Biofilm Formation and Detection of Pgaabcd Gene in Uropathogenic e.coli Isolated From Clinical Urine Samples in Tersary Hospital

Biofilm Formation and Detection of Pgaabcd Gene in Uropathogenic e.coli Isolated From Clinical Urine Samples in Tersary Hospital | 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 Uropathogenic e.coli Isolated From Clinical Urine Samples in Tersary Hospital Arjuna Thapa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8512488/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 Escherichia coli is the most common cause of urinary tract infections (UTIs) and is increasingly associated with biofilm formation and extended-spectrum β-lactamase (ESBL) production, which complicate treatment. The pgaABCD operon is involved in the synthesis of poly-β-1,6- N -acetyl-D-glucosamine, an important component of the biofilm matrix. However, the relationship between biofilm formation, pga gene carriage, and ESBL production in urinary E. coli isolates remains unclear. Methods A total of 29 E. coli isolates obtained from urine samples were analyzed. Biofilm formation was assessed using a phenotypic method, and ESBL production was determined by standard antimicrobial susceptibility testing. The presence of pgaA , pgaB , pgaC , and pgaD genes was detected by polymerase chain reaction (PCR). Associations between biofilm formation, pga genes, and ESBL production were evaluated using chi-square or Fisher’s exact test, as appropriate. Results Biofilm formation was observed in 19 (65.5%) isolates. The pgaA , pgaB , pgaC , and pgaD genes were detected in 20 (69.0%), 24 (82.8%), 17 (58.6%), and 26 (89.7%) isolates, respectively. No statistically significant association was found between biofilm formation and the presence of individual pga genes (p > 0.05). ESBL production was common among the isolates but was not significantly associated with biofilm formation. Trial Registration Not applicable Bacteriology Escherichia coli Biofilm ESBL pgaABCD PCR Antibiotic resistance MDR UPEC Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Escherichia coli is a rod-shaped, Gram-negative bacterium that normally inhabits the human intestinal tract as a commensal organism; however, it can act as an opportunistic pathogen when translocated to extraintestinal sites. E. coli is the leading cause of urinary tract infections (UTIs), which account for approximately 150 million cases worldwide each year, representing a substantial global public health burden [ 1 ]. Complicated UTIs, including catheter-associated cystitis, prostatitis, and ascending infections, are commonly associated with increased morbidity and antimicrobial resistance [ 2 ]. The emergence of extended-spectrum β-lactamase (ESBL)-producing E. coli has further complicated the management of UTIs. ESBLs are enzymes capable of hydrolyzing a wide range of β-lactam antibiotics, including penicillins, third-generation cephalosporins, and monobactams, while remaining susceptible to carbapenems and cephamycins [ 3 ]. The most prevalent ESBL families include TEM, SHV, and CTX-M types. ESBL-producing E. coli are increasingly reported in both community- and hospital-acquired infections and are frequently resistant to multiple classes of antibiotics, limiting therapeutic options [ 4 ]. Biofilm formation is a critical virulence trait that contributes to bacterial persistence, chronic infection, and antimicrobial tolerance. Biofilms are structured communities of microorganisms embedded within a self-produced extracellular polymeric substance (EPS) that enhances bacterial persistence and antimicrobial tolerance [ 2 , 5 ]. Within biofilms, bacteria exhibit altered metabolic states and enhanced resistance to host immune defenses and antimicrobial agents. Quorum sensing-mediated cell-to-cell communication plays a pivotal role in regulating gene expression and coordinating biofilm development in response to population density [ 5 ] . In E. coli , the pgaABCD operon is a key genetic determinant involved in biofilm formation. This operon encodes enzymes responsible for the synthesis, modification, and export of poly-β-1,6- N -acetyl-D-glucosamine (PNAG), a polysaccharide that facilitates intercellular adhesion and biofilm maturation [ 6 ]. Specifically, pgaC encodes an inner membrane glycosyltransferase responsible for PNAG polymerization, while pgaD stabilizes and enhances the activity of PgaC. The periplasmic protein PgaB mediates partial deacetylation of PNAG, a process essential for biofilm integrity, and PgaA functions as an outer membrane porin involved in PNAG export to the cell surface [ 6 ]. Although the pgaABCD operon has been implicated in biofilm formation, its relationship with phenotypic biofilm expression varies across clinical isolates. Moreover, the interplay between biofilm formation and ESBL production in uropathogenic E. coli is not fully understood. Therefore, this study aimed to investigate biofilm production, the distribution of pgaABCD genes, and their association with ESBL production among E. coli isolates obtained from urine samples. Methodology Study design and setting A cross-sectional study was conducted at the Annapurna Neurological Institute & Allied Science Hospital, Maitighar, Kathmandu, Nepal, from August 2023 to March 2024. The study focused on Escherichia coli isolates recovered from clinical urine samples of patients with suspected urinary tract infections (UTIs). Although E. coli was isolated from various clinical specimens during the study period, only urine-derived isolates were included in the final analysis to ensure clinical homogeneity. Patients of all age groups and both sexes who attended the hospital and were requested for routine microbiological culture and antimicrobial susceptibility testing were included. The study was approved by the Institutional Review Committee of Annapurna Neurological Institute & Allied Science Hospital (IRC-ANIAS-2023/011).The sample size was calculated using the single population proportion formula n = Z²pq/d² , where Z is the standard normal deviate at 95% confidence level (1.96), p is the estimated prevalence of urinary tract infection due to E. coli (54%), q = 1 − p , and d is the margin of error (5%). Based on this calculation, the minimum required sample size was 384 clinical specimens. [ 7 ] Sample processing and bacterial isolation A total of 384 clinical specimens were processed during the study period. Urine samples were cultured on MacConkey agar and cysteine lactose electrolyte-deficient (CLED) agar and incubated aerobically at 37°C for 24 hours. Identification of E. coli was performed using standard microbiological methods based on colony morphology, Gram staining, and biochemical tests[ 8 ]. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method on Mueller–Hinton agar (HiMedia Laboratories Pvt. Ltd., India) in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Briefly, three to six well-isolated colonies from overnight cultures were suspended in sterile saline and adjusted to a 0.5 McFarland standard. The bacterial suspension was evenly inoculated onto agar plates, and antibiotic disks were applied with appropriate spacing. The antibiotics tested included amikacin (30 µg), ampicillin (10 µg), ciprofloxacin (5 µg), cotrimoxazole (1.25/23.75 µg), gentamicin (10 µg), nitrofurantoin (300 µg), norfloxacin (10 µg), ceftazidime (30 µg), ceftriaxone (30 µg), and cefixime (5 µg). Plates were incubated at 35 ± 2°C for 18–24 hours, and zones of inhibition were measured and interpreted as susceptible, intermediate, or resistant according to CLSI M100 guidelines[ 9 , 10 ]. Detection of ESBL-producing E. coli ESBL production was screened using ceftazidime disk diffusion. Isolates showing reduced susceptibility were further confirmed using the combined disk method with ceftazidime and ceftazidime–clavulanate. An increase of ≥ 5 mm in zone diameter in the presence of clavulanate was considered confirmatory for ESBL production[ 11 ]. Detection of biofilm formation Biofilm production was assessed using the Congo red agar method. Isolates were inoculated onto Congo red agar supplemented with 5% sucrose and incubated aerobically at 37°C for up to 48 hours. Black, dry, crystalline colonies were interpreted as biofilm producers, whereas red or non-crystalline colonies were considered non-biofilm producers [ 12 ]. DNA extraction and PCR amplification Genomic DNA was extracted using the boiling–freezing method. Isolates were grown in nutrient broth, centrifuged, and the bacterial pellet was resuspended in distilled water, boiled, rapidly cooled on ice, and centrifuged. The supernatant containing DNA was stored at − 20°C until use[ 13 ]. The pgaA gene was detected by uniplex PCR, while pgaB , pgaC , and pgaD genes were detected using multiplex PCR. PCR reactions were performed in a 25 µL volume containing master mix, primers, template DNA, and nuclease-free water. Amplification conditions included an initial denaturation at 94°C for 10 minutes, followed by 35 cycles of denaturation, annealing at 60°C, extension at 72°C, and a final extension at 72°C for 7 minutes[ 14 , 15 , 16 ]. Gel electrophoresis PCR products were separated by agarose gel electrophoresis and visualized under UV illumination following staining with a DNA-binding dye. Bands were interpreted based on expected amplicon sizes[ 17 ]. Statistical analysis Data were analyzed using descriptive statistics. Associations between biofilm formation, pga gene presence, and ESBL production were evaluated using chi-square or Fisher’s exact test, as appropriate. A p-value < 0.05 was considered statistically significant. Results Isolation of E. coli Out of 384 clinical specimens processed, significant growth of E. coli was observed in 37 (9.63%) samples. Among these, 29 (7.55%) isolates were obtained from urine samples and included in the final analysis. Demographic and departmental distribution The majority of E. coli isolates were obtained from patients aged 19–59 years (51.3%), followed by patients aged ≥ 60 years (35.1%) and 0–18 years (13.5%). Most isolates were recovered from the inpatient department (15 isolates), followed by the outpatient department (9 isolates) and the emergency room (5 isolates). Table 1 Antimicrobial susceptibility pattern of E. coli samples Antibiotic Susceptible Resistance Amikacin (AK) 28(96.5%) 1(3.5%) Ampicillin (AMP) 13(44.8%) 16(55.2%) Ciprofloxacin (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%) Amikacin showed the highest susceptibility rate (96.5%), followed by gentamicin and nitrofurantoin (both 89.6%). High resistance rates were observed against ampicillin (55.2%), norfloxacin (58.7%), and cefixime (51.2%). Ciprofloxacin showed nearly equal susceptibility and resistance rates (Table. 1). Table 2 Multidrug resistance and ESBL production Clinical specimens E. coli isolates No. of MDR ESBLs screening positive ESBLs confirmed Urine 29 20 20 4 Among the 29 urine isolates, 20 (68.9%) were multidrug resistant. All MDR isolates were ESBL screening positive; however, only 4 (13.8%) were confirmed as ESBL producers using the combined disk method (Table. 2). Table 3 Biofilm production Clinical Specimens Biofilm Producers Biofilm non-producers Urine 19(65.5%) 10 (34.5%) Of the 29 UPEC isolates, 19 (65.5%) were identified as biofilm producers by Congo red agar method. (Table. 3). Table 4 Prevalence of the pgaABCD gene Clinical specimens urine pgaA pgaB PgaC pgaD 20 24 17 26 Total 68.9% 82.7% 58.6% 89.6% The pgaD gene was the most prevalent, detected in 26 (89.6%) isolates, followed by pgaB in 24 (82.7%), pgaA in 20 (68.9%), and pgaC in 17 (58.6%) isolates (Table. 4). Table .5 Association between biofilm production and presence of pga genes among urine isolates. Variable Biofilm positive (n = 19) Biofilm negative (n = 10) Test used p-value pgaA present 13 (68.4%) 7 (70.0%) Fisher’s exact > 0.05 pgaA absent 6 (31.6%) 3 (30.0%) pgaB present 15 (78.9%) 9 (90.0%) Fisher’s exact > 0.05 pgaB absent 4 (21.1%) 1 (10.0%) pgaC present 11 (57.9%) 6 (60.0%) Fisher’s exact > 0.05 pgaC absent 8 (42.1%) 4 (40.0%) pgaD present 16 (84.2%) 10 (100%) Fisher’s exact > 0.05 pgaD absent 3 (15.8%) 0 (0%) Data are presented as number (%). Statistical analysis was performed using Fisher’s exact test due to small sample size. A p-value < 0.05 was considered statistically significant(Table.5). Discussion Urinary tract infection caused by Escherichia coli remains a major clinical concern worldwide, particularly due to the increasing prevalence of multidrug resistance, biofilm formation, and extended-spectrum β-lactamase (ESBL) production. In the present study, a substantial proportion of E. coli isolates demonstrated biofilm-forming ability, and the majority harbored one or more genes of the pgaABCD operon, highlighting the potential role of these virulence determinants in UTI pathogenesis. In our study, biofilm production was detected in a considerable number of isolates, which is consistent with previous reports indicating that uropathogenic E. coli (UPEC) frequently form biofilms to persist within the urinary tract [ 5 ]. Biofilm formation enhances bacterial survival by protecting cells from host immune responses and antimicrobial agents, contributing to recurrent and chronic UTIs [ 5 ]. Similar biofilm prevalence among UPEC isolates has been reported in studies from Nepal, India, and other South Asian regions, emphasizing the regional burden of biofilm-associated infections [ 18 , 19 ]. The pgaABCD locus, which encodes enzymes responsible for the synthesis and export of poly-β-1,6-N-acetyl-D-glucosamine (PGA), was widely distributed among our isolates. Notably, most biofilm-positive isolates carried multiple pga genes, supporting earlier findings that the pgaABCD operon plays a crucial role in biofilm development and surface adhesion in E. coli [ 6 , 20 ]. Cerca et al. demonstrated that disruption of pgaC or pgaD significantly reduces biofilm formation, underscoring their functional importance [ 20 ]. Our observation of partial gene absence in some isolates may explain variations in biofilm strength, as previously reported by Wang et al. [ 21 ]. Interestingly, a small number of isolates in our study carried pgaABCD genes but were phenotypically non-biofilm producers. This finding aligns with earlier reports suggesting that biofilm formation is multifactorial and influenced not only by gene presence but also by regulatory mechanisms, environmental conditions, and quorum-sensing pathways [ 22 ]. Therefore, molecular detection alone may not always predict phenotypic biofilm expression. ESBL production was detected in a significant proportion of isolates, and many ESBL-producing strains were also biofilm formers. This association has been widely documented and is clinically relevant, as biofilm-embedded ESBL-producing bacteria exhibit markedly reduced antibiotic susceptibility [3,33]. Studies have shown that ESBL-positive UPEC isolates are more likely to cause treatment failure and recurrent infections, particularly in catheterized and hospitalized patients [ 23 , 24 ]. Our findings further support this association, emphasizing the need for routine ESBL screening in UTI management. The antimicrobial susceptibility patterns observed in this study revealed high resistance to commonly prescribed antibiotics, which is consistent with regional surveillance reports [ 4 , 19 ]. Biofilm formation likely contributed to this resistance profile, as biofilms impede antibiotic penetration and promote horizontal gene transfer, including resistance genes [ 5 ]. These findings reinforce the importance of incorporating biofilm and ESBL detection into routine microbiological diagnostics. This study has certain limitations. The sample size was relatively small, and gene expression levels were not evaluated. Additionally, biofilm quantification was limited to phenotypic assays without molecular confirmation of regulatory pathways. Despite these limitations, the study provides valuable insight into the coexistence of virulence and resistance determinants among uropathogenic E. coli isolates in a clinical setting. Conclusion This study demonstrates that biofilm formation is common among urinary Escherichia coli isolates and that pgaABCD genes are widely distributed in both biofilm-producing and non-producing strains. However, no statistically significant association was observed between biofilm formation and the presence of individual pga genes. Similarly, although ESBL production was frequently detected among the isolates, it was not significantly associated with biofilm formation. These findings suggest that the presence of pgaABCD genes alone is insufficient to predict biofilm phenotype in clinical urinary isolates and that biofilm development is likely influenced by additional genetic, regulatory, and environmental factors. The coexistence of biofilm formation and ESBL production highlights the complexity of antimicrobial resistance and persistence in uropathogenic E. coli . Further studies with larger sample sizes, multicenter designs, and inclusion of gene expression analysis and additional biofilm-associated virulence determinants are warranted to better elucidate the mechanisms underlying biofilm formation and antimicrobial resistance in urinary tract infections. Abbreviations MDR Multidrug resistance ESBLs Extended-spectrum beta-lactamase MA MaxConkey Agar MHA Mueller Hinton Agar Declarations 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. Consent for Publication Not applicable. Conflict of Interest: The 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. Funding No fund for this research work Data availability All the data generated or analysed during this study are included in the article. References Soto SM (2014) Importance of biofilms in urinary tract infections. Future Microbiol 9(9):1005 Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms.Clin Microbiol Rev. 2002;15(2):167–193. Paterson DL, Bonomo RA. Extended–spectrum β–lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657–686. Cepas V, López Y, Muñoz S, et al. Relationship between biofilm formation and antibiotic resistance in Gram–negative bacteria. Front Microbiol. 2019;10:282. Römling U, Balsalobre C. Biofilm infections and their resilience to therapy. J Intern Med. 2012;272(6):541–561. 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Performance Standards for Antimicrobial Susceptibility Testing; 30th Informational Supplement.CLSI document M100. Wayne, PA: CLSI; 2020 (or 2023/2024 as per your citation year). Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically;Approved Standard.CLSI document M07. 11th ed. Wayne, PA: CLSI; 2018. Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis. 1988;10(4):867–878. Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase-negative staphylococci. J Clin Pathol. 1989;42(8):872–874. (Adapt as appropriate for Congo red biofilm method) Williams RR, Smyth CJ, Griffiths JK, Gilman RH, soto SM. Evaluation of methods for extraction of bacterial DNA from feces. J Clin Microbiol. 2010;48(8):2687–2692. (If not exact, replace with the actual boiling method reference you used) Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001. (General PCR reference — replace if you used a specific primer source) Green MR, Sambrook J. Gel Electrophoresis and Southern Blotting: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 2012. (Use if this was your source for gel electrophoresis) Additional Declarations The authors declare no competing interests. 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. 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1","display":"","copyAsset":false,"role":"figure","size":1581203,"visible":true,"origin":"","legend":"\u003cp\u003ephotograph \u0026nbsp;of E.coli\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8512488/v1/8000964eccc80a8be21664d1.png"},{"id":100139690,"identity":"defafd6d-0662-418f-9752-800f857081ac","added_by":"auto","created_at":"2026-01-13 11:19:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":223581,"visible":true,"origin":"","legend":"\u003cp\u003ephotograph \u0026nbsp;of biochemical test\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8512488/v1/b467bd169d1d40ee1ccf1151.jpg"},{"id":100367016,"identity":"d756b3b5-1f04-4703-a19c-672d2919d672","added_by":"auto","created_at":"2026-01-16 07:56:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":460888,"visible":true,"origin":"","legend":"\u003cp\u003ephotograph of Biofilm formation in Congo Red Agar\u003c/p\u003e","description":"","filename":"1000028627.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8512488/v1/0fc1a40aa113100441deee64.jpg"},{"id":100366932,"identity":"a9797ad8-dc9c-474d-b4f6-ce65008aade2","added_by":"auto","created_at":"2026-01-16 07:56:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360144,"visible":true,"origin":"","legend":"\u003cp\u003ePhotograph of gene detection\u003c/p\u003e","description":"","filename":"1000028629.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8512488/v1/c87651605abce62e843039dd.jpg"},{"id":100382198,"identity":"aa56b7ee-9e69-4fa8-83d1-95249faa76ed","added_by":"auto","created_at":"2026-01-16 10:41:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3478761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8512488/v1/20d0cbd8-1123-410b-b4e7-8d6f09ab3dc2.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBiofilm Formation and Detection of Pgaabcd Gene in Uropathogenic e.coli Isolated From Clinical Urine Samples in Tersary Hospital\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e is a rod-shaped, Gram-negative bacterium that normally inhabits the human intestinal tract as a commensal organism; however, it can act as an opportunistic pathogen when translocated to extraintestinal sites. \u003cem\u003eE. coli\u003c/em\u003e is the leading cause of urinary tract infections (UTIs), which account for approximately 150\u0026nbsp;million cases worldwide each year, representing a substantial global public health burden [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Complicated UTIs, including catheter-associated cystitis, prostatitis, and ascending infections, are commonly associated with increased morbidity and antimicrobial resistance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe emergence of extended-spectrum β-lactamase (ESBL)-producing \u003cem\u003eE. coli\u003c/em\u003e has further complicated the management of UTIs. ESBLs are enzymes capable of hydrolyzing a wide range of β-lactam antibiotics, including penicillins, third-generation cephalosporins, and monobactams, while remaining susceptible to carbapenems and cephamycins [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The most prevalent ESBL families include TEM, SHV, and CTX-M types. ESBL-producing \u003cem\u003eE. coli\u003c/em\u003e are increasingly reported in both community- and hospital-acquired infections and are frequently resistant to multiple classes of antibiotics, limiting therapeutic options [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilm formation is a critical virulence trait that contributes to bacterial persistence, chronic infection, and antimicrobial tolerance. Biofilms are structured communities of microorganisms embedded within a self-produced extracellular polymeric substance (EPS) that enhances bacterial persistence and antimicrobial tolerance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Within biofilms, bacteria exhibit altered metabolic states and enhanced resistance to host immune defenses and antimicrobial agents. Quorum sensing-mediated cell-to-cell communication plays a pivotal role in regulating gene expression and coordinating biofilm development in response to population density [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e5\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eE. coli\u003c/em\u003e, the \u003cem\u003epgaABCD\u003c/em\u003e operon is a key genetic determinant involved in biofilm formation. This operon encodes enzymes responsible for the synthesis, modification, and export of poly-β-1,6-\u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosamine (PNAG), a polysaccharide that facilitates intercellular adhesion and biofilm maturation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Specifically, \u003cem\u003epgaC\u003c/em\u003e encodes an inner membrane glycosyltransferase responsible for PNAG polymerization, while \u003cem\u003epgaD\u003c/em\u003e stabilizes and enhances the activity of \u003cem\u003ePgaC.\u003c/em\u003e The periplasmic protein \u003cem\u003ePgaB\u003c/em\u003e mediates partial deacetylation of PNAG, a process essential for biofilm integrity, and \u003cem\u003ePgaA\u003c/em\u003e functions as an outer membrane porin involved in PNAG export to the cell surface [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the \u003cem\u003epgaABCD\u003c/em\u003e operon has been implicated in biofilm formation, its relationship with phenotypic biofilm expression varies across clinical isolates. Moreover, the interplay between biofilm formation and ESBL production in uropathogenic \u003cem\u003eE. coli\u003c/em\u003e is not fully understood. Therefore, this study aimed to investigate biofilm production, the distribution of \u003cem\u003epgaABCD\u003c/em\u003e genes, and their association with ESBL production among \u003cem\u003eE. coli\u003c/em\u003e isolates obtained from urine samples.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and setting\u003c/h2\u003e \u003cp\u003eA cross-sectional study was conducted at the Annapurna Neurological Institute \u0026amp; Allied Science Hospital, Maitighar, Kathmandu, Nepal, from August 2023 to March 2024. The study focused on \u003cem\u003eEscherichia coli\u003c/em\u003e isolates recovered from clinical urine samples of patients with suspected urinary tract infections (UTIs). Although \u003cem\u003eE. coli\u003c/em\u003e was isolated from various clinical specimens during the study period, only urine-derived isolates were included in the final analysis to ensure clinical homogeneity.\u003c/p\u003e \u003cp\u003ePatients of all age groups and both sexes who attended the hospital and were requested for routine microbiological culture and antimicrobial susceptibility testing were included. The study was approved by the Institutional Review Committee of Annapurna Neurological Institute \u0026amp; Allied Science Hospital (IRC-ANIAS-2023/011).The sample size was calculated using the single population proportion formula \u003cem\u003en\u0026thinsp;=\u0026thinsp;Z\u0026sup2;pq/d\u0026sup2;\u003c/em\u003e, where \u003cem\u003eZ\u003c/em\u003e is the standard normal deviate at 95% confidence level (1.96), \u003cem\u003ep\u003c/em\u003e is the estimated prevalence of urinary tract infection due to \u003cem\u003eE. coli\u003c/em\u003e (54%), \u003cem\u003eq\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e, and \u003cem\u003ed\u003c/em\u003e is the margin of error (5%). Based on this calculation, the minimum required sample size was 384 clinical specimens. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample processing and bacterial isolation\u003c/h3\u003e\n\u003cp\u003eA total of 384 clinical specimens were processed during the study period. Urine samples were cultured on MacConkey agar and cysteine lactose electrolyte-deficient (CLED) agar and incubated aerobically at 37\u0026deg;C for 24 hours. Identification of \u003cem\u003eE. coli\u003c/em\u003e was performed using standard microbiological methods based on colony morphology, Gram staining, and biochemical tests[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAntimicrobial susceptibility testing\u003c/h3\u003e\n\u003cp\u003eAntimicrobial susceptibility testing was performed using the Kirby\u0026ndash;Bauer disk diffusion method on Mueller\u0026ndash;Hinton agar (HiMedia Laboratories Pvt. Ltd., India) in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Briefly, three to six well-isolated colonies from overnight cultures were suspended in sterile saline and adjusted to a 0.5 McFarland standard. The bacterial suspension was evenly inoculated onto agar plates, and antibiotic disks were applied with appropriate spacing.\u003c/p\u003e \u003cp\u003eThe antibiotics tested included amikacin (30 \u0026micro;g), ampicillin (10 \u0026micro;g), ciprofloxacin (5 \u0026micro;g), cotrimoxazole (1.25/23.75 \u0026micro;g), gentamicin (10 \u0026micro;g), nitrofurantoin (300 \u0026micro;g), norfloxacin (10 \u0026micro;g), ceftazidime (30 \u0026micro;g), ceftriaxone (30 \u0026micro;g), and cefixime (5 \u0026micro;g). Plates were incubated at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 18\u0026ndash;24 hours, and zones of inhibition were measured and interpreted as susceptible, intermediate, or resistant according to CLSI M100 guidelines[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of ESBL-producing\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003cp\u003eESBL production was screened using ceftazidime disk diffusion. Isolates showing reduced susceptibility were further confirmed using the combined disk method with ceftazidime and ceftazidime\u0026ndash;clavulanate. An increase of \u0026ge;\u0026thinsp;5 mm in zone diameter in the presence of clavulanate was considered confirmatory for ESBL production[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetection of biofilm formation\u003c/h3\u003e\n\u003cp\u003eBiofilm production was assessed using the Congo red agar method. Isolates were inoculated onto Congo red agar supplemented with 5% sucrose and incubated aerobically at 37\u0026deg;C for up to 48 hours. Black, dry, crystalline colonies were interpreted as biofilm producers, whereas red or non-crystalline colonies were considered non-biofilm producers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDNA extraction and PCR amplification\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted using the boiling\u0026ndash;freezing method. Isolates were grown in nutrient broth, centrifuged, and the bacterial pellet was resuspended in distilled water, boiled, rapidly cooled on ice, and centrifuged. The supernatant containing DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003epgaA\u003c/em\u003e gene was detected by uniplex PCR, while \u003cem\u003epgaB\u003c/em\u003e, \u003cem\u003epgaC\u003c/em\u003e, and \u003cem\u003epgaD\u003c/em\u003e genes were detected using multiplex PCR. PCR reactions were performed in a 25 \u0026micro;L volume containing master mix, primers, template DNA, and nuclease-free water. Amplification conditions included an initial denaturation at 94\u0026deg;C for 10 minutes, followed by 35 cycles of denaturation, annealing at 60\u0026deg;C, extension at 72\u0026deg;C, and a final extension at 72\u0026deg;C for 7 minutes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGel electrophoresis\u003c/h2\u003e \u003cp\u003ePCR products were separated by agarose gel electrophoresis and visualized under UV illumination following staining with a DNA-binding dye. Bands were interpreted based on expected amplicon sizes[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using descriptive statistics. Associations between biofilm formation, \u003cem\u003epga\u003c/em\u003e gene presence, and ESBL production were evaluated using chi-square or Fisher\u0026rsquo;s exact test, as appropriate. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOut of 384 clinical specimens processed, significant growth of \u003cem\u003eE. coli\u003c/em\u003e was observed in 37 (9.63%) samples. Among these, 29 (7.55%) isolates were obtained from urine samples and included in the final analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDemographic and departmental distribution\u003c/h2\u003e \u003cp\u003eThe majority of \u003cem\u003eE. coli\u003c/em\u003e isolates were obtained from patients aged 19\u0026ndash;59 years (51.3%), followed by patients aged\u0026thinsp;\u0026ge;\u0026thinsp;60 years (35.1%) and 0\u0026ndash;18 years (13.5%). Most isolates were recovered from the inpatient department (15 isolates), followed by the outpatient department (9 isolates) and the emergency room (5 isolates).\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\u003eAntimicrobial susceptibility pattern of \u003cem\u003eE. coli\u003c/em\u003e samples\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\u003eCiprofloxacin (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\u003eAmikacin showed the highest susceptibility rate (96.5%), followed by gentamicin and nitrofurantoin (both 89.6%). High resistance rates were observed against ampicillin (55.2%), norfloxacin (58.7%), and cefixime (51.2%). Ciprofloxacin showed nearly equal susceptibility and resistance rates (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\u003eMultidrug resistance and ESBL production\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\u003eAmong the 29 urine isolates, 20 (68.9%) were multidrug resistant. All MDR isolates were ESBL screening positive; however, only 4 (13.8%) were confirmed as ESBL producers using the combined disk method (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\u003eBiofilm production\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\u003eOf the 29 UPEC isolates, 19 (65.5%) were identified as biofilm producers by Congo red agar method. (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\u003e\u003cb\u003ePrevalence of\u003c/b\u003e \u003cb\u003ethe pgaABCD\u003c/b\u003e \u003cb\u003egene\u003c/b\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 urine\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\u0026nbsp;\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\u003eThe \u003cem\u003epgaD\u003c/em\u003e gene was the most prevalent, detected in 26 (89.6%) isolates, followed by \u003cem\u003epgaB\u003c/em\u003e in 24 (82.7%), \u003cem\u003epgaA\u003c/em\u003e in 20 (68.9%), and \u003cem\u003epgaC\u003c/em\u003e in 17 (58.6%) isolates (Table. 4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable .5 Association between biofilm production and presence of\u003c/b\u003e \u003cem\u003epga\u003c/em\u003e \u003cb\u003egenes among urine isolates.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiofilm positive (n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiofilm negative (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTest used\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\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\u003epgaA present\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13 (68.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (70.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaA absent\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6 (31.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (30.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaB present\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15 (78.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (90.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaB absent\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4 (21.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (10.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaC present\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11 (57.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (60.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaC absent\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8 (42.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaD present\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16 (84.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epgaD absent\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3 (15.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\"\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData are presented as number (%). Statistical analysis was performed using Fisher\u0026rsquo;s exact test due to small sample size. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant(Table.5).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUrinary tract infection caused by \u003cem\u003eEscherichia coli\u003c/em\u003e remains a major clinical concern worldwide, particularly due to the increasing prevalence of multidrug resistance, biofilm formation, and extended-spectrum β-lactamase (ESBL) production. In the present study, a substantial proportion of \u003cem\u003eE. coli\u003c/em\u003e isolates demonstrated biofilm-forming ability, and the majority harbored one or more genes of the \u003cem\u003epgaABCD\u003c/em\u003e operon, highlighting the potential role of these virulence determinants in UTI pathogenesis.\u003c/p\u003e \u003cp\u003eIn our study, biofilm production was detected in a considerable number of isolates, which is consistent with previous reports indicating that uropathogenic \u003cem\u003eE. coli\u003c/em\u003e (UPEC) frequently form biofilms to persist within the urinary tract [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Biofilm formation enhances bacterial survival by protecting cells from host immune responses and antimicrobial agents, contributing to recurrent and chronic UTIs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similar biofilm prevalence among UPEC isolates has been reported in studies from Nepal, India, and other South Asian regions, emphasizing the regional burden of biofilm-associated infections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003epgaABCD\u003c/em\u003e locus, which encodes enzymes responsible for the synthesis and export of poly-β-1,6-N-acetyl-D-glucosamine (PGA), was widely distributed among our isolates. Notably, most biofilm-positive isolates carried multiple \u003cem\u003epga\u003c/em\u003e genes, supporting earlier findings that the \u003cem\u003epgaABCD\u003c/em\u003e operon plays a crucial role in biofilm development and surface adhesion in \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Cerca et al. demonstrated that disruption of \u003cem\u003epgaC\u003c/em\u003e or \u003cem\u003epgaD\u003c/em\u003e significantly reduces biofilm formation, underscoring their functional importance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our observation of partial gene absence in some isolates may explain variations in biofilm strength, as previously reported by Wang et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, a small number of isolates in our study carried \u003cem\u003epgaABCD\u003c/em\u003e genes but were phenotypically non-biofilm producers. This finding aligns with earlier reports suggesting that biofilm formation is multifactorial and influenced not only by gene presence but also by regulatory mechanisms, environmental conditions, and quorum-sensing pathways [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, molecular detection alone may not always predict phenotypic biofilm expression.\u003c/p\u003e \u003cp\u003eESBL production was detected in a significant proportion of isolates, and many ESBL-producing strains were also biofilm formers. This association has been widely documented and is clinically relevant, as biofilm-embedded ESBL-producing bacteria exhibit markedly reduced antibiotic susceptibility [3,33]. Studies have shown that ESBL-positive UPEC isolates are more likely to cause treatment failure and recurrent infections, particularly in catheterized and hospitalized patients [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our findings further support this association, emphasizing the need for routine ESBL screening in UTI management.\u003c/p\u003e \u003cp\u003eThe antimicrobial susceptibility patterns observed in this study revealed high resistance to commonly prescribed antibiotics, which is consistent with regional surveillance reports [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Biofilm formation likely contributed to this resistance profile, as biofilms impede antibiotic penetration and promote horizontal gene transfer, including resistance genes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These findings reinforce the importance of incorporating biofilm and ESBL detection into routine microbiological diagnostics.\u003c/p\u003e \u003cp\u003eThis study has certain limitations. The sample size was relatively small, and gene expression levels were not evaluated. Additionally, biofilm quantification was limited to phenotypic assays without molecular confirmation of regulatory pathways. Despite these limitations, the study provides valuable insight into the coexistence of virulence and resistance determinants among uropathogenic \u003cem\u003eE. coli\u003c/em\u003e isolates in a clinical setting.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that biofilm formation is common among urinary \u003cem\u003eEscherichia coli\u003c/em\u003e isolates and that \u003cem\u003epgaABCD\u003c/em\u003e genes are widely distributed in both biofilm-producing and non-producing strains. However, no statistically significant association was observed between biofilm formation and the presence of individual \u003cem\u003epga\u003c/em\u003e genes. Similarly, although ESBL production was frequently detected among the isolates, it was not significantly associated with biofilm formation.\u003c/p\u003e \u003cp\u003eThese findings suggest that the presence of \u003cem\u003epgaABCD\u003c/em\u003e genes alone is insufficient to predict biofilm phenotype in clinical urinary isolates and that biofilm development is likely influenced by additional genetic, regulatory, and environmental factors. The coexistence of biofilm formation and ESBL production highlights the complexity of antimicrobial resistance and persistence in uropathogenic \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFurther studies with larger sample sizes, multicenter designs, and inclusion of gene expression analysis and additional biofilm-associated virulence determinants are warranted to better elucidate the mechanisms underlying biofilm formation and antimicrobial resistance in urinary tract infections.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cp\u003eMDR Multidrug resistance\u003c/p\u003e \u003cp\u003eESBLs Extended-spectrum beta-lactamase\u003c/p\u003e \u003cp\u003eMA MaxConkey Agar\u003c/p\u003e \u003cp\u003eMHA Mueller Hinton Agar\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \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 \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of Interest:\u003c/strong\u003e \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 \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo fund for this research work\u003c/p\u003e \u003cp\u003eData availability\u003c/p\u003e \u003cp\u003eAll the data generated or analysed during this study are included in the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSoto SM (2014) Importance of biofilms in urinary tract infections. Future Microbiol 9(9):1005\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms.Clin Microbiol Rev. 2002;15(2):167\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaterson DL, Bonomo RA. Extended\u0026ndash;spectrum β\u0026ndash;lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657\u0026ndash;686.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCepas V, L\u0026oacute;pez Y, Mu\u0026ntilde;oz S, et al. Relationship between biofilm formation and antibiotic resistance in Gram\u0026ndash;negative bacteria. Front Microbiol. 2019;10:282.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026ouml;mling U, Balsalobre C. Biofilm infections and their resilience to therapy. J Intern Med. 2012;272(6):541\u0026ndash;561.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoh Y, Wang X, Hinnebusch BJ, Preston JF, Romeo T. Poly\u0026ndash;N\u0026ndash;acetylglucosamine is a component of the extracellular matrix in E. coli biofilms. J Bacteriol. 2008;190(13):3829\u0026ndash;3835.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLwanga SK, Lemeshow S. \u003cem\u003eSample size determination in health studies: a practical manual\u003c/em\u003e. World Health Organization; 1991.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheesbrough M. \u003cem\u003eDistrict laboratory practice in tropical countries\u003c/em\u003e. 2nd ed. Cambridge University Press; 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. \u003cem\u003eAm J Clin Pathol\u003c/em\u003e. 1966;45:493\u0026ndash;496.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClinical and Laboratory Standards Institute (CLSI). \u003cem\u003ePerformance standards for antimicrobial susceptibility testing\u003c/em\u003e. CLSI supplement M100. CLSI; 2020\u0026ndash;2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. \u003cem\u003eRev Infect Dis\u003c/em\u003e. 1988;10:867\u0026ndash;878.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase-negative staphylococci. \u003cem\u003eJ Clin Pathol\u003c/em\u003e. 1989;42:872\u0026ndash;874. \u003cem\u003e(Widely accepted for Congo red agar biofilm detection)\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDashti AA, Jadaon MM, Abdulsamad AM, Dashti HM. Heat treatment of bacteria: a simple method of DNA extraction for molecular techniques. \u003cem\u003eKuwait Med J\u003c/em\u003e. 2009;41:117\u0026ndash;122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Preston JF, Romeo T. The \u003cem\u003epgaABCD\u003c/em\u003e locus of \u003cem\u003eEscherichia coli\u003c/em\u003e promotes the synthesis of a polysaccharide adhesin required for biofilm formation.\u003cem\u003eJ Bacteriol\u003c/em\u003e. 2004;186:2724\u0026ndash;2734.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoh Y, Rice JD, Goller C, et al. Roles of \u003cem\u003epgaABCD\u003c/em\u003e genes in synthesis, modification, and export of the \u003cem\u003eE. coli\u003c/em\u003e biofilm adhesin PGA. \u003cem\u003eJ Bacteriol\u003c/em\u003e. 2008;190:3670\u0026ndash;3680.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerca N, Jefferson KK, Oliveira R, Pier GB, Azeredo J. Comparative analysis of \u003cem\u003epga\u003c/em\u003e genes involved in biofilm formation. \u003cem\u003eMicrobiology\u003c/em\u003e. 2006;152:165\u0026ndash;175.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSambrook J, Russell DW. \u003cem\u003eMolecular cloning: a laboratory manual\u003c/em\u003e. 3rd ed. Cold Spring Harbor Laboratory Press; 2001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 30th Informational Supplement.CLSI document M100. Wayne, PA: CLSI; 2020 (or 2023/2024 as per your citation year).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically;Approved Standard.CLSI document M07. 11th ed. Wayne, PA: CLSI; 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. \u003cem\u003eRev Infect Dis.\u003c/em\u003e 1988;10(4):867\u0026ndash;878.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase-negative staphylococci. \u003cem\u003eJ Clin Pathol.\u003c/em\u003e 1989;42(8):872\u0026ndash;874. \u003cem\u003e(Adapt as appropriate for Congo red biofilm method)\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams RR, Smyth CJ, Griffiths JK, Gilman RH, soto SM. Evaluation of methods for extraction of bacterial DNA from feces. \u003cem\u003eJ Clin Microbiol.\u003c/em\u003e 2010;48(8):2687\u0026ndash;2692. \u003cem\u003e(If not exact, replace with the actual boiling method reference you used)\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001. \u003cem\u003e(General PCR reference \u0026mdash; replace if you used a specific primer source)\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen MR, Sambrook J. Gel Electrophoresis and Southern Blotting: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 2012. \u003cem\u003e(Use if this was your source for gel electrophoresis)\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Annapurna Neurological Institute and Allied Sciences","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Escherichia coli, Biofilm, ESBL, pgaABCD, PCR, Antibiotic resistance, MDR, UPEC","lastPublishedDoi":"10.21203/rs.3.rs-8512488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8512488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e is the most common cause of urinary tract infections (UTIs) and is increasingly associated with biofilm formation and extended-spectrum β-lactamase (ESBL) production, which complicate treatment. The \u003cem\u003epgaABCD\u003c/em\u003e operon is involved in the synthesis of poly-β-1,6-\u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosamine, an important component of the biofilm matrix. However, the relationship between biofilm formation, \u003cem\u003epga\u003c/em\u003e gene carriage, and ESBL production in urinary \u003cem\u003eE. coli\u003c/em\u003e isolates remains unclear.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 29 \u003cem\u003eE. coli\u003c/em\u003e isolates obtained from urine samples were analyzed. Biofilm formation was assessed using a phenotypic method, and ESBL production was determined by standard antimicrobial susceptibility testing. The presence of \u003cem\u003epgaA\u003c/em\u003e, \u003cem\u003epgaB\u003c/em\u003e, \u003cem\u003epgaC\u003c/em\u003e, and \u003cem\u003epgaD\u003c/em\u003e genes was detected by polymerase chain reaction (PCR). Associations between biofilm formation, \u003cem\u003epga\u003c/em\u003e genes, and ESBL production were evaluated using chi-square or Fisher\u0026rsquo;s exact test, as appropriate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBiofilm formation was observed in 19 (65.5%) isolates. The \u003cem\u003epgaA\u003c/em\u003e, \u003cem\u003epgaB\u003c/em\u003e, \u003cem\u003epgaC\u003c/em\u003e, and \u003cem\u003epgaD\u003c/em\u003e genes were detected in 20 (69.0%), 24 (82.8%), 17 (58.6%), and 26 (89.7%) isolates, respectively. No statistically significant association was found between biofilm formation and the presence of individual \u003cem\u003epga\u003c/em\u003e genes (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). ESBL production was common among the isolates but was not significantly associated with biofilm formation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTrial Registration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNot applicable\u003c/p\u003e","manuscriptTitle":"Biofilm Formation and Detection of Pgaabcd Gene in Uropathogenic e.coli Isolated From Clinical Urine Samples in Tersary Hospital","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 11:19:02","doi":"10.21203/rs.3.rs-8512488/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":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61058753,"name":"Bacteriology"}],"tags":[],"updatedAt":"2026-01-13T11:19:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 11:19:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8512488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8512488","identity":"rs-8512488","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}