Molecular screening of extended-spectrum β-lactamase and carbapenemase resistance genes in hospital wastewater from Eastern Odisha: Predominance of extended-spectrum β-lactamase determinants and absence of blaNDM and blaKPC

INTRODUCTION

Hospital wastewater (HWW) originates from various healthcare settings, including patient care areas, operating rooms, laboratories, and the disposal of pharmaceutical waste, along with wastewater produced by administrative departments, laundries, kitchens, and other sections of the hospital.^[1,2] A failure to appropriately treat HWW can cause the release of antibiotic-resistant bacteria (ARB) into municipal sewage systems, rivers, and other water bodies, which can then spread into the environment and into human populations.^[3]

HWW, an essential but frequently ignored factor in the transmission of antimicrobial resistance (AMR), acts as a major source and pathway for antimicrobial- resistant bacteria (ARB).^[4] Hospital effluent is a key site for antimicrobial-resistant bacteria (ARB) because of the elevated concentrations of antibiotics and various pharmaceuticals, which greatly contribute to the emergence of antimicrobial-resistant pathogenic bacteria and the global spread of infections.^[5,6] As a result of presenting sub-lethal concentrations of antibiotics into HWW, AMR can develop in these environments. As a result, wastewater from hospitals serves as a crucial pathway for the source of antibiotics entering the environment; it includes a variety of biological materials, including human waste that still contains metabolized antimicrobials.^[7] HWW is home to numerous pathogenic microbes such as Escherichia coli, Pseudomonas aeruginosa, and Enterococcus spp., some of which are transmitted directly to patients by personnel in the healthcare field, or via contaminated medical equipment.^[8] Based on information from the World Health Organization (WHO), HWW is a major global contributor to the emergence of AMR in pathogenic bacteria.^[9]

Antibiotic resistance is a critical global public health issue that could become a leading cause of illnesses in the near future.^[10] This danger is exacerbated by the widespread use of antibiotics in medical facilities, where a large percentage (30– 90%) of these drugs are excreted by patients and then released into the environment. The ongoing release of antibiotics into the environment, often at sub-lethal levels within HWW, has the capacity to trigger and perpetuate antibiotic resistance in bacteria.^[11,12] There is an increasing interest in examining the existence of these bacteria in the environment, particularly in connection with human activities. HWW is recognized as a major source of antibiotic-resistant bacteria and acts as a connection between human communities and their environmental ecosystems.^[13-15] In this environment, bacterial populations engage with each other and can obtain, modify, and share resistance genes. The transmission of these genes may occur via different mechanisms, particularly through horizontal gene transfer facilitated by plasmids.^[16] Before being released into water bodies, wastewater is treated at wastewater treatment plants (WWTPs) to remove pollutants and microbes. Chlorine is frequently utilized for disinfection in accordance with WHO guidelines and local standards. Nonetheless, treatment failures can still lead to the presence of antibiotic-resistant bacteria.^[17-19]

There is a rising worry about the escalating prevalence of environment-based AMR bacteria (ARB). Releasing sewage from hospitals consists of a mixture that includes the waste materials and excretions of individuals receiving treatment, along with wastewater that harbors ARB from medical settings. There are a number of reasons why sewage is regarded as a reservoir for antibiotic resistance. The reason for this is that it is crucial for horizontal gene transfer, which promotes the dissemination of resistant genes for antibiotics.^[20]

Wastewater contains antibiotic residues, pollutants, and nutrients, which serve as selective pressures that encourage microorganisms to develop resistance. These mutations and horizontal gene transfers may result in the dissemination of resistance.^[21] Beta-lactam defenses against many different infections can be compromised by extended-spectrum β-lactamase (ESBL) enzymes, which destroy virtually all beta-lactams. With the emergence of blaCTX-M as the most common ESBL, it is responsible for the development of resistance to antibiotics such as penicillins and oxyimino- cephalosporins in combination with blaSHV and blaTEM.^[22] In the past, carbapenems and cephamycins were preferred for the treatment of ESBL bacteria. However, in light of the rising prevalence of bacteria such as blaOXA, blaKPC, and the newly identified blaNDM, these drugs are no longer effective.

A significant and escalating issue for human health pertains to the rise and swift dissemination of antibiotic resistance.^[23] Antibiotics with beta-lactam residues are commonly prescribed around the world as both humans and animals use them to treat infections.^[24] A major concern regarding these medications is the quick development of beta-lactamases, particularly among Gram-negative bacteria (GNB); this creates every fresh medication to lose its effectiveness in a brief period.^[25] In the beta-lactam category, carbapenem antibiotics are considered the most reliable final option for treating infections caused by bacteria that produce ESBLs.^[26] On a global scale, carbapenem resistance represents a significant public health issue. This is due to the production of carbapenemase. Penicillins, cephalosporins, and carbapenems can all be broken down by carbapenemases, which limits the choice of antibiotics.^[27] Research indicates that serious infections caused by carbapenem-resistant Enterobacteriaceae are associated with higher mortality rates among hospitalized patients.^[28,29] There have been several types of carbapenemases developed, with the most commonly identified enzymes being Ambler class A beta- lactamases (such as KPC), class B beta-lactamases/metallo- beta-lactamases (such as NDM and IMP), and class D oxacillinases (such as OXA-48 and OXA-58).^[30] Genes that encode carbapenemases are typically located on mobile genetic elements, which are capable of being transferred from one species of bacterium to another.^[31] In particular, organisms within the Enterobacteriaceae family and Acinetobacter typically act as significant carriers for the spread of beta-lactamase genes within naturally occurring bacterial ecosystems.^[32,33]

Although HWW has increasingly been recognized as an important reservoir of AMR genes (ARGs), most studies from India have been conducted in selected metropolitan regions, and data from eastern India, particularly Odisha, remain scarce. Furthermore, information regarding the distribution of clinically important ESBL and carbapenemase genes in HWW from Odisha is limited. The absence of such regional surveillance data restricts our understanding of the environmental dissemination of AMR and hinders the development of targeted mitigation strategies. Therefore, this study aimed to investigate the prevalence of ARB and characterize major ESBL and carbapenemase genes (blaTEM, blaCTX-M, blaSHV, blaOXA, blaKPC, and blaNDM) in wastewater collected from six tertiary-care hospitals in Bhubaneswar, Odisha. The findings provide baseline evidence on the environmental burden of resistance genes in eastern India and contribute to ongoing efforts to strengthen AMR surveillance using a one health approach.

MATERIALS AND METHODS

RESULTS

In total, 164 bacterial isolates were collected from HWW. The isolates comprised a diverse range of bacteria, including K. pneumoniae n = 102 (62%), E. coli n = 41 (25%), P. aeruginosa n = 17 (10%) and Enterococcus n = 4 (3%). The drain exhibited the highest level of contamination, indicating that both healthcare workers and patients who cleanse their hands in the basin contribute to the transfer of clinical bacteria into the drainage, which eventually flows into the sewage lines.

Antibiotic resistance pattern of hospital wastewater: The most resistant antibiotics were CXM, PIT, OF, NX, NIT, CPT, and COT which were 100, 99, 98, 97, 90, 87% resistant, respectively. Least resistance was found in PB, Amoxicillin- clavulanic acid (AMC), AK, C, and HLG, antibiotics which were 35, 47, 52, 56, 60% resistant, respectively [Table 3].

Among the eight distinct classes of antibiotics evaluated, the highest resistance was noted for penicillins, while the lowest resistance was reported for the miscellaneous category, with C identified as the most potent in this group, as illustrated in Figure 1. Overall, AK emerged as the most effective antibiotic, demonstrating a resistance rate of 85 (52%) among 164 isolates.

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Figure 1: Illustrates the antibiotic resistance of 164 isolates of Enterobacterales collected from hospital wastewater samples. AK: Amikacin, C: Chloramphenicol, CAZ: Ceftazidime, CIP: Ciprofloxacin, CL: Colistin, CTR: Ceftriaxone, HLG: Gentamicin, IC: Imipenem/cilastatin, MRP: Meropenem, PIT: Piperacillin/tazobactam.

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The isolates exhibited a diverse occurrence of the 6 beta- lactam genes, with blaTEM being the most common at 34%, closely succeeded by blaCTX-M at 26%. The gene blaOXA was detected in 18% of the isolates, while blaSHV appeared in 13%. No presence of the blaKPC or blaNDM genes was observed [Table 4, Figures 2 and 3].

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Figure 2: Genes detected in selected isolates are shown in this chart. CTX-M: Cefotaximase-München, SHV: Sulfhydryl variable, TEM: Temoniera, KPC: Klebsiella pneumoniae carbapenemase, OXA: Oxacillinase, NDM: New Delhi metallo-β-lactamase.

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Figure 3: Identification of the gene responsible for bacterial resistance that encodes beta-lactamase in total DNAs extracted from hospital wastewater. Lane 1: DNA ladder; Lane 2: Negative control; Lanes 3, 4, 5, 6, 7, and 8 represent samples from hospital wastewater. CTX-M: Cefotaximase-München, SHV: Sulfhydryl variable, TEM: Temoniera, KPC: Klebsiella pneumoniae carbapenemase, OXA: Oxacillinase, NDM: New Delhi metallo-β-lactamase.

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DISCUSSION

The setting of a hospital is a complex system that has often been ignored as a possible source of bacteria; however, with the increasing incidence of hospital-acquired infections, it has become an important focus for microbial research.^[37] In addition, the rising patterns of antibiotic resistance in microbes have compelled the research community to explore the sources of such pathogens and the ways in which they acquire resistance in various environmental contexts. Bodies of water have also been identified as a source of antibiotic resistance, particularly because they promote interactions between pathogenic and non-pathogenic bacteria, contributing to the rise in resistance levels.^[38]

In the present study, a total of 164 bacterial isolates were obtained from HWW. The collection comprised various bacteria, including K. pneumoniae (n = 102, 62%), followed by E. coli (n = 41, 25%), P. aeruginosa (n = 17, 10%), and Enterococcus (n = 4, 3%). A similar bacterial distribution was reported by Aleem et al.^[39] who examined 162 isolates, with 127 classified as Gram-negative and 35 as Gram- positive. Their findings included a range of bacteria such as Klebsiella spp. (13%), Pseudomonas spp. (10%), E. coli (9%), and Enterococcus spp. Duran-Bedolla et al.^[40] documented comparable outcomes, identifying 243 Gram- negative isolates across 21 distinct bacterial species. Among these, E. coli accounted for 32.9% (80/243) and Enterobacter spp. for 24.6% (60/243), representing a cumulative total of 71.9%. In a related study by Zhang et al.^[41] it was noted that 62 out of 104 isolates (59.6%) were classified as Enterobacteriaceae, with E. coli making up 35 (56.5%), K. pneumoniae 17 (27.4%), Enterobacter spp. 5 (8.1%), and Pseudomonas spp. 6 (5.8%). Similar proportions were noted in further research that assessed the variety of bacteria found in hospital environments, highlighting the common occurrence of GNB.^[42]

In terms of the resistance patterns found in HWW, the antibiotics exhibiting the highest resistance included CXM, PIT, OF, NX, NIT, CPT, and COT, with resistance rates of 100%, 99%, 98%, 97%, 90%, 87%, respectively. The antibiotics showing the least resistance were PB, Amoxiclav (AMC), AK, C, and HLG, which had resistance rates of 35%, 47%, 52%, 56%, and 60%, respectively. A study conducted by Duran- Bedolla et al.^[40] found similar observations, reporting 100% resistance against CAZ, while cefepime, imipenem, and MRP also showed high resistance. The lower resistance was noted with AK and levofloxacin, which had resistance rates of 50% and 42%, respectively. Another related investigation by Aleem et al.^[39] reported higher resistance levels, with 90% resistance to CIP, cefoxitin, and erythromycin, 80% resistance to CTR, and 40% resistance concerning carbapenems. These findings align with our research, which demonstrated 100% resistance to AMP and over 80% resistance to amoxicillin, aminoglycosides, and macrolides. A similar conclusion was highlighted by Endalamaw et al.^[43] revealing that all K. pneumoniae and more than half of E. coli showed resistance to the third-generation cephalosporins like CAZ. Furthermore, in the wastewater from government hospitals in Addis Ababa, 43.3% of E. coli demonstrated resistance to CTR.^[44] There is either a lack of understanding about how these medications should be used or inappropriate disposal practices for antimicrobials.^[45] In addition, it may result from the release of sub-therapeutic levels of antibiotics from hospitals into the ecosystem, potentially exerting selective pressure on vulnerable bacteria in that setting. Consequently, it has been documented that wastewater from hospitals promotes the growth of antibiotic-resistant bacteria within healthcare settings.

Additionally, in this study, beta-lactam resistance genes were examined in wastewater samples collected from hospitals. It was revealed that during the study period, both ESBL and carbapenem resistance genes were present in these samples. The resistance patterns identified are primarily associated with the increased activity of beta-lactam in these strains, which is related to the beta-lactamase genes. The isolates we tested contained extended-spectrum beta-lactamase genes: blaTEM, blaCTXM, blaOXA, and blaSHV, with frequencies calculated at 34%, 26%, 18%, and 13%, respectively. No blaKPC or blaNDM genes were detected. In a different study conducted by Aleem et al., blaTEM was found to be the most common gene at 68%,^[39] closely followed by blaSHV at 66%. The presence of blaCTX-M was recorded at 29%, with blaNDM at 18% and blaOXA at 16%. Similarly, no blaKPC gene was identified, aligning with our results. In another related investigation by Duran-Bedolla et al., the identified β-lactamases included blaTEM at 33.1%, blaCTX-M at 25.4%, blaKPC also at 25.4%, blaNDM at 8.8%, blaSHV at 5.3%, and blaOXA-48 at 1.1%.^[39,40]

There is an increasing health concern associated with the identification of various multidrug-resistant opportunistic pathogens in WWTPs and in river environments, including E. coli, K. pneumoniae, Enterobacter species, and Acinetobacter species. It is crucial to increase efforts focused on improving the removal of microorganisms that are resistant to antimicrobials from wastewater in hospitals.

The methods used in wastewater treatment may affect the prevalence of genes associated with antibiotic resistance in samples of wastewater.^[46] According to earlier research,^[47-49] there is an increase in the number of ESBL-producing E. coli and K. pneumoniae in the community. According to these findings, E. coli and K. pneumoniae were found in wastewater samples from clinical settings.

A notable strength of the present study is that it provides region-specific data from Odisha, a geographical area for which published information on ESBL and carbapenemase genes in HWW is limited. The predominance of blaTEM and blaCTX-M and the absence of and blaNDM blaKPC among the analyzed isolates suggest a resistance gene distribution pattern that may differ from reports from other parts of India, highlighting the importance of local AMR surveillance.

A limitation of this study relates to antimicrobial susceptibility testing methodology. Although susceptibility testing was performed using the Kirby–Bauer disk diffusion method according to CLSI guidelines, certain antibiotics included in the testing panel may not be routinely recommended for interpretation in Enterococcus spp. Furthermore, susceptibility testing of PB by disk diffusion is not recommended because of the poor diffusion characteristics of polymyxins in agar media; broth microdilution is considered the preferred method. Therefore, the PB results should be interpreted with caution. In addition, the very high resistance rates observed for several antibiotics may reflect the substantial antimicrobial selective pressure present in HWW environments; however, confirmation through larger multicenter studies and complementary susceptibility testing methods would strengthen these findings.

CONCLUSIONS

This study demonstrates the widespread occurrence of multidrug-resistant bacterial species and ESBL-associated resistance genes in HWW from Odisha. Among the screened isolates, blaTEM was the most prevalent resistance gene, followed by blaCTX-M, blaOXA, and blaSHV. Notably, no blaNDM or blaKPC carbapenemase genes were detected in the analyzed isolates. These findings highlight HWW as a potential environmental reservoir for AMR and underscore the importance of continuous surveillance, effective wastewater treatment, antimicrobial stewardship, and infection-control measures to limit the dissemination of resistant bacteria and resistance genes into the environment.

This study found that bacterial identification was based on conventional morphological and biochemical methods. Although these methods are widely used for routine microbiological identification, they may provide lower taxonomic resolution than advanced techniques such as Matrix assisted laser Desorption/Ionization -Time of flight mass spectrometry (MALDI-TOF) or 16S rRNA gene sequencing. Due to resource limitations, these molecular identification approaches were not employed in the present study. Future investigations should incorporate advanced identification methods to improve species-level accuracy and strengthen the characterization of AMR determinants.