This paper is in the following e-collection/theme issue:

Proposals (non-eHealth) (44) Public Health (185) Health Services Research (457) Antimicrobial Stewardship and Antibiotic Resistance (40)

Published on in Vol 12 (2023)

Preprints (earlier versions) of this paper are available at https://preprints.jmir.org/preprint/39022, first published .
Prevalence of Antibiotic-Resistant Pathogenic Bacteria and Level of Antibiotic Residues in Hospital Effluents in Selangor, Malaysia: Protocol for a Cross-sectional Study

Prevalence of Antibiotic-Resistant Pathogenic Bacteria and Level of Antibiotic Residues in Hospital Effluents in Selangor, Malaysia: Protocol for a Cross-sectional Study

Prevalence of Antibiotic-Resistant Pathogenic Bacteria and Level of Antibiotic Residues in Hospital Effluents in Selangor, Malaysia: Protocol for a Cross-sectional Study

Authors of this article:

Sophia Karen Bakon1 Author Orcid Image ;   Zuraifah Asrah Mohamad1 Author Orcid Image ;   Mohd Azerulazree Jamilan2 Author Orcid Image ;   Hazimah Hashim3 Author Orcid Image ;   Mohamed Yazid Kuman4 Author Orcid Image ;   Rafiza Shaharudin1 Author Orcid Image ;   Norazah Ahmad5 Author Orcid Image ;   Nor Asiah Muhamad6 Author Orcid Image
Article Authors Cited by Tweetations (8) Metrics

    Protocol

    1Environmental Health Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, Shah Alam, Malaysia

    2Nutritional, Metabolic and Cardiovascular Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, Shah Alam, Malaysia

    3Pharmacy Practice and Development Division, Ministry of Health Malaysia, Petaling Jaya, Malaysia

    4Engineering Service Division, Ministry of Health Malaysia, Putrajaya, Malaysia

    5Infectious Disease Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, Shah Alam, Malaysia

    6Evidence Based Unit, National Institutes of Health, Ministry of Health Malaysia, Shah Alam, Malaysia

    *these authors contributed equally

    Corresponding Author:

    Sophia Karen Bakon, BSc, MSc

    Environmental Health Research Centre

    Institute for Medical Research

    National Institutes of Health, Ministry of Health Malaysia

    Persiaran Setia Murni, Setia Alam

    Shah Alam, 40170

    Malaysia

    Phone: 60 +60333627774

    Email: sophia.karen@moh.gov.my


    Background: Antimicrobial resistance (AMR) has emerged as a major global public health challenge due to the overuse and misuse of antibiotics for humans and animals. Hospitals are among the major users of antibiotics, thereby having a large contribution to AMR.

    Objective: The aim of this study is to determine the prevalence of antibiotic-resistant pathogenic bacteria and the level of antibiotic residues in the hospital effluents in Selangor, Malaysia.

    Methods: A cross-sectional study will be performed in the state of Selangor, Malaysia. Tertiary hospitals will be identified based on the inclusion and exclusion criteria. The methods are divided into three phases: sample collection, microbiological analysis, and chemical analysis. Microbiological analyses will include the isolation of bacteria from hospital effluents by culturing on selective media. Antibiotic sensitivity testing will be performed on the isolated bacteria against ceftriaxone, ciprofloxacin, meropenem, vancomycin, colistin, and piperacillin/tazobactam. The identification of bacteria will be confirmed using 16S RNA polymerase chain reaction (PCR) and multiplex PCR will be performed to detect resistance genes (ermB, mecA, blaNDM-L, blaCTX-M, blaOXA-48, blaSHV, VanA, VanB, VanC1, mcr-1, mcr-2, mcr-3, Intl1, Intl2, and qnrA). Finally, the level of antibiotic residues will be measured using ultrahigh-performance liquid chromatography.

    Results: The expected outcomes will be the prevalence of antibiotic-resistant Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter (ESKAPE) bacterial species from the hospital effluents, the occurrence of antibiotic resistance genes (ARGs) from the isolated ESKAPE bacteria, and the level of antibiotic residues that may be detected from the effluent. Sampling has been conducted in three hospitals. Data analysis from one hospital showed that as of July 2022, 80% (8/10) of E. faecium isolates were resistant to vancomycin and 10% (1/10) were resistant to ciprofloxacin. Further analysis will be conducted to determine if the isolates harbor any ARGs and effluent samples are being analyzed to detect antibiotic residues. Sampling activities will be resumed after being suspended due to the COVID-19 pandemic and are scheduled to end by December 2022.

    Conclusions: This study will provide the first baseline information to elucidate the current status of AMR of highly pathogenic bacteria present in hospital effluents in Malaysia.

    International Registered Report Identifier (IRRID): DERR1-10.2196/39022

    JMIR Res Protoc 2023;12:e39022

    doi:10.2196/39022

    Keywords

    ESKAPE; antimicrobial resistance; hospital effluent; antibiotics; health care; antibiotic resistance; antimicrobial; hospital setting; antibiotic residues; wastewater 


    Background

    Antibiotics have been the most crucial medications for the treatment of infections since the 1940s. However, some bacteria have developed resistance to practically all of the widely accessible antibiotics, which makes them capable of causing serious illness, resulting in a significant public health issue. Due to the selective pressure caused by the overuse of antibiotics, antibiotic-resistant bacteria (ARB) have developed and spread among human and animal populations worldwide [1].

    The overuse and abuse of antibiotics have contributed to the global epidemic of antimicrobial resistance (AMR). Environmental contamination with antibiotic residues and resistant microorganisms and genes due to human activity has been demonstrated in pharmaceutical plants and may be a leading driver of the spread of ARB. Insufficient wastewater management by bulk drug manufacturing facilities has led to unprecedented levels of contamination of water resources with antimicrobial pharmaceuticals, which seems to be associated with the selection and dissemination of carbapenemase-producing pathogens [2]. In India, the concentration of the most abundant drug, ciprofloxacin (up to 31,000 μg/L), was reported to exceed the toxicity level by over 1000-fold [3].

    Several hospital-acquired outbreaks caused by gram-negative bacteria such as Acinetobacter baumannii and Pseudomonas aeruginosa have been associated with contaminated water distribution systems [4-6]. The presence of Escherichia coli has been used as an indicator of fecal contamination in drinking water. Multidrug-resistant E. coli have been isolated from food sources [7] and urinary samples [8], as well as from hospital and municipal effluents [9]. However, the relationship between environmentally driven and clinically acquired resistant bacteria remains unclear; therefore, it is important to determine whether this effect is either strictly environmental or clinical.

    The hospital setting has been the focus of AMR as one of the primary sources owing to the high usage of a wide range of antibiotics and the spread of antibiotic resistance genes (ARGs) that may be transferred by the ARB released into the environment from hospital effluents [10]. Some antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), and vancomycin-resistant enterococci (VRE) may pose significant risks to human health [11-13]. Ignorance that these possible effluent sources such as hospitals, including ARB or ARGs from clinical sources, can be dispersed and even thrive in the environment is likely to accelerate the development of multiple antibiotic–resistant bacteria.

    Although antibiotics remain effective at treating many bacterial infections, some strains are extremely difficult to treat, and therapeutic options are becoming increasingly scarce. The term “ESKAPE” encompasses six such pathogens with growing multidrug resistance and virulence: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species [14]. ESKAPE pathogens are responsible for most nosocomial infections and are capable of resisting the biocidal action of antimicrobial agents. This phenomenon is caused by bacterial evolution; thus, AMR has increasingly become a problem with rapid rates of development and spread, and we are lacking new drugs to challenge these new superbugs.

    The World Health Organization (WHO) has called for coordinated actions to minimize the emergence and spread of AMR by providing technical assistance for countries to develop national health action plans and urging more research and development on AMR [15]. In Malaysia, the collaborative effort between the Ministry of Health and the Ministry of Agriculture and Agro-based Industry resulted in the Malaysian Action Plan on Antimicrobial Resistance (MyAP-AMR) 2017-2021. The MyAP-AMR details plans to minimize AMR in both health care practices and agricultural sectors, including increasing awareness of antimicrobials, appropriate usage of antibiotics in clinical settings, and pollution influenced by the direct use of antibiotics in poultry farms [16].

    The Bacteriology Unit, Infectious Diseases Research Centre in the Institute for Medical Research has performed profiling of antibiotic resistance from a poultry farm and clinical samples in Malaysia. Some published studies also reported the presence of pharmaceutical residues from surface water in Malaysia. Al-Odaini et al [17] documented the presence of human pharmaceutical products—including antidiabetic, antihypertensive, and hypolipidemic agents; β2-adrenergic receptor agonists; antihistamines; analgesics; and sex hormones—in river water and sewage effluents, although no antibiotics were measured. Praveena et al [18] reported the presence of pharmaceutical residues, including antibiotics, in three rivers in Selangor, Malaysia. The researchers detected ciprofloxacin in all samples, which was the most common pollutant in the rivers. Recently, Bong et al [19] found that multiple antibiotic-resistant phenotypes of E. coli, harboring the tet and sul resistance genes, were more prevalent in wastewater effluents than in the river waters. A concerted effort has been carried out to address the issue of AMR in clinical settings and the misuse of antibiotics in the current practice of poultry farming and pharmaceutical manufacturing industries. However, Malaysia lacks research on environmental aspects that may contribute to AMR, particularly the possibility of ARB and ARGs that may be released from hospitals into the environment through various modes. Moreover, no study has investigated whether antibiotic residues present in the hospital effluent contribute to the AMR problem in Malaysia. Since hospitals are the major users of antibiotics, it is important to determine the status of the effluent water from our hospitals that is released into the environment. However, to date, there has been no monitoring of the antibiotic residues under wastewater management in Malaysia. Therefore, this proposed study represents a first step to fill this gap to better address the AMR issues in Malaysia.

    Dissemination of Antibiotic Resistance

    Antibiotics can enter the environment through several different routes. Antibiotics and their metabolites are released from hospitals with urine and feces from patients as hospital wastewater effluent [20]. Similarly, antibiotics are released into the wastewater treatment system from people taking antibiotics in their homes [21]. Antibiotics are also used therapeutically or as growth promoters in livestock and poultry in Malaysia. Antibiotics and their metabolites will spread through animal excrement and end up in fields and groundwater, or in the case of antibiotic use in fish farms directly into the aquatic environment [22] (Figure 1). Regardless of the routes for antibiotics disseminating in the environment, it is also likely that resistant bacteria follow the same routes of dispersal [23]. This results in an environment where antibiotics, ARGs, resistant bacteria, and the environmental bacterial flora are mixed. However, the environment is likely becoming a resistance hotspot where ARGs can proliferate and undergo a process of evolution to result in the development of new resistant strains. Pathogens can acquire an ARG from the environmental gene pool by either conjugation, transformation, or transduction. The most important genetic elements capable of being transferred by conjugation are plasmids and integrative conjugative elements, which may carry ARGs and potential virulence genes.

    ‎
    Figure 1. Schematic flow of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from hotspots of evolution and transmission (red circles) to the environment (green circle). Blue circles indicate possible vectors that may aid transmission between specific environments, including air, surface waters, humans, and other animal vectors. Black arrows indicate known flows of ARB and ARGs. The grey arrow indicates a possible transmission route from a contaminated environment back to the general populace. CAFO: concentrated animal feeding operation; WWTP: wastewater treatment plant (reproduced from Kraemer et al [22], which is published under Creative Commons Attribution 4.0 International License [24]).

    Scale of the Problem

    A total of 88 pathogen-drug combinations were used to quantify the worldwide burden of drug-resistant diseases in 2019. This resulted in an estimated 495 million fatalities, of which 127 million deaths were directly attributed to drug resistance [25]. By 2050, it is estimated that approximately 10 million lives will be at risk annually as a consequence of the rise in drug-resistant infections [26].

    Furthermore, antibiotic resistance is a substantial economic burden worldwide. In 2006, approximately 50,000 Americans died due to two common hospital-acquired infections (HAIs), namely pneumonia and sepsis, costing approximately US $8 billion [27]. In Europe, the levels of antibiotic resistance are varied. The common causes of infections are used as indicators to focus on AMR in S. aureus, specifically against methicillin (ie, MRSA) as a common cause of severe infections in health facilities and the community. MRSA is among the resistant pathogens that are widely recognized to pose an urgent or serious threat to human health. CRE such as K. pneumoniae and E. coli are also recognized as major causes of HAIs, including pneumonia. The WHO has listed MRSA as a high-priority (priority 2) pathogen for research and development of new antibiotics, while CRE have been listed as critically important (priority 1) [28].

    In Asia, India has been documented as one of the largest burdens of drug-resistant pathogens worldwide, including the highest burden of multidrug-resistant tuberculosis, along with alarmingly high resistance rates among gram-negative and gram-positive bacteria even to newer antimicrobials such as carbapenems and faropenem since its introduction in 2010 [29]. Regional studies in India reported high AMR among pathogens such as Salmonella typhi, Shigella, Pseudomonas, and Acinetobacter [30].

    In Thailand, approximately 88,000 infections were attributed to AMR, resulting in at least 3.24 million additional days in the hospital and 38,000 deaths [31]. The Global Antimicrobial Resistance Surveillance System (GLASS) launched by the WHO was implemented in Siriraj Hospital in Bangkok, reporting that in-hospital mortality was significantly higher in patients with antibiotic-resistant bacteremia than in patients with antibiotic nonresistant bacteremia (40.5% vs 28.5%, P<.001) [32]. The same study concluded that patients with antibiotic-resistant bacteremia utilized more resources than those with antibiotic nonresistant bacteremia [32].

    MRSA emerged in Singapore in the 1970s [33], rapidly becoming endemic in local health care facilities, with 35.3% of all S. aureus isolates reported to be methicillin-resistant in 2006 [34]. In another study, the prevalence of MRSA was found to be significantly higher in intermediate (29.9%) and long-term (20.4%) care facilities than in acute-care hospitals (11.8%) [35], and the organism has also been sporadically reported in the community as well as in local retail food and animals [36-38].

    Nonetheless, there are limited studies examining the role of hospital effluents in contributing to the development of drug-resistant pathogens in the environment. Hospitals are the major users of antibiotics and may become one of the major contributors to AMR in the environment. Furthermore, the levels of antibiotic resistance in hospital effluents may be different from those of other water environments such as homes or agriculture. Therefore, the general aim of this study is to determine the prevalence of antibiotic-resistant pathogenic bacteria and the level of antibiotic residues in hospital effluents in Selangor, Malaysia. This study will also be determining the prevalence of ARB (specifically ESKAPE) present in the effluents of hospitals with an individual sewage treatment system, the occurrence of ARGs among ESKAPE bacteria, and the residual concentrations of antibiotics based on their usage at the selected hospitals. The findings from this study will provide important scientific evidence relevant to stakeholders that can help to reduce or interrupt the spread of AMR from clinical settings into the environment.


    Study Design

    A cross-sectional study will be performed at preidentified hospitals with three specific objectives as depicted in the workflow diagram in Figure 2. Objective 1 is to obtain resistant isolates of ESKAPE bacteria from the hospital effluent and to perform antibiotic susceptibility testing (AST). Objective 2 is to extract the genomic DNA of the resistant isolates from objective 1 to identify the isolates and the ARGs present in the isolates. Objective 3 is to quantify the level of targeted antibiotic residues from the hospital effluent samples. The targeted antibiotics that will be quantified are ceftriaxone, ciprofloxacin, meropenem, vancomycin, colistin, piperacillin, and tazobactam, which are the most highly used antibiotics in the hospital setting. The outcome of activities in objective 1 and 2 will provide estimates of the prevalence of the ARB in the hospital effluent, while the outcome of objective 3 will determine the level of antibiotic residues in the hospital effluent to address the overall aim of this study.

    ‎
    Figure 2. Summary of the workflow for this study. ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae; gDNA: genomic DNA; PCR: polymerase chain reaction.

    Selection of Study Area

    We will identify all the tertiary hospitals in Selangor, one of the states in Malaysia, prior to effluent sample collection, based on the following inclusion and exclusion criteria.

    Hospitals with individual sewage treatment plants will be included. An individual sewage treatment plant is a system whereby the sewage from the hospital is the only source that enters the sewage treatment plant, as depicted in Figure 3. Hospitals with nonindividual sewage treatment plants will be excluded. A nonindividual sewage treatment plant is defined as a communal sewage treatment plant whereby the sewage entering the sewage treatment plant is coming from various sources such as housing, farming, and manufacturing industries, as illustrated in Figure 4.

    ‎
    Figure 3. The sampling point for the effluent sample to be collected is at the final effluent for the disposal outlet, as depicted in the diagram. This is the outlet after the treatment before entering the river system that is going to be treated by the Indah Water Treatment Plant to be used as a clean water source for the public.
    ‎
    Figure 4. The waste from multiple human activities such as farming, domestic, school, factory, and hospital activities is concentrated into same sewage treatment plant before release to the river to be treated by Indah Water Treatment Plant as a clean water source for the public.

    Sample Size Calculation

    Calculation of sample size is based on the prevalence using the following formula [39]: N=Z2p(1 – p)/d2, where N is the sample size, Z is the statistic for a level of confidence, and p is the expected prevalence or proportion (considering a total proportion of 100%, if 20% then p=0.2), and d is precision. For this study in proportion of 100%, if 5%, d=0.05. Considering a conventional 95% CI as the level of confidence, the Z value is 1.96. The sample size was determined from prevalence estimates of previous studies of ESKAPE isolates from the hospital or wastewater treatment plant with the 95% CIs, as shown in Table 1. This calculation served as the guidance for the number of plates to be used to isolate the targeted ESKAPE bacteria.

    Table 1. Prevalence of antibiotic-resistant bacteria from related studies.
    Study and bacterial speciesPrevalence, %Sample size (number of plates to isolate ESKAPEa bacteria)
    Haller et al [40]

    		Pseudomonas spp.28.2312

    		Klebsiella spp.28.2312

    		Enterobacter spp.18.3230

    		Citrobacter spp.11.3155
    Hrenovic et al [41]: Acinetobacter baumannii57.0377
    Thompson et al [42]: methicillin-resistant Staphylococcus aureus81.0237
    Varela et al [43]

    		Enterococcus spp. on vancomycin0.711

    		Enterococcus spp. on ciprofloxacin3.454
    Karimi et al [44]

    		Enterococcus faecium6.0388

    		Enterococcus faecalis0.6310

    aESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter.

    Sample Collection and Preparation

    In total, two bottles of effluent water will be collected. Samples for objective 1 and objective 2 will be collected in a 1-L sterile screw-capped Schott Duran (Germany) amber bottle. In addition, samples for antibiotics quantification (objective 3) will be collected in a 1-L high-density polyethylene (Kris Plastic Industries, Malaysia) sample bottle. All samples will be kept in an ice-packed cooler box maintained at 4°C and transported back to the Environmental Microbiology Laboratory, Institute for Medical Research, for immediate processing. The samples will be collected at the final effluent disposal point of the sewage treatment system before reaching the Indah Water Treatment Plant Malaysia (Figure 3).

    Microbiological Analysis

    Isolation of ESKAPE ARB

    Ten-fold serial dilutions for each water sample will be prepared in 50-mL sterile saline solution and 10 mL of the sample will be filtered through sterile 0.45-µm pore size cellulose acetate filters (Sartorious Singapore Pte Ltd) in triplicate (Figure 5). The filter will be then placed onto the selective media to isolate ESKAPE pathogens. The selective media to be used in this process of isolation are listed in Table 2. Identification of the ESKAPE bacteria will be carried out using the 16S RNA polymerase chain reaction (PCR) technique with a universal primer pair (forward, CCT ACG GGA GGC AGC AG; reverse, ATT ACC GCG GCT GCT GG) under the following condition: polymerase activation at 94°C for 2 minutes, denaturation at 94°C for 5 seconds, annealing at 60°C for 10 seconds, extension at 72°C for 20 seconds, and holding at 4°C.

    The positive colonies will be subjected to AST using the disk diffusion method (Kirby Bauer) referring to the Clinical Laboratory Standards Institute (CLSI) guidelines. The antibiotics that will be used for AST are ceftriaxone (30 µg), ciprofloxacin (5 µg), meropenem (10 µg), vancomycin (30 µg), colistin (10 µg), and piperacillin/tazobactam (110 µg). For the bacteria that are not available in CLSI guidelines, measurement will refer to European Committee of Antimicrobial Susceptibility Testing (EUCAST) guidelines. The minimal inhibitory concentration of colistin will be determined using a liquid broth dilution test as recommended by EUCAST with cation-adjusted Mueller-Hinton broth (NutriSelect Plus, Germany), because the disk diffusion method is not reliable for this antibiotic since its large molecular size results in poor diffusion into the agar medium, leading to inaccurate results with a high error rate and poor reproducibility [45].

    ‎
    Figure 5. The number of plates to be used to isolate ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acitenobacter baumannii, Pseudomonas aeruginosa, and Enterobacter) pathogens from the hospital effluent based on the calculated sample size. See Table 2 for the selective media used for each species.
    Table 2. Selective media and the incubation condition to isolate pathogens from the effluent samples.
    Selective organismMediumIncubation condition
    Enterococcus faeciumChromogenic E. faecium Agar (Himedia, India)37°C, aerobic, 24-48 hours
    Staphylococcus aureusMannitol Salt Agar (BD, Germany)37°C, aerobic, 24-48 hours
    Klebsiella pneumoniaeEosin Methylene Blue Agar Modified (HHT) (BD-BBL, USA)37°C, aerobic, 24-48 hours
    Acinetobacter baumanniiChromagar Acinetobacter (CHROMagar, Paris France)37 °C, aerobic, 24-48 hours
    Pseudomonas aeruginosaCetrimide Agar (Neogen USA)37°C, aerobic, 24-48 hours
    EnterobacterMacConkey II Agar (BD-BBL, USA)37°C, aerobic, 24 hours
    Detection of ARGs Among ESKAPE Isolates

    Each colony found to be resistant to any of the tested antibiotics will be cultured and inoculated in tryptone soy broth medium (CM0129, Oxoid). The culture will be incubated at 37°C for 18-24 hours. The genomic DNA will be extracted using the PureGene Bacteria Kit (Qiagen, Germany) following the manufacturer’s instructions. The concentration of the extracted DNA will be measured using a Multiskan SkyHigh microplate spectrophotometer (Thermo Scientific) before multiplex PCR amplification. Primers were selected to amplify the targeted ARGs based on published findings [46-56] (see Multimedia Appendix 1). The extracted DNA will be amplified using multiplex PCR following the single-template PCR method. The PCR conditions will be optimized according to published methods. The size of the amplified products will be confirmed by 3% agarose gel (NextGene) electrophoresis at 90 V for 90 minutes. The PCR products will be sent to an outsourcing service for Sanger sequencing. The sequencing results will be analyzed using the Basic Local Alignment Search Tool nucleotide (BLASTn) tool in the National Center for Biotechnology Information database to identify the correct amplified relevant genes.

    Chemical Analysis

    Quantification of the Concentrations of Selected Antibiotics

    An ultrahigh-performance liquid chromatography (UHPLC) system will be used to quantify the levels of antibiotics from all samples following the method reported by Kim et al [57]. Samples will be collected and treated by preconcentration using a nitrogen evaporator. The samples will then be cleaned by an automated solid-phase extraction system. The analysis will be performed using Q-exactive liquid chromatography-mass spectrometry. External standards of antibiotics will include ceftriaxone, ciprofloxacin, meropenem, vancomycin, colistin, piperacillin, and tazobactam. The internal standards will be ciprofloxacin-d8 (molecular weight: 339.38) and piperacillin-d5 (molecular weight: 522.59). The analytical column will be Waters Xbridge C18 Column (50 mm × 2.1 mm, 2.5 µm inner diameter) run at 35°C. Two types of solvents will be used: 0.1% formic acid (solvent A) in water and 100% acetonitrile (solvent B). The solvent gradients with a flow rate of 0.2 mL/min for this experiment are shown in Table 3.

    Table 3. Solvent gradients with a flow rate of 0.2 mL/min.
    Time (minutes)Solvent A (0.1% formic acid), %Solvent B (100% acetonitrile), %
    0.0982
    4.93565
    13.5298
    21.0298
    21.5982
    26.098 2

    Data Analysis

    The prevalence of ARB will be calculated using the following formula:

    Prevalence (%)=(number of resistant isolates/number of resistant isolates + number of susceptible isolates) × 100

    To detect ARGs carried by ESKAPE bacteria, nucleotide results from the sequencing will be checked using Chromas, a DNA sequencing software to view and edit the chromatograms (traces) from automated Sanger sequencers. The edited chromatograms from the sequencing results will be sent to the BLASTn database to confirm the amplified genes.

    The concentration of the antibiotic residues quantified using UHPLC will be compared to the antibiotic usage in the hospitals.

    Ethics Considerations

    No primary data will be collected and thus no ethical approval is required. The study protocol was approved by the Medical Research and Ethical Committee, Ministry of Health Malaysia. This study is also registered under the National Medical Research Registry (NMRR-19-3637-51680) Malaysia.


    The primary outcome measure for this study is the prevalence of resistant ESKAPE bacteria isolated from antibiotic-selective media. The bacteria will be identified by 16S RNA PCR to determine species. The secondary outcome of this study is the occurrence of ARGs that may be discovered from the resistant ESKAPE bacteria isolates as the likely cause of the resistance toward the antibiotics (Table 2). The multiplex PCR method will be applied to screen the ARGs present. As of July 2022, effluent sampling has been conducted in three hospitals and analysis of samples is ongoing to determine the level of antibiotic residues. ESKAPE bacteria were isolated and AST is ongoing to determine the resistant isolates. Some preliminary findings for E. faecium from Hospital A showed that 80% (8/10) were resistant to vancomycin and 20% (2/10) were susceptible, whereas only 10% (1/10) were resistant, 60% (6/10) had intermediate resistance, and 30% (3/10) were susceptible to ciprofloxacin. Moreover, 100% (10/10) of E. faecium isolates from Hospital B were resistant toward vancomycin but susceptible toward ciprofloxacin. Resistant isolates will be processed for further screening of ARGs using PCR. Effluent sampling activities were suspended for a few months due to the COVID-19 pandemic, which caused a delay in the downstream analysis. The findings of the study will be disseminated through peer-reviewed journals and scientific conferences.


    VRE represent an issue of both medical and public health importance, as these bacteria are commonly associated with multidrug-resistant infections and persistent colonization in patients [58]. From our early findings, the prevalence of vancomycin-resistant E. faecium from two hospitals was more than 80%, which was higher than the prevalence of 11% reported in a study performed in the Czech Republic on isolates collected from Military Hospital Olomouc and University Hospital Olomouc [59].

    The outcomes of this study will fill the gap of important information for clinical practitioners to determine whether the hospitals in Malaysia are main contributors to the AMR problem in the country. This study will provide baseline information on the prevalence of antibiotic resistance from hospital effluents in Malaysia to assist in hospital wastewater management and improve antibiotic stewardship in Malaysia.

    This study will have an impact on the improvement of hospital sewage treatment because the data will provide information to the Engineering Service Division of the Ministry of Health that reflects the efficiency of the current sewage treatment in Ministry of Health hospitals. This should facilitate the hospital’s sewage manager in the Engineering Service Division in effective service improvement.

    Moreover, the findings from this study will help in the optimization of antimicrobial stewardship programs by recommending strategies for infectious disease physicians and pharmacists in their prospective audit and feedback of antimicrobial prescriptions to clinicians, formulary restriction, education, and guidelines. Antimicrobial stewardship programs are aiming to optimize antimicrobial selection, dosing, route, and duration of therapy to maximize a clinical cure or prevention of infection, while limiting unintended consequences such as the emergence of resistance, adverse drug events, and extra costs.

    Ultimately, this study will contribute to the improvement in public health. The improvement in the hospital sewage system will eventually lead to controlling antibiotics pollution in the environment by reducing the spread of clinically driven ARB and ARGs into the environment. This will in turn reduce the unnecessary demand for stronger antibiotics or even failure to treat many types of resistant infections.

    Acknowledgments

    We thank the Director General of Health, Ministry of Health, Malaysia for permission to publish this report. We also thank the Director of the Institute for Medical Research and Manager, National Institutes of Health Malaysia for their permission to conduct this study.

    Data Availability

    The authors confirm that the data supporting the findings of this study are available within the article and supplementary materials.

    Conflicts of Interest

    None declared.

    Multimedia Appendix 1

    Published primers representing the occurrence of bacterial DNA and antibiotic resistance genes.

    DOCX File , 19 KB
    1. Xiong W, Sun Y, Ding X, Wang M, Zeng Z. Selective pressure of antibiotics on ARGs and bacterial communities in manure-polluted freshwater-sediment microcosms. Front Microbiol 2015;6:194 [https://europepmc.org/abstract/MED/25814986] [CrossRef] [Medline]
    2. Lübbert C, Baars C, Dayakar A, Lippmann N, Rodloff AC, Kinzig M, et al. Environmental pollution with antimicrobial agents from bulk drug manufacturing industries in Hyderabad, South India, is associated with dissemination of extended-spectrum beta-lactamase and carbapenemase-producing pathogens. Infection 2017 Aug;45(4):479-491 [CrossRef] [Medline]
    3. Larsson DJ, de Pedro C, Paxeus N. Effluent from drug manufactures contains extremely high levels of pharmaceuticals. J Hazard Mater 2007 Sep 30;148(3):751-755 [CrossRef] [Medline]
    4. Umezawa K, Asai S, Ohshima T, Iwashita H, Ohashi M, Sasaki M, et al. Outbreak of drug-resistant Acinetobacter baumannii ST219 caused by oral care using tap water from contaminated hand hygiene sinks as a reservoir. Am J Infect Control 2015 Nov;43(11):1249-1251 [https://linkinghub.elsevier.com/retrieve/pii/S0196-6553(15)00699-9] [CrossRef] [Medline]
    5. Breathnach A, Cubbon M, Karunaharan R, Pope C, Planche T. Multidrug-resistant Pseudomonas aeruginosa outbreaks in two hospitals: association with contaminated hospital waste-water systems. J Hosp Infect 2012 Sep;82(1):19-24 [CrossRef] [Medline]
    6. Peleg AY, Hooper DC. Hospital-acquired infections due to gram-negative bacteria. N Engl J Med 2010 May 13;362(19):1804-1813 [https://europepmc.org/abstract/MED/20463340] [CrossRef] [Medline]
    7. Rasheed MU, Thajuddin N, Ahamed P, Teklemariam Z, Jamil K. Antimicrobial drug resistance in strains of Escherichia coli isolated from food sources. Rev Inst Med Trop Sao Paulo 2014;56(4):341-346 [https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46652014000400341&lng=en&nrm=iso&tlng=en] [CrossRef] [Medline]
    8. Morrill HJ, Morton JB, Caffrey AR, Jiang L, Dosa D, Mermel LA, et al. Antimicrobial resistance of Escherichia coli urinary isolates in the Veterans Affairs Health Care System. Antimicrob Agents Chemother 2017 May;61(5):e02236-16 [https://europepmc.org/abstract/MED/28193660] [CrossRef] [Medline]
    9. Korzeniewska E, Korzeniewska A, Harnisz M. Antibiotic resistant Escherichia coli in hospital and municipal sewage and their emission to the environment. Ecotoxicol Environ Saf 2013 May;91:96-102 [CrossRef] [Medline]
    10. Brown KD, Kulis J, Thomson B, Chapman TH, Mawhinney DB. Occurrence of antibiotics in hospital, residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico. Sci Total Environ 2006 Aug 01;366(2-3):772-783 [CrossRef] [Medline]
    11. Xiao M, Wang H, Zhao Y, Mao L, Brown M, Yu Y, et al. National surveillance of methicillin-resistant Staphylococcus aureus in China highlights a still-evolving epidemiology with 15 novel emerging multilocus sequence types. J Clin Microbiol 2013 Nov;51(11):3638-3644 [https://europepmc.org/abstract/MED/23985906] [CrossRef] [Medline]
    12. Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011 Oct;17(10):1791-1798 [https://europepmc.org/abstract/MED/22000347] [CrossRef] [Medline]
    13. Remschmidt C, Behnke M, Kola A, Peña Diaz LA, Rohde AM, Gastmeier P, et al. The effect of antibiotic use on prevalence of nosocomial vancomycin-resistant enterococci- an ecologic study. Antimicrob Resist Infect Control 2017;6:95 [https://aricjournal.biomedcentral.com/articles/10.1186/s13756-017-0253-5] [CrossRef] [Medline]
    14. Rice L. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 2008 Apr 15;197(8):1079-1081 [CrossRef] [Medline]
    15. Library of AMR national action plans. World Health Organization. URL: https:/​/www.​who.int/​teams/​surveillance-prevention-control-AMR/​national-action-plan-monitoring-evaluation/​library-of-national-action-plans [accessed 2023-04-19]
    16. Malaysian Action Plan on Antimicrobial Resistance (MyAP-AMR) 2017-2021. Ministry of Health, Malaysia and Ministry of Agriculture & Agro-Based Industry Malaysia. 2017. URL: https://faolex.fao.org/docs/pdf/mal189905.pdf [accessed 2023-04-19]
    17. Al-Odaini NA, Zakaria MP, Yaziz MI, Surif S. Multi-residue analytical method for human pharmaceuticals and synthetic hormones in river water and sewage effluents by solid-phase extraction and liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010 Oct 29;1217(44):6791-6806 [CrossRef] [Medline]
    18. Praveena SM, Shaifuddin SNM, Sukiman S, Nasir FAM, Hanafi Z, Kamarudin N, et al. Pharmaceuticals residues in selected tropical surface water bodies from Selangor (Malaysia): occurrence and potential risk assessments. Sci Total Environ 2018 Nov 15;642:230-240 [CrossRef] [Medline]
    19. Bong CW, Low KY, Chai LC, Lee CW. Prevalence and diversity of antibiotic resistant Escherichia coli from anthropogenic-impacted Larut River. Front Public Health 2022;10:794513 [https://europepmc.org/abstract/MED/35356018] [CrossRef] [Medline]
    20. Singer AC, Shaw H, Rhodes V, Hart A. Review of antimicrobial resistance in the environment and its relevance to environmental regulators. Front Microbiol 2016;7:1728 [https://europepmc.org/abstract/MED/27847505] [CrossRef] [Medline]
    21. Mughini-Gras L, Dorado-García A, van Duijkeren E, van den Bunt G, Dierikx CM, Bonten MJM, ESBL Attribution Consortium. Attributable sources of community-acquired carriage of Escherichia coli containing β-lactam antibiotic resistance genes: a population-based modelling study. Lancet Planet Health 2019 Aug;3(8):e357-e369 [https://linkinghub.elsevier.com/retrieve/pii/S2542-5196(19)30130-5] [CrossRef] [Medline]
    22. Kraemer SA, Ramachandran A, Perron GG. Antibiotic pollution in the environment: from microbial ecology to public policy. Microorganisms 2019 Jun 22;7(6):180 [https://www.mdpi.com/resolver?pii=microorganisms7060180] [CrossRef] [Medline]
    23. Stanton IC, Bethel A, Leonard AFC, Gaze WH, Garside R. Existing evidence on antibiotic resistance exposure and transmission to humans from the environment: a systematic map. Environ Evid 2022;11(1):8 [https://europepmc.org/abstract/MED/35308196] [CrossRef] [Medline]
    24. Attribution 4.0 International (CC BY 4.0). Creative Commons. URL: https://creativecommons.org/licenses/by/4.0/
    25. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022 Feb 12;399(10325):629-655 [https://linkinghub.elsevier.com/retrieve/pii/S0140-6736(21)02724-0] [CrossRef] [Medline]
    26. O'Neill J. Tackling drug-resistant infections globally: final report and recommendations. The Review on Antimicrobial Resistance. 2016 May. URL: https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf [accessed 2023-04-19]
    27. Infectious Diseases Society of America (IDSA), Spellberg B, Blaser M, Guidos R, Boucher H, Bradley J, et al. Combating antimicrobial resistance: policy recommendations to save lives. Clin Infect Dis 2011 May;52 Suppl 5(Suppl 5):S397-S428 [https://europepmc.org/abstract/MED/21474585] [CrossRef] [Medline]
    28. WHO publishes list of bacteria for which new antibiotics are urgently needed. World Health Organization. 2017 Feb 27. URL: https:/​/www.​who.int/​news/​item/​27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed [accessed 2023-04-19]
    29. Gandra S, Klein EY, Pant S, Malhotra-Kumar S, Laxminarayan R. Faropenem consumption is increasing in India. Clin Infect Dis 2016 Apr 15;62(8):1050-1052 [CrossRef] [Medline]
    30. Kakkar M, Walia K, Vong S, Chatterjee P, Sharma A. Antibiotic resistance and its containment in India. BMJ 2017 Sep 05;358:j2687 [CrossRef] [Medline]
    31. Sumpradit N, Wongkongkathep S, Poonpolsup S, Janejai N, Paveenkittiporn W, Boonyarit P, et al. New chapter in tackling antimicrobial resistance in Thailand. BMJ 2017 Sep 05;358:j3415 [http://www.bmj.com/lookup/pmidlookup?view=long&pmid=28874352] [CrossRef] [Medline]
    32. Tangcharoensathien V, Sommanustweechai A, Chanthong B, Sumpradit N, Sakulbumrungsil R, Jaroenpoj S, Thai SAC Working Group. Surveillance of antimicrobial consumption: methodological review for systems development in Thailand. J Glob Health 2017 Jun;7(1):010307 [https://europepmc.org/abstract/MED/28702173] [CrossRef] [Medline]
    33. Esuvaranathan K, Kuan Y, Kumarasinghe G, Bassett D, Rauff A. A study of 245 infected surgical wounds in Singapore. J Hosp Infect 1992 Jul;21(3):231-240 [CrossRef] [Medline]
    34. Hsu L, Tan T, Jureen R, Koh T, Krishnan P, Tzer-Pin Lin R, et al. Antimicrobial drug resistance in Singapore hospitals. Emerg Infect Dis 2007 Dec;13(12):1944-1947 [https://europepmc.org/abstract/MED/18258055] [CrossRef] [Medline]
    35. Chow A, Lim V, Khan A, Pettigrew K, Lye D, Kanagasabai K, et al. MRSA transmission dynamics among interconnected acute, intermediate-term, and long-term healthcare facilities in Singapore. Clin Infect Dis 2017 May 15;64(suppl_2):S76-S81 [https://europepmc.org/abstract/MED/28475785] [CrossRef] [Medline]
    36. Aung KT, Hsu LY, Koh TH, Hapuarachchi HC, Chau ML, Gutiérrez RA, et al. Prevalence of methicillin-resistant (MRSA) in retail food in Singapore. Antimicrob Resist Infect Control 2017;6:94 [https://aricjournal.biomedcentral.com/articles/10.1186/s13756-017-0255-3] [CrossRef] [Medline]
    37. Shankar N, Chow ALP, Oon J, Hsu LY, Ang B, Pang J, et al. The epidemiology and transmission of methicillin-resistant Staphylococcus aureus in the community in Singapore: study protocol for a longitudinal household study. BMC Infect Dis 2017 Oct 11;17(1):678 [https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-017-2793-y] [CrossRef] [Medline]
    38. Sergio D, Koh T, Hsu L, Ogden B, Goh A, Chow PKH. Investigation of meticillin-resistant Staphylococcus aureus in pigs used for research. J Med Microbiol 2007 Aug;56(Pt 8):1107-1109 [CrossRef] [Medline]
    39. Daniel WW. Biostatistics: a foundation for analysis in the health sciences, 7th edition. New York: John Wiley & Sons; 1999.
    40. Haller L, Chen H, Ng C, Le TH, Koh TH, Barkham T, et al. Occurrence and characteristics of extended-spectrum β-lactamase- and carbapenemase- producing bacteria from hospital effluents in Singapore. Sci Total Environ 2018 Feb 15;615:1119-1125 [CrossRef] [Medline]
    41. Hrenovic J, Goic-Barisic I, Kazazic S, Kovacic A, Ganjto M, Tonkic M. Carbapenem-resistant isolates of Acinetobacter baumannii in a municipal wastewater treatment plant, Croatia, 2014. Euro Surveill 2016 Apr 14;21(15):30195 [http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=21440] [CrossRef] [Medline]
    42. Thompson J, Gündoğdu A, Stratton H, Katouli M. Antibiotic resistant Staphylococcus aureus in hospital wastewaters and sewage treatment plants with special reference to methicillin-resistant Staphylococcus aureus (MRSA). J Appl Microbiol 2013 Jan;114(1):44-54 [https://onlinelibrary.wiley.com/doi/10.1111/jam.12037] [CrossRef] [Medline]
    43. Varela AR, Ferro G, Vredenburg J, Yanık M, Vieira L, Rizzo L, et al. Vancomycin resistant enterococci: from the hospital effluent to the urban wastewater treatment plant. Sci Total Environ 2013 Apr 15;450-451:155-161 [CrossRef] [Medline]
    44. Karimi F, Samarghandi MR, Shokoohi R, Godini K, Arabestani MR. Prevalence and removal efficiency of enterococcal species and vancomycin-resistant enterococci of a hospital wastewater treatment plant. Avicenna J Environ Health Eng 2016 Dec 27;3(2):e8623 [CrossRef]
    45. Humphries RM. Susceptibility testing of the polymyxins: where are we now? Pharmacotherapy 2015 Jan;35(1):22-27 [CrossRef] [Medline]
    46. Bergeron S, Raj B, Nathaniel R, Corbin A, LaFleur G. Presence of antibiotic resistance genes in raw source water of a drinking water treatment plant in a rural community of USA. Int Biodeterior Biodegrad 2017 Oct;124:3-9 [CrossRef]
    47. Shanthi M, Sekar U, Kamalanathan A, Sekar B. Detection of New Delhi metallo beta lactamase-1 (NDM-1) carbapenemase in Pseudomonas aeruginosa in a single centre in southern India. Indian J Med Res 2014 Oct;140(4):546-550 [https://europepmc.org/abstract/MED/25488450] [Medline]
    48. Marti E, Jofre J, Balcazar JL. Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant. PLoS One 2013;8(10):e78906 [https://dx.plos.org/10.1371/journal.pone.0078906] [CrossRef] [Medline]
    49. Mlynarcik P, Roderova M, Kolar M. Primer evaluation for PCR and its application for detection of carbapenemases in Enterobacteriaceae. Jundishapur J Microbiol 2016 Jan;9(1):e29314 [https://europepmc.org/abstract/MED/27099689] [CrossRef] [Medline]
    50. Azimian A, Havaei SA, Fazeli H, Naderi M, Ghazvini K, Samiee SM, et al. Genetic characterization of a vancomycin-resistant Staphylococcus aureus isolate from the respiratory tract of a patient in a university hospital in northeastern Iran. J Clin Microbiol 2012 Nov;50(11):3581-3585 [https://europepmc.org/abstract/MED/22933598] [CrossRef] [Medline]
    51. Kafil HS, Asgharzadeh M. Vancomycin-resistant enteroccus faecium and enterococcus faecalis isolated from education hospital of iran. Maedica (Bucur) 2014 Dec;9(4):323-327 [https://europepmc.org/abstract/MED/25705299] [Medline]
    52. Saadat S, Solhjoo K, Norooz-Nejad M, Kazemi A. VanA and VanB positive vancomycin-resistant Staphylococcus aureus among clinical isolates in Shiraz, South of Iran. Oman Med J 2014 Sep;29(5):335-339 [https://europepmc.org/abstract/MED/25337309] [CrossRef] [Medline]
    53. Praharaj I, Sujatha S, Parija SC. Indian J Med Res 2013 Oct;138(4):549-556 [https://europepmc.org/abstract/MED/24434263] [Medline]
    54. Zhang J, Wang J, Chen L, Yassin AK, Kelly P, Butaye P, et al. Housefly (Musca domestica) and blow fly (Protophormia terraenovae) as vectors of bacteria carrying colistin resistance genes. Appl Environ Microbiol 2018 Jan 01;84(1):e01736-17 [https://europepmc.org/abstract/MED/29030447] [CrossRef] [Medline]
    55. Goldstein C, Lee MD, Sanchez S, Hudson C, Phillips B, Register B, et al. Incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. Antimicrob Agents Chemother 2001 Mar;45(3):723-726 [https://europepmc.org/abstract/MED/11181350] [CrossRef] [Medline]
    56. Wang M, Sahm DF, Jacoby GA, Hooper DC. Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States. Antimicrob Agents Chemother 2004 Apr;48(4):1295-1299 [https://europepmc.org/abstract/MED/15047532] [CrossRef] [Medline]
    57. Kim C, Ryu H, Chung EG, Kim Y. Determination of 18 veterinary antibiotics in environmental water using high-performance liquid chromatography-q-orbitrap combined with on-line solid-phase extraction. J Chromatogr B Analyt Technol Biomed Life Sci 2018 May 01;1084:158-165 [CrossRef] [Medline]
    58. Lee T, Pang S, Abraham S, Coombs GW. Antimicrobial-resistant CC17 Enterococcus faecium: the past, the present and the future. J Glob Antimicrob Resist 2019 Mar;16:36-47 [CrossRef] [Medline]
    59. Hricová K, Röderová M, Fryčák P, Pauk V, Kurka O, Mezerová K, et al. Prevalence of vancomycin-resistant enterococci and antimicrobial residues in wastewater and surface water. Life 2021 Dec 15;11(12):1403 [https://www.mdpi.com/resolver?pii=life11121403] [CrossRef] [Medline]

    ‎
    AMR: antimicrobial resistance
    ARB: antimicrobial-resistant bacteria
    ARG: antibacterial resistance gene
    AST: antibiotic susceptibility testing
    BLASTn: Basic Local Alignment Search Tool nucleotide
    CLSI: Clinical Laboratory Standards Institute
    CRE: carbapenem-resistant Enterobacteriaceae
    ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae
    EUCAST: European Committee of Antimicrobial Susceptibility Testing
    GLASS: Global Antimicrobial Resistance Surveillance System
    HAI: hospital-acquired infection
    MRSA: methicillin-resistant Staphylococcus aureus
    MyAP-AMR: Malaysian Action Plan on Antimicrobial Resistance
    PCR: polymerase chain reaction
    UHPLC: ultrahigh-performance liquid chromatography
    VRE: vancomycin-resistant enterococci
    WHO: World Health Organization

    Edited by A Mavragani; submitted 29.08.22; peer-reviewed by G Penalva, N Mohamad Jamil, P Wiśniewski; comments to author 30.09.22; revised version received 24.02.23; accepted 28.02.23; published 29.05.23

    Copyright

    ©Sophia Karen Bakon, Zuraifah Asrah Mohamad, Mohd Azerulazree Jamilan, Hazimah Hashim, Mohamed Yazid Kuman, Rafiza Shaharudin, Norazah Ahmad, Nor Asiah Muhamad. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 29.05.2023.

    This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included.