the environmental resistome - bsac.org.uk · all representative types tested for ... studying the...
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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
Dr Fiona Walsh/Biology Department
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
Dr Fiona Walsh/Biology Department
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
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45
50
55
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65
70
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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
Dr Fiona Walsh/Biology Department
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
Dr Fiona Walsh/Biology Department
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
Dr Fiona Walsh/Biology Department
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?
Dr Fiona Walsh/Biology Department
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
PT1PT1PT2PT2DE1DE1DE2DE2CY1CY1CY2CY2ES1ES1NO1NO1IE1IE1IE2IE2
Mean log mFC + AML faecal coliforms
mFC + AML mFC
‐2.00 0.00 2.00 4.00
PT1PT1PT2PT2DE1DE1DE2DE2CY1CY1CY2CY2ES1ES1NO1NO1IE1IE1IE2IE2
Mean log mFC + TET faecal coliforms
mFC + TET mFC
‐2.00 0.00 2.00 4.00 6.00
PT1PT1PT2PT2DE1DE1DE2DE2CY1CY1CY2CY2ES1ES1NO1NO1IE1IE1IE2IE2
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
Dr Fiona Walsh/Biology Department
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
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