 2017), already published [1]. The bioinformatics Polarity Profile method [2] can identify the four sub-groups of β-lactamase proteins: TEM, SHV, CTX, and OXA from the antimicrobial proteins, and intrinsically disorder protein groups. According to these results, the Polarity Index Method has a high level of efficiency identifying each of these four β-lactamases groups; therefore, it can be used for in-depth analysis of databases. Keywords: β-lactamase; Gram-negative bacteria; Gram-positive bacteria; antimicrobial peptides; proteins; structural proteomics; bioinformatics; polarity index method [1] Polanco C. Buhse T, Uversky VN, Samaniego Mendoza JL, Vizcaino G (2018) A computational evaluation of the potential composition of the prebiotic proteins based on the composition of the CM2 class meteorites Curr Top Pept Protein Res 18:79-89, 8, 202; DOI: /mi [2] Polanco, C. (2016) Polarity index in Proteins-A Bioinformatics Tool 2016, DOI: / , eisbn: , ISBN: Bentham Science Publishers ed, Sharjah, U.A.E. 31

42 Biological and molecular characterization of South African bacteriophages infective against Streptococcus uberis, a predominant causal agent of bovine mastitis C. Pillay, I.H. Basdew and M.D. Laing Discipline of Plant Pathology, School of Agriculture, Earth and Environmental Sciences, Faculty of Science and Agriculture, University of KwaZulu-Natal, 1 Carbis Road, Scottsville, Pietermaritzburg, 3201, KwaZulu-Natal, South Africa Mastitis is an inflammatory disease of bovine mammary glands and is the most economically important disease to dairy herds in South Africa, and globally. The incidence and history of mastitis has not been documented in South Africa, but studies in sub-saharan Africa revealed 50% of dairy herds to be infected (Petzer et al., 2009). The aim of this study was to isolate and classify robust lytic bacteriophages from unpasteurized milk of dairy cows, with the ability to stop the growth of a recurring antibiotic-resistant causal agent of bovine mastitis, Streptococcus uberis. Streptococcus uberis has been identified as a prominent causal agent of disease from dairy herds in KwaZulu-Natal, SA (Petzer et al., 2009). Eleven strains of Strep. Uberis were isolated and identified serologically, and genomically by sequencing of the ribosomal 16s rrna. The extent of antibiotic resistance was demonstrated using the Kirby-Bauer antibiotic sensitivity assay against the following antibiotics: Amoxicillin, Ampicillin, Cefalexin, Erythromycin, Penicillin G, Oxacillin, Streptomycin, Tetracycline, Tobramycin and Vancomycin. The strains showed completely different levels of susceptibility and resistance compared to each other. One strained demonstrated complete resistance to all antibiotics, another showed complete susceptibility. Five strains revealed varying ranges of susceptibility and resistance to 2-5 antibiotics from the 10 screened. After screening 1307 mastitic milk samples from dairy farms, 28 phages were isolated. The isolated phages are currently under characterization in the following categories: host range, lethal dose activity, shelf life durability, temperature stability, ph sensitivity, and morphology. Phage therapy has not been passed by legislation in any country for bovine mastitis treatment. This in vitro study will pave the way for phage therapy against South African strains of streptococcal etiological agents of bovine mastitis. Keywords: bovine mastitis; bacteriophages; biological control; antibiotic resistance [1] Petzer, I., Karzis, J., Watermeyer, J., Van Der Schans, T., Van Reenen, R. (2009). Trends in udder health and emerging mastitogenic pathogens in South African dairy herds. Journal of the South African Veterinary Association 80 (1), pp

43 Coating of titanium surface with Ag 2 WO 4 inhibits methicillin-resistant Staphylococcus aureus adhesion J. Andrés ; C.C. De Foggi ; M. Assis ; A. Kubo ; L.C.L. Santana*; C.E. Vergani*; A. C. Catto and E. Longo. CDMF, LIEC, Chemistry Department of the Federal University of São Carlos (UFSCar), P.O. Box 676, São Carlos, SP, Brazil Department of Analytical and Physical Chemistry, University Jaume I (UJI), Castelló 12071, Spain * São Paulo State University (Unesp), School of Dentistry, Araraquara, SP, Brazil Staphylococcus aureus is one of the major microorganisms associated to orthopedic implants contamination in total hip arthroplasties. The biofilm formation can be divided into a sequence of events: attachment of bacteria to the solid surface, bacteria accumulation and irreversible attachment, biofilm maturation and detachment of some bacteria for the colonization of new surfaces [1]. To prevent bacterial adhesion and contamination of commercially pure titanium (cpti), a coating with silver tungstate (Ag 2 WO 4 ) was developed. Briefly, Ag 2 WO 4 microcrystals were deposited onto cpti surface by spin-coating method, and the antibacterial activity against methicillin-resistant Staphylococcos aureus (MRSA, ATCC 33591) was evaluated. The microorganism was cultured in TSB culture medium until it reached the mid-log phase of growth. The inoculum was standardized at a final concentration of 10 8 cells per ml and thereafter it was planted onto titanium samples for 90 minutes to assess the initial MRSA cell adhesion. After this period, the specimens were washed with PBS and prepared for SEM and CLSM analysis. Scanning micrographs showed significant reduction of adhered cells in the specimens coated with Ag 2 WO 4 compared to uncoated samples. Data obtained from confocal laser microscopy images corroborate to SEM images. Absence of cells labeled with fluorides Syto 9 (green - live cells) and propidium iodide (red dead cells) indicate that there was no microbial adhesion on the surface of the treated specimens. These results suggest the potential use of Ag 2 WO 4 to prevent the contamination of titanium materials. [1] Ferraris, Sara, and S. Spriano. "Antibacterial titanium surfaces for medical implants." Materials Science and Engineering: C 61 (2016):

44 Comparison of antimycotic efficacy of biosynthesized selenium and tellurium nanoparticles against grapevine (Vitis vinifera) pathogenic fungi M. Andreolli, S. Lampis and G. Vallini Department of Biotechnology, University of Verona, Verona, Italy Grapevine is the most cultivated fruit plant in the world as well as the first economically important tree crop whose berries are mainly processed for making wine. However, growth of fungal pathogens both during grape ripening in plant and post-harvest fruit handling is the main cause of noticeable economic loss. Nanotechnology is an emerging research field with the potential to generate new innovative materials. Nanotechnology approaches to control fungal diseases in plants have recently registered an impressive burst. Studies have focused so far mostly on silver, zinc oxide, copper and gold nanoparticles [1]. Moreover, traditional physico-chemical synthesis of nanoparticles has been severely limited due to high production cost of and generation of toxic byproducts [2]. Therefore, development of eco-friendly processes for the synthesis of nanoparticles is regarded with growing interest. The natural capability of selected bacterial strains to change the oxidation state of metal/metalloid salts represents a valid and sustainable alternative for nanoparticles biogenesis. In this study we focused on the potential of antifungal activity of selenium and tellurium nanoparticles biologically synthesized by Ochrobactrum sp. MPV1 against four phytopathogenic fungi, namely Botrytis cinerea (the causative agent of grey mould), Neofusicoccum parvum (the Botryosphaeriaceae disease agent), Phaeomoniella chlamydospora and Phaeoacremonium aleophilum (both associated to the tracheomycosis/esca disease). Scanning Electron Microscopy (SEM) analysis indicated that either SeNPs or TeNPs produced were all spherically shaped. Moreover, EDX microanalysis of the purified SeNPs exhibited the characteristic absorption peak at 1.37, while,tenps showed the characteristic Te absorption peak at Eventually, SeNPs and TeNPs exhibited an average diameter of 215 nm and 83 nm respectively. The inhibition tests evidenced that SeNPs did not affect the growth of all the fungal strains considered even at the highest concentration of 500 mg l -1. On the other hand, TeNPs showed inhibitory activity against the fungal strains herein tested. The addition of 10 and 50 mg l -1 of TeNPs in the growth medium resulted in 91.5 and 100% of inhibition percentage respectively against B. cinerea. Furthermore, the treatment with 100 mg l -1 TeNPs showed 53.0% inhibition against N. parvum. Increasing of TeNPs concentration in the medium resulted in a progressive reduction of fungal growth up to 500 mg l -1 with complete inhibition of N. parvum. Similarly, the presence of 100 mg l -1 of TeNPs resulted in 46.2 and 47.2% of inhibition of P. chlamydospora and P. aleophilum respectively. Therefore, TeNPs treatment with 400 mg l -1 resulted in the total inhibition of P. chlamydospora, and the presence of 500 mg l -1 of TeNPs evidenced a 100% of inhibition towards P. aleophilum. The effects of TeNPs on fungal mycelia were analyzed by SEM. B. cinerea and N. parvum revealed that the presence of nanoparticles in the growth medium induced an morphological alteration of hyphae in comparison to the control. In fact, the mycelium in controls appeared organized in regular structure with a uniform hiphal shape. On the other hand, the mycelium treated with TeNPs appeared completely overlapped, disorganized, with unusual bulged and with higher hiphae density in comparison to the control. SEM analysis carried out on P. chlamydospora showed that the presence of TeNPs in the medium strongly reduced the formation of hyphal lateral branches in comparison to the control mycelia. Detection of reactive oxygen species (ROS) was performed by means of H 2 DCFDA, which is a cell-permeable indicator of ROS. The results showed a strong production of ROS in fungal hyphae treated with of TeNPs. ROS production was also induced by the presence of 500 mg l -1 of SeNPs, nevertheless with lower fluorescence intensity when compared to fungi exposed to TeNPs. In conclusion, this study reports on first evidences of antimycotic efficacy of biogenic TeNPs against phytopagenic fungi. Moreover, the results showed that TeNPs enhance the formation of reactive oxygen species in filamentous fungi and induce morphological changes during hyphal growth. Keywords: phytopathogenic fungi; metallic nanoparticles [1] He L, Liu Y, Mustapha A, Lin M (2011) Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiological research 166; [2] Narayanan KB and Sakthivel N (2010) Biological synthesis of metal nanoparticles by microbes. Adv Colloid Interface Sci. 156:

45 Comparison of sporulation and germination between clinical and hospital environment strains of Clostridium difficile and Clostridium perfringens Krzysztof Sacha, Jakub Orszulak, Aleksandra Orszulak and Alan Osuch Department of Medical Microbiology, Medical University of Silesia, Poland Hospital environment containing many hospital pathogens including: C. perfringens and C. difficile which can cause severe forms of infection in vulnerable patients. Spreading of these bacteria is facilitate due to sporulation. Spores are more resistant to unfavourable conditions i.a. oxygen, high temperature or chemicals like-alcoholbased disinfectants which can even potentiate sporulation. The aim was to compare sporulation and germination of C. difficile and C. perfringens strains isolated from clinical sample obtained from patients and hospital environment. Environmental strains were taken from one hospital located in Silesian region and cultured using C diff Banana BrothTM. Clinical strains were obtained from fecal samples of patient with antibiotic associated diarrhea (AAD), hospitalized in the same hospital. All strains were cultured on Columbia agar, and in the BHI broth, with 1% horse serum and in the Ellner medium. After growth of colonies alcohol shock (96% ethanol, 1:1) was performed for 30, 60 and 90 min. At the end, presence of alive bacteria and spores was controlled by Gram and Schaeffer Fulton staining. To check the ability of germination 96 hours after alcohol shock bacteria growth in BHI and Ellner medium were cultured on Columbia agar. Strains of C. perfringens and C. difficile isolated from fecal samples patients with AAD demonstrated better sporulation than environmental strains. Dependence between time of alcohol shock exposure and sporulation was observed. All strains from Ellner medium except one germinated very well. Growth of C. perfringens strains was abundant than C. difficile strains. Ellner medium provided better conditions for germination independently of source of strains. Germination of C. perfringens was more intensive than C. difficile especially among strains isolated from fecal samples of patients. 35

46 Copper alloys door handles: weapons against biofilms formation? G. Carré, F. Klingelschmitt and S. Gangloff UFR Pharmacie, Université de Reims Champagne-Ardenne, Laboratoire de Biomatériaux et Inflammation en Site Osseux (EA 4691), Reims, France Environmental surfaces as room doorknobs are a likely reservoir for potential pathogens and play a role in the acquisition of healthcare associated infection. Micro-organisms such as methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, from contaminated surfaces may then be transmitted via hands to other inanimate objects or to patients. To reduce the spread of bacteria, Le Bronze Alloys, a French company equipped several healthcare facilities / retirement homes with door knobs / door push plates in copper alloys. While the antimicrobial activity of these components is clear, a question was raised regarding the biofilm formation: is it possible to form a biofilm on copper alloys? The aim of this study was to evaluate the anti-biofilm properties of different copper alloys while also keeping its aesthetic appearance during time. Static MRSA biofilm cultures on these materials were lab evaluated in different simulations of environmental conditions with a low organic contamination (in peptone water) or with a high density of organic contamination (in tryptic soy broth). Although these environmental conditions are extreme, our results demonstrated that the biofilm formation is directly dependent to the composition of environments. The biofilm formation is reduced in poorly environment whereas in the rich media, the biofilm formation is favoured. Moreover, we observed by count on agar plates and by SEM, that the biofilm formation is also well dependent to the composition of the copper alloys. These artificially contaminations and extreme environmental conditions highlight the anti-biofilm properties of copper alloys developed by Le Bronze Alloys. However, as cumulative soiling and cleaning procedures can cause damage the anti-biofilm efficiency of materials, this anti-biofilm effect will also be evaluated under tarnishing conditions. Keywords: Copper alloys; MRSA; antimicrobial activity; biofilm Acknowledgments The project Cuprocare is funded by the European Union, FEDER and by the Region Grand Est. 36

47 Designer polymeric biocides C.A. Hae Cho 1, J. Jin 1 and S. Swift 2 1 School of Chemistry, University Of Auckland, Private Bag 92019, Auckland 1142, New Zealand 2 Department of Molecular Medicine and Pathology, University Of Auckland, Private Bag 92019, Auckland 1142, New Zealand The antimicrobials and biocides currently available now suffer from inactivity because of increasing rates of resistance developed in target organisms. In some cases there are further human and environmental toxicity issues that restrict use. One potential solution is the synthesis of new designer polymeric antimicrobials and biocides. The designer process uses an approach that allows for a modular synthesis of polymer backbone, with the addition of biocidal groups and ancillary groups with the facility for tuning each group to increase both potency and applicability. We have applied this strategy to the synthesis of an antimicrobial with an aliphatic polycarbonate polymer backbone and guanidine biocide groups. We have tuned the synthesis to give polymer chains of differing lengths, with different densities of the biocidal group, and different charge densities of the final polymer by the inclusion of ancillary groups on the polymer backbone. The success of this approach is measured by lowering the minimum inhibitory and lethal concentrations for Gram-positive and Gram-negative bacteria, reducing the time-to-kill for target bacteria, and increasing the concentrations required to show the potential for harmful effects in mammalian toxicity assays. Keywords: polymer biocide; designer biocide; polycarbonate; guanidine 37

48 Detection of marker proteins for bacteria antimicrobial resistance by mass spectrometry Y. Zhu and H.H. Girault Laboratoire d Electrochimie Physique et Analytique, École Polytechnique Fédérale de Lausanne, Rue de l'industrie 17, CH Sion, Switzerland Misuse and overuse of antimicrobial drugs over many years have led to the emergence of antimicrobial resistance among microbes worldwide[1]. For the fast diagnosis and efficient treatment of microbial infections, it is crucial to perform quick pathogens identification and rapid analysis of their antimicrobial resistance phenotypes. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for identification of microbes, especially bacteria. It can generate characteristic mass spectral fingerprints directly from intact bacteria and complete bacteria identification within minutes. This technique has received the clearance from US Food and Drug Administration (FDA)[2] and related equipments have been installed in many hospitals. Proteins encoded by antimicrobial resistance genes are directly involved in bacterial resistance process against antimicrobial drugs[3]. Antimicrobial resistance can be analysed by tracing these resistance-associated proteins within bacterial cells. Ideally, they should be read out directly from MALDI-TOF MS fingerprint patterns of intact bacteria without any sample pre-treatment, a useful procedure that would be comparable to fast bacteria identification. But many of those proteins are large ones (> 15,000 Da) expressed in low abundance, and are difficult to detect directly from intact cells by classic MALDI-TOF MS measurements, which typically focus on smaller proteins (< 15,000 Da) expressed in high abundance[4]. To enhance the performance of MALDI-TOF MS in intact bacteria analysis, we have developed a type of titanium dioxide-modified MALDI target plates.the plates were designed to photocatalytically destroy the bacterial envelope structure and improve the ionization efficiency of intracellular components, thereby promoting the measurable mass range and the achievable detection sensitivity. By generating high quality bacterial fingerprint patterns, the titanium dioxide-modified target plates could greatly boost the reliability of bacteria identification, which is based on fingerprint patterns matching. More importantly, it facilitates the extraction of more bacterial cellular information, and enables the detection of large molecular weight and low abundant bacterial components. Accordingly, a method for rapid detection of antimicrobial resistance-associated proteins, conferring bacterial resistance against antimicrobial drugs, was established by mass spectrometric fingerprinting of intact bacteria without the need of any sample pre-treatment. This approach of resistance proteins detection directly from intact bacteria by mass spectrometry is useful for fast discrimination of antimicrobial-resistant bacteria from their non-resistant counterparts whilst performing species identification. Also, It could be used as a rapid and convenient way for initial determination of the underlying resistance mechanisms. Keywords: bacteria; antimicrobial resistance proteins; MALDI-TOF MS; titanium dioxide-modified traget plates [1] M. Sharland, P. Saroey and E. N. Berezin, J Pediat-Brazil, 2015, 91, [2] P. Seng, M. Drancourt, F. Gouriet, B. La Scola, P. E. Fournier, J. M. Rolain and D. Raoult, Clin Infect Dis, 2009, 49, [3] J. M. A. Blair, M. A. Webber, A. J. Baylay, D. O. Ogbolu and L. J. V. Piddock, Nat Rev Microbiol, 2015, 13, [4] S. Sauer and M. Kliem, Nat Rev Microbiol, 2010, 8,

49 Determining the rate and cost of in vitro resistance development in Methicillin Resistant Staphylococcus aureus against moenomycin A D.G. Lloyd 1, E. Taylor 1, M. Goddard 1, I. Singh 2, C. Bolt 1, A. Parmar 2 and A. Iyer 2,3 1 School of Life Sciences, Beevor Street, University of Lincoln, Lincoln, UK 2 School of Pharmacy, Beevor Street, University of Lincoln, Lincoln, UK 3 Organic and Biomimetic Chemistry, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium Background: Resistance to all available antimicrobial drugs is rising and the ongoing development of resistance is one of the greatest threats to human health. First discovered in 1965, Moenomycin A is a naturally occurring, potent inhibitor of bacterial cell wall synthesis. Moenomycin A has broad spectrum activity against Grampositive bacteria. The mechanism of action operates via competition with Lipid II for the active site of the transglycosylase responsible for catalysing peptidoglycan synthesis [1]. This compromises the ability of the cell to regulate the cell wall synthesis and re-modelling, ultimately leading to cell death [2]. Despite widespread use in agriculture, there are only limited reports of developed resistance [3]. Furthermore, low-level laboratory resistance has previously only been induced via phage mediated point mutations [4]. Results: Six independent populations of methicillin-resistant Staphylococcus aureus were evolved from a single ancestor through continual passage culturing under Moenomycin A selection. After ~300 generations a >400-fold increase in tolerance to Moenomycin A was observed across all six replicates. This high level of resistance has not previously been reported in an initially susceptible cell line. Following removal of this selective pressure, the tolerance of Moenomycin A resistant strains had dropped to only 20x the initial susceptibility within 200 generations. A control experiment using rifampicin was run in parallel; these cell lines saw a >5000x increase in resistance over the same period. Examination of susceptibility in generated strains to a panel of antibiotics uncovered low level cross-resistance to other cell wall antimicrobials, such as vancomycin and bacitracin, to be associated with moenomycin resistance. Growth curves were plotted for each resistant strain across a range of conditions to highlight links between stress tolerance and acquired resistance. When subjected to each environmental stress, strains resistant to Moenomycin A saw a smaller impact on growth rate when compared with control strains. Except for when challenged by a sublethal concentration of Moenomycin A, leading to a significant reduction of growth rate of Moenomycin A resistant cells compared with the control. Conclusions: We have for the first time demonstrated the evolution of significant resistance to Moenomycin A in a previously susceptible cell line. Our results suggest that compared with rifampicin, this resistance comes at a low fitness cost to the cell. Low levels of cross resistance to other cell wall inhibitors may provide insight into potential mechanisms of resistance, which will be subject to genetic study. Keywords: Antibiotic resistance; Moenomycin; Staphylococcus aureus; In vitro resistance [1] Ostash B, Walker S. Moenomycin family antibiotics: chemical synthesis, biosynthesis, biological activity. Natural product reports. 2010;27(11): [2] Wallhausser, K. H., Nesemann, G., Prave, P., & Steigler, A. (1965). Moenomycin, a new antibiotic. I. Fermentation and isolation. Antimicrobial agents and chemotherapy, 5, 734. [3] Pfaller, M. A. (2006). Flavophospholipol use in animals: Positive implications for antimicrobial resistance based on its microbiologic properties. Diagnostic microbiology and infectious disease, 56(2), [4] Rebets, Y., Lupoli, T., Qiao, Y., Schirner, K., Villet, R., Hooper, D.,... & Walker, S. (2013). Moenomycin resistance mutations in Staphylococcus aureus reduce peptidoglycan chain length and cause aberrant cell division. ACS chemical biology, 9(2),

50 Development of novel disinfectants against pathogens occur in the hospital environment J. Marek 1, M. Benkova 1,2, D. Malinak 2, R. Dolezal 2, V. Bostikova 1, P. Bostik 1, L. Hobzova 2, L. Ryskova 2, D. Jun 3 and O. Soukup 2,3 1 Department of Epidemiology, Faculty of Military Health Sciences, University of Defence in Brno, Czech Republic 2 Biomedical Research Centre, University Hospital Hradec Kralove, Czech Republic 3 Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence in Brno, Czech Republic In this project, we would like to develop new compounds based on quaternary ammonium salts with a strong disinfectant potential against nosocomial infections in hospital environment both civil and military, thus bacterial, fungal and viral pathogens. Quaternary ammonium salts bearing 12 to 16 methylen units are well known cationic surfactants, whose mechanism of action is mostly related to the disruption of the microbial membrane. They were found to be effective against both bacteria and fungi. Furthermore, QAS were reported to be effective also against both encapsulated and non-encapsulated viruses. We have prepared 66 analogues of quaternary ammonium salts (QAS) containing quaternary nitrogen and carbon chain of C12, C14, and C16. Here we report the antimicrobial efficacy of newly prepared compounds against various pathogens (Gram positive strains, Gramm negative strains, fungi and VZ virus). We show the correlation of the antimicrobial effect with the chain length and with the lipophilicity which may be the major parameter for the interaction with the membrane leading to microbicidal action. We also show unique and potent antiviral action of some compounds. Due to low toxicity and safety for the operating personnel, we believe such compounds may become an interesting possibility for the disinfection in the hospital environment, especially in the form of water-based wide-spectrum mixtures. Such mixtures, whose spectrum can be tuned by the efficacy of individual components, may reduce the risk of microbial resistance due to unspecific mechanism of action. Keywords: Antimicrobial activity; synthesis; quaternary ammonium salts [1] Marek, J.; Malinak, D.; Dolezal, R.; Soukup, O.; Benkova, M.; Nachtigal, P.; Vavra, P.; Kuca, K. Novel Series of Quaternary Ammonium Surfactants Based on 2,3-Dihydro[2,3-b]pyridine-7-ol Ring: Synthesis, Analysis and Antimicrobial Evaluation, Lett Org Chem, 2018, 15, 3-9 [2] Marek, J.; Joskova, V.; Dolezal, R.; Soukup O.; Benkova, M.; Fucikova, A.; Malinak, D.; Bostikova, V.; Kuca, K. Synthesis, Antimicrobial Effect and Surface Properties of Hydroxymethyl-substituted Pyridinium Salts. 2017, Lett Drug Des Discov, 14, in press 40

51 Effect of an organic acids based feed additive and enrofloxacin on the prevalence of antibiotic-resistant E. coli in broilers N. Roth 1,3, S. Mayrhofer 1, M. Gierus 1, C. Weingut 1, C. Schwarz 1, B. Doupovec 2, R. Berrios 2 and K.J. Domig 1 1 Department of Food Science and Technology, Institute of Food Science, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria 2 Department of Agrobiotechnology, Institute of Animal Nutrition, Livestock Products, and Nutrition Physiology, BOKU - University of Natural Resources and Life Sciences, Vienna Austria 3 BIOMIN Holding GmbH, Austria The increase in antibiotic resistance is a global concern for human and animal health. Antibiotic use in animal production leads to high resistance prevalence. Antibiotics used for therapy, metaphylaxis or prophylaxis in animal husbandry belong often to the same classes as those used in human medicine. Further actions to reduce the spread of antibiotic resistance are of upmost importance. Reduction of antibiotic use is the key point in order to achieve lower resistance rates of bacteria. An approach that combines proper nutrition, biosecurity, hygiene, genetics, health and good farm management practices is needed. Innovative feed additives may contribute to the solution of the challenge of antibiotic resistance by decreasing the prevalence of antibiotic-resistant bacteria. Escherichia coli is regarded as an indicator organism of antibiotic resistance determination in Gram-negative bacteria. A study with broilers was evaluating the effect of an acidbased feed additive, as well as fluoroquinolone antibiotic on the prevalence of antibiotic-resistant E. coli. A total of 480 broiler chickens (Ross 308) were randomly assigned to three treatment groups (eight replicates per group): a control group receiving a basal diet; a group additionally receiving a feed additive (FA) based on formic acid, acetic acid and propionic acid; and group obtaining antibiotic enrofloxacin (AB) was provided via drinking water for three days. A pooled fecal sample of one-day-old chicks was collected upon arrival at the experimental farm. On day 17 and day 38 of the trial, cecal samples of three chicks from each of the eight replicate pens were taken, and the count of E. coli and antibiotic-resistant E.coli was determined. The results of this study showed a high prevalence of antibiotic-resistant E. coli in one-day-old chicks. Supplementation of the diet with FA or the treatment of broilers with AB did not have any significant influence on the total number of E. coli in the cecal content on day 17 and day 38 of the trial. Supplementation with FA contributed to a better growth performance and to a significant decrease (P 0.05) in E. coli resistant to ampicillin and tetracycline compared to the control and AB groups, as well as to a decrease (P 0.05) in sulfamethoxazole and ciprofloxacin-resistant E. coli compared to the AB group at the end of the trial. Compared to the control group, the average daily weight gain, but also the number of E. coli resistant to ciprofloxacin, streptomycin, sulfamethoxazole and tetracycline were significantly higher (P 0.05) in the AB group on day 38. The prudent use of antibiotics achieved by disseminating relevant information, raising awareness and implementing good farm management, nutrition and biosecurity practices is a main tool for the reduction of antibiotic resistance. However, also other strategies are required. Within this study promising results for the tested feed additive concerning the decline of antibiotic-resistant E. coli in poultry were obtained. In addition, it would be necessary to elucidate the underlying mechanism for the maintenance and loss of resistance. 41

52 Effect of Isothiocyanates on cholera toxin s gene expression Klaudyna Krause*, Dariusz Nowicki, Anna Herman-Antosiewicz and Agnieszka Szalewska-Pałasz University of Gdansk, Faculty of Biology, ul. Wita Stwosza 59, Gdańsk, Poland * Corresponding author: Klaudyna.Krause@phdstud.ug.edu.pl Introduction: Vibrio cholerae is a Gram-negative aquatic bacterium responsible for diarrheal disease cholera, which is still current in many developing countries. Vibrio cholerae persist in aquatic environments and infect humans via ingestion of contaminated water of food. The major virulence factors in V. cholerae are cholera toxin (CT) encoded by the ctxab cause a massive flux of fluid into the intestinal lumen and toxin-coregulated pili (TCP) form bundled filaments at the bacterial surface encoded by tcpa genes. Expressions of virulence factors are regulated by TcpP/TcpH and ToxR/ToxS, which activate the espression of ToxT- master regulator of virulence gene expression. ToxT protein attaches to toxbox region at upstream of ctxab genes and induces ctxab toxin expression. The use of natural compounds as inhibitory agents of virulence factors is a new idea to overcome increased antimicrobial resistance in pathogenic bacteria. Isothiocyanates are secondary plant metabolites, hydrolysis products of glucosinolates that are found in Brassica vegetables. There are limited evidence about molecular mechanism of antimicrobial properties of ITCs. Previously, we showed that ITC are promising agents against enterohemorrhagic Escherichia coli (EHEC) strains due to impairment of Shiga-toxin harboring bacteriophage development. In this study we examined if isothiocyanates can repress the cholera toxin (CT). Methods: The mechanism of ITC antimicrobial activities were tested according to CLSI standard methodology. MIC (minimal inhibitory concentration) were assessed by the twofold broth microdilution methods. Growth inhibition kinetics was determined spectrophotometrically. The influence of genes expressions were tested by Real-time quantitative reverse transcription-pcr assay. Total RNA extraction and reverse transcription was performed for making genes cdna (Evoscript universal cdna master Roche). Results was investigated by Western blotting analyses. The blot were subsequently probed with anti-cholera Toxin Alpha and anti-cholera Toxin Beta followed by horseradish peroxidase (HPR)-conjugated anti-rabbit IgG. Results: We determined MIC and MBC (Minimal Bactericidal Concentration) for phenethyl isothiocyanate (PEITC), sulforaphane (SFN. We observed the growth inhibition by ITCs, varying for different compounds. We found that isothiocyanates effectively repressed the transcription of ctxab, toxt, toxr, toxs, tcpi genes. These results suggest that isothiocyanetes might act as a potential repressor for CT production. Keywords: Vibrio cholera; virulence factors; isothiocyanates; MIC; ITC; cholera 42

53 Effects of cerium oxide nanoparticles on bacterial outer membrane permeability and their potential application as antibiotic adjuvant in MDR pathogenic bacteria P. Bellio 1, C. Luzi 1, A. Mancini 1, S. Cracchiolo 1, M. Passacantando 2, L. Di Pietro 1, M. Perilli 1, G. Amicosante 1, S. Santucci 2 and G. Celenza 1 1 Department of Biotechnological and Applied Clinical Sciences, University of l Aquila, via Vetoio- loc. Coppito, 67100, l Aquila, Italy 2 Department of Physical and Chemical Sciences, University of L'Aquila, via Vetoio-loc. Coppito, 67100, l Aquila, Italy Background. The phenomenon of antimicrobial resistance has emerged among pathogenic bacteria since the beginning of the antibiotic era as consequence of the selective pressure generated by the use, abuse and misuse of antibiotics for human and veterinary medicine. Therapeutic options against Multi Drug Resistant (MDR) pathogens are limited and the overall strategy would be the development of adjuvants able to enhance the activity of available antibiotics. The development of antibiotic adjuvants does not necessarily imply the discovery of new targets in bacterial cells, on the contrary, most of the known cell targets can be exploited (e.g. beta-lactamase inhibitors), or even unspecific compounds like outer membrane permeabilizer can be used to increase the activity of antibiotics. The outer membrane in Gram-negative bacteria is a semipermeable barrier conferring inherent resistance to most antibiotics. Unspecific outer membrane permeabilizer, like metal-oxide nanoparticles, can be used to increase the activity of antibiotics in drug-resistant pathogens. The study aims to investigate the effect of cerium oxide nanoparticles (CeO 2 NPs) on bacterial outer membrane permeability and their potential application in increasing the antibacterial activity of antibiotics in MDR pathogens. Methods. The ability of CeO 2 NPs to permeabilize Gram-negative bacterial outer membrane was investigated by several methodologies. To verify the ability of cerium oxide NPs to induce perturbation of membrane permeability, multilayer liposomes were loaded with calcein, which fluorescence is quenched as the molecules are confined within the liposomal vesicles. The extent of the damage was evaluated using lipid vesicles loaded with FITC-dextran probes. In this study, three different FITC-dextrans have been used: D-4, D-20 and D-70 with an average molecular weight of about 4, 20 and 70 kda, respectively. The difference in molecular weights allows to determine the extent of the damages to the membrane by observing the size-dependent release of liposomeentrapped probes. The effect on bacterial outer membrane was evaluated by measuring the coefficient of permeability of the outer membrane of Escherichia coli at increasing concentrations of CeO 2 NPs. To measure the C parameter, the recombinant E. coli HB101 producing TEM-1 β-lactamase, has been used as model. With the purpose of exploiting the possible application in the field of antimicrobial resistance, cerium oxide NPs were tested in combination with therapeutically available β-lactam antibiotics by chequerboard assay, against multidrug resistant pathogenic K. pneumoniae clinical isolate expressing high levels of resistance against those antibiotics. Results. The percentage of permeabilised liposomes increases in a dose-response manner when treated with increasing concentrations of cerium oxide NPs. The maximum percentage of calcein leakage is reached at 400 µg/ml of cerium oxide NPs with almost the 80% of permeabilised liposomes. The highest leakage can be observed in lipid vesicles containing D-4 probe, with a percentage of dextran leakage about 15% at the highest concentration of cerium oxide NPs (600 µg/ml). In D-20 and D-70 loaded liposomes, even at the highest cerium oxide NPs concentration, no significant leakage can be observed. In our experimental settings, the E. coli outer membrane permeability coefficient determined at several cerium oxide NPs concentrations increases as the concentration of cerium oxide NPs becomes higher. In absence of CeO 2 NPs the value of C is equal to ± s -1, while it becomes twice at 100 µg/ml of cerium oxide (C= ± s -1 ). Native CeO 2 NPs are not behaving as antibacterial since no reduction in bacterial growth has been observed at a concentration of 256 µg/ml, the maximum solubility obtained. Synergistic activity was observed when CeO 2 NPs were combined with cefotaxime, imipenem, amoxicillin, and its combination with clavulanate. Conclusion. Any significant alteration of outer membrane permeability, in the meaning of augmentation, would increase the total uptake of antibiotics into the periplasmic space, which, despite the presence of hydrolytic enzymes, can exert their antimicrobial action. In this study, we have shown as inactive cerium oxide NPs, are able to increase the effectiveness of antimicrobials which activity has been compromised by the presence of drug resistance mechanisms. It is plausible to suppose that the synergistic effect is the result of the interaction between cerium oxide NPs and the outer membrane of bacterial cell. Moreover, the demonstrated low toxicity of CeO 2 NPs, makes them attractive as potential antibiotic adjuvants against MDR pathogenic organisms. The new 43

54 frontier of antimicrobial chemotherapy is, nowadays, to try to revert antibacterial resistance by combination of drugs, to make old and inactive molecules useful in clinical practice. The idea that the efficacy of a lost antibiotic can be retrieved, seems to be today the only rescue strategy capable of fighting infections sustained by MDR pathogenic bacteria. Keywords: CeO 2 nanoparticles; drug synergism; antimicrobial resistance; Bliss independence theory; KPC beta-lactamase; K. pneumoniae 44

55 Effects on cell viability, growth and morphology of C. albicans SC5314 biofilms after kinpen 09 plasma treatment O. Handorf 1, *, T. Weihe 1, S. Bekeschus 1, A. Graf 2, U. Schnabel 1, K. Riedel 2 and J. Ehlbeck 1 1 Leibniz Institute for Plasma Science and Technology e.v., Felix-Hausdorff-Str. 2, Greifswald, Germany 2 Ernst Moritz Arndt University, Microbial Physiology and Molecular Biology, Felix-Hausdorff-Str. 8, Greifswald, Germany The major form of microbial life is the biofilm, where cells live in dense communities and can survive lethal conditions for planktonic cells. The aim of the present study was to investigate in vitro the susceptibility of Candida albicans biofilms to plasma treatment with the commercial non-thermal plasma device kinpen 09, in terms of growth, survival and cell viability (Fig. 1 A). For these effects, 24 h grown C. albicans biofilms, strain SC5314 (ATCC MYA-2876), were treated with plasma in five different treatment times. The fungicidal effects of the plasma treatment were assessed by colony forming units, fluorescence and XTT assays. With these experiments, it was shown a high impact on the viability and metabolism of the cells. In the second part, it was discussed if the insights provided by fluorescence microscopy (Fig. 1 B-C), confocal laser scanning microscopy and atomic force microscopy, supported the indications for strong changes in morphology within 5 minutes of plasma treatment of the biofilms with the kinpen 09. These in vitro studies demonstrated that the plasma treatment of C. albicans SC5314 biofilms with kinpen 09 may also have a major impact on cell viability and growth within 5 minutes of treatment. Because of the biochemical-mechanical influence of the plasma on the biofilm, resistances of microbiological structures against plasma treatment are unknown at this juncture. Keywords: biofilms; C. albicans SC5314; decontamination; fluorescence microscopy; kinpen 09 Acknowledgments The Leibniz Institute for Plasma Science and Technology, INP in Greifswald, Germany, supported this work financially. Fig. 1 A) kinpen 09 non thermal atmospheric plasma jet B) fluorescence microscopy of C. albicans reference biofilm C) fluorescence microscopy of C. albicans biofilm after 60 s of plasma treatment with the kinpen 09 in a distance of 18 mm. 45

56 Engineering of Next-Generation Antimicrobial Peptides (AMPs) I: mechanisms behind how AMPs act and how bacteria develop resistance against them Jefferson Shaw 1, Qingchun Shi 1, Ed Stover 1, Jeanette Velasquez 2, Roel Rabara 2, Loan Huynh 2, Shujian Zhang 2 and Goutam Gupta 2 1 U. S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA 2 New Mexico Consortium Biolab, 100 Entrada Drive, Los Alamos, NM Antimicrobial peptides (AMPs) are an important component of host innate immune system, which offers the first line of defense against pathogen attack. After their discovery, researchers and physicians hoped that the host AMPs would be a viable alternative to antibiotics against antimicrobial resistant bacteria. However, this hope was short lived. Bacteria were able to develop resistance also against the host AMPs. First, we conducted theory and experiment to determine the mechanisms of how AMPs act and how bacteria develop resistance against them. For this, we chose a host amphipathic helical 11 amino acid long peptide (named P11) as a model AMP. We performed molecular dynamics (MD) simulations to analyze the contributions of the three key steps critical to P11 action, i.e., attachment to the polar heads of a bilayer lipid membrane, subsequent insertion into the hydrophobic interior, and finally rupture of the membrane. We also generated an E. coli strain resistant to P11. We sequenced the genome of the resistant strain to determine the gene mutations that affect the membrane attachment, insertion, and rupture by P11. We identified mutations in as many as 12 genes in the resistant strain. We also experimentally determined that mutations in all 12 genes (and not on one or a few) are needed for the observed resistance. Mutations in these genes led to impaired membrane attachment and insertion, which, in turn, inhibited membrane rupture. This is the first demonstration that multiple gene mutations are absolutely required for the observed bacterial resistance against the host AMPs. We are currently performing transcriptome (RNA-seq) analysis of the P11-suscptible and P11-resistant E. coli strains with or without P11 to identify the pathways altered in the resistant strain due to mutations in 12 genes. Acknowledgement This work was funded by USDA and Industries. 46

57 Engineering of Next-Generation Antimicrobial Peptides (AMPs) II: nextgeneration AMPs with high bactericidal activity and low susceptibility to bacterial resistance Jefferson Shaw 1, Qingchun Shi 1, Ed Stover 1, Jeanette Velasquez 2, Roel Rabara 2, Loan Huynh 2, Shujian Zhang 2 and Goutam Gupta 2 1 U. S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA 2 New Mexico Consortium Biolab, 100 Entrada Drive, Los Alamos, NM Based upon MD simulations, we predicted that a helix-turn-helix, in which two endogenous amphipathic P11 helices are joined by a turn, would have higher bactericidal activity due to more efficient membrane attachment, insertion, and rupture than the single helix. Bactericidal assays demonstrated that P26 not only was more active on human and plant bacteria P11 but also was able to overcome the E. coli resistance developed against P11. We are now designing several members of the helix-turn-helix scaffold to further improve the bactericidal activity and at the same time overcome bacterial resistance developed against endogenous single helical peptides and common antibiotics. Finally, we showed that P26 is also active on bacterial biofilms, which are prevalent in community- and hospital-associated infections. In summary, we report that understanding the mechanisms of host AMP action and bacterial resistance against them allow us to engineer novel AMPs that are superior to endogenous AMPs and are active on antibioticresistant bacteria and biofilms. Acknowledgement This work was funded by USDA and Industries. 47

58 Essential oils of wild Algerian endemic Lamiaceae as antibacterial against opportunistic bacteria involved in nosocomial infections L. Bousmaha-Marroki 1,2, Ahmed Marroki 1, Ange Bighelli 3, Felix Tomi 3 and Joseph Casanova 3 1 Department of Biology, Faculty of Natural and Life Sciences, University Djillali Liabes, Sidi Bel Abbès 22000, Algeria 2 Microbiology and Natural Product Team, Laboratory of Vegetal Biodiversity - Faculty of Natural and Life Sciences, University Djillali Liabes, Sidi Bel Abbès 22000, Algeria 3 University of Corsica, UMR-CNRS 6134, Route des Sanguinaires, Ajaccio, France The emergence of bacterial strains showing resistance to the antibiotic molecules had followed the introduction of antibiotic therapy as clinical practice in real time. The ancestral use of herbal medicine against infectious diseases, strengthen assumptions of researchers considering metabolism of plants the real future of antimicrobial molecules. Essential oils are complex volatile mixture produced by secondary metabolism of aromatic plants. Our work was focused on chemical characterization and antimicrobial screening of Algerian endemic plants essential oil's to suggest their use as antimicrobial agents in hospital hygienic and disinfection practices. Chemical composition of Thymus ciliatus and Lavandula dentata oils were achieved by C 13 RMN of the mixtures and CPG. Thymus ciliatus is carvacrol rich oil. Oils of L. dentata shows tow chemotyps β-pinen/transpinocarveol and β-pinen/1,8-cineol. We assess in vitro the inhibitory effect of oils on opportunistic bacterial strains isolated from patient s wounds showing nosocomial infections: Staphylococcus aureus; Pseudomonas aeruginosa; Escherichia coli; Proteus mirabilis; Enterobacter cloeaceae; Cirobacter frundii. Antimicrobial assays of T. ciliatus and the tow L. dentata chemotyps oils were performed by using direct contact (liquid oil fraction) and microatmosphere (vapor oil fraction) methods. High activity was noted against S. aureus strains by vapor and liquid part of the tree oil samples. Thyme oil exhibited a significant inhibition of enterobacteria and Pseudomonas aeruginosa. On the fact that tested strains were hospital acquired, possible use of essential oils in hospitals as natural antimicrobial agents in hygienic practices may be suggested. The additive effect of major and minor constituents of essential oils reinforces their lethal action towards a wide spectrum of microorganism. Keywords: Essential oils; Endemic Lamiaceae; C 13 RMN of the mixtures; antibacterial activity; opportunistic bacteria; nosocomial infections [1] Leila Bousmaha-Marroki, Fewzia Atik-Bekkara and Félix Tomi and Joseph Casanova. (2007) Chemical Composition and Antibacterial Activity of the Essential Oil of Thymus ciliatus (Desf.) Benth. ssp. eu-ciliatus Maire from Algeria. J. Essent. Oil Res., 19, [2] Leila Bousmaha, Jean Brice Boti, Fewzia Atik Bekkara, Vincent Castola and Joseph Casanova (2006). Infraspecific chemical variability of the essential oil of Lavandula dentata L. from Algeria. Flavour Fragr. J.; 21: [3] Leila Bousmaha, Fewzia Atik Bekkara., Felix Tomi and Joseph Casanova (2003). Advances in the Chemical Composition of Lavandula dendata L. Essential Oil from Algeria. J. Essent. Oil Res., 17,

59 Estimation of Non-Specific Immunity of Patients with Patients with Inflammatory Diseases of Large Salivary Glands A.I. Goncharova, V.U. Zemko and A.K. Kakoichankava Relevance: Determination of lysozyme content in serum, saliva and other biological fluids in various diseases gives opportunities for the development of new methods of diagnosis and treatment. In clinical laboratory practice, the determination of the level of lysozyme in biological fluids can be useful as a monitoring of the course of infectious and inflammatory diseases. Lysozyme is present in all body fluids and is an important factor of bactericidal activity. The amount of lysozyme in serum is from 30 μg / ml [1]. Its serum content increases with acute and chronic myelogenous and monocytic leukemias, neutrophilic leukocytosis, tuberculosis, sarcoidosis, acute bacterial infection and decreases in chronic bacterial infections, sepsis, peritonitis, and also with bone marrow hypoplasia. By cleaving the bacterial wall peptidoglycan, lysozyme protects the oral mucosa from pathogenic bacteria. The source of lysozyme in the oral fluid is the parotid and submandibular salivary glands. The content of the enzyme in the secretion of the submaxillary glands is higher than in the parotid glands. In the oral liquid, lysozyme contains more than in other human fluids. Oral fluid contains approximately 200 μg / ml of lysozyme, but a particularly high lysozyme content in the lacrimal fluid is about 7000 μg / ml [2]. Goal: Estimate the concentration of lysozyme in biological fluids in patients with siloadenitis. Materials and methods of research: To study the amount of lysozyme of oral fluid, 60 patients with sialadenitis were examined. Patients were on inpatient treatment in the department of maxillofacial surgery of the Vitebsk Regional Clinical Hospital. The comparison group was 20 people without purulent-inflammatory diseases of the maxillofacial region and pathology of the salivary glands. The average age of patients with inflammatory diseases of the large salivary glands was ± years, in the demographic structure, men predominated - 55%, women - 45%. The duration of hospitalization was from 3 to 13 days, an average of 6.46 ± 2.46 days. Before the treatment, oral fluid was taken one hour before meals into sterile test tubes. In patients with sialoadenitis two times during the period of hospitalization: 1 sample - on the day of admission to the hospital before antibiotic therapy, 2 test - on the first day of clinical recovery, coinciding with the patient's discharge from the hospital. Results of the study: Isolation of peptidoglycan (PG) from the cell wall of Gram-positive bacteria was carried out according to the procedure proposed by Lvov VL, Pinegin BV, Khaitov RM. in our modification. Micrococcus lysodeikticus ATCC 4698 was used as the culture. The resulting PG was bound with a 2% Congo red solution. For the formulation of the method, peptidoglycan labeled with 2% Congo red (PMC), oral liquid, phosphate buffer solution (PBS) ph 6.0 and 0.9% NaCl. The oral liquid was centrifuged for 1.5 minutes at 10,000 rpm for 10 minutes to precipitate suspended particles. One series of eppendorfes was introduced sequentially: 300 μl of PBS, 100 μl of PMK solution and 100 μl of oral liquid. Controls were samples containing a phosphate buffer solution ph 6.0 in an amount of 300 μl, 100 μl 0.9% NaCl and 100 μl of oral liquid. Further incubation of samples in a thermostat was performed at t = 370C for 24 hours. After incubation, the samples were removed from the thermostat and centrifuged to precipitate the remaining unbroken PMC. From the supernatant, 150 μl of the solution was taken in duplicate and transferred to the wells of a 96-well polystyrene plate. The plate was placed in a multichannel spectrophotometer F300, where the optical density in the wells was determined at a wavelength of 492 nm. The intermediate result was calculated as the difference between the optical densities of the experimental samples and the corresponding control samples. To recalculate the final result of lysozyme activity in μg / ml, a formula was obtained after performing the correlation regression analysis and constructing a matched calibration schedule for the diluted lysozyme, which reflects the dependence of lysozyme concentration on the release of Congo red solution from the substrate. The equation of the function was as follows: X = * (Eop-Eopk) 2.26 where X is the amount of lysozyme, in μg / ml; Eop is the optical density of the sample; Eopk is the optical density of the control. The results of the study of the amount of lysozyme in the oral fluid are presented in Table 1. 49

60 N Patient groups N Concentration of lysozyme, μg / ml Me; LQ - UQ 1 Comparison group ,198; 97,93 303,42 2 Sialoadenite (sample 1) ,71; 387,8 884,8 3 Sialoadenite (sample 2) ,83; 204,69 334,35 Note: The differences between the groups are valid (p 1-2 <0,01; p 2-3 <0,01; p 1-3 <0,05) To assess the sensitivity (SEN) and the specificity (SP) of the method for diagnosing sialoadenitis by the level of lysozyme in the oral fluid, an ROC analysis of the data was performed. Diagnostic criterion (DC) and diagnostic efficiency (DE) of this method are determined. The sensitivity of the method for the diagnosis of sialoadenitis by the level of lysozyme in the oral fluid was 90.0%, the specificity was 76.9%. the area under the ROC curve is Analysis of the results and application of ROC analysis in the course of the study allows us to classify the patients examined with a lysozyme level in the oral fluid above 340 μg / ml to the group of patients with sialadenitis and to use the level of lysozyme in the oral fluid of patients with siladenites as an additional diagnostic criterion of the disease. Conclusions: A significant increase in the level of lysozyme of the oral fluid was revealed in the development of the inflammatory process in the large salivary glands (559.71, μg / ml). During the reconvalescence, the lysozyme content in the oral fluid is normalized (286.83, μg / ml). With the development of purulent-inflammatory processes of the salivary glands during the exacerbation of chronic sialoadenitis, a higher level of lysozyme is observed as an adaptation factor. [1] Kovanyi, G. Urine and blood serum muramidase (lysozyme) in patients with urogenital tumors / G.Kovanyi K.Letnansky - European Journal of Cancer - Volume 7, Issue 1. [2] Zelenova, E.G. Microflora of the oral cavity: norm and pathology / M.I. Zaslavskaya, E.V. Salina, S.P. Rassanov.- Nizhny Novgorod,

61 Evaluation of antibacterial properties of copper alloys surfaces in long-term geriatric care facilities M. Colin 1, F. Klingelschmitt 1, E. Charpentier 1, F. Reffuveille 1, C. De Champs 2 and S. C. Gangloff 1 1 EA 4691 BIOS, Faculty of Pharmacy, University of Reims Champagne-Ardenne, Reims, France 2 Laboratory of Bacteriology Virology Hospital hygiene, CHU Reims, Reims, France During the last decades, healthcare-associated infections (HAIs) emerged as a major threat for public health, especially in the hospital environment. In France, HAIs concern around 1 on 20 patients during their hospitalization [1]. Cross-transmission of microorganism from inanimate surfaces, on which they can persist for months [2], to patients or health workers may have a significant role in HAIs [3]. Among these surfaces, door handles appear as evident source of microorganisms cross-transmission. Copper antimicrobial properties are known since antiquity. Nevertheless, recent interests have grown to create antimicrobial copper touch surfaces, in order to counteract microbial spread in the healthcare environment. In this study, 5 long-term geriatric care facilities (located in Marne, France) were at 50% outfitted with copper alloys door handles and handrails. A first in vitro study on copper alloys door handles from these facilities demonstrated a 2.5 log reduction of the number of meticillin-resistant Staphylococcus aureus. Related to the environmental bacterial contamination, 1400 samples were carried out on copper and control elements, 1.5 and 3 years after copper installation. Swabs were transferred into sterile peptone water. 100 µl of the suspension were inoculated onto various agar plates and incubated at 37 C, for Colony Forming Unit (CFU) enumeration. Identification of bacterial species was performed using MALDI-TOF technology. In comparison to control surfaces, copper door handles and handrails revealed significantly lower contamination levels. A maximal bacterial burden reduction of 94.3% for copper door handle and 70.5% for handrail was observed. Indicators species such as S. aureus, E. faecium, E. faecalis and member of the Enterobacteriaceae family were rarely observed. For example, S. aureus was observed on 0.46% of copper elements vs 1.38% of control elements. Staphylococcus and Micrococcus were the most predominant genera. Both genera were less frequently observed on copper elements, with a significant difference for Staphylococcus. Copper alloys elements show lower levels of contaminations. Box plot representation. Thick line in boxes: median. Boxes with horizontal lines: control elements. Boxes with vertical lines: copper elements. This study shows that copper elements could be successful in controlling environmental bacterial contamination. Furthermore, antibacterial properties of copper alloys elements are preserved through years, supporting the strong interest of introducing copper touch surfaces in long-term geriatric care facilities or healthcare facilities. Keywords: Copper; Antimicrobial; Healthcare-associated infection; Long-term geriatric care facilities; MALDI-TOF [1] Enquête nationale de prévalence des infections nosocomiales et des traitements anti-infectieux en établissements de santé, France, Réseau d alerte, d investigation et de surveillance des infections nosocomiales (Raisin), Ed : Institut de veille sanitaire, ISSN : , ISBN NET : [2] Kramer, A., Schwebke, I., & Kampf, G. (2006). How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC infectious diseases, 6(1), 130. doi: / [3] Otter, J. A., Yezli, S., Salkeld, J. A., & French, G. L. (2013). Evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings. American journal of infection control, 41(5), S6-S11. doi: /j.ajic Acknowledgments The project is funded by Region Grand Est and the European Union, program A , convention N , MC is the recipient of a fellowship from the French Association Nationale de la Recherche et de la Technologie (ANRT). The copper alloys have been installed by Lebronze alloys, France. 51

62 Extensive drug resistant (XDR) Acinetobacter baumannii in coronary care units of a hospital in Kuwait Ali A. Dashti 1, Sara Shamsah 1, Ishaq Mohmid 2, Sherief El-Shazly 1 and Leila Vali 1 1 Department of Medical Laboratory sciences, Faculty of Allied Health Sciences, Kuwait University, Kuwait 2 Department of Laboratory, Kuwait Oil Company Hospital, Kuwait Introduction: Extensive drug resistant (XDR) Acinetobacter baumannii are resistant to at least three classes of antimicrobials as well as to carbapenems. Here we examined seven XDR clinical A. baumannii isolates obtained from one of the largest secondary hospitals in Kuwait managed by Kuwait Oil Company (KOC). Materials and methods: Seven XDR A. baumannii isolates were isolated from different clinical specimens including blood, sputum, pus, wound, and central line tip from patients admitted to ICU, CCU, and coronary and surgical wards in The average age of the patients was 72.2 yrs. Patients primary diagnosis included internal perforation, facial swelling, diabetes, Left ventricular failure in coronary artery disease, investigational fever after surgery, tracheostomy fever and intestinal blockage surgery. Antimicrobial susceptibility testing was performed for ampicillin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, sulfamethoxazole/trimethoprim, tetracycline, erythromycin, piperacillin/tazobactam, amoxicillin/clavulanate, ceftazidime/ceftazidime clavulanate and colistin. Pulse-field gel electrophoresis (PFGE) was carried out to study the genetic typing of the isolates. Results: All 7 isolates were resistant to all the antibiotics tested except for colistin. Analyzing the PFGE patterns demonstrated 4 PF types. Four of the isolates obtained from CCU and the coronary ward showed indistinguishable PF pattern suggesting the presence of an epidemic. The source of the epidemic was not identified. Conclusions: Carbapenem-resistant A. baumannii isolates found in ICU, CCU and surgical wards of a hospital raise serious concern especially among immune-compromised patients. Nevertheless KOC hospital is one of the rare health facilities that resistance to colistin has not yet been identified in clinical A. baumannii that circulate the wards. Although in this hospital infection control practices is adhered to, it is essential to vigilantly monitor the affected patients. Keywords: Extensive drug resistant (XDR); Acinetobacter baumannii 52

63 Fmoc Chemical Synthesis of a Cyclotide with Antibacterial Activity against Aquaculture Pathogens Claudio A. Álvarez 1,2,3, Paula A. Santana 4, Omar Luna 4, Constanza Cárdenas 4, Fernando Albericio 5,6, María S. Romero 2 and Fanny Guzmán 4 1 Laboratorio de Fisiología y Genética Marina, Centro de Estudios Avanzados en Zonas Áridas, Larrondo 1281, , Coquimbo, Chile 2 Facultad de Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, , Coquimbo, Chile 3 Centro AquaPacífico, Larrondo 1281, , Coquimbo, Chile 4 Núcleo de Biotecnología de Curauma, Pontificia Universidad Católica de Valparaíso, Avenida universidad 330, , Valparaíso, Chile 5 CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, and Department of Organic Chemistry, University of Barcelona, C/ Adolf Florensa, 8, 08028, Barcelona, Spain 6 School of Chemistry, University of KwaZulu-Natal, 4041, Durban, South Africa Most peptides show a linear structure with open ends, which makes them targets for proteolytic enzymes, thus decreasing their bioavailability [1,2]. Measuring amino acid residues in length, cyclotides are the largest known family of cyclic peptides [3]. A cyclized backbone, together with multiple disulfide bonds, confers the peptide exceptional stability against protease digestion and thermal denaturation [4]. Thus, the ability to chemically synthesize cyclotides is an important goal, both with respect to understanding their mechanism of action mechanism and studying new pharmaceutical applications. Here we used the tea-bag Fmoc-SPPS chemical synthesis approach on 2-chlorotrytil chlorine (CTC) resin to generate a bioactive cyclotide. MALDI- TOF mass spectrometry determined the correct peptide sequence and the cyclization sites-critical in this multicyclic compound. The cyclotide showed antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens present in Chilean aquaculture. The highest antimicrobial activity was found to be against F. psychrophilum. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy. We postulate that this compound exerts its mechanism of action by disrupting the bacterial membrane. Keywords: Cyclotide; Fmoc-SPSS; Gram negative bacteria; membrane disruption [1] A.S. Gokhale, S. Satyanarayanajois. Peptides and peptidomimetics as immunomodulators. Immunotherapy (2014) 6: [2] J.H. Hamman, G.M. Enslin, A.F. Kotzé. Oral delivery of peptide drugs: barriers and developments. BioDrugs (2005) 19: [3] D.C. Ireland, M.L. Colgrave, P. Nguyencong, N.L. Daly, D.J. Craik. Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins, J. Mol. Biol. (2006) 357: [4] D.J. Craik, R.J. Clark, N.L. Daly, Potential therapeutic applications of the cyclotides and related cystine knot miniproteins, Expert Opin. Investig. Drugs. (2007) 16:

64 Garlic essential oil as an antimicrobial agent towards mycotoxigenic fungi K.C.P. Bocate, A.G. Evangelista, J.A.F. Corrêa and F.B. Luciano Graduate Program in Animal Science, Pontifical Catholic University of Paraná, R. Imaculada Conceição, 1155, , Curitiba, Brazil Brazil is one of the largest exporters of grains in the world, producing million tons in the 2016/2017 harvest season. It is estimated that 25% of grains produced worldwide are contaminated with mycotoxins. According to the International Agency for Research on Cancer (IARC), mycotoxins can present carcinogenic, nephrotoxic, immunotoxic, teratogenic, and other activities in biological systems. In addition, physical chemical conditions, such as high humidity and temperature, and inadequate cultivation, harvesting, and storage can predispose grains for mycotoxin contamination. Alternative measures are proposed to reduce the growth of toxigenic fungi and consequent production of toxins, such as the use of treatments with natural compounds, which have been well received by consumers. Garlic essential oil has proven antioxidant, antihypertensive, antiarrhythmogenic, antifungal, and antibacterial activities, and one of the major compounds responsible for these activities is known as allicin. The objective of this work was to evaluate the antifungal action of garlic essential oil (51% allicin) against the mycotoxigenic fungi Aspergillus parasiticus CECT 2681, Fusarium verticillioides CECT 2983, and Giberella zeae CECT 2150, respective producers of aflatoxins, fumonisins, and zearalenone. The microdilution technique standardized by the Clinical and Laboratory Standards Institute (CLSI) protocol, was used to determine the minimum inhibitory concentration (MIC) of garlic oil for the three fungal species. In addition, the gaseous antifungal activity of the oil was tested in hermetic glass jars introduced with 50 mm petri dishes containing 10 4 spores of either fungal species. The MIC of the garlic essential oil was determined as 15.62, 125, and μl/l for A. parasiticus, F. verticillioides, and G. zeae, respectively. When in volatilization, there was a significant decrease for A. parasiticus to 0.5 μl/l with 29% inhibition, and total growth inhibition at 4 μl/l. For F. verticillioides and G. zeae there was a significant growth inhibition at the concentration of 2.5 μl/l, with total inhibition at 10 and 20 μl/l, respectively. The results demonstrated the efficiency of garlic essential oil against the evaluated mycotoxigenic fungi, and evaluation tests of its efficiency in food matrices are now required. Keywords: Natural antifungals; Bioactive natural compounds; Garlic essential oil; Mycotoxins 54

65 Graphene-based Oleo-polyurethane Nanocomposites with Biocidal Function as Active Food Packaging Materials: Synthesis and Mechanism of Antimicrobial Action Younes Ahmadi and Sharif Ahmad Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi , India Nano-materials having antibacterial activity are the potential candidates in the food-packaging industry due to their ability in preventing the growth of food-borne pathogenic bacteria, improving the quality and shelf life of food materials along with reducing the environmental hazards. Therefore, in present study we developed oilbased biodegradable antimicrobial polyurethane nanocomposites (PU-NCs) using different graphene-based (Gr, graphene oxide [GO], and reduced graphene oxide [RGO]) via in-situ polymerization. The morphology, mechanical, thermal, antibacterial, packaging (barrier), migration, and soil degradation properties of PU-NCs were investigated. These studies revealed that homogeneous dispersion of NPs within PU decreased the crystallinity of the matrix that improved its flexibility. The PU-NCs exhibited greater strength, glass transition temperature, and toughness, while they showed decrease in water and simulants uptake compared to the plain PU, which is associated with the strong matrix NPs interfacial interaction achieved through hydrogen and/or chemical bonding. The antimicrobial activity of these NCs were evaluated by the well diffusion technique against two human pathogen bacteria, E. coli and S. aureus, which revealed an effective biocidal result by GO- PU and RGO-PU NCs. To understand their antibacterial activity, we conducted the scanning electron microscopy (SEM) on these bacteria samples after incubating with GO-PU and RGO-PU NCs that suggested higher antimicrobial effect of GO-PU compared to RGO-PU NCs. This can be ascribed to the size and better dispersion of GO-NPs within the matrix, which increased the chance of contact between bacteria cells and GO-NPs, causing their cell membrane disruption. These results revealed that fabricated oleo-pu-nc (GO and RGO) films have potential scope to be used as novel antimicrobial films in food packaging industry. 55

66 High prevalence of extended-spectrum ß-lactamase producing Enterobacteriaceae among clinical isolates from cats and dogs in Switzerland M. Nüesch-Inderbinen 1, A.L. Zogg 1, S. Simmen 1, K. Zurfluh 1, S.N. Schmitt 2 and R. Stephan 1 1 National Centre for Enteropathogenic Bacteria and Listeria, Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zürich, Winterthurerstrasse 272, 8057 Zürich, Switzerland 2 Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zürich, Winterthurerstrasse 272, 8057 Zürich, Switzerland Background: One of the most important mechanisms of antimicrobial resistance in Enterobacteriaceae is the production of plasmid-mediated extended-spectrum ß-lactamases (ESBLs) including TEM-, SHV- or CTX-Mgroup enzymes. ESBL producing Enterobacteriaceae in companion animals constitutes an increasing challenge to infection management in veterinary therapy. Moreover, multidrug resistant, highly virulent human related clonal lineages of Enterobacteriaceae such as Escherichia coli belonging to sequence type (ST)131 and ST648, or Klebsiella pneumoniae ST11, ST15, and ST147 have been isolated from companion animals [1].There is growing concern that ESBL producers in companion animals pose a potential health hazard to humans, either through direct transmission of resistant pathogens or indirectly through transmission of resistance genes [2]. This study aimed to identify and characterize ESBL producing Enterobacteriaceae among 346 non-duplicate clinical samples of companion animals that were presented for treatment at the Veterinary Clinic of the University of Zürich, Switzerland. Methods: Isolates (n=346) were collected between 2012 and 2016 from diseased cats (n=115) and dogs (n=231). Characterization of ESBL producers included antimicrobial resistance profiling, PCR based identification of bla ESBL,, plasmid-mediated quinolone resistance (PMQR) genes and the azithromycin resistance gene mph(a) and sequencing of bla genes. Further genotyping included multilocus sequence typing of K. pneumoniae and E. coli isolates, phylogenetic grouping, identification of mutations in the quinolone resistance determining regions (QRDR) of gyra and parc in E. coli isolates and screening for virulence associated genes in E. coli isolated from urine. Results: Among the 346 isolates, 72 (20.8%) ESBL producers were identified (58 E. coli, 11 Klebsiella pneumoniae, and three Enterobacter cloacae). The strains originated from urine, skin and skin wounds, abscesses, surgical sites, bile, and other sites (Figure). A total of 73 bla ESBL genes were identified, including bla CTX-M-1, 14, 15, 27, 55, and bla SHV-12 (Figure). Further resistance genes included qnrb (4.2% of the isolates), qnrs (9.7%), aac(6 )-Ib-cr (47.2%), and mph(a) (38.9%). Seventeen (23.6%) isolates belonged to the major lineages of human pathogenic K. pneumoniae ST11, ST15, and ST147 and E. coli ST131. The most prevalent ST was E. coli ST410 belonging to phylogenetic group C. Conclusions: The prevalence of ESBL producing clinical Enterobacteriaceae from cats and dogs in Switzerland is high. The detection of highly virulent human related K. pneumoniae and E. coli clones raises concern about transmission from animals to humans, and presents a challenge to infection management and prevention in veterinary medicine. Fig. 1 ESBL producing Enterobacteriaceae isolated from cats and dogs, Switzerland, Keywords: extended-spectrum ß-lactamase; clinical; genotypes; cats; dogs [1] Bogaerts, P., Huang, T. D., Bouchahrouf, W., Bauraing, C., Berhin, C., El Garch, F. et al. (2015). Characterization of ESBL- and AmpC-producing Enterobacteriaceae from diseased companion animals in Europe. Microb Drug Resist, 21, [2] Pomba, C., Rantala, M., Greko, C., Baptiste, K. E., Catry, B., van Duijkeren, E. et al. (2017). Public health risk of antimicrobial resistance transfer from companion animals. J Antimicrob Chemother, 72,

67 How different sterols contribute to cytotoxic action of sea cucumber saponins Emily J.S. Claereboudt 1,2, Igor Eeckhaut 2, Magali Deleu 1 and Laurence Lins 1 1 Lab of molecular biophysics at interfaces, GBX ABT, Université de Liège, Gembloux, Belgium 2 Lab of marine biology and biomimetics, Research Institute for Biosciences, University of Mons, Belgium Saponins form a diverse class of secondary metabolites found in both plants and some marine invertebrates. Holothuroids, or sea cucumbers, produce these molecules as a chemical defense against predators and parasites, but interestingly, tolerate the cytotoxic nature of these chemicals. This tolerance is poorly understood. The aim of this study was therefore to elucidate the mechanisms behind the tolerance of holothuroid cells to the cytotoxic saponins (e.g. Frondoside A) they produce. This investigation was conducted using a suite of complementary biophysical tools, firstly using in vitro techniques such as Isothermal Titration Calorimetry (ITC) and calcein leakage experiments, for measuring interactions between lipid models of various compositions and the saponin Frondoside A, then using in silico approaches such as docking methods and insertion into implicit lipid bilayers, to provide a molecular point of view on our observations. Interactions between Frondoside A and cholesterol were more favorable than those with the holothuroid Δ7 and Δ9(11) sterols. Liposomes containing cholesterol resulted in exothermic interactions with the holothuroid saponin Frondoside A whereas liposomes containing the Δ7 sterol resulted in endothermic interactions. Lipid phase simulations using settings previously developed for plant saponins revealed that the holothuroid saponin Frondoside A has an agglomerating effect on cholesterol domains, similar to that previously observed for the plant saponin α-hederin. However, when interacting with the Δ7 sterols, the sterol domains were fragmented into small clusters. A significantly lower leakage was observed with liposomes containing the Δ7 holothuroid sterol than that with liposomes containing cholesterol. Our results suggest that the structural peculiarities of holothuroid sterols provide the organisms with a mechanism to mitigate the sterol-agglomerating effect of saponins on the cell membranes, and therefore to protect sea cucumber cells from the cytotoxicity of the saponins they produce. Keywords: saponin; triterpene glycoside; biocide; plasma membrane; sterols; auto toxicity; molecular biophysics; molecular modeling 57

68 Identification of compound with microbial activity extracted from the cyanobacteria Arthrospira maxima. R. Olvera-Ramírez 1, N.B. Medina-Jaritz 1 and J. M. Vega-López 2 1 Phisiology Plant Laboratory, Department of Botany, National School of Biological Sciences, National Polytechnic Institute, Prol. Carpio s/n, Mexico City, 11320, Mexico 2 Departament of Inorganic Chemistry, National School of Biological Sciences, National Polytechnic Institute, Prol. Carpio s/n, Mexico City, 11320, Mexico In the last 20 years the resistance to antibiotics by microorganisms has increased due to several factors among which are: natural processes as a result of a secondary metabolism, response to different abiotic factors, as well as the processes of genetic recombination, and the indiscriminate use of antibiotics [1]. Due to the above, the need has arisen to find new organisms that synthesize compounds that present antimicrobial activity; among these are the cyanobacteria that are microorganisms capable of producing substances with different structures and characteristics, for this reason they have been able to adapt to extreme conditions in different natural environments, and make them susceptible to great biotechnological exploitation [2]. One of the best known cyanobacteria is Arthrospira (Spirulina) maxima, native to the central region of Mexico and used since the Aztecs as a source of food and currently as a food supplement. The aim of this work was the extraction of a compound with activity from cultivated A. maxima biomass. A. maxima was grown in 20 L plastic bags in modified Zarrouk mineral medium, the biomass was harvested by filtration and extractions were made using different solvents (methanol-acetic acid, hexane, ethyl acetate, chloroform, tetrahydrofuran and methanol) and purified by silica gel column chromatography, different mobile phases were tested for the separation of the compounds having antimicrobial activity. Three fractions were obtained by column chromatography, identified as fraction 1 with low polarity, the second as fraction 2 with medium polarity and the third as fraction 3 with high polarity. Antimicrobial activity of the extracts was evaluated using the ATCC strains of Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Salmonella typhi ATCC 6534, Escherichia coli ATCC and Pseudomonas aeruginosa ATCC 9027 and tests were carried out using Mueller Hinton agar plates. Antimicrobial activity was presented in fraction 2 with medium polarity and this activity was presented against P. aeruginosa, S. aureus, S. typhi and E. coli The compounds of the fractions were identified with different spectroscopy techniques Keywords: Cyanobacterias; Arthrospira maxima; antimicrobial activity [1] Gilbert DN, Guidos RJ, Boucher HW, Talbot GH, Spellberg B, Edwards JE Jr, Scheld WM, Bradley JS, Bartlett JG. (2010) The 10 x '20 Initiative: pursuing a global commitment to develop 10 new antibacterial drugs by Clin Infect Dis. 50(8): [2] Kreitlow S, Mundt S, Lindequist U. (1999) Cyanobacteria: a potential source of new biologically active substances. J. Biotechnol. 70 (1-3):

69 In vitro Efficacy of Ocimum Basilicum Essential oil extracted by Supercritical Carbon Dioxide on Lactococcus garviae. Preliminary Results R. Barbero 1, C. Vercelli 2, D. Dezzutto 1, S. Bergagna 1, S. Gennero 1, A. Botto 3, C. Colombo 3, M. Caramelli 1 and G.Re 2 1 Istituto Zooprofilattico Sperimentale Piemonte, Liguria e Valle d Aosta; via Bologna Turin, Italy 2 Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, Largo Braccini Grugliasco (Turin), Italy 3 Separeco Srl, Via Rivarossa 15, Piscina (Turin), Italy Introduction: Inappropriate and irrational use of antimicrobial drugs provided favourable conditions for emergence and spread of resistant microorganisms. As a consequence greater need for alternative treatments arised. Moreover, imprudent drug use has lead to increasing environmental pollution levels. The aim of the present study is to evaluate the use of Ocinum basilicum, a natural antimicrobial compound, in rainbow trout (Oncorhynchus mykiss) affected by Lactococcus garvieae. In vitro efficacy evaluation have been performed by measuring antimicrobial activities. Several essential oils extracted from aromatic plants are known to generate biological activity, in particular they exhibit antibacterial and antioxidant effects [1]. These substances can be extracted using supercritical fluid extraction (SFE) [2,3]. SFE with carbon dioxide can extract natural compounds, especially those unstable at high temperature. It is the most widely method used in food and pharmaceutical industry and the extracts contain no organic residues. Furthermore the extraction process can be carried out with low temperature and moderate pressure. Material & Methods: In order to obtain essential oils extracted from basil, a method was developped to optimize the performance of the prototype. Samples of fresh basil, after rapid drying, was subjected to the action of the supercritical CO 2 for the extraction of essential oils (about 1-2%). During the study, until complete standardization of the method, a significant number of samples (N=10) obtained by this technique was analyzed through HPLC-MS /MS. In vitro efficacy of Ocimum basilicum essential oil versus antimicrobial drugs generally used in rainbow trout infected by Lactococcus garvieae (e.g.erythromycin, florfenicol) was evaluated using microbiological methods (Kirby-Bauer, semi-quantitative method). Results: The Kirby-Bauer method was used to compare the antimicrobial susceptibility of Lactococcus garvieae isolated from rainbow trouts. Several samples (N=30) of L.garviae strains were tested with essential oils. In the 89% of samples, they showed a zone of inhibition > 15 mm. The same test, with erythromycin and florfenicol showed also the zone of inhibition (range conformed to CLSI Standard: erythromycin S > 18 mm and florfenicol S > 20 mm). Discussion: Preliminary results of our research group show the activity of Ocimum basilicum essential oils extracted by SFE on Lactococcus garvieae in rainbow trout. Several essential oils extracted from aromatic plants are known to exhibit strong antibacterial effects, particularly the essential oil from Ocimum basilicum. Supercritical fluid extraction (SFE) was used with success to obtain extracts. L.garviae showed, with Kirby- Bauer method, a susceptibility to the essential oil. The size of this zone depends on how effective the antibiotic is at stopping the growth of the bacterium. A stronger antibiotic will create a larger zone, because a lower concentration of the antibiotic is enough to stop growth. However, at present we cannot yet compare the zones of inhibition measured to determine the actual effectiveness of the essential oil. Keywords: essential oil; Ocimum basilicum; supercritical fluid extraction; Lactococcus garvieae; rainbow trout [1] Opalchenova G et al Comparative studies on the activity of basil an essential oil from Ocimum basilicum L. against multidrug resistant clinical isolates of the genera Staphylococcus, Enterococcus and Pseudomonas by using different test methods. Journal of Microbiological Methods 2003; 54: [2] Filip S et al Fractionation of non-polar compounds of basil (Ocimum basilicum L.) by supercritical fluid extraction (SFE) The Journal of Supercritical Fluids 2014; 86: [3] Fornari T et al Isolation of essential oil from different plants and herbs by supercritical fluid extraction. Journal of Chromatography A 2012; 1250:

70 Insects novel source for antimicrobial natural products N. Berezina* Ynsect, 1 rue P. Fontaine, Evry, France * Corresponding author: nbe@ynsect.com After their discovery in 1922 by A. Fleming, the antimicrobial peptides (AMPs) were somehow neglected until the 1960s due to the emergence of antibiotics in the meantime. However, when the bacteria started to show resistance to the antibiotics, AMPs gained in interest. Insects are, among others, known AMPs producers, in this case it is even more interesting because the insects almost do not have induced immunity, and the native AMPs are protecting them from different bacterial and fungal aggressions [1]. The most studied among the insects AMPs are cecropins, active against a range of Gram-negative bacteria; attacins, mainly active against E. coli and a restricted number of Gram-negative bacteria; lebocins and other small proline-rich peptides, these substances have been found to be active against both Gram-negative and Grampositive bacteria; and moricins, active not only against Gram-positive and Gram-negative bacteria but also against some fungi and yeast [2, 3]. Some of the insects AMPs are excreted, such as AMPs found in honey of bees Apis carnica or Apis florae [4], but most of them need to be extracted from the haemolymph of the insects. Also, the amount and the availability of the AMPs is often an issue, therefore until now these molecules were only rarely present on the market. The recent development of the insect industry and of the subsequent entorefineries, may change this statement, as huge amounts of insects are to be produced for feed and food applications. However, the insects, mainly studied for the entorefinery, were, until now, rarely reported in the studies involving AMPs evaluation. One of the biggest challenges to be discussed here is the combination of the mass production, i.e. entorefinery approach, with the production of high-added values molecules, i.e. insects AMPs. Several, among the most popular biorefinery species, such as H. illucens, T. molitor or G. mellonella will be presented for this double aspect of mass and high-added value molecules production. Keywords: insects; entorefinery; induction of the antimicrobial activity [1] Zhang, L-J., Gallo, R.L., Curr.Biology, 2016, 26, [2] Yi, H-Y., Chowdhury, Huang, Y-D., Appl. Microbiol. Biotechnol., 2014, 98, [3] Hegedus, N., Marx, F., Fungal Biol. Rev., 2013, 26, [4] Ayaad, T.H., Shaker, G.H., Almuhnaa, A.M., J. King Saud Univ. Science, 2012, 24,

71 Insight into the metabolomics inventory and exploring metabolic adaptation of Streptococcus pneumoniae to antibiotic stresses A. Leonard and M. Lalk Department of Metabolomics, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, Greifswald, Germany The Gram-positive bacterium Streptococcus pneumoniae can cause a broad range of severe diseases like pneumonia, septicaemia etc. Deciphering its metabolome would improve our understanding of its pathophysiology and its dependency on host-derived nutrients. We aimed for a metabolomics approach that would allow us to investigate basic metabolic characteristics during growth in a chemical defined medium. To analyze the complex variety of metabolites we used analytical techniques like 1 H-NMR, HPLC-MS and GC-MS. We monitored important nutrients like glucose, adenine and asparagine but we detected also accumulation of fermentation products, and other metabolites [1]. With the first sampling protocol for intracellular metabolome analysis for S. pneumoniae in hand, we can now investigate a basal snapshot of the intracellular pneumococcal metabolome. By using HPLC-MS and GC-MS, nearly 100 intracellular metabolites, including amino acids, carbohydrates, nucleotides and organic acids, were identified. The intracellular metabolic profile shows high abundances for ATP and Frc-1.6-bP, most likely due to the high glycolytic metabolic flux. Other high abundant metabolites are UTP and UTP-dependent precursors of peptidoglycan synthesis probably to fuel cell-wallmetabolism in growing cells [1]. Alternations in essential metabolic pathways may point to new targets in the drug development against pathogenic bacteria such as S. pneumoniae. Metabolomics experiments are also important tools to identify and monitor affected catabolic or anabolic reactions, for example during drug treatment. The standardization of cultivation and sampling conditions achieved by our work will be used in a follow-up study, in which we want to study the metabolic response of S. pneumoniae to limiting growth conditions as well as during treatment with known and novel antibiotic compounds to identify metabolic adaptation processes. This approach might also uncover metabolic strategies that are required to cope with the host environment during infection. Keywords: Streptococcus pneumoniae; metabolism; glycolysis; antibiotic; metabolic strategies [1] Leonard A, Gierok P, Methling K, Gómez-Mejia A, Hammerschmidt S, Lalk M (2018) Metabolic inventory of Streptococcus pneumoniae growing in a chemical defined environment. International Journal of Medical Microbiology /j.ijmm

72 Investigating levels of evolved Arg 10 -teixobactin resistance in methicillin-resistant Staphylococcus aureus Daniel Lloyd 1, Anish Parmar 2, Matthew Goddard 1, Rajamani Lakshminarayanan 3, Ishwar Singh 2 and Edward Taylor 1, * 1 School of Life Sciences, University of Lincoln, United Kingdom 2 School of Pharmacy, University of Lincoln, United Kingdom 3 Singapore Eye Research Institute, The Academia, Discovery Tower Level 6, 20 College Road, Singapore * Corresponding author: Etaylor@lincoln.ac.uk Resistance to all available antimicrobial drugs is increasing, and the rapid development of resistance is one of the greatest threats to human health [1]. The discovery of the antibiotic teixobactin in 2015 [2] is a significant advance in the race against resistance [3]. Teixobactin is a naturally occurring antimicrobial peptide produced by an unculturable soil bacteria [4]. We have produced a synthetic library of teixobactin analogues which has been used to investigate the structure activity relationship, antimicrobial activity and in vivo antimicrobial activity [5-7]. To further understand the bacterial response to these compounds, the rate at which de novo resistance evolves in Staphylococcus aureus (MRSA) was assessed. Six independent populations of MRSA were cultured through continuous passage from a single ancestor in the presence of Arg 10 -teixobactin. After ~300 generations a fourfold increase in resistance was recorded. This low level resistance is conveyed through an unidentified, high cost mechanism. The resistance is lost after 35 generations when selection is withdrawn. The rifampicin control saw a >2000-fold increase in resistance across six separate replicates, at what appears to be a lower evolutionary cost. We have for the first time induced resistance to a teixobactin analogue. Our results suggest the low levels of resistance observed compare favourably to many currently licenced antibiotics. We speculate the cost of this resistance is likely to place a significant burden upon the MRSA model and these evolutionary trade-offs may reduce virulence. Keywords: teixobactin-analogues; resistance; antimicrobial [1] Blair JMA, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJV. Molecular mechanisms of antibiotic resistance. Nat Rev Micro. 2015;13: [2] Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015;517: [3] Choices. AbBEbN. New 'game-changing' antibiotic discovered [4] Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015;517: [5] Parmar A, Iyer A, Vincent CS, Van Lysebetten D, Prior SH, Madder A, et al. Efficient total syntheses and biological activities of two teixobactin analogues. Chemical Communications. 2016;52: [6] Parmar A, Prior SH, Iyer A, Vincent CS, Van Lysebetten D, Breukink E, et al. Defining the molecular structure of teixobactin analogues and understanding their role in antibacterial activities. Chemical Communications. 2017;53: [7] Parmar A, Lakshminarayanan R, Iyer A, Mayandi V, Leng Goh ET, Lloyd DG, et al. Design and Syntheses of Highly Potent Teixobactin Analogues against Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), and Vancomycin-Resistant Enterococci (VRE) in Vitro and in Vivo. J Med Chem. 2018;61:

73 Involvement of Oligopeptidase B in Bacterial Resistance against the Antimicrobial Peptide Bac7 I. Wuethrich, S. Panke and M. Held Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058 Basel, Switzerland Proline/arginine-rich ribosomally synthesized peptides (PRAMPs) are part of the immune defense system employed by mammals and insects for fending off invading bacteria. PRAMPs are ribosomally synthesized by the host from a DNA template and in turn effectively inhibit protein translation at the bacterial ribosome. Surprisingly, not much is known about the importance of host peptidases for the reduction of bacterial vulnerability or development of resistance towards PRAMPs. The high proline content of PRAMPs attributes rigidity to PRAMPs, which provides increased stability 1. However, specialized or promiscuous proteases such as the cytosolic oligopeptidase B (OpdB) recognize and cleave at unprotected arginine residues, which are a feature of PRAMPs. A correlation of OpdB presence with resistance against PRAMPs has previously been proposed 2. We set out to study the influence of OpdB on the vulnerability of Escherichia coli towards the bovine PRAMP Bac7. Using cell-free extracts of wild-type E. coli and a corresponding ΔopdB strain, we confirmed by mass spectrometry that Bac7 is indeed specifically degraded by OpdB. Moreover, we provide bacterial growth data that show involvement of OpdB in defense against the peptide. OpdB is widely prevalent among Gram-negative and certain Gram-positive bacteria, including the human pathogens Pseudomonas aeruginosa, Salmonella enterica, and Mycobacterium tuberculosis 3. We found that this is also true for important pathogens affecting animal husbandry, such as Streptococcus uberis and Corynebacterium bovis. Taken together, these results suggest that inhibition or circumvention of pathogen-specific proteases would be required to prohibit resistance formation against PRAMP-based antibiotics. Keywords: antimicrobial peptides; resistance factor; Bac7; Oligopeptidase B [1] Adzhubei, A. A., Sternberg, M. J. E. & Makarov, A. A. Polyproline-II helix in proteins: structure and function. J. Mol. Biol. 425, (2013). [2] Mattiuzzo, M. et al. Proteolytic activity of Escherichia coli oligopeptidase B against proline-rich antimicrobial peptides. J. Microbiol. Biotechnol. 24, (2014). [3] Morty, R. E., Fülöp, V. & Andrews, N. W. Substrate recognition properties of oligopeptidase B from Salmonella enterica serovar typhimurium. J. Bacteriol. 184, (2002). 63

74 Low-molecular amphiphilic compounds regulation of pore-forming activity of cecropins Svetlana S. Efimova* and Olga S. Ostroumova Institute of Cytology of the Russian Academy of Sciences, Tikhoretsky av.4, St. Petersburg , Russia * Corresponding author: efimova@incras.ru Antibiotics have been used to treat bacterial and fungal infections for hundred years, however the number of resistant strains continues increase. Antibacterial peptides are currently considered as a basis to develop new effective antibacterial drugs. Cecropins are the first identified antimicrobial peptides that characterized by a wide range of the antibacterial activity. The molecular mechanisms of interaction of the peptides with cellular membranes have not fully understood. The low-molecular amphiphiles are capable to adsorb and incorporate into membrane and to change the physico-chemical properties of the bilayers. These tools can be effectively used to study the membranotropic action of cecropins. The addition of 1 mm bupivacaine, benzocaine or 0.3 mm tetracaine to the membrane bathing solution decreases the mean conductance of single cecropin channels (i sc ) and the steady-state macroscopic transmembrane current induced by cecropin A (I ). The introduction of 1 mm lidocaine or procaine in the membrane-bathing solutions does not practically affect i sc and I. The data are not in agreement with the results of the measurements of the boundary potential of negatively charged membranes in the presence of local anesthetics. Taking into account the significant decrease of the temperature or cooperativety of the main phase transition of DPPC in the presence of bupivacaine, benzocaine or tetracaine it can be concluded that the observed changes in i sc and I were caused by the modulation of the elastic properties of the membranes. The work was supported by the RFBR (# ). S.Efimova was awarded by scholarship SP Keywords: cecropins; low-molecular amphiphilies; lipid bilayers 64

75 Methicillin resistant Staphylococcus aureus (MRSA) in raw buffalo milk from Italy: preliminary results E. Spinelli 1, M. Caruso 2, G. Santagada 2, A. Parisi 2, A. Barlaam 1 and G. Normanno 1 1 Department of Science of Agriculture, Food and Environment, University of Foggia, 25, via Napoli, Foggia, Italy 2 Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, via Manfredonia, 20, Foggia, Italy Methicillin resistant Staphylococcus aureus (MRSA) has become one of the major public health challenges. MRSA is a widespread antimicrobial resistant bacterium able to cause a wide range of human pathologies. S. aureus becomes methicillin resistant by the acquisition of the meca gene that encodes a penicillin binding protein (PBP2a) with a low affinity for β lactams. MRSA has long been considered a major hospital multi-drug resistant pathogen and it is highly prevalent in the community and livestock worldwide. In addition, recently Larsen provided evidence of the foodborne transmission of some MRSA strains isolated from poultry meat [1]. The transmission of zoonotic MRSA to humans via either animal contact or eating and handling contaminated food is well documented or hypothesized [2]. Quite a number of studies focused on cow milk [3], even though the milk produced by other animals such as buffaloes are essential in human diet in different parts of the world. Water buffaloes provide the most important source of non-cattle milk worldwide (13.2%) [4]. In Italy it is almost all transformed into mozzarella. Buffalo mozzarella is a fresh and stringy-textured Italian cheese softer and more appetizing compared by other kind. The appreciation of this buffalo mozzarella by consumers is demonstrated by the Mozzarella di Bufala Campana PDO (Protected Designation of Origin) recognition obtained in The high consumptions of buffalo drinking milk and dairy products worldwide involve an elevated number of consumers of all ages. And this is crucial considering the potential role of raw buffalo milk in the transmission of foodborne pathogens. Despite of the potential hazard for human health there is a lack of date on prevalence of MRSA in some foods, such as buffalo milk and buffalo dairy products. This study aimed to investigate, for the first time in Europe, the prevalence and characteristics of MRSA isolated from raw buffalo milk produced in Apulia and Basilicata regions (Southern Italy). Almost the 90% of dairy buffalo farms present in this geographical area were investigated and among 39 raw buffalo milk samples processed, one (2.56%) MRSA strain was detected by standard culture methods. To identify MRSA, the isolated were tested by real time PCR targeting the meca gene and the sau gene. The isolate resulted susceptible to gentamycin, cephalothin, clindamycin, chloramphenicol, amikacin, vancomycin, tobramycin, enrofloxacin and resistant to amoxicillin, ampicillin, cefoxitin, doxycycline, erithromycin, kanamycin, oxacillin, penicillin, streptomycin, sulfisoxazole, tetracyclin, sulfamethoxazole-trimethoprim. The molecular characterization of the strain, in order to assign the spa-type, the ST-type and the SCC-mec- type are ongoing. Our finding cleary demonstrates that MRSA could be present in raw-buffalo milk and that attention must be payed to this new potential treat for the food safety and the operators of the milk production chain. Keywords: MRSA; raw buffalo milk; antimicrobial resistance; food safety [1] Larsen, J., Stegger, M., Andersen, P.S., Petersen, A., Larsen, A.R., Westh, H., Agersø, Y., Fetsch et al. Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus, Clinical Infectious Diseases, 63, 10, , [2] EFSA. Scientific Opinion of the Panel on Biological Hazards on a request from the European Commission on Assessment of the Public Health significance of meticillin resistant Staphylococcus aureus (MRSA) in animals and foods. The EFSA Journal, 993, 1 73, [3] Parisi, A., Caruso, M., Normanno, G., Latorre, L., Sottili, R., Miccolupo, A., Fraccalvieri, R., Santagada, G. Prevalence, antimicrobial susceptibility and molecular typing of Methicillin-Resistant Staphylococcus aureus (MRSA) in bulk tank milk from southern Italy, Food Microbiology, 58, 36-42, [4] FAO report, c_refk8 01/10/

76 Microorganisms in nurses hands, clinical uniforms, and pocket medical devices S. Alarico 1, V. Oliveira 2,3,4, P. Costa 3,4, E. Fernandes 4, C. Santos 4, H. Albano 5, J. Graveto 4 and N. Osório 2,6 1 Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 2 Department of Biomedical Laboratory Sciences, Coimbra Health School - IPC, Coimbra, Portugal 3 The Health Sciences Research Unit: Nursing, Coimbra, Portugal 4 Nursing School of Coimbra, Coimbra, Portugal 5 Catholic University of Portugal - Centre for Biotechnology and Fine Chemistry, Porto, Portugal 6 University of Coimbra - Molecular Physical-Chemistry, Coimbra Portugal Health care associated infections is a type of infection acquired within the hospital setting [1]. The main microorganisms associated with this type of infection and at the same time represent greater risks due to the resistance patterns, conditioning the therapeutic alternatives were grouped into an acronym known as ESKAPE, including Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas and Escherichia coli species [2-4]. This study intends to understand the contribution of the health professional (nurses) through their hands, clinical uniforms and pocket medical devices (fomites) in the health care associated infections. A total of 50 nurses were sampled from the clinical uniforms, hands and pocket medical devices. A directed research was done to the microorganisms called ESKAPE, using selective medium. The isolates obtained in each medium were enumerated and the morphologically different were isolated. Then they were characterized by GRAM staining, oxidase, catalase and coagulase tests. The antimicrobial sensitivity tests were performed using the disk diffusion method. The clinical uniforms were the samples with the most positive for the presence of the microorganisms (22%), when we evaluated the presence of the ESKAPE microorganisms, the Staphylococcus had the greatest representativity (55%) and the species S. aureus appears with greater prevalence (39.3%). Regarding the susceptibility profile in Staphylococcus genus, we observed a higher number of isolates with oxacillin and cefoxitin resistance, contributing for a huge presence of methicillin resistance. In this study, we observed that the health professionals adopting inadequate procedures could promote health care associated infections, contributing to this serious problem. Therefore, it will be important to appeal to awareness for the adoption of preventive and control measures at this level. Keywords: Health care associated infections; Microorganisms ESKAPE; Antimicrobial resistance [1] Monteiro J. Infeções nosocomiais. Alguns aspectos. Acta medica portuguesa. 1993; [2] Santajit S, Indrawattana N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. Biomed Res Int. 2016;2016:8. [3] Moreira RAS. Infeções Nosocomiais e bactérias implicadas neste tipo de infeção. Universidade de Coimbra; [4] Aftab H Bin, Zia B, Zahid MF, Raheem A, Beg MA. Knowledge, attitude, and practices of healthcare personnel regarding the transmission of pathogens via fomites. Open Forum Infect Dis. 2016;3(1):

77 Mo-W-O system: A study on the mechanisms of antibacterial action via protons release Cezarina Cela Mardare 1,2,3, Zuzana Gajarska 1,3, Karl Christian Zelenka 2,3, Dominik Recktenwald 1,3, Norbert Müller 4 and Achim Walter Hassel 1,2,3 1 Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) at Institute for Chemical Technology of Inorganic Materials, Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria 2 CEST Competence Center for Electrochemical Surface Technology, Viktor Kaplan Str. 2, 2700 Wiener Neustadt, Austria 3 Institute for Chemical Technology of Inorganic Materials, Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria 4 Institute of Organic Chemistry, Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria Nosocomial infections together with the development of microorganisms which show increasing resistance to antibiotics represent a major problem in healthcare settings. Annually, these infections cause hundreds of thousands of possibly avoidable deaths and billions of euros spent on patient treatments [1]. Microorganisms spread fast via touch surfaces (e.g. doorknobs, trays, light switches etc.) with which patients come in contact, thus enabling facilitated diseases transmission. One route to decrease the contamination of inanimate surfaces is to functionalize them, to the end of either preventing the adhesion of microorganisms or inhibiting their growth and proliferation. In the class of bactericidal agents, a new type was discovered recently which is based on molybdenum oxide [2]. The mechanism of action is assumed to be based on proton-release and subsequent acidification of the surrounding environment. When MoO 3 comes into contact with environmental humidity, molybdic acid is obtained and subsequent protons and molybdate species are formed. Though working efficiently, MoO 3 has the issue of high solubility, which eventually may lead to its complete dissolution and consequently the loss of the antibacterial capability. Our approach is to combine MoO 3 with another material that is insoluble [3-5], in order to form mixed oxides which preserve the antibacterial feature while decreasing the solubility. In the current research, we are focusing on the synthesis, characterization and antibacterial testing together with experiments to proof the mechanism of antibacterial feature of powders from the Mo-W-O system. Powders with discrete ratio of Mo/W ranging from 100 % MoO 3 to 100 % WO 3 with a stepwise increase of 5 to 10 wt.% of W were synthesized by spray drying method, and subsequently calcinated. Spherical hollow particles with a broad size distribution were formed. The crystalline phases changed as a function of composition, with mixed oxides and/or mixed oxides together with pure ones being obtained. Antibacterial tests performed on a model gram-negative bacterium (Escherichia coli) correlate the composition variation and the phases present to the antibacterial activity, simultaneously providing a detailed screening of the activity versus composition transition. Furthermore, the mechanism of action is proven by correlating media acidification and the inability of bacteria to live at low ph values [2]. The solubility measurements of powders in solution performed by inductively coupled plasma optical emission spectroscopy prove a steep decrease of solubility with the addition of W, while still preserving the antibacterial feature. These powders show adequate features for being implemented in hybrid inorganic-polymer systems or varnishes that can be used in healthcare settings for inanimate surfaces. Keywords: molybdenum-tungsten mixed oxides; powder synthesis; antibacterial properties; inductively coupled plasma optical emission spectroscopy; ph measurements [1] Annual epidemiological report Antimicrobial resistance and healthcare-associated infections European Center for Disease Prevention and Control (2014). [2] C. Zollfrank, K. Gutbrod, P. Wechsler and J. P. Guggenbichler: Mater. Sci. Eng. C 32 (2012), p.47. [3] C.C. Mardare, A.W. Hassel: ACS Comb. Sci. 16 (2014), p [4] C.C. Mardare, D. Tanasic, A. Rathner, N. Müller, A.W. Hassel: Phys. Status Solidi A 213 (2016), p [5] D. Tanasic, A. Rathner, J.P. Kollender, P. Rathner, N. Müller, K.C. Zelenka, A.W. Hassel, C.C. Mardare: Biointerphases 12 (2017) p. 05G

78 MRSA in horses and humans working in contact with them A. Parisi 2, M. Caruso 2, E. Spinelli 1, G. Normanno 1 and G. Santagada 2 1 Department of Science of Agriculture, Food and Environment, University of Foggia, 25, via Napoli, Foggia, Italy 2 Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, via Manfredonia, 20, Foggia, Italy Methicillin-resistant Staphylococcus aureus (MRSA) is a widespread pathogen able to cause serious infections in humans both in the hospital care environment and in the community [1]. In addition, particular clones of MRSA are associated with farm and companion animals and there are able to cause infections in humans [2]. The presence of MRSA in horses has important implications both in veterinary and human medicine. In fact, MRSA could be responsible for several types of infections in horses, especially respiratory, urinary, joint, wound and post-surgery infections [3]. On the other hand, the colonization of horses by MRSA could result in infections in humans working in close contact with them, such as veterinarians and stable workers [4]. The spread of MRSA in horses intended for meat production, could result in colonization of slaughterhouse operators and represents a potential food safety concern [5]. Italy is the biggest EU consumer of horse meat: every year about 170,000 horses are slaughtered and the production of horse meat is approximately 51 thousand tonnes per year. In addition, the habit of consuming some dishes made with raw meat still persists in some Italian regions. Our study aimed to investigate the prevalence and characteristics of MRSA in horses used for recreational purposes and for meat production and the prevalence and characteristics of MRSA in humans working in close contact with horses. Out of 388 nasal swabs from horses, we detected an overall prevalence of 7% of MRSA, with values of 15% among the slaughtered horses, 1% among the livestock horses and 0% in racehorses. The percentage of isolation recorded in slaughtered horses was significantly higher than in the ones used for recreational purposes. As for the people working in close contact with horses, the percentage of MRSA isolation was higher in the slaughterhouse staff (12%) than in the operators of farms or riding stables (5%) and racecourses (7%). The results of our study have shown that while a low MRSA prevalence both in equines and workers in contact with them is recorded on farms or riding stables and racecourses, a high prevalence is found in slaughterhouses. In addition, the detection of MRSA in horses belonging to the human transmissible genotypes (ST398, ST1) and the isolation of strains with identical genetic profile in a horse and a worker of the same slaughterhouse, confirm the potential zoonotic risk associated with direct contact with animals or handling and consumption of raw horse. This abstract is based on previously published data [6]. Keywords: MRSA; horses; humans [1] Kluytmans-Vandenbergh, M.F. and Kluytmans, J.A. Community-acquired methicillin resistant Staphylococcus aureus: current perspectives. Clin. Microbiol, 12: 9-15, [2] Cuny, C., Friedrich, A., Kozytska, S., Layer, F., Nubel, U., Ohlsen, K., Strommenger, B., Walther, B., Wieler, L., Witte, W. Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species. Int. J. Med. Microbiol., , [3] Leonard, F.C. and Markey, B.K. Methicillin-resistant Staphylococcus aureus in animals: a review. Vet. J., 175:27-36, [4] van Duijkeren, E., Moleman, M., Sloet van Oldruitemborgh-Oosterbaan, Multem, J., Troelstra, A. et al. Methicillinresistant Staphylococcus aureus in horses and horse personnel: an investigation of several outbreaks. Vet. Microbiol., 141 (1-2):96-102, [5] EFSA. Scientific opinion of the panel on biological hazards on a request from the European commission on Assessment of the public health significance of methicillin-resistant Staphylococcus aureus (MRSA) in animal and foods. The EFSA J., 993, 1-73, [6] Parisi, A., Caruso, M., Normanno, G., Latorre, L. Miccolupo, A., Fraccalvieri, R., Intini, F., Manginelli, T., Santagada, G. High occurrence of methicillin-resistant Staphylococcus aureus in horses at slaughterhouses compared with those for recreational activities: a professional and food safety concern? Foodborne Pathog Dis. 14(12): ,

79 New antibiotic leads targeting bacterial RNA polymerase holoenzyme assembly identified with a BRET drug discovery platform S. Sartini 1, E. Levati 1, M. Maccesi 2, S. Bach 3, S. Ottonello 1, S. Rivara 4 and B. Montanini 1 1 Laboratory of Biochemistry and Molecular Biology, Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy 2 Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy 3 Sorbonne Universités, UPMC Univ Paris 06, CNRS USR3151, Protein Phosphorylation and Human Disease Unit, Station Biologique de Roscoff, Roscoff Cedex, France 4 Department of Food and Drug, University of Parma, Parma, Italy Bacterial resistance is a major health problem worldwide, that is being actively addressed through the discovery of novel antibiotics acting on thus far unexploited targets. Transcription is an essential process for pathogen propagation and represents an attractive, yet poorly exploited, target mechanism for the development of novel antibacterial drugs. To date, only two antibacterial drugs targeting RNA polymerase (RNAP) are used in clinic: the rifamycins and the lipiarmycins (e.g., the fidaxomicin), both susceptible to resistance phenomena due to mutation of the respective binding sites on the RNAP core enzyme [1]. The interaction between the RNAP core enzyme (β subunit) and the σ factor is essential for transcription initiation and may thus represent an alternative target for transcription inhibition. All previous attempts to disrupt the σ-β interaction have been conducted with the use of in vitro assays, which yielded candidate inhibitors with a high potency in vitro, but a rather poor antibacterial activity, mainly due to low internalization by bacterial cells [2, 3]. To address the permeability issue from the very beginning, we relied for our screening on the yeast Bioluminescent Resonance Energy Transfer (ybret) assay, an in vivo methodology we have recently optimized and which has been already successfully used in screening campaigns to identify protein-protein interaction inhibitors [4-6]. The ybret system, reproducing the Escherichia coli σ70-β interaction, was used to screen a total of compounds: (i) a set of compounds, pre-selected from a wider library through in silico evaluation; (ii) a high diversity set of synthetic and natural compounds. Hits and structural analogues were validated using an ELISA-based secondary assay, in order to confirm their ability to disrupt the σ70-β interaction. The efficacy of the selected lead-like compounds in inhibiting RNAP holoenzyme formation was evaluated in an in vitro transcription assay. Lead compounds displayed antimicrobial activity against Gram-positive (Bacillus subtilis) and Gram-negative (E. coli) bacteria, thus demonstrating the versatility and discovery potential of the ybret platform. Based on the molecular structures of compounds able to interfere with the σ70-β interaction, a pharmacophore model was also proposed, which will pave the way to the identification of novel hits and to the guided design of modified derivatives with improved antibacterial properties. Keywords: antibiotic; BRET; protein-protein interaction; RNA polymerase [1] Brown ED, Wright GD: Antibacterial drug discovery in the resistance era. Nature 2016, 529: [2] Ma C, Yang X, Kandemir H, et al. Inhibitors of bacterial transcription initiation complex formation. ACS Chem Biol. 2013;8(9): [3] Ma C, Yang X, Lewis PJ. Bacterial Transcription Inhibitor of RNA Polymerase Holoenzyme Formation by Structure- Based Drug Design: From in Silico Screening to Validation. ACS Infect Dis. 2016;2(1):39-46 [4] Corbel C, Sartini S, Levati E, et al. Screening for Protein-Protein Interaction Inhibitors Using a Bioluminescence Resonance Energy Transfer (BRET) Based Assay in Yeast. SLAS Discov. 2017;22(6). [5] Bacart J, Corbel C, Jockers R, Bach S, Couturier C. The BRET technology and its application to screening assays. Biotechnol J. 2008;3(3): [6] Corbel C, Wang Q, Bousserouel H, et al. First BRET-based screening assay performed in budding yeast leads to the discovery of CDK5/p25 interaction inhibitors. Biotechnol J. 2011;6(7):

80 New antimicrobial compounds from microorganisms P. Sanguiñedo 1, S. Raffaelli 1,2, M.B. Estevez 1, A. Vázquez 2, S. Alborés 1 1 Department of Bioscience, Facultad de Química, Universidad de la República, Avda. Gral. Flores 2124, Montevideo, Uruguay 2 Department of Organic Chemistry, Facultad de Química, Universidad de la República, Avda. Gral. Flores 2124, Montevideo, Uruguay Despite the great advances in chemotherapeutics, infectious diseases are still one of the leading causes of death worldwide. Although a great array of antibacterial and antifungal drugs are in clinical use, the appearance of resistant organisms makes them sometimes ineffective or leads to recurrence, as stated by the WHO. Among some of the most problematic clinically relevant pathogens, Staphylococcus aureus ranks as one of the most difficult bacteria to treat [1]. Tracing of active molecules from natural sources is the traditional path in the antimicrobial area, leading to the development of modern anti infectious chemotherapy. Particularly important is the study of microorganism metabolites, as they can naturally produce antimicrobials, in order to compete for nutrients [2]. Furthermore, silver nanoparticles have been used as antimicrobial agents in the clinical area, proving effective in the treatment of infectious diseases. This antimicrobial activity was demonstrated against resistant bacteria in in vitro experiments and in animal models [3,4].The biological synthesis of nanoparticles is of great interest over other physical and chemical methods because the use of toxic chemicals and drastic reaction conditions is avoided, and stabilized nanoparticles can be obtained. The extracellular biosynthesis of nanoparticles using fungi could also make downstream processing much easier than the intracellular biosynthesis [5]. In this work, we have carried out two different strategies to search for new antistaphylococcal compounds using microorganisms isolated from samples collected in Uruguay. First, we report the screening of the antistaphylococcal activity of extracts from extracellular broth of microorganisms isolated from soils. Second, we have studied the ability of fungal strains to synthesize silver nanoparticles and evaluated the antibacterial activity of these biogenic nanoparticles. Both extracts from microorganisms and biogenic silver nanoparticles were able to inhibit S. aureus growth, showing their potential in the clinical area. Acknowledgments CSIC - Universidad de la República, FCE-ANII and PEDECIBA Química, Uruguay. Keywords: antibacterial activity; natural compounds; biogenic silver nanoparticles [1] Barneche, S., Alborés, S.,Borthagaray, G., Cerdeiras, M.P., Vázquez, A. (2017). Anti-MRSA Activity of Fruiting Body Extracts of Spectacular Rustgill Mushroom, Gymnopilus junonius (Agaricomycetes). International Journal of Medicinal Mushrooms, 19(3): [2] Bologa, C., Ursu, O., Tudor, O., Melacon, C., Tegos, G.P. (2013). Emerging trends in the discovery of natural product antibacterials. Current Opinion in Pharmacology, 13(5): [3] Shanthi, S., Jayaseelan, B.D., Velusamy, P., Vijayakumar, S., Chih, C.T., Vaseehran, B. (2016). Biosynthesis of silver nanoparticles using a probiotic Bacillus licheniformis Dahb 1 and their antibiofilm activity and toxicity effects in Ceriodaphnia cornuta. Microb. Pathogen. 93: [4] Hajipour, M.J., Fromm, K.M., Akbar Ashkarran, A., de Larramendi, I.R., Rojo, T., Serpooshan, V., Parak, W.J., Mahmoudi, M. (2012). Antimicrobial properties of nanoparticles. Trends Biotechnol. 30(10): [5] Duran, N.; Duran M.; de Jesus M.B., Seabra, A.B., Fávaro, W.J., Nakazato, G. (2016). Silver nanoparticles: a new view on mechanistic aspects on antimicrobial activity. Nanomed. Nanotechnol. Biol. Med., 12(3):

81 Photo-inactivation of Gram-positive and Gram-negative bacteria by an octahedral molybdenum cluster complex K. Kirakci 1, J. Zelenka 2, T. Ruml 2 and K. Lang 1 1 Institute of Inorganic Chemistry of the CAS, Husinec-Řež 1001, Řež, Czech Republic 2 University of Chemistry and Technology, Technická 5, Praha, Czech Republic The worldwide increase of bacterial resistance to antibiotics requires the development of new antibacterial strategies. The photo-inactivation of pathogens, mediated by photosensitizers of the singlet oxygen, O 2 ( 1 g ), constitutes a promising approach that has demonstrated its efficiency against various Gram-positive and Gramnegative bacteria. 1 In this respect, octahedral molybdenum clusters (Mo 6 ) have recently emerged as relevant O 2 ( 1 g ) photosensitizers (PSs) for biological applications thanks to attractive chemical and photophysical properties. Upon excitation from the ultra-violet up to the green spectral region, these complexes form long-lived triplet states that are efficiently quenched by oxygen leading to the formation of O 2 ( 1 g ) in a high yield up to 92 %. 2 In contrast to commonly used organic PSs such as porphyrins which are losing their sensitizing activity upon aggregation, the Mo 6 complexes remain good O 2 ( 1 g ) sensitizers even in the solid state. The deliberate selection of ligands can lead to an optimization of the properties towards a projected function. For instance, the coordination of carboxylate ligands to the {Mo 6 I 8 } 4+ core provides relatively stable complexes with good light absorption in the visible spectral region, high O 2 ( 1 g ) productivity, and allows for additional functionalization. 3 Recently, several Mo 6 cluster-based nanostructured systems were successfully employed in the context of photoinactivation of bacteria. 4 Herein, we report on the photophysics in water solutions of a cationic cluster complex based on the {Mo 6 I 8 } 4+ core associated with (4-carboxybutyl)triphenylphosphonium ligands (see Fig.1), as well as its bacterial photo-inactivation properties against several Gram-positive and Gram-negative bacteria, namely E.coli, E. faecalis, P. aeruginosa, B. subtilis, B. megaterium, P. vulgaris, and P. mirable. Fig. 1 Schematic representation of the molecular structure of [Mo 6 I 8 (OCOC 4 H 8 PPh 3 ) 6 ] 4+ Keywords: bacteria; photo-inactivation; singlet oxygen [1] Y. Feng et al. J. Mater. Chem. B 2017, 5, [2] K. Kirakci et al. Dalton Trans. 2013, 42, [3] K. Kirakci et al. Eur. J. Inorg. Chem. 2014, 2014, 2331; K. Kirakci et al. Inorg. Chem. 2014, 53, [4] A. Beltrán et al. J. Mater. Chem. B, 2016, 4, 5975; C. Felip-León et al. J. Mater. Chem. B, 2017, 5,

82 Potential uses of genetically engineered bacteriocins L. Rutledge School of Pharmacy, University of Lincoln, UK, LN6 7TS Novel antibiotics are urgently needed to replace the arsenal of traditional antibiotics which is being depleted, this requires research into sources previously disregarded 1. Bacteriocins are ribosomally synthesised antimicrobial peptides produced by bacteria 2. Bacteriocins are an attractive alternative to antibiotics and are non-toxic to humans as their antimicrobial activity is focused on bacterial target 3. Klebsiella pneumoniae is an ESKAPE pathogen, part of a group of multi-drug resistant Gram-negative bacteria 4. Bacteriocins produced by Gramnegative bacteria such as the klebicins produced by Klebsiella pneumoniae may be particularly valuable clinically as a source of narrow spectrum treatment against related ESKAPE pathogens 3. Klebicins are colicinlike bacteria, compromised of a transmembrane domain, a receptor binding domain and a nuclease domain 5. The modular structure exhibited by colicin-like bacteriocins including klebicins would allow bioengineering of domains to create a chimera with the salient receptor binding and cytotoxic action to precisely combat individual infections 4. The objective of this study was to identify bacteriocin producing nosocomial strains of Klebsiella pneumoniae and to purify the bacteriocins. Once purified mass spectrometry was used to obtain the amino acid sequence this was then used to generate primers. The genes responsible for bacteriocin production were amplified by PCR and inserted into E. coli to allow increased expression of bacteriocin without use of pathogenic bacteria. Keywords: Bacteriocin; antibiotic; Gram-negative; antimicrobial peptide; antimicrobial activity [1] Waldetoft., K. and Brown, S. (2017) Alternative therapeutics for self-limiting infections-an indirect approach to the antibiotic resistance challenge. PLoS biology, 15(12) e [2] Chikindas, M., Weeks, R., Drider, D., Chistyakov, V. and Dicks, L. (2018) Functions and emerging applications of bacteriocins. Current opinion in biotechnology, 49, [3] Yang, S., Lin, C., Sung, C. and Fang, J. (2014) Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Frontiers in microbiology, 5 (241) [4] Behrens, H., Six, A., Walker, D. and Kleanthous, C. (2017) The therapeutic potential of bacteriocins as protein antibiotics. Emerging topics in life sciences, 1, [5] Sharp, C., Bray, J., Housden, N., Maiden, M. and Kleanthous, C. (2017) Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models. PLoS Comput Biol 13(7): e

83 Quorum sensing and Quorum quenching in Pseudomonas spp.: role of antibiotics as quorum quenchers at subclinical concentrations A. Peña Pita, M. Robas Mora 1, A. Probanza 2 and P.A. Jiménez Gómez 1 1 Section of Microbiology, Facultad de Farmacia; Departamento de Ciencias Farmaceúticas y de la Salud, Universidad San Pablo CEU, Campus de Montepríncipe, Ctra. Boadilla del Monte Km 5.300, Boadilla del Monte, Madrid, Spain 2 Section of Plant Biology, Facultad de Farmacia; Departamento de Ciencias Farmaceúticas y de la Salud, Universidad San Pablo CEU, Campus de Montepríncipe, Ctra. Boadilla del Monte Km 5.300, Boadilla del Monte, Madrid, Spain Antibiotics are a group of drugs considered as a keystone in modern Medicine. Despite that, increasing emergence of bacterial strains containing novel resistance mechanisms, represents a serious hazard to public health at a global scale [1]. Thus, the deficit in new antibiotic family s discovery makes very challenging fight against emerging multi-resistant bacterial species, justifying the need of new approaches that enable the treatment of different infectious processes [2]. Quorum sensing (QS) are cell to cell communication processes that encompasses different mechanisms that enables communication within a bacterial population and regulates the expression of multiple virulence factors that may facilitate the development of an infectious disease [3]. On the other hand, Quorum quenching (QQ) are inhibitory mechanisms that interfere the naturally regulated by QS, and thus, stands as a novel therapeutical methodology, based on the interference exerted upon the pathogenicity and virulence, to face infections. So far, a great number of molecules have been evaluated as potential quorum quenchers [4]. Within the high variability of potential quorum quencher molecules, it s important to highlight the capacity of certain antibiotics, administrated at subclinical doses. Pseudomonas aeruginosa is an opportunistic pathogen that naturally exhibits a high antibiotic resistance rate. It s one of the most prevalent bacteria in hospitals, were they produce a wide range of nosocomial infections. In this work, a thorough bibliography review is done in order to evaluate the potential use of different antibiotics, at subclinical doses, as an inhibitor molecule of the QS process in Pseudomonas aeruginosa. Among all, azithromycin is the antibiotic that exhibits a higher inhibitory capacity against different mechanisms regulated by QS, such as biofilm formation and other virulence factors. On the other hand, those forming part of the β-lactam family, have not yet been sufficiently studied. Administration of quorum quencher molecules in combination with other first-line treatments could increase success rate of such treatments and help reduce the increasing trend emergence of new resistance mechanisms in the different microorganisms. This novel approach could allow the development of new antimicrobial treatments. Keywords: Pseudomonas aeruginosa; Quorum sensing; Quorum quenching; antibiotics; subclinical dose [1] World Health Organization (2014). Antimicrobial Resistance. Global Report on Surveillance Reference. Available from: F3CE6EA9D937?sequence=1. Last visit 16/04/2018. [2] Lewis, K. (2012) Antibiotics: recover the lost art of drug discovery. Nature 485: [3] Moradali, M. F. et al. (2017). Pseudomonas aeruginosa Lifestyle: a paradigm for adaptation, survival, and persistence. Front. Cell. Infect. Microbiol. 7:39. [4] B. La Sarre, M.J. Federle (2013). Exploiting quorum sensing to confuse bacterial pathogens. Microbiol. Mol. Biol. Rev. 77:

84 Remineralization effect of hydroxyapatite and its potential for substituting fluoride in dentifrice I. Maric 1, K. Peros 2 and I. Sutej 2 1 Student of 4 th year School of Dental Medicine, University of Zagreb, Gunduliceva 5, Zagreb, Croatia 2 Department of Pharmacology, School of Dental Medicine, University of Zagreb, Salata 11, Zagreb, Croatia Prevention is still main focus and the best method for fighting the caries. The lack of education makes this approach more difficult and leads to the increase use of unfluoridated dentifrice. Fluoride is known as a successful remineralization agent. However, lack of remineralization in hyposalivation conditions, lower efficiency of mineralization of fissure caries and danger of dental fluorosis motivated scientists to find a remineralization agent that could substitute its role. Consequently, today many dentifrices contain newly found remineralization agents like calcium and phosphate ions and compounds such as casein phosphopeptideamorphous calcium phosphate (CPP-ACP), tricalcium phosphate (TCP), xylitol and hydroxyapatite. The composition of unfluoridated dentifrice with an emphasis on remineralization agents and remineralisation effects were investigated by literature search. Due to the increased number of promising results in the revised researches, inquiry was emphasised on the hydroxyapatite. Literature search was conducted on PubMed website with key words: enamel and remineralization. Given the limitation of last 10 years and excluding the reviews, search showed total of 887 results of clinical and preclinical studies. More than half of investigations, involved fluoride as reminerallization agent, followed by agents containing calcium phostphate compounds. Lastly, researches with hydroxyapatite as most recent agent used for enamel remineralization investigated, was used in 112 articles. Studies of CPP-ACP, tricalcium phosphate xylitol and hydroxyapatite showed promising results in remineralization. Moreover, hydroxyapatite was distinguished as a better alternative to fluoride compared to other calcium compounds Hydroxyapatite has demonstrated exceptional biocompatibility and affinity to dental enamel. The main disadvantages of hydroxyapatite are lack of strength, fragility and poor solubility in neutral ph. For this reason, particles of hydroxyapatite were synthesized in various forms, sizes and combinations with other elements such as zinc and strontium. In many studies, it has shown lesser or equal remineralization effects compared to fluorine. However, it is necessary to highlight that the results varied, where hydroxyapatite showed better results when biofilm or saliva was integrated in the remineralization model. At the same time remineralization proved to be more successful at lower ph values. There is a need for further research of mechanisms of hydroxyapatite remineralization given that potential exists. During the literature research, diversity in methodology has been observed, from tooth selection to choice of analytical method. This diversity makes it difficult to compare studies and make conclusions about the real remineralization effect. Therefore, more in vivo research and better consent of scientists in selection of methods are needed. Please prepare your abstract using Microsoft Word (or compatible formats like RTF). Abstracts must be formatted according to this template. Do not alter the margins and use the predefined styles for title, author(s), affiliation(s), abstract text, keywords Abstracts must begin with the title in lower case letters except for proper nouns, certain abbreviations, physical quantities etc. It will be followed by the names of all authors (with first name initials or full names) and the corresponding institutions (from the smallest to the largest unit, e.g. group, department, and university) with addresses (street or P.O. box, city with zip codes and country), as given in the above example. Keywords must be provided separated by semicolon. Keywords: remineralization; enamel; hydroxyapatite 74

85 Study of the chemical component and evaluation of essential oils of Daucus carota (Apiaceae) and Chenopodium ambrosioïdes (Chenopodiaceae) A. Amechrouq, M. Elhourri and M. El Idrissi Laboratory of Molecular Chemistry and Natural Substance, Moulay Ismail University, Faculty of Science, B.P , Zitoune, Meknes, Morocco The objective of our work is to determine the chemical component of essential oils of Chenopodium ambrosioïdes (L.) in the four regions of Morocco: Salé, Agadir, Meknes and Mrirt; and Daucus carota (L.) ssp. carota the Ain El Orma region located 20km from Meknes. Essential oils thus obtained are used first, as insecticides against pests Sitophilus oryzae, Bruchus lentis, Melolontha melolontha and Dysaphis plantaginea, and secondly, as inhibitors of germination of Lepidium sativum. For four essential oils of Chenopodium ambrosioïdes (L.), we note that there are very significant differences in percentage component particularly for Terpinolene, p-cymene and p-cymen-7-ol. The percentage of α- Terpinene from the essential oil of Mrirt and Salé regions is similar, while for the other regions a significant difference of content was recorded. We found that the essential oils of flowers Daucus carota (L.) ssp. carota contain mainly: the β-asarone (46.5%), 10S,11S-Himachala-3(12),4-diene (29.9%) and β-cubebene (4.5%). The β-asarone (63.4%) is the main compound found in essential oils seeds, followed by α-himachalène (18.5%) and β-bourbonène (8.1%) [1]. The insecticidal activity of essential oils of these plants was tested by fumigation under the conditions of the breeding of the insect studied. Throughout the duration of exposure, concentration and time lethal required for 50% and 99% mortality, were determined. Keywords: Chenopodium ambrosioïde (L.); Daucus Carota (L.) ssp. Carota; GC/MS, Spectroscopic Techniques; β- Asarone; Sitophilus oryzae; Bruchus lentis; Melolontha melolontha; Dysaphis plantaginea; Lepidium sativum [1] M. El Idrissi, M. Elhourri, A. Amechrouq, Phys. Chem. News, 2013, 69, [2] M. Elhourri, A. Amechrouq, M. El idrissi, A. Boughdad, ScienceLib Editions Mersenne, 2014, 6, N ISSN [3] M. El idrissi, M. Elhourri, A. Amechrouq, A. Boughdad, J. Mater. Environ. Sci., 2014, 5 (4), [4] M. El idrissi, M. Elhourri, A. Amechrouq, R. Zouhair, ScienceLib Editions Mersenne, 2014, 6, N ISSN [5] M. El Idrissi, M. Elhourri, A. Amechrouq, A. Lemrhari, World journal of pharmacy and pharmaceutical sciences, 2016, 5, issue 3,

86 Surface-Active, Nonleaching Antimicrobial Boron-Modified Hyperbranched Polyurethane Younes Ahmadi and Sharif Ahmad Material Research Laboratory, Department of Chemistry, Jamia Millia Islamia- New Delhi, India The rapid growth of pathogenic bacteria on surfaces and their resistance to the bactericidal agents has led to an urgent need to control their growth by the application of surface-active polymer coatings. Therefore, in present study we report a novel synthesis of surface-active nonleaching antimicrobial coating by simple etherification of boric acid (BA) and monoglyceride of vegetable oil (MG) to reduce the use of volatile organic compounds. The structural elucidation of boron-hyperbranched polyurethane (BHPU) was investigated by FT-IR, 11 B NMR, 1 H NMR, and 13 C NMR spectroscopies. Anti-microbial activities of these polymers were investigated by well diffusion and soil burial (210 days) techniques that were confirmed by the appearance of clear zone of inhibition and coatings weight loss respectively. The antimicrobial mechanism of BHPU was further investigated by Scanning Electron Microscopy, which displayed the permanent damage caused by BHPU on direct contact with Gram-positive and Gram-negative bacteria after incubation that occurred through creating a distortion in their cell wall. The physico-mechanical, contact angle, and thermal stability of BHPUs showed high mechanical properties, good hydrophobicity, and satisfactory thermal behavior respectively. Therefore, these results evidenced that BHPUs exhibited effective surface-active antimicrobial activity with excellent performance. 76

87 Synergistic antibacterial activity of phenolic compounds with commercial antibiotics Jeong Woo Kang*, Md Akil Hossain, Hae-Chul Park and Kwang-Jick Lee Veterinary Drugs & Biologics Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea * Corresponding author: hijach@korea.kr Bacteria have a remarkable ability to acquire resistance against antibiotics by several mechanisms. New strategies are needed to block the development of resistance and to prolong the life of traditional antibiotics. Thus in this study, we intended to increase the efficacy of commercially available antibiotics by combining with opportunistic phenolic compounds. Eight commercial antibiotics (Ampicillin, Amoxicillin, Ceftiofur, Penicillin G, Cefataxime, Marbofloxacin, Erythromycin and Theamphenicol) and 5 phenolic compounds (Gallic Acid, Hamamelitannin, Epicatechin, Epicatechine Gallate and Epigallocatechin) were used against Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus to evaluate the antibacterial combination effect. Phenolic compounds demonstrated good antimicrobial activity varied with minimum inhibitory concentration (MIC) from 32 g/ml to 2048 g/ml depending on compounds and strains. The 5 phenolic compounds were investigated for quorum sensing inhibition by disc agar diffusion assay, but none of them showed positive result. Fractional inhibitory concentration (FIC) index of 40 sets of combination against Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus ranged from to Three combinations were selected for further investigation depending on the critically important antibiotics list of World Health Organization and the FIC index of our study. Inhibition rates of Salmonella Typhimurium in presence of Gallic Acid + Ceftiofur, and Escherichia coli in presence of Hamamelitannin + Erythromycin, and Gallic Acid + Ampicillin demonstrated improved efficacy at different time points compared to the efficacy of those antimicrobials alone. The effect of those three combinations on the cell morphology (e.g.: Cell division, cell wall rupture, etc.) of Salmonella Typhimurium and Escherichia coli were evaluated and found that those antimicrobial combinations have no effect on cell morphology. All of the three combinations showed different degrees of biofilm inhibition potential. Among them Hamamelitannin + Erythromycin combination demonstrated better inhibition potential of Escherichia coli biofilm. Based on the result of this study, it is concluded that the therapy of these combinations can be more effective than the conventional antibiotics in controlling Salmonella Typhimurium, Escherichia coli associated infections. Further investigations are recommended to determine the safety profile and combination antimicrobial effect in in vivo system. Keywords: Phenolic Compounds; Antibactrial activity; Synergy; Antimicrobial Resistance; Tannin [1] Md. Akil Hossain, Seung-Jin Lee, Na-Hye Park, Abraham Fikru Mechesso, Biruk Tesfaye Birhanu, JeongWoo Kang, Md. Ahsanur Reza, Joo-Won Suh, Seung-Chun Park. Impact of phenolic compounds in the acyl homoserine lactonemediated quorum sensing regulatory pathways. Scientific Reports 2017; 7(10618):1-16. [2] Md. Akil Hossain, Ji-Yong Park, Jin-Yoon Kim, Joo-Won Suh, Seung-Chun Park. Synergistic Effect and Antiquorum Sensing Activity of Nymphaea tetragona (Water Lily) Extract. BioMed Research International 2014; 2014(562173):

88 Synthesis and in-vitro biological activity of some novel 2-pyazinecarboxylic acid derivatives Hossein Mostafavi 1, Zahara Agazadeh 1 and Fatemeh Khodaei 2 1 Department of organic Chemistry & Biochemistry, Faculty of Chemistry, University of Tabriz P.O Box , Tabriz, Iran 2 Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran A diversity of biological activities and pharmaceutical uses have been attributed to pyazinecarboxylic acid derivatives such as antituberculosis antibacterial, antifungal, anticancer (1,2). A series of 2-pyazinecarboxylic acid derivatives were synthesized and their structure confirmed by FT-IR, 1 HNMR, 13 CNMR, elemental analysis. In vitro biological activity of compounds was evaluated by employing 24 hours cultures of Escherichia coli ATCC (8739), Klebsiella sp. ATCC (700834), bacillus subtilis ATCC (6051) and Staphylococus aureus ATCC (6538). Antibacterial susceptibility tests were done by use of the paper disc diffusion method on Mueller Hinton agar (Merck). Gentamicin was standard reference antibiotics. The inhibition zone was determined by measuring the minimum dimensions of the zone of no microbial growth around the disc. The Minimum Inhibitory Concentrations (MIC) were determined by serial dilution technique according to CLSI. The MIC values of active anti-bacterial compounds 2 and 4 were in the range of 25 to 50µg/ml concentrations against common bacterial infection causing species. Keywords: 2-pyazinecarboxylic acid; derivatives; antibacterial; antibiotics; inhibition [1] Sayahi, H.; Zimhony, O.; Jacobs, W.R.; Shekthman, A.; Welch, J.T. Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I. Bioorg. Med. Chem. Lett. 2011, 21, [2] Shi, W.; Chen, J.; Feng, J.; Cui, P.; Zhang, S.; Weng, X.; Zhang, W.; Zhang, Y. Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis. Emerg. Microbes Infect. 2014, 3, e58. 78

89 The effects of adequate education through the implementation of antibiotic stewardship programs in long-term care facilities on successful reduction of antibiotic usage, antibiotic resistance, and Clostridium difficile infections Sarah Henderson Columbus State University; 4225 University Ave; Columbus, Georgia; Antibiotic overuse, antibiotic resistance, and Clostridium difficile infections are rising problems across the globe, particularly in long-term care facilities. These issues have a cause and effect relationship that earned them the label of the trifecta. Healthcare costs, patient safety matters, and morbidity and mortality rates are associated consequences of these combined issues. Antibiotic stewardship programs are a developing way to manage the trifecta and evade major medicinal regression. Knowledge deficits involving the components and definition of these programs are identified throughout the literature as well as research deficits on effective implementation methods. The primary commonality for implementation in the literature is education. Effective education is recognized as the most crucial component to implementation with tools including informational handouts, written guides and communication tools, audits, electronic learning, and in-services pertaining to all methods. Evaluation of effectiveness was monitored via reflective statements, questionnaires, audits, and tracking and trending of antibiotic use. Keywords: antibiotic stewardship programs; antibiotic resistance; clostridium difficile; education; long-term care 79

90 Typing Burkholderia cepacia complex isolated from non-cystic fibrosis patients in Kuwait Ali A. Dashti 1, Dana Al-Kayyali 1, Khaled Al Obaid 2, Fatma Failakawi 3, Ishaq Mohmid 4 and Leila Vali 1 1 Department of Medical Laboratory sciences, Faculty of Allied Health, Kuwait University, Kuwait 2 Microbiology Unit, Amiri Hospital, Kuwait 3 Microbiology Unit Mubarak Al-Kabeer Hospital, Kuwait 4 Department of Laboratory, Kuwait Oil Company Hospital, Kuwait Introduction: Burkholderia cepacia complex (Bcc) is a group of opportunistic pathogens comprising of at least 20 closely related species that are phenotypically similar yet genetically distinct. It commonly affects patients with cystic fibrosis (CF), however there are several cases of nosocomial Bcc infections in immuno-compromised patients with no history of CF. Lately, the life-threatening sporadic cases of infections caused by Bcc among ICU patients in Kuwaiti hospitals has been observed. The focus on Bcc species in this study is driven by their pathological significance and the lack of precise reports concerning their prevalence in Kuwait. Objectives: Our objectives were to characterize multidrug resistant Bcc isolates from hospitalized patients in Kuwait and to analyze their genetic relatedness. Materials and Methods: A total of eight isolates were collected within six months (2015) from two hospitals with no history of cystic fibrosis from different sites of infection. Phenotypic identification was performed using VITEK 2 compact system and API20NE kit. Genotypic identification methods including PCR for reca gene, PCR-RFLP for reca were performed. For typing Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST) were used. Antimicrobial susceptibility testing using E-Test strips and agar dilution methods were performed to determine the MICs of antibiotics recommended by CLSI 2016 guidelines. Results: All isolates were identified as Bcc and produced a PCR product of 1000 bp Bcc-specific reca gene. Three different patterns were observed by PCR-RFLP for reca. B. cenocepacia IIIA (n=6), B. cepacia (n=1) and B multivorans (genomovar II) (n=1) MLST profiles demonstrated different sequence types (ST). We found three new STs: B. cepacia ST1288, B. cenocepacia IIIA ST1282 and B. cenocepacia IIIA Other STs included 306 (n=3), ST964 (n=1) and B multivorans (genomovar II) ST646. ST306. B. cenocepacia IIIA was the most prevalent type isolated. PFGE using SpeI restriction endonuclease illustrated the genomic diversity of the isolates. Not all the isolates belonging to the same ST shared identical PFGE pattern. Interpretation of MIC values showed all isolates were resistant to at least one group of antibiotics: ceftazidime (n=2), meropenem (n=3), minocycline (n=7), levofloxacin (n=3), TMP-sulfa (n=1), and chloramphenicol (n=8). Discussion: This is the first study performed to characterize the Bcc strains in Kuwait. The results will contribute to better understanding of Bcc bacteria and the genetic relatedness of Kuwaiti isolates to other regions of the world. The resistance of most isolates to several antimicrobial agents necessitates the appropriate selection of antimicrobial therapy. Keywords: Burkholderia cepacia; (MLST) 80

91 Ureolitic and arginolitic activity of dental plaque and saliva and the influence over the bacteria abundance in samples taken from children with or without caries P. Palma 1, I. Yévenes 2 and G. Moncada 3 1 Dept. of Pathology, Faculty of Dentistry, University of Chile. Sergio Livingstone 943, Santiago, Chile 2 Investigation Institute, Faculty of Dentistry, University of Chile. Sergio Livingstone 943, Santiago, Chile 3 Dental School, Universidad de los Andes, ons. Álvaro del Portillo , Santiago, Chile Objective: To determine the abundance of alkalogenic / acidogenic bacteria and the urease / arginine deiminase activity in samples of bacterial plaque and saliva of Chilean children of 7 years old of age with and without decay Materials and methods: Study was approved by Ethical Scientific Committee of the Faculty of Dentistry, 78 children of 7 years of the Metropolitan Region with different socioeconomic level, were selected according to inclusion and exclusion criteria. A dental examination (by trained and calibrated examiner Kappa 0.77) was carried out using WHO and ICDAS criteria for the diagnosis of lesions. Saliva and supragingival dental plaque samples were obtained to quantify the specific activity of urease and the arginine deiminase system by means of the quantitative spectrophotometric method. From these same samples the abundance of Streptococcus sanguinis, Streptococcus gordonii, Streptococcus salivarius, Streptococcus mutans and Actinomyces naeslundii were determined by q-pcr. Results: An inverse, moderate and significant correlation was found between the specific activity of urease and the arginine deiminase system and the presence of caries lesions. The abundance of S. sanguinis in plaque and saliva was higher for the caries-free group. For the group with caries (CA) S. gordonii in plaque and S salivarius in saliva were more abundant and with significant differences compared to the group without caries (CF). S mutanswas more abundant in the samples of the CA group, both plaque and saliva (p <0.05). Conclusions: A greater activity of the arginine deiminase system in saliva and supragingival plaque is associated with a lower number of caries lesions according to the WHO and ICDAS criteria in 7-year-old children of the Metropolitan Region. The levels of alkalogenic bacteria such as S sanguinis could be related to lower cariogenic activity, suggesting that the formation of alkalis could act as a regulator of the acidity produced by acidogenic bacteria such as S. mutans, which was more abundant in a patient with caries. Keywords: Alkali; Arginine; Dental plaque; Saliva; Urease; Dental caries [1] Ástvaldsdóttir Á, Naimi-Akbar A, Davidson T, Brolund A, Lintamo L, AttergrenGranath A, Tranæus S, Östlund P (2016). Arginine and Caries Prevention: A Systematic Review. Caries Res 50: [2] Gordan V, Garvan C, Ottenga M, Schulte R, Harris P, McEdward D y cols. (2011). Could Alkali Production Be Considered an Approach for Caries Control? Caries Res 44: [3] Moncada G, Duperat LC, Palma P, Corsini G, Neira M, Gordan V, Yévenes I. Técnica de reacción de polimerasa en cadena (QPCR) en tiempo real para la identificación y cuantificación de streptococcus mutans en saliva y biopelícula dentaria de niños. Rev Fac Odontol Univ Antioq 2016; 28(1):

92 Use of marine and freshwater cyanobacteria as biological control agents in agriculture F. Suárez-Estrella, M.J. López, M.M. Jurado, J.A. López-González and J. Moreno Area of Microbiology, Department of Biology and Geology, University of Almería, La Cañada de San Urbano, S/N, Almería, Spain Cyanobacteria belong to a diverse group of photosynthetic prokaryotes. They are increasingly used as biofertilizers in agriculture due to their role in fixing atmospheric nitrogen, improving the physical and chemical properties of the soil, and the ability to compete with the native microbiota of this type of habitat. Cyanobacteria are producers of a large number of biologically active substances. The existing literature suggests that cyanobacteria may play a potential role in the protection of crops as biopesticide agents that produce antimicrobial substances 1. The commercial development of this type of substances for non-biomedical applications includes bioactive compounds that show a significant biocidal activity against devastating plant diseases. The aim of this work was to investigate the biostimulant and suppressive capacity of 52 different microalgae: 36 cyanobacteria from a collection of fresh water algae (Mosonmagyaróvár Algal Culture Collection, MACC, Hungary) and other 16 marine strains from the Spanish Bank of Algae (SBA). The study was carried out in order to select the more suitable aqueous algal extract that could be applied for crop growth improvement and disease control in an economic and environmental-friendly manner. Two in vitro tests were performed. On one side, algal extracts in a concentration of 10 mg.ml 1 were ultrasonicated and then searched for biological activity against four damping-off producing fungi using an agar confrontation method 2. The phytotoxicity of extracts on water cress seeds was also analyzed 3. In this case, ultrasonicated algal extracts were used in a concentration of 2 mg.ml 1 and 0.5 mg.ml -1. Several cyanobacteria presented a wide antagonistic range, as they inhibited all the four phytopathogen fungi tested. On the other hand, the phytotoxic effect on treated seeds was eliminated when the concentration of the extracts was reduced up to 0.5 mg.ml -1. Acknowledgement This study was made in the framework of the European Project SABANA (proposal , H2020- BG ) Keywords: cyanobacteria; biological control; damping off [1] Michalak et al., Eng Life Sci. 17, [2] Landa et al Phytoparasitica. 25 (4), [3] Zucconi et al Elsev App Sci. pp

93 Vancomycin loaded superparamagnetic MnFe 2 O 4 nanoparticles coated with PEGylated chitosan to enhance antibacterial activity Akbar Esmaeili 1, * and Sepideh Ghobadian Pour 2 1Department of Chemical Engineering, North Tehran Branch, Islamic Azad University, PO Box 19585/936, Tehran, Iran 2Department of Pharmacognosy, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran * Corresponding author: akbaresmaeili@yahoo.com Background and aims: Increasing prevalence of antibiotic-resistant and failed-treatment make more investigations to deal with these problems. Hence new therapeutic approaches for effective treatment are necessary. Ferrite superparamagnetic nanoparticles have potentially antibacterial activity. Methods: In this study we prepared MnFe 2 O 4 superparamagnetic nanoparticles as core by precipitation method and used chitosan crosslinked by glutaraldehyde as shell, then modified with PEG to increase stability of particles against RES. Results: Chitosan coating not only improves the properties of ferrit nanoparticles but also has antibacterial activity. FT-IR confirmed this surface modification; XRD and SEM were developed to demonstrate particle size and characteristics of crystal structure of these nanoparticles. Final particle size was reported approximately 25 nm. Magnetic properties of nanoparticles were evaluated by VSM. Actual drug loading and releasing were examined by (UV-Vis) spectroscopy method. Conclusions: We employed liquid broth dilution method to assessment antibacterial activity of nanoparticles against microorganisms. Significant antibacterial effect against gram negative bacteria was developed. Keywords: Vancomycin; superparamagnetic; PEGylated; chitosan 83