The German Zoonose research network VibrioNet: First findings

Transcription

1 International Symposium Pathogenic Vibrio spp. in Northern European Waters 31. Mai / 1. Juni 2012, Koblenz The German Zoonose research network VibrioNet: First findings Gunnar Gerdts Alfred Wegener Institute for Polar and Marine Research

2 International Symposium Pathogenic Vibrio spp. in Northern European Waters 31. Mai / 1. Juni 2012, Koblenz VibrioNet: Climate warming and the emergence of seafood and waterborne vibrioses Motivation Structure of the network & objectives Results

3 Vibrio Infections in Europe Cholera Eurosurveillance (ECDC) all diseases imported by travellers Diarrhoea caused by Vibrio spp. Few cases reported from Italy, Spain, France, UK Wound infections caused by Vibro spp. In hot summers infections reported from Baltic (SE, DK, DE) and North Sea (NL) caused by V. vulnificus, V. alginolyticus, V. parahaemolyticus, V. cholerae non O1,non O 139 Few reports from other European countries

4 Vibrio Infections in Germany Cholera 15 cases since 2001 all diseases were travel related Diarrhea caused by Vibrio spp. Few suspected cases in Germany in the last 10 years Notification not required (Protection against Infection Act) Wound infections caused by Vibrio spp. (since 2002) In hot summers three deadly infections reported caused by Vibrio vulnificus after contact with seawater Seven wound infections without lethal outcome were reported Notification not required

5 Why Vibrio Research in Germany? Wound infections Death cases of elderly immunocompromised persons (V. vulnificus) Economic impact for coastal regions feared (tourism) Climate change will affect Vibrio populations

6 The german zoonose research network VibrioNet Funding commenced on November 2010 (and later)

7 The german zoonose research network VibrioNet National partners Federal Institute for Risk Assessment (BfR) Medical Microbiology, Technical University Dresden (TUD) Institute for Food Hygiene, Free University Berlin (IFH) Institute for Fish and Fishery Products (IFF) Alfred Wegener Institute for Polar and Marine Research (AWI) Robert Koch Institute (RKI) Q Bioanalytic (QBA)

8 The german zoonose research network VibrioNet Foreign partners Bangladesh: International Center for Diarrhoeal Disease Resesearch, Dhaka (ICDDR) Chile: Institute of Nutrition and Food Technology, University Santiago (INTA) India: National Institute of Oceanography, Goa (NIO) Thailand: Department of Veterinary Public Health, Chiang Mai University (CMU) Vietnam: Institute of Oceanography, Nha Trang (VNIO)

9 The german zoonose research network VibrioNet Main objectives 1 To assess the occurrence of Vibrio spp. pathotypes in environment, retail food and of human disease To define environmental parameters promoting the emergence, proliferation, and transmission of pathogenic Vibrio spp. with special reference to climate driven processes To define problematic food processing and transportation practices that favour the emergence, proliferation and transmission of pathogenic Vibrio spp.

10 The german zoonose research network VibrioNet Main objectives 2 To identify marker genes of host specific and strain specific pathogenicity that allow for the rapid detection of Vibrio spp. To develop rapid, accurate and affordable detection systems for Vibrio spp.

11 The german zoonose research network VibrioNet 12 subprojects

12 The german zoonose research network VibrioNet Subproject 1 (BfR, IFH) Global aspects of Vibrio spp. as emerging zoonotic agents: An international approach To establish and strengthen collaborative links on the epidemiology, the population dynamics and the pathogenesis of Vibrio spp. between national and international partners Training and education in microbiological analysis, molecular biological approaches and data analysis

13 The german zoonose research network VibrioNet Subproject 2 (AWI) Seasonality of pathogenic Vibrio spp. in seawater, plankton, and shellfish of North and Baltic Sea To desribe the current status of pathogenic Vibrio spp. populations in German waters To estimate the possible future status of pathogenic Vibrio spp. populations in German waters To evaluate analysis schemes for pathogenic Vibrio spp. populations in German waters To describe and evaluate the environmental factors influencing the occurrence of pathogenic Vibrio spp. in German waters

14 Subproject 2 (AWI) Seasonality of pathogenic Vibrio spp. in seawater, plankton, and shellfish of North and Baltic Sea Characterization of over 800 environmental isolates from the North and Baltic Sea Main focus: V. parahaemolyticus, V. vulnificus & V. cholerae Sources: Environmental agencies & own sampling campaigns Current Vibrio spp. strain collection at BAH Helgoland (liquid N 2 ) Analyses: PCRs (species, virulence factors) rpob sequence analysis ERIC PCR fingerprints MALDI TOF

15 MALDI TOF (Bruker Biotyper system) Acquiring mass spectra of isolates Environmental Sampling (North & Baltic Sea) rpob Phylogeny MALDI TOF reference database Alignment Acquiring mass spectra of single colonies Poster: Analyzing Vibrio Populations by MALDI-TOF

16 MALDI TOF (Bruker Biotyper system) Sub-species level Species level MSP Dendrogram MALDI-TOF V. alginolyticus 2 V. alginolyticus 1 Vibrio alginolyticus VN-2626 Vibrio alginolyticus VN-2604 Vibrio alginolyticus VN-2594 Vibrio alginolyticus VN-2628 Vibrio alginolyticus VN-2613 Vibrio alginolyticus VN-2611 Vibrio alginolyticus VN-2584 Vibrio alginolyticus VN-2624 Vibrio alginolyticus VN-2583 Vibrio alginolyticus VN-2637 Vibrio alginolyticus VN-2586 Vibrio alginolyticus VN-2514 Vibrio alginolyticus VN-2630 Vibrio alginolyticus VN-2508 Vibrio alginolyticus VN-2517 Vibrio alginolyticus VN-2620 Vibrio alginolyticus VN-2619 rpob Distance Level

17 Monthly transects (German Bight) Environmental gradient (Elbe estuary to open sea): Salinity? Temperature? X? Yearly cruises (North vs. Baltic Sea) Vibrio biogeography Microcosm experiments with isolates from different locations with different waters Growth / mortality rates

18 Cruise 2011 getting an overview New isolates Cruise in August 2011 Vibrio spp. counts Seawater (ChromAgar) North Sea Baltic Sea Overgrowth

19 Cruise 2012 Detailed Vibrio spp. community analysis Culture dependent & independent approaches on board & off board analyses Contextual data Samples Seawater Zooplankton Phytoplankton Microplastic* (Coop.: Univ. Hull, UK) Identifying single colonies without subcultivation (MALDI TOF) Detection of species / virulence genes in filter enriched biomass (QPCR & Array) Community analysis from filter enriched biomass (rpob DHPLC)

20 The german zoonose research network VibrioNet Subproject 3 (IFF) Detection of Vibrio spp. in bivalve mollusc harvesting areas in the Lower Saxony Wadden Sea region Qualitative and quantitative analysis of blue mussels (Mytilus edulis) of harvesting areas for pathogenic Vibrio spp. by cultural methods Qualitative and quantitative analysis of the pacific oyster (Crassotrea gigas) for pathogenic Vibrio spp. by cultural methods

21 The german zoonose research network VibrioNet Subproject 4 (IFH) Detection and proliferation of pathogenic Vibrio spp. in retail seafood Screening of seafood samples Development of quantitative real time PCR assays for quantification of Vibrio spp. in seafood Application of the quantitative real time PCR assays on spiked food samples Comparative analysis of the Vibrio load in retail food samples using phenotypic methods and quantitative real time PCR Growth and survival of Vibrio spp. in seafood samples until and beyond date of minimum durability

22 Subproject 4 (IFH) Detection and proliferation of pathogenic Vibrio spp. in retail seafood Number of different V. spp. per sample V. spp. composition n=226 positive (%) Mussels Europe (70%), Atlantic (20%) Prawns Asia (65%), Atlantic (20%) Seawater Baltic Sea (85%)

23 MLST Analysis of V. parahaemolyticus West Asia Central Asia East Asia South America Europe 78 isolates 88% from Asia Distribution independent of geographic origin high diversity no dominant ST Sri Lanka Bangladesh Ecuador Thailand Vietnam Pacific Baltic Sea Japan China Honduras India Poster: MLST for genotyping of Vibrio parahaemolyticus

24 The german zoonose research network VibrioNet Subproject 5 (BfR) Toxigenic potential of Vibrio spp. from environmental, food and clinical sources in Germany DNA based classification and subtyping (MLST, PCR Virulotyping) Virulence and pathogenic potential of Vibrio isolates (Phenotyping) Transcriptional and Proteome Analyses (e.g. Toxin production) Immunological assay development Central strain collection

25 The german zoonose research network VibrioNet Subproject 6a (BfR) Molecular adaptations of pathogenic Vibrio spp. to bivalve and mammalian hosts in vitro cell culture systems (cytotoxicity, cell adherence, cell invasion assays) in vivo infection models (mussel, oyster)

26 Subproject 5 (BfR) Toxigenic potential of Vibrio spp. from environmental, food and clinical sources in Germany Cell free synthesis of toxins (Cooperation with Fraunhofer Institute Potsdam) TDH of Vibrio parahaemolyticus Genomic DNA of pandemic strain O3:K6, cloned tdh2 gene as PCR templates Cell lysates of E.coli, PCR generated templates for transcription/translation Advantage: No inhibition of expression by host Expression in cell free system is 1000fold higher Supernatant toxins can be easily purified and used for tests 1: tdh1/2 + signal sequence 2: tdh1/2 signal sequence 3 : tdh2 + signal sequence 4 : tdh2 signal sequence 5: lysate alone

27 Characterization of environmental and clinical Vibrio vulnificus isolates from the Baltic sea Strains supplied by LAGUS Rostock (Dr. Hauk / Dr. Duty) Clinical and environmental strains (period 1994 to 2011) What are the characteristics of Baltic sea isolates? Genotypic and phenotypic assays MLST, 16S rrna, vcg locus, hemolysis, serum resistance, cytotoxicity Poster: "Phenotypic and genotypic characterisation of environmental and clinical Vibrio vulnificus isolates from the Baltic sea"

28 The german zoonose research network VibrioNet Subproject 6b (TUD) Transcriptome profiling of pathogenic Vibrio spp. via Microarrays Establish Transcriptomics assays (Microarray) Identification of temperature induced genes in vitro Verification of temperature induced genes in vitro via wild type mutants Verification of Microarrays: Transcriptome studies via quantitative real time RT PCR

29 Subproject 6b (TUD) Transcriptome profiling of pathogenic Vibrio spp. via Microarrays V. parahaemolyticus RIMD Agilent 8x15k Microarray e.g. at different temperatures Upregulation: Downregulation: n 221: e.g. groes, groel 518: e.g. cspa 271: e.g. cspa 170: e.g. groes, groel n

30 The german zoonose research network VibrioNet Subproject 6c (TUD) Molecular characterisation of virulence determinants of pathogenic Vibrio spp. by whole genome sequencing Complete sequencing and annotation of selected Vibrio strains Comparative genome analysis to identify further virulence markers/ determinants for species identification/typing

31 Subproject 6c (TUD) Molecular characterisation of virulence determinants of pathogenic Vibrio spp. by whole genome sequencing V. vulnificus strains Origin Vv V48/10 (VN 0101) Vv V52/10 (VN 0104) Vv V57/10 (VN 0108) Vv V63/10 (VN 0112) Vv va35905 (VN 0092) Sequencing of selected Vibrio strains Water (Binz) Water (Binz) Wound infection Wound infection Wound infection (Greifswald)

32 The german zoonose research network VibrioNet Subproject 7 (RKI) Pilot studies on the human burden of wound and diarrhoeal Vibrio spp. infections in Germany Case collection in a national data base Investigation of specific risk factors Analysis of a correlation between incidence of infection and water temperature in Baltic and North Sea

33 Subproject 7 (RKI) Pilot studies on the human burden of wound and diarrhoeal Vibrio spp. infections in Germany incidence of infections caused by non cholera Vibrio spp. in Germany unclear since reported cases of wound infections 11 cases on the Baltic Sea, 1 case on the North Sea 83% with pre existing conditions like diabetes or liver disease 83% male 33% lethality 2004 two cases of gastroenteritis

34 Reasons for unknown incidence in Germany Surveillance lack of notification requirement in the German Protection against Infection Act (IfSG) known cases voluntarily reported or collected from publications Missed cases due to non diagnosis AB therapy pre. sample taking in wound infections not looking for Vibrio in diarrheal infections Biased targeted diagnostics Wound infections focus on risk patients geographically differential awareness Diarrheal infections focus on imported infections

35 Pilot studies on wound infections retrospective evaluation of case data (hospital, physicians and laboratory records) of the past 10 years for infections caused by non cholera Vibrio spp. along German coasts and nationally prospective case collection in a national data base investigation of specific risk factors analysis of a correlation between incidence of infection and (high) water temperature in Baltic Sea and North Sea

36 The german zoonose research network VibrioNet Subproject 8 (TUD) Incidence of Vibrio spp. isolates in human stool specimens and evaluation of their virulence potential in in vivo model systems Assessing the incidence of Vibrio spp. in routine diagnostic stool samples from patients of a university hospital Development of a custom designed DNA subset micro array to establish molecular fingerprints of human (and environmental & food) isolates of Vibrio spp. Development of an in vivo infection model to evaluate the virulence of well characterized V. parahaemolyticus strains Functional characterization of selected virulence determinants from V. parahaemolyticus in the established in vivo infection model

37 Subproject 8 (TUD) Incidence of Vibrio spp. isolates in human stool specimens and evaluation of their virulence potential in in vivo model systems Isolation of Vibrio spp. from human stool samples Which specimens are tested for presence of Vibrio spp.? MVZ (ambulatory healthcare center) or outpatient clinics at the University Hospital Dresden (UKD) UKD sampling 48h after admission

38 Development and evaluation of a pathotyping array for Vibrio spp. Array construction Version 1.0: Definition of test conditions (DNA extraction, DNA labeling, hybridisation, visualisation) 24 target genes (e.g. virulence and resistance markers, type 3 secretion systems, regulator genes) Mapped by 68 hybridisation probes Coverage: 5 fold

39 Array operation DNA extraction Linear amplification Array coating Hybridisation and washing steps Addition of HRP conjugate and substrate precipitation Array readout: ArrayMate from Alere Technologies (Jena)

40 First experiments with 4 completly sequenced Vibrio strains: Vp RIMD Vv CMCP6 Vfur NCTC Vv M06 24/O (negative control)

41 The german zoonose research network VibrioNet Subproject 9 (QBA) Development of rapid, accurate and affordable PCR based detection systems for pathogenic Vibrio spp. Validation of real time PCR tests Development of robust and cost effective detection tools (dipstick / NALFIA) Supporting Project Mass Spectrometry Development of a MALDI TOF (Biotyper) database for Vibrio spp. (AWI)

42 VibrioNet Project C 9 Development of rapid, accurate and affordable PCR based detection systems for pathogenic Vibrio spp.

43 Real Time PCR kits under development and validation Vibrio Vibrio cholerae Vibrio Cholerae (ctx) Vibrio parahaemolyticus Vibrio parahaemolyticus (trh) Vibrio parahaemolyticus (tdh) Vibrio vulnificus Vibrio alginolyticus 43

44 Validation of tests Real Time PCR Vibrio para. Vibrio cholerae : 44

45 Inclusivity und Exclusivity DNA from 40 strains were sucessfully tested 20 Vibrio para. 17 Vibrio chol (non-tox) 3 Vibrio chol (tox) Three different primer and probe sets were tested with target strain and with the other strains as non target strains. All targets were detected and nontargets were tested as negative. 45

46 The german zoonose research network VibrioNet

47 Thank you for your attention!