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transcript
Surveillance and Risk Assessment of Antibiotic Resistance in Urban
Water Cycle
Le Thai Hoang, PhD Lecturer, Environmental Engineering
International University - Vietnam National University HCMC
ProSPER.Net Young Researchers’ School 6 to 15 March, 2017
May, 2016
Jan, 2017
Annual death by Antimicrobial resistance (AMR)
Tetanus 60,000
Car traffic accidences 1,200,000
Cholera 100,000-120,000
Diarrhea diseases 1,300,000
Measles 130,000
Diabetes 1,500,000
AMR 700,000
AMR in 2050 10,000,000
Cancers 8,200,000
WHO. Review on Antimicrobial Resistance. 2014
Timeline of antibiotic resistance
1930 1935 1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005
Sulfonamides Penicillin
Streptomycin chloramphenicol
Tetracycline
Erythromycin
Vancomycin
Methicillin
Ampicillin Cephalosporins
Linezolid Daptomycin
Antibiotic deployment
Sulfonamides Penicillin Streptomycin
Tetracycline
Erythromycin
Vancomycin
Methicillin
Chloramphenicol Ampicillin
Cephalosporins
Linezolid
Daptomycin
Antibiotic resistance observed
Nature Chemical Biology 3, 541 - 548 (2007)
Antimicrobial uses by category
ACS Infectious Diseases, 2015
Antibiotic in environment Antibiotics
Livestock uses Human uses
Excretion
Treatment of manure
Land application
Excretion Flushing unused
Topical application
Municipal WW
Runoff Treated discharge
Leach to ground water
Contaminate surface water
Potential drinking water sources
Why is resistance monitoring important?
Baseline Spread Trend
Source tracking
Veterinary/human use
Risk factors
Education Policy
ANTIBIOTIC RESISTANCE
16 ARGs & class 1
integrons
20 Antibiotic residues
Pathogenic ARBs
Diversity of ARB & ARG
Risk assessment
Quantitative PCR
LC-MS/MS Culture-based method
Integrated approach
Metagenomics
Quantitative microbial risk assessment
Application
Baseline A. Reservoirs &
catchments
B. Hospital Wastewater
C. Domestic wastewater
10.0 km
Reservoirs & Catchments
Catchments & Reservoirs
K PKR1 (R) WQ4A (C) WQ5 (C) WQ6 (C)
M RmbA (R) CmbB (C) CmbH (C)
U RUSA (R) RUSF (R) RUSH (R)
FRESHWATER
Mc RmcA (R)
K U
Mc M
Hospital Wastewater
Hospital wastewater
S6 S7
H1 H2
• Reservoirs for pathogenic bacteria • High usage of antibiotics • Concern with transmission and
long term survival in the environment
• Discharged into domestic sewage system without any treatments routes of dissemination to environment
Domestic wastewater
Influent
Effluent
Primary Clarifiers
Anoxic/Aerobic tank
Secondary Clarifiers
Return Activated Sludge
Effluent
Primary Clarifiers
Anoxic/Aerobic tank
Membrane Bioreactor
Return Activated Sludge
INF
A1 A2
B1 B2
CAS treatment process
MBR treatment process
Occurrences of AMR in reservoirs, hospital wastewater, and domestic
wastewater
Target analytes
Min (ppb) (n=8) Median (ppb) Max (ppb)
(n=8) MDL (ppt)
CFZ <DL <DL <DL 268 MER 0.10 0.82 0.94 15 CAP <DL Only one detection 0.44 19 CIPX <DL 2.44 71.94 70 LIN <DL <DL <DL 1.5 CLI <DL 0.83 1.14 28 ERY <DL <DL <DL 17 AZT 0.12 0.30 1.23 2 CLAR 1.02 2.63 56.77 2.4 TYL <DL <DL <DL 243 SMZ <DL <DL <DL 8 SMX 1.00 14.34 24.72 22 TMP 0.81 9.51 61.18 5
TET <DL <DL <DL 50 MIN <DL <DL <DL 94 CTC <DL <DL <DL 12 OXY <DL <DL <DL 74 VAN 0.15 6.94 42.59 5
Concentrations of AB in hospital wastewaters
1.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+09
MPN
\100m
l
E.coli
Enterococci
Pseudomonas aeruginosa
Reservoirs and catchments: - Enterococci: between 1.76 x 101 and 2.54 x 103 MPN/100mL - E.coli: between 2.11 x 101 and 4.28 x 103 MPN/100mL Reservoirs water quality all below thresholds recommended by USEPA.
Biological indicators for WQ
Concentrations of ARB
ARB concentrations (geometric means): Hospital wastewaters - (1.40 x 105 CFU/mL) Domestic wastewaters – (5.94 x 105 CFU/mL) Freshwaters – (5.14 x 102 CFU/mL)
Relative abundance of ARGs
Relative abundance of ARGs (geometric means): Hospital wastewaters – Average (8.91x10-2) Domestic watewaters – Average (3.62x10-2 ) Freshwaters – Average (8.67x10-4) 1. ARG abundance in freshwaters 2 magnitudes lower 2. All 4 bla-gene targets found in freshwaters (10-5-10-7) , however at least a magnitude lower than in wastewaters (10-3-10-5)
Phylogenetic composition of ARB
Dominant AR bacteria: Wastewaters: Aeromonas, Enterobacteriaceae (Klebsiella, Enterobacter, E.coli), Pseudomonas, Acinetobacter Freshwaters: Flectobacillus, Pseudomonas, Acinetobacter, Flavobacterium, Aeromonas
Risk assessment of Antibiotic resistant E. coli O157H7 in Recreational Health
Risks
Hazard Identification
Dose Response Assessment
Exposure Assessment
DALYs
Probability of infection/illness
Reservoirs water
Treated water • Sewage
• Hospital effluent
ARB at MIC
Indicator organism
Antibiotics ARGs/Integrons
ARB pathogens (e.g., E. coli, K.
pneumoniae, etc.)
Library of ARB
• Frequency • Severity (e.g., last resort AB, pathogen, virulence factor)
Risk
Risk Controll
QMRA approach for Antibiotic resistance
MIC/MDR
ARGs Virulence genes
Occurrence of Antibiotic resistant E. coli
<100 CFU/100ml <10,000 CFU/100ml
• Prevalence of E. coli in agricultural and urbanized area > 100 times in reservoirs • Among 4 reservoirs, Marina is the highest prevalence of AMR E. coli. • CIP and SXT are the most prevalent. AMK was the least. • Average concentration of E. coli in reservoirs < EPA guideline (200 EC/100ml).
Concentration of E. coli O157H7
Eco CEFT-Eco CIP-Eco SXT-Eco MEM-Eco Average 108.58 0.02 0.34 1.01 0.05 Median 1.6 0 0.01 0.01 0 Mode 0.04 0 0 0 0
SD 7,537.19 0.23 12.74 91.98 1.08 Distribution lnorm lnorm lnorm lnorm lnorm
E. coli : E. coli O157H7 = 1: 0.08 Reference: Haas et al., 1999; Howard et al., 2006;
Assumption AR E. coli : AR E. coli O157H7 = 1:0.08
Exposure and dose-response parameters
Distribution Parameters References Exposure duration (h) PERT(minimum, likeliest, maximum) (0.25, 0.5, 2) Mcbridge 2013
Ingestion rate (ml/h) PERT(minimum, likeliest, maximum) (2,10, 20) Dorevitch 2010,
2011
Dose-response model
Beta-poison model: 𝑃𝑃 = 1 − (1 + 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 × 21𝛼𝛼−1𝑁𝑁50
)−𝛼𝛼
Exposure for 2nd contact activities (Rowing, canoeing, kayaking)
alpha N50 illness/infection rate Reference
E.coli O157H7 2.10E-01 1.12E+03 0.35 Hass 1999, Horward and Pedley 2004
Assumption Susceptible and resistant E. coli O157H7 have the same ability to infect to human.
Probability of Gastrointestinal illness
EPA guideline (2012): 36 illnesses/ 1000 cases
Number of GI cases per 1000 recreators
2.9%
0.02%
Statistics Eco157 CAZ-Eco157 CIP-Eco157 SXT-Eco157 MEM-Eco157 Mean 4.04 0.00397 0.0317 0.0953 0.00626
Median 0.167 0.000185 0.000817 0.00109 0.000164 Minimum 0.0006 0 0 0 0 Maximum 219 5.49 15.9 69.3 6.49
EPA guideline (2012): 36 illnesses/ 1000 cases
Frequency of exceeding the EPA guideline 2012
Removal of Antibiotic Resistance in Domestic Wastewater by The
Membrane Bioreactor Treatment
Influent
Effluent
Primary Clarifiers
Anoxic/Aerobic tank
Secondary Clarifiers
Return Activated Sludge
Effluent
Primary Clarifiers
Anoxic/Aerobic tank
Membrane Bioreactor
Return Activated Sludge
INF
A1 A2
B1 B2
CAS treatment process
MBR treatment process
Membrane bioreactor treatment
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• Introduced in late 1960s
• Is the combination of a membrane process with a suspended growth bioreactor
• is now widely used for wastewater treatment.
• Advantages over the activated sludge treatment:
• high quality of effluent: low turbidity, bacteria, TSS, BOD
• can operate at high concentration of MLSS, low reactor volume
OBJECTIVE: To evaluate the removal efficiency of AB, ARB, and ARG in the MBR process compared to the CAS process.
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𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑹𝑹𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝑹𝑹𝒆𝒆𝒆𝒆𝒆𝒆 % =𝑪𝑪𝑰𝑰𝑰𝑰𝑰𝑰 − 𝑪𝑪 × 𝟏𝟏𝟏𝟏𝟏𝟏
𝑪𝑪𝑰𝑰𝑰𝑰𝑰𝑰
Removal of Antibiotic residues
• On average, about 75% and 80% AB were removed in CAS and MBR processes.
• Both Secondary clarifier and MBR treatment did not efficiently remove AB.
ng/l CAS MBR
High >200
Chlotetracycline Chlotetracycline Oxytetracycline Amoxicilin
Tetracycline Oxytetracycline Azithromycin Clarithromycin
Clarithromycin Sulfamethaxazole
Ciprofloxacin Tetracycline Sulfamethaxazole Ciprofloxacin
Medium 10-200
Trimethoprim Azithromycin Sulfamethazine Erythromycin Erythromycin Sulfamethazine Meropenem Trimethoprim Lincomycin Meropenem Vancomycin Lincomycin
Vancomycin
Low <10
Clindamycin Clindamycin Minocycline Minocycline
Chloramphenicol Chloramphenicol Ceftazidime Ceftazidime
Tylosin Tylosin Amoxicilin
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Removal of Antibiotic resistant bacteria
• Prevalence of ARB in the effluent were from 102 to 104 CFU/ml in CAS, and under detection limit in MBR.
• Average log removal of ARB in final effluent were about 2.3 in CAS, and 5.5 in MBR.
• MBR treatment was highly efficient in removal of ARB.
𝑳𝑳𝑹𝑹𝑳𝑳 𝒓𝒓𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 (𝑨𝑨𝑹𝑹𝑨𝑨) = −log𝟏𝟏𝟏𝟏𝑪𝑪
𝑪𝑪𝑰𝑰𝑰𝑰𝑰𝑰
*
* P=0.016
P=2.5x10-9
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• Average log removal of ARG were approximately 1.5 in CAS, and 3.0 in MBR.
• Compared to the CAS, MBR showed a better efficiency in removal of ARG genes.
CFU/ml CAS MBR
High >1000
16S 16S sul1 sul1 tetO tetO aac6 int1
ermB
Medium <1000
qnrB ermB blaCTX-M qnrB
tetM blaCTX-M blaSHV tetM blaKPC
Low <100
qnrA qnrA vanA int1 dfrA vanA sul2 dfrA cfr blaKPC
blaNDM1 aac6 sul2 cfr
blaSHV blaNDM1
*
Removal of Antibiotic resistant genes
Overall summary
∗ Antibiotic resistance (AB, ARB, ARG) is already a global concern threatening environmental and community health, not something in future.
∗ Surveillance effort, especially on aquatic environment, need to be raised worldwide to understand the current status, baseline, and guideline for further management.
∗ Culture-based method, qPCR, LC-MSMS, and metagenomics are demonstrated a good method to detect and analyze AR.
∗ There need to be a specific treatment of AR in WWTP to increase removal of AR factors (AB, ARB, ARG)
∗ Burden of disease for AR pathogen needs to evaluate.
Acknowledgements
A/Prof. Karina Gin Dr. Ng Charmaine
Dr. Laurence Haller
National Research Foundation (NRF)
International University HCMC RCE ESD Southern Vietnam