Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Producing E. coli and Klebsiella spp. in Sewage Effluents of Dharan, Nepal

Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Producing E. coli and Klebsiella spp. in Sewage Effluents of Dharan, Nepal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Producing E. coli and Klebsiella spp. in Sewage Effluents of Dharan, Nepal Prashant Dahal, Rojina Rai, Madhav Raj Sharma, Lalit Narayan Chaudhary, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1509782/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The prevalence of extended-spectrum \(\beta\) -lactamase (ESBL) and AmpC \(\beta\) -lactamase (ABL) producing Enterobacteriaceae is increasing rapidly across the world. Members of Enterobacteriaceae like E. coli and Klebsiella spp . exhibit antimicrobial resistance mainly due to the production of beta-lactamase enzymes like extended-spectrum βlactamases, AmpC β-lactamases, and carbapenemases. These bacteria are frequently reported in sewage effluents of hospital and municipal sewerage systems indicating sewage as a promising source for dissemination of such drug-resistant pathogens. However, in most of the developing countries including Nepal, the major portion of sewage is discharged in water sources without proper treatment and disinfection. This study was undertaken to access the prevalence of ESBL and ABL producing E. coli and Klebsiella spp . in sewage effluents of Dharan, Nepal. Results A total of 235 bacteria were isolated, out of which 103 (43.83%) were E. coli and 132 (56.17%) were Klebsiella spp . ESBL production was seen in 157 (66.81%) isolates. Among them, 89 (56.69%) were Klebsiella spp . and 68 (43.31%) were E. coli. 66.02% of total isolated E. coli and 67.42% of total isolated Klebsiella spp . showed production of ESBL enzymes. ABL production was seen in 133 (56.59%) isolates. Among them, 54 (40.60%) were E. coli and 79 (59.40%) were Klebsiella spp. 52.43% of the isolated E. coli and 59.85% of isolated Klebsiella spp . were found producing ABL enzyme. Conclusions The results indicate that there is a high prevalence of ESBL and ABL producing E. coli and Klebsiella spp . in sewage effluents of Dharan. Effective treatment of sewage effluents must be ensured before discharging the sewage into the environment. National guidelines for discharging the municipal sewage must be immediately amended and an effective treatment system before discharge must be implemented. Dissemination of such drug-resistant bacteria in the human population leading to severe public health emergency is likely to occur from sewage contamination, so further study and surveillance and effective prevention and control measures are necessary. ESBL ABL Enterobacteriaceae E. coli Klebsiella spp Sewage effluent Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Sewage is the collection of wastewater in a municipal piper system or sewerage channel. It is mostly a combination of domestic effluents from toilets, bathing, and kitchen, water from commercial establishments and institutions including hospitals and clinics, hotels and restaurants, shops and markets, schools, colleges, and different offices, industrial effluents, and surface runoff water (Mateo-Sagasta et al., 2015 ). Sewage water contains a different variety of organic and inorganic wastes and nutrients merged from different sources. Hospital sewage water is loaded with several pathogenic microorganisms with antimicrobial resistance capacity, partially metabolized pharmaceutical substances like antimicrobial, pharmaceutical, disinfectants, and un-metabolized drugs, radioactive elements, and other toxic substances. Non-hospital sewage is loaded with microorganisms from human and animal feces and environmental sources. This will make sewage more prone to pathogenic organisms including antibiotic-resistant bacteria (Mahato et al., 2019 ). Different studies have shown the presence of antibiotic-resistant bacteria and genes which have even escaped treatment systems and disseminated in wastewater (Nasser et al., 2019 ). Wastewater treatment systems can reduce contaminants including microorganisms to a minimum limit, but only 5% of generated wastewater is treated appropriately while the remaining is directly connected to water bodies in Nepal. This has resulted in the transmission of microorganisms and water-borne infections (Jha & Bajracharya, 2014 ). Enterobacteriaceae covers a large part of hospital sewage microbiota (Korzeniewska & Harnisz, 2013 ). Among the sewage pathogens, coliforms are responsible for frequent human infections (Doi et al., 2017 ). Coliforms are aerobic or facultative, Gram-negative, non-sporing lactose fermenting bacteria. They were traditionally represented by 4 genera viz. Escherichia, Klebsiella, Citrobacter , and Enterobacter , but now contain over 20 bacterial genera (Masiello et al., 2016 ). Enterobacteriaceae includes different Gram-negative bacteria including pathogenic genera like Escherichia, Salmonella, Shigella, Klebsiella , and Serratia (Rock & Donnenberg, 2014 ). Klebsiella pneumoniae and E. coli of Enterobacteriaceae have notable antibiotic resistance to a wide variety of antibiotics typically used in the treatment of their infection (Le et al., 2016 ; Kazemian et al., 2019 ). Members of Enterobacteriaceae like E. coli and Klebsiella spp. exhibit antimicrobial resistance mainly due to the production of beta-lactamase enzymes like extended-spectrum β-lactamases, AmpC β-lactamases, and carbapenemases (Ojer-Usoz et al., 2013 ). Enterobacteriaceae were found to produce intrinsic chromosomal encoded beta-lactamases but due to the transfer of such gene to transferable plasmid gene, there is rapid dissemination of Enterobacteriaceae resistant to a wide variety of beta-lactam antibiotics (Susić, 2004 ).ESBLs are \(\beta\) -lactamases capable of developing resistance against \(\beta\) -lactam drugs like penicillins, extended-spectrum cephalosporins, and monobactams except for cephamycins and carbapenems and susceptible to \(\beta\) -lactam inhibitor clavulanic acid (Raut et al., 2015 ). ABL is characterized by the hydrolysis of broad-spectrum cephalosporins like cefotaxime and ceftazidime and cephamycins (7-α-methoxy cephalosporins) like cefoxitin and cefotetan, including monobactam, and the β-lactam/β-lactam inhibitor combination, but are sensitive to cefepime and carbapenems (Dehkharghani et al., 2020 ). Different clinical isolates of E. coli and Klebsiella spp . are found to be antibiotic resistant and produce ESBL and ABL enzymes for conferring the resistance against \(\beta\) -lactam antibiotics. (Shakya et al., 2017 ) concluded on the higher prevalence of ESBL producing E. coli and Klebsiella spp . in Nepal by examining 2209 non-repetitive MSU samples at a tertiary hospital of Nepal. Similarly, (Baral et al., 2013 ) and (Aryal et al., 2020 )concluded the high prevalence of ABL producers in clinical isolates. (Mahato et al., 2019 ) studied sewage effluents of different hospitals of Biratnagar, Nepal, and concluded the prevalence of MDR and ESBL producing E. coli and Klebsiella spp . in hospital sewage effluents. Sewage effluents have high potentiality as an environmental reservoir of a wide variety of pathogenic organisms including ESBL and ABL producing E. coli and Klebsiella spp. Unfortunately, scanty research is done on sewage effluents as a source of such pathogens. The present study was undertaken to reduce the research gap on assessment of sewage microbiota with the sole focus on determining the prevalence of ESBL and ABL producing E. coli and Klebsiella spp. in sewage effluents of Dharan, Nepal. Results Distribution of E. coli and Klebsiella spp . in the Samples Among the total of 20 samples analyzed 16 samples (80%) showed growth of either E. coli or Klebsiella spp . or both and 4 samples (20%) did not show growth of E. coli and Klebsiella spp .(Table 1 ). Out of 16 positive samples, 13 (81.25%) showed growth of both E. coli and Klebsiella spp ., 2 (12.5%) showed growth of Klebsiella spp . only and 1 (6.25%) showed growth of E. coli only. All of the hospital-sewage effluents (6 out of 6) and 8 of the non-hospital sewage effluents (8 out of 14) showed growth of both E. coli and Klebsiella spp .(Table 1 ). This showed that 70% of the samples contained E. coli and 75% of the samples contained Klebsiella spp . Table 1 Prevalence of E. coli and Klebsiella spp . in each sample Sample Location Bacteria Isolated E. coli Klebsiella spp. 1 BPKIHS, Dharan + + 2 BPKIHS, Dharan + + 3 BPKIHS, Dharan + + 4 BPKIHS, Dharan + + 5 Bijaypur Hospital Pvt.Ltd + + 6 Bijaypur Hospital Pvt. Ltd + + 7 Residential area – 1 - - 8 Residential area – 2 + + 9 Residential area – 3 - + 10 Residential area – 4 - - 11 Market area – 1 + + 12 Market area – 2 + + 13 Market area – 3 + + 14 Commercial area – 1 + + 15 Commercial area – 2 + + 16 Commercial area – 3 + - 17 Commercial area – 4 - - 18 Commercial area – 5 - + 19 Commercial area – 6 - - 20 Commercial area – 7 + + A total of 235 bacteria were identified as either E. coli or Klebsiella spp . Out of them, 103 isolates (43.83%) were E. coli and 132 isolates (56.17%) were Klebsiella spp . The prevalence of Klebsiella was found to be higher. (Fig. 1 ) Antibiotic Susceptibility Pattern of Isolated E. coli and Klebsiella spp . Antibiotic sensitivity test of selected antibiotics against the isolated E. coli and Klebsiella spp . showed the highest sensitivity to Azithromycin (63.82%) and the lowest sensitivity to Cefoxitin (1.28%) (Table 2 ). Table 2 Antibiotic Susceptibility Pattern of Isolated E. coli and Klebsiella spp . Antibiotics Disc Content (in \(\varvec{\mu }\varvec{g}\) ) Susceptibility Pattern Sensitive n (%) Resistant n (%) Ampicillin 10 27 (11.49%) 208 (88.51%) Azithromycin 15 150 (63.83%) 85 (36.17%) Aztreonam 30 55 (23.40%) 180 (76.60%) Ciprofloxacin 5 113 (48.09%) 122 (51.91% Co-Trimoxazole 25 146 (62.13%) 89 (37.87%) Ceftriaxone 30 45 (19.15%) 190 (80.85%) Ceftazidime 30 39 (16.59%) 196 (83.41%) Cefotaxime 30 41 (17.45%) 194(82.55%) Cefoxitin 30 3 (1.28%) 232 (98.72%) Imipenem 10 140 (59.57%) 95 (40.43%) Nitrofurantoin 300 127 (54.04%) 108 (45.96%) Beta-lactam antibiotics; Ampicillin, Aztreonam, Ceftriaxone, Ceftazidime, Cefotaxime, and Cefoxitin showed very poor sensitivity patterns showing the sensitive result to only 11.49%, 23.40%, 19.15%, 16.59%, 17.45%, and 1.28% of tested bacteria respectively. Ciprofloxacin showed sensitivity to 48.09% of the tested bacterial species, while Co-Trimoxazole showed sensitivity to 62.13% of the tested bacterial species. Imipenem showed a sensitive result on 59.57% of the tested bacteria. Nitrofurantoin showed a sensitive result on 54.04% of the tested bacteria. Antibiotic susceptibility test of E. coli revealed that 84.47% of them were sensitive to Nitrofurantoin, while only 2.91% were sensitive to Cefoxitin. Ampicillin, Azithromycin, Aztreonam, Ciprofloxacin, Co-Trimoxazole, Ceftriaxone, Ceftazidime, Cefotaxime, and Imipenem showed sensitivity against 15.53%, 55.34%, 31.07%, 48.54%, 68.93%, 31.07%, 25.24%, 20.39%, and 58.25% of the tested E. coli respectively. On other hand, 70.45% of Klebsiella spp . were sensitive to Azithromycin and 0% of Klebsiella spp . were sensitive to Cefoxitin. Ampicillin, Aztreonam, Ciprofloxacin, Co-Trimoxazole, Ceftriaxone, Ceftazidime, Cefotaxime, Imipenem, and Nitrofurantoin showed sensitivity against 8.33%, 17.42%, 47.73%, 56.82%, 9.85%, 9.85%, 15.15%, 60.61%, and 30.31% of the tested Klebsiella spp . respectively. Distribution of ABL Producing E. coli and Klebsiella spp . 133 isolates (56.59%) were found positive for ABL production. Among 133, 54 (40.60%) were E. coli and 79 (59.40%) were Klebsiella spp . (Fig. 2 ) This showed a higher prevalence of ABL producing Klebsiella spp. than E. coli in sewage effluents of Dharan. Distribution of ESBL Producing E. coli and Klebsiella spp . ESBL production was seen in 157 isolates (66.81%) while testing 235 isolated bacteria. Out of 157 ESBL producing 89 (56.69%) were Klebsiella spp . and 68 (43.31%) were E. coli . (Fig. 3 ) This showed a higher prevalence of ESBL producing Klebsiella spp . than E. coli in sewage effluent of Dharan. Distribution of ABL and ESBL Co-producing E. coli and Klebsiella spp . A total of 118 isolates showed the production of both ABL and ESBL enzymes. This accounted for 50.21% of total isolated bacteria. Out of 118 co-producers, 51 (43.22%) were E. coli , and the remaining 67 (56.78%) were Klebsiella spp . (Fig. 4 ) Prevalence of ABL and ESBL co-production was comparatively higher in Klebsiella spp . than in E. coli. Discussion Beta-lactam antibiotics are among the most widely used antibiotic class(Bush & Bradford, 2016 ). Bacteria have developed resistance to \(\beta\) -lactam antibiotics by evolving their ability to synthesize several types of \(\beta\) -lactamase enzymes capable of hydrolyzing \(\beta\) -lactam ring. Among the \(\beta\) -lactamase enzymes ESBL and ABL are the most commonly encountered types (Bush & Bradford, 2020 , Bush & Jacoby, 2010 ). Resistance to \(\beta\) -lactam antibiotics by producing ESBL and ABL enzymes is increasing rapidly among members of Enterobacteriaceae across the world(Caron et al., 2018 ) (De Angelis et al., 2020 ). This study was conducted to detect the prevalence of ABL and ESBL producing E. coli and Klebsiella spp. in sewage effluents of Dharan. A total of 20 samples were analyzed in the Molecular and Microbiology Laboratory of Sunsari Technical College, Dharan-1 from May 21, 2021, to October 29, 2021. The phenotypic method was used to confirm the production of AmpC Beta-lactamase and Extended Spectrum Beta-lactamase enzymes in the isolated species using the methods recommended by CLSI(Patel & Clinical and Laboratory Standards Institute, 2017) Among the total of 20 samples analyzed 16 samples (80%) showed growth of either E. coli or Klebsiella spp . or both and 4 samples (20%) did not show growth of E. coli and Klebsiella spp . Among the studied sample, 70% (14 out of 20) showed the presence of E. coli , and 75% (15 out of 20) showed the presence of Klebsiella spp . Mahato et al., 2019 had reported that 70% of sewage effluents contained E. coli while only 60% of the sewage effluents contained Klebsiella spp . This variation may be since Mahato et al., 2019 had studied hospital sewage effluents only while we studied both hospital and municipal sewage. In the study, 235 bacteria were identified as either E. coli or Klebsiella spp. 103 (43.83%) of them were E. coli , whereas 132 (56.17%) of them were Klebsiella spp . Results obtained by Mahato et al., 2019 were different. They had shown that 53.85% of their isolates were E. coli , while 46.15% were Klebsiella spp . (Fadare & Okoh, 2021 ) had shown that 65.71% of their isolates were Klebsiella spp . and only 10% were E. coli . Ampicillin resistance was seen in 84.47% of E. coli . It was 44.66% for Azithromycin, 68.93% for Aztreonam, 51.46% for Ciprofloxacin, 31.07% for Co-Trimoxazole, 68.93% for Ceftriaxone, 74.76% for Ceftazidime, 79.61% for Cefotaxime, 97.09% for Cefoxitin, 41.75% for Imipenem, and 15.53% for Nitrofurantoin. This result was similar to Mahato et al., 2019 for Ampicillin, Aztreonam, and Cefoxitin. They had shown 100% resistance to these antibiotics. But, for Co-Trimoxazole, Ceftazidime, Ceftriaxone, and Cefotaxime they had shown resistance to 85.7% which was higher than the result obtained in this research. Similarly, they had shown that resistance to Nitrofurantoin was 42.9%, which was higher in comparison to this research. For Azithromycin, they had shown that non-of them (0%) were resistant, but 44.66% were found resistant in this research. They had reported that 14.3% were found to be resistant to Ciprofloxacin but it was 51.46% in this research. This study showed that 91.67% of Klebsiella spp . were resistant to Ampicillin. It was 29.55% for Azithromycin, 82.58% for Aztreonam, 52.27% for Ciprofloxacin, 43.18% for Co-Trimoxazole, 90.15% for Ceftriaxone, 90.15% for Ceftazidime, 84.85% for Cefotaxime, 100% for Cefoxitin, 39.39% for Imipenem, and 69.69% for Nitrofurantoin. The result was shown by Mahato et al., 2019 indicates comparatively higher resistance against Ampicillin and Co-Trimoxazole, and lower to the other antibiotics. ESBL production was found higher in Klebsiella spp . than in E. coli . Out of 157 ESBL producers, 43.31% (68) were E. coli . and 56.69% (89) were Klebsiella spp . Mahato et al., 2019 showed higher ESBL production in E. coli (66.67%) than in Klebsiella spp . (33.33%). Similarly, Zaatout et al., 2021 also had reported a higher prevalence of ESBL E. coli in sewage effluents than that of Klebsiella spp . Fadare & Okoh, 2021 on the other hand had shown a higher prevalence of ESBL producing Klebsiella spp . than E. coli in hospital wastewater. ESBL production was seen in 81.59% of total tested E. coli and 85.61% of total tested Klebsiella spp . A higher prevalence of ESBL producing E. coli was found by (Reinthaler et al., 2009 ). ABL production was also found to be higher in Klebsiella spp . than in E. coli . Among 133 ABL producers, 59.40% (79) were Klebsiella spp . and 40.60% (54) were E. coli . Among 57 wastewater samples tested by (Ben Said et al., 2016 ), 24 were found to be positive for ESBL-Eb or pAmpC-Eb producing Enterobacteriaceae . They detected ESBL-Eb in 20 samples, and pAmpC-Eb in 4 samples. No relevant literature on ABL producing E. coli and Klebsiella spp . in sewage effluents were found for further comparison. ESBL and ABL co-production was seen in 50.21% (total 118) of the isolates. 75% of the ESBL producing E. coli were found to produce ABL also, and 75.28% of the ESBL producing Klebsiella spp . were found to produce ABL also. This result was higher than that of Rizi et al., 2020. They had shown co-production in only 30% of the isolated E. coli and Klebsiella spp . Similarly, co-production was seen in 29% of the isolated E. coli and Klebsiella pneumoniae in research by (Hertz et al., 2019 ). Co-production was seen in 9.38% of isolated E. coli and non (0%) of isolated Klebsiella spp . by (Shrestha et al., 2019 ). These variations may be because all these studies are on clinical isolates. Conclusions Based on the finding of the present study, it can be concluded that sewage effluents of Dharan have a higher prevalence of ESBL and ABL producing E. coli and Klebsiella spp . This suggests that there may be a direct link between human or animal feces in the sewage system. The result is an alarming indicator of a probable outbreak of life-threatening infection by \(\beta\) -lactamase producing E. coli and Klebsiella spp . if sewage is contaminated with drinking water channels or food in the Dharan area. There is an urgent need for further research in sewage microflora and the probability of their dissemination to human populations. Government bodies should prepare effective regulatory measures to control the irrational use of antibiotics and monitor the antimicrobial resistance status of different suspected bacteria. Effective alternatives for the treatment of ESBL and ABL producing E. coli and Klebsiella spp . may be needed soon. Immediate and effective response from concerned authorities for controlling the dissemination of such bacteria from sewage to the environment or community is required. Regular monitoring and study of antibiotic susceptibility patterns of several bacteria in different aspects of the environment like sewage is needed. Methods Design and Setting of Study A laboratory-based cross-sectional quantitative study was designed. The study was conducted in Dharan sub-metropolitan city. It is a city in Koshi zone’s Sunsari district of Province-1, Nepal with co-ordinates 26 0 49 \(\text{'}\) 0 \(\text{'}\text{'}\) N 87 0 17 \(\text{'}\) 0 \(\text{'}\text{'}\) E. The study was conducted from May 21, 2021, to October 29, 2021. Sewage effluents, directly from the municipal sewage channel, were taken as samples for the study. Non-probability convenience sampling method was followed for sample collection. Sample Collection and Transportation A total of 20 samples were taken from different sites of Dharan city. Samples were taken considering that it would cover almost every part of the city. Out of 20 samples, 6 samples (30%) were from the hospital area, and the remaining 14 samples (70%) were from the non-hospital area. Samples were collected directly from the center of the sewage flow channel. For hospital sewages, samples were collected from the point where hospital sewage is connected to the municipal sewerage channel. A pre-sterilized sterile glass bottle was used to collect the sample from the flow channel with the help of a sampling rod. About 100 mL of sewage effluents were taken from one site. Each bottle was labeled with the date, sample location, and time of collection. Collected samples were transported to the laboratory immediately maintaining a cold chain in the ice-box to inhibit the growth of microorganisms. (Mahato et al., 2019 ) Sample Processing and Inoculation Samples were serially diluted in sterilized distilled water (SDW) up to tenfold. Eosin-methylene blue (EMB) agar plates and MacConkey (MAC) agar plates were prepared and solidified properly about 3–4 hours before inoculation. 5 plates of each media were prepared and labeled as E10 − 2 , E10 − 4 , E10 − 5 , E10 − 6 , and Econtrol on EMB plates and M10 − 2 , M10 − 4 , M10 − 5 , M10 − 6, and Mcontrol for MAC plates. From the serially diluted samples in the tube, 100 \(\mu\) L of the sample was pipetted out after mixing properly and inoculated on the agar plates using the spread-plate (lawn culture) method. Dilution 10 − 2 , 10 − 4 , 10 − 5 , and 10 − 6 were inoculated. The plates were incubated aerobically at 37 0 C for 24 hours. Isolation and Identification of E. coli and Klebsiella spp . Following incubation colonies were isolated from the plate with well-isolated colonies. MAC was used for isolation of Klebsiella spp . and EMB was used for isolation of E. coli . (Mahato et al., 2019 b) From MAC plates, well-isolated colonies which were showing characters of Klebsiella spp. were marked and streaked on Nutrient Agar (NA) plates for purification. Similarly, well-isolated colonies which were showing characters of E. coli in EMB plates were marked and isolated in NA plates (Anderson et al., 2019 ). The isolated pure colonies on NA plates were Gram-stained and observe under a microscope for morphological characterization. After Gram staining, biochemical tests were performed. IMViC test, catalase test, oxidase test, motility test, H 2 S production test, and TSI test were performed to confirm the identification of isolates as E. coli and Klebsiella spp (Rosa et al., 2016 ). Antimicrobial Susceptibility Testing Antimicrobial susceptibility tests of the isolates were performed by Kirby-Bauer disc diffusion method on Muller Hilton Agar (Hi-Media, India) according to CLSI 2017 guidelines (Patel & Clinical and Laboratory Standards Institute, 2017). Amoxicillin(10 µg), Aztreonam(30 µg), Azithromycin (15 µg ), Ciprofloxacin (5 µg ), Co-Trimoxazole (25 µg ), Cefoxitin(30 µg), Ceftazidime(30 µg), Ceftriaxone(30 µg), Cefotaxime(30 µg), Imipenem(10 µg), and Nitrofurantoin (300 µg) were used for the AST. Following incubation, zone size was measured and compared with the AST chart provided by CLSI for E. coli and Klebsiella spp . for the tested antibiotic discs. All the isolated E. coli and Klebsiella spp . were tested for the production of ESBL and ABL enzymes. Phenotypic Confirmation of ESBL Production The combination disc method was used to phenotypically confirm the ESBL production by the isolated E. coli and Klebsiella spp . Two combination discs, Cefotaxime (CTX) 30 \(\mu g\) vs. Cefotaxime + Clavulanic acid (CEC) 30/10 \(\mu g\) , and Ceftazidime (CAZ) 30 \(\mu g\) vs. Ceftazidime + Clavulanic acid (CAC) 30/10 \(\mu g\) were used. Those isolated E. coli and Klebsiella strains which showed an increase in zone size by \(\ge\) 5 mm in combination discs (CAC and CEC) than those of CTX and CAZ discs alone were confirmed as ESBL producers (Paterson & Bonomo, 2005 ). Phenotypic Confirmation of ABL Production The combination disc method was used for phenotypic confirmation of ABL production among isolated E. coli and Klebsiella spp . Combination disc Cefoxitin (CX) 30 \(\mu g\) vs. Cefoxitin + Cloxacillin (CC) 30/200 \(\mu g\) was used. Those isolated E. coli and Klebsiella strains which showed an increase in zone size by \(\ge\) 4 mm in combination discs CC than that of CX alone was confirmed as ABL producers (Polsfuss et al., 2011 ). Abbreviations ESBL – Extended Spectrum Beta-Lactamase ABL – AmpC Beta-Lactamase BPKIHS- BP Koirala Institute of Health Science MSU – Mid Stream Urine MDR – Multi Drug-Resistant CLSI – Clinical and Laboratory Standards Institute ESBL - Eb- Extended Spectrum Beta-Lactamase Producing Enterobacteriaceae pAmpC-Eb- Plasmid Mediated AmpC Producing Enterobacteriaceae SDW - Sterile Distilled Water MAC- MacConkey EMB- Eosin Methylene Blue CTX- Cefotaxime CEC- Cefotaxime + Clavulanic acid CAZ- Ceftazidime CAC- Ceftazidime + Clavulanic acid CX- Cefoxitin CC- Cefoxitin + Cloxacillin Declarations Ethical Approval and Consent to Participate Not applicable Consent for Publication Not applicable Availability of Data and Materials The datasets supporting the conclusion of this article are included within the article. The raw data will be available from the corresponding author on a reasonable request. Competing Interests The authors declare that they have no competing interests Funding Self-funded Author’s Contribution PD and RR conceived the topic and designed the proposal. PD, RR, and MRS prepared the conceptual framework and methodology for experiments. LNC, PNC, PD, and MP performed the field works and experiments. RR and NK managed and analyzed the data. RR and PD wrote the paper. All authors read and approved the final manuscript. Acknowledgments We would like to express our sincere gratitude to Mr. Sagar Aryal for his technical support, guidance, and manuscript editing. References Anderson, D. G., Salm, S. N., Allen, D., Nester, E. W., & Nester, E. W. (Eds.). (2019). Nester’s microbiology: A human perspective (Ninth edition). McGraw-Hill Education. Aryal, S. C., Upreti, M. K., Sah, A. K., Ansari, M., Nepal, K., Dhungel, B., Adhikari, N., Lekhak, B., & Rijal, K. R. (2020). Plasmid-Mediated AmpC β-Lactamase CITM and DHAM Genes Among Gram-Negative Clinical Isolates. 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The Mechanism of Resistance in AmpC-Producing Escherichia coli Isolated from Urinary Tract Infections: Archives of Medical Laboratory Sciences , 6 , 1–9 (e6). https://doi.org/10.22037/amls.v6.32573 Doi, Y., Iovleva, A., & Bonomo, R. A. (2017). The ecology of extended-spectrum β-lactamases (ESBLs) in the developed world. Journal of Travel Medicine , 24 (suppl_1), S44–S51. https://doi.org/10.1093/jtm/taw102 Fadare, F. T., & Okoh, A. I. (2021). Distribution and molecular characterization of ESBL, pAmpC β-lactamases, and non-β-lactam encoding genes in Enterobacteriaceae isolated from hospital wastewater in Eastern Cape Province, South Africa. PLoS ONE , 16 (7), e0254753. https://doi.org/10.1371/journal.pone.0254753 Hertz, F., Jansåker, F., Okon, K., Sulaiman, A., Onah, J., Ladan, J., & Knudsen, J. (2019). ESBL-production in Escherichia coli and Klebsiella pneumoniae isolates from Nigeria. MicrobiologyOpen , 8 . https://doi.org/10.1002/mbo3.816 Jha, A. K., & Bajracharya, T. R. (2014). Wastewater Treatment Technologies in Nepal . 7. Kazemian, H., Heidari, H., Ghanavati, R., Ghafourian, S., Yazdani, F., Sadeghifard, N., Valadbeigi, H., Maleki, A., & Pakzad, I. (2019). Phenotypic and Genotypic Characterization of ESBL-, AmpC-, and Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli Isolates. Medical Principles and Practice , 28 (6), 547–551. https://doi.org/10.1159/000500311 Korzeniewska, E., & Harnisz, M. (2013). Beta-lactamase-producing Enterobacteriaceae in hospital effluents. Journal of Environmental Management , 123 , 1–7. https://doi.org/10.1016/j.jenvman.2013.03.024 Le, T.-H., Ng, C., Chen, H., Yi, X. Z., Koh, T. H., Barkham, T. M. S., Zhou, Z., & Gin, K. Y.-H. (2016). Occurrences and Characterization of Antibiotic-Resistant Bacteria and Genetic Determinants of Hospital Wastewater in a Tropical Country. Antimicrobial Agents and Chemotherapy , 60 (12), 7449–7456. https://doi.org/10.1128/AAC.01556-16 Mahato, S., Mahato, A., Pokharel, E., & Tamrakar, A. (2019). Detection of extended-spectrum beta-lactamase-producing E. coli and Klebsiella spp. In effluents of different hospitals sewage in Biratnagar, Nepal. BMC Research Notes , 12 . https://doi.org/10.1186/s13104-019-4689-y Masiello, S. N., Martin, N. H., Trmčić, A., Wiedmann, M., & Boor, K. J. (2016). Identification and characterization of psychrotolerant coliform bacteria isolated from pasteurized fluid milk. Journal of Dairy Science , 99 (1), 130–140. https://doi.org/10.3168/jds.2015-9728 Mateo-Sagasta, J., Raschid-Sally, L., & Thebo, A. (2015). Global Wastewater and Sludge Production, Treatment and Use. In P. Drechsel, M. Qadir, & D. Wichelns (Eds.), Wastewater (pp. 15–38). Springer Netherlands. https://doi.org/10.1007/978-94-017-9545-6_2 Nasser, A. M., Fawaqa, H., & Nitzan, Y. (2019). The Role of Wastewater Treatment Plants in the Environmental Dissemination of Antibiotic Resistant Bacteria (ARB) and Resistance Genes (ARG). Journal of Water Resource and Protection , 11 (08), 981–994. https://doi.org/10.4236/jwarp.2019.118058 Ojer-Usoz, E., González, D., Vitas, A. I., Leiva, J., García-Jalón, I., Febles-Casquero, A., & Escolano, M. de la S. (2013). Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in meat products sold in Navarra, Spain. Meat Science , 93 (2), 316–321. https://doi.org/10.1016/j.meatsci.2012.09.009 Patel, J. B. & Clinical and Laboratory Standards Institute. (2017). Performance standards for antimicrobial susceptibility testing . Paterson, D. L., & Bonomo, R. A. (2005). Extended-Spectrum β-Lactamases: A Clinical Update. Clinical Microbiology Reviews , 18 (4), 657–686. https://doi.org/10.1128/CMR.18.4.657-686.2005 Polsfuss, S., Bloemberg, G. V., Giger, J., Meyer, V., Böttger, E. C., & Hombach, M. (2011). Practical Approach for Reliable Detection of AmpC Beta-Lactamase-Producing Enterobacteriaceae ▿. Journal of Clinical Microbiology , 49 (8), 2798–2803. https://doi.org/10.1128/JCM.00404-11 Raut, S., Gokhale, S., & Adhikari, B. (2015). Prevalence of Extended Spectrum Beta-Lactamases among Escherichia coli and Klebsiella spp isolates in Manipal Teaching Hospital, Pokhara, Nepal. Journal of Microbiology and Infectious Diseases , 5 (2). https://doi.org/10.5799/ahinjs.02.2015.02.0179 Reinthaler, F., Feierl, G., Galler, H., Haas, D., Leitner, E., Mascher, F., Melkes, A., Posch, J., Winter, I., Zarfel, G., & Marth, E. (2009). ESBL-producing E. coli in Austrian sewage sludge. Water Research , 44 , 1981–1985. https://doi.org/10.1016/j.watres.2009.11.052 Rock, C., & Donnenberg, M. S. (2014). Human Pathogenic Enterobacteriaceae. In Reference Module in Biomedical Sciences . Elsevier. https://doi.org/10.1016/B978-0-12-801238-3.00136-7 Rosa, B., Victor, T., Vivas-reyes, R., Montes, A., & Arzuza, O. (2016). Anti-biofilm activity of ibuprofen and diclofenac against some biofilm producing Escherichia coli and Klebsiella pneumoniae uropathogens. African Journal of Microbiology Research , 10 , 1675–1684. https://doi.org/10.5897/AJMR2016.8039 Shakya, P., Shrestha, D., Maharjan, E., Sharma, V. K., & Paudyal, R. (2017). ESBL Production Among E. coli and Klebsiella spp. Causing Urinary Tract Infection: A Hospital Based Study. The Open Microbiology Journal , 11 (1). https://doi.org/10.2174/1874285801711010023 Shrestha, B., Acharya, J., Chhetri, J., Gurung, K., & Khaling Rai, M. (2019). Distrubution of Extended Spectrum β-Lactamase and AMPC-β Lactamase Among Bacteria Isolated From Urine Samples. Journal of Health and Allied Sciences , 5 (1), 21–24. https://doi.org/10.37107/jhas.28 Susić, E. (2004). [Mechanisms of resistance in Enterobacteriaceae towards beta-lactamase antibiotics]. Acta Medica Croatica: Casopis Hravatske Akademije Medicinskih Znanosti , 58 (4), 307–312. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1509782","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":96006874,"identity":"846f89b5-e0f7-4d6b-82a3-4b6017cb5a71","order_by":0,"name":"Prashant Dahal","email":"data:image/png;base64,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","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Prashant","middleName":"","lastName":"Dahal","suffix":""},{"id":96006875,"identity":"7dc3e6d1-eb60-41f6-bd9f-77b0d96e448e","order_by":1,"name":"Rojina Rai","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rojina","middleName":"","lastName":"Rai","suffix":""},{"id":96006876,"identity":"39590239-2ef6-4634-b085-d33a646efc57","order_by":2,"name":"Madhav Raj Sharma","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Madhav","middleName":"Raj","lastName":"Sharma","suffix":""},{"id":96006877,"identity":"31effa2c-9249-4a52-beea-2face25be9e7","order_by":3,"name":"Lalit Narayan Chaudhary","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lalit","middleName":"Narayan","lastName":"Chaudhary","suffix":""},{"id":96006878,"identity":"91a4de20-0829-4d82-9160-375e71e8a372","order_by":4,"name":"Prabha Nand Chaudhary","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Prabha","middleName":"Nand","lastName":"Chaudhary","suffix":""},{"id":96006879,"identity":"9e1e41af-d324-4136-b8e2-e467b24d554f","order_by":5,"name":"Niku Khadka","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Niku","middleName":"","lastName":"Khadka","suffix":""},{"id":96006880,"identity":"5be0f0d2-8f91-42e6-8c7d-3ec34b4143ee","order_by":6,"name":"Mausam poudel","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mausam","middleName":"","lastName":"poudel","suffix":""},{"id":96006881,"identity":"8d270c26-e85e-41c9-b9c1-ab90327aed0e","order_by":7,"name":"Rabin Rai","email":"","orcid":"","institution":"Department of Microbiology, Sunsari Technical College, Dharan, Province-1, Nepal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rabin","middleName":"","lastName":"Rai","suffix":""}],"badges":[],"createdAt":"2022-03-31 14:59:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1509782/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1509782/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20032059,"identity":"9497a730-5e5f-4612-a0ea-ca62bead9a8f","added_by":"auto","created_at":"2022-04-06 16:35:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProportion of isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinedrawingimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1509782/v1/b29d799f4ee25b88d628c8f1.png"},{"id":20032058,"identity":"7042bfd6-9d82-4109-a05d-a362bbea359e","added_by":"auto","created_at":"2022-04-06 16:35:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of ABL Producing \u003cem\u003eE. coli \u003c/em\u003eand \u003cem\u003eKlebsiella spp\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinedrawingimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1509782/v1/148d34010ed6a408d6e1e487.png"},{"id":20032057,"identity":"64df855c-29a2-4172-8ca9-b4582edcec9c","added_by":"auto","created_at":"2022-04-06 16:35:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of ESBL Producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinedrawingimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1509782/v1/48ffe6aafbf2a86ccbe9d496.png"},{"id":20032060,"identity":"7defe22d-7a7b-416e-9a01-409b8c3a3acb","added_by":"auto","created_at":"2022-04-06 16:35:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of ABL and ESBL Co-producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinedrawingimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1509782/v1/7e43fbbe3442d3f84d0b2c06.png"},{"id":22928830,"identity":"163f9aea-5c5e-43a1-8267-391e10c5ad92","added_by":"auto","created_at":"2022-06-22 09:59:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":531399,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1509782/v1/b90015e8-b14f-4595-a4c5-7a343cab5ece.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Producing E. coli and Klebsiella spp. in Sewage Effluents of Dharan, Nepal","fulltext":[{"header":"Background","content":"\u003cp\u003eSewage is the collection of wastewater in a municipal piper system or sewerage channel. It is mostly a combination of domestic effluents from toilets, bathing, and kitchen, water from commercial establishments and institutions including hospitals and clinics, hotels and restaurants, shops and markets, schools, colleges, and different offices, industrial effluents, and surface runoff water (Mateo-Sagasta et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Sewage water contains a different variety of organic and inorganic wastes and nutrients merged from different sources. Hospital sewage water is loaded with several pathogenic microorganisms with antimicrobial resistance capacity, partially metabolized pharmaceutical substances like antimicrobial, pharmaceutical, disinfectants, and un-metabolized drugs, radioactive elements, and other toxic substances. Non-hospital sewage is loaded with microorganisms from human and animal feces and environmental sources. This will make sewage more prone to pathogenic organisms including antibiotic-resistant bacteria (Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Different studies have shown the presence of antibiotic-resistant bacteria and genes which have even escaped treatment systems and disseminated in wastewater (Nasser et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Wastewater treatment systems can reduce contaminants including microorganisms to a minimum limit, but only 5% of generated wastewater is treated appropriately while the remaining is directly connected to water bodies in Nepal. This has resulted in the transmission of microorganisms and water-borne infections (Jha \u0026amp; Bajracharya, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnterobacteriaceae\u003c/em\u003e covers a large part of hospital sewage microbiota (Korzeniewska \u0026amp; Harnisz, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Among the sewage pathogens, coliforms are responsible for frequent human infections (Doi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Coliforms are aerobic or facultative, Gram-negative, non-sporing lactose fermenting bacteria. They were traditionally represented by 4 genera viz. \u003cem\u003eEscherichia, Klebsiella, Citrobacter\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e, but now contain over 20 bacterial genera (Masiello et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eEnterobacteriaceae\u003c/em\u003e includes different Gram-negative bacteria including pathogenic genera like \u003cem\u003eEscherichia, Salmonella, Shigella, Klebsiella\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003e (Rock \u0026amp; Donnenberg, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e have notable antibiotic resistance to a wide variety of antibiotics typically used in the treatment of their infection (Le et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kazemian et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMembers of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e like \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e spp. exhibit antimicrobial resistance mainly due to the production of beta-lactamase enzymes like extended-spectrum β-lactamases, AmpC β-lactamases, and carbapenemases (Ojer-Usoz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eEnterobacteriaceae\u003c/em\u003e were found to produce intrinsic chromosomal encoded beta-lactamases but due to the transfer of such gene to transferable plasmid gene, there is rapid dissemination of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e resistant to a wide variety of beta-lactam antibiotics (Susić, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).ESBLs are \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactamases capable of developing resistance against \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactam drugs like penicillins, extended-spectrum cephalosporins, and monobactams except for cephamycins and carbapenems and susceptible to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactam inhibitor clavulanic acid (Raut et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). ABL is characterized by the hydrolysis of broad-spectrum cephalosporins like cefotaxime and ceftazidime and cephamycins (7-α-methoxy cephalosporins) like cefoxitin and cefotetan, including monobactam, and the β-lactam/β-lactam inhibitor combination, but are sensitive to cefepime and carbapenems (Dehkharghani et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent clinical isolates of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. are found to be antibiotic resistant and produce ESBL and ABL enzymes for conferring the resistance against \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactam antibiotics. (Shakya et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) concluded on the higher prevalence of ESBL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. in Nepal by examining 2209 non-repetitive MSU samples at a tertiary hospital of Nepal. Similarly, (Baral et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and (Aryal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)concluded the high prevalence of ABL producers in clinical isolates. (Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) studied sewage effluents of different hospitals of Biratnagar, Nepal, and concluded the prevalence of MDR and ESBL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. in hospital sewage effluents.\u003c/p\u003e \u003cp\u003eSewage effluents have high potentiality as an environmental reservoir of a wide variety of pathogenic organisms including ESBL and ABL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp.\u003c/em\u003e Unfortunately, scanty research is done on sewage effluents as a source of such pathogens. The present study was undertaken to reduce the research gap on assessment of sewage microbiota with the sole focus on determining the prevalence of ESBL and ABL producing \u003cem\u003eE. coli and Klebsiella spp.\u003c/em\u003e in sewage effluents of Dharan, Nepal.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDistribution of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e. \u003cb\u003ein the Samples\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the total of 20 samples analyzed 16 samples (80%) showed growth of either \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eKlebsiella spp\u003c/em\u003e. or both and 4 samples (20%) did not show growth of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e.(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Out of 16 positive samples, 13 (81.25%) showed growth of both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e., 2 (12.5%) showed growth of \u003cem\u003eKlebsiella spp\u003c/em\u003e. only and 1 (6.25%) showed growth of \u003cem\u003eE. coli\u003c/em\u003e only. All of the hospital-sewage effluents (6 out of 6) and 8 of the non-hospital sewage effluents (8 out of 14) showed growth of both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e.(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This showed that 70% of the samples contained \u003cem\u003eE. coli\u003c/em\u003e and 75% of the samples contained \u003cem\u003eKlebsiella spp\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevalence of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. in each sample\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eBacteria Isolated\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp.\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPKIHS, Dharan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPKIHS, Dharan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPKIHS, Dharan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPKIHS, Dharan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBijaypur Hospital Pvt.Ltd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBijaypur Hospital Pvt. Ltd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResidential area \u0026ndash; 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResidential area \u0026ndash; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResidential area \u0026ndash; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResidential area \u0026ndash; 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarket area \u0026ndash; 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarket area \u0026ndash; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarket area \u0026ndash; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial area \u0026ndash; 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA total of 235 bacteria were identified as either \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eKlebsiella spp\u003c/em\u003e. Out of them, 103 isolates (43.83%) were \u003cem\u003eE. coli\u003c/em\u003e and 132 isolates (56.17%) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. The prevalence of \u003cem\u003eKlebsiella\u003c/em\u003e was found to be higher. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibiotic Susceptibility Pattern of Isolated\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAntibiotic sensitivity test of selected antibiotics against the isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. showed the highest sensitivity to Azithromycin (63.82%) and the lowest sensitivity to Cefoxitin (1.28%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAntibiotic Susceptibility Pattern of Isolated\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDisc Content\u003c/p\u003e \u003cp\u003e(in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\varvec{\\mu }\\varvec{g}\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSusceptibility Pattern\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSensitive\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003en (%)\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eResistant\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003en (%)\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27 (11.49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e208 (88.51%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzithromycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e150 (63.83%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85 (36.17%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAztreonam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55 (23.40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e180 (76.60%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e113 (48.09%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e122 (51.91%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCo-Trimoxazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e146 (62.13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89 (37.87%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftriaxone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45 (19.15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e190 (80.85%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39 (16.59%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e196 (83.41%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41 (17.45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e194(82.55%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefoxitin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3 (1.28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e232 (98.72%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e140 (59.57%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95 (40.43%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrofurantoin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e127 (54.04%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e108 (45.96%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBeta-lactam antibiotics; Ampicillin, Aztreonam, Ceftriaxone, Ceftazidime, Cefotaxime, and Cefoxitin showed very poor sensitivity patterns showing the sensitive result to only 11.49%, 23.40%, 19.15%, 16.59%, 17.45%, and 1.28% of tested bacteria respectively. Ciprofloxacin showed sensitivity to 48.09% of the tested bacterial species, while Co-Trimoxazole showed sensitivity to 62.13% of the tested bacterial species. Imipenem showed a sensitive result on 59.57% of the tested bacteria. Nitrofurantoin showed a sensitive result on 54.04% of the tested bacteria.\u003c/p\u003e \u003cp\u003eAntibiotic susceptibility test of \u003cem\u003eE. coli\u003c/em\u003e revealed that 84.47% of them were sensitive to Nitrofurantoin, while only 2.91% were sensitive to Cefoxitin. Ampicillin, Azithromycin, Aztreonam, Ciprofloxacin, Co-Trimoxazole, Ceftriaxone, Ceftazidime, Cefotaxime, and Imipenem showed sensitivity against 15.53%, 55.34%, 31.07%, 48.54%, 68.93%, 31.07%, 25.24%, 20.39%, and 58.25% of the tested \u003cem\u003eE. coli\u003c/em\u003e respectively.\u003c/p\u003e \u003cp\u003eOn other hand, 70.45% of \u003cem\u003eKlebsiella spp\u003c/em\u003e. were sensitive to Azithromycin and 0% of \u003cem\u003eKlebsiella spp\u003c/em\u003e. were sensitive to Cefoxitin. Ampicillin, Aztreonam, Ciprofloxacin, Co-Trimoxazole, Ceftriaxone, Ceftazidime, Cefotaxime, Imipenem, and Nitrofurantoin showed sensitivity against 8.33%, 17.42%, 47.73%, 56.82%, 9.85%, 9.85%, 15.15%, 60.61%, and 30.31% of the tested \u003cem\u003eKlebsiella spp\u003c/em\u003e. respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution of ABL Producing\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e133 isolates (56.59%) were found positive for ABL production. Among 133, 54 (40.60%) were \u003cem\u003eE. coli\u003c/em\u003e and 79 (59.40%) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) This showed a higher prevalence of ABL producing \u003cem\u003eKlebsiella spp.\u003c/em\u003e than \u003cem\u003eE. coli\u003c/em\u003e in sewage effluents of Dharan.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution of ESBL Producing\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eESBL production was seen in 157 isolates (66.81%) while testing 235 isolated bacteria. Out of 157 ESBL producing 89 (56.69%) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. and 68 (43.31%) were \u003cem\u003eE. coli\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) This showed a higher prevalence of ESBL producing \u003cem\u003eKlebsiella spp\u003c/em\u003e. than \u003cem\u003eE. coli\u003c/em\u003e in sewage effluent of Dharan.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution of ABL and ESBL Co-producing\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eA total of 118 isolates showed the production of both ABL and ESBL enzymes. This accounted for 50.21% of total isolated bacteria. Out of 118 co-producers, 51 (43.22%) were \u003cem\u003eE. coli\u003c/em\u003e, and the remaining 67 (56.78%) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) Prevalence of ABL and ESBL co-production was comparatively higher in \u003cem\u003eKlebsiella spp\u003c/em\u003e. than in \u003cem\u003eE. coli.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBeta-lactam antibiotics are among the most widely used antibiotic class(Bush \u0026amp; Bradford, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Bacteria have developed resistance to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactam antibiotics by evolving their ability to synthesize several types of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e -lactamase enzymes capable of hydrolyzing \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactam ring. Among the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactamase enzymes ESBL and ABL are the most commonly encountered types (Bush \u0026amp; Bradford, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Bush \u0026amp; Jacoby, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Resistance to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactam antibiotics by producing ESBL and ABL enzymes is increasing rapidly among members of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e across the world(Caron et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (De Angelis et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This study was conducted to detect the prevalence of ABL and ESBL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp.\u003c/em\u003e in sewage effluents of Dharan. A total of 20 samples were analyzed in the Molecular and Microbiology Laboratory of Sunsari Technical College, Dharan-1 from May 21, 2021, to October 29, 2021. The phenotypic method was used to confirm the production of AmpC Beta-lactamase and Extended Spectrum Beta-lactamase enzymes in the isolated species using the methods recommended by CLSI(Patel \u0026amp; Clinical and Laboratory Standards Institute, 2017)\u003c/p\u003e \u003cp\u003eAmong the total of 20 samples analyzed 16 samples (80%) showed growth of either \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eKlebsiella spp\u003c/em\u003e. or both and 4 samples (20%) did not show growth of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. Among the studied sample, 70% (14 out of 20) showed the presence of \u003cem\u003eE. coli\u003c/em\u003e, and 75% (15 out of 20) showed the presence of \u003cem\u003eKlebsiella spp\u003c/em\u003e. Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e had reported that 70% of sewage effluents contained \u003cem\u003eE. coli\u003c/em\u003e while only 60% of the sewage effluents contained \u003cem\u003eKlebsiella spp\u003c/em\u003e. This variation may be since Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e had studied hospital sewage effluents only while we studied both hospital and municipal sewage.\u003c/p\u003e \u003cp\u003eIn the study, 235 bacteria were identified as either \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eKlebsiella spp.\u003c/em\u003e 103 (43.83%) of them were \u003cem\u003eE. coli\u003c/em\u003e, whereas 132 (56.17%) of them were \u003cem\u003eKlebsiella spp\u003c/em\u003e. Results obtained by Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e were different. They had shown that 53.85% of their isolates were \u003cem\u003eE. coli\u003c/em\u003e, while 46.15% were \u003cem\u003eKlebsiella spp\u003c/em\u003e. (Fadare \u0026amp; Okoh, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) had shown that 65.71% of their isolates were \u003cem\u003eKlebsiella spp\u003c/em\u003e. and only 10% were \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAmpicillin resistance was seen in 84.47% of \u003cem\u003eE. coli\u003c/em\u003e. It was 44.66% for Azithromycin, 68.93% for Aztreonam, 51.46% for Ciprofloxacin, 31.07% for Co-Trimoxazole, 68.93% for Ceftriaxone, 74.76% for Ceftazidime, 79.61% for Cefotaxime, 97.09% for Cefoxitin, 41.75% for Imipenem, and 15.53% for Nitrofurantoin. This result was similar to Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e for Ampicillin, Aztreonam, and Cefoxitin. They had shown 100% resistance to these antibiotics. But, for Co-Trimoxazole, Ceftazidime, Ceftriaxone, and Cefotaxime they had shown resistance to 85.7% which was higher than the result obtained in this research. Similarly, they had shown that resistance to Nitrofurantoin was 42.9%, which was higher in comparison to this research. For Azithromycin, they had shown that non-of them (0%) were resistant, but 44.66% were found resistant in this research. They had reported that 14.3% were found to be resistant to Ciprofloxacin but it was 51.46% in this research.\u003c/p\u003e \u003cp\u003eThis study showed that 91.67% of \u003cem\u003eKlebsiella spp\u003c/em\u003e. were resistant to Ampicillin. It was 29.55% for Azithromycin, 82.58% for Aztreonam, 52.27% for Ciprofloxacin, 43.18% for Co-Trimoxazole, 90.15% for Ceftriaxone, 90.15% for Ceftazidime, 84.85% for Cefotaxime, 100% for Cefoxitin, 39.39% for Imipenem, and 69.69% for Nitrofurantoin. The result was shown by Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e indicates comparatively higher resistance against Ampicillin and Co-Trimoxazole, and lower to the other antibiotics.\u003c/p\u003e \u003cp\u003eESBL production was found higher in \u003cem\u003eKlebsiella spp\u003c/em\u003e. than in \u003cem\u003eE. coli\u003c/em\u003e. Out of 157 ESBL producers, 43.31% (68) were \u003cem\u003eE. coli\u003c/em\u003e. and 56.69% (89) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e showed higher ESBL production in \u003cem\u003eE. coli\u003c/em\u003e (66.67%) than in \u003cem\u003eKlebsiella spp\u003c/em\u003e. (33.33%). Similarly, Zaatout et al., 2021 also had reported a higher prevalence of ESBL \u003cem\u003eE. coli\u003c/em\u003e in sewage effluents than that of \u003cem\u003eKlebsiella spp\u003c/em\u003e. Fadare \u0026amp; Okoh, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e on the other hand had shown a higher prevalence of ESBL producing \u003cem\u003eKlebsiella spp\u003c/em\u003e. than \u003cem\u003eE. coli\u003c/em\u003e in hospital wastewater. ESBL production was seen in 81.59% of total tested \u003cem\u003eE. coli\u003c/em\u003e and 85.61% of total tested \u003cem\u003eKlebsiella spp\u003c/em\u003e. A higher prevalence of ESBL producing \u003cem\u003eE. coli\u003c/em\u003e was found by (Reinthaler et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eABL production was also found to be higher in \u003cem\u003eKlebsiella spp\u003c/em\u003e. than in \u003cem\u003eE. coli\u003c/em\u003e. Among 133 ABL producers, 59.40% (79) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. and 40.60% (54) were \u003cem\u003eE. coli\u003c/em\u003e. Among 57 wastewater samples tested by (Ben Said et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), 24 were found to be positive for ESBL-Eb or pAmpC-Eb producing \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. They detected ESBL-Eb in 20 samples, and pAmpC-Eb in 4 samples. No relevant literature on ABL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. in sewage effluents were found for further comparison.\u003c/p\u003e \u003cp\u003eESBL and ABL co-production was seen in 50.21% (total 118) of the isolates. 75% of the ESBL producing \u003cem\u003eE. coli\u003c/em\u003e were found to produce ABL also, and 75.28% of the ESBL producing \u003cem\u003eKlebsiella spp\u003c/em\u003e. were found to produce ABL also. This result was higher than that of Rizi et al., 2020. They had shown co-production in only 30% of the isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. Similarly, co-production was seen in 29% of the isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in research by (Hertz et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Co-production was seen in 9.38% of isolated \u003cem\u003eE. coli\u003c/em\u003e and non (0%) of isolated \u003cem\u003eKlebsiella spp\u003c/em\u003e. by (Shrestha et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These variations may be because all these studies are on clinical isolates.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBased on the finding of the present study, it can be concluded that sewage effluents of Dharan have a higher prevalence of ESBL and ABL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. This suggests that there may be a direct link between human or animal feces in the sewage system. The result is an alarming indicator of a probable outbreak of life-threatening infection by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactamase producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. if sewage is contaminated with drinking water channels or food in the Dharan area. There is an urgent need for further research in sewage microflora and the probability of their dissemination to human populations. Government bodies should prepare effective regulatory measures to control the irrational use of antibiotics and monitor the antimicrobial resistance status of different suspected bacteria. Effective alternatives for the treatment of ESBL and ABL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. may be needed soon. Immediate and effective response from concerned authorities for controlling the dissemination of such bacteria from sewage to the environment or community is required. Regular monitoring and study of antibiotic susceptibility patterns of several bacteria in different aspects of the environment like sewage is needed.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDesign and Setting of Study\u003c/h2\u003e \u003cp\u003eA laboratory-based cross-sectional quantitative study was designed. The study was conducted in Dharan sub-metropolitan city. It is a city in Koshi zone\u0026rsquo;s Sunsari district of Province-1, Nepal with co-ordinates 26\u003csup\u003e0\u003c/sup\u003e49\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{\u0026#039;}\\)\u003c/span\u003e\u003c/span\u003e0\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{\u0026#039;}\\text{\u0026#039;}\\)\u003c/span\u003e\u003c/span\u003eN 87\u003csup\u003e0\u003c/sup\u003e17\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{\u0026#039;}\\)\u003c/span\u003e\u003c/span\u003e0\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{\u0026#039;}\\text{\u0026#039;}\\)\u003c/span\u003e\u003c/span\u003eE. The study was conducted from May 21, 2021, to October 29, 2021. Sewage effluents, directly from the municipal sewage channel, were taken as samples for the study. Non-probability convenience sampling method was followed for sample collection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection and Transportation\u003c/h2\u003e \u003cp\u003eA total of 20 samples were taken from different sites of Dharan city. Samples were taken considering that it would cover almost every part of the city. Out of 20 samples, 6 samples (30%) were from the hospital area, and the remaining 14 samples (70%) were from the non-hospital area.\u003c/p\u003e \u003cp\u003eSamples were collected directly from the center of the sewage flow channel. For hospital sewages, samples were collected from the point where hospital sewage is connected to the municipal sewerage channel. A pre-sterilized sterile glass bottle was used to collect the sample from the flow channel with the help of a sampling rod. About 100 mL of sewage effluents were taken from one site. Each bottle was labeled with the date, sample location, and time of collection. Collected samples were transported to the laboratory immediately maintaining a cold chain in the ice-box to inhibit the growth of microorganisms. (Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample Processing and Inoculation\u003c/h2\u003e \u003cp\u003eSamples were serially diluted in sterilized distilled water (SDW) up to tenfold. Eosin-methylene blue (EMB) agar plates and MacConkey (MAC) agar plates were prepared and solidified properly about 3\u0026ndash;4 hours before inoculation. 5 plates of each media were prepared and labeled as E10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, E10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, E10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, E10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e, and Econtrol on EMB plates and M10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, M10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, M10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, M10\u003csup\u003e\u0026minus;\u0026thinsp;6,\u003c/sup\u003e and Mcontrol for MAC plates. From the serially diluted samples in the tube, 100 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eL of the sample was pipetted out after mixing properly and inoculated on the agar plates using the spread-plate (lawn culture) method. Dilution 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e were inoculated. The plates were incubated aerobically at 37\u003csup\u003e0\u003c/sup\u003eC for 24 hours.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation and Identification of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. coli\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eKlebsiella spp\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFollowing incubation colonies were isolated from the plate with well-isolated colonies. MAC was used for isolation of \u003cem\u003eKlebsiella spp\u003c/em\u003e. and EMB was used for isolation of \u003cem\u003eE. coli\u003c/em\u003e. (Mahato et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003eb) From MAC plates, well-isolated colonies which were showing characters of \u003cem\u003eKlebsiella spp.\u003c/em\u003e were marked and streaked on Nutrient Agar (NA) plates for purification. Similarly, well-isolated colonies which were showing characters of \u003cem\u003eE. coli\u003c/em\u003e in EMB plates were marked and isolated in NA plates (Anderson et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe isolated pure colonies on NA plates were Gram-stained and observe under a microscope for morphological characterization. After Gram staining, biochemical tests were performed. IMViC test, catalase test, oxidase test, motility test, H\u003csub\u003e2\u003c/sub\u003eS production test, and TSI test were performed to confirm the identification of isolates as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e(Rosa et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial Susceptibility Testing\u003c/h2\u003e \u003cp\u003eAntimicrobial susceptibility tests of the isolates were performed by Kirby-Bauer disc diffusion method on Muller Hilton Agar (Hi-Media, India) according to CLSI 2017 guidelines (Patel \u0026amp; Clinical and Laboratory Standards Institute, 2017). Amoxicillin(10 \u0026micro;g), Aztreonam(30 \u0026micro;g), Azithromycin (15 \u0026micro;g ), Ciprofloxacin (5 \u0026micro;g ), Co-Trimoxazole (25 \u0026micro;g ), Cefoxitin(30 \u0026micro;g), Ceftazidime(30 \u0026micro;g), Ceftriaxone(30 \u0026micro;g), Cefotaxime(30 \u0026micro;g), Imipenem(10 \u0026micro;g), and Nitrofurantoin (300 \u0026micro;g) were used for the AST. Following incubation, zone size was measured and compared with the AST chart provided by CLSI for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. for the tested antibiotic discs. All the isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. were tested for the production of ESBL and ABL enzymes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic Confirmation of ESBL Production\u003c/h2\u003e \u003cp\u003eThe combination disc method was used to phenotypically confirm the ESBL production by the isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. Two combination discs, Cefotaxime (CTX) 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu g\\)\u003c/span\u003e\u003c/span\u003e vs. Cefotaxime\u0026thinsp;+\u0026thinsp;Clavulanic acid (CEC) 30/10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu g\\)\u003c/span\u003e\u003c/span\u003e, and Ceftazidime (CAZ) 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu g\\)\u003c/span\u003e\u003c/span\u003e vs. Ceftazidime\u0026thinsp;+\u0026thinsp;Clavulanic acid (CAC) 30/10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu g\\)\u003c/span\u003e\u003c/span\u003e were used.\u003c/p\u003e \u003cp\u003eThose isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e strains which showed an increase in zone size by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\ge\\)\u003c/span\u003e\u003c/span\u003e5 mm in combination discs (CAC and CEC) than those of CTX and CAZ discs alone were confirmed as ESBL producers (Paterson \u0026amp; Bonomo, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic Confirmation of ABL Production\u003c/h2\u003e \u003cp\u003eThe combination disc method was used for phenotypic confirmation of ABL production among isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. Combination disc Cefoxitin (CX) 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu g\\)\u003c/span\u003e\u003c/span\u003e vs. Cefoxitin\u0026thinsp;+\u0026thinsp;Cloxacillin (CC) 30/200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu g\\)\u003c/span\u003e\u003c/span\u003e was used. Those isolated \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e strains which showed an increase in zone size by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\ge\\)\u003c/span\u003e\u003c/span\u003e4 mm in combination discs CC than that of CX alone was confirmed as ABL producers (Polsfuss et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eESBL \u0026ndash;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Extended Spectrum Beta-Lactamase\u003c/p\u003e\n\u003cp\u003eABL \u0026ndash; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;AmpC Beta-Lactamase\u003c/p\u003e\n\u003cp\u003eBPKIHS-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;BP Koirala Institute of Health Science\u003c/p\u003e\n\u003cp\u003eMSU \u0026ndash;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; Mid Stream Urine\u003c/p\u003e\n\u003cp\u003eMDR \u0026ndash;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; Multi Drug-Resistant\u003c/p\u003e\n\u003cp\u003eCLSI \u0026ndash; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Clinical and Laboratory Standards Institute\u003c/p\u003e\n\u003cp\u003eESBL - Eb- \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Extended Spectrum Beta-Lactamase Producing Enterobacteriaceae\u003c/p\u003e\n\u003cp\u003epAmpC-Eb-\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Plasmid Mediated AmpC Producing Enterobacteriaceae\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSDW - \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Sterile Distilled Water\u003c/p\u003e\n\u003cp\u003eMAC-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;MacConkey\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEMB-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Eosin Methylene Blue\u003c/p\u003e\n\u003cp\u003eCTX-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cefotaxime\u003c/p\u003e\n\u003cp\u003eCEC-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cefotaxime + Clavulanic acid\u003c/p\u003e\n\u003cp\u003eCAZ-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ceftazidime\u003c/p\u003e\n\u003cp\u003eCAC-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ceftazidime + Clavulanic acid\u003c/p\u003e\n\u003cp\u003eCX-\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cefoxitin\u003c/p\u003e\n\u003cp\u003eCC- \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Cefoxitin + Cloxacillin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusion of this article are included within the article. The raw data will be available from the corresponding author on a reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelf-funded\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePD and RR conceived the topic and designed the proposal. PD, RR, and MRS prepared the conceptual framework and methodology for experiments. LNC, PNC, PD, and MP performed the field works and experiments. \u0026nbsp;RR and NK managed and analyzed the data. RR and PD wrote the paper. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to Mr. Sagar Aryal for his technical support, guidance, and manuscript editing. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnderson, D. G., Salm, S. N., Allen, D., Nester, E. W., \u0026amp; Nester, E. W. (Eds.). (2019). \u003cem\u003eNester\u0026rsquo;s microbiology: A human perspective\u003c/em\u003e (Ninth edition). McGraw-Hill Education.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAryal, S. C., Upreti, M. K., Sah, A. K., Ansari, M., Nepal, K., Dhungel, B., Adhikari, N., Lekhak, B., \u0026amp; Rijal, K. R. (2020). 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[Mechanisms of resistance in Enterobacteriaceae towards beta-lactamase antibiotics]. \u003cem\u003eActa Medica Croatica: Casopis Hravatske Akademije Medicinskih Znanosti\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e(4), 307\u0026ndash;312.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ESBL, ABL, Enterobacteriaceae, E. coli, Klebsiella spp, Sewage effluent","lastPublishedDoi":"10.21203/rs.3.rs-1509782/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1509782/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe prevalence of extended-spectrum \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactamase (ESBL) and AmpC \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\beta\\)\u003c/span\u003e\u003c/span\u003e-lactamase (ABL) producing \u003cem\u003eEnterobacteriaceae\u003c/em\u003e is increasing rapidly across the world. Members of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e like \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. exhibit antimicrobial resistance mainly due to the production of beta-lactamase enzymes like extended-spectrum βlactamases, AmpC β-lactamases, and carbapenemases. These bacteria are frequently reported in sewage effluents of hospital and municipal sewerage systems indicating sewage as a promising source for dissemination of such drug-resistant pathogens. However, in most of the developing countries including Nepal, the major portion of sewage is discharged in water sources without proper treatment and disinfection. This study was undertaken to access the prevalence of ESBL and ABL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. in sewage effluents of Dharan, Nepal.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 235 bacteria were isolated, out of which 103 (43.83%) were \u003cem\u003eE. coli\u003c/em\u003e and 132 (56.17%) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. ESBL production was seen in 157 (66.81%) isolates. Among them, 89 (56.69%) were \u003cem\u003eKlebsiella spp\u003c/em\u003e. and 68 (43.31%) were \u003cem\u003eE. coli.\u003c/em\u003e 66.02% of total isolated \u003cem\u003eE. coli\u003c/em\u003e and 67.42% of total isolated \u003cem\u003eKlebsiella spp\u003c/em\u003e. showed production of ESBL enzymes. ABL production was seen in 133 (56.59%) isolates. Among them, 54 (40.60%) were \u003cem\u003eE. coli\u003c/em\u003e and 79 (59.40%) were \u003cem\u003eKlebsiella spp.\u003c/em\u003e 52.43% of the isolated \u003cem\u003eE. coli\u003c/em\u003e and 59.85% of isolated \u003cem\u003eKlebsiella spp\u003c/em\u003e. were found producing ABL enzyme.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results indicate that there is a high prevalence of ESBL and ABL producing \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eKlebsiella spp\u003c/em\u003e. in sewage effluents of Dharan. Effective treatment of sewage effluents must be ensured before discharging the sewage into the environment. National guidelines for discharging the municipal sewage must be immediately amended and an effective treatment system before discharge must be implemented. Dissemination of such drug-resistant bacteria in the human population leading to severe public health emergency is likely to occur from sewage contamination, so further study and surveillance and effective prevention and control measures are necessary.\u003c/p\u003e","manuscriptTitle":"Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Producing E. coli and Klebsiella spp. in Sewage Effluents of Dharan, Nepal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-06 16:35:31","doi":"10.21203/rs.3.rs-1509782/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93fe654f-b26d-40fa-b3d7-94fe57b18f06","owner":[],"postedDate":"April 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-22T09:59:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-06 16:35:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1509782","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1509782","identity":"rs-1509782","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}