Environmental reservoirs of antibiotic resistance genes

Transcription

1 Environmental reservoirs of antibiotic resistance genes

2 Antibiotic resistance in the environment: soil, sediments, water bodies Environment acts as an reservoir for antibiotic resistance genes: -associated with antibiotic biosynthesis clusters - in closely related non-producers - in unrelated non-producers indigenous soil bacteria - in unrelated non-producers exotic bacteria = pathogens/commensals added to soil Potential for selection of resistance -pollution HGT of resistance genes- mobilome Pathogens can survive in soil -Acquire integrons/plasmids -Act as source of antibiotic resistance

3 Flow of antibiotic resistance genes, antibiotics and pathogens in the environment Veterinary antibiotics Farm animals Clinical antibiotics Antibiotics produced by soil bacteria Pollutants Disinfectants Surfactants Soil Water Food Hospital People

4 Studying the environmental gene pool: Antibiotic resistance Cultivation from environmental samples: Isolation and PCR Metagenomics- expression screening Gene capture: mobilomes Integrons -gene cassettes- gene capture Transposons Plasmids- exogenous isolation host capture Phage

5 Studying the environmental gene pool: Antibiotic resistance Resistance gene expression can vary and up regulation commonly occurs Co-selection results from linkage of several resistance genes, resistance to pollutants, heavy metals, disinfectants, detergents Horizontal gene transfer, adaptive genes form part of the mobilome: Integrons -gene cassettes- gene capture Transposons Plasmids Phage

6 Non-producers Tetracycline Non-producers Gentamicin Producers Streptomycin Non-producers Streptomycin Reservoirs of antibiotic resistance genes in diverse environments: RESERVOIR survey Soil Rhizosphere Manure Sewage Seawater Prevalence aph3 aph6-id ant3 adenylase aph6-ic aph6-ic (deg) stra aphd strb1 stsc aac(3)-i aac(3)-ii/vi aac(3)-iii/iv aac(6 )-II/Ib ant(2 )-I aph(2 )-I teta tetb tetc tetd tete tetg teth tetk tetl tetm teto tett Soil Rhizosphere Manure Sewage Seawater

7 Antibiotic class General behaviour Chloramphenicol impersistent/ 2,4- diaminopyridines Occurrence of antibiotics in the natural environment, fish, crops and drinking water from published studies mobile persistent/ Sewage sludge River water Groundwat er Drinking water Fish Soil Crops Example compounds monitored - X immobile X X - trimethoprim Fluoroquinolones persistent/ immobile X X - - ciprofloxacin, norfloxacin, ofloxacin -lactams impersistent mobile - X X X amoxicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V Macrolides slightly persistent/ slightly mobile X azithromycin, clarithromycin, lincomycin, roxithromycin, spyramycin, tylosin Sulfonamides persistent/ mobile X - sulfamethoxazole, sulfadiazine, sulfamerazine, sulfamethazine, sulfapyridine Tetracyclines persistent/ immobile - X X chlortetracycline, doxycycline, oxytetracycline, tetracycline A tick means that it has been monitored for and detected and a cross means that it has been monitored for and not detected. No entry means that no monitoring has been done yet (Alistair Boxall)

8 Sewage treatment and disposal Application of sewage sludge to land: what is the impact on antibiotic resistance soil?

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10 Schematic map of the complex class 1 integron carrying the bla CTX-M-14 gene on plasmid paje0508 gene on plasmid paje0508 Bae et al., AAC, Aug 2007,

11 Sampling polluted environments: Diagram of mill and reed bed system Reed bed system for mill effluent remediation oceans-esu Ltd

12 Relative resistance to DTDMAC and CTAB at 50 µg/ml CTAB cetyltrimethylammonium chloride DTDMAC ditallowdimethylammonium chloride Gaze et al., AAC, May 2005,

13 Incidence of int11, qae, and qaceδ : Reed bed and river sediment Gaze et al., AAC, May 2005,

14 prevalence (%) Integron and qac gene prevalence: comparison of environmental samples inti1 qace 1 qace qacg RB, Reed bed sediment from textile mill - QAC pollution only SS, Fully digested sewage sludge - QAC pollution and antibiotic residues Pig slurry - antibiotic residues only CW, Fallowed agricultural soil - no QACs or antibiotics 4 qach RB SS PS CW sample site Under strong QAC selection IS elements were maintained in the 5 region of class 1 intergons where they acted as powerful promoters for gene cassette expression Gaze et al., Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment. ISME

15 prevalence % 90 million tons animal faecal slurry added to UK soils per year class 1 integron prevalence following pig manure application to land preapplication day 1 day 21 day 90 day 289 days after slurry application Bailey-Byrne K, Gaze WH, Zhang L, Hawkey P, Wellington EMH Class 1 integron and class 2 integron diversity in agricultural soil amended with pig slurry. AEM, Jan;77(2):684-7.

16 Schematic diagram of class 1 integron from Arthrobacter aritaii (strain C361), putatively carried on a transferable plasmid P2 inti1 5 conserved region/atti1 aada9 59bp element qaceδ1 suli 3 conserve d region 500bp 750bp 450bp 6000bp atti1 integrase binding site aada9 streptomycin/spectinomycin resistance gene, 99% blast homology with aada9 from Corynebacterium glutamicum qaceδ1 quaternary ammonium compound resistance gene, 98% blast homology with E.coli suli sulphonamide resistance gene, 98% blast homology with Salmonella enterica Byrne-Bailey et al Appl Environ Microbiol. 77, Bailey-Byrne et al., Antimicrob.Agents Chemotherap. 53,

17 Resistance to β-lactam antibiotics- culture independent Reed Bed soil Sewage Cake 1 Month Cake Applied Control Soil Grass Land Soil FYM Applied Grass Soil No. of clones Average insert size (Kb) Clones with inserts (%) Coverage (Gb) No of cefotaxime resistance No of ceftazidime resistance No of imipenem resistance No of amp res clones ? 2? ? 5? 1? Hit rate 1/80 1/150 1/

18 Waste water treatment plants as a reservoir for antibiotic resistance Waste Water treatment plants Hotspot for Horizontal Gene Transfer (HGT) as waste received from various sources Little is known about the impacts of effluent further downstream in the river or the possible role of co-selection of antibiotic resistant determinants via quaternary ammonium compounds (QACs) (Gaze et al., AAC 2005, ISMEJ 2011)

19 Case study of bla CTX-M prevalence 3 rd generation cephalosporin (3GC) resistance necessitates the use of last resort antibiotics P Extended Spectrum β-lactamases (ESBLs) ISEcP1 bla 48bp CTX-M-15 ORF477 bla CTX-M is most dominant. CTX-M-15 is the major dominant enzyme type in the UK, and is reported worldwide P 48bp bla CTX-M is carried on several plasmids. Genetic context can vary and is important in understanding the dissemination of bla CTX-M IS26 24bp ISEcP1 remnant bla CTX-M-15 ORF477

20 WWTP effluent acts as an input and or/selects for mobile antibiotic resistance determinants 3 sediment cores were taken at 3 x 500m intervals below (DS) and above (US) Finham sewage works on the River Sowe Samples taken a year apart in late 2009 and early 2011 Cultivation on Chromocult and PCR screening 3 rd generation cephalosporin (3GC) resistance gene abundance and diversity

21 Resistance prevalence / (%) * Flow of resistance genes into the rivers: Waste Water treatment plants Resistance Quotients Coliforms * DS US Downstream and upstream of WWTP 2009 and 2011 * P< * * * * * Antibiotic selection 4.48 x 10 5 coliforms / g DS 2.07 x 10 5 coliforms / g US Amos et al., 2013

22 Prevalence / (%) 3GC resistance gene analysis A subset of E. coli and other Enterobacteriaceae were taken from 2011 samples for further analysis 708 bla CTX-M carrying presumptive coliforms / g DS bla CTX-M carrying presumptive coliforms / g US DS US Sequencing of bla CTX-M revealed all belonged to the genotype bla CTX-M-15 Amos et al., CTX-M TEM SHV inti1 Gene

23 bla CTX-M genetic context Prevalence bla CTX-M was carried on 13 genetic contexts, including the international genetic context P Eleven were novel, 8 were found DS, 3 were found US and 2 were found DS and US, simultaneously 60 ISECP1 bla 48bp CTX-M-15 ORF Different genetic contexts were carried on different plasmids and one genetic context could be seen on multiple plasmids Amos et al., FIA FIB FIIA HI2 A/C I1/IY IncK Plasmid rep type DS US

24 Mobilisation of bla CTX-M bp spacer 256 bp, ISeCP1 IR CTX-M-15 promoter, spacer CTX-M bp ORF bp IS26 tnpa 716 bp ISeCP1 tnpa 181 bp CTX-M bp One DS E. coli carrying FIA, one DS E. coli carrying HI 2. One US E. coli carrying FIB and HI 2. One DS C. Freundii carrying FIB + K and one DS C. Freundii carrying FIB and I1/IY 124 bp spacer Is26 IRL 80 bp, IS26 IR CTX-M-15 promoter, spacer CTX-M bp IS26 tnpa 716 bp CTX-M bp CTX-M-15 is carried throughout a wide range of genetic contexts and plasmids Contexts were seen in human pathogens, including several novel genetic contexts The environment may mobilise CTX-M-15 between plasmids and species and WWTP effluent may drive this process Amos et al., 2013

25 Introduction or selection or both? More detailed typing of the E. coli (MLST) was used to determine if E. coli were of the same origin upstream and downstream Upstream the E. coli sequence types were mainly uncharacterized (80 %), indicative of a more environmental origin Downstream the sequences types were split between the clinically familiar ST131, ST167, ST3103, ST1421 and environmental STs

26 Prevalence / (%) 4 Integron prevalence based on real time PCR data 3,5 3 2,5 2 1,5 1 inti1 qace 1 qace qach 0,5 0 DS1 DS2 DS3 US1 US2 US3 Sample site Down stream of WWTP Upstream of WWTP

27 Contribution of WWTP effluent to integron levels in a whole river system River Thames catchment area: Collaboration with Wallingford CEH, meta-data available 13 sites samples every 3 months for a year: analysed for integron prevalence and 3GC resistance counts

28 Integron Prevalence / (%) Integron prevalence 3,5 3 2,5 2 Significant difference between summer months (May and August, and Winter months November and February P = t-test May August February 1,5 November 1 0,5 0 Sample site

29 Evaluating the impact of WWTPs model development

30 Actual log integron prevalence Output WWTP only Predicted log integron prevalence Explained 49 % of variance: R 2 adjusted (0.49) P < 0.01

31 actual log integron prevalence All metadata included Explained % of variance : R 2 adj (0.83) P < WWTP: Main positive driver 0.0 Coniferous Woodland -ve:rainfall Neutral Grassland: Rainfall Rough Grassland - ve Acid Grassland : Season Heather Grassland - ve: Season Inland rock : Season Urban : Season Suburban : Season Predicted log integron prevalence

32 Model validated using 6 samples from independent river basin : Could predict integron prevalence within a 20 % range of actual integron value

33 The connectivity of potential sources of antibiotic-resistant bacteria

34 Acknowledgements University of Warwick Dr William Gaze Dr Greg Amos Dr Andrew Mead Dr Lihong Zhang Dr Leonides Calvo-Bado Helen Green Abigail Carter Shruthi Sankaranaryanan University of Birmingham Professor Peter Hawkey Claire Murray University of York Professor Alistair Boxall

35 CONCLUSIONS Both resistance genes and integrons recovered in bacteria indigenous to soil and water environments Enteric bacteria survive in soil and water, can transfer genes to both G+ and G- indigenous bacteria Integrons and ESBLs present in environmental metagenomes Pollutants, sewage, WWTP effluent associated with increased resistance- anthropogenic effects QUESTIONS What are the risks of environmental exposure? Which mitigation strategies are possible? How can efficiency of interventions be measured?