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Antimicrobial resistance is a known global public health threat. In addition, it brings serious economic consequences to agriculture. Antibiotic resistance in humans, animals, and environment is interconnected, as proposed in the tricycle surveillance by the World Health Organization. In Malaysia, research and surveillance of antimicrobial resistance are mainly performed in clinical samples, agricultural settings, and surface waters, but no surveillance of the drinking water systems has been performed yet. Hence, this policy-driven study is a combined effort of microbiologists and engineers to provide baseline data on the magnitude of antimicrobial resistance in the drinking water systems of Malaysia.
The aim of this study was to study the baseline level of antibiotic-resistant bacteria in the drinking water distribution systems of Malaysia by collecting samples from the pretreatment and posttreatment outlets of water treatment plants in a selected state of Malaysia. We aimed to determine the prevalence of antibiotic-resistant bacteria, the occurrence of antibiotic-resistant genes, and the level of antibiotics present in the drinking water systems.
This is a laboratory-based, cross-sectional study in a selected state of Malaysia. Water samples from 6 drinking water treatment plants were collected. Samples were collected at 3 sampling points, that is, the intake sampling station, service reservoir outlet station, and the distribution system sampling station. These were tested against 7 types of antibiotics in triplicates. Samples were screened for antibiotic-resistant bacteria and antibiotic-resistant genes and quantified for the level of antibiotics present in the drinking water treatment plants.
We will show the descriptive statistics of the number of bacterial colonies harvested from water samples grown on Reasoner’s 2A agar with or without antibiotics, the occurrence of antibiotic-resistant genes, and the level of antibiotics detected in the water samples. The sampling frame was scheduled to start from November 2021 and continue until December 2022. Data analysis is expected to be completed by early 2023, and the results are expected to be published in mid-2023.
This study provides baseline information on the status of the antimicrobial-resistant bacteria, the presence of resistance genes as contaminants, and the level of antibiotics present in the drinking water systems of Malaysia, with the aim of demonstrating to policymakers the need to consider antimicrobial resistance as a parameter in drinking water surveillance.
DERR1-10.2196/37663
Access to clean and safe water is of the utmost importance worldwide. For general well-being, human beings use water for daily consumption and hygiene purposes. Water also serves as an important factor for maintaining a sustainable environment and plays an important role in climate change issues. Unfortunately, environmental water also plays a prominent role in the spread of antibiotic-resistant genes (ARGs) and antibiotic-resistant bacteria (ARB). Over 80% of all wastewater in low-income countries is estimated to be discharged untreated directly into rivers, lakes, or the oceans [
Route of antibiotic-resistant bacteria and antibiotic-resistant genes from various anthropogenic sources to a drinking water source. Adapted from Stalder et al [
Antimicrobial resistance (AMR) occurs when microorganisms develop the ability to resist antimicrobial treatment designed to inhibit their growth and kill them. Bacteria, as the most extensively studied for AMR in both clinical and environmental settings, can exchange resistance genes between species in a microbial population [
Tracking of the spread of antibiotic-resistant microorganisms provides insights into the transmission of antibiotic-resistant strains and resistance genes from humans or animals to the environmental reservoir and back to human use and consumption [
The World Health Organization calls for coordinated actions to minimize the emergence and spread of AMR by providing technical assistance to countries to develop national health action plans and urging more research and development on AMR. As of October 2021, 148 countries have finalized their National Action Plan, which aligns with the objectives of the Global Action Plan on Antimicrobial Resistance [
A few published studies in Malaysia have reported the presence of pharmaceutical residues from surface water in Malaysia. The presence of human pharmaceutical products, which include antidiabetic agents, antihypertensive agents, hypolipidemic agents, β-2 adrenergic receptor agonists, antihistamines, analgesics, and sex hormones, in river water and sewage effluent were reported, but no antibiotics were detected [
Drinking water treatments have previously been shown to act as a source of antibiotic resistance, and water distribution systems could serve as an important reservoir for microbial resistance [
The presence of biofilm-producing bacteria such as
Outbreaks in hospitals caused by multidrug-resistant
Although there is no conclusive evidence on the “safe limit” of ARB or ARGs in drinking water worldwide, several studies have been conducted in other countries, including China, which have indicated the presence of antibiotic residues, ARB, and ARGs in their water systems. These data have been used to warrant further research and put up new efforts to improve their DWTP systems (eg, to improve the filtration system). For example, Liu et al [
Several hospital-acquired outbreaks caused by gram-negative bacteria such as
AMR is an important global issue that needs to be addressed holistically so as to prevent people from dying owing to ineffective antibiotic treatment and to avoid antibiotic resistance eventually, leading us to the postantibiotic era [
The aim of this study is to address the abovementioned gaps by determining the prevalence of ARB and the occurrence of resistant genes (ARGs) in a natural water source, drinking water treatment system, and water distribution outlets in Malaysia. We will do this by investigating the most common microbial species and their phylogenetic relationships before the water treatment and after the water treatment and in different DWTPs by determining the occurrence of ARB and ARGs and by quantifying the level of antibiotics.
This is a cross-sectional study using laboratory-based methodologies (
We will use simple random sampling to choose a sample. We will randomly generate a number for each DWTP by using Excel and identify the sample. We assume that all the DWTPs are homogenous (
Flow of the experimental work. AMR: antimicrobial resistance; ARB: antibiotic-resistant bacteria; ARG: antibiotic-resistant gene; BLAST: Basic Local Alignment Search Tool; HPC: heterotrophic plate count; NCBI: National Center for Biotechnology Information; PCR: polymerase chain reaction; R2A: Reasoner's 2 agar.
Schematic diagram of a drinking water treatment plant in Malaysia (Source: Engineering Services Division, Ministry of Health, Malaysia).
Sampling size was calculated using prevalence [
Number of DWTPs in Selangor = 34
Prevalence range of heterotrophic ARB in a drinking water plant [
Assuming the prevalence (p) would be 40%, p=0.40;
Where n=sample size, z=1.96 for a confidence level (α) of 95%, p=assumed prevalence, and d=precision (corresponding to effect size).
All DWTPs are monitored under the National Drinking Water Quality Program, Selangor State Health Department, Ministry of Health of Malaysia. There are no exclusion criteria.
Samples will be handled properly by trained laboratory personnel to ensure aseptic techniques, and other procedures will be performed according to the quality management system for microbiological analysis. Water sampling will be performed following the Standard Methods for the Examination of Water and Wastewater [
We will be using culture-dependent methods and molecular techniques to determine the prevalence of ARB, identify them, and investigate the occurrence of ARGs present in DWTPs to answer each specific objective. A heterotrophic plate count on Reasoner’s 2A agar (Oxoid) will be used to determine the ARB in the collected water samples. Bacteria in river water (sources) are expected to be present at much higher concentrations at approximately 350 colony-forming units (CFUs)/100 mL [
The polymerase chain reaction method will be used for the rapid and accurate identification of bacteria by using universal primers targeting the 16S rRNA gene [
List of the published primers representing the occurrence of bacterial DNA and antibiotic-resistant genes [
Gene name | Resistance mechanism |
16S rRNA gene | N/Aa |
|
Ribosomal protection |
|
Enzymatic modification |
|
Efflux |
|
Ribosomal protection |
|
Enzymatic modification |
|
β-lactam binding protein |
|
Hydrolysis |
|
Enzymatic degradation |
|
Efflux |
|
Hydrolysis |
|
Amino acid cleavage |
aN/A: not applicable.
Samples for antibiotic quantification (
Before quantification, samples will be preconcentrated and eluted to achieve satisfactory accuracies and sensitivities [
Sample collection and treatment
Sample preconcentration using a nitrogen evaporator
Sample clean-up by automated solid-phase extraction
Analysis using liquid chromatography–mass spectrometry (Q Exactive Orbitrap Mass Spectrometer, Thermo Fisher Scientific)
Amoxicillin-d4 (Toronto Research Chemicals)
Amoxicillin trihydrate (Dr Ehrenstorfer GmbH)
Ciprofloxacin hydrochloride (Dr Ehrenstorfer GmbH)
Vancomycin hydrochloride (Dr Ehrenstorfer GmbH)
Gentamycin sulfate (Dr Ehrenstorfer GmbH)
(+/-) chloramphenicol (Cambridge Isotope Labs Inc)
Sulfamethoxazole (Dr Ehrenstorfer GmbH)
Tetracycline hydrochloride (Dr Ehrenstorfer GmbH)
Waters Xbridge C18 Column 50 mm×2.1 mm id, 2.5 µm at 35 °C
Solvent gradient
A: Water liquid chromatography-mass spectrometry grade (Merck)
B: Acetonitrile with 0.1% (v/v) formic acid (Sigma-Aldrich)
Flow: 0.8 mL/min
Solvent gradient for the determination of antibiotics by high-performance liquid chromatography.
Retention time (min) | Flow rate (mL/min) | Mobile phase A (water liquid chromatography-mass spectrometry grade) (%) | Mobile phase B (acetonitrile with 0.1% (v/v) formic acid) (%) |
0 | 0.80 | 95 | 5 |
1 | 0.80 | 95 | 5 |
12 | 0.80 | 70 | 30 |
13 | 0.80 | 0 | 100 |
17 | 0.80 | 0 | 100 |
17.1 | 0.80 | 95 | 5 |
23 | 0.80 | 95 | 5 |
No clinical samples will be collected. This study has been exempted from ethics approval by the Medical Research and Ethics Committee, Malaysia. Data will be disseminated to the Drinking Water Quality Surveillance Program (DWQSP), Engineering Services Division, Ministry of Health, as our main stakeholders and collaborator, and through a peer-reviewed publication or presentation following approval from the Ministry of Health, Malaysia.
A paired 2-sided
Data will be recorded in Microsoft Excel. DNA sequences will be kept in FASTA files. All positive ARB samples will be recorded for resistance profiles and will be kept as environmentally derived ARB culture collections for future studies.
This project was funded in June 2020. The sampling frame was scheduled to start from November 2021 and continue until December 2022. To date (July 2022), we have sampled 4 DWTPs at 3 points, that is, the river (source), posttreatment outlet (DWTP), and the main distribution tap. Results will be reported as the prevalence of ARB in the drinking water system and the level of antibiotics present in the drinking water system a
The prevalence of heterotrophic ARB in source water will be compared with that in other sampling points. Only strains that are able to grow on Reasoner’s 2A agar containing antibiotics will be subjected to analysis for antibiotic susceptibility and ARGs. Each antibiotic-resistant strain will be inoculated on Muller-Hinton media (Oxoid) for testing. Results from the antibiotic susceptibility test (Kirby-Bauer disk assay) will be compared to Clinical and Laboratory Standards Institute disk measurement standards for specific bacteria and antibiotics. However, for isolates that are not included in the Clinical and Laboratory Standards Institute list, results will be interpreted by adopting the European Committee on Antimicrobial Susceptibility Testing database or by considering inhibition diameters ≥10 mm as susceptible [
Resistance rates (prevalence%) = Number of bacteria that grow on antibiotic plates/number of bacteria that grow on plates containing no antibiotics
Multiresistance index = Number of antibiotics to which isolate is resistant/number of antimicrobials to be tested
The presence of ARGs will be marked by determining the amplification by the specific primers of each gene of interest. DNA will be visualized using 2% agarose gel. Positive bands of sample DNA using 16S rRNA primer sets indicate the presence of bacterial DNA, while positive bands in samples using specific ARG primer sets show the presence of ARGs at the particular sampling points.
The preconcentration technique will be used for each antibiotic to achieve parts per trillion detection. The concentrations of the antibiotics will be quantified and determined whether the levels correspond with the ARB at each site.
From our preliminary analysis in 3 DWTPs, we found multidrug-resistant
The outcomes of this study would provide a basis for holistic research in AMR by determining the extent of AMR present in Malaysia and the likely paths of transmission from the environment to the public. The prevalence data on antibiotic resistance generated from this study will also help the relevant stakeholders in Malaysia to steer evidence-based policies to control and prevent the possible transmission of ARB in drinking water systems to humans [
The DWTP in Malaysia consists of a river catchment as the water source, water treatment plant, service reservoir, and finally, the distribution outlet from where it goes to every house (
We have identified several strengths in this study; first, this study provides baseline data on ARB in the drinking water system of Malaysia. Second, this study will illustrate the burden of ARGs in the drinking water treatment systems in urban areas. Third, these results will be used to provide evidence and increase the precision of the quantitative estimates of exposure related to ARGs in drinking water and finally provide baseline data for AMR in national safe drinking water. We anticipate limitations such as the recovery of bacteria from low nutrient media when cultivated in enriched media, which will require optimization of the incubation period and temperature, and a lack of reference data on minimum inhibitory concentrations to interpret all types of bacteria, especially the less commonly known bacteria present in a water system.
There are several impacts anticipated from this study. First, we may elucidate the magnitude of the problem by providing baseline information on the level of ARB and ARGs present in our drinking water system in Selangor, Malaysia. Second, we may improve the water treatment system to be more sustainable, safe, and clean, particularly for long-term daily water consumption [
Findings from this study will be used to determine whether AMR is a problem in our drinking water by providing information on the status of ARB and the presence of ARGs in drinking water systems. Depending on the magnitude of AMR in the drinking water system, further actions/key measures can be taken (future research or by providing evidence to the stakeholders) to reduce or eliminate the contaminants from entering our drinking water system. This study would help to draw a holistic picture of the occurrence of antibiotic resistance in the environment, apart from other anthropogenic sources such as human effluents that may contribute to antibiotic resistance, by considering water as a potential reservoir for humans to be exposed to antibiotic resistance. This study will provide information on the status of ARB and the presence of resistance genes as contaminants in drinking water systems, which are not being monitored in the National Drinking Water Quality program by the Ministry of Health at present. Further actions and key measures can be taken for future research or expert advice to reduce or eliminate contaminants from entering our drinking water system. This study will broaden a research niche in AMR concerning the environment and human health risks in Malaysia.
AMR is a public health challenge that requires the concerted efforts of multiple agencies. AMR management in clinical and agricultural settings is more established compared to that in DWTPs in Malaysia. The output from this study will provide evidence and key measures to benefit and assist the current DWQSP to reduce the risk of antibiotic resistance transmission to the public. Rationally, if the resistance is decreased at the local level, it can help prevent the global antibiotic resistance crisis from growing even bigger.
Data from this study will be used to embark on future research based on the tricycle (human, food and environment) approach by incorporating the possible sources of pollutants. This will enable us to determine the prevalence of AMR within the same localities and thus enable researchers to elucidate the possible route of transmission of AMR in a defined setting, especially into the rivers as drinking water sources.
antimicrobial resistance
antibiotic-resistant bacteria
antibiotic-resistant gene
colony-forming unit
drinking water treatment plant
drinking water quality surveillance program
Malaysian Action Plan on Antimicrobial Resistance
This study was funded by the Ministry of Health of Malaysia and is managed by Clinical Research Malaysia. This study has been registered with the National Medical Research Register, Malaysia. We thank the Director-General of Health for the permission to publish this report. We wish to thank the Engineering Division of the Ministry of Health of Malaysia and Selangor State Health Department for detailed information on the drinking water treatment plants as well as expert panels involved throughout the development of this study. Last but not least, we thank Catie Williams at the Pathogen Genomics Unit, Public Health Wales for proofreading this manuscript.
Data will be kept in the Ministry of Health and will be disseminated following approval from the Director-General of Health, Malaysia.
ZAM initiated the study, applied for funding, and is the principal investigator. SKB and MAJJ were involved in the development of laboratory techniques. LC was involved in giving expert advice on antimicrobial resistance and molecular techniques. NA gave expert advice on antimicrobial resistance in Malaysia. NAM provided important statistical contributions. All authors contributed to the content of this study.
None declared.