INTRODUCTION
Antimicrobial resistance (AMR), the capacity of bacteria to resist antimicrobial treatments, is recognized as a critical global health challenge that requires urgent monitoring as of 2021 (WHO, 2020) The worldwide rise and dissemination of AMR pose a significant threat, leading to a shortage of effective treatments for infections caused by resistant bacteria The severity of AMR is underscored by predictions estimating that it could result in 10 million deaths by 2050 (O’Neill, 2014).
AMR will continue remaining as a key challenge to human health in the years ahead
To address the challenge of antimicrobial resistance (AMR), the United Nations advocates for the One Health approach, which emphasizes collaboration among agencies focused on human, animal, and environmental health The environment, particularly water, significantly contributes to the emergence and spread of AMR, serving as both a reservoir for AMR discharge from humans and animals and a source for agricultural irrigation and recreation Notably, inadequate wastewater treatment exacerbates this issue, with only 13% of wastewater in Vietnam being treated, while the remaining 87% is released untreated into the environment (Bộ Tài nguyên và Môi trường, 2018).
Antimicrobial resistance (AMR) is a One Health issue that necessitates a comprehensive multisectoral surveillance system, which is currently inadequate To address this gap, the World Health Organization (WHO) has introduced the Tricycle protocol, focusing on the surveillance of extended-spectrum β-lactamase-producing Escherichia coli (ESBL E coli), a specific bacterium with a defined resistance mechanism The Tricycle approach emphasizes data collection across three sectors: human health, the food chain (animal health), and environmental monitoring However, it is important to note that ESBL E coli alone does not fully capture the global AMR landscape, as numerous other infectious microorganisms and resistance mechanisms also contribute to the problem.
2 resistant traits However, ESBL E coli were selected as the target of the surveillance protocol based on the following reasons (World Health Organization, 2021):
(i) Existence of great variation in the rate of ESBL E coli colonization in humans and among countries, as well as the prevalence over time
(ii) Existence of ESBL E coli among farm animals
There is evidence indicating that certain human deaths associated with ESBL E coli can be attributed to antibiotic use in food production or the presence of ESBL E coli in the environment.
(iv) Interventions that aim to decrease antibiotics use or exposure in human and animals have been accompanied with the decline in in ESBL E coli occurrence rates
(v) ESBL production is an important resistant mechanism that makes critically important antimicrobials ineffective
Figure 1.1 Transmission of AMR in One Health approach
Research on ESBL E coli in Vietnam has primarily concentrated on its presence in humans and animals, with limited attention given to the water environment Notably, only two studies have documented the occurrence of ESBL E coli in pig farms and slaughterhouses.
Recent studies on wastewater in Vietnam have highlighted the presence of ESBL E coli isolates (Hinenoya et al., 2018; Nguyen et al., 2021) However, the limited number of isolates examined has left the overall occurrence of ESBL in the country's water environment still unclear.
This thesis research in Environmental Engineering investigates the characteristics of extended-spectrum β-lactamase-producing Escherichia coli (ESBL E coli) in the urban water environment of Northern Vietnam, aligning with the WHO Tricycle Project to address existing research gaps The study focuses on cefotaxime-resistant E coli, which are identified as ESBL E coli, and aims to provide valuable insights into their prevalence and implications for public health.
(i) Determine the characteristics of ESBL E coli in different urban water environment in Northern Vietnam,
(ii) Evaluate the role of wastewater treatment plant to reduce ESBL E coli discharge into the water environment h
LITERATURE REVIEW
Antimicrobial resistance
Antimicrobials, commonly known as antibiotics, are effective treatments for bacterial infections by either killing bacteria or inhibiting their growth These antibiotics are classified according to their action on specific sites within the bacterial cell, primarily targeting components such as the cell wall, cell membrane, ribosomes, and nucleic acids (Sauberan and Bradley, 2018) The specific targets of each antibiotic are illustrated in Figure 2.1.
Figure 2.1 Antibiotics and its target sites on bacterial cells
The discovery of antibiotics in the early 20th century marked a significant milestone in human history, revolutionizing the treatment of bacterial infections and saving countless lives However, the effectiveness of antibiotics has diminished over time due to the emergence of antibiotic-resistant bacteria, posing a serious challenge to public health.
Antimicrobial resistance (AMR) refers to the ability of bacteria to survive despite the presence of antibiotics While antibiotics effectively eliminate susceptible strains, resistant bacteria thrive without competition, leading to a rapid increase in resistance to these drugs.
Although development of resistance in bacteria is a natural selection process, it is accelerated by misuse and overuse of antibiotics in human and food animal production
The emergence of antimicrobial resistance (AMR) is outpacing the development of new antibiotics, with discoveries plummeting since the 1980s This situation has led to a significant shortage of effective treatments for drug-resistant bacterial infections, forcing doctors to prescribe previously avoided antibiotics, such as colistin, despite their severe side effects, including kidney failure Alarmingly, resistance to colistin has already been reported Moreover, while resistant infections were once primarily confined to hospital settings, they have increasingly been observed in community environments over the past decade.
Antimicrobial resistance (AMR) is a significant global health challenge, leading to approximately 700,000 deaths annually due to infections caused by drug-resistant bacteria The economic burden of AMR is substantial, with infections resulting in longer hospital stays and increased treatment costs In the United States alone, two million AMR infections contribute an excess of $20 billion to the healthcare system each year.
2.1.2 Molecular genetics of antimicrobial resistance
Antimicrobial resistance genes (ARG) can be found in both chromosomal and plasmid DNA The emergence of these genes occurs through three main processes: microevolutionary changes, macroevolutionary changes, and the acquisition of large segments of foreign DNA.
Microevolutionary changes, such as point mutations, can alter the target site or enzyme-substrate specificity of antibiotics For instance, mutations in traditional β-lactamase genes like blaTEM-1 and blaSHV-1 have led to the emergence of extended-spectrum β-lactamases (ESBLs) (Opal and Pop-Vicas, 2014).
Macroevolutionary change occurs when large segments of DNA undergo significant rearrangements in a single event, which can include inversions, duplications, insertions, or transpositions Notably, transpositions are driven by specific genetic elements known as transposons.
The final stage in the development of antimicrobial resistance involves the acquisition of substantial segments of foreign DNA, which can be transported by plasmids, bacteriophages, free DNA sequences, or mobile genetic elements This process, known as horizontal gene transfer (HGT), refers to the release and uptake of foreign DNA (Opal and Pop-Vicas, 2014) HGT encompasses several mechanisms that facilitate the spread of resistance traits among bacteria.
(i) Transformation: Bacteria uptakes of DNA from the surroundings, these free
DNA are released from dead bacterial cell (see in Figure 2.2) (Opal and Pop-Vicas, 2014)
Bacteria can acquire foreign DNA through two primary mechanisms: transduction and conjugation In transduction, bacteriophages act as carriers, transferring genetic material from a donor cell to a recipient cell Conversely, during conjugation, bacteria exchange plasmid DNA directly through a mating bridge that connects the two cells.
Figure 2.2 Horizontal gene transfer in bacteria h
HGT is largely responsible for the wide spread of AMR Once antimicrobial resistance genes (ARGs) are emerged, they can be spread among bacteria through HGT
Plasmids are circular double-stranded DNA molecules prevalent in bacteria, capable of existing in multiple copies or different types within a single bacterial cell They can replicate independently of chromosomal DNA, and smaller plasmids can be transferred between bacteria through conjugation This unique ability contributes to the rapid spread of plasmid-borne antibiotic resistance genes among bacterial populations.
Transposon is a kind of transposable genetic element They are small pieces of DNA that encode functional genes, for example, antibiotic resistance It can translocate (or
“jump”) between the chromosome and plasmid and vice versa (Opal and Pop-Vicas,
Transposons, which cannot replicate independently, rely on replicons like chromosomes or plasmids for their existence Their transposition is a continuous process that significantly contributes to bacterial evolution and plays a crucial role in the maintenance of antibiotic resistance genes (ARG).
Integrons, also known as gene cassettes, are genetic elements capable of integrating, exchanging, and expressing specific DNA sequences They can incorporate multiple gene cassettes, allowing them to carry various antimicrobial resistance genes within a single integron Although integrons themselves are not mobile genetic elements due to the absence of self-mobility genes, they can be mobilized when located on plasmids or transposons Additionally, integrons may also reside on chromosomes and can be acquired through transformation.
Until now, eight mechanisms of antibiotic resistance have been discovered in bacteria (Opal and Pop-Vicas, 2014), which are:
(i) enzyme inactivation: bacteria produce enzyme that can inactivate the drug, h
(ii) decrease membrane permeability: bacteria change the permeability of its cell membrane (either outer membrane or inner membrane or both) so that the drug cannot enter the cell,
Bacterial cells utilize active efflux pumps as a membrane transport system to expel drugs immediately after their entry These pumps can be either universal, capable of removing multiple classes of antibiotics, or specific to certain drugs Efflux pumps play a significant role in the development of multi-drug resistance in bacteria.
Bacteria can alter the target sites of drugs, such as ribosomal binding sites, cell wall binding sites, or target enzymes, preventing the drugs from effectively binding and reacting with these targets.
(v) protection of target site: the bacteria cell produces an enzyme that prevent the drug from binding to the target site,
(vi) overproduction of target: bacteria cell produces an excess amount of the target so that the drug cannot bind to all these targets,
Antimicrobial resistance in the water environment
The water environment plays a crucial role in the reservoir and transmission of antimicrobial resistance (AMR), receiving antibiotic residues, antibiotic-resistant genes (ARG), and antimicrobial-resistant bacteria (ARB) primarily from human and animal sources The use of antibiotics, along with their residues in food, exerts selection pressure on intestinal bacteria, fostering the development of AMR (Amarasiri et al., 2020) Additionally, consuming food contaminated with ARG or ARB can facilitate horizontal gene transfer (HGT) with normal organisms in the gastrointestinal tract (Amarasiri et al., 2020) Ultimately, these AMR elements are excreted in feces and find their way into wastewater systems.
Wastewater treatment plants – hot spots of AMR
Wastewater treatment plants (WWTPs) are identified as hotspots for the emergence of new antimicrobial resistance (AMR) mechanisms due to their receipt of wastewater from various sources, including domestic, hospital, and agricultural discharges The presence of contaminants such as microbes, antibiotic residues, and metals creates selection pressure that facilitates horizontal gene transfer Research indicates that WWTPs are unable to fully eliminate antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARG), and antibiotic residues, resulting in these AMR elements being released into surface water This release contributes to the development of antibiotic resistance among natural microorganisms.
Currently, wastewater treatment plants (WWTPs) primarily focus on controlling bacteria, often overlooking antibiotic-resistant genes (ARGs) The control techniques can be categorized into removal processes, such as pretreatment, filtration, and coagulation-flocculation, and inactivation processes, which utilize strong oxidizing agents like chlorine, chlorine dioxide, ozone, or UV light Disinfection plays a vital role in reducing microbial presence in WWTPs The efficiency of these removal techniques varies based on factors like chemical dosages and contact time, making the selection of appropriate treatment processes essential for minimizing antimicrobial resistance (AMR) loads released into the environment This is particularly important as reclaimed water is utilized for various purposes, including agricultural irrigation, aquaculture, and recreational activities, potentially exposing humans to AMR elements through sports, irrigation, or consumption of food irrigated with reclaimed water.
Occurrence of extended-spectrum -lactamase-producing Escherichia coli (ESBL E coli)
2.4.1 Extended-spectrum -lactamase-producing Escherichia coli
Escherichia coli (E coli), a member of the Enterobacteriaceae family, can be categorized into three main groups: (i) harmless commensal strains that contribute to the normal microbiota of the gastrointestinal tract, (ii) pathogenic strains responsible for diarrheal diseases, and (iii) strains that can cause urinary tract infections and other serious health issues.
12 intestinal disease, and (iii) strains that cause extraintestinal infections (Poolman, 2016)
E coli are the main agent causing diarrheal diseases, which accounts for around 9% children death worldwide (Poolman, 2016) It is also estimated that approximately 80% of bacterial-related diarrheal disease in developing countries are caused by the diarrheagenic E coli (Poolman, 2016) Infections caused by E coli are caused by several ways: via contact between person affected or via transmission from animals, food chains or unsanitary water
The genes determining AMR in E coli are likely to be found in pathogenicity island (which commensal E coli are lack of) and mobile genetic elements (MGEs) (Poolman,
2016) These AMR-encoding elements have been found in other pathogenic species, which suggest the history of genetic transfer and/or exchange (Poolman, 2016)
ESBL E coli is a critical antibiotic-resistant bacterium that exhibits resistance to essential antibiotics The rise of ESBL E coli is largely due to the extensive use of extended-spectrum β-lactam antibiotics (Chong et al., 2018) ESBL refers to a group of enzymes capable of inactivating β-lactam antibiotics, which are discussed in more detail in section 2.4.3.
β-lactams are crucial antibiotics that inhibit bacterial cell wall formation by targeting the β-lactam ring, which is essential for their activity This ring disrupts cell wall synthesis by binding to penicillin-binding proteins (PBPs), the enzymes responsible for constructing the cell wall The bacterial cell wall is vital for maintaining cellular structure, protecting organelles, and facilitating cell division Consequently, when β-lactams bind to PBPs, the assembly and maintenance of the cell wall are hindered, ultimately leading to bacterial cell death.
β-lactam antibiotics, like other antibiotics, have been modified to broaden their activity spectrum, improve pharmacokinetics, and combat the rise of antimicrobial resistance These antibiotics are categorized according to their functional groups.
There are 13 groups that attach to the β-lactam ring, leading to the classification of four main subclasses of β-lactam antibiotics used in humans: penicillins, cephalosporins, carbapenems, and monocyclic β-lactams The chemical structures of these antibiotics are illustrated in Figure 2.3.
Figure 2.3 lactam ring (i) and its subclasses: (ii) Penicillins, (iii) Cephalosporins, (iv) Carbapenems, and (v) Monocyclic -lactams
Penicillins, the first discovered β-lactams, are regarded as one of the safest antimicrobial agents alongside cephalosporins These non-toxic antibiotics feature β-lactam rings that are susceptible to hydrolysis by various β-lactamases, enzymes that can degrade the β-lactam structure While penicillins have a narrow spectrum of activity, they are particularly effective against Gram-positive bacteria compared to Gram-negative bacteria.
Cephalosporins are categorized into generations according to their spectrum of activity and stability against β-lactamases While later generations may show reduced effectiveness against Gram-positive bacteria, they enhance their bactericidal properties against Gram-negative bacteria.
14 and higher generations are relatively stable to -lactamases Detail of the spectrums of each generation are expressed in Table 2.2
Table 2.2 Classification of cephalosporins (Nguyễn, 2011)
- More effective against Gram-positive bacteria, somewhat effective against some Gram-negative bacteria
- Unstable, easily being hydrolyzed by -lactamases
- Effective against both Gram-negative and Gram-positive bacteria
- Less effect against Gram-positive bacteria compared to 1 st generation
- Broad-spectrum, less active than 1 st generations against
- More active to MDR bacteria
- More stable to -lactamases than 2 nd generation
- Similar effect on Gram-negative as 3 rd generation, more effectively against Gram-positive in comparison with 3 rd generation
- Stable to a wide range of -lactamases
Carbapenems are potent antibiotics that can bind to multiple types of penicillin-binding proteins (PBPs), enhancing their bactericidal effects and reducing the likelihood of resistance development Recognized as "last-resort" antibiotics, they are crucial for treating infections caused by multidrug-resistant bacteria Notably, carbapenems exhibit remarkable stability against nearly all β-lactamases, with the exception of carbapenemases.
Monocyclic β-lactams, commonly referred to as monobactams, include the FDA-approved antibiotic Aztreonam, which is effective against aerobic enteric bacteria This antibiotic demonstrates stability against most β-lactamases, with the exception of extended-spectrum β-lactamases (ESBLs) and carbapenemases.
In the 1960s, scientists began developing compounds to inhibit β-lactams in response to the rise of β-lactamase-producing pathogens (Bush and Bradford, 2016) Currently, four main β-lactamase inhibitors are utilized in human medicine: clavulanic acid, sulbactam, tazobactam, and avibactam These inhibitors share structural similarities with penicillin but have a weaker impact on bacterial cell wall formation When combined with other β-lactams, they can irreversibly bind to β-lactamases, protecting β-lactams from hydrolysis However, the effectiveness and substrate range of these inhibitors vary significantly (Toussaint and Gallagher, 2015).
A global report on antibiotic consumption across 55 countries revealed that β-lactams are the most commonly used class of antibiotics, according to the World Health Organization (2018) In the United States, β-lactams represent 65% of prescribed injectable antibiotics, with nearly half of these prescriptions being for cephalosporins (Bush and Bradford).
2016) In Vietnam, -lactams are also the most prescribed antibiotics in the hospital h
Second and third generation cephalosporins, along with carbapenems, are classified as β-lactams (Binh et al., 2018) In Vietnam, the practice of using medications without a prescription is prevalent, with 71% of patients having taken antibiotics prior to hospital visits, and 76% of these antibiotics being β-lactams (Binh et al., 2018).
Resistance to β-lactam antimicrobials primarily arises from the production of β-lactamases, enzymes that disrupt the β-lactam ring structure of these drugs, rendering them ineffective The rapid proliferation of β-lactamases is largely due to their presence on mobile genetic elements, including plasmids and transposons.
β-lactamases existed in the natural environment long before the introduction of β-lactam antibiotics in humans Initially, these "old" β-lactamases were rare, but their prevalence increased significantly with the widespread use of β-lactams in both humans and animals (Wilke et al., 2005) Extended spectrum β-lactamases (ESBL) are now recognized as the "new" variants of these enzymes.
METHODS
Sampling
In accordance with the Tricycle project's guidelines, water samples were collected in two cities, Hanoi and Bac Ninh, following the surveillance protocols outlined in Working Package 3 by the World Health Organization (2021).
Three types of water were collected:
(i) River water in upstream: this sample represents the pre-city impact
(ii) River water in downstream: this sample represents the impact of the city (iii) Human communal wastewater: this sample represents the intestinal microbiome of the city’s population
Extended samples of effluent from wastewater treatment plants were collected during each sampling event In Bac Ninh, samples were taken from the stabilization pond prior to discharge into the agricultural canal, with no disinfection applied at this facility In Hanoi, samples were collected immediately after discharge into surface water Detailed information about the sampling sites can be found in Table 3.1.
Table 3.1 Sampling points in Hanoi and Bac Ninh
Hanoi Urban drainage To Lich river
Kim Nguu river (before WWTP) Upstream surface water Nhue river upstream
Downstream surface water Nhue river in urban residence Effluent WWTP Kim Nguu river (after WWTP) h
Bac Ninh Urban drainage Influent WWTP
Upstream surface water Ngu Huyen Khe river (upstream) Downstream surface water Ngu Huyen Khe river (downstream)
Location of the sampling sites are shown in Figure 3.1
Figure 3.1 Sampling sites in Hanoi and Bac Ninh
In Northern Vietnam, a study was conducted to compare the occurrence of ESBL E coli, which involved regular sampling in Hanoi and Bac Ninh, along with the collection of surface water samples from additional cities including Ninh Binh, Nam Dinh, Thai Binh, and Hung Yen between September and October 2020 Detailed information regarding the samples is provided in Table A3.
In order to study how much ESBL E coli can be removed from wastewater, besides the samples described in Table 3.1, extended sampling events were caried out from h
From October to December 2020, influent and effluent samples were collected from Yen So WWTP in Hanoi, Bay Mau WWTP in Hanoi, and Vinh Niem WWTP in Hai Phong, as detailed in Table A4 Yen So WWTP utilized UV light disinfection, whereas Vinh Niem and Bay Mau WWTPs employed chlorine disinfection methods.
From September 2020 to May 2021, sampling was conducted on a monthly basis in Hanoi and every two months in Bac Ninh However, the sampling event in Bac Ninh was canceled in May 2021 due to the outbreak of Covid-19 in the community.
Autoclaved Duran ® original GL45 bottles (500 mL or 250 mL) or SPL ® Wide-mouth
PP bottles (500 mL or 250 mL), tissue paper, insulated bag, cooling gel ice, label, pH strips, waterproof conductivity meter AS650, clean bucket tied with rope, clean water, gloves
To ensure accurate sampling, begin by collecting a water sample using a clean bucket while wearing gloves to reduce infection risk Take the sample from approximately 20 cm below the surface and transfer it into an autoclaved bottle, sealing it tightly to prevent leakage Rinse the exterior of the bottle with clean water and dry it with tissue paper Record critical parameters such as temperature, conductivity, and pH from the remaining water in the bucket, and label the bottle with this information Finally, place the sample in an insulated bag with gel ice packs and transport it to the laboratory promptly, ensuring the temperature remains below 4°C, and aim to analyze the sample within 24 hours for optimal results.
Note: to prevent the cross-contamination from the previous sample, rinse the collecting bucket with the water from the subsequent sampling point, prior to sampling.
Quantification of E coli and ESBL E coli (cefotaxime-resistant E coli)
The concentration of ESBL E coli and E coli in the water samples were determined by the membrane filtration method
E coli and ESBL E coli quantification is performed within 24 hours of sample collection Initially, samples undergo a 10-fold serial dilution, followed by membrane filtration to capture the bacteria on the filter's surface The filter is then incubated on a suitable medium to allow for enumeration the following day.
In this experiment, two types of agars are used for each sample:
(i) For quantification of E coli: Tryptone Bile X-glucuronide (TBX) agar
(ii) For quantification of ESBL E coli: TBX supplemented with cefotaxime
The concentration of CTX used in TBX medium is set at 4 µg/mL, which aligns with the minimum inhibitory concentration (MIC) breakpoint established by the Clinical and Laboratory Standards Institute (CLSI) for verifying ESBL E coli (CLSI, 2021).
Material o Phosphate buffer saline (PBS) solution: Phosphate Buffer Saline powder (Wako Pure Chemical Industries, Ltd.) is used to prepare PBS solution with the ratio of
For each liter of double-distilled water, add one bag of PBS solution, dispensing 9 mL into each 25-mL test tube Seal the test tubes and autoclave them at 121°C for 15 minutes Allow the test tubes to cool and store them at room temperature until needed Additionally, ensure the use of sterilized pipette tips and a micropipette, both with a capacity of 1000 µL.
To prepare the sample for analysis, begin by removing it from the insulated cooling bag or refrigerator and thoroughly mix it to resuspend any sediments Next, pipette 1 mL of the sample into 9 mL of PBS and mix the dilution by hand Discard the pipette tip and use a new one to transfer 1 mL of this dilution for the next dilution, repeating this process until all expected dilutions are prepared.
The study utilized an autoclaved membrane filtration apparatus, vacuum pump, and sterile materials, including a 0.45 µm pore size membrane filter and sterile pipette tips, to conduct serial dilutions of samples on Chromocult TBX agar plates Each sample underwent two to four ten-fold serial dilutions, while a cefotaxime stock solution was prepared by dissolving 160 mg of cefotaxime sodium salt in 40 mL of double-distilled water, filtered through a 0.2 µm membrane filter, and stored in a refrigerator Fresh stock solutions were made monthly The agar plates were created by dissolving 31.6 g of Chromocult TBX agar powder in 1 liter of double-distilled water, boiling the mixture, autoclaving it at 120°C for 15 minutes, and then pouring it into plates after cooling to about 50°C For plates supplemented with cefotaxime, the stock solution was added to achieve a final concentration of 4 µg/mL in the agar.
30 when the medium has cooled down to 50-55°C, agitate to dissolve evenly, then pour the medium into sterile plates (12-15 mL/plate)
To perform the filtration procedure, begin by using sterile forceps to position a 0.45 µm filter, grid side up, onto the porous filter base, ensuring no air is trapped beneath it Attach the funnel to the filter base and stabilize the system with a clamp If filtering less than 10 mL, add 10 mL of sterile PBS solution to the filter and close the vacuum valve Next, using the highest dilution, pipette the sample onto the filter without contacting the tip to the surface, and apply vacuum until the sample is fully filtered, avoiding vacuum application when no liquid remains Aseptically remove the funnel and place the membrane filter, grid side up, onto an agar plate, filtering each dilution in triplicate and rinsing the funnel and filter base with sterile PBS Repeat this process for the next dilution with a new sterile filtration system After filtering, invert the agar plates and incubate them overnight at 37°C for 16-18 hours Post-incubation, E coli colonies will appear blue or blue-green on TBX agar, while ESBL E coli will show the same coloration on TBX agar supplemented with cefotaxime Record the number of colonies, and calculate the colony-forming unit (CFU) concentration as CFU/100 mL from plates with countable colonies (greater than 10).
Figure 3.2 E coli appears as blue-green colony on TBX agar plate
The resistance ratio of each sample is calculated by dividing the concentration of ESBL E coli (cefotaxime-resistant E coli) to the concentration of E coli
Blue-green colonies growing on TBX agar is purified for further experiment using streak plate method Each colonies is purified at least twice
Blue-green colonies on TBX agar are analyzed for species identification to verify the presence of ESBL E coli The identification process utilizes MALDI-TOF MS technology.
MALDI-TOF mass spectrometry is an innovative technology that enables the swift and accurate identification of bacterial species This system analyzes the distinctive ribosomal protein peaks of bacteria and identifies their species by comparing these peaks to a comprehensive reference library.
Bruker MALDI Biotyper (Model: Microflex LT/SH)
MALDI Biotyper Compass Software o Experimental consumables:
Clean reusable MALDI target plates
Sterile wooden applicator sticks (toothpicks)
To utilize the Bruker MALDI Biotyper (Microflex LT/SH) for colony identification, begin by preparing a fresh overnight culture of the isolates Using a sterile toothpick, apply a small amount of the tested colony onto a designated spot on the target plate Next, add 1µL of 70% formic acid to the spot and place the plate on a heater to facilitate quick evaporation of the acid Once dry, add 1µL of matrix to the spot and allow it to dry completely Finally, insert the target plate into the MALDI-TOF MS machine and initiate the analysis for colony identification, followed by interpreting the results.
Table 3.2 MALDI-TOF MS result interpretation
0.00 – 1.69 No organism identification possible red
Figure 3.4 Result of species identification by MALDI-TOF MS
Antimicrobial susceptibility testing
The antimicrobial susceptibility of ESBL E coli isolates is assessed using the disk diffusion method, a standardized technique for evaluating the susceptibility of bacterial isolates to selected antimicrobial agents This experiment specifically tests the susceptibility of ESBL E coli isolates obtained from urban wastewater against 14 different antibiotics.
Liquid culture: Inoculate the tested colony in autoclaved Tryptone soya broth (4-5 mL) and incubate it at 35±2℃, in shaking condition (120 rpm) for 2-6 h in h
34 thermal water bath Then, measure and adjust McFarland standard turbidity at 0.5 by using sterilized saline or Tryptone soya broth
To prepare agar plates, add 38 g of Muller-Hinton agar (Eiken Chemical Co., LTD) to the medium Once the mixture cools to a temperature between 45-50℃, pour it onto the plate, ensuring that the depth of the agar is approximately 4 mm.
Antibiotics disks of 14 antibiotics (Eiken Chemical Co.,LTD) Details of the antibiotic and its concentration are shown in Table 3.3
Table 3.3 Antibiotic disks used for susceptibility testing
Antibiotic class Antibiotic group Antibiotic Concentration
Beta-lactams Aminopenicillin Ampicillin (ABP) 25
Beta-lactams Cephalosporins Cefotaxime (CTX) 30
Beta-lactams Cephalosporins Ceftazidime (CAZ) 30
Beta-lactams Cephalosporins Cefidnir (CFN) 5
Beta-lactams Cephalosporins Cefpirome (CPR) 30
Beta-lactams Carbapenem Imipenem (IPM) 10
Beta-lactams Carbapenem Meropenem (MPM) 10
Sulfonamides Sulfa in combination with other AM
To initiate the culture spread on Mueller-Hinton agar plates, use a cotton swab to evenly distribute the prepared liquid culture within 15 minutes Rotate the plate at a 60° angle during the spreading process to ensure uniform coverage After the initial spread, rotate the plate again at the same angle Allow the agar surface to settle for 3-5 minutes before proceeding to the next step.
Step 2: Antimicrobial disks are placed by disk dispenser on the Muller-Hinton agar within 15 min after culture spread
Step 3: Incubate at 37 °C for 16-18 hours
Step 4: Measure the inhibitory zone using a digital caliper
Step 5: Result interpretation involves assessing the susceptibility of E coli to various antibiotics by measuring the inhibition zone diameter, as detailed in Table 3.4 and illustrated in Figure 3.5 This diameter determines whether E coli is classified as resistant, intermediate, or susceptible to the antibiotic tested, following CLSI guidelines (2021) A susceptible isolate shows significant inhibition, indicating high clinical efficacy; an intermediate isolate exhibits partial inhibition, reflecting lower clinical efficacy; while a resistant isolate demonstrates no inhibition, leading to potential treatment failure.
The interpretation of inhibition zone diameters is guided by the Clinical and Laboratory Standards Institute (CLSI-M100 31st edition, 2021) and KB Disk EIKEN standards, which are largely similar, with the exception of levofloxacin (LVX) As the antibiotic disks are manufactured by Eiken, the KB Disk EIKEN standards are utilized Additionally, CLSI does not provide susceptibility judgment information for cefpirome (CPR).
Figure 3.5 Growth of bacteria on the surface of agar plate after overnight incubation
(16 hours) Table 3.4 Criteria of susceptibility of E coli
Persistence of ESBL E coli in oligotrophic water environment
The purpose of the experiment is to examine how long ESBL E coli can survive in the water environment with poor nutrient (oligotrophic) condition
1 purified non-ESBL E coli isolate recovered from upstream Nhue river sample collected in December 2020
1 ESBL E coli isolate recovered from upstream Nhue river sample collected in December 2020 h
To prepare a sterile phosphate-buffered saline (PBS) solution, dissolve one bag of PBS powder from Wako Pure Chemical Industries, Ltd in one liter of double-distilled water Autoclave the mixture at 121°C for 15 minutes to ensure sterility.
To prepare a liquid medium, combine 3 g of Pearlcore Trypto-Soy Broth (Eiken Chemical Co., LTD) with 100 mL of double distilled water in a 250 mL Duran® original GL45 bottle Mix thoroughly and autoclave at 121°C for 15 minutes Once the medium cools to 50-55°C, incorporate 100 µL of cefotaxime stock solution (4 mg/mL) into the mixture.
TBX agar plates, along with TBX agar supplemented with cefotaxime at a concentration of 4 µg/mL, were utilized to quantify both non-ESBL and ESBL E coli The preparation method for these agar plates follows the same protocol outlined in section 3.2.
Corning® 50mL centrifuge tubes, water bath, inoculum loops, alum foil
To prepare liquid cultures of the tested isolates, inoculate the colony into autoclaved Pearlcore Trypto-Soy Broth Incubate the cultures at 40℃ with shaking at 120 rpm for 16 hours in a thermal water bath until clear turbidity is observed.
Step 2: Centrifuge the culture at 3,500 rpm in 15 minutes, discard the supernatant
Step 3: Add 25mL PBS, shake well, centrifuge at 3,500 rpm in 15 minutes, discard the supernatant
Step 4: Add 40 mL PBS, shake well, centrifuge at 4,000 rpm in 15 minutes, discard the supernatant
Step 5: Add 25 mL PBS, shake well
Step 6: Adjust McFarland standard turbidity of the cultures to 2.0 (~ 600×10 8 cells/100mL) by using sterilized PBS
Step 7: Add the adjusted cultures to the autoclaved Duran bottles containing
200 mL PBS solution Wrap the bottles in alum foil and keep in the incubator at 25℃ h
To determine the concentration of E coli and ESBL E coli, take 1 mL of the incubated solution after 1, 3, 5, 10, 15, and 40 days The quantification method used is the spread method, utilizing TBX agar and TBX agar supplemented with cefotaxime at 4 µg/mL, as detailed in section 3.2 Incubate the agar plates at 37°C for 16-18 hours for accurate results.
Genotyping of ESBL-encoding genes
Polymerase chain reaction (PCR) is utilized to identify ESBL-encoding genes in ESBL-producing E coli A literature review indicates that blaCTX-M or CTX-M genes are the most common ESBL-encoding genes Consequently, the experiment aims to investigate the prevalence of each blaCTX-M group, which includes four major categories: group 1, group 2, group 9, and group 8/25 Multiplex PCR is employed for the detection of these CTX-M groups.
M group 1, group 2, group 9 simultaneously, while monoplex PCR is used to detect CTX-M group 8/25 (Dallenne et al., 2010)
One ESBL E coli colony is mixed in 30 l of distilled water in tube/plate The plate is then slightly centrifuge and heat at 95℃ for 10 min Then centrifuge the tube/plate
To prepare DNA templates, centrifuge at 4000 rpm for 10 minutes and carefully transfer the supernatant to a new tube or plate These DNA templates can be prepared ahead of time and stored in a refrigerator at -20°C for up to three months until needed.
Multiplex PCR for detection of blaCTX-M group 1, 2, 9
A multiplex PCR was conducted using a primer mixture from CTX-M groups 1, 2, and 9, as detailed in Table 3.5 These primers were derived from the previous research conducted by Dallenne et al (2010) Specifically, the primers for CTX-M group 1 target CTX-M 1 and CTX-M 3.
M 15 variants, while those for CTX-M group 2 are able to detect CTX-M 2 variant, and primers for CTX-M group 9 target CTX-M 9 and CTX-M 14 variants h
Table 3.5 Primer set for multiplex PCR CTX-M group 1, 2, 9 (Dallenne et al., 2010)
CTX-M group Primer Primer sequence
*Y=T or C; R=A or G; S=G or C; D=A or G or T
The primer mixture contains 3 primer sets for detection of group 1, 2 and 9 The component of primer mixture is presented in Table 3.6
Table 3.6 Primer mixture CTX-M group 1, 2, 9
CTX-M group Primer PCR product
The multiplex PCR procedure begins with the preparation of a 25 µL reaction mix in Thermo Scientific 24-well PCR plates, consisting of 2 µL DNA template and 23 µL PCR mixture, as detailed in Table 3.7 A negative control is created by substituting the DNA template with 2 µL nuclease-free water Following this, the PCR plate is flash centrifuged to eliminate any residual reaction mixture on the walls or caps The amplification process is conducted in a Bio-Rad T100 Thermal Cycler, involving 30 cycles with denaturation at 94°C for 40 seconds, annealing at 60°C for 40 seconds, and extension at 72°C for 60 seconds, concluding with a final extension at 72°C for 5 minutes The resulting multiplex PCR products are subsequently analyzed through gel electrophoresis.
Table 3.7 PCR mixture for multiplex PCR
Monoplex PCR for detection of blaCTX-M group 8/25
The below primers are used for detection of CTX-M group 8/25 The -lactamses targeted by these primers were CTX-M-8, CTX-M-25, CTX-M-26, CTX-M-39, CTX- M-40, CTX-M-41 (Dallenne et al., 2010) h
Table 3.8 Primer set for multiplex PCR CTX-M group 8/25 (Dallenne et al., 2010)
*Y=T or C; R=A or G; S=G or C; D=A or G or T
The PCR procedure utilizes Thermo Scientific 24-well PCR plates, where a multiplex PCR assay is conducted with a 25 µL reaction mix for one sample This reaction mixture comprises 2 µL of DNA template and 23 µL of the PCR mixture, detailed in Table 3.9 Additionally, a negative control is created by incorporating 2 µL of nuclease-free water.
To prepare a 23 µL PCR mixture, first flash centrifuge the PCR plate to eliminate any residual reaction mixture from the inner walls or cap Next, perform the PCR using a Bio-Rad T100 Thermal Cycler, following a protocol that includes 30 cycles of denaturation at 94°C for 40 seconds, annealing at 60°C for 40 seconds, and extension at 72°C for 60 seconds Conclude the reaction with a final extension at 72°C for 5 minutes Finally, analyze the PCR product through gel electrophoresis.
Table 3.9 PCR mixture for monoplex PCR
Gel electrophoresis o Preparation of 2% agarose gel:
To make the gel, use Agarose S (Nippon Gene) and TAE buffer, with the ratio of 2g Agarose S and 100 mL TAE buffer in a Duran Laboratory bottle
Microwave the solution on Medium High power for 1-3 minutes, ensuring that Agarose S fully dissolves without overboiling To prevent overboiling, periodically pause the microwave to swirl the bottle.
Let the solution cool down to 50°C
Pour the solution into the gel tray with the well comb in place, let the gel solidify
Gently remove the comb, be careful not to tear up the well, put the gel in TAE buffer until use o Run of gel electrophoresis
Dilute 1 L of loading dye in 5 L multiplex PCR product on parafilm, using sterile pipette tip to completely mix the mixture
Place the gel in electrophoresis unit
Load the mixture, gene ladder (Nippon Gene Gene Ladder Wide 1 (0.1-20 kbp)), and negative control on the well of the agarose gel
Run electrophoresis at 100 voltages for 20-30 minutes
Soak the gel on Ethidium bromide solution for 20-25 minutes
Soak the gel briefly in distilled water and observe the PCR product under UV transillumination in WSE-5300Printgraph CMOS I h
Statistical analysis
The Kruskal-Wallis one-way analysis of variance is utilized to determine the significance of differences among multiple data sets In contrast, the paired samples Student’s t-test is employed to assess the differences between two related data sets A p-value is calculated to evaluate the statistical significance of these findings.
Statistical analysis is performed in Python language using Google Colab (https://colab.research.google.com/) h
RESULT AND DISCUSSION
Occurrence of ESBL E coli
The TBX medium, utilized for the enumeration and isolation of E coli, features X-glucuronide, a component that specifically targets the β-glucuronidase enzyme produced by E coli This enzyme hydrolyzes β-glucuronides, resulting in the characteristic blue-green color of E coli cells Isolates that develop on TBX agar are confirmed as E coli To ensure the reliability of this identification method, a total of 109 blue-green isolates were randomly selected and analyzed for species detection using MALDI-TOF MS.
Figure 4.1 Composition of isolates identified by MALDI-TOF MS
Figure 4.1 confirms that 108 isolates were identified as E coli, with only one remaining unidentified This high accuracy rate of 99% demonstrates that TBX agar is highly selective for E coli, making it a reliable tool for its detection Consequently, the presence of blue-green colonies on TBX agar can be directly interpreted as E coli.
4.1.2 Occurrence of ESBL E coli in urban drainage
In Hanoi, water samples were collected from the To Lich River, which flows through the urban area from Nghia Do in Cau Giay district to Cau Set in Hoang Mai district The river primarily receives water from domestic wastewater, rain, and surface runoff, classifying it as "urban drainage." Additionally, in Bac Ninh, water samples obtained from wastewater treatment plants (WWTP) are also categorized as urban drainage samples.
The study found that the concentration of ESBL E coli in wastewater samples ranged from 2.9×10^4 to 3.5×10^4 CFU/100mL in Hanoi and from 3.6×10^4 to 5.4×10^4 CFU/100mL in Bac Ninh Total E coli concentrations were significantly higher in Hanoi, ranging from 4.5×10^5 to 2.0×10^6 CFU/100mL compared to Bac Ninh's range of 1.7×10^5 to 5.1×10^5 CFU/100mL Statistical analysis revealed no significant difference (p = 0.08) between the E coli concentrations in samples from both locations, although Hanoi's levels were approximately 2.9 times higher These findings suggest a greater presence of fecal contamination in Hanoi's water, likely due to poor septic tank conditions and illegal discharge of untreated waste Additionally, the stagnant state of the To Lich River may contribute to prolonged retention times of E coli in the water.
In a recent study, the concentration of ESBL E coli in Hanoi's wastewater was found to be 3.8 times higher than that in Bac Ninh, with a significant difference noted (paired t-test, p = 0.01) This elevated level in Hanoi can be linked to its higher population density compared to Bac Ninh, according to the General Statistics Office (2020) However, the comparison may be limited due to a lack of data on the total wastewater volume received by the rivers in both regions.
The concentration of ESBL E coli in wastewater treatment plant influent varies significantly by geographic region, with lower levels observed in the Netherlands (8.2×10^3 CFU/100mL) compared to higher levels in the USA (2.3-3.1×10^5 CFU/100mL) This variation is influenced by factors such as antibiotic usage patterns and onsite wastewater treatment technologies.
Figure 4.2.Abundance of ESBLE coli and total E coli and resistance ratios in different water samples in Hanoi and Bac Ninh (from Sep 47 2020 to May 2021) h
E coli occurrence in the domestic wastewater reflects its presence in the intestine of the residents discharging that wastewater, as E coli is widely used as a fecal contamination indicator Occurrence of ESBL E coli in the wastewater indicates its presence in the healthy residents Untreated wastewater carrying high loads of ESBL E coli contaminates the natural water environment that it enters In Vietnam, this is a great issue since 87% domestic wastewater from urban area is untreated and discharged directly into the environment (Bộ Tài nguyên và Môi trường, 2018) The water in To Lich rivers were used directly for irrigation, which implies that those ESBL E coli are circulated back to the human via consumption of the food irrigated by those water
4.1.3 Occurrence of ESBL E coli in river water
The concentration of ESBL E coli in Hanoi's river varied from 3.2×10^1 to 8.0×10^3 CFU/100mL upstream and 4.1×10^2 to 5.7×10^3 CFU/100mL downstream In Bac Ninh, the levels ranged from 1.0×10^1 to 4.0×10^4 CFU/100mL upstream and 1.4×10^4 to 1.2×10^5 CFU/100mL downstream Comparatively, the Tama River in Tokyo showed a stable concentration around 1.0×10^2 CFU/100mL, while lake and river water in the Netherlands exhibited lower levels between 0 and 15 CFU/100mL Both Japan and the Netherlands collected samples from rivers receiving wastewater treatment plant (WWTP) effluents, with the Netherlands lacking disinfection at the WWTP Notably, the concentrations in the downstream samples of our study were higher than those in the other studies, underscoring the critical role of WWTPs in mitigating ESBL E coli discharge into the aquatic environment.
In Bac Ninh, the concentration of ESBL E coli at upstream sampling points was found to be significantly higher than in Hanoi, with variations ranging from 2 to 13 times, excluding January samples Conversely, downstream samples in Hanoi exhibited concentrations that were 5 to 15 times greater than those in Bac Ninh during the same period Notably, the concentration of ESBL E coli increased dramatically from upstream to downstream, with Hanoi samples showing an increase of 6.5 to 1454.9 times, while Bac Ninh samples ranged from 1.5 to 41.1 times, excluding January samples.
In March, Bac Ninh's upstream water samples exhibited elevated levels of E coli and ESBL E coli compared to downstream samples This increase in bacterial concentration is attributed to contamination from the backward flow of drainage from agricultural canals.
The concentration of ESBL E coli in the downstream water of the Hanoi River is significantly higher than in Bac Ninh, attributed to Hanoi's urban population being 20 times greater than that of Bac Ninh city (General Statistics Office, 2020).
4.1.4 Resistance ratios of ESBL E coli in water environments
Resistance ratios in each water samples varied within the sampling period (Figure 4.3)
In upstream waters, resistance ratios were observed to range from 6.2% to 24.2% in Hanoi and 6.8% to 14.3% in Bac Ninh In downstream locations, these ratios varied from 9.3% to 16.5% in Hanoi and 6.9% to 12.5% in Bac Ninh Comparatively, lower resistance ratios of 2.9% to 5.3% were reported in river water in Japan (Tsutsui and Urase, 2019), while in the Netherlands, resistance ratios in lakes and rivers ranged from 0.05% to 1% (Blaak et al., 2014).
Figure 4.3 Resistance ratios in different water samples in Hanoi and Bac Ninh
No statistically significant difference exists among the resistance ratios among 3 kinds of sample in two cities (Kruskal Wallis ANOVA test, p = 0.187) It means that, h
Although the concentrations of E coli and ESBL E coli varied, the resistance ratios in both upstream and downstream river samples, as well as in wastewater, were similar This indicates that the upstream water had already been contaminated with E coli, likely originating from domestic or livestock wastewater discharges.
Extended sampling events in Northern provinces such as Nam Dinh, Thai Binh, Hung Yen, Bac Ninh, and Ninh Binh revealed that the resistance ratios in both upstream and downstream water environments were comparable to those observed in Hanoi and Bac Ninh This finding was supported by a Kruskal Wallis ANOVA test, which indicated no significant difference (p = 0.07).
Figure 4.4 Resistance ratios in upstream and downstream water in Hanoi, Bac Ninh and other Northern provinces
The findings indicate that resistance ratios in the water environments of Northern cities in Vietnam are consistent, suggesting widespread fecal contamination E coli serves as a key indicator of this contamination, allowing us to evaluate the antimicrobial resistance (AMR) situation in the population based on these resistance ratios A higher resistance ratio correlates with an increased prevalence of resistant bacteria in humans Additionally, the resistance rates of E coli in these water environments can be utilized to assess the AMR status, as E coli can persist in the environment for extended periods.
Antimicrobial susceptibility of ESBL E coli
85 ESBL E coli isolates were subjected to test for antimicrobial susceptibility against
13 antibiotics belonging to 6 antibiotic classes These isolates were recovered from: wastewater (WW) in urban drainage in Hanoi (n@), urban drainage in Bac Ninh (n5), effluent WWTP in Bac Ninh (n)
All 100% of isolates from three sources demonstrated resistance to ampicillin (ABP), consistent with findings from previous studies on ESBL E coli, including those from healthy humans and various raw meats Notably, resistance was observed in chicken, pig, and surface water samples, as well as pig farm wastewater However, it's important to note that not all ESBL E coli isolates from surface water in China exhibited resistance to ABP.
All isolates demonstrated resistance to CTX and CFN, both of which are third-generation cephalosporins Resistance levels to CAZ, another third-generation cephalosporin, were notably lower compared to CTX and CFN Among the three samples analyzed, isolates from Hanoi exhibited higher resistance rates than those from Bac Ninh, which is associated with urban drainage and effluent wastewater treatment plants Within the third-generation cephalosporins, the resistance ratio to CAZ is lower than that of CTX, as noted in studies by Tsutsui and Urase (2019) and Hinenoya et al (2018).
Resistance to the 4 th generation cephalosporins – CPR – is also high in all sample types (Figure 4.5) The proportion of resistance and intermediate resistance of ESBL
E coli to CPR were, respectively, as follow: Hanoi urban drainage (87.5%, 5%), Bac Ninh urban drainage (83%, 13%), effluent WWTP (50%, 50%) In Japan 90% and 95% ESBL E coli from river water and wastewater, respectively, were reported to be resistant to CPR (Tsutsui and Urase, 2019) h
Figure 4.5 Antibiotic resistance profile of ESBL E coli isolated from (i) Hanoi urban drainage; (ii) Bac Ninh urban drainge; (iii) WWTP effluent
Antibiotics tested: ABP = Ampicillin, CTX = Cefotaxime, CAZ = Ceftazidime, CFN Cefdinir, CPR = Cefpirome, IPM = Imipenem, MPM = Meropenem, TC Tetracycline, GM = Gentamycin, KM = Kanamycin, LVX = Levofloxacin, ST Sulfamethoxazole + Trimethoprim, CP = Chloramphenicol
(ii) Bac Ninh urban drainage
ESBL E coli exhibited significant resistance to antibiotics, including TC, GM, KM, LVX, ST, and CP All sample types showed a resistance rate of 60% or higher to TC and ST Additionally, resistance to other drugs ranged from 15% to 57.5% in wastewater samples, while treated samples displayed a higher resistance rate of 60% to 76%.
WW Urban drainages samples exert lowest resistance rate to KM (Hanoi: 15%, Bac Ninh: 26%)
All tested isolates exhibited resistance to a minimum of four antibiotics, with some showing resistance to as many as eleven (Table 4.1) The resistance range varied by sample, with isolates from Hanoi wastewater (WW) resistant to 5-11 antibiotics, Bac Ninh WW resistant to 4-11, and WWTP effluent isolates also resistant to 5-11 antibiotics (Table 4.1) Notably, the majority of tested isolates displayed resistance to 6-9 antibiotics.
Table 4.1 Numbers of ESBL E coli isolates tested and percentages of isolates resistant to at least 4 antibiotics
Percentages of isolates resistant to n antibiotics (Rn) R4
WWTP 10 0 10.0 20.0 0 10.0 30.0 20.0 10.0 100 Total 85 1.0 8.6 19.4 15.8 18.3 20.6 10.4 6.0 100 Rn: Resistance to n antibiotics
The research reveals a multidrug-resistant phenotype, indicating the presence of additional antimicrobial resistance (AMR) genes The tested isolates, obtained from wastewater and effluent at wastewater treatment plants (WWTP), suggest that multidrug-resistant extended-spectrum beta-lactamase (ESBL) E coli is present in healthy individuals across two cities Furthermore, the findings imply that WWTPs are ineffective in eliminating these multidrug-resistant strains.
Multidrug-resistant ESBL E coli has been identified in various environmental sources, including water in Japan (Yamashita et al., 2017), wastewater from pig farms in Southern Vietnam (Hinenoya et al., 2018), and surface water in China (Liu et al., 2018).
Genotyping of ESBL-encoding genes in ESBL E coli
A study analyzed 116 ESBL E coli isolates from urban drainage samples using multiplex PCR to detect blaCTX-M groups 1, 2, and 9, along with monoplex PCR for blaCTX-M group 8/25 The samples from Hanoi were collected in October 2020, November 2020, December 2020, and March 2021, while Bac Ninh samples included isolates obtained in November 2020 and March 2021.
The results of the multiplex PCR gel electrophoresis, as illustrated in Figure 4.1, revealed the presence of blaCTX-M group 1 (688bp) and blaCTX-M group 9 (561bp) in the tested ESBL E coli isolates, as detailed in Table 4.2.
Figure 4.6 Image of gel electrophoresis of blaCTX-M group 1, group 2, and group 9 h
In a comprehensive analysis of blaCTX-M genes across various samples, blaCTX-M group 1 emerged as the most prevalent, with the exception of the December sample from Hanoi The data revealed that the percentage of isolates containing any blaCTX-M group was 74% in Hanoi, 95% in Bac Ninh, and 85% across both cities Specifically, the frequencies for blaCTX-M group 1 were 42% in Hanoi and 74% in Bac Ninh, while blaCTX-M group 9 was found in 32% of Hanoi samples and 21% of Bac Ninh samples.
Figure 4.7 Result of genotyping blaCTX-M-type ESBL-encoding gene in ESBL E coli
Table 4.2 Genotyping of ESBL E coli isolated from urban drainage
Any blaCTX-M blaCTX-M group 1 blaCTX-M group 2 blaCTX-M group 9 blaCTX-M group 8/25 n % n % n % n % n %
Oct Nov Dec Mar Nov Mar
CTX-M group 1 CTX-M group 2 CTX-M group 9 CTX-M group 8/25 bla CTX-M group 1 bla CTX-M group 9 bla CTX-M group 2 bla CTX-M group 8/25 h
The study reveals that blaCTX-M group 1 and blaCTX-M group 9 are the most common ESBL-encoding genes found in ESBL E coli within Vietnam's water environment These blaCTX-M groups are also prevalent among healthy humans, chickens, pigs, and pig farm wastewater in the country However, their prevalence varies across different subjects; for instance, group 9 is predominantly detected in chickens from Ba Vi in Northern Vietnam, while group 1 is more frequently found in Southern Vietnam's chickens Additionally, research shows inconsistencies in the prevalence rates of these groups among humans in the same region Overall, the findings highlight the complex distribution of blaCTX-M groups across
In the Netherlands, the most prevalent ESBL-encoding genes in ESBL E coli isolated from wastewater and surface water were blaCTX-M group 1 and blaCTX-M group 9 (Blaak et al., 2014) Among blaCTX-M-positive ESBL E coli found in river and lake water, blaCTX-M group 9 was the most frequently detected, followed closely by blaCTX-M group 1.
1 In Japan, the rates of harboring these gene groups were similar in ESBL E coli in river water and wastewater (Tsutsui and Urase, 2019)
Figure 4.8 Relationship of ESBL-encoding genes and number of antibiotics resistance
Number of antibiotics resistance bla CTX-M group 1 bla CTX-M group 9 bla CTX-M group 1+9 others h
Figure 4.8 illustrates the correlation between CTX-M-type ESBL genes and antibiotic resistance in isolates Isolates positive for blaCTX-M group 1 exhibited resistance to 4-11 antibiotics, whereas those positive for blaCTX-M group 9 showed resistance to 5-8 antibiotics Notably, a single isolate contained both group 1 and group 9 blaCTX-M genes, demonstrating resistance to 9 antibiotics.
Persistence of ESBL E coli in oligotrophic water environment
In an oligotrophic water environment at 25°C and under dark conditions, resembling stagnant water, ESBL E coli exhibited a faster decay rate compared to non-ESBL E coli, with a significant difference noted (paired t-test, p=0.01) Both strains experienced a dramatic reduction of 3 log10 units in concentration within the first five days, followed by a gradual decline up to day 40 By day 40, the log reduction values were 5.2 for ESBL E coli and 3.7 for non-ESBL E coli.
Figure 4.9 Log reduction of ESBL E coli and non-ESBL E coli in oligotrophic water with time
In this study, both ESBL E coli and non-ESBL E coli were found to survive for over 40 days in water, highlighting the potential for ESBL E coli to exchange genetic material with other bacteria in the environment Additionally, the contamination of food by ESBL E coli is a significant concern, as surface water is commonly utilized for irrigation and aquaculture.
By consumption of those food, healthy human is exposed to ESBL E coli and some might get infection.
Removal of ESBL E coli by wastewater treatment plant
Due to the long persistence of ESBL E coli in aquatic environments, wastewater treatment plants (WWTP) must effectively eliminate these bacteria from wastewater prior to environmental discharge Figure 4.10 illustrates the concentrations of E coli and ESBL E coli in both influent and effluent samples.
The study found that without disinfection, the log reduction value (LRV) for total E coli and ESBL E coli ranged from 0.11 to 0.79 and 0.22 to 0.82, respectively In contrast, the application of UV light disinfection significantly increased the LRV for total E coli to between 1.11 and 6.51 and for ESBL E coli to between 1.01 and 5.59 Additionally, chlorine disinfection achieved an LRV removal ranging from 2.80 to 6.68.
Disinfection can achieve a log reduction value of E coli that is 1-5 logs higher compared to processes without disinfection However, it is important to note that disinfection does not eliminate E coli entirely Given that E coli can persist in aquatic environments for extended periods, careful application of disinfection methods is essential to prevent its discharge from effluent wastewater treatment plants (WWTP).
Figure 4.10 Concentration of E coli and ESBL E coli in influent and effluent of WWTP
No disinfection UV light disinfection Chlorine disinfection
E c ol i co nc en tr at io n (C F U /1 00 m L )
E coli in influent E coli in effluent ESBL E coli in influent ESBL E coli in effluent
ESBL E coli in influent ESBL E coli in effluent
E coli in influent E coli in effluent h
A paired t-test indicated no significant difference in the log reduction value (LRV) between E coli and ESBL E coli (p>0.05), suggesting that both strains are reduced at the same rate This correlation allows for the estimation of ESBL E coli LRV based on E coli LRV data (Figure 4.11) Consequently, any effective disinfection method capable of eliminating E coli will also be effective against ESBL E coli.
Figure 4.11 Correlation of log reduction value of E coli and ESBL E coli without disinfection and with disinfection
1 The characteristics of ESBL E coli in urban water environment in Northern Vietnam
ESBL E coli has been detected across various water environments in Northern Vietnam, with the highest concentrations found in wastewater samples The presence of ESBL E coli in river water increases as it flows through urban areas The resistance ratios across all water environments are consistent, ranging from 6.2% to 32.4%, indicating a common source for these bacteria This study offers essential insights for One Health data related to water environments.
ESBL E coli were also resistant to other classes of antibiotics, suggesting the co- existence of other AMR genes
Group 1 and 9 of blaCTX-M is the most prevalent ESBL-encoding genes in ESBL E coli, which is similar to the ESBL-encoding genes in human and animals in Vietnam
ESBL E coli can survive in water with poor nutrient condition for long time, posing the threat of transmission into the food chain
2 Role of wastewater treatment plant to reduce ESBL E coli load
The careful application of disinfection methods can significantly lower the levels of ESBL E coli in wastewater effluent By effectively targeting these bacteria prior to discharge, we can improve the overall safety and quality of water released into the environment.
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