Environmental reservoirs of antibiotic resistance genes

(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

Hospital Soil People Water Food Clinical antibiotics Antibiotics produced by soil bacteria Veterinary antibiotics Farm animals Pollutants Disinfectants Surfactants

(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)

N o n -p ro d u cers St rep to m y ci n N o n -p ro d u cers G en tam ici n N o n -p ro d u cers T et racy cl in e 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 Pro d u cers St rep to m y ci n strA aphD strB1 stsC

Soil Rhizosphere Manure Sewage Seawater

Reservoirs of antibiotic resistance genes in diverse environments: RESERVOIR survey

(7)

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, 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

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)

(8)

Application of sewage sludge to land:

what is the impact on antibiotic resistance soil?

(9)

(10)

Schematic map of the complex class 1

integron carrying the

bla

_CTX-M-14

gene on

plasmid pAJE0508

gene on plasmid pAJE0508

(11)

Sampling polluted environments: Diagram of mill and

reed bed system

Reed bed system for mill effluent remediation oceans-ESU Ltd

(12)

CTAB cetyltrimethylammonium chloride

DTDMAC ditallowdimethylammonium chloride

Relative resistance to DTDMAC and CTAB at 50

µg/ml

(13)

Incidence of int11, qaE, and qacEΔ : Reed bed

and river sediment

(14)

Integron and qac gene prevalence: comparison of environmental samples 0 1 2 3 4 5 6 7 8 9 RB SS PS CW sample site pr e v a le nc e ( % ) intI1 qacE∆1 qacE qacG qacH

Gaze et al., 2011. Impacts of anthropogenic activity on the ecology of class

1 integrons and integron-associated genes in the environment. ISME Journal.

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

• 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

(15)

• 90 million tons animal faecal slurry added to UK soils per year

Bailey-Byrne K, Gaze WH, Zhang L, Hawkey P, Wellington EMH. 2011. Class 1 integron and class 2 integron diversity in agricultural soil amended with pig slurry. AEM, Jan;77(2):684-7.

class 1 integron prevalence following pig manure application to land

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

p re v a le n c e %

(16)

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), putatively carried on

a transferable plasmid

Byrne-Bailey et al. 2009. Appl Environ Microbiol. 77, 684-7.

(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 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 0 2 1 0 0 0 No of ceftazidime resistance 2 1 0 0 0 0 No of imipenem resistance 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

(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

3rd_{generation cephalosporin (3GC) resistance}

necessitates the use of last resort antibiotics Extended Spectrum β-lactamases (ESBLs)

bla_CTX-M is most dominant. CTX-M-15 is the major

dominant enzyme type in the UK, and is reported worldwide

bla_CTX-M is carried on several plasmids.

Genetic context can vary and is important in understanding the dissemination of bla_CTX-M

P

ISEcP1 _48bp blaCTX-M-15 _ORF477

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

(20)

•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 3rd_{generation cephalosporin}

(3GC) resistance gene abundance and diversity

WWTP effluent acts as an input and or/selects for

mobile antibiotic resistance determinants

(21)

Resistance Quotients Coliforms 4.48 x 105 _{coliforms / g DS 2.07 x 10}5 _{coliforms / g US} * P<0.05 0 5 10 15 20 25 Re sis tance p re valence / (% ) Antibiotic selection DS

US Downstream and _{upstream of WWTP 2009}

and 2011 * * * * * _* _*

Flow of resistance genes into the rivers:

Waste Water treatment plants

(22)

3GC resistance gene analysis

A subset of E. coli and other Enterobacteriaceae were taken from 2011 samples for further analysis

Sequencing of bla_CTX-M

revealed all belonged to the genotype bla_CTX-M-15 0 10 20 30 40 50 60 70 80 90 100 CTX-M TEM SHV intI1 P re val ence / (% ) Gene DS US 708 bla_CTX-M carrying presumptive coliforms / g DS 141 bla_CTX-M carrying presumptive coliforms / g US Amos et al., 2013

(23)

bla

_CTX-M

genetic context

P

ISECP1 _48bp blaCTX-M-15 _ORF477

bla_CTX-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

P

re

v

a

lence

Plasmid rep type

DS US

(24)

Mobilisation of

bla

_CTX-M-15

One DS E. coli carrying FIA, one DS E. coli carrying HI₂. 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 IS26 tnpa 716 bp CTX-M-15 875 bp ORF47 7 151 bp 47 bp spacer ISeCP1 tnpa 181 bp 256 bp, ISeCP1 IR CTX-M-15 promoter, spacer CTX-M-15 875bp IS26 tnpa 716 bp

124 bp spacer Is26 IRL 80 bp, IS26 IR CTX-M-15 promoter, spacer

CTX-M-15 875 bp

(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

(26)

0 0,5 1 1,5 2 2,5 3 3,5 4

DS1 DS2 DS3 US1 US2 US3

Pr eva le nce / (% ) Sample site intI1 qacE∆1 qacE qacH

Integron prevalence based on real time PCR

data

(27)

Collaboration with Wallingford CEH, meta-data available

13 sites samples every 3 months for a year: analysed for integron prevalence and 3GC resistance counts

Contribution of WWTP effluent to integron levels

in a whole river system

(28)

Integron prevalence

0 0,5 1 1,5 2 2,5 3 3,5 Integ ro n P rev a lence / ( % ) Sample site May August February

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

(29)

Evaluating the impact of WWTPs –

model development

(30)

Output WWTP only

Explained 49 % of variance: R2_adjusted_(0.49)_{P < 0.01}

0.5 1.5 2.5 -2.0 0.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 2.0 Ac tual lo g in tegr on pr ev ale nce

(31)

Explained 82. 9 % of variance :  R2_{adj (0.83)}

P < 0.01

WWTP: Main positive driver

Coniferous Woodland -ve:Rainfall Neutral Grassland: Rainfall

Rough Grassland - ve Acid Grassland : Season

Heather Grassland - ve: Season Inland rock : Season

Urban : Season Suburban : Season -1.5 -0.5 0.5 -2.0 -2.0 -1.5 -1.0 -0.5 0.0 0.5 -1.0 0.0 ac tu al log i n teg ron p re valenc e

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

(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