Antimicrobial resistance: a global problem The connectivity of - - PowerPoint PPT Presentation

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

Antibiotic class General behaviour Sewage sludge River water Groundwat er Drinking water Fish Soil Crops Example compounds monitored

Chloramphenicol

impersistent/ mobile

• 

X

• 2,4-

diaminopyridines

persistent/ immobile

  X X

• 

 trimethoprim

Fluoroquinolones

persistent/ immobile

  X X

• 
• ciprofloxacin, norfloxacin,
• floxacin

-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

Occurrence of antibiotics in the natural environment, fish, crops and drinking water from published studies

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)

Non-producers Streptomycin Non-producers Gentamicin Non-producers Tetracycline

aac(3)-I aac(3)-II/VI aac(3)-III/IV aac(6’)-II/Ib ant(2”)-I aph(2”)-I aph3 aph6-Id ant3 adenylase aph6-Ic aph6-Ic (deg) tetA tetB tetC tetD tetE tetG tetH tetK tetL tetM tetO tetT

Producers Streptomycin

strA aphD strB1 stsC Soil Rhizosphere Manure Sewage Seawater

Soil Rhizosphere Manure Sewage Seawater

Reservoirs of antibiotic resistance genes in diverse EU environments

pig slurry pre-app year1 day1 year1 day21 year1 day90 year1 day289

25 50 2 4 6 8 10 12 14

Resistance Quotient (%) ul/ml

RQ values for sulphachloropyridazine over year 1

μg/ml

Pig slurry applied to land- tylosin fed pigs

0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016

pre- application day 1 day 21 day 90 day 289

days after slurry application prevalence %

Class 1 integron prevalence following pig slurry application

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 intI1 500bp P2 5’conserved region/attI1 aadA9 750bp 59bp element qacEΔ1 sulI 3’conserve d region 450bp 6000bp

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

Transfer

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
• f 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

Byrne-Bailey et al. 2009. Appl Environ Microbiol. 77, 684-7. Bailey-Byrne et al., 2011. Antimicrob.Agents Chemotherap. 53, 696-702.

A1

Slide 9 A1

Administrator, 09/12/2004

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

Cattle and β-lactam resistance

• S. aureus (LGA251)

Staphylococcal communities in milk

• 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

Oxacillin-resistant staphylococcal species in organic and conventional farms

2 4 6 8 10 12 14 16 S . a u r e u s S . c a s e

• l

y t i c u s S . c h r

• m
• g

e n e s S . e p i d e r m i d i s S . e q u

• r

u m S . f l e u r e t t i S . h a e m

• l

y t i c u s S . l e n t u s S . p a s t e u r i S . s a p r

• p

h y t i c u s S . s c i u r i S . s u c c i n u s S . v i t u l i n u s S , x y l

• s

u s Species Number of farms Conventional Organic

Resistance to other clinically important antibiotics in oxacillin-resistant staphylococci

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% C i p r

• f

l

• x

a c i n C l i n d a m y c i n E r y t h r

• m

y c i n F u s i d i c a c i d G e n t a m y c i n L i n e z

• l

i d M u p i r

• c

i n R i f a m p i c i n T e t r a c y c l i n e T

• b

r a m y c i n T r i m e t h

• p

r i m V a n c

• m

y c i n Antibiotic Percentage of isolates resistant 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

Resistance to other clinically important antibiotics in oxacillin-resistant staphylococci

O’Neill et al., 2013

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%

• f 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.

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

J Clin Microbiol 50, 2147-2151 2012

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

Sewage treatment and disposal

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 Ligate sample DNA with blunt ended/dephos plasmid pCF430 Transform E coli competent cell Analyse efficiency and estimate the coverage Amplified and store the libraries Screen for new phenotype 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

2 1

No of ceftazidime resistance

2 1

No of imipenem resistance

1 ? 2 ?

No of amp res clones

4 ? 5? 1 ?

Hit rate

1/80 1/150 1/900

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

• f 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 blaCTX-M prevalence

3rd generation cephalosporin (3GC) resistance necessitates the use of last resort antibiotics Extended Spectrum β-lactamases (ESBLs) blaCTX-M is most dominant. CTX-M-15 is the major dominant enzyme type in the UK, and is reported worldwide blaCTX-M is carried on several plasmids. Genetic context can vary and is important in understanding the dissemination of blaCTX-M

P ISEcP1 blaCTX-M-15 ORF477 48bp P 24bp ISEcP1 remnant blaCTX-M-15 ORF477 48bp IS26

• 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 3rd generation cephalosporin (3GC) resistance gene abundance and diversity

WWTP effluent acts as an input and or/selects for mobile antibiotic resistance determinants

Resistance Quotients

Coliforms

4.48 x 105 coliforms / g DS 2.07 x 105 coliforms / g US

* P<0.05

5 10 15 20 25

Resistance prevalence / (%)

Antibiotic selection DS US

Downstream and upstream of WWTP 2009 and 2011

* * * * * * *

Flow of resistance genes into the rivers: Waste Water treatment plants

Amos et al., 2013

3GC resistance gene analysis

A subset of E. coli and other Enterobacteriaceae were taken from 2011 samples for further analysis Sequencing of blaCTX-M revealed all belonged to the genotype blaCTX-M-15

10 20 30 40 50 60 70 80 90 100 CTX-M TEM SHV intI1

Prevalence / (%) Gene

DS US

708 blaCTX-M carrying presumptive coliforms / g DS 141 blaCTX-M carrying presumptive coliforms / g US

Amos et al., 2013

blaCTX-M genetic context

P ISECP1 blaCTX-M-15 ORF477 48bp

blaCTX-M was carried on 13 genetic contexts, including the ‘international genetic context’ Eleven were novel, 8 were found DS, 3 were found US and 2 were found DS and US, simultaneously Different genetic contexts were carried on different plasmids and one genetic context could be seen on multiple plasmids

10 20 30 40 50 60 FIA FIB FIIA HI2 A/C I1/IY IncK Prevalence Plasmid rep type DS US

Amos et al., 2013

Mobilisation of blaCTX-M-15

blaCTX-M-15 ORF477 IS26 IS26 blaCTX-M-15 One DS E. coli carrying FIA, one DS E. coli carrying HI2. One US E. coli carrying FIB and HI2. 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

• rigin 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

0.5 1 1.5 2 2.5 3 3.5 4 DS1 DS2 DS3 US1 US2 US3

Prevalence / (%) Sample site

intI1 qacE∆1 qacE qacH

Integron prevalence based on real time PCR data

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

0.5 1 1.5 2 2.5 3 3.5 Integron Prevalence / (%) Sample site

May August November February

Significant difference between summer months (May and August, and Winter months November and February P = 0.004 t-test

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