Antimicrobial resistance: a global problem

Antimicrobial resistance: a global problem

The connectivity of potential sources of antibioticresistant bacteria Wellington et al. Lancet Infect Dis 13, 155-65 2013

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

Antibiotic class Occurrence of antibiotics in the natural environment, fish, crops and drinking water from published studies Chloramphenicol 2,4- diaminopyridines General behaviour impersistent/ 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)

Reservoirs of antibiotic resistance genes in diverse EU environments aph3 aph6-id ant3 adenylase aph6-ic aph6-ic (deg) stra aphd strb1 stsc Non-producers Streptomycin Producers Streptomycin Soil Rhizosphere Manure Sewage Seawater 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 Non-producers Gentamicin Non-producers Tetracycline Soil Rhizosphere Manure Sewage Seawater

Pig slurry applied to land- tylosin fed pigs RQ values for sulphachloropyridazine over year 1 14 Resistance Quotient (%) 12 10 8 6 4 2 0 pig slurry pre-app year1 day1 year1 day21 year1 day90 year1 day289 50 25 μg/ml ul/ml

Class 1 integron prevalence following pig slurry application 0.016 0.014 0.012 prevalence % 0.01 0.008 0.006 0.004 0.002 0 preapplication day 1 day 21 day 90 day 289 days after slurry application

Schematic diagram of class 1 integron from Arthrobacter aritaii (strain C361), carried on a transferable plasmid P2 Transfer 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

Main conclusion for pig slurry application Resistance seen over two year study period may be due to; 1. Survival of enteric bacteria 2. Transfer of resistance to the indigenous population- integrons Pig slurry contains many highly resistant bacteria. Resistance to SCP observed up to 50μg ml -1 at year 1 day 289, despite lack of selective pressure, SCP conc. decreased rapidly in soil Resistance seen is patchy-maybe due to characteristics of clay soil Resistance to tylosin constant throughout both years A1 Byrne-Bailey et al. 2009. Appl Environ Microbiol. 77, 684-7. Bailey-Byrne et al., 2011. Antimicrob.Agents Chemotherap. 53, 696-702.

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Cattle and β-lactam resistance Garcia-Alvarez et al. 2011 Lancet Infect Dis 11, 595 603 Meticillinresistant Staphylococcus aureus with a novel meca homologue in human and bovine populations in the UK and Denmark: a descriptive study S. aureus (LGA251)

Staphylococcal communities in milk Oxacillin-resistant staphylococcal species in organic and conventional farms 16 14 Number of farms 12 10 8 6 4 2 Conventional Organic 0 S. aureus S. caseolyticus S. chromogenes S. epidermidis S. equorum S. fleuretti S. haemolyticus S. lentus S. pasteuri S. saprophyticus S. sciuri S. succinus S. vitulinus S, xylosus Species 47% of conventional farms and 28% of organic farms harboured meticillin resistant staphylococci Conventional farms: S. sciuri > S. epidermidis > S. aureus Organic farms: S. fleuretti was the most common species, with S. aureus and S. epidermidis being relatively rare O Neill et al., 2013

Percentage of isolates resistant 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Resistance to other clinically important antibiotics in oxacillin-resistant Resistance to other clinically important antibiotics in oxacillin-resistant staphylococci Ciprofloxacin Clindamycin Erythromycin Fusidic acid staphylococci O Neill et al., 2013 Gentamycin Linezolid Mupirocin Antibiotic Rifampicin Tetracycline Tobramycin Trimethoprim Vancomycin Isolates from conventional farms (n=70) Isolates from organic farms (n=27) S. epidermidis in conventional farms reservoir of resistance and increased pathogenic genotypes e.g. ST2 nosocomial strain

A Multidrug-Resistant Staphylococcus epidermidis Clone (ST2) Is an Ongoing Cause of Hospital-Acquired Infection in a Western Australian Hospital Micael Widerström,Cheryll A. McCullough,Geoffrey W. Coombs,Tor Monsen,and Keryn J. Christiansen Department of Clinical Bacteriology, Umeå University, Umeå, Sweden,a and Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine, Royal Perth Hospital, Western Australia, Australiab Report the molecular epidemiology of 27 clinical multidrug-resistant Staphylococcus epidermidis (MDRSE) isolates collected between 2003 and 2007 in an Australian teaching hospital. The dominant genotype (sequence type 2 [ST2]) accounted for 85% of the isolates tested and was indistinguishable from an MDRSE genotype identified in European hospitals, which may indicate that highly adaptable health care-associated genotypes of S. epidermidis have emerged and disseminated worldwide in the health care setting. J Clin Microbiol 50, 2147-2151 2012

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

Flow chart of metagenomic approaches DNA extracted from samples by soil plug methods Gel-Fractionate 4-10 kb fragments End-repair to make DNA blunt-ended Analyse efficiency and estimate the coverage Amplified and store the libraries Transform E coli competent cell Screen for new phenotype Ligate sample DNA with blunt ended/dephos plasmid pcf430 Transposon mutagenesis and sequencing Culture independent focus on 3 GCs resistance Gaze et al., 2013

Resistance to β-lactam antibiotics Reed Bed soil- QAC polluted Sewage Cake 1 Month Cake Applied Control Soil Grass Land Soil FYM Applied Grass Soil No. of clones 400000 386000 500000 170000 630000 210000 Average insert size (Kb) 4.64 4.12 4.40 3.70 2.85 3.71 Clones with inserts (%) 65 65 85 50 75 85 Coverage (Gb) 0.63 1.59 1.87 0.32 1.53 1.47 No of cefotaxime resistance No of ceftazidime resistance No of imipenem resistance No of amp res clones 0 2 1 0 0 0 2 1 0 0 0 0 1? 2? 0 0 0 0 4? 5? 1? 0 0 0 Hit rate 1/80 1/150 1/900 0 0 0

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)

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 bla CTX-M-15 24bp ISEcP1 remnant ORF477

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) sewage works on the River 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

25 20 * Flow of resistance genes into the rivers: Waste Water treatment plants Resistance Quotients Coliforms Resistance prevalence / (%) 15 10 5 0 * * * DS US Downstream and upstream of WWTP 2009 and 2011 * P<0.05 * * * Antibiotic selection 4.48 x 10 5 coliforms / g DS 2.07 x 10 5 coliforms / g US Amos et al., 2013

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 100 90 80 141 bla CTX-M carrying presumptive coliforms / g US Prevalence / (%) 70 60 50 40 30 DS US Sequencing of bla CTX-M revealed all belonged to the genotype bla CTX-M-15 0 20 10 Amos et al., 2013 CTX-M TEM SHV inti1 Gene

bla CTX-M genetic context bla CTX-M was carried on 13 genetic contexts, including the international genetic context P ISECP1 bla 48bp CTX-M-15 ORF477 Eleven were novel, 8 were found DS, 3 were found US and 2 were found DS and US, simultaneously 60 50 Different genetic contexts were carried on different plasmids and one genetic context could be seen on multiple plasmids Amos et al., 2013 Prevalence 40 30 20 10 0 FIA FIB FIIA HI2 A/C I1/IY IncK Plasmid rep type DS US

Mobilisation of bla CTX-M-15 bla CTX-M-15 ORF477 IS26 IS26 bla CTX-M-15 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 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

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 pathogenic ST131, ST167, ST3103, ST1421

4 Integron prevalence based on real time PCR data 3.5 Prevalence / (%) 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

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 Amos et al., 2013

Integron prevalence Integron Prevalence / (%) 3.5 3 2.5 2 1.5 Significant difference between summer months (May and August, and Winter months November and February P = 0.004 t-test May August November February 1 0.5 0 Sample site

CONCLUSIONS Gut microflora survive in soil and water, can transfer genes to both G+ and G- indigenous bacteria Integrons and ESBLs present in environmental metagenome associated with farming activities Slurry, manure, pollutants, sewage, WWTP effluent associated with increased resistance- anthropogenic effects

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