Surveillance and Risk Assessment of Antibiotic Resistance in the Urban Water Cycle, Le Thai Hoang

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

Presenter

Presentation Notes

Hello everyone, my name is …, lecturer of environmental engineering, IU VN Today I am glad to have this opportunity to talk about one of the aspect of water contamination, antibiotic resistance. Title “…” In this presentation, I will focus on the effect of antibiotic on aquatic environment, 2- how to detect and test their presence, quantify them, 3-occurrence of the AMR in some water body in Singapore, 4- estimate the risk they may pose to environmental and community health.

May, 2016

Jan, 2017

Presenter

Presentation Notes

http://www.cbsnews.com/news/woman-dies-from-superbug-resistant-to-all-available-antibiotic-in-u-s/ http://abcnews.go.com/Health/deadly-superbug-infection-resistant-fda-approved-antibiotics/story?id=44806424 https://www.sciencenews.org/article/bacteria-resistant-last-resort-antibiotic-appears-us Antibiotic resistance is not a new issue, there are lots of warning from the scientist, medical doctors about this problem because of their serious consequences. However, nowadays this issue seem to be a global concern, receiving more n more attention from the stakeholder and policy maker. In may 2016, a woman, 49 y old was infected with an pathogenic e. coli resistant to the last-resort antibiotic. That is collistin. This ab has adverse side effect to kidney and liver, and the doctor is very cautious to use unless there is no other option. Isolated e coli was confirmed with the mcr-1 gene which cause resistance to collistin. Last month, another case was reported in US, a woman died from a superbug that s resistant to 26 different kind of antibiotic. Unfortunately, the resistance is transferable because of the resistant genes. These gene can transfer from a bacteria to another via the mechamism called horizontal gene transfer. and so more and more scientists and stake holder try to make more effort on research to fight with this super bugs.

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

Presenter

Presentation Notes

According to a WHO report in 2014, death by AMR was about 700 k, among top 8 reasons caused total death in the world, much higher than tetanus, cholera or measles. And if the problem is not controlled, an estimation about 10 million death in 2050, cost more than 100 trillion $

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)

Presenter

Presentation Notes

The year each antibiotic was deployed is depicted above the timeline, and the year resistance to each antibiotic was observed is depicted below the timeline (with the caveat that the appearance of antibiotic resistance does not necessarily imply that a given antibiotic has lost all clinical utility)

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

Presenter

Presentation Notes

Human input Excretion of therapeutic antibiotics and metabolites > 40% of ingested dose remain in human excretion Disposal of unused antibiotics Flushing down the toilet Washing topically applied antibiotics All go to septic systems or municipal WWTPs Inefficient removal Breeding grounds for antibiotic resistant organisms (ARO) Veterinarian input Antibiotics used in livestock operations since the early 1950s AB use in livestock operations: Therapy: Antibiotics for treatment of infectious disease Prevention: Antibiotics used in animals considered “at risk”, but where individuals do not show signs of disease Growth Promotion: Antibiotics administered over a period of time, usually as a feed additive, to growing animals Large fraction of drug remain unchanged in animal waste released into aquatic environment

Why is resistance monitoring important?

Baseline Spread Trend

Source tracking

Veterinary/human use

Risk factors

Education Policy

Presenter

Presentation Notes

USFDA. National Antimicrobial Resistance Monitoring System Document resistance levels in different reservoirs (Baselines) Describe the spread of resistant bacterial strains and resistance genes (Spread) Identify temporal and spatial trends in resistance (Trends) Generate hypotheses about sources and reservoirs of resistant bacteria (Attribution) Understand the association between use practices and resistance (Veterinary Use) Identify risk factors and clinical outcomes of infections caused by antimicrobial resistant bacteria Provide data for education on current and emerging hazards (Education) Guide evidence-based policies and guidelines to control antimicrobial use in hospitals, communities, agriculture, aquaculture, and veterinary medicine (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


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)

Presenter

Presentation Notes

Quantification of more than 10 ARB show higher abundance in ww than in freshwater, about >1000 times

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)

Presenter

Presentation Notes

Relative abundance was used to compare ARG among 3 kind of samples. Generally, ARG presence was higher in wastewater than in freshwater. Between, domestic ww and hos ww, hos ww contain more bla gene.

Phylogenetic composition of ARB

Dominant AR bacteria: Wastewaters: Aeromonas, Enterobacteriaceae (Klebsiella, Enterobacter, E.coli), Pseudomonas, Acinetobacter Freshwaters: Flectobacillus, Pseudomonas, Acinetobacter, Flavobacterium, Aeromonas

Presenter

Presentation Notes

Bacteria in ww are more pathogenic In fresh water, environmental bacteria is more

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


Effluent

Primary Clarifiers

Anoxic/Aerobic tank

Membrane Bioreactor


INF

A1 A2

B1 B2

CAS treatment process

MBR treatment process

Membrane bioreactor treatment

27

• 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.

28

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

29

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

30

• 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