Biofilm Formation and Detection of Pgaabcd Gene in Clinical Isolates of Extended-spectrum Betalactamase E.coli

Biofilm Formation and Detection of Pgaabcd Gene in Clinical Isolates of Extended-spectrum Betalactamase E.coli | 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 Biofilm Formation and Detection of Pgaabcd Gene in Clinical Isolates of Extended-spectrum Betalactamase E.coli Arjuna Thapa¹, Jyoti Amatya, Anil Kumar Shah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7617318/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 Biofilm formation is a critical virulence factor in Escherichia coli , especially in multidrug-resistant and extended-spectrum beta-lactamase (ESBL) producing strains. The pgaABCD operon plays a vital role in synthesizing poly-beta-1,6-N-acetyl-D-glucosamine, a key exopolysaccharide in biofilms. This study aimed to detect pgaABCD genes and evaluate biofilm formation in ESBL-producing E. coli from clinical isolates. Methods A cross-sectional study was conducted from August 2023 to March 2024. A total of 384 clinical samples were cultured and E. coli isolates were tested for antimicrobial susceptibility using the Kirby-Bauer method per CLSI 2020 guidelines. ESBL production was screened using the combined disk method. Biofilm production was determined using Congo Red Agar. The presence of pgaABCD genes was assessed using conventional PCR and gel electrophoresis. Results Among 384 clinical samples, 29 (7.55%) E. coli isolates were recovered from urine. Of these, 65.5% were biofilm producers, and 67% exhibited multidrug resistance (MDR). ESBL production was confirmed in 4 isolates. The pgaD gene was the most prevalent (89.6%), followed by pgaB (82.7%), pgaA (68.9%), and pgaC (58.6%). Conclusions The high prevalence of biofilm-forming and MDR E. coli isolates harboring pgaABCD genes underscores the clinical significance of these genes in antibiotic resistance and persistence. Understanding their distribution contributes to inform targeted therapeutic and infection control strategies. Trial Registration Not applicable Escherichia coli Biofilm ESBL pgaABCD PCR Antibiotic resistance MDR Figures Figure 1 Figure 2 Figure 3 Figure 4 Background E. coli is rod shaped Gram negative bacteria which is found as the normal flora of colon in humans but acts as opportunistic parasite when trans-located. Urinary tract infections (UTIs) account for around 150 million new instances of diagnosis each year worldwide. Thus, it shows the health issues that warrants actual attention ( 1 ). Fulminant prostatitis, biliary tract infection, and urinary catheter cystitis are the primary causes of UTI ( 2 ). Extended-spectrum beta-lactamases (ESBLs) are enzymes produced by E. coli , which can break down and confer resistance to a wide variety of beta-lactam antibiotics including cephalosporins, monobactams, penicillins, but not carbapenems and cephamycins. ESBLs hydrolyze the beta-lactam ring present in many antibiotics rendering them ineffective and the most common ESBLs belong to TEM, SHV ( 3 ). E. coli strains, as the most frequent etiological representative of community & hospital acquired infection which is often resistant to numerous antibiotics previously prescribed to treat such infections ( 4 ). In many natural and artificial habitats, bacteria from sessile communities are known as biofilm ( 5 ). Biofilms are ordered collections of microorganisms that stick to both biotic and abiotic surfaces. They are enclosed in a matrix made of self-produced extracellular polymeric substance (EPS). Bacterial populations use a technique called quorum sensing (QS) to communicate with one another and coordinate gene expression in response to changes in cell density. For E. coli to synthesize biofilm and perform other harmful functions, the pgaABCD locus is necessary ( 2 , 6 ). Poly-beta-1,N-acetyl-D-glucosamine (PGA) is a particular polysaccharide that is synthesized, modified, and exported by means of this operon. The periplasmic protein pgaB is in charge of deacetylating PGA, which is essential for its correct function in biofilm formation ( 6 ). PgaC is an inner membrane glycosyltransferase that catalyzes the polymerization of N-acetyglucosamine into PGA. PgaD is an inner membrane protein that supports PgaC 's activity and stabilizes the complex. PgaA role in outer membrane porin that is included in the export of PGA to the exterior of the cell ( 6 ). Antibiotic resistance in Escherichia coli represents a growing public health crisis, with biofilm formation emerging as a key adaptive strategy that can enhance bacterial survival by up to 1,000-fold compared to free-floating cells.Central to this phenomenon is the pgaABCD operon, which mediates the synthesis and export of poly-β-1,6-N-acetyl-D-glucosamine (PGA), a foundational component of the biofilm extracellular matrix ( 2 , 6 ). Although this operon has been linked to biofilm resilience, its prevalence and functional impact within multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL) producing clinical E. coli remain under-characterized, representing a critical gap in our understanding of biofilm-mediated antibiotic evasion. To address this gap, the present study aims to detect pgaABCD genes in clinical E. coli isolates and evaluate their association with biofilm formation, MDR, and ESBL production. By elucidating the role of the pgaABCD operon in biofilm-driven persistence and antibiotic resistance, this work provides crucial genetic insights that may enable the development of targeted anti-biofilm interventions and strengthen infection control strategies against resilient E. coli pathogens. Methodology A cross-sectional study was carried out in the Annapurna Neurological Institute & Allied Science Maitighar (Microbiology Laboratory ), Kathmandu from August 2023 to March 2024. The variables of the study are clinical specimens, age, sex, E. coli isolates in clinical samples as well as ESBLs, biofilm production and antibiotic susceptibility profile of the isolates. The study population were patients visiting at Annapurna Neurological Institute & Allied Science Hospital, suspected to have bacterial infections and requested for microbiological culture and antimicrobial susceptibility testing of any age and sex within the study period was considered as population. The study units were different clinical samples (Urine) requested for routine microbiological culture from the patients visiting Neurological Institute & Allied Science Hospital, Maitighar, Kathmandu. The research study was approved by the Institutional Review Committee (IRC-ANIAS-2023/011) of Annapurna Neurological Institute & Allied Science Hospital. The size of the population was determined using the following formula: n = z 2 pq/d 2 , where n is the sample size, p the prevalence, q is 1-p, z is the statistic level of confidence (1.96), d is precision (0.05). p -value was considered as 54% ( 7 ). Hence, the sample size was obtained as 384. The non-duplicate different aerobic bacterial isolates obtained from patients with any kind of infection or having major surgeries of having catheters processed at the hospital were included. After routine culture testing, only gram negative E.coli isolates were processed. Contaminated, unlabeled, mislabeled samples, sample after 4 hours of collection without any preservation and specimen collection procedure other than guided by standard microbiological procedures were rejected for this study. 384 samples were cultured in MacConkey Agar and Cysteine lactose and electrolyte Deficient and incubated at 37°C for 24 hrs. Antibiotic susceptibility test was performed by Kirby-Bauer disk diffusion method following the guidelines of Clinical and Laboratory Standard Institute (CLSI) ( 8 ). Antibiotic susceptibility test The Kirby-Bauer disk diffusion method was utilized to assess the antibiotic susceptibility of E. coli isolates on Muller-Hinton Agar (MHA) plates (HiMedia Laboratories Pvt. Ltd., India) in accordance with the Clinical and Laboratory Standards Institute (CLSI). For inoculum preparation, three to six morphologically uniform colonies from overnight nutrient agar cultures were suspended in sterile saline and adjusted to 0.5 McFarland turbidity (≈ 1×10⁸ CFU/mL). Using a sterile cotton swab, the standardized suspension was uniformly streaked over the entire agar surface to produce a confluent bacterial lawn. After brief drying (≤ 15 minutes), commercial antibiotic disks were applied—specifically: amikacin (AK, 30 µg), ampicillin (AMP, 10 µg), ciprofloxacin (CIP, 5 µg), cotrimoxazole (COT, 1.25/23.75 µg), gentamicin (GEN, 10 µg), nitrofurantoin (NIT, 300 µg), norfloxacin (NX, 10 µg), ceftazidime (CAZ, 30 µg), ceftriaxone (CTR, 30 µg), and cefixime (CFM, 5 µg). Disks were placed with ≥ 24 mm spacing (center-to-center) on 100 mm plates. According to CLSI (2024), Plates were incubated in ambient air at 35 ± 2°C for 18–24 hours. Zones of inhibition were measured to the nearest millimetre using a ruler or calipers; interpretation into susceptible (S), intermediate (I), or resistant (R) categories followed CSLI M100 (2020) breakpoints ( 8 , 9 ). Detection of ESBLs producing E. coli After the identification of multidrug resistance among E. coli isolates, Combined disk method was performed for the screening of ESBLs isolated by measure of Ceftazidime zone of inhibition > 22mm and confirmed by increases of zone of inhibition of size > 5mm by Ceftazidime + clavuanic ( 10 ). Detection of Biofilm production by Congo Red Agar A solid medium that was inoculated with Congo red, supplemented with 5% sucrose, and incubated aerobically at 37°C up to 48 hrs was used to incubate the brain heart infusion broth. Black colonies with a dry crystalline quality are indicative of a favorable outcome. Poor biofilm producers often don't change color; nevertheless, occasionally, colonies' centers have been seen to darken. The outcome is shown by a darkening of the colonies without a dry crystalline colonial morphology ( 11 ). Bacterial DNA Extraction and PCR DNA was extracted from the isolates by using the boiling-freezing method. The isolates were inoculated in nutrient broth and incubated at 37ºC for 24 hours. After incubation, 1000 µl of sample was taken in a micro-centrifuge tube. The samples were centrifuged at 15,000 rpm for 15 minutes. Then the supernatant was discarded and the pellet was re-suspended in double distilled water. It was again centrifuged at 15,000 rpm for 10 minutes. The supernatant was discarded again and the pellet was re-suspended in 50 µl of double distilled water and then subjected to boiling at 100ºC in a water bath for 10 minutes. After boiling in the water bath it was immediately cooled on ice for 5–10 minutes. The tubes were centrifuged at 15,000 rpm for 10 seconds and were stored at -20ºC for the PCR ( 12 ). Using a conventional boiling-freezing technique, the genomic DNA was recovered from E. coli isolates. PgaA primer was used for uniplex pcr to identify pagA gene while, pgaB, pgaC , and pgaD primers was use for multiplex PCR to identify the pagB, pgaC , and pgaD genes. To maintain a 25 µl PCR mixture, 12.5 µl of Master Mix (Simply,USA) was added first, followed by 0.5 µl of each primer (Macrogen, Inc., South Korea) of all genes, and 2 µl of DNA from the bacterial strains and 9 µl nuclease free water for pgaB, pgaC and pgaD gene and 10µl nuclease free water for pgaA gene. The initial denaturation step of 10 minutes at 94°C was maintained, as were the 35 cycles of 1 minute at 94°C, annealing temperature was maintain for 1 minute at 60°C, and 45 seconds at 72°C, followed by a final extension step of 7 minutes at 72°C ( 13 ). Gel electrophoresis This technique is used to separate DNA fragments according to their size. DNA samples were loaded into wells at one end of a gel, and an electric current was applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. When a gel was stained with a DNA-binding dye, the DNA fragments were seen as bands, each representing a group of same-sized DNA fragments ( 14 ). Results A total number of 384 clinical samples were collected from patients of different age group and gender visiting in Annapurna Neurological Institute & Alliance Science Hospital and the samples were inoculated for culture. Out of 384 clinical samples, significant bacterial growth of E. coli was obtained in 37 (9.63%) and 29(7.55%) was isolated from UTI, others showed no growth or other organism. Throughout the investigation, more than 200 urine sample were collected and further process was done in microbiology laboratory. Age wise distribution pattern of E. coli in clinical specimen Among the positive isolates more number of E. coli was present in the age group of 19–59 i.e 51.3%. Similarly followed by the age group of 60 above which is 35.13% and followed by age of 0–18 which is 13.5%. Table 1 Distribution pattern of E. coli isolates from department of hospital Department of hospital Number of E.coli isolates IPD 15 OPD 7 ER 4 A total of E. coli isolates were collected from different hospital departments among them, the majority were obtained from the Inpatient Department (IPD) with 15 isolates, followed by the Outpatient Department (OPD) with 7 isolates. In addition, 4 isolates were collected from the Emergency Room (ER), indicating that E. coli was present across all major service areas of the hospital(Table.1). Table 2 Antibiotic susceptibility pattern of E. coli Antibiotic Susceptible Resistance Amikacin(AK) 28(96.5%) 1(3.5%) Ampicillin(AMP) 13(44.8%) 16(55.2%) Ciproflaxacine(CIP) 15(51.7%) 14(48.3%) Cotrimoxazole(COT) 21(72.4%) 8(27.6%) Gentamicin(GEN) 26(89.6%) 3(10.4%) Nitrofurantoin(NIT) 26(89.6%) 3(10.4%) Norfloxacin(NX) 12(41.3%) 17(58.7%) Ceftazidime(CAZ) 18(62%) 11(38%) Ceftriaxone(CTR) 20(68.9) 9(31.1%) Cefixime(CFM) 13(44.8%) 16(51.2%) The antibiotic susceptibility pattern of E. coli isolates revealed that Amikacin exhibited the highest activity (96.5%), followed by Gentamicin and Nitrofurantoin (both 89.6%). In contrast, a high level of resistance was detected against Ampicillin (55.2%), Norfloxacin (58.7%), and Cefixime (51.2%). Notably, Ciprofloxacin demonstrated nearly equal proportions of susceptibility and resistance (51.7% vs. 48.3%), highlighting a concerning trend of reduced fluoroquinolone efficacy(Table.2). Table 3 Multi-Drugs Resistant distribution pattern in E. coli Clinical specimens E. coli isolates No. of MDR ESBLs screening positive ESBLs confirmed Urine 29 20 20 4 Out of 29 isolates of E. coli 20 (68.97%) were shown multi drug resistant in two or more then two classes of antibiotics group, as well as EBSLs screening positive, 4 were confirmed as extended-spectrum beta-lactamase (ESBL) producers, indicating the presence of resistant strains with potential clinical significance. Highest rate of antibiotic resistance is the concern of public people in the world(Table. 3). Table 4 Distribution pattern of biofilm producer among the clinical isolates of E. coli Clinical Specimens Biofilm Producers Biofilm non-producers Urine 19(65.5%) 10 (34.5%) Among the E. coli isolates from urine samples, 19 (65.5%) were identified as biofilm producers, while 10 (34.5%) were non-producers, indicating a predominance of biofilm-forming strains.(Table. 4) Table 5 Clinical specimen wise distribution pattern of pgaABCD gene in E. coli Clinical specimens pgaA pgaB PgaC pgaD Urine 20 24 17 26 Total 68.9% 82.7% 58.6% 89.6% Prevalence of pgaABCD gene in E. coli The study was conducted to deal with different types of genes responsible for biofilm production in E. coli. It was found that the most dominant gene responsible was pgaD which was obtained in 26 isolates which include 89.6% of total isolates. The second most obtained responsible gene was pgaB gene including 24 isolates followed by pgaA and p gaC including number of isolates 20 and 17 respectively.(Table. 5) Significant growth of E. coli in MA after 24hrs of incubation at 37oC(Figure. 1). From left to right: Indol + ve, MR + ve, VP -ve, Citrate -ve, TSI A|A, gas, motility, Urease -ve, O/F fermentative.(Figure. 2) Black color colony with black background show the Biofilm producer in Cargo Red Agar(Figure. 2). Under UV radiation in 60 volt for 1.5 hrs. From left to right well 1- ladder, well 2- NC, well 3- PC, well 4- NS, well 5,6,7- PS, well 8- NS.(NS- Negative Sample, PS- Positive Sample)(Figure. 4). DISCUSSION Ecoli is found as a normal flora of colon in human but acts as opportunistic parasite when trans-located. Despite the reality that the most of its strains are benign and even helpful to humans, a few dangerous variants have drawn notice because of a bacterium that is fundamental to both clinical medicine and microbiology. UTI are the most prevalent infections in both the community as well as in hospitals. Most of E.coli isolates was found from uropathogen and ability of uropathogenic E.coli strain to colonized the human urinary tract is facilitated by number of virulence factors such as fimbriae(P and type 1), K antigens, siderophores, haemolysins, serum resistance, urease production, motility, fast growth and biofilm formation ( 15 ). The screening of virulence characteristics and multidrug resistance in E.coli from clinical isolates can be helpful in the prevention and treatment of infection caused by these bacteria. A total of 384 clinically significant E.coli collected during the study period were studied. E.coli are commensals as well as pathogens of human beings and because of their versatile character, they are isolated from different clinical sample. In this study, E. coli was confirmed in only 29 (7.55%) of the collected isolates comparable to the low rates reported by ( 16 ) yet significantly lower than several other regional studies: 22.7% in urine cultures examined by ( 17 ) at a tertiary teaching hospital in Kathmandu (67/300 positive), 16.9% and 16% in the earlier works by ( 18 , 19 ) respectively. The majority of positive isolates (over 50%) were recovered from individuals in the 19–59-year age group, a demographic widely exposed to public settings such as workplaces, transit hubs, and eateries environments that pose higher cross-contamination risks as characterized by ( 20 ) . Different kind of infection is caused by E.coli like UTIs, Gasteroenteritis, RTIs, Hemolytic Uremic syndrome (HUS), Neonatal Meningitis, Septicemia, Pneumonia and so on. Different antibiotics are used to treat infections. Antimicrobial susceptibility profiles revealed substantial resistance: ampicillin (≈ 55%), ciprofloxacin (~ 48%), cefixime (~ 51%), and norfloxacin (≈ 59%), aligning with broader observations of elevated fluoroquinolone and penicillin-class resistances among E. coli uropathogens in LMIC settings, including a 64.9% MDR rate in 739 paediatric isolates at a Nepalese tertiary centre (resistance exceeding 80% to ampicillin and ciprofloxacin). According to study in England the resistance rates for ampicillin were 55.2%, ciprofloxacin were 11.9%, and nitrifurantoin were 5.9% ( 21 ) conducted a comparable investigation like this study. The ampicillin resistivity in UTis patient is most likely owing to continued usage of it for many years ( 22 ). Antimicrobial susceptibility profiles revealed substantial resistance: ampicillin (≈ 55%), ciprofloxacin (~ 48%), cefixime (~ 51%), and norfloxacin (≈ 59%), aligning with broader observations of elevated fluoroquinolone and penicillin-class resistances among E. coli uropathogens in LMIC settings, including a 64.9% MDR rate in 739 paediatric isolates at a Nepalese tertiary centre (resistance exceeding 80% to ampicillin and ciprofloxacin). At the same centre, sensitivity remained high for gentamicin , and nitrofurantoin a pattern mirrored in our findings and earlier studies ( 23 ). Nitrifurantoin is an effective drug for urine infection that is not used to treat other types of infections. It do not show any effect while using antibiotics for other infection ( 24 ). In this study, only 10.72% of the isolates were resistant to nitrifurantoin. This data is agreed with antimicrobial resistivity is natural biological phenomena which seen when microbes respond selectively to antimicrobial medicines. Resistance may be inherent ( 25 ). The term "multi-drug resistant" (MDR) refers to obtain resistance to at least one antimicrobial agent across three plus categories. Clinical isolates showed 18(67%) MDR in this study. Out of the 232 E. coli in 214 (92.2%) by ( 26 ) in Sudan. Out of 739 E. coli isolates 64.9% were MDR by ( 27 ) in Nepal. Ceftazidime(CAZ) was used in the combined disk approach for screening Extended spectrum beta-lactamase(ESBLs) in accordance with the 2022 CLSI guidelines. The ESBLs isolate was found using the combined disk method, which measured the ceftazidime zone of inhibition > 22 mm and verified the results by increasing the zone of inhibition > 5 mm with ceftazidime + clavuanic acid(CAC). 2022 CLSI guidelines. ESBL producers comprised 13.7% of our 29 isolates, a lower proportion compared to the 13.5% confirmed in 444 E. coli isolates at another Kathmandu hospital by ( 28 ) and community prevalence ranging 10–43% across hospital settings in Nepal and Thailand. Numerous previous research primarily focusing on adult patients, examined the prevalence rate of ESBL pathogens among patients. Research has indicated that the frequency of ESBL organisms varies. For example, in Pokhara, ( 29 ) found a prevalence of 27.7%, whereas in Kathmandu ( 30 ) found a prevalence of 18%, whereas in pediatric hospital in Kathmandu found prevalence of 43% ( 31 ). Furthermore, 35.9% of E. coil isolates among outpatients at a tertiary care hospital in Kathmandu had ESBL, according to another study. ( 32 ) found prevalence of 6.8% in Lalitpur district. According to ( 30 ) research has revealed that the prevalence of ESBLs varies greatly throughout hospital patients and equipment settings, ranging from 10% to 43%. According to ( 33 ) from Thailand have showed 10% of ESBLs positive which is similar to this study. ( 34 ) 44% from Cambodia showed highest ESBLs. Additionally, our investigation revealed that 10.86% of E. coli from urine samples of clinical isolates. 65.5% of our E. coli strains formed biofilms on Congo Red Agar , consistent with Ugandan surveillance (62.5% of 200 isolates) linking biofilm formation to ESBL and MDR phenotypes—and highlighting pgaD. ( 35 ) Study in Uganda found that 125 (62.5%), among 200 E. coli isolates produce biofilms. In congo red agar method, 49% out of 100 E. coli isolates were positive in India ( 36 ). Out of 208 isolates 51.9% showed biofilm producer ( 37 ) in Nepal. The pgaABCD gene cluster is crucial for PGA production and export, polysaccharide essential for biofilm production in E. coli. This gene cluster consists of four genes pgaA, pgaB, pgaC and pgaD each genes are playing a specific role in the production and export of PGA. pgaA is an outer membrane porin involved in the export of PGA to the cell surface, pgaB is a periplasmic protein responsible for the deacetylation of PGA, which is crucial for its proper function in biofilm formation, pgaC is an inner membrane glycosyltransferase that catalyzed the polymerization of N-acetyglucosamine into PGA, pgaD is an inner membrane protein that assists pgaC in its activity and stabilizes the complex ( 6 ). The pgaD gene was the most prevalent (89.6%), followed by pgaB (82.7%), pgaA (68.9%), and pgaC (58.6%). pgaD as the dominant gene in biofilm regulation in our genotypic assays. Collectively, these findings affirm that while overall E. coli prevalence may appear low in certain populations, the share of high-risk, drug-resistant and biofilm-forming strains remains substantial, underscoring the need for ongoing surveillance, judicious antibiotic use, and targeted public health interventions to curb transmission in community and healthcare settings. CONCLUSIONS Our findings underscore that, despite a relatively low overall prevalence of E. coli (7.6%) among clinical isolates, there is a disproportionately high presence of multidrug-resistant (67%) , ESBL-producing (13.7%) , and biofilm-forming (65.5%) strains. These high-risk phenotypes are concentrated in the 19–59 year age group , suggesting an elevated transmission dynamic potentially linked to social and occupational exposures. The study reveals alarmingly high resistance to ampicillin, ciprofloxacin, cefixime, and norfloxacin , all exceeding 50% resistance rates; meanwhile, sensitivity remains robust for amikacin, gentamicin, and nitrofurantoin affirming their current therapeutic value for uncomplicated urinary tract infections. Genotypic analysis also highlights pgaA–D operon , especially pgaD , as a significant contributor to biofilm formation, pointing to potential targets for adjunctive interventions aimed at disrupting biofilm-associated persistence. Taken together, these data emphasize the urgent need for, first, strengthened antimicrobial stewardship particularly in curtailing fluoroquinolone and third-generation cephalosporin overuse and second, routine phenotypic and genotypic surveillance to monitor resistance trends and virulence markers. Finally, exploring biofilm-disrupting agents and refining local antibiotic susceptibility guidelines will be critical steps in effectively managing E. coli infections in both community and hospital settings. Abbreviations MDR Multidrug resistance ESBLs Extended-spectrum beta-lactamase MA MaxConkey Agar MHA Mueller Hinton Agar Declarations Funding No fund for this research work Data availability All the data generated or analysed during this study are included in the article. Ethics approval and consent to participate The research study was approved by the Institutional Review committee (IRC-ANIAS 2023/011) of Annapurna Neurological & Allied Science Hospital. Informed written consent to use the sample was obtained from each participant or the legal guardian/nearest relative for minor in this study. All the methods were carried out in accordance with the principle stated in the Declaration of Helsinki. Trial Registration Not applicable Consent for publication Not applicable. 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Yansouni CP, Dendukuri N, Liu G, Fernandez M, Frenette C, Paraskevas S, et al. Positive Cultures of Organ Preservation Fluid Predict Postoperative Infections in Solid Organ Transplantation Recipients. Infect Control & Hosp Epidemiol. 2012;33(7):672–680. Fox-Lewis A, Takata J, Miliya T, Lubell Y, Soeng S, Sar P, et al. Antimicrobial resistance in invasive bacterial infections in hospitalized children, Cambodia, 2007–2016. Emerg Infect Dis. 2018;24(5):841. Katongole P, Nalubega F, Florence NC, Asiimwe B, Andia I. Biofilm formation, antimicrobial susceptibility and virulence genes of Uropathogenic Escherichia coli isolated from clinical isolates in Uganda. BMC Infect Dis. 2020 Jun;20(1):453. M. N, Jayakumar K, Aravazhi A. The effectiveness of antibiotics against a major uropathogen- Escherichia coli and its biofilm assay by phenotypic methods. Int J Res Med Sci. 2016;4(11):4820–8. Shrestha S, Baniya L, Shrestha B, Shrestha P. Biofilm and Extended Spectrum Beta Lactamase ( ESBL ) Producing Multi Drug Resistant Bacterial Uropathogens : A Challenge to Antibiotic Therapy in Nepal Biofilm and Extended Spectrum Beta Lactamase ( ESBL ) Producing Multi Drug Resistant Bacterial Uropath. 2024;(August). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:06:13","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118853,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7617318/v1/b6877922d30b7dc5b3afe842.html"},{"id":95529416,"identity":"d61f6010-0c2e-4dc8-aa36-6fc84a6f27d6","added_by":"auto","created_at":"2025-11-10 10:17:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":529984,"visible":true,"origin":"","legend":"\u003cp\u003eSignificant Growth of \u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7617318/v1/0f468546d976e65cbb5b037f.png"},{"id":95519259,"identity":"168585a0-df13-434c-8a3f-1df9f18c7d7e","added_by":"auto","created_at":"2025-11-10 09:06:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":589799,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical test for E.coli\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7617318/v1/d7e4f9e5a5afd8fe5694f1c1.png"},{"id":95519254,"identity":"e9bdcff3-9e10-4096-ae88-409effd5c93b","added_by":"auto","created_at":"2025-11-10 09:06:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":319094,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm producer\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7617318/v1/08e3fde3c09ca2902eb1e83a.png"},{"id":95529267,"identity":"832e14c2-35d5-4a2c-b950-c55191c78396","added_by":"auto","created_at":"2025-11-10 10:16:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":190763,"visible":true,"origin":"","legend":"\u003cp\u003eBand formation of \u003cem\u003epgaB\u003c/em\u003e and \u003cem\u003ePgaD \u003c/em\u003egene with bp 100 \u0026amp;182 in Electrophoresis\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7617318/v1/ac71af79c7f2b9c28047181d.png"},{"id":97250395,"identity":"b85bdf92-075e-46c5-8200-001f5649a9c7","added_by":"auto","created_at":"2025-12-02 13:14:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3403992,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7617318/v1/87f36d95-1c29-44cc-857d-39d0d2f6309e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eBiofilm Formation and Detection of Pgaabcd Gene in Clinical Isolates of Extended-spectrum Betalactamase E.coli\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e is rod shaped Gram negative bacteria which is found as the normal flora of colon in humans but acts as opportunistic parasite when trans-located. Urinary tract infections (UTIs) account for around 150\u0026nbsp;million new instances of diagnosis each year worldwide. Thus, it shows the health issues that warrants actual attention (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Fulminant prostatitis, biliary tract infection, and urinary catheter cystitis are the primary causes of UTI (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExtended-spectrum beta-lactamases (ESBLs) are enzymes produced by \u003cem\u003eE. coli\u003c/em\u003e, which can break down and confer resistance to a wide variety of beta-lactam antibiotics including cephalosporins, monobactams, penicillins, but not carbapenems and cephamycins. ESBLs hydrolyze the beta-lactam ring present in many antibiotics rendering them ineffective and the most common ESBLs belong to TEM, SHV (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eE. coli\u003c/em\u003e strains, as the most frequent etiological representative of community \u0026amp; hospital acquired infection which is often resistant to numerous antibiotics previously prescribed to treat such infections (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn many natural and artificial habitats, bacteria from sessile communities are known as biofilm (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Biofilms are ordered collections of microorganisms that stick to both biotic and abiotic surfaces. They are enclosed in a matrix made of self-produced extracellular polymeric substance (EPS). Bacterial populations use a technique called quorum sensing (QS) to communicate with one another and coordinate gene expression in response to changes in cell density.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003eE. coli\u003c/em\u003e to synthesize biofilm and perform other harmful functions, the \u003cem\u003epgaABCD\u003c/em\u003e locus is necessary (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Poly-beta-1,N-acetyl-D-glucosamine (PGA) is a particular polysaccharide that is synthesized, modified, and exported by means of this operon. The periplasmic protein \u003cem\u003epgaB\u003c/em\u003e is in charge of deacetylating PGA, which is essential for its correct function in biofilm formation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). \u003cem\u003ePgaC\u003c/em\u003e is an inner membrane glycosyltransferase that catalyzes the polymerization of N-acetyglucosamine into PGA. \u003cem\u003ePgaD\u003c/em\u003e is an inner membrane protein that supports \u003cem\u003ePgaC\u003c/em\u003e's activity and stabilizes the complex. \u003cem\u003ePgaA\u003c/em\u003e role in outer membrane porin that is included in the export of PGA to the exterior of the cell (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAntibiotic resistance in \u003cem\u003eEscherichia coli\u003c/em\u003e represents a growing public health crisis, with biofilm formation emerging as a key adaptive strategy that can enhance bacterial survival by up to 1,000-fold compared to free-floating cells.Central to this phenomenon is the \u003cem\u003epgaABCD\u003c/em\u003e operon, which mediates the synthesis and export of poly-β-1,6-N-acetyl-D-glucosamine (PGA), a foundational component of the biofilm extracellular matrix (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough this operon has been linked to biofilm resilience, its prevalence and functional impact within multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL) producing clinical \u003cem\u003eE. coli\u003c/em\u003e remain under-characterized, representing a critical gap in our understanding of biofilm-mediated antibiotic evasion. To address this gap, the present study aims to detect \u003cem\u003epgaABCD\u003c/em\u003e genes in clinical \u003cem\u003eE. coli\u003c/em\u003e isolates and evaluate their association with biofilm formation, MDR, and ESBL production. By elucidating the role of the \u003cem\u003epgaABCD\u003c/em\u003e operon in biofilm-driven persistence and antibiotic resistance, this work provides crucial genetic insights that may enable the development of targeted anti-biofilm interventions and strengthen infection control strategies against resilient \u003cem\u003eE. coli\u003c/em\u003e pathogens.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003eA cross-sectional study was carried out in the Annapurna Neurological Institute \u0026amp; Allied Science Maitighar\u003cb\u003e(Microbiology Laboratory\u003c/b\u003e), Kathmandu from August 2023 to March 2024. The variables of the study are clinical specimens, age, sex, \u003cem\u003eE. coli\u003c/em\u003e isolates in clinical samples as well as ESBLs, biofilm production and antibiotic susceptibility profile of the isolates. The study population were patients visiting at Annapurna Neurological Institute \u0026amp; Allied Science Hospital, suspected to have bacterial infections and requested for microbiological culture and antimicrobial susceptibility testing of any age and sex within the study period was considered as population. The study units were different clinical samples (Urine) requested for routine microbiological culture from the patients visiting Neurological Institute \u0026amp; Allied Science Hospital, Maitighar, Kathmandu. The research study was approved by the Institutional Review Committee (IRC-ANIAS-2023/011) of Annapurna Neurological Institute \u0026amp; Allied Science Hospital. The size of the population was determined using the following formula: n\u0026thinsp;=\u0026thinsp;z\u003csup\u003e2\u003c/sup\u003epq/d\u003csup\u003e2\u003c/sup\u003e, where n is the sample size, p the prevalence, q is 1-p, z is the statistic level of confidence (1.96), d is precision (0.05). \u003cem\u003ep\u003c/em\u003e-value was considered as 54% (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Hence, the sample size was obtained as 384.\u003c/p\u003e\u003cp\u003eThe non-duplicate different aerobic bacterial isolates obtained from patients with any kind of infection or having major surgeries of having catheters processed at the hospital were included. After routine culture testing, only gram negative \u003cem\u003eE.coli\u003c/em\u003e isolates were processed. Contaminated, unlabeled, mislabeled samples, sample after 4 hours of collection without any preservation and specimen collection procedure other than guided by standard microbiological procedures were rejected for this study. 384 samples were cultured in MacConkey Agar and Cysteine lactose and electrolyte Deficient and incubated at 37\u0026deg;C for 24 hrs. Antibiotic susceptibility test was performed by Kirby-Bauer disk diffusion method following the guidelines of Clinical and Laboratory Standard Institute (CLSI) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAntibiotic susceptibility test\u003c/h2\u003e\u003cp\u003eThe Kirby-Bauer disk diffusion method was utilized to assess the antibiotic susceptibility of \u003cem\u003eE. coli\u003c/em\u003e isolates on Muller-Hinton Agar (MHA) plates (HiMedia Laboratories Pvt. Ltd., India) in accordance with the Clinical and Laboratory Standards Institute (CLSI). For inoculum preparation, three to six morphologically uniform colonies from overnight nutrient agar cultures were suspended in sterile saline and adjusted to 0.5 McFarland turbidity (\u0026asymp;\u0026thinsp;1\u0026times;10⁸ CFU/mL). Using a sterile cotton swab, the standardized suspension was uniformly streaked over the entire agar surface to produce a confluent bacterial lawn. After brief drying (\u0026le;\u0026thinsp;15 minutes), commercial antibiotic disks were applied\u0026mdash;specifically: amikacin (AK, 30 \u0026micro;g), ampicillin (AMP, 10 \u0026micro;g), ciprofloxacin (CIP, 5 \u0026micro;g), cotrimoxazole (COT, 1.25/23.75 \u0026micro;g), gentamicin (GEN, 10 \u0026micro;g), nitrofurantoin (NIT, 300 \u0026micro;g), norfloxacin (NX, 10 \u0026micro;g), ceftazidime (CAZ, 30 \u0026micro;g), ceftriaxone (CTR, 30 \u0026micro;g), and cefixime (CFM, 5 \u0026micro;g). Disks were placed with \u0026ge;\u0026thinsp;24 mm spacing (center-to-center) on 100 mm plates. According to CLSI (2024), Plates were incubated in ambient air at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 18\u0026ndash;24 hours. Zones of inhibition were measured to the nearest millimetre using a ruler or calipers; interpretation into susceptible (S), intermediate (I), or resistant (R) categories followed CSLI M100 (2020) breakpoints (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of ESBLs producing\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter the identification of multidrug resistance among \u003cem\u003eE. coli\u003c/em\u003e isolates, Combined disk method was performed for the screening of ESBLs isolated by measure of Ceftazidime zone of inhibition\u0026thinsp;\u0026gt;\u0026thinsp;22mm and confirmed by increases of zone of inhibition of size\u0026thinsp;\u0026gt;\u0026thinsp;5mm by Ceftazidime\u0026thinsp;+\u0026thinsp;clavuanic (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetection of Biofilm production by Congo Red Agar\u003c/h3\u003e\n\u003cp\u003eA solid medium that was inoculated with Congo red, supplemented with 5% sucrose, and incubated aerobically at 37\u0026deg;C up to 48 hrs was used to incubate the brain heart infusion broth. Black colonies with a dry crystalline quality are indicative of a favorable outcome. Poor biofilm producers often don't change color; nevertheless, occasionally, colonies' centers have been seen to darken. The outcome is shown by a darkening of the colonies without a dry crystalline colonial morphology (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBacterial DNA Extraction and PCR\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from the isolates by using the boiling-freezing method. The isolates were inoculated in nutrient broth and incubated at 37\u0026ordm;C for 24 hours. After incubation, 1000 \u0026micro;l of sample was taken in a micro-centrifuge tube. The samples were centrifuged at 15,000 rpm for 15 minutes. Then the supernatant was discarded and the pellet was re-suspended in double distilled water. It was again centrifuged at 15,000 rpm for 10 minutes. The supernatant was discarded again and the pellet was re-suspended in 50 \u0026micro;l of double distilled water and then subjected to boiling at 100\u0026ordm;C in a water bath for 10 minutes. After boiling in the water bath it was immediately cooled on ice for 5\u0026ndash;10 minutes. The tubes were centrifuged at 15,000 rpm for 10 seconds and were stored at -20\u0026ordm;C for the PCR (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUsing a conventional boiling-freezing technique, the genomic DNA was recovered from \u003cem\u003eE. coli isolates. PgaA\u003c/em\u003e primer was used for uniplex pcr to identify \u003cem\u003epagA\u003c/em\u003e gene while, \u003cem\u003epgaB, pgaC\u003c/em\u003e, and \u003cem\u003epgaD\u003c/em\u003e primers was use for multiplex PCR to identify the \u003cem\u003epagB, pgaC\u003c/em\u003e, and \u003cem\u003epgaD\u003c/em\u003e genes. To maintain a 25 \u0026micro;l PCR mixture, 12.5 \u0026micro;l of Master Mix (Simply,USA) was added first, followed by 0.5 \u0026micro;l of each primer (Macrogen, Inc., South Korea) of all genes, and 2 \u0026micro;l of DNA from the bacterial strains and 9 \u0026micro;l nuclease free water for \u003cem\u003epgaB, pgaC\u003c/em\u003e and \u003cem\u003epgaD\u003c/em\u003e gene and 10\u0026micro;l nuclease free water for \u003cem\u003epgaA\u003c/em\u003e gene. The initial denaturation step of 10 minutes at 94\u0026deg;C was maintained, as were the 35 cycles of 1 minute at 94\u0026deg;C, annealing temperature was maintain for 1 minute at 60\u0026deg;C, and 45 seconds at 72\u0026deg;C, followed by a final extension step of 7 minutes at 72\u0026deg;C (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGel electrophoresis\u003c/h3\u003e\n\u003cp\u003eThis technique is used to separate DNA fragments according to their size. DNA samples were loaded into wells at one end of a gel, and an electric current was applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. When a gel was stained with a DNA-binding dye, the DNA fragments were seen as bands, each representing a group of same-sized DNA fragments (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total number of 384 clinical samples were collected from patients of different age group and gender visiting in Annapurna Neurological Institute \u0026amp; Alliance Science Hospital and the samples were inoculated for culture. Out of 384 clinical samples, significant bacterial growth of \u003cem\u003eE. coli\u003c/em\u003e was obtained in 37 (9.63%) and 29(7.55%) was isolated from UTI, others showed no growth or other organism. Throughout the investigation, more than 200 urine sample were collected and further process was done in microbiology laboratory.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAge wise distribution pattern of\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003ein clinical specimen\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong the positive isolates more number of \u003cem\u003eE. coli was\u003c/em\u003e present in the age group of 19\u0026ndash;59 i.e 51.3%. Similarly followed by the age group of 60 above which is 35.13% and followed by age of 0\u0026ndash;18 which is 13.5%.\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\u003eDistribution pattern of \u003cem\u003eE. coli\u003c/em\u003e isolates from department of hospital\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDepartment of hospital\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber of \u003cem\u003eE.coli\u003c/em\u003e isolates\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIPD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOPD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eER\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\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 \u003cem\u003eE. coli\u003c/em\u003e isolates were collected from different hospital departments among them, the majority were obtained from the Inpatient Department (IPD) with 15 isolates, followed by the Outpatient Department (OPD) with 7 isolates. In addition, 4 isolates were collected from the Emergency Room (ER), indicating that \u003cem\u003eE. coli\u003c/em\u003e was present across all major service areas of the hospital(Table.1).\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\u003eAntibiotic susceptibility pattern of \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibiotic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eResistance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAmikacin(AK)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28(96.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1(3.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAmpicillin(AMP)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13(44.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16(55.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCiproflaxacine(CIP)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15(51.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14(48.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCotrimoxazole(COT)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21(72.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8(27.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGentamicin(GEN)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26(89.6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3(10.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNitrofurantoin(NIT)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26(89.6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3(10.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNorfloxacin(NX)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12(41.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17(58.7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCeftazidime(CAZ)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18(62%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11(38%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCeftriaxone(CTR)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20(68.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9(31.1%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCefixime(CFM)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13(44.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16(51.2%)\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\u003eThe antibiotic susceptibility pattern of \u003cem\u003eE. coli\u003c/em\u003e isolates revealed that Amikacin exhibited the highest activity (96.5%), followed by Gentamicin and Nitrofurantoin (both 89.6%). In contrast, a high level of resistance was detected against Ampicillin (55.2%), Norfloxacin (58.7%), and Cefixime (51.2%). Notably, Ciprofloxacin demonstrated nearly equal proportions of susceptibility and resistance (51.7% vs. 48.3%), highlighting a concerning trend of reduced fluoroquinolone efficacy(Table.2).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMulti-Drugs Resistant distribution pattern in \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical specimens\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo. of MDR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eESBLs screening positive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eESBLs confirmed\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eUrine\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\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\u003eOut of 29 isolates of \u003cem\u003eE. coli\u003c/em\u003e 20 (68.97%) were shown multi drug resistant in two or more then two classes of antibiotics group, as well as EBSLs screening positive, 4 were confirmed as extended-spectrum beta-lactamase (ESBL) producers, indicating the presence of resistant strains with potential clinical significance. Highest rate of antibiotic resistance is the concern of public people in the world(Table. 3).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution pattern of biofilm producer among the clinical isolates of \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical Specimens\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBiofilm Producers\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiofilm non-producers\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eUrine\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19(65.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (34.5%)\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\u003eAmong the \u003cem\u003eE. coli\u003c/em\u003e isolates from urine samples, 19 (65.5%) were identified as biofilm producers,\u003c/p\u003e\u003cp\u003ewhile 10 (34.5%) were non-producers, indicating a predominance of biofilm-forming\u003c/p\u003e\u003cp\u003estrains.(Table. 4)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical specimen wise distribution pattern of \u003cem\u003epgaABCD\u003c/em\u003e gene in \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical\u003c/p\u003e\u003cp\u003especimens\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003epgaA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003epgaB\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePgaC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003epgaD\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eUrine\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e89.6%\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\u003e\u003cb\u003ePrevalence of\u003c/b\u003e \u003cb\u003epgaABCD\u003c/b\u003e \u003cb\u003egene in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study was conducted to deal with different types of genes responsible for biofilm production in \u003cem\u003eE. coli.\u003c/em\u003e It was found that the most dominant gene responsible was \u003cem\u003epgaD\u003c/em\u003e which was obtained in 26 isolates which include 89.6% of total isolates. The second most obtained responsible gene was \u003cem\u003epgaB\u003c/em\u003e gene including 24 isolates followed by \u003cem\u003epgaA\u003c/em\u003e and p\u003cem\u003egaC\u003c/em\u003e including number of isolates 20 and 17 respectively.(Table. 5)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSignificant growth of \u003cem\u003eE. coli\u003c/em\u003e in MA after 24hrs of incubation at 37oC(Figure. 1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFrom left to right: Indol\u0026thinsp;+\u0026thinsp;ve, MR\u0026thinsp;+\u0026thinsp;ve, VP -ve, Citrate -ve, TSI A|A, gas, motility, Urease -ve, O/F fermentative.(Figure. 2)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBlack color colony with black background show the Biofilm producer in Cargo Red Agar(Figure. 2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnder UV radiation in 60 volt for 1.5 hrs. From left to right well 1- ladder, well 2- NC, well 3- PC, well 4- NS, well 5,6,7- PS, well 8- NS.(NS- Negative Sample, PS- Positive Sample)(Figure. 4).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cem\u003eEcoli\u003c/em\u003e is found as a normal flora of colon in human but acts as opportunistic parasite when trans-located. Despite the reality that the most of its strains are benign and even helpful to humans, a few dangerous variants have drawn notice because of a bacterium that is fundamental to both clinical medicine and microbiology. UTI are the most prevalent infections in both the community as well as in hospitals. Most of \u003cem\u003eE.coli\u003c/em\u003e isolates was found from uropathogen and ability of uropathogenic \u003cem\u003eE.coli\u003c/em\u003e strain to colonized the human urinary tract is facilitated by number of virulence factors such as fimbriae(P and type 1), K antigens, siderophores, haemolysins, serum resistance, urease production, motility, fast growth and biofilm formation (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). The screening of virulence characteristics and multidrug resistance in \u003cem\u003eE.coli\u003c/em\u003e from clinical isolates can be helpful in the prevention and treatment of infection caused by these bacteria.\u003c/p\u003e\n\u003cp\u003eA total of 384 clinically significant \u003cem\u003eE.coli\u003c/em\u003e collected during the study period were studied. \u003cem\u003eE.coli\u003c/em\u003e are commensals as well as pathogens of human beings and because of their versatile character, they are isolated from different clinical sample.\u003c/p\u003e\n\u003cp\u003eIn this study, \u003cstrong\u003eE. coli\u003c/strong\u003e was confirmed in only \u003cstrong\u003e29 (7.55%)\u003c/strong\u003e of the collected isolates comparable to the low rates reported by (\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e) yet significantly lower than several other regional studies: \u003cstrong\u003e22.7%\u003c/strong\u003e in urine cultures examined by (\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e) at a tertiary teaching hospital in Kathmandu (67/300 positive), \u003cstrong\u003e16.9%\u003c/strong\u003e and \u003cstrong\u003e16%\u003c/strong\u003e in the earlier works by (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e) respectively. The majority of positive isolates (over 50%) were recovered from individuals in the \u003cstrong\u003e19\u0026ndash;59-year\u003c/strong\u003e age group, a demographic widely exposed to public settings such as workplaces, transit hubs, and eateries environments that pose higher cross-contamination risks as characterized by (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e) .\u003c/p\u003e\n\u003cp\u003eDifferent kind of infection is caused by \u003cem\u003eE.coli\u003c/em\u003e like UTIs, Gasteroenteritis, RTIs, Hemolytic Uremic syndrome (HUS), Neonatal Meningitis, Septicemia, Pneumonia and so on. Different antibiotics are used to treat infections. Antimicrobial susceptibility profiles revealed substantial resistance: \u003cstrong\u003eampicillin\u003c/strong\u003e (\u0026asymp;\u0026thinsp;55%), \u003cstrong\u003eciprofloxacin\u003c/strong\u003e (~\u0026thinsp;48%), \u003cstrong\u003ecefixime\u003c/strong\u003e (~\u0026thinsp;51%), and \u003cstrong\u003enorfloxacin\u003c/strong\u003e (\u0026asymp;\u0026thinsp;59%), aligning with broader observations of elevated fluoroquinolone and penicillin-class resistances among \u003cstrong\u003eE. coli\u003c/strong\u003e uropathogens in LMIC settings, including a \u003cstrong\u003e64.9% MDR\u003c/strong\u003e rate in 739 paediatric isolates at a Nepalese tertiary centre (resistance exceeding 80% to ampicillin and ciprofloxacin). According to study in England the resistance rates for ampicillin were 55.2%, ciprofloxacin were 11.9%, and nitrifurantoin were 5.9% (\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e) conducted a comparable investigation like this study. The ampicillin resistivity in UTis patient is most likely owing to continued usage of it for many years (\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAntimicrobial susceptibility profiles revealed substantial resistance: \u003cstrong\u003eampicillin\u003c/strong\u003e (\u0026asymp;\u0026thinsp;55%), \u003cstrong\u003eciprofloxacin\u003c/strong\u003e (~\u0026thinsp;48%), \u003cstrong\u003ecefixime\u003c/strong\u003e (~\u0026thinsp;51%), and \u003cstrong\u003enorfloxacin\u003c/strong\u003e (\u0026asymp;\u0026thinsp;59%), aligning with broader observations of elevated fluoroquinolone and penicillin-class resistances among \u003cstrong\u003eE. coli\u003c/strong\u003e uropathogens in LMIC settings, including a \u003cstrong\u003e64.9% MDR\u003c/strong\u003e rate in 739 paediatric isolates at a Nepalese tertiary centre (resistance exceeding 80% to ampicillin and ciprofloxacin). At the same centre, sensitivity remained high for \u003cstrong\u003egentamicin\u003c/strong\u003e, and \u003cstrong\u003enitrofurantoin\u003c/strong\u003e a pattern mirrored in our findings and earlier studies (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e). Nitrifurantoin is an effective drug for urine infection that is not used to treat other types of infections. It do not show any effect while using antibiotics for other infection (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). In this study, only 10.72% of the isolates were resistant to nitrifurantoin. This data is agreed with antimicrobial resistivity is natural biological phenomena which seen when microbes respond selectively to antimicrobial medicines. Resistance may be inherent (\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe term \u0026quot;multi-drug resistant\u0026quot; (MDR) refers to obtain resistance to at least one antimicrobial agent across three plus categories. Clinical isolates showed 18(67%) MDR in this study. Out of the 232 \u003cem\u003eE. coli\u003c/em\u003e in 214 (92.2%) by (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e) in Sudan. Out of 739 \u003cem\u003eE. coli\u003c/em\u003e isolates 64.9% were MDR by (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e) in Nepal.\u003c/p\u003e\n\u003cp\u003eCeftazidime(CAZ) was used in the combined disk approach for screening Extended spectrum beta-lactamase(ESBLs) in accordance with the 2022 CLSI guidelines. The ESBLs isolate was found using the combined disk method, which measured the ceftazidime zone of inhibition\u0026thinsp;\u0026gt;\u0026thinsp;22 mm and verified the results by increasing the zone of inhibition\u0026thinsp;\u0026gt;\u0026thinsp;5 mm with ceftazidime\u0026thinsp;+\u0026thinsp;clavuanic acid(CAC). 2022 CLSI guidelines. ESBL producers comprised \u003cstrong\u003e13.7%\u003c/strong\u003e of our \u003cstrong\u003e29\u003c/strong\u003e isolates, a lower proportion compared to the \u003cstrong\u003e13.5%\u003c/strong\u003e confirmed in \u003cstrong\u003e444\u003c/strong\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003cstrong\u003eisolates\u003c/strong\u003e at another Kathmandu hospital by (\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e) and community prevalence ranging \u003cstrong\u003e10\u0026ndash;43%\u003c/strong\u003e across hospital settings in Nepal and Thailand. Numerous previous research primarily focusing on adult patients, examined the prevalence rate of ESBL pathogens among patients. Research has indicated that the frequency of ESBL organisms varies. For example, in Pokhara, (\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e) found a prevalence of 27.7%, whereas in Kathmandu (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e) found a prevalence of 18%, whereas in pediatric hospital in Kathmandu found prevalence of 43% (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e). Furthermore, 35.9% of \u003cem\u003eE. coil\u003c/em\u003e isolates among outpatients at a tertiary care hospital in Kathmandu had ESBL, according to another study. (\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e) found prevalence of 6.8% in Lalitpur district. According to (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e) research has revealed that the prevalence of ESBLs varies greatly throughout hospital patients and equipment settings, ranging from 10% to 43%. According to (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e) from Thailand have showed 10% of ESBLs positive which is similar to this study. (\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e) 44% from Cambodia showed highest ESBLs. Additionally, our investigation revealed that 10.86% of \u003cem\u003eE. coli\u003c/em\u003e from urine samples of clinical isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e65.5%\u003c/strong\u003e of our \u003cstrong\u003eE. coli\u003c/strong\u003e strains formed \u003cstrong\u003ebiofilms\u003c/strong\u003e on \u003cstrong\u003eCongo Red Agar\u003c/strong\u003e, consistent with Ugandan surveillance (62.5% of 200 isolates) linking biofilm formation to ESBL and MDR phenotypes\u0026mdash;and highlighting pgaD. (\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e) Study in Uganda found that 125 (62.5%), among 200 \u003cem\u003eE. coli\u003c/em\u003e isolates produce biofilms. In congo red agar method, 49% out of 100 \u003cem\u003eE. coli\u003c/em\u003e isolates were positive in India (\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e). Out of 208 isolates 51.9% showed biofilm producer (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e) in Nepal.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003epgaABCD\u003c/em\u003e gene cluster is crucial for PGA production and export, polysaccharide essential for biofilm production in \u003cem\u003eE. coli.\u003c/em\u003e This gene cluster consists of four genes \u003cem\u003epgaA, pgaB, pgaC\u003c/em\u003e and \u003cem\u003epgaD\u003c/em\u003e each genes are playing a specific role in the production and export of PGA. \u003cem\u003epgaA\u003c/em\u003e is an outer membrane porin involved in the export of PGA to the cell surface, \u003cem\u003epgaB\u003c/em\u003e is a periplasmic protein responsible for the deacetylation of PGA, which is crucial for its proper function in biofilm formation, \u003cem\u003epgaC\u003c/em\u003e is an inner membrane glycosyltransferase that catalyzed the polymerization of N-acetyglucosamine into PGA, \u003cem\u003epgaD\u003c/em\u003e is an inner membrane protein that assists \u003cem\u003epgaC\u003c/em\u003e in its activity and stabilizes the complex (\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e). The \u003cem\u003epgaD\u003c/em\u003e gene was the most prevalent (89.6%), followed by \u003cem\u003epgaB\u003c/em\u003e (82.7%), \u003cem\u003epgaA\u003c/em\u003e (68.9%), and pgaC (58.6%). \u003cstrong\u003epgaD\u003c/strong\u003e as the dominant gene in biofilm regulation in our genotypic assays. Collectively, these findings affirm that while overall \u003cstrong\u003eE. coli\u003c/strong\u003e prevalence may appear low in certain populations, the share of high-risk, drug-resistant and biofilm-forming strains remains substantial, underscoring the need for ongoing surveillance, judicious antibiotic use, and targeted public health interventions to curb transmission in community and healthcare settings.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eOur findings underscore that, despite a relatively low \u003cb\u003eoverall prevalence of\u003c/b\u003e \u003cem\u003eE. coli\u003c/em\u003e (7.6%) among clinical isolates, there is a disproportionately high presence of \u003cb\u003emultidrug-resistant (67%)\u003c/b\u003e, \u003cb\u003eESBL-producing (13.7%)\u003c/b\u003e, and \u003cb\u003ebiofilm-forming (65.5%)\u003c/b\u003e strains. These high-risk phenotypes are concentrated in the \u003cb\u003e19\u0026ndash;59 year age group\u003c/b\u003e, suggesting an elevated transmission dynamic potentially linked to social and occupational exposures.\u003c/p\u003e\u003cp\u003eThe study reveals \u003cb\u003ealarmingly high resistance to ampicillin, ciprofloxacin, cefixime, and norfloxacin\u003c/b\u003e, all exceeding 50% resistance rates; meanwhile, sensitivity remains robust for \u003cb\u003eamikacin, gentamicin, and nitrofurantoin\u003c/b\u003e affirming their current therapeutic value for uncomplicated urinary tract infections. Genotypic analysis also highlights \u003cb\u003epgaA\u0026ndash;D\u003c/b\u003e \u003cb\u003eoperon\u003c/b\u003e, especially \u003cb\u003epgaD\u003c/b\u003e, as a significant contributor to biofilm formation, pointing to potential targets for adjunctive interventions aimed at disrupting biofilm-associated persistence.\u003c/p\u003e\u003cp\u003eTaken together, these data emphasize the urgent need for, first, \u003cb\u003estrengthened antimicrobial stewardship\u003c/b\u003e particularly in curtailing fluoroquinolone and third-generation cephalosporin overuse and second, \u003cb\u003eroutine phenotypic and genotypic surveillance\u003c/b\u003e to monitor resistance trends and virulence markers. Finally, exploring \u003cb\u003ebiofilm-disrupting agents\u003c/b\u003e and refining local antibiotic susceptibility guidelines will be critical steps in effectively managing \u003cem\u003eE. coli\u003c/em\u003e infections in both community and hospital settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMDR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Multidrug resistance\u003c/p\u003e\n\u003cp\u003eESBLs \u0026nbsp; \u0026nbsp; \u0026nbsp; Extended-spectrum beta-lactamase\u003c/p\u003e\n\u003cp\u003eMA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;MaxConkey Agar\u003c/p\u003e\n\u003cp\u003eMHA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Mueller Hinton Agar\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo fund for this research work\u003c/p\u003e\n\u003cp\u003eData availability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the data generated or analysed during this study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research study was approved by the Institutional Review committee (IRC-ANIAS 2023/011) of Annapurna Neurological \u0026amp; Allied Science Hospital. Informed written consent to use the sample was obtained from each participant or the legal guardian/nearest relative for minor in this study. All the methods were carried out in accordance with the principle stated in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial Registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConflict of \u0026nbsp; Interest:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHarwalkar A, Sataraddi J, Gupta S, Yoganand R, Rao A, Srinivasa H. The detection of ESBL-producing Escherichia coli in patients with symptomatic urinary tract infections using different diffusion methods in a rural setting. J Infect Public Health. 2013;6(2):108\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eWang X, Preston III JF, Romeo T. The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation. J Bacteriol. 2004;186(9):2724\u0026ndash;34. \u003c/li\u003e\n\u003cli\u003eZhong Y, Guo S, KLG S, GOH M, Schlundt J. Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Jurong Lake, Singapore with Whole-Genome-Sequencing. LID - 10.3390/ijerph18030937 [doi] LID - 937. Nanyang Technological University Food Technology Centre (NAFTEC), 62 Nanyang Drive, Singapore 637459, Singapore.; 2021. \u003c/li\u003e\n\u003cli\u003eAnsari S, Nepal HP, Gautam R, Shrestha S, Neopane P, Gurung G, et al. Community acquired multi-drug resistant clinical isolates of Escherichia coli in a tertiary care center of Nepal. Antimicrob Resist Infect Control. 2015;4(1):15. \u003c/li\u003e\n\u003cli\u003eCosterton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. 1995; \u003c/li\u003e\n\u003cli\u003eItoh Y, Rice JD, Goller C, Pannuri A, Taylor J, Meisner J, et al. Roles of pgaABCD genes in synthesis, modification, and export of the Escherichia coli biofilm adhesin poly-\u0026beta;-1, 6-N-acetyl-D-glucosamine. J Bacteriol. 2008;190(10):3670\u0026ndash;80. \u003c/li\u003e\n\u003cli\u003eHunduma D, Amenu K, Desta H, Grace D, Agga GE, Kerro Dego O. Prevalence and antimicrobial resistance of Escherichia coli O157: H7 and Salmonella, and the prevalence of Staphylococcus aureus in dairy cattle and camels under pastoral production system. Antibiotics. 2023;13(1):26. \u003c/li\u003e\n\u003cli\u003eWayne P. CLSI performance standards for antimicrobial susceptibility testing. CLSI Suppl M. 2020;100:20\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eWalker RD. Standards for antimicrobial susceptibility testing. Vol. 60, American journal of veterinary research. 1999. 1034 p. \u003c/li\u003e\n\u003cli\u003eJarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum \u0026beta;-lactamases conferring transferable resistance to newer \u0026beta;-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Clin Infect Dis. 1988;10(4):867\u0026ndash;78. \u003c/li\u003e\n\u003cli\u003ePanda PS, Chaudhary U, Dube SK. Comparison of four different methods for detection of biofilm formation by uropathogens. Indian J Pathol Microbiol [Internet]. 2016 Apr 1 [cited 2025 Aug 14];59(2):177\u0026ndash;9. Available from: https://journals.lww.com/ijpm/fulltext/2016/59020/comparison_of_four_different_methods_for_detection.8.aspx\u003c/li\u003e\n\u003cli\u003eQueipo-Ortu\u0026ntilde;o MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P. 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J Chitwan Med Coll. 2013;3(1):14\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eGuragain N, Pradhan A, Dhungel B, Banjara MR, Rijal KR, Ghimire P. Extended Spectrum Beta-lactamase Producing Gram Negative Bacterial Isolates from Urine of Patients Visiting Everest Hospital, Kathmandu, Nepal. Tribhuvan Univ J Microbiol [Internet]. 2019;6:26\u0026ndash;31. Available from: https://www.nepjol.info/index.php/tujm/article/view/26575\u003c/li\u003e\n\u003cli\u003ePatel P, Garala RN. Bacteriological profile and antibiotic susceptibility pattern (antibiogram) of urinary tract infections in paediatric patients. J Resear Med Dent Sci. 2014;2(1). \u003c/li\u003e\n\u003cli\u003ePoudyal S, Bhatta DR, Shakya G, Upadhyaya B, Dumre SP, Buda G, et al. Extended spectrum \u0026acirc;-lactamase producing multidrug resistant clinical bacterial isolates at National Public Health Laboratory, Nepal. Nepal Med Coll J. 2011;13(1):34\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eMead R, Curnow RN, Hasted AM. Statistical methods in agriculture and experimental biology: Third edition. Stat Methods Agric Exp Biol Third Ed. 2017 Jan 1;1\u0026ndash;472. \u003c/li\u003e\n\u003cli\u003eBean DC, Krahe D, Wareham DW. Antimicrobial resistance in community and nosocomial Escherichia coli urinary tract isolates, London 2005\u0026ndash;2006. Ann Clin Microbiol Antimicrob. 2008;7(1):13. \u003c/li\u003e\n\u003cli\u003eYeganeh BY, Mahmoudvand G, Abadi RNS, Heidarian M, Rouzbahani AK. Antibiotic resistance pattern of urinary tract infections among patients with recent antibiotic use: a cross-sectional study. Int J Infect. 2023;10(1). \u003c/li\u003e\n\u003cli\u003eMorawska M. Annual acknowledgement of manuscript reviewers. BMC Ear, Nose Throat Disord [Internet]. 2014;14(1):1. Available from: https://doi.org/10.1186/1472-6815-14-1\u003c/li\u003e\n\u003cli\u003eDescalzi O, Cartes C, Cisternas J, Brand HR. Exploding dissipative solitons: The analog of the Ruelle-Takens route for spatially localized solutions. Phys Rev E [Internet]. 2011 May;83(5):56214. Available from: https://link.aps.org/doi/10.1103/PhysRevE.83.056214\u003c/li\u003e\n\u003cli\u003eNoor MN, Shakoor S, Hasan R. Tracing linkages between pharmaceutical incentivisation, antibiotic prescribing and antimicrobial resistance in Pakistan. In: The Elgar Companion to Health and the Sustainable Development Goals. Edward Elgar Publishing; 2025. p. 361\u0026ndash;72. \u003c/li\u003e\n\u003cli\u003eAssob JCN, Nde PF, Nsagha DS, Njimoh DL, Nfor O, Njunda AL, et al. The incidence of feco-oral parasites in street-food vendors in Buea, south-west region Cameroon. Afr Health Sci [Internet]. 2012 [cited 2025 Aug 31];12(3):376\u0026ndash;80. Available from: https://www.ajol.info/index.php/ahs/article/view/84463\u003c/li\u003e\n\u003cli\u003eRaya GB, Dhoubhadel BG, Shrestha D, Raya S, Laghu U, Shah A, et al. Multidrug-resistant and extended-spectrum beta-lactamase-producing uropathogens in children in Bhaktapur, Nepal. Trop Med Health [Internet]. 2020;48(1):65. Available from: https://doi.org/10.1186/s41182-020-00251-6\u003c/li\u003e\n\u003cli\u003eChander A, Shrestha CD. Prevalence of extended spectrum beta lactamase producing Escherichia coli and Klebsiella pneumoniae urinary isolates in a tertiary care hospital in Kathmandu, Nepal. BMC Res Notes. 2013;6(1):487. \u003c/li\u003e\n\u003cli\u003ePrevalence of Urinary Tract Infection among Suspected Female Patients Attending Manipal Teaching Hospital, Pokhara, Nepal | Request PDF [Internet]. [cited 2025 Aug 31]. Available from: https://www.researchgate.net/publication/280244658_Prevalence_of_Urinary_Tract_Infection\n_among_Suspected_Female_Patients_\nAttending_Manipal_Teaching_\nHospital_Pokhara_Nepal\u003c/li\u003e\n\u003cli\u003eRai S, Yadav UN, Pant ND, Yakha JK, Tripathi PP, Poudel A, et al. Bacteriological Profile and Antimicrobial Susceptibility Patterns of Bacteria Isolated from Pus/Wound Swab Samples from Children Attending a Tertiary Care Hospital in Kathmandu, Nepal. Int J Microbiol. 2017;2017:2529085. \u003c/li\u003e\n\u003cli\u003ePradhan MM, Sharma S, Poudyal S, Gnyawali D, Adhikari S, Chapagain S, et al. Prevalence and Antimicrobial Susceptibility of Multi-Drug Resistant Uropathogens in a Tertiary Hospital in Nepal: A One-Year Audit. J Nepal Med Assoc. 2025;63(287):523\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eSubedi M, Thapaliya S, Thapa S. Prevalence and Antibiotic Susceptibility Pattern Of Uropathogens From Urinary Tract Infection Suspected Patients Visiting Tertiary Care Hospital of Nepal. Amrit Res J. 2024;5(1):114\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eYansouni CP, Dendukuri N, Liu G, Fernandez M, Frenette C, Paraskevas S, et al. Positive Cultures of Organ Preservation Fluid Predict Postoperative Infections in Solid Organ Transplantation Recipients. Infect Control \u0026amp;#38; Hosp Epidemiol. 2012;33(7):672\u0026ndash;680. \u003c/li\u003e\n\u003cli\u003eFox-Lewis A, Takata J, Miliya T, Lubell Y, Soeng S, Sar P, et al. Antimicrobial resistance in invasive bacterial infections in hospitalized children, Cambodia, 2007\u0026ndash;2016. Emerg Infect Dis. 2018;24(5):841. \u003c/li\u003e\n\u003cli\u003eKatongole P, Nalubega F, Florence NC, Asiimwe B, Andia I. Biofilm formation, antimicrobial susceptibility and virulence genes of Uropathogenic Escherichia coli isolated from clinical isolates in Uganda. BMC Infect Dis. 2020 Jun;20(1):453. \u003c/li\u003e\n\u003cli\u003eM. N, Jayakumar K, Aravazhi A. The effectiveness of antibiotics against a major uropathogen- Escherichia coli and its biofilm assay by phenotypic methods. Int J Res Med Sci. 2016;4(11):4820\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eShrestha S, Baniya L, Shrestha B, Shrestha P. Biofilm and Extended Spectrum Beta Lactamase ( ESBL ) Producing Multi Drug Resistant Bacterial Uropathogens : A Challenge to Antibiotic Therapy in Nepal Biofilm and Extended Spectrum Beta Lactamase ( ESBL ) Producing Multi Drug Resistant Bacterial Uropath. 2024;(August). \u003c/li\u003e\n\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":true,"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":"Escherichia coli, Biofilm, ESBL, pgaABCD, PCR, Antibiotic resistance, MDR","lastPublishedDoi":"10.21203/rs.3.rs-7617318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7617318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eBiofilm formation is a critical virulence factor in \u003cem\u003eEscherichia coli\u003c/em\u003e, especially in multidrug-resistant and extended-spectrum beta-lactamase (ESBL) producing strains. The \u003cem\u003epgaABCD\u003c/em\u003e operon plays a vital role in synthesizing poly-beta-1,6-N-acetyl-D-glucosamine, a key exopolysaccharide in biofilms. This study aimed to detect \u003cem\u003epgaABCD\u003c/em\u003e genes and evaluate biofilm formation in ESBL-producing \u003cem\u003eE. coli\u003c/em\u003e from clinical isolates.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA cross-sectional study was conducted from August 2023 to March 2024. A total of 384 clinical samples were cultured and \u003cem\u003eE. coli\u003c/em\u003e isolates were tested for antimicrobial susceptibility using the Kirby-Bauer method per CLSI 2020 guidelines. ESBL production was screened using the combined disk method. Biofilm production was determined using Congo Red Agar. The presence of \u003cem\u003epgaABCD\u003c/em\u003e genes was assessed using conventional PCR and gel electrophoresis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAmong 384 clinical samples, 29 (7.55%) \u003cem\u003eE. coli\u003c/em\u003e isolates were recovered from urine. Of these, 65.5% were biofilm producers, and 67% exhibited multidrug resistance (MDR). ESBL production was confirmed in 4 isolates. The \u003cem\u003epgaD\u003c/em\u003e gene was the most prevalent (89.6%), followed by \u003cem\u003epgaB\u003c/em\u003e (82.7%), \u003cem\u003epgaA\u003c/em\u003e (68.9%), and \u003cem\u003epgaC\u003c/em\u003e (58.6%).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe high prevalence of biofilm-forming and MDR \u003cem\u003eE. coli\u003c/em\u003e isolates harboring \u003cem\u003epgaABCD\u003c/em\u003e genes underscores the clinical significance of these genes in antibiotic resistance and persistence. Understanding their distribution contributes to inform targeted therapeutic and infection control strategies.\u003c/p\u003e\u003ch2\u003eTrial Registration\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e","manuscriptTitle":"Biofilm Formation and Detection of Pgaabcd Gene in Clinical Isolates of Extended-spectrum Betalactamase E.coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 09:06:07","doi":"10.21203/rs.3.rs-7617318/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":"1c187450-06d8-4a85-90c5-04de57f6957b","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T14:53:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-10 09:06:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7617318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7617318","identity":"rs-7617318","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}