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

Groundwater

Drinking water

Fish Soil Crops Example compoundsmonitored

Chloramphenicolimpersistent/

mobile- X - - - - -

2,4-diaminopyridines

persistent/

immobile X X - trimethoprim

Fluoroquinolonespersistent/

immobile X X - -

ciprofloxacin, norfloxacin, ofloxacin

-lactamsimpersistent

mobile- X X X - - -

amoxicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V

Macrolidesslightly persistent/

slightly mobile

X - - - -

azithromycin, clarithromycin, lincomycin, roxithromycin, spyramycin, tylosin

Sulfonamidespersistent/

mobile X -

sulfamethoxazole, sulfadiazine, sulfamerazine, sulfamethazine, sulfapyridine

Tetracyclinespersistent/

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

-pro

duce

rs

Stre

ptom

ycin

Non

-pro

duce

rsG

enta

mic

inN

on-p

rodu

cers

Tetra

cycl

ine

aac(3)-Iaac(3)-II/VI

aac(3)-III/IVaac(6’)-II/Ib

ant(2”)-Iaph(2”)-I

aph3aph6-Id

ant3adenylase

aph6-Icaph6-Ic (deg)

tetAtetBtetCtetDtetEtetGtetHtetKtetL

tetMtetOtetT

Prod

ucer

sSt

rept

omyc

in

strAaphDstrB1stsC

Soil Rhizosphere Manure Sewage Seawater

Soil Rhizosphere Manure Sewage SeawaterReservoirs of antibiotic resistance genes in diverse EU environments

pigslurry

pre-app year1day1

year1day21

year1day90

year1day289

2550

0

2

4

6

8

10

12

14

Res

ista

nce

Quo

tient

(%)

ul/ml

RQ values for sulphachloropyridazine over year 1

μg/ml

Pig slurry applied to land- tylosin fed pigs

0

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

prev

alen

ce %

Class 1 integron prevalence following

pig slurry application

attI1 integrase binding site

aadA9 streptomycin/spectinomycin resistance gene, 99% blast homology with aadA9 from Corynebacterium glutamicumqacEΔ1 quaternary ammonium compound resistance gene, 98% blast homology with E.colisulI sulphonamide resistance gene, 98% blast homology withSalmonella enterica

intI1

500bp

P2

5’conserved region/attI1

aadA9

750bp

59bp element

qacEΔ1 sulI3’conserved region

450bp 6000bp

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

transferable plasmidTransfer

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

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 mecAhomologue in human and bovine populations in the UK andDenmark: 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

02468

10121416

S. aure

usS. c

aseolyt

icus

S. chrom

ogen

esS. e

piderm

idis

S. equ

orum

S. fleure

ttiS. h

aemoly

ticus

S. lentu

sS. p

asteu

riS. s

aproph

yticu

sS. s

ciuri

S. succ

inus

S. vitu

linus

S, xylo

sus

Species

Num

ber o

f far

ms

ConventionalOrganic

Resistance to other clinically important antibiotics in oxacillin-resistant staphylococci

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Ciproflox

acinClin

damyc

inEryt

hromyc

inFus

idic a

cidGen

tamycin

Linezo

lidMup

irocin

Rifampic

inTetr

acyc

line

Tobram

ycin

Trimeth

oprim

Vanco

mycin

Antibiotic

Perc

enta

ge o

f iso

late

s re

sist

ant

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% 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. epidermidishave emerged and disseminated worldwide in the health care setting.

A Multidrug-Resistant Staphylococcus epidermidis Clone (ST2) Is anOngoing Cause of Hospital-Acquired Infection in a WesternAustralian HospitalMicael Widerström,Cheryll A. McCullough,Geoffrey W. Coombs,Tor Monsen,and Keryn J. ChristiansenDepartment 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

Transposonmutagenesis and sequencing

Culture independent focus on 3 GCs resistanceGaze et al., 2013

Resistance to β-lactam antibioticsReed Bedsoil- QACpolluted

Sewage Cake

1 Month Cake Applied

Control Soil

GrassLand Soil

FYMApplied 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 cefotaximeresistance

0 2 1 0 0 0

No of ceftazidimeresistance

2 1 0 0 0 0

No of imipenemresistance

1 ? 2 ? 0 0 0 0

No of amp res clones

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 plantsHotspot 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 blaCTX-M prevalence3rd 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 ORF47748bp

P

24bp ISEcP1remnant

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

0

5

10

15

20

25

Res

ista

nce

prev

alen

ce /

(%)

Antibiotic selection

DSUS Downstream and

upstream of WWTP 2009and 2011

*

*

* ** * *

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

Amos et al., 2013

3GC resistance gene analysisA subset of E. coli and other Enterobacteriaceae were taken from 2011 samples for further analysis

Sequencing of blaCTX-Mrevealed all belonged to the genotype blaCTX-M-15

0

10

20

30

40

50

60

70

80

90

100

CTX-M TEM SHV intI1

Prev

alen

ce /

(%)

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 contextP

ISECP1 blaCTX-M-15 ORF47748bp

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

0

10

20

30

40

50

60

FIA FIB FIIA HI2 A/C I1/IY IncK

Prev

alen

ce

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 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

0

0.5

1

1.5

2

2.5

3

3.5

4

DS1 DS2 DS3 US1 US2 US3

Prev

alen

ce /

(%)

Sample site

intI1qacE∆1qacEqacH

Integron prevalence based on real time PCR data

Down stream of WWTP Upstream of WWTP

Contribution of WWTP effluent to integronlevels in a whole river system

River Thames catchment area:

Collaboration with Wallingford CEH, meta-data available13 sites samples every 3 months for a year: analysed for integron prevalence and 3GC resistance counts Amos et al., 2013

Integron prevalence

0

0.5

1

1.5

2

2.5

3

3.5In

tegr

on P

reva

lenc

e / (

%)

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 WarwickDr William Gaze Dr Greg AmosDr Andrew MeadDr Lihong ZhangDr Colette O’NeillDr Leonides Calvo-BadoHelen GreenAbigail CarterShruthi Sankaranaryanan

University of BirminghamProfessor Peter HawkeyClaire Murray

University of YorkProfessor Alistair Boxall

Antibiotic Action