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The environmental resistome

Dr Fiona Walsh

Where does AR originate?

Dr Fiona Walsh/Biology Department

Recent years

Antibiotics in medicine

Antibiotic resistance research

Billions ofyears ago

Bacteria live in the environment together producing andresisting antibiotics, bioactive compounds and toxins

Bacteria emergefrom premordialsoup

Concepts to date relating to environment


Soil = Reservoir of antibiotic resistance

Manure increases the abundance and diversity of resistance genes

Faecal pollution of water causes antibiotic resistance pollution of water (Joseph Kelly image of Lough Mask

Antibiotic resistance researchKnowledge


Human105000 papers

Animal24000 papers

Food8000 papers

Water3700 papers

Soil1400 papers

Wild animal1400 papers

An approximate overview of the number of papers published

Treatment options

Antibiotic resistance outside pathogens

Antibiotic resistance in pathogens

Large proportion bacteria do not grow

Molecular biology tools especially next generation sequencing enabled the analysis of total populations

Antibiotic resistance research

Tools for investigating resistance in the environment

Functional metagenomics Metagenomics

qPCR

PCR

Culture

Epicentre

IMGM Laboratories

Whole genome sequencing

ProteomicsRT: 0.97 - 130.01

10 20 30 40 50 60 70 80 90 100 110 120 130Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

46.68

78.0971.7574.81 78.22

105.07

105.3336.90 50.13

39.46

100.0243.53

53.32 59.3395.77

36.02 129.3953.74 59.66

63.61 113.9179.6155.02 88.9870.02 113.78

112.7291.3267.42

83.17107.59

33.14 129.1232.80 84.88

32.57

29.50 124.41114.5027.89117.4626.8422.03

18.001.90 15.777.42

NL:3.83E9Base Peak MS N2_2

Amplicon sequencing

SoilSource of Antibiotic Resistance


Antibiotics sources: Soil microorganisms

Antibiotics resistance sources: Soil microorganisms?

Erythrobacter litoralis blaNDM

Kluyvera species blaCTX‐M

Clinically identified resistance genes

Zheng et al., 2011, Oliver et al., 2001

Identifying antibiotic resistance in the environment: Culture


Soil Suspension(125 g/5 ml)

Deep‐well culture plates20 mg/L antibiotic in LB

7 days

Plated onto antibiotic media

All representative types tested for MDR against all 23 antibiotics

Growing resistant bacteria

10 different soils (Switzerland)

Mountains, urban, farms, orchard, lakeside

Fiona Walsh/Dept of Biology

0

10

20

30

40

50

60

70

80

90

100

Penicillin

Dicloxacillin

Cefotaxime

Ceph

alexin

Cfx+Clavulanic acid

Vancom

ycin

Amikacin

Streptom

ycin

Kanamycin

Gen

tamicin

Sisomicin

Levoflo

xacin

Ciprofloxacin

Naladixic Acid

Chloramph

enicol

D‐cycloserine

Novob

iocin

Trim

etho

prim

Tetracycline

Sulfametho

xazole

Sulfisoxazole

Erythrom

ycin

Colistin

Rifampicin

% Resistance

Antibiotics coloured by class

Selected Resistome and % Mediated by Efflux

Soil contains resistant bacteria

But are the resistance mechanisms the same as in human pathogens?

Mobile resistance genes:

• Extended spectrum beta‐lactamasesblaTEM, blaSHV, blaOXA, blaCTX‐M Groups 1, 2, 8, 9 and 25, blaVEB, blaPER, blaGES, blaNDM

• Plasmid mediated quinolone resistance genesaac(6’)Ib‐cr, qnrA, qnrB, qnrS, qep

None present


Relevance of novel environmental resistances

Will these resistance mechanisms ever transfer to human pathogens?

Mobility – plasmids transfer AR genes between bacteriaStability – can they survive under stressful conditions?

• Do they maintain the resistance phenotype in pathogens?

• Will they ever emerge from the soil to cause clinical problems?

blaNDM, blaCTX‐M, blaSHV, qnr…….

Environment as sink of antibiotic resistance

How do our activities influence antibiotic resistance in the environment?


Human waste


www.waterworld.com

Human bacteria

WWTP effluent across EUStudying the effluents as sources of antibiotics, AR genes and bacteria

Detected across EUResistant bacteriaMobile resistance genesAntibiotics at varying concentrations

WWTP effluents are sources of antibiotics, AR genes and bacteria – no regulations

‐4.00 ‐2.00 0.00 2.00 4.00 6.00

PT1PT1PT2PT2DE1DE1DE2DE2CY1CY1CY2CY2ES1ES1NO1NO1IE1IE1IE2IE2

Mean log mFC faecal coliforms

mFC

‐2.00 0.00 2.00 4.00 6.00


Mean log mFC + AML faecal coliforms

mFC + AML mFC

‐2.00 0.00 2.00 4.00


Mean log mFC + TET faecal coliforms

mFC + TET mFC

‐2.00 0.00 2.00 4.00 6.00


Mean log mFC +CIP faecal coliforms

mFC + CIP mFC

Total and amoxicillin, tetracycline and ciprofloxacinnon‐susceptible/resistant coliforms

Antibiotic concentrations

Antibiotics identified across EU partner countries:

Antibiotic class Antibiotic

Cephalosporins Cefalexin

Trimethoprim Trimethoprim

Quinolones/Fluoroquinolones Nalidixic acid, Ciprofloxacin, Ofloxacin (enrofloxacin)

Lincosamides Clindamycin

Macrolides Azithromycin, clarithromycin

Nitromidazole Methronidazole

Penicillins Ampicillin

Sulfonamides Sulfamethoxazole, sulfapyridine

Tetracyclines Tetracycline

Selected antibiotic resistance genes detected in relatively high abundances (qPCR)

Gene Resistance

blaOXA, cfxA Beta‐lactams e.g. penicillin

Int1 Mobility element

qacA Quaternary ammonium compounds (disinfectants)

sul1 Sulphonamides

tetQ, tetC Tetracyclines

aadA, strB Aminoglycosides – Spectinomycin, Streptomycin

ermF Macrolides e.g. erythromycin

Waste & Water

Regulations for water quality:Chemicals (toxic chemicals)Indicator bacteria (coliforms, enterococci)

No regulations for WWTP discharge into water or water quality :Antibiotic concentrationsAntibiotic resistant bacteriaAntibiotic resistance genes


Plasmid mediated resistance

• Currently characterising >30 plasmids

• Both exogenous extraction from samples directly and from isolated bacteria

• MDR and all resistance phenotypes identified on plasmids

Conclusions

• If environment is a major source of novel/known AR mechanisms we need to identify them and their methods of transmission or barriers to their transfer• Then can identify risks to animal/human/environmental health

• We need to measure the impact of our activities, climate change and changing environments on the environmental resistome in order to measure changing risks to animal/human/environmental health

AcknowledgementsSwiss Federal Department of Agriculture

EU Leonardo stiftung

EPA through Water JPI project StARE (WATER/JPI/0001/2013)

COST Actions:

TD0803 DARE, ES1403 NEREUS

the environmental resistome - bsac.org.uk · all representative types tested for ... studying the...

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