Antimicrobial resistance (AMR) has emerged as one of the most serious global public health threats of the twenty-first century. The increasing prevalence of resistant microorganisms compromises the effectiveness of antimicrobial agents, resulting in prolonged illness, increased healthcare costs, treatment failures, and elevated mortality rates. According to the landmark analysis by Murray et al., bacterial AMR was directly responsible for approximately 1.27 million deaths worldwide and associated with nearly 4.95 million deaths in 2019, highlighting its substantial burden on global health systems [1]. The World Health Organization (WHO) has consistently identified AMR as a critical public health challenge requiring coordinated action across human, animal, and environmental sectors [2].

The emergence and dissemination of antimicrobial resistance are driven by multiple factors, including inappropriate antibiotic use, inadequate infection control practices, environmental contamination, and the horizontal transfer of resistance determinants among microbial populations [3]. Urban healthcare facilities, particularly hospitals, are recognized as major reservoirs and amplifiers of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs). The intensive use of antibiotics in healthcare settings exerts strong selective pressure on microorganisms, facilitating the development and persistence of resistance mechanisms. Consequently, hospital effluents often contain high concentrations of antibiotics, resistant pathogens, and ARGs, which are discharged into municipal wastewater systems and eventually released into the environment [4].

Wastewater has increasingly gained recognition as an important environmental reservoir of antimicrobial resistance. It receives inputs from hospitals, residential communities, pharmaceutical industries, and agricultural sources, creating a complex mixture of microorganisms, antimicrobial compounds, and genetic elements associated with resistance [5]. Wastewater treatment plants (WWTPs) represent critical interfaces between human populations and the environment, where resistant bacteria and ARGs can accumulate, persist, and potentially disseminate through treated effluents, sludge, and receiving water bodies [6]. The presence of mobile genetic elements such as plasmids, integrons, and transposons further enhances the potential for horizontal gene transfer, facilitating the spread of resistance across diverse bacterial species [7].

Traditional AMR surveillance relies primarily on clinical isolates obtained from infected patients. Although clinical surveillance remains indispensable, it is often limited by sampling bias, delayed reporting, and the inability to capture asymptomatic carriers or community-wide resistance patterns [8]. In contrast, wastewater surveillance offers a complementary and population-level approach for monitoring AMR. By analyzing wastewater samples, researchers can assess the collective burden of resistant microorganisms and ARGs shed by large populations, providing valuable insights into resistance trends without the need for individual patient testing [9].

The successful implementation of wastewater-based epidemiology during the COVID-19 pandemic has significantly accelerated interest in wastewater surveillance as a public health tool. Similar approaches are now being applied to monitor AMR, enabling the detection of emerging resistance determinants and the evaluation of temporal and spatial trends within communities [10]. Wastewater surveillance has demonstrated the ability to identify clinically important resistance genes, including extended-spectrum β-lactamase (ESBL) genes, carbapenemase genes such as blaKPC, blaNDM, and blaOXA-48, as well as genes conferring resistance to colistin and other last-resort antibiotics [11]. Such information can support early warning systems and guide targeted infection prevention and antimicrobial stewardship interventions.

Urban healthcare settings are particularly suitable targets for wastewater-based AMR surveillance due to their high patient density, intensive antibiotic consumption, and concentration of multidrug-resistant organisms. Monitoring hospital wastewater can provide real-time information regarding resistance dynamics within healthcare facilities, allowing for the identification of emerging threats before they become evident through routine clinical surveillance [12]. Furthermore, surveillance of healthcare-associated wastewater can help evaluate the effectiveness of infection control measures, antibiotic stewardship programs, and wastewater treatment processes aimed at reducing environmental dissemination of resistance determinants [13].

Advances in molecular technologies have enhanced the sensitivity and scope of wastewater surveillance. Quantitative polymerase chain reaction (qPCR), digital PCR, metagenomic sequencing, and whole-genome sequencing enable comprehensive characterization of resistomes and microbial communities present in wastewater samples [14]. These methods facilitate the detection of both culturable and non-culturable microorganisms and provide detailed insights into the diversity, abundance, and transmission pathways of ARGs. Consequently, wastewater surveillance is increasingly viewed as a key component of the One Health framework, which recognizes the interconnectedness of human, animal, and environmental health in addressing AMR [15].

Despite its considerable potential, wastewater surveillance of AMR faces several challenges, including methodological standardization, data interpretation, variability in wastewater composition, and the establishment of meaningful correlations between wastewater findings and clinical resistance patterns [9]. Nevertheless, growing evidence suggests that wastewater monitoring can serve as a cost-effective, non-invasive, and scalable strategy for AMR surveillance, particularly in densely populated urban healthcare environments [6,10].

In this context, wastewater surveillance of antimicrobial resistance genes in urban healthcare settings represents a promising approach for understanding the environmental dimensions of AMR transmission, strengthening public health surveillance systems, and supporting evidence-based interventions aimed at mitigating the global burden of antimicrobial resistance.

Study Design and Setting A cross-sectional observational study was conducted to investigate the occurrence and distribution of antimicrobial resistance genes (ARGs) in wastewater collected from urban healthcare settings. The study was carried out at the Telangana Territory Centre and College, Telangana, India. Wastewater samples were collected from healthcare-associated wastewater discharge points and associated sewage collection systems serving the institution and surrounding urban healthcare facilities. The study was conducted from January 2020 to September 2020. A total surveillance period of 9 months was used to capture representative wastewater characteristics and antimicrobial resistance gene profiles. Sample Size A total of 160 wastewater samples were included in the study. The sample size was determined to provide adequate representation of wastewater sources across the study area and to enable reliable estimation of the prevalence and diversity of antimicrobial resistance genes. Samples were collected from multiple designated sampling locations within the healthcare wastewater network to ensure comprehensive coverage of the surveillance system. Sample Collection Wastewater samples were collected using sterile polypropylene sampling containers following standard environmental microbiology protocols. Grab samples of approximately 500 mL were obtained from selected wastewater discharge points, including hospital effluents, sewage collection chambers, and wastewater conveyance systems. Samples were collected during peak operational hours to maximize representativeness of healthcare-associated wastewater. All samples were transported to the laboratory in insulated containers maintained at 4°C and processed within 24 hours of collection. Sample Processing and Concentration Upon arrival at the laboratory, wastewater samples were homogenized and subjected to preliminary filtration to remove large particulate matter. Microbial biomass was concentrated by membrane filtration using 0.22-µm pore-size filters. The filters containing concentrated microbial material were aseptically transferred to sterile tubes for subsequent molecular analysis. All procedures were performed under standardized laboratory conditions to minimize contamination and ensure reproducibility. DNA Extraction Total genomic DNA was extracted from concentrated wastewater samples using a commercially available environmental DNA extraction kit according to the manufacturer’s instructions. DNA quantity and purity were assessed using spectrophotometric analysis (NanoDrop™ or equivalent system). Extracted DNA samples were stored at −20°C until molecular testing. Negative extraction controls were included throughout the extraction process to monitor potential contamination. Detection of Antimicrobial Resistance Genes The presence of antimicrobial resistance genes was determined using quantitative polymerase chain reaction (qPCR). Target genes included clinically relevant resistance determinants associated with β-lactam, carbapenem, tetracycline, sulfonamide, and colistin resistance. The selected ARGs included blaTEM, blaCTX-M, blaNDM, blaOXA-48, tetA, tetM, sul1, sul2, and mcr-1. Gene-specific primers reported in previous validated studies were employed for amplification. Each qPCR reaction was performed in a final reaction volume of 20 µL containing template DNA, gene-specific primers, master mix, and nuclease-free water. Amplification was conducted using a real-time PCR system under optimized cycling conditions. Positive and negative controls were included in each run to ensure assay validity. Samples were considered positive when amplification occurred within the predefined cycle threshold (Ct) range. Quality Assurance and Quality Control Strict quality assurance procedures were implemented throughout the study. Sterile sampling equipment was used during sample collection, and field blanks were included to assess environmental contamination. DNA extraction controls, no-template controls, and positive amplification controls were incorporated into molecular analyses. Laboratory personnel followed standardized operating procedures, and all assays were performed in duplicate to ensure analytical reliability. Outcome Measures The primary outcome was the prevalence of antimicrobial resistance genes in wastewater samples. Secondary outcomes included the distribution of specific ARGs across sampling locations and the frequency of multidrug resistance gene co-occurrence. Quantitative gene abundance was expressed as gene copy numbers per unit volume of wastewater where applicable. Statistical Analysis Data were entered into Microsoft Excel and analyzed using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize the prevalence and distribution of antimicrobial resistance genes. Continuous variables were presented as mean ± standard deviation (SD) or median with interquartile range (IQR), depending on data distribution. Categorical variables were expressed as frequencies and percentages. Associations between sampling locations and ARG prevalence were evaluated using the Chi-square test or Fisher’s exact test where appropriate. Differences in gene abundance among sampling sites were assessed using independent-samples t-tests or one-way analysis of variance (ANOVA) for normally distributed data and non-parametric equivalents for skewed distributions. A p-value <0.05 was considered statistically significant. Ethical Considerations The study protocol was reviewed and approved by the Institutional Ethics Committee of Telangana Territory Centre and College. As the investigation involved environmental wastewater samples and did not include human participants or identifiable personal information, individual informed consent was not required. All laboratory procedures were conducted in accordance with institutional biosafety guidelines and national regulations governing environmental microbiological research.

A total of 160 wastewater samples were collected and analyzed from healthcare-associated wastewater discharge points at Telangana Territory Centre and College. Antimicrobial resistance genes (ARGs) were detected in 147 (91.9%) samples. The most frequently identified genes were sul1 (70.0%), blaTEM (65.0%), and tetA (60.0%), while carbapenem resistance genes such as blaNDM and blaOXA-48 were detected in 31.3% and 25.0% of samples, respectively.

Table 1. Distribution of Wastewater Samples According to Sampling Location (n=160)

Table 1 presents the distribution of the 160 wastewater samples collected from various healthcare-associated wastewater sources. The majority of samples were obtained from hospital effluent outlets (37.5%), followed by sewage collection chambers (25.0%), wastewater conveyance systems (21.9%), and wastewater treatment plant inlets (15.6%). The sampling strategy ensured adequate representation of different points within the healthcare wastewater network, facilitating comprehensive surveillance of antimicrobial resistance genes across the study area.

Table 2. Prevalence of Antimicrobial Resistance Genes in Wastewater Samples (n=160)

Table 2 illustrates the prevalence of antimicrobial resistance genes detected in the wastewater samples. Among the investigated genes, sul1 exhibited the highest prevalence (70.0%), followed by blaTEM (65.0%) and tetA (60.0%). Extended-spectrum β-lactamase gene blaCTX-M was identified in 55.0% of samples, while carbapenem resistance genes blaNDM and blaOXA-48 were detected in 31.3% and 25.0% of samples, respectively. The colistin resistance gene mcr-1 demonstrated the lowest prevalence (15.0%). These findings indicate widespread dissemination of clinically important resistance determinants within healthcare-associated wastewater systems.

Table 3. Comparison of ARG Detection Across Sampling Locations

Chi-square = 4.86; df = 3; p = 0.182

Table 3 compares the frequency of antimicrobial resistance gene-positive samples across different wastewater collection sites. The highest proportion of ARG-positive samples was observed in hospital effluent outlets (96.7%), followed by sewage collection chambers (92.5%), wastewater conveyance systems (88.6%), and wastewater treatment plant inlets (84.0%). Although hospital effluents showed a greater burden of resistance genes, statistical analysis revealed no significant difference among the sampling locations (χ² = 4.86, p = 0.182). This finding suggests that antimicrobial resistance genes were widely distributed throughout the wastewater network.

Table 4. Mean Abundance of Selected Resistance Genes in Wastewater Samples

Table 4 summarizes the quantitative abundance of selected antimicrobial resistance genes measured by qPCR. The highest mean gene abundance was observed for sul1 (8.5 × 10⁴ copies/mL), followed by blaTEM (7.8 × 10⁴ copies/mL) and tetA (6.9 × 10⁴ copies/mL). Lower concentrations were recorded for carbapenem resistance genes blaNDM and blaOXA-48, while the colistin resistance gene mcr-1 exhibited the lowest abundance. These findings indicate that sulfonamide, β-lactam, and tetracycline resistance determinants represent the dominant components of the wastewater resistome in the study setting.

Table 5. Co-occurrence of Multiple Resistance Genes in Wastewater Samples (n=147 Positive Samples)

Table 5 demonstrates the co-occurrence of multiple antimicrobial resistance genes among positive wastewater samples. Approximately 36.7% of samples harbored three to four resistance genes, while 28.6% contained five to six genes. Notably, 15.7% of samples carried seven or more resistance genes simultaneously, reflecting a substantial burden of multidrug resistance determinants. The frequent occurrence of multiple ARGs within individual samples highlights the potential for horizontal gene transfer and the emergence of multidrug-resistant bacterial populations in wastewater environments.

Table 6. Association Between Sampling Location and Carbapenem Resistance Genes

Chi-square = 8.74; df = 3; p = 0.033*

Table 6 evaluates the distribution of carbapenem resistance genes (blaNDM and blaOXA-48) across different wastewater sampling locations. Hospital effluent outlets demonstrated the highest prevalence of carbapenem resistance genes (46.7%), followed by sewage collection chambers (37.5%), wastewater conveyance systems (28.6%), and wastewater treatment plant inlets (20.0%). Statistical analysis revealed a significant association between sampling location and the presence of carbapenem resistance genes (χ² = 8.74, p = 0.033). This finding suggests that hospital wastewater serves as a major reservoir of clinically significant carbapenem resistance determinants and may contribute substantially to their environmental dissemination.

Figure 1. A total of 160 wastewater samples were collected from four healthcare-associated sampling locations. Hospital effluent outlets represented the largest sampling category (n = 60, 37.5%), followed by sewage collection chambers (n = 40, 25.0%), wastewater conveyance systems (n = 35, 21.9%), and wastewater treatment plant inlets (n = 25, 15.6%). The sampling design ensured representative coverage of the healthcare wastewater continuum for antimicrobial resistance gene surveillance.

Figure 2. Prevalence of antimicrobial resistance genes identified in healthcare wastewater samples. The sulfonamide resistance gene sul1 was the most frequently detected determinant (70.0%), followed by the β-lactam resistance gene blaTEM (65.0%) and the tetracycline resistance gene tetA (60.0%). Extended-spectrum β-lactamase-associated blaCTX-M and sulfonamide resistance gene sul2 were detected in more than half of the samples. Carbapenem resistance genes (blaNDM and blaOXA-48) were identified in 31.3% and 25.0% of samples, respectively, while the colistin resistance gene mcr-1 exhibited the lowest prevalence (15.0%). These findings demonstrate a substantial burden of clinically relevant resistance determinants within healthcare-associated wastewater systems.

Figure 3. revalence of carbapenem resistance genes (blaNDM and/or blaOXA-48) across different healthcare-associated wastewater sampling locations. Hospital effluent outlets demonstrated the highest prevalence of carbapenem resistance determinants (46.7%; 28/60 samples), followed by sewage collection chambers (37.5%; 15/40), wastewater conveyance systems (28.6%; 10/35), and wastewater treatment plant inlets (20.0%; 5/25). The progressive decline in prevalence along the wastewater pathway suggests dilution and partial removal of resistant microbial populations; however, clinically significant carbapenem resistance genes remained detectable throughout the wastewater network. Statistical analysis revealed a significant association between sampling location and carbapenem resistance gene occurrence (χ² = 8.74, p = 0.033), indicating that hospital wastewater represents a major environmental reservoir of high-priority antimicrobial resistance determinants.

The present study investigated the occurrence, prevalence, and abundance of antimicrobial resistance genes (ARGs) in healthcare-associated wastewater collected from Telangana Territory Centre and College. The findings revealed a high overall prevalence of ARGs, with 91.9% of wastewater samples harboring at least one resistance determinant. This observation highlights the significant role of healthcare wastewater as an environmental reservoir of antimicrobial resistance and supports growing evidence that hospital effluents contribute substantially to the dissemination of resistance genes into urban wastewater systems. Similar findings have been reported globally, where healthcare-associated wastewater consistently demonstrates elevated concentrations of antimicrobial-resistant bacteria and resistance determinants compared to municipal wastewater sources [16,17].

Among the investigated resistance genes, sul1 exhibited the highest prevalence (70.0%), followed by blaTEM (65.0%) and tetA (60.0%). The predominance of sul1 is consistent with previous studies identifying this gene as one of the most ubiquitous resistance determinants in wastewater environments. sul1 is frequently associated with class 1 integrons, which facilitate horizontal gene transfer and contribute to the persistence and dissemination of antimicrobial resistance in aquatic ecosystems [18]. Hendriksen et al. reported the widespread occurrence of sulfonamide resistance genes in urban sewage systems across multiple countries, suggesting their value as indicators of anthropogenic antimicrobial pressure [14]. The high prevalence of blaTEM and tetA observed in the present study may reflect extensive historical and current use of β-lactam and tetracycline antibiotics in clinical practice, resulting in sustained selective pressure favoring resistant microbial populations [19].

The detection of extended-spectrum β-lactamase (ESBL)-associated blaCTX-M in 55.0% of samples further emphasizes the public health significance of healthcare wastewater. ESBL-producing organisms are recognized as priority pathogens due to their capacity to confer resistance to third-generation cephalosporins and other clinically important antibiotics. Similar prevalence rates of blaCTX-M have been reported in hospital wastewater from Asia and Europe, indicating the widespread dissemination of ESBL resistance determinants within healthcare environments [20]. The persistence of these genes within wastewater systems raises concerns regarding environmental transmission and the potential emergence of resistant infections in both community and healthcare settings.

A particularly important finding of this study was the detection of carbapenem resistance genes. The prevalence of blaNDM (31.3%) and blaOXA-48 (25.0%) demonstrates the circulation of clinically significant carbapenemase-producing organisms within healthcare wastewater. Carbapenems are considered last-resort antibiotics for the treatment of severe multidrug-resistant infections, and the emergence of carbapenem resistance represents a major threat to global health. Previous investigations have similarly reported the occurrence of blaNDM and blaOXA-48 in hospital effluents, highlighting wastewater systems as important reservoirs and transmission pathways for these critical resistance determinants [21,22]. The detection of these genes in the present study reinforces concerns regarding environmental dissemination of carbapenem resistance and underscores the importance of wastewater surveillance as an early warning tool.

The colistin resistance gene mcr-1 was identified in 15.0% of samples. Although its prevalence was lower than that of other ARGs, the presence of mcr-1 remains clinically significant because colistin is frequently regarded as a last-line therapeutic option for infections caused by carbapenem-resistant organisms. The global emergence of plasmid-mediated colistin resistance has generated considerable concern due to its potential for rapid horizontal transmission among bacterial populations [23]. Detection of mcr-1 in healthcare wastewater indicates that resistance to critically important antibiotics may already be established within environmental reservoirs.

Quantitative analysis revealed that sul1, blaTEM, and tetA were also the most abundant resistance genes, exhibiting mean concentrations of 8.5 × 10⁴, 7.8 × 10⁴, and 6.9 × 10⁴ gene copies/mL, respectively. These findings are consistent with metagenomic investigations demonstrating that sulfonamide, β-lactam, and tetracycline resistance determinants constitute major components of wastewater resistomes [24]. The elevated abundance of these genes may result from continuous antibiotic exposure, high bacterial densities, and the presence of mobile genetic elements that facilitate resistance gene maintenance and dissemination. Quantitative assessments provide important information beyond simple prevalence data by indicating the magnitude of resistance gene burdens within wastewater systems.

Analysis of sampling locations demonstrated that hospital effluent outlets exhibited the highest prevalence of ARG-positive samples (96.7%), followed by sewage collection chambers (92.5%), wastewater conveyance systems (88.6%), and wastewater treatment plant inlets (84.0%). Although differences in overall ARG prevalence were not statistically significant, the observed trend suggests gradual dilution of resistant microorganisms as wastewater moves through the collection network. Similar spatial patterns have been reported by studies demonstrating that hospital discharge points contain the highest concentrations of antimicrobial-resistant bacteria and ARGs before mixing with municipal wastewater streams [17,19].

The distribution of carbapenem resistance genes showed a significant association with sampling location (p = 0.033). Hospital effluent outlets demonstrated the highest prevalence of blaNDM and blaOXA-48 (46.7%), whereas wastewater treatment plant inlets exhibited substantially lower prevalence (20.0%). These findings support previous observations that healthcare facilities serve as major point sources of carbapenem resistance determinants [21]. The statistically significant reduction in prevalence across the wastewater continuum may reflect dilution effects, microbial competition, and partial removal processes occurring during wastewater transport. Nevertheless, the continued detection of carbapenem resistance genes at downstream locations indicates that these determinants remain environmentally persistent and capable of dissemination beyond healthcare settings.

An additional notable finding was the frequent co-occurrence of multiple resistance genes within individual samples. Approximately 44.3% of ARG-positive samples contained five or more resistance genes, while 15.7% harbored seven or more genes simultaneously. The occurrence of complex resistance profiles suggests extensive horizontal gene transfer and the accumulation of multiple resistance determinants within bacterial communities. Similar multidrug resistance patterns have been documented in wastewater environments where mobile genetic elements, including plasmids, integrons, and transposons, facilitate the exchange of resistance genes among diverse bacterial species [18,25]. Such conditions create opportunities for the emergence of highly resistant bacterial strains with significant clinical and environmental implications.

The findings of this study further support the growing application of wastewater-based epidemiology for antimicrobial resistance surveillance. Wastewater monitoring offers a population-level approach capable of capturing resistance trends across large communities while complementing conventional clinical surveillance systems. Recent studies have demonstrated that wastewater surveillance can provide early warning signals for emerging resistance threats and assist in evaluating the effectiveness of infection prevention and antimicrobial stewardship programs [9]. Furthermore, the integration of environmental surveillance within the One Health framework provides a comprehensive strategy for understanding and mitigating the spread of antimicrobial resistance across human, environmental, and microbial interfaces [6].

Overall, the present study demonstrates that healthcare-associated wastewater represents an important reservoir of antimicrobial resistance genes, including clinically significant carbapenem and colistin resistance determinants. The high prevalence, abundance, and co-occurrence of ARGs observed in this investigation underscore the need for continuous wastewater surveillance, improved hospital wastewater management strategies, and integration of environmental monitoring within One Health approaches to combat antimicrobial resistance.

The present study demonstrated a high prevalence and abundance of antimicrobial resistance genes in healthcare-associated wastewater from Telangana Territory Centre and College, highlighting the critical role of hospital wastewater as a reservoir of antimicrobial resistance. The widespread detection of sul1, blaTEM, tetA, and blaCTX-M, together with the presence of clinically important carbapenem resistance genes (blaNDM and blaOXA-48) and the colistin resistance gene (mcr-1), indicates substantial dissemination of resistance determinants within the wastewater environment. Hospital effluent outlets exhibited the highest burden of resistance genes, emphasizing healthcare facilities as major contributors to environmental antimicrobial resistance. The frequent co-occurrence of multiple resistance genes further suggests the potential for horizontal gene transfer and the emergence of multidrug-resistant bacterial populations. These findings underscore the importance of integrating wastewater surveillance into antimicrobial resistance monitoring programs, strengthening hospital wastewater management practices, and adopting a One Health approach to mitigate the environmental spread of antimicrobial resistance and protect public health.

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