Antimicrobial resistance: a global problem
The connectivity of potential sources of antibiotic- resistant 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
Occurrence of antibiotics in the natural environment, fish, crops and drinking water from published studies Antibiotic class General Sewage River Groundwat Drinking Fish Soil Crops Example compounds behaviour sludge water er water monitored impersistent/ Chloramphenicol - X - - - - - mobile persistent/ 2,4- immobile X X - trimethoprim diaminopyridines persistent/ ciprofloxacin, norfloxacin, Fluoroquinolones X X - - ofloxacin immobile amoxicillin, cloxacillin, impersistent dicloxacillin, methicillin, -lactams - X X X - - - nafcillin, oxacillin, penicillin mobile G, penicillin V slightly azithromycin, clarithromycin, lincomycin, persistent/ Macrolides X - - - - roxithromycin, spyramycin, slightly mobile tylosin persistent/ sulfamethoxazole, Sulfonamides X - sulfadiazine, sulfamerazine, mobile sulfamethazine, sulfapyridine persistent/ chlortetracycline, Tetracyclines - X X doxycycline, oxytetracycline, immobile 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 Non-producers Soil Rhizosphere Manure Sewage Seawater Streptomycin aph3 aph6-Id ant3 adenylase aph6-Ic aph6-Ic (deg) Streptomycin Producers strA aphD strB1 stsC aac(3)-I Non-producers Gentamicin aac(3)-II/VI aac(3)-III/IV aac(6’)-II/Ib ant(2”)-I aph(2”)-I tetA tetB tetC tetD Non-producers Tetracycline tetE tetG tetH tetK tetL tetM tetO tetT Soil Rhizosphere Manure Sewage Seawater
Pig slurry applied to land- tylosin fed pigs RQ values for sulphachloropyridazine over year 1 14 12 Resistance Quotient (%) 10 8 6 4 2 μ g/ml 0 50 pig ul/ml slurry pre-app 25 year1 year1 year1 day1 year1 day21 day90 day289
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 pre- day 1 day 21 day 90 day 289 application days after slurry application
Schematic diagram of class 1 integron from Arthrobacter aritaii (strain C361), carried on a transferable plasmid Transfer P2 intI1 aadA9 qacE Δ 1 sulI 5’conserved 59bp 3’conserve element d region region/ attI1 500bp 750bp 450bp 6000bp attI1 integrase binding site aadA9 streptomycin/spectinomycin resistance gene, 99% blast homology with aadA9 from Corynebacterium glutamicum qacE Δ 1 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 50 μ g ml -1 at year 1 day 289, despite lack of selective pressure, SCP conc. decreased rapidly in soil A1 Resistance seen is patchy-maybe due to characteristics of clay soil • Resistance to tylosin constant throughout both years • Byrne-Bailey et al. 2009. Appl Environ Microbiol . 77, 684-7. Bailey-Byrne et al., 2011. Antimicrob.Agents Chemotherap. 53, 696-702 .
Slide 9 Administrator, 09/12/2004 A1
Cattle and β -lactam resistance Garcia-Alvarez et al. 2011 Lancet Infect Dis 11, 595–603 Meticillin- resistant 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 Conventional 8 Organic 6 4 2 0 m s s i s s s i s i s s t s s r r u e t u u i u u u u u u u d u e n e c t e c n c r i n s i r e m n c r o u i i i i t i o t t l u t e s s c g y y u y u e l r a l a c y e l h t l o q l . o o f . S u i x p p v m d e . m S . s S e o , i S . . p s o . S S S e r . S a r e p S a h c a . h c S s . . S . S S . S 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
Resistance to other clinically important antibiotics in oxacillin-resistant Resistance to other clinically important antibiotics in oxacillin-resistant staphylococci staphylococci O’Neill et al., 2013 90% Percentage of isolates resistant 80% 70% 60% 50% 40% 30% 20% 10% 0% n n m n d n n n n e n d i i i i i i i n c i i c c i i c c l c c c r c o a i y y y o i p a y p l y z c x m m m m r o m m e y o c i h p a a n o c o l i a a d u t f d i a r o r t e c L f b i M h n i r n m n r s R t o t e p i y u e a l T C G i i F T r C r V T E Antibiotic 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 End-repair to Gel-Fractionate from samples by make DNA 4-10 kb soil plug methods blunt-ended fragments Analyse efficiency Ligate sample Transform E coli and estimate the DNA with blunt competent cell coverage ended/dephos plasmid pCF430 Transposon Amplified and Screen for new mutagenesis store the libraries phenotype and sequencing Culture independent focus on 3 GCs resistance Gaze et al., 2013
Resistance to β -lactam antibiotics Reed Bed Sewage 1 Month Control Grass FYM soil- QAC Cake Cake Soil Land Soil Applied polluted Applied Grass Soil No. of clones 400000 386000 500000 170000 630000 210000 Average insert size 4.64 4.12 4.40 3.70 2.85 3.71 (Kb) 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 0 2 1 0 0 0 resistance No of ceftazidime 2 1 0 0 0 0 resistance No of imipenem 1 ? 2 ? 0 0 0 0 resistance No of amp res 4 ? 5? 1 ? 0 0 0 clones 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) IS EcP1 bla CTX-M-15 48bp 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. IS26 bla CTX-M-15 Genetic context can vary and is important in ORF477 understanding the dissemination of bla CTX-M 24bp IS EcP1 remnant
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