Mrk D and fim H Biofilm Genes Detection and Antibiotic Susceptibility: A Study on Uropathogenic Biofilm Producing Klebsiella pneumoniae in Sana'a City, Yemen

Mrk D and fim H Biofilm Genes Detection and Antibiotic Susceptibility: A Study on Uropathogenic Biofilm Producing Klebsiella pneumoniae in Sana'a City, Yemen | 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 Article Mrk D and fim H Biofilm Genes Detection and Antibiotic Susceptibility: A Study on Uropathogenic Biofilm Producing Klebsiella pneumoniae in Sana'a City, Yemen Safa’a Mohammed M Darwiesh, Dekra A El-Aghbary, Khaled Abdulkarim Al-Moyed, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9321938/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Urinary tract infections (UTIs) caused by antibiotic-resistant strains of Klebsiella pneumoniae ( K. pneumoniae ) are one of the most important causes of morbidity and health care spending affecting people in Yemen, including young women, children, and the elderly. Biofilm formation has become a major factor among those uropathogenic strains. Objectives This study aimed to investigate the biofilm-forming ability of K. pneumoniae isolated from patients with UTIs, to determine the antibiotic susceptibility of those biofilm producers, and to assess the association between fimbrial virulence genes ( fimH and mrkD ) and biofilm strength. Methods The standard procedures for identifying and isolating bacteria from UTIs were used. The ability of the isolated K. pneumoniae to form biofilms was examined using tissue culture plates. The Kirby-Bauer disc diffusion method was used to determine the antibiogram susceptibility pattern of uropathogens. The fimbriae virulence genes ( fimH and mrkD ) for the biofilm-producing K. pneumoniae were found using conventional PCR. Results From UTI patients, 79(32.8%) of K. pneumoniae were isolated. 39 UTI patients (49.4%) had K. pneumoniae that did not produce a biofilm, although 40 (50.6%) of the bacteria are capable of doing so. Among those biofilm-forming strains, 19 (47.7%) were able to produce strong biofilms, while 21 (52.5%) produced moderate biofilms. PDR (5%), on the other hand, had the lowest percentage of antibiotic resistance, whereas XDR (47.5%) and MDR (32.5%) had the greatest percentages. The mrkD gene was detected in 40 (100%) of the biofilm-forming K. pneumoniae , and fimH had a significant association with strong biofilm (p = 0.031). Conclusion The presence of fimbriae virulence genes ( mrkD and fimH ) was significantly correlated with multi-drug resistance (MRD) biofilm-producing K. pneumoniae isolated from UTI patients. Health sciences/Diseases Health sciences/Medical research Biological sciences/Microbiology Urinary tract infections K. pneumoniae biofilm formation fimbriae virulence genes (mrkD and fimH) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction One of the most prevalent clinical Gram-negative bacteria, K. pneumoniae , can cause serious infections, including wound infections, pneumonia, and urinary tract infections [ 1 , 2 , 3 ]. Due to the high death rate from K. pneumoniae infections, particularly in children and patients with impaired immune systems, a great deal of research has focused on the development of antibiotic resistance [ 4 ]. Indeed, it has been documented that K. pneumoniae is a common cause of infections in people who have indwelling urinary catheters [ 5 , 6 , 7 ]. Multiple processes allow this bacterium to become resistant to antibiotics, leading to the development of multidrug-resistant (MDR) strains that pose a severe threat to patient safety and medical center architecture. The production of biofilms is a significant element in the initiation and propagation of infection [ 8 ]. The ability of bacteria to live in unfavorable environments is known as biofilm development [ 9 ]. This biofilm shields the bacteria from antibiotics and the human immune response, making the bacteria within highly resistant to antibiotic treatments [ 10 ]. Multidrug-resistant organisms (MDRO) are able to spread more easily in biofilms because they offer an environment that is resistant to antibiotic penetration and allows for the horizontal transfer of virulence genes [ 11 ]. Biofilm-forming bacteria are responsible for about 80% of urinary tract infections (UTIs), which is a severe medical concern. In addition, biofilm contributes significantly to recurring UTIs and catheter-associated UTIs [ 12 ]. Since biofilm formation by pathogenic bacteria may be a virulence factor in individual attacks, it is currently of interest. This emphasizes the need for ongoing assessment of pathogen development [ 13 ]. The virulence elements primarily responsible for K. pneumoniae's capacity to form a biofilm include the type 1 or type 3 fimbriae, the capsule, and the lipopolysaccharides. It has been shown that type 3 fimbriae are the main appendages that mediate biofilm formation on abiotic surfaces and adhesion to endothelium and bladder epithelial cell lines [ 14 ]. Specifically, the type 3 fimbrial protein mrkA promotes K. pneumoniae growth on abiotic surfaces, while the type 3 fimbrial adhesin mrkD is necessary for development on surfaces covered with human extracellular matrix [ 15 ]. After E. coli, K. pneumoniae is the second pathogen that causes UTIs in Yemen [ 6 , 7 , 16 , 17 ]. Although UTIs pose a significant threat to Yemeni society, little is known about the expression of virulence genes in biofilm-forming K. pneumoniae UTIs and how these genes relate to antibiotic resistance. To ascertain the antibiotic susceptibility of K. pneumoniae isolates from UTI patients in Sana'a City, our study aimed to investigate the biofilm-forming capacity of K. pneumoniae isolated from patients with UTIs, to determine the antimicrobial susceptibility patterns of biofilm-producing isolates, and to detect the prevalence of common fimbrial virulence genes ( fimH and mrkD ), as well as to evaluate the association between the presence of these genes and the strength of biofilm formation. Subjects and Methods This descriptive cross-sectional study was conducted at Al-48 Modern General Hospital and the University of Science and Technology Hospital in Sana'a City. The laboratory works were performed at the Department of Microbiology and Molecular Biology at the National Centre of Public Health Laboratories (NCPHL), Sana’a city, Yemen, during a period of one year, starting in February 2024 and ending in February 2025. About 10 to 20 ml of midstream urine was collected in a sterile screw-capped wide-mouth container from all participating UTI patients. Isolation and identification of K. pneumoniae The urine sample with leukocyte counts > 10 WBCs/HPF was cultured using a standard calibrated loop to contain 0.01 ml of urine on cysteine-lactose-electrolyte-deficient (CLED) agar media. Inoculated plates were incubated at 37°C aerobically for 18–24 hours. After incubation, the plates were examined, and growth characteristics were noted. Samples were usually inoculated before cell content analysis to reduce the risk of sample contamination. Colonies were counted on the inoculated medium and multiplied by the loop volume to determine the bacterial count (significant growth > 10 5 CFU/mL). Bacteria were identified using the automated Vitek2 system, conducted according to the manufacturer's instructions (BioMérieux, France). Biofilm detection The isolated bacteria were tested for biofilm production on a tissue culture plate. The classification of bacterial adherence by the tissue culture plate method into three categories: average optical densities (OD) value 0.240 (strong) biofilm formation (Fig. 1 ). Determination of the antibiogram susceptibility pattern The antibiogram susceptibility pattern of uropathogens to different antibacterial agents was determined in vitro by employing a modified disk diffusion method described by Kirby-Bauer [ 18 ]. The procedure involved measuring the diameter of the zone of inhibition that results from the diffusion of the antibiotic agent into the medium surrounding the disc. The interpretation of the obtained results was based on the Clinical Laboratory Standards Institute (CLSI) guidelines [ 19 ]. Identification of K. pneumonia Genes The conventional polymerase chain reaction (PCR) was used to detect the sequences of virulence genes ( mrkD and fimH ) for the biofilm-forming K. pneumoniae using specific forward (F) and reverse (R) primers that were previously used in a study conducted by Shah et al . [ 20 ] (Table 1 ). Table 1 Primers used to detect the sequences of the mrkD and fimH gene this study Gene Primer Sequence (5–3) Expected size (bp) fim H F: 5´- ATG AAC GCC TGG TCC TTT GC -3´ 688 R:5´-GCT GAA CGC CTA TCC CCT GC -3´ mrk D F: 5´- CCA CCA ACT ATT CCC TCG AA -3´ 240 R: 5´- ATG GAA CCC ACA TCG ACA TT -3´ Statistical analysis The data was analyzed using SPSS Version 26. The qualitative variables were presented as frequencies and percentages. Associations between the positive fimbriae virulence genes and the biofilm-producing capacity of K. pneumoniae were tested using chi-square tests, where a p-value < 0.05 was considered a significant result. The relative risk with a 95% confidence interval was also determined. Results A total of 241 urine samples from UTI patients were collected in this study. The rate of K. pneumoniae was 79 (32.8%) isolates in UTI cases ( Fig. 2 ). Among 79 isolates, 40 (50.6%) of K. pneumoniae can form a biofilm, while non-biofilm-producing K. pneumonia e were found in 39 (49.4%) of UTI patients. The biofilm-producing K. pneumoniae gives a strong biofilm formation in 19 (47.7%) and moderate in 21 (52.5%) of isolates, while no cases show weak biofilm ( Fig. 3 A and B). Regarding the antibiogram susceptibility pattern among the isolated biofilm producing K. pneumoniae, the highest percentage of antibiotic resistance was for XDR (47.5%), followed by MDR (32.5%), while the lowest percentage of antibiotic resistance was for PDR (5%) (Fig. 4). However, the sensitivity to antibiotics among the biofilm-producing K . pneumoniae was only 6 (15%). In this study, all 40 isolates (100%) of biofilm-producing K. pneumoniae appeared to express the mrk D gene; 19 (47.5%) and 21 (52.5%) were among the strong and moderate biofilm-producing K. pneumoniae , respectively. While the fim H gene was found in 35of 40 (87.5%) isolates, 19 (54.3%) and 16 (45.7%) of high and moderate biofilm-producing K. pneumonia , respectively. The statistically significant association with a significant risk of more than two times between positive fim H gene and strong biofilm-formation (RR: 2.19; CI: 1.5–3.1; p = 0031) (Table 2 ). MDR: multidrug-resistant, XDR: extensively drug-resistant, PDR: pan-drug-resistant Figure 4: The antibiogram susceptibility pattern among the isolated biofilm-producing K. pneumoniae Table 2 The prevalence of fimH and mrkD genes among the isolated biofilm-producing K. pneumoniae and their associations with biofilm strength. Fimbriae virulence genes Biofilm producer PR (CI 95%) p -value Moderate Strong No. % No. % Fim H (n = 35) 16 45.7 19 54.3 2.19(1.5–3.1) 0.031 Mrk D (n = 40) 21 52.5 19 47.5 UD UD CI : Confidence interval; PR Prevalence ratio; UD: Undefined Discussion K. pneumonia has the capability of biofilm formation, which protects it from host immune system responses and antibiotics [ 20 ] and promotes its persistence on the epithelial tissues and external surfaces of indwelling devices. This study showed that 50.6% of K. pneumoniae isolates were capable of forming biofilm, which gives strong biofilm production in 47.7% and moderate biofilm production in 52.5% of isolates. Several global studies recorded higher than our findings. Makhrmash et al. reported that all isolated K. pneumonia 100% were biofilm producers, with 57% of isolates producing strong biofilm [ 21 ]. Hamam et al . showed that the majority (81%) of the isolates in their studies had the capacity to form biofilm [ 22 ]. Another related study conducted in Iran showed that 79.6% of K. pneumoniae isolated from urine specimens were biofilm producers, where 23% of them could produce strong biofilm [ 23 ]. Research from Nigeria revealed that 68.18% of isolates were biofilm producers [ 24 ]. The formation of biofilm protects K. pneumoniae against the host immune responses, the action of antibiotics, and enhances its persistence [ 25 ]. The discrepancy in the biofilm formation demonstration between our results and the previous literature may be due to different bacterial strains, resistance profiles, genes, variations in the sites of isolates, and the culture conditions and detection method [ 21 , 26 ]. Our findings showed that XDR was found in 22 (55%), followed by MDR, which represented 15 (37.5%), and PDR, which counted 2 (5%) of all biofilm-producing K. pneumoniae . However, the sensitivity to antibiotics among the biofilm-producing K . pneumoniae was only 6 (15%). This result is similar to reports from Pakistan, which showed all isolates of K. pneumoniae were highly resistant to the majority of antibiotics [ 24 ]. Two studies conducted elsewhere in which antibiotic resistance was significantly high among biofilm-producing K. pneumoniae [ 27 , 28 ]. In general, this may be because bacterial biofilms are often associated with long-term persistence of organisms in various environments, decreased bacterial growth rate in a biofilm, expression of resistance genes, and restricted penetration of antibiotics into the biofilm. Furthermore, the proximity of cells within a biofilm can facilitate plasmid exchange and hence enhance the spread of antimicrobial resistance [ 29 ]. Regarding the fimbriae virulence genes, this data showed a significant association between the positive fim H gene and the strong biofilm-formation (p = 0.031). Three studies were in agreement with our finding [ 21 , 22 , 30 , 31 ]. The FimH has a significant role in pathogenicity and resistance among strains of K. pneumoniae due to enhancing biofilm formation [ 32 ]. Meanwhile, the mrkD gene was discovered to be present in all biofilm-producing K. pneumoniae isolated from UTIs 100% in this investigation, which determined the virulence genes ( mrkD and fimH ) among biofilm-producing K. pneumoniae . This finding aligned with two studies conducted in Iraq that demonstrated that all biofilm-forming K. pneumoniae isolates from UTIs 100% tested positive for the mrkD genes [ 14 , 33 ]. Numerous investigations have demonstrated that type 3 fimbriae ( mrkD gene) appear to play important roles in K. pneumoniae biofilm formation, which is consistent with our results [ 15 , 34 ]. The observation that mutant K. pneumoniae strains lacking this gene cluster produced noticeably less biofilm in the presence of lung surfactant, with cholesterol and phosphatidylcholine identified as surfactant components promoting type III fimbriae expression [ 35 ]. Among the limitations of the present study include; the study design, a cross-sectional study in which data was collected at one point of time and the causative are not detected and for financial issues such as the tested panel of antibiotic was not including all antibiotics used in treatment of urinary tract isolates and detection of only mrkD and fimH as two biofilm-associated genes without involving other complex genetic determinants may affect the findings. Conclusion The presence of fimbriae virulence genes (mrkD and fimH) was significantly correlated with multidrug-resistant biofilm-producing K. pneumoniae isolated from UTI patients. Declarations Ethics approval and consent form Ethical approval: The ethical approval of this study was obtained from the Medical Ethical Committee of the Faculty of Medicine and Health Science, Sana'a University; No. (287). Ethical approval was obtained in accordance with the standards outlined in the Helsinki Declaration. Written informed consent forms were obtained from all participated UTIs patients before any research procedure was performed. Competing interests The authors declare that there is no conflict of interest. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution HAA supervision and revision, and editing of the manuscript. SMMD collected data and samples from all patients and performed the laboratory procedures for the samples. DAE and KAA wrote of introduction and methodology. HAA and ANMG analyzed the data, prepared tables and figures, and discussed the results. All authors arranged the referencing style, read, and made the final revision of the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors express their gratitude to Yemen and the Sana'a University Faculty of Dentistry for their kind assistance. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviewers invited by journal 22 Apr, 2026 Editor invited by journal 14 Apr, 2026 Editor assigned by journal 08 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 04 Apr, 2026 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9321938","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":618221975,"identity":"850e3c84-de06-49ed-8165-52ff13038cb6","order_by":0,"name":"Safa’a Mohammed M Darwiesh","email":"","orcid":"","institution":"Sana'a University","correspondingAuthor":false,"prefix":"","firstName":"Safa’a","middleName":"Mohammed M","lastName":"Darwiesh","suffix":""},{"id":618221976,"identity":"8163ccbb-faf0-4025-beaf-54029fb88705","order_by":1,"name":"Dekra A El-Aghbary","email":"","orcid":"","institution":"Sana'a University","correspondingAuthor":false,"prefix":"","firstName":"Dekra","middleName":"A","lastName":"El-Aghbary","suffix":""},{"id":618221977,"identity":"4db8d109-e698-4b52-9ad0-b62dc0a61abd","order_by":2,"name":"Khaled Abdulkarim Al-Moyed","email":"","orcid":"","institution":"Sana'a University","correspondingAuthor":false,"prefix":"","firstName":"Khaled","middleName":"Abdulkarim","lastName":"Al-Moyed","suffix":""},{"id":618221980,"identity":"e5f57a90-3169-4216-8e79-8799398a30dd","order_by":3,"name":"Hassan Abdulwahab Al-Shamahy","email":"","orcid":"","institution":"Sana'a University","correspondingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"Abdulwahab","lastName":"Al-Shamahy","suffix":""},{"id":618221981,"identity":"306926da-1277-4794-879c-8032403052dc","order_by":4,"name":"Ali N. M. Gubran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie3RMUvDQBTA8XcGXpdru144TL7CC4FCwQ+TLE5ugpPgSeFc1L1gyVeom+OVQLKIc8eKa4W6FKUqJlXEJdeOBe8/Hcf7cRwPwOXaydi5AQgAWwrE7yW3Es9UJAbk5ofgRoLJmoBItiRdxSnnpxR0/KfH6csdpFmm2OxZNxNhalJQjPIw7g/vIR0X4EUjC4E1wfdUy6Qn27oiCFgfGgtNd5HzTzrTfrmUHxXJNLRWNkL1K21NCQrek6wiqgD0bCTKOU1G1xRpfnTcv9IiHhfpwL95aCZBeRnN5ksKw4vydvqmD/azQT5ZzE8s3/cA9v5soV4NU5b579jrxhGXy+X6130Biy5NtjXDuKwAAAAASUVORK5CYII=","orcid":"","institution":"University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"N. M.","lastName":"Gubran","suffix":""}],"badges":[],"createdAt":"2026-04-04 16:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9321938/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9321938/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106348758,"identity":"7bbc2ebe-751a-4e50-9ecb-35dbd66152e3","added_by":"auto","created_at":"2026-04-07 16:50:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52640,"visible":true,"origin":"","legend":"\u003cp\u003eTissue culture plate\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9321938/v1/2d49c09eec1723243aeb0b08.jpg"},{"id":106348759,"identity":"43c3d2f0-028c-438d-8a49-c1c68fbcd1f2","added_by":"auto","created_at":"2026-04-07 16:50:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22897,"visible":true,"origin":"","legend":"\u003cp\u003eThe rate of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from UTI cases (n=241).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9321938/v1/62e793a62dc572a015b95a51.png"},{"id":106404572,"identity":"be50175d-ad87-4fc0-961a-8ebe2189f8ad","added_by":"auto","created_at":"2026-04-08 09:16:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41077,"visible":true,"origin":"","legend":"\u003cp\u003eA) The rate of biofilm-producing K. pneumoniae, B) The strength of biofilm among biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9321938/v1/89c75247428b60c9d58f40eb.png"},{"id":106348760,"identity":"e4394170-96a9-414f-ad5c-deb2d9541413","added_by":"auto","created_at":"2026-04-07 16:50:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25438,"visible":true,"origin":"","legend":"\u003cp\u003eThe antibiogram susceptibility pattern among the isolated biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9321938/v1/8974ca3eaf80856f078a7bea.png"},{"id":106415126,"identity":"695ded9d-97c0-4fff-bbfe-0661f888ef15","added_by":"auto","created_at":"2026-04-08 10:33:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4331785,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9321938/v1/5bfebebb-41d4-491e-a663-9a224b967aee.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mrk D and fim H Biofilm Genes Detection and Antibiotic Susceptibility: A Study on Uropathogenic Biofilm Producing Klebsiella pneumoniae in Sana'a City, Yemen","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne of the most prevalent clinical Gram-negative bacteria, \u003cem\u003eK. pneumoniae\u003c/em\u003e, can cause serious infections, including wound infections, pneumonia, and urinary tract infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Due to the high death rate from \u003cem\u003eK. pneumoniae\u003c/em\u003e infections, particularly in children and patients with impaired immune systems, a great deal of research has focused on the development of antibiotic resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Indeed, it has been documented that \u003cem\u003eK. pneumoniae\u003c/em\u003e is a common cause of infections in people who have indwelling urinary catheters [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Multiple processes allow this bacterium to become resistant to antibiotics, leading to the development of multidrug-resistant (MDR) strains that pose a severe threat to patient safety and medical center architecture. The production of biofilms is a significant element in the initiation and propagation of infection [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ability of bacteria to live in unfavorable environments is known as biofilm development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This biofilm shields the bacteria from antibiotics and the human immune response, making the bacteria within highly resistant to antibiotic treatments [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Multidrug-resistant organisms (MDRO) are able to spread more easily in biofilms because they offer an environment that is resistant to antibiotic penetration and allows for the horizontal transfer of virulence genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Biofilm-forming bacteria are responsible for about 80% of urinary tract infections (UTIs), which is a severe medical concern. In addition, biofilm contributes significantly to recurring UTIs and catheter-associated UTIs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Since biofilm formation by pathogenic bacteria may be a virulence factor in individual attacks, it is currently of interest. This emphasizes the need for ongoing assessment of pathogen development [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe virulence elements primarily responsible for \u003cem\u003eK. pneumoniae's\u003c/em\u003e capacity to form a biofilm include the type 1 or type 3 fimbriae, the capsule, and the lipopolysaccharides. It has been shown that type 3 fimbriae are the main appendages that mediate biofilm formation on abiotic surfaces and adhesion to endothelium and bladder epithelial cell lines [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Specifically, the type 3 fimbrial protein \u003cem\u003emrkA\u003c/em\u003e promotes \u003cem\u003eK. pneumoniae\u003c/em\u003e growth on abiotic surfaces, while the type 3 fimbrial adhesin \u003cem\u003emrkD\u003c/em\u003e is necessary for development on surfaces covered with human extracellular matrix [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter \u003cem\u003eE. coli, K. pneumoniae\u003c/em\u003e is the second pathogen that causes UTIs in Yemen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although UTIs pose a significant threat to Yemeni society, little is known about the expression of virulence genes in biofilm-forming \u003cem\u003eK. pneumoniae\u003c/em\u003e UTIs and how these genes relate to antibiotic resistance. To ascertain the antibiotic susceptibility of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from UTI patients in Sana'a City, our study aimed to investigate the biofilm-forming capacity of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from patients with UTIs, to determine the antimicrobial susceptibility patterns of biofilm-producing isolates, and to detect the prevalence of common fimbrial virulence genes (\u003cem\u003efimH\u003c/em\u003e and \u003cem\u003emrkD\u003c/em\u003e), as well as to evaluate the association between the presence of these genes and the strength of biofilm formation.\u003c/p\u003e"},{"header":"Subjects and Methods","content":"\u003cp\u003eThis descriptive cross-sectional study was conducted at Al-48 Modern General Hospital and the University of Science and Technology Hospital in Sana'a City. The laboratory works were performed at the Department of Microbiology and Molecular Biology at the National Centre of Public Health Laboratories (NCPHL), Sana\u0026rsquo;a city, Yemen, during a period of one year, starting in February 2024 and ending in February 2025. About 10 to 20 ml of midstream urine was collected in a sterile screw-capped wide-mouth container from all participating UTI patients.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation and identification of\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe urine sample with leukocyte counts\u0026thinsp;\u0026gt;\u0026thinsp;10 WBCs/HPF was cultured using a standard calibrated loop to contain 0.01 ml of urine on cysteine-lactose-electrolyte-deficient (CLED) agar media. Inoculated plates were incubated at 37\u0026deg;C aerobically for 18\u0026ndash;24 hours. After incubation, the plates were examined, and growth characteristics were noted. Samples were usually inoculated before cell content analysis to reduce the risk of sample contamination. Colonies were counted on the inoculated medium and multiplied by the loop volume to determine the bacterial count (significant growth\u0026thinsp;\u0026gt;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e CFU/mL). Bacteria were identified using the automated Vitek2 system, conducted according to the manufacturer's instructions (BioM\u0026eacute;rieux, France).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm detection\u003c/h2\u003e \u003cp\u003eThe isolated bacteria were tested for biofilm production on a tissue culture plate. The classification of bacterial adherence by the tissue culture plate method into three categories: average optical densities (OD) value\u0026thinsp;\u0026lt;\u0026thinsp;0.120 (non-adherent), 0.120\u0026ndash;0.240 (Moderate), and \u0026gt;\u0026thinsp;0.240 (strong) biofilm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of the antibiogram susceptibility pattern\u003c/h3\u003e\n\u003cp\u003eThe antibiogram susceptibility pattern of uropathogens to different antibacterial agents was determined \u003cem\u003ein vitro\u003c/em\u003e by employing a modified disk diffusion method described by Kirby-Bauer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The procedure involved measuring the diameter of the zone of inhibition that results from the diffusion of the antibiotic agent into the medium surrounding the disc. The interpretation of the obtained results was based on the Clinical Laboratory Standards Institute (CLSI) guidelines [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eK. pneumonia\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe conventional polymerase chain reaction (PCR) was used to detect the sequences of virulence genes (\u003cem\u003emrkD\u003c/em\u003e and \u003cem\u003efimH\u003c/em\u003e) for the biofilm-forming K. pneumoniae using specific forward (F) and reverse (R) primers that were previously used in a study conducted by Shah \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used to detect the sequences of the mrkD and \u003cem\u003efimH\u003c/em\u003e gene this study\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer Sequence (5\u0026ndash;3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExpected size (bp)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003efim\u003c/em\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026acute;- ATG AAC GCC TGG TCC TTT GC -3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e688\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR:5\u0026acute;-GCT GAA CGC CTA TCC CCT GC -3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003emrk\u003c/em\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026acute;- CCA CCA ACT ATT CCC TCG AA -3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026acute;- ATG GAA CCC ACA TCG ACA TT -3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data was analyzed using SPSS Version 26. The qualitative variables were presented as frequencies and percentages. Associations between the positive fimbriae virulence genes and the biofilm-producing capacity of \u003cem\u003eK. pneumoniae\u003c/em\u003e were tested using chi-square tests, where a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a significant result. The relative risk with a 95% confidence interval was also determined.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 241 urine samples from UTI patients were collected in this study. The rate of \u003cem\u003eK. pneumoniae\u003c/em\u003e was 79 (32.8%) isolates in UTI cases \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Among 79 isolates, 40 (50.6%) of \u003cem\u003eK. pneumoniae\u003c/em\u003e can form a biofilm, while non-biofilm-producing \u003cem\u003eK. pneumonia\u003c/em\u003ee were found in 39 (49.4%) of UTI patients. The biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e gives a strong biofilm formation in 19 (47.7%) and moderate in 21 (52.5%) of isolates, while no cases show weak biofilm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003eand B).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRegarding the antibiogram susceptibility pattern among the isolated biofilm producing K. pneumoniae, the highest percentage of antibiotic resistance was for XDR (47.5%), followed by MDR (32.5%), while the lowest percentage of antibiotic resistance was for PDR (5%) \u003cb\u003e(Fig.\u0026nbsp;4).\u003c/b\u003e However, the sensitivity to antibiotics among the biofilm-producing \u003cem\u003eK\u003c/em\u003e. \u003cem\u003epneumoniae\u003c/em\u003e was only 6 (15%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, all 40 isolates (100%) of biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e appeared to express the \u003cem\u003emrk\u003c/em\u003eD gene; 19 (47.5%) and 21 (52.5%) were among the strong and moderate biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e, respectively. While the \u003cem\u003efim\u003c/em\u003eH gene was found in 35of 40 (87.5%) isolates, 19 (54.3%) and 16 (45.7%) of high and moderate biofilm-producing \u003cem\u003eK. pneumonia\u003c/em\u003e, respectively. The statistically significant association with a significant risk of more than two times between positive \u003cem\u003efim\u003c/em\u003eH gene and strong biofilm-formation (RR: 2.19; CI: 1.5\u0026ndash;3.1; p\u0026thinsp;=\u0026thinsp;0031) (Table\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMDR: multidrug-resistant, XDR: extensively drug-resistant, PDR: pan-drug-resistant\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;4: The antibiogram susceptibility pattern among the isolated biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe prevalence of fimH and mrkD genes among the isolated biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e and their associations with biofilm strength.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFimbriae virulence genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eBiofilm producer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePR (CI 95%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eStrong\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\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\u003cem\u003eFim\u003c/em\u003e H (n\u0026thinsp;=\u0026thinsp;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.19(1.5\u0026ndash;3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMrk\u003c/em\u003e D\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eUD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eCI\u003c/em\u003e: Confidence interval; \u003cem\u003ePR\u003c/em\u003e Prevalence ratio; UD: Undefined\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eK. pneumonia\u003c/em\u003e has the capability of biofilm formation, which protects it from host immune system responses and antibiotics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and promotes its persistence on the epithelial tissues and external surfaces of indwelling devices. This study showed that 50.6% of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were capable of forming biofilm, which gives strong biofilm production in 47.7% and moderate biofilm production in 52.5% of isolates. Several global studies recorded higher than our findings. Makhrmash et al. reported that all isolated \u003cem\u003eK. pneumonia\u003c/em\u003e 100% were biofilm producers, with 57% of isolates producing strong biofilm [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Hamam \u003cem\u003eet al\u003c/em\u003e. showed that the majority (81%) of the isolates in their studies had the capacity to form biofilm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Another related study conducted in Iran showed that 79.6% of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from urine specimens were biofilm producers, where 23% of them could produce strong biofilm [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Research from Nigeria revealed that 68.18% of isolates were biofilm producers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The formation of biofilm protects \u003cem\u003eK. pneumoniae\u003c/em\u003e against the host immune responses, the action of antibiotics, and enhances its persistence [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The discrepancy in the biofilm formation demonstration between our results and the previous literature may be due to different bacterial strains, resistance profiles, genes, variations in the sites of isolates, and the culture conditions and detection method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings showed that XDR was found in 22 (55%), followed by MDR, which represented 15 (37.5%), and PDR, which counted 2 (5%) of all biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e. However, the sensitivity to antibiotics among the biofilm-producing \u003cem\u003eK\u003c/em\u003e. \u003cem\u003epneumoniae\u003c/em\u003e was only 6 (15%). This result is similar to reports from Pakistan, which showed all isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e were highly resistant to the majority of antibiotics [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Two studies conducted elsewhere in which antibiotic resistance was significantly high among biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In general, this may be because bacterial biofilms are often associated with long-term persistence of organisms in various environments, decreased bacterial growth rate in a biofilm, expression of resistance genes, and restricted penetration of antibiotics into the biofilm. Furthermore, the proximity of cells within a biofilm can facilitate plasmid exchange and hence enhance the spread of antimicrobial resistance [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the fimbriae virulence genes, this data showed a significant association between the positive \u003cem\u003efim\u003c/em\u003eH gene and the strong biofilm-formation (p\u0026thinsp;=\u0026thinsp;0.031). Three studies were in agreement with our finding [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The FimH has a significant role in pathogenicity and resistance among strains of K. \u003cem\u003epneumoniae\u003c/em\u003e due to enhancing biofilm formation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Meanwhile, the \u003cem\u003emrkD\u003c/em\u003e gene was discovered to be present in all biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from UTIs 100% in this investigation, which determined the virulence genes (\u003cem\u003emrkD\u003c/em\u003e and \u003cem\u003efimH\u003c/em\u003e) among biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e. This finding aligned with two studies conducted in Iraq that demonstrated that all biofilm-forming \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from UTIs 100% tested positive for the \u003cem\u003emrkD\u003c/em\u003e genes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Numerous investigations have demonstrated that type 3 fimbriae (\u003cem\u003emrkD\u003c/em\u003e gene) appear to play important roles in \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilm formation, which is consistent with our results [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The observation that mutant \u003cem\u003eK. pneumoniae\u003c/em\u003e strains lacking this gene cluster produced noticeably less biofilm in the presence of lung surfactant, with cholesterol and phosphatidylcholine identified as surfactant components promoting type III fimbriae expression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the limitations of the present study include; the study design, a cross-sectional study in which data was collected at one point of time and the causative are not detected and for financial issues such as the tested panel of antibiotic was not including all antibiotics used in treatment of urinary tract isolates and detection of only mrkD and fimH as two biofilm-associated genes without involving other complex genetic determinants may affect the findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe presence of fimbriae virulence genes (mrkD and fimH) was significantly correlated with multidrug-resistant biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from UTI patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent form\u003c/b\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical approval:\u003c/strong\u003e \u003cp\u003eThe ethical approval of this study was obtained from the Medical Ethical Committee of the Faculty of Medicine and Health Science, Sana'a University; No. (287). Ethical approval was obtained in accordance with the standards outlined in the Helsinki Declaration. Written informed consent forms were obtained from all participated UTIs patients before any research procedure was performed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHAA supervision and revision, and editing of the manuscript. SMMD collected data and samples from all patients and performed the laboratory procedures for the samples. DAE and KAA wrote of introduction and methodology. HAA and ANMG analyzed the data, prepared tables and figures, and discussed the results. All authors arranged the referencing style, read, and made the final revision of the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to Yemen and the Sana'a University Faculty of Dentistry for their kind assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data will be available to anyone upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbinaya, M., Gayathri, M. \u0026amp; Khanna, V. G. Evaluation of in-vitro biological and antibiofilm activities of various leaf extracts of \u003cem\u003eAerva lanata\u003c/em\u003e. \u003cem\u003eInt. J. Pharm. Sci. Rev. Res.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 65\u0026ndash;72 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Shehari, M. M. et al. 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Immun.\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, e00135\u0026ndash;e00118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/IAI.00135-18\u003c/span\u003e\u003cspan address=\"10.1128/IAI.00135-18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Urinary tract infections, K. pneumoniae, biofilm formation, fimbriae virulence genes (mrkD and fimH)","lastPublishedDoi":"10.21203/rs.3.rs-9321938/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9321938/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUrinary tract infections (UTIs) caused by antibiotic-resistant strains of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (\u003cem\u003eK. pneumoniae\u003c/em\u003e) are one of the most important causes of morbidity and health care spending affecting people in Yemen, including young women, children, and the elderly. Biofilm formation has become a major factor among those uropathogenic strains.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThis study aimed to investigate the biofilm-forming ability of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from patients with UTIs, to determine the antibiotic susceptibility of those biofilm producers, and to assess the association between fimbrial virulence genes \u003cb\u003e(\u003c/b\u003e\u003cem\u003efimH\u003c/em\u003e and \u003cem\u003emrkD\u003c/em\u003e\u003cb\u003e)\u003c/b\u003e and biofilm strength.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe standard procedures for identifying and isolating bacteria from UTIs were used. The ability of the isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e to form biofilms was examined using tissue culture plates. The Kirby-Bauer disc diffusion method was used to determine the antibiogram susceptibility pattern of uropathogens. The fimbriae virulence genes (\u003cem\u003efimH\u003c/em\u003e and \u003cem\u003emrkD\u003c/em\u003e) for the biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e were found using conventional PCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFrom UTI patients, 79(32.8%) of \u003cem\u003eK. pneumoniae\u003c/em\u003e were isolated. 39 UTI patients (49.4%) had \u003cem\u003eK. pneumoniae\u003c/em\u003e that did not produce a biofilm, although 40 (50.6%) of the bacteria are capable of doing so. Among those biofilm-forming strains, 19 (47.7%) were able to produce strong biofilms, while 21 (52.5%) produced moderate biofilms. PDR (5%), on the other hand, had the lowest percentage of antibiotic resistance, whereas XDR (47.5%) and MDR (32.5%) had the greatest percentages. The \u003cem\u003emrkD\u003c/em\u003e gene was detected in 40 (100%) of the biofilm-forming \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003efimH\u003c/em\u003e had a significant association with strong biofilm (p\u0026thinsp;=\u0026thinsp;0.031).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe presence of fimbriae virulence genes (\u003cem\u003emrkD\u003c/em\u003e and \u003cem\u003efimH\u003c/em\u003e) was significantly correlated with multi-drug resistance (MRD) biofilm-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from UTI patients.\u003c/p\u003e","manuscriptTitle":"Mrk D and fim H Biofilm Genes Detection and Antibiotic Susceptibility: A Study on Uropathogenic Biofilm Producing Klebsiella pneumoniae in Sana'a City, Yemen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 16:50:03","doi":"10.21203/rs.3.rs-9321938/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"145172022639595555656972145414350772850","date":"2026-05-18T07:38:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T12:50:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-14T11:13:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T09:41:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T09:40:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-04T16:21:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3394ed37-90cf-4f82-80c4-beda8858bc36","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"145172022639595555656972145414350772850","date":"2026-05-18T07:38:43+00:00","index":88,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65784785,"name":"Health sciences/Diseases"},{"id":65784786,"name":"Health sciences/Medical research"},{"id":65784787,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-22T12:53:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 16:50:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9321938","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9321938","identity":"rs-9321938","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}