Detection of antibiotic-resistant bacteria and resistance genes SHV, TEM, and CTX-M in drinking waters in Tehran, Iran

The discharge of agricultural, urban, livestock, and industrial effluents into waterways endangers public health, resulting in high rates of pathogenic and other environmental pollution substances (Sarmah et al. 2006; Alipour et al. 2014). Human and animal infectious bacteria are constantly introduced into the water system by wastewater (Baquero et al. 2008; Yang et al. 2022). Bathing in fecally infected recreational waters has been linked to an increased risk of infectious disease transmission, including gastroenteritis and gastrointestinal, skin, eye, and ear infections (Alipour et al. 2014). There is also an increasing health risk that antimicrobial-resistant bacteria (ARB) and antimicrobial resistance (AMR) genes in feces could contaminate water sources with untreated or treated wastewater, affecting people who drink, bathe, wash, and use water for primary contact recreation and irrigation (WHO 2014). The use of antibiotics for feeding in aquaculture to increase the growth of fish, crustaceans, and mollusks, or to prevent bacterial diseases causes the spread of antibiotics in aquatic environments (Voitsitskiy et al. 2019).

Antibiotic-resistant bacteria are common in places where drugs are involved, but they are also on the rise in aquatic ecosystems (Schwartz et al. 2003; Pan et al. 2022). Many bacterial genera contain antibiotic resistance genes (ARGs), which have been subsequently integrated into genetic mobile platforms (plasmids, transposons, and integrons) that can be transmitted among waterbodies by bacteria. Water is used not only to distribute antibiotic-resistant bacteria among humans and wildlife (drinkable water is provided from surface water), but also to introduce resistance genes into microbial communities (Yang et al. 2023). In the context of the urban water cycle, ubiquitous bacteria that can inhabit various types of water are of particular importance for assessing possible AMR dissemination (Figueira et al. 2011).

With the exception of virgin sites in the mountain ranges before flowing water passes across metropolitan or agricultural areas, it is difficult to find a place in which antibiotics cannot be observed in water bodies (Yang & Carlson 2003; Wang et al. 2022). As a result, the marine environment has been regarded as a vital reservoir of antibiotic-resistant bacteria and ARGs (Zhang et al. 2009). The new water treatment process is unable to fully remove all antibiotics from potable water (Figueira et al. 2011; Guo et al. 2023). Antibiotics in the marine ecosystem have sparked two worries. The major problem is the possible toxicity of these materials to aquatic species and humans via treated wastewater. Furthermore, there is a growing concern that releasing antibiotics into the environment correlates with the appearance of pathogenic bacteria that are tolerant to high concentrations of these drugs. One of those resistant factors is the existence of extended-spectrum beta-lactamase (ESBLs) and metallo-beta-lactamase (MBLs) genes in bacterial species (Duan et al. 2022). This is a pressing issue, as these genes provide resistance to a wide range of beta-lactams (Devarajan et al. 2017). The majority of research on antibiotic resistance in the environmental setting has been conducted on surrounding liquids and does not represent the circumstances in drinking and mineral water. The aim of this study was to investigate the presence of genes isolated from bla SHV, bla TEM, and bla CTX-M beta-lactamase from possible microorganisms present in the drinking water of Tehran, Iran.

This research looked at the heterogeneity, propagation, and antibiotic resistance of diverse microbial families distributed in drinking and mineral waters. It was a longitudinal study that aimed at the diversity and antibiotic resistance of culturable bacteria from various categories found in specific zones within the urban water cycle.

To diagnose P. aeruginosa in different water samples, membrane filters were incubated on cetrimide agar media enriched with 15 mg/L nalidixic acid for 24 h at 37 °C. Colonies with blue- or green-tinged coloration or fluorescence beneath UV light were considered potential P. aeruginosa isolates (364 nm). On nutrient agar plates, bacterial isolates of suspected cultures were incubated for 22 ± 2 h at 37 °C (Herath et al. 2014). For confirmation of P. aeruginosa, not only were suspected colonies cultured on nutrient agar plates separately and the plates were incubated at 37 °C for 22 ± 2 h, but also Pseudomonas primers (Ps-F and Ps-R) targeting the 16S rRNA gene were utilized to test bacteria relevant assays of ISO 16266:2006.

For the detection of coliform bacteria and E. coli in drinking water samples, membrane filters were incubated on Chromogenic Coliform Agar (CCA) at 36 ± 2 °C for 21 ± 3 h. The membrane filters were examined and all colonies giving a positive β-d-galactosidase reaction (pink to red) were counted as presumptive coliform bacteria that were not E. coli. All colonies giving a positive β-d-galactosidase and β-X-glucuronidase reaction were counted (dark blue to violet) as E. coli (ISO 9308-1 2014). Both groups of colonies would be approved by 16S rRNA, lacZ, and wecG (Maheux et al. 2014).

Enterococcus spp. was purified and counted through membrane filtration. Before being deposited on KF-Streptococcus agar containing 1 mL 2,3,5-triphenyltetrazolium chloride (TTC) per 100 mL, 250 mL samples were filtered through a 0.45 μm (47 mm grid) CHMLAB Group sterilized membrane filter (CHMLAB Group Barcelona, Spain). For 48 h, the KF-Streptococcus agar cultures were kept at 37 °C. Single well-isolated pink colonies were sterilized, subcultured three times on nutrient agar, and incubated overnight at 37 °C using the streak plate technique (Molale & Bezuidenhout 2016).

For the detection of spores of sulfite-reducing anaerobes (Clostridia) in specimens of drinkable water, the test was treated with heat treatment (75 °C for 15 min). Filters were incubated on sulfite iron agar in an anaerobic jar for 24 h at 37 °C (ISO 15213 2003; ISO 6461-2 2018). All varieties were stored at −80 °C in Luria–Bertani broth (20% glycerol) for future use.

Antibiotic tests were carried out by the Kirby–Bauer method (Bauer et al. 1959; Andrews 2001; Hudzicki 2009), a method according to Clinical and Laboratory Standards Institute Guidelines 2018/M100S28 (CLSI 2018). All bacterial isolates were tested for resistance to eight antibiotics. The antibiotics tested were imipenem (IMI, 10 μg); gentamicin (GM, 10 μg); amikacin (AK, 30 μg); ceftriaxone (CRO, 30 μg); trimethoprim/sulfamethoxazole (SXT, 75 μg); cefixime (CFM, 5 μg); colistin (CO, 10 μg); and cefalexin (CFLEX, 30 μg). The diameters of the minimum inhibitory concentration were measured in millimetres, and the results were classified as sensitive or resistant. This approach classified microbes as resistant if they were intermediate. Quality control was done using P. aeruginosa ATCC 27853.

Polymerase chain reaction (PCR) assays were used to check for the existence of bla TEM, bla SHV, and bla CTX-M in all P. aeruginosa isolates. Table 3 lists the primers included in this analysis (Bhattacharjee et al. 2008; Sidjabat et al. 2009). The amplification was carried out in a thermocycler (Eppendorf, Germany) in a 25 μL reaction solution containing 2.5 μL 10× PCR buffer, 0.6 μL 50 mM MgCl, 0.4 μL dNTP mix (10 mM each), 0.5 μL 20 pmol/μL each primer, 1 μL template DNA, 1 μL Taq DNA polymerase (5 U/μL), and 18.5 μL nuclease-free water. Each primer received its own reaction solution (Table 3). DNA Green Viewer staining was used after electrophoresis of the PCR product at 80 V, 380 mA in a 1.0% agarose gel.

The phenol–chloroform method was used to extract bacterial DNA. Cells (5 mL) were precipitated using a centrifuge at 5,000 rpm. The collected cells were entered into the instructions. First, the cell digestion process was performed in 400 μL of standard STE buffer, 2% sodium dodecyl sulfate (SDS), and 20 μL of proteinase K at 56 °C for 2 h. Nucleic acids were separated by phenol–chloroform–ischemyl alcohol solution (25-24-1) and 12,000g centrifugation. Finally, DNA was dissolved in 50 μL of distilled water free of any DNase and evaluated qualitatively by 1% agarose electrophoresis and nanodrop (Thermo 2000c, USA). Random amplification of polymorphic DNA (RAPD) analysis is used to type strains. RAPD primer 7 was used to type all of the isolates (Pérez-Brocal et al. 2020; Liu et al. 2022).

Seventeen non-duplicative P. aeruginosa isolates were collected from 400 samples of drinking water (300 samples of drinking water and 100 samples of mineral water) collected over a 12-month period in Tehran Province. There were no other forms of bacteria. Most of the contamination is related to the samples collected from the southern regions of Tehran. Resistance to eight antimicrobials was evaluated in all strains. The resistance rates to IMI and SXT were 11.76% and 88.24%, respectively (Table 4). GM, AK, and CO were all sensitive to isolates. As a result, these antibiotics are the most effective choice. SXT was found to have the highest resistance rate. The results of agarose gel electrophoresis showed that all isolates except the T22 isolate had the TEM gene, and only the P7 isolate had the CTX gene. None of the isolates showed SHV status.

The presence of healthy water and management in controlling water pollution is always decisive in protecting the human environment (Wang et al. 2021). With the emergence of the city and urbanization, the issue of healthful water supply, as well as the purification and disposal of municipal wastewater, has been a characteristic of measuring the progress of urban communities. Chemical, biological, and organic analyses are used to determine the consistency of drinking water. Drinkable water effluents can host ARGs and a multitude of pathogens due to their relationships with pollution from fertilized soil (Udikovic-Kolic et al. 2014; Han et al. 2018), and municipal and hospital wastewater (Le et al. 2018; Manaia et al. 2018). However, due to their resistance to disinfectants, some bacteria can develop in aqueous media. The use of chlorine to clean drinking water has been shown in studies to promote the development of antibiotic-resistant bacteria in aqueous media (Ribas et al. 2000). The Tehran metropolis is facing a lack of proper drinking water supply due to the growth of its population by immigration (Liu et al. 2023). Community members’ wellbeing is jeopardized by the existence of large numbers of bacteria as well as ARGs. Water treatment may enhance antibiotic resistance in living bacteria, and water supply systems may function as a major reservoir for opportunistic pathogens to acquire antibiotic resistance (Baquero et al. 2008; Xi et al. 2009).

Since drinking water treatment processes cannot completely remove ARGs from drinking water sources, drinking water supply systems may be the first point of entry for ARGs from the atmosphere into the host, posing potential health risks (Han et al. 2020). The arrival of sewage from villages, livestock centers, military centers, and recreational centers upstream of dams has led to a decline in the quality of surface water supply resources (Tian et al. 2023). The entry of residential, commercial, industrial, and hospital wastewater has also reduced the quality of groundwater resources. Chlorination can destroy Methylophilus, Limenobacter, and Polynucleobacter at the genus level, whereas the relative abundance of Pseudomonas, Acidovorax, and Sphingomonas increases Plesiomonas and Undibacterium in drinking water (Jia et al. 2015). Chlorination greatly altered the bacterial community's overall habits, with possible-host research revealing Pseudomonas as the most likely host of stable ARGs (Jia et al. 2015). Multidrug resistance genes have also been found in drinkable water, and their relative abundance increases significantly after chlorination (Jia et al. 2015). According to previous studies (Bergeron et al. 2015), beta-lactamase multidrug resistance genes are present in drinking water sources, and multidrug resistance genes are predominant in filtered drinking water (Han et al. 2018). In this research, bacterial contamination, patterns of antibiotic resistance, and the prevalence of antibiotic-resistant genes in drinking water specimens from Tehran were investigated. According to the findings, P. aeruginosa contamination was observed in 5.6% of drinking water samples. A study by Sajadi et al. (2017) found that the highest contamination belongs to the southern areas of Tehran Province, which indicates that most of these areas were contaminated due to contamination of groundwater aquifers with municipal, industrial, and hospital wastewater.

In addition, the four antibiotics gentamicin, amikacin, ceftriaxone, and colisitin are extremely susceptible to this bacterium. Trimethoprim (88.24%) and imipenem (11.76%) have the greatest resistance, which is consistent with previous research (Kittinger et al. 2016; Devarajan et al. 2017). Imipenem resistance was observed in 15% and 33% of Swiss and Indian strains, respectively. In addition, they discovered that 1.2% of the Pseudomonas strains isolated from the Danube River were resistant to imipenem in a report on antibiotic resistance. They also discovered that aminoglycoside resistance was extremely rare, with only two isolates resistant to amikacin and gentamicin, respectively (Kittinger et al. 2016). P. aeruginosa has been indicated as a replacement measure for the existence of other opportunistic pathogens in the case of water contamination (Herath et al. 2014). Antibiotic overuse and indiscriminate use are currently two of the leading causes of antibiotic resistance in various parts of the world (Pan et al. 2022). The spread of MBL-producing bacteria is caused by an excessive rise in broad-spectrum beta-lactam drugs. Antibiotics called beta-lactams are often used to treat infections in humans and animals, and their use has resulted in resistance to beta-lactamases like AmpC (Fernando et al. 2016). According to recent reports (Jia et al. 2015), ARGs of various forms have been discovered in drinking water around the world. Metallo-beta-lactamase-producing bacteria are commonly multidrug-resistant, resulting in severe infections that are difficult to treat. These enzymes can hydrolyze beta-lactam antibiotics, which are mainly found in Pseudomonas and Acinetobacter bacteria (Walsh 2005). The study's ultimate aim was to determine the function of these bacteria as antibiotic resistance reservoirs and to identify the resistant genes SHV, TEM, and CTX-M in drinking water. In general, the results indicate the importance of vertical transfer in the development of antibiotic resistance in aqueous media. In fact, if horizontal gene transfer is involved, the resistance pattern is associated with general types of water; in contrast, in the case of vertical transfer, the resistance pattern is species-related, therefore, some environmental factors may support the development of specific species or phenotypes and cause the vertical translocation of antibiotic resistance phenotypes (Vaz-Moreira et al. 2012).

The CTX-M gene was found in a small number of isolates (three isolates) by Pappa et al. (2016), demonstrating the appearance of broad-spectrum beta-lactamase (ESBL) genes in aqueous media. In this analysis, it was discovered that 94.1% of the samples had the TEM gene, with only one isolate having the CTX-M gene and none having the SHV gene. In the study on Pseudomonas resistance in tropical aquatic environments, the presence of the bla SHV NDM gene was shown in 3%, 8%, and 13% of isolates from CH, DRC, and IN, respectively (Devarajan et al. 2017). Mulet et al. (2010) inferred the same results by inferring the isolated phylogeny of Pseudomonas, including 107 species, which supports the reliable phylogenetic analysis of Pseudomonas. In patients with blood infections caused by resistance to Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia, and Escherichia coli, antibacterial therapy with inadequate experience to which the pathogen is not responsive has been linked to increased mortality (Tenover 2006). Drinking water safety hinges on the collection and preservation of drinking water supplies.

Antibiotic resistance is linked to the microbial community, and ARG occurrence is linked to the prevalence of many opportunistic pathogens (Yang et al. 2022). Antibiotics are also correlated with the bacterial community. As a result, implementing stringent policies to safeguard drinking water supplies from antibiotic and biological pollution could be a realistic step to control drinking water resources in the future. Because of the wide range of antibiotic contamination in water supplies, precise management methods should be considered when treating drinking water. Monitoring drinking water may effectively assess the different health risks raised by ARGs and pathogens, and sources of drinking water with a significant risk of biological contamination may undergo additional disinfection or membrane treatment in addition to normal water purification processes. Understanding antibiotic resistance in drinking water supplies may help with drinking water management strategies. Since ARG has such a wide range of driving factors in drinking water supplies, comprehensive data mining of ARG and related variables is critical (Han et al. 2020). The study's ultimate aim was to determine the function of these bacteria as antibiotic resistance reservoirs in drinking water. ARGs can be passed from one environment to another through specific bacteria, especially pathogens. The findings show that vertical transfer has a significant impact on antibiotic resistance production in aqueous media. In fact, if horizontal gene transfer is involved, the resistance pattern is associated with sites (general types of water). In contrast, in the case of vertical transfer, the resistance pattern is related to the species, as observed. As a result, certain external conditions can encourage the production of specific species or phenotypes and result in the vertical transmission of antibiotic resistance phenotypes. Furthermore, the presence of certain antibiotic resistance phenotypes exclusively in tap water indicates the entry of resistant bacteria at an unspecified stage of the source hazard for milk. The significance of these results, on the other hand, must be believed (Vaz-Moreira et al. 2012).

The emergence of possible antibiotic resistance in drinkable water delivery systems in some countries or regions necessitates additional monitoring in order to assess risk and develop mitigation measures to safeguard public health (Zhang et al. 2009). Sewage systems in communities should be such that existing treatment options take into account the appropriate conditions for disinfecting and killing ARG-containing bacteria prior to discharge into the sewer, hence the need to monitor resistance and its importance. Aquatic ecosystems are the determinants of antibiotic resistance because, in many parts of the Earth's surface water, they act as the end point for the treated or untreated effects of wastewater treatment (Devarajan et al. 2017).

Although some antibiotics still work well against P. aeruginosa, many cannot or will not be used soon. Transposable genes that are resistant to antibiotics are thought to be the main cause of drug resistance. However, it is important to remember that the widespread use of antibiotics has led to the selection of bacteria that are resistant to drugs. Therefore, choosing the right antibiotic based on an accurate and timely antibiotic program will play an important role in preventing the spread of antibiotic resistance. It should be mentioned that, due to the complexity of the effective factors in the phenomenon of drug resistance in aquatic environments, repeated research should be carried out to monitor the development of resistance. Antibiotics for pathogenic bacteria are necessary to maintain human health.

We would like to thank the Microbiology Department of the Standard Research Institute (SIR) and the General Directorate of Food and Drug Control Laboratories (FDCL) of the Ministry of Health and Medical Education (MOH) for their cooperation in collecting samples and performing the test. We also thank Cell Probe Company for its cooperation in conducting molecular genetic testing.

Conceptualization and Methodology, M.A., S.S.; Validation and Investigation, M.A., N.R.; Analysis, S.S., B.P.; Resources, M.A., R.N.M.; Writing original, M.A., S.S.; Review and editing. All authors have read and agreed to the published version of the manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.