Vitamin C is an effective biofilm prevention agent against uropathogenic Escherichia coli on urinary catheter tubes

Vitamin C is an effective biofilm prevention agent against uropathogenic Escherichia coli on urinary catheter tubes | 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 Short Report Vitamin C is an effective biofilm prevention agent against uropathogenic Escherichia coli on urinary catheter tubes Anisha Paul, Amal Razak, Shafa Thekkekara, Rima Rafi, Michael Magaogao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4532112/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 Objective Uropathogenic Escherichia coli (UPEC) is the major cause of catheter associated urinary tract infections (UTI). As the production of biofilm is usually associated with enhanced antibiotic resistance, formation of biofilm by UPEC poses a challenge against its eradication. Vitamin C is showing great promise as an antimicrobial agent in recent studies. In this study, we explored antibacterial and biofilm prevention property of vitamin C against biofilm produced by fresh UPEC strains on urinary catheter tubes. As expression of fimbriae can enhance biofilm formation by UPEC, the capacity of the strains for heamagglutionation with human O + ve red blood cells and detection of fimbrial gene by PCR were also explored. Results The average minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vitamin C for UPEC strains were 3.90 mg/ml and 7.81 mg/ml, respectively. Increased biofilm formation on plastic surfaces and on silicone catheter tubes were noted at sub-MIC concentrations of vitamin C. Biofilm prevention concentration (BPC) of vitamin was calculated to be 6.24 mg/ml. Gene detection by PCR revealed UPEC strains possessed type1-fimbriae ( fimH ) gene and were also capable of hemagglutinating human O + ve red blood cells, indicating the presence of fimbriae. Taken together, these findings indicate that vitamin C possess antibacterial and biofilm prevention properties against UPEC strains both on plastic surfaces and on silicone catheter tubes. UPEC Biofilm Vitamin C MIC MBC BPC Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Urinary tract infection (UTI), which involves infection in any part of the urinary system such as urethra, bladder, ureter or kidney is the most common type health-care associated infection ( 1 ). Urinary catheter is a tube inserted into the bladder to drain urine. Approximately 75%-80% of the hospital acquired UTIs are associated with use of urinary catheter ( 2 ). The presence of catheter tube offers the uropathogens an opportunity to colonize its surface, creating a pathway for the organism to reach the urinary tract and cause infection. Predominant organisms associated with catheter associated UTI are uropathogenic E. coli (UPEC) as this pathogen accounts for more than 80% of the cases both in the hospital and community environments ( 3 ). UPEC contain different types of fimbriae and adhesins which facilitie its adherence to host cells and cause infection ( 4 ). Different strategies were adopted to reduce the incidence of catheter associated infection, which included frequent changing the catheter tube and following aseptic techniques. But the prevalence of UPEC infection continues to be high. did not been reduced which demands consideration of alternative strategies ( 5 ). Biofilm is a colony of bacteria enclosed in a self-produced extracellular material, consisting of proteins, carbohydrates and nucleic acids, protecting them from biotic and abiotic stressors ( 6 ). Formation of biofilm by bacteria is induced under stressed conditions as a survival strategy such as nutritional scarcity, exposure to antibiotics and immune challenge posed by the host ( 6 , 7 ) and thus is considered as a virulence attribute of pathogens ( 8 ). For a variety of reasons, antibiotics given to patients may not achieve optimal concentration at the site of infection; also administered antibiotics may become sub-MIC after a specific time period ( 9 , 10 ). Sub-MIC dose of antibiotics alter physiology of bacteria, resulting in modulation of gene expression, virulence and sensitivity to antimicrobial agents and are responsible for emerging of new antibiotic resistance. Moreover, exposure of bacteria sub-MIC antimicrobial agents lead to biofilm production ( 9 , 10 , 11 , 12 ). Type 1 fimbriae is involved in two important virulence attributes of UPEC i.e. adherence to urinary epithelial cells and formation of biofilm ( 8 , 13 14 ). Cell to cell communication is established in UPEC via quorum sensing in biofilm, enabling the bacteria to further strengthening their resistance against antibiotics ( 15 , 16 ). Eradication of UPEC using the conventional antibiotics has dramatically decreased over the past decade ( 17 ) which has led researchers to find alternative means for treatment ( 18 , 19 , 20 ). Vitamic C has emerged as a promising candidate as an antibacterial agent and has led researchers to explore their antibacterial as well as antibiofilm properties against UPEC, in addition to other pathogens including methicillin-resistant Staphylococcus spp. (MRSA) and Pseudomonas aeruginosa ( 20 , 21 , 22 , 23 , 24 ). In addition, anti-oxidant property of vitamin C helps in killing the microbes present inside the phagocytic cells by generating reactive oxidative species ( 25 ). In this study we evaluated the antibacterial and biofilm prevention property of both vitamin C against UPEC to explore the possibility of repurposing their usage as an alternative and effective approach to reduce the pathogen burden. We explored whether vitamin C can interfere with the process of biofilm formation on urinary catheter tubes, which is a common vehicle of infection in UTI patients. We also determined the association of fimH gene of type 1 fimbriae with biofilm formation in UPEC strains. Materials and Methods Sample collection and processing Fully characterized and preserved 4 UPEC strains were used. These strains were isolated from UTI patients attending a local hospital of Ras Al Khaimah, UAE for treatment. MIC and MBC estimation The minimal inhibitory concentration (MIC) of vitamin C against all strains was determined by micro-dilution method, according to the clinical and laboratory standard institute (CLSI) guidelines Clinical and Laboratory Standard Institute ( 26 ), using 96-well microtiter plates. The plates were processed as described earlier ( 22 ). The first column received 180 ul of 50 mg/ml vitamin C. Then, two-fold serial dilution was prepared in brain heart infusion (BHI) broth, so that concentration of vitamin C ranged from 25 mg/ml to 0.39 mg/ml in 90 ul. Ten microliter of overnight bacterial culture in BHI broth was added to the test wells. Uninoculated BHI broth served as negative control. The microtiter plates was then kept in the incubator at 37°C for 24 h. The microtiter plates were visually inspected to determine the MIC by checking the wells lacking growth. The overall average MIC was calculated by averaging the MICs of all the four strains of UPEC we tested. For MBC estimation, 5 ul of culture from each well of MIC assay plates were spotted on Mueller Hinton agar plates and incubated for at 37°C in a for 24 h. Afterward, MBC was visually interpreted as the vitamin C concentration showing no growth. The overall average MBC was calculated by averaging the MBCs of all the strains tested Biofilm assay on plastic surfaces Biofilm assay was carried as described earlier ( 27 ). Briefly, BHI broth in microtiter plates (200 µL/well) and in plastic tubes (2 ml/tube) were inoculated with overnight culture of bacterial strains (5 µL/well and 50 µL /tube) adjusted at 0.5 McFarland standard and inoculated at 37°C for 48 hours for biofilm formation. Bacterial cultures were then carefully removed, washed with normal saline (3X) and stained 0.1%. crystal violet solution for 10 min. Biofilm on tube appeared as purple coloured ring (Fig. 2 ). The crystal violet dye retained by the purple coloured ring in tubes or microtiter plates were extracted with 95% ethanol (2 ml/tube and 200 µL/well) and absorbance was read at 560 nm using a spectrophotometer or ELISA reader, as needed. Biofilm formation on catheter tubes Biofilm production and assay was carried out as described earlier ( 28 ), with little modification. Briefly, biofilms were allowed to be formed on the test tubes in 2 ml of growth media containing vitamin C in a series of 2-fold dilutions ranging from 25 mg/ml to 0.39 mg/ml. The silicone catheter tubes were cut into 1 cm long pieces, keeping the package still intact to avoid contamination. Using sterile forceps, the catheter pieces were added to the test tube and the tubes were inoculated UPEC strains (10 µL from overnight culture), adjusted to 0.5 McFarland standard and allowed to grow for 48 hours at 37°C. UPEC strains were inoculated in triplicate. After 48 hours, the catheter tube pieces were taken out of the tubes using the forceps, gently washed three times with normal saline. The biofilm formed on the catheter tube pieces were subjected to crystal violet staining and quantitation as described in the previous section. Determination of Biofilm prevention concentration (BPC) on urinary catheter tubes BPC is the minimum concentration of vitamin C which can inhibit the formation of biofilm. This is due to sufficient reduction in viable bacteria leading to prevention of biofilm formation. BPC was determined by adding a fixed number of bacteria to a series of concentrations of vitamin C (25 mg/ml to 0.39 mg/ml) which sufficiently represented concentrations of vitamin C both sub-MIC and supra-MIC levels and measuring formation of biofilm at each test point. Quantitation of biofilm formation on catheter tubes was carried as described in the earlier section. The minimum concentration of the vitamin C which reduced the biofilm formation, as measured by OD at 560 nm, to that of uninoculated control tubes is considered as BPC. Hemagglutination assay Hemagglutination assay was carried out on glass slides with human O + ve red blood cell (RBC) with little modification ( 28 ). Freshly collected blood was washed (3X) with normal saline and made up to a 1% suspension in normal saline. UPEC strains were grown at 37°C for 48 hours for full fimbriation. One drop of RBC suspension was added to a drop of the broth culture on a glass slide and slide was rocked at room temperature for 5 min. Presence of clumping was noted as positive for hemagglutination. fimH Gene Detection Commercially designed primers for fimH genes were adapted from a previous study ( 29 ). The amplification of fimH gene was carried out in a Thermal Cycler (Eppendorf Master Cycler) under the following PCR conditions; denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 60 s, annealing at 63°C for 30 s, and extension at 72°C for 90 s, with a final extension at 72°C for 5 min. The amplified PCR products were visualized by 1.5% ethidium bromide staining after gel electrophoresis; size for fimH gene was 564 bp. Ethics approval The research proposal was approved by the RAK Medical and Health Sciences University Research and Ethics Committee; approval number- RAKMHSU-REC-076-2021/22-UG-M Statistical analysis We used SPSS software (statistical package for the social sciences, version 26, International Business Machines Corporation, Armonk, NY, USA) to carry out the statistical tests, all of which were two-sample t-tests, and the significance level was determined at p < 0.05. We compared OD at 560 nm as an index of biofilm formation with vitamin C concertation of 0 mg/ml with 3.12 mg/ml, at which concentration biofilm production reached maximum level. We used two two-sample (paired) t-test and estimated the range of difference between their means by measuring the confidence interval; it was found to significant at p < 0.05 Results MIC and MBC of vitamin C The minimum inhibitory concentration (MIC) is defined as the lowest concentration of compound needed to inhibit the growth of bacteria as evidenced by absence of visible growth after an overnight incubation. Minimum bactericidal concentration (MBC) is the lowest concentration of that agent required to kill the bacterial inoculums over a fixed period of time as evidenced by the absence of growth of the microorganism in antibiotic-free media. The overall average of MIC of vitamin C for the 4 strains of UPEC were 3.90 mg/ml and MBC 7.81 mg/ml. The results are presented in (Fig. 1 ; Table 1) Table-1. Profile of the UPEC strains used in this study. UPEC Strains MIC mg / ml MBC mg / ml BPC mg / ml Biofilm production Hem- agglutination fimH gene Ec 5 6.25 12.5 9.37 3+ +ve +ve Ec 12 3.12 6.25 ND 0 +ve +ve Ec 45 3.12 6.25 4.68 1+ +ve +ve Ec 48 3.12 6.25 4.68 1.5+ +ve +ve Average 3.90 Average 7.81 Average 6.24 BPC of the strain Ec 12 was not determined as it did not produce any detectable biofilm. Details of each assay is given in the materials and methods section. Biofilm production by UPEC strains and BPC of vitamin C UPEC Strains were screened for biofilm production microtiter plates and plastic tubes. Strain Ec 5 routinely produced higher amounts of biofilm; while strain Ec12 did not produce any detectable biofilm. Strains Ec 45 and Ec 48 produced intermediate levels of biofilm (Fig. 2 ; Table-1). Biofilm production potential was graded from 0–4 + on an arbitrarily scale in comparison to biofilm forming potential of a known biofilm producer strain of Staphylococcus aureus (4 + biofilm producer) (Table 1). UPEC strain, which was negative for biofilm production. not included in BPC assays. For BPC assay on catheter tubes, we quantitated biofilm production as a function of the concentration of vitamin C ranging from sub-MIC to supra-MIC levels. This allowed us to determine at which concentration biofilm production was prevented. It was interesting to note that biofilm production reached maximum level at 3.12 mg/ml; then it sharply dropped to the level of negative control at 6.25 mg/ml, at the next dilution of the 2-fold dilution series. As at 3.12 mg/ml concentration, biofilm formation reached the maximum levels, the dropped to negative level at 6.25 mg/ml, we inferred that BPC is more than > 3.12 but < 6.25. To arrive at a fixed BPC value, we considered the average of these two values (Fig. 3 ). From the BPC value of each strains, the average BPC of vitamin C against the strains tested was calculated (Table 1). Gene detection Fimbrial gene fimH was present in all 4 UPEC samples tested (Fig. 4 ). The primers used to detect the fimbrial genes are presented in supplementary data-1. The amplicon size was 564 bp. Hemagglutination The result of hemagglutination is presented in Table-1 along with biofilm production and the presence of fimbrial gene, fimH . Presence of fimbrial gene correlated with hemagglutination property but not with biofilm production as the strain Ec 12 which was positive for fimH gene and caused hemagglutination but did not produce any detectable biofilm. Microscopic image of hemagglutination is presented in the supplementary data-2. Discussion In this study, the primary research question was whether vitamin C can inhibit biofilm formation on silicone catheter tube by fresh clinical strains of UPEC. In addressing this question, we carried out experiments for determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm prevention concentration (BPC) of vitamin C. To the best of our knowledge, our study investigated for the first time the effect of vitamin C on biofilm formation by UPEC strains on silicone catheter tubes. In addition, as production of fimbriae by UPEC is critical for biofilm formation, we also explored heamagglutination of human o + ve blood to detect fimbriation and used PCR for detection of fimbrial genes in these test strains. It is evident from the results obtained in this study that vitamin C possess antibacterial effect against UPEC. The MIC and MBC data obtained for vitamin C against UPEC strains (Fig. 1 ; Table-1) in this study was comparable with those obtained in other studies in which other bacteria were tested. Antimicrobial and anti-biofilm effect of vitamin C either singly or in combination with different antibiotics have been demonstrated against different pathogens such as Pseudomonas aeruginosa, Streptococcus mutans , Proteus sp., Enterococcus sp, and Citrobacter sp. ( 8 , 20 , 22 ). For determination of antibiofilm effect of different antimicrobials and non-antimicrobial compounds, determination of BPC is an important assay ( 20 , 30 ). As pathogenic bacteria commonly exist in biofilm at infection site, BPC is more clinically relevant than MIC. In determining the BPC, we assayed biofilm formation as a function of vitamin C centration ranging from a series of sub- MIC concentration to a series of supra-MIC concentrations. Interestingly, we observed that UPEC produced increasingly higher amounts of biofilm (as determined by the amount of crystal violet dye binding, assayed spectrophotometrically), as the concentration of the vitamin increased (Fig. 3 ). It is reported that antimicrobial agents induce biofilm production at sub- MIC concentrations by different bacteria such as E. coli, P. aeruginosa and XXX ( 12 , 31 ). It is apparent that from the Fig. 3 that biofilm production peaked at a highest sub-MIC concentration (3.12 mg/ml), closest to the MIC (3.90 mg/ml) but not exceeding it. Then there was massive drop in biofilm production as the concentration of vitamin C reached near MIC value. This sudden drop can be interpreted with reference to previous studies ( 9 , 10 , 11 , 12 ). As biofilm production is considered as a stress response phenotype, it is possible that at sub-MIC concentration of vitamin C, the bacteria are sufficiently stressed (but not inhibited or killed) as the vitamin C concentration increased. However, when the vitamin C concentration reached the critical concentration of BPC, biofilm production dropped to the range of negative control (uninoculated tubes). The drop in biofilm formation was most likely due to sufficient reduction in viable bacteria to form biofilm. One important observation is that one strain (Ec 12), although did not produce any detectable biofilm, had identical MIC and MBC as those of two other strains (Ec 45 and Ec 48). Moreover, this strain was genetically and phenotypically positive for the presence of fimH gene and hemagglutination assays (Fig-4, Table-1). Taken together, these findings indicate that multiple mechanisms may be operative linking fimbriation and biofilm production. Lastly, the gene associated with biofilm formation fimH was found to be present in all 4 samples of the clinical strains. This type1 fimbriae are responsible for virulence of UPEC and it is present in more than 95% of E. coli and it contributes towards adherence bacteria to target cells formation of biofilm ( 14 , 17 , 19 ). How vitamin C exerts its antibacterial effect of UPEC is not known at present. It has been shown that Mycobacterium tuberculosis is killed by vitamin C through generation of oxidative radicals and (p)ppGpp mediated stress response ( 31 ). Similar mechanism may be operative against UPEC; further studies are needed to answer this question. Our findings add UPEC to the growing list of pathogens against which vitamin C possess antimicrobial and antibiofilm activity. Potential of vitamin C as therapeutic adjunct need to be explored. Conclusion The antibacterial and antibiofilm properties of vitamin C has been clearly demonstrated against fresh clinical strains of UPEC. The potential of vitamin C to inhibit biofilm formation on catheter tubes at sub-MIC strength exhibits its potential as antibiofilm agent in UTI cases where prolonged use of urinary catheter needed. It is possible that Vitamin C may be routinely prescribed alone or along antimicrobial agents as antibiotic modifier / therapeutic adjunct to treat UTI infections in clinical settings as this combination may shorten the antibiotic course. Limitation of the study Limited number of strains have been used. It needs to be tested with larger number fresh clinical strains to completely realize the applicability of the findings. Abbreviations UTIs: Urinary tract infections; MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration; BPC: Biofilm prevention concentration; UPEC: Uropathogenic Escherichia coli. Declarations Acknowledgements The authors acknowledge the support extended by RAK Medical and Health Sciences University, Ras Al Khaimah, UAE in conducting this research. Author contributions Conceptualization: AH Research work, Original draft preparation: AP, AR, RR, ST, MM Data analysis: HA, MAP, AH Review and editing: HA, MAP, AH The final version of the manuscript was approved by all authors. Funding The research was supported by the Department of Medical Microbiology of RAK Medical and Health Sciences University. Availability of data and materials Additional files available. The datasets used and/or analysed during this study are available from the corresponding author ( [email protected] ) on reasonable request. Competing interests The authors declare no competing interest Author details AP, AR, ST, RR, MM, HA and AH: Department of Medical Microbiology and Immunology, RAK Medical College; RAK Medical and Health Sciences University (RAKMHSU). MSP: Department of Public Health, RAK College of Medicine; RAK Medical and Health Sciences University (RAKMHSU), Ras Al Khaimah, UAE. References CDC. 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Hemagglutination and biofilm formation as virulence markers of uropathogenic Escherichia coli in acute urinary tract infections and urolithiasis. Ind J Urol. 2013;29:277–281. doi.org/10.4103/0970-1591.120093 Zhao R, Shi J, ShenY, Li Y, Han Q, Zhang, X., Gu, G., Xu, J. 2015. Phylogenetic distribution of virulence genes among ESBL-producing uropathogenic Escherichia coli isolated from long-term hospitalized patients. J Clin Diag Res 2015;9: DC01–DC04. doi: 10.7860/JCDR/2015/13234.6157 Noach N, Lavy E, Reifen, R. et al. Zinc chloride is effective as an antibiotic in biofilm prevention following septoplasty. Sci Rep. 2023;13: 8344. doi.org/10.1038/s41598-023-35069-9 Syal K. Chatterji D. Vitamin C: A natural Inhibitor of cell wall functions and stress response in Mycobacteria . Adv Expt Med Biol. 2018; 1112: 321–332. doi.org/10.1007/978-981-13-3065-0_22 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4532112","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":314404215,"identity":"0a563209-df33-4ac1-a7fb-c191d210f675","order_by":0,"name":"Anisha Paul","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Anisha","middleName":"","lastName":"Paul","suffix":""},{"id":314404216,"identity":"db86aacb-ea37-40d3-817f-7c6f07f6a674","order_by":1,"name":"Amal Razak","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Amal","middleName":"","lastName":"Razak","suffix":""},{"id":314404217,"identity":"bbbfc2b6-19ce-4586-9a74-6e489679830a","order_by":2,"name":"Shafa Thekkekara","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Shafa","middleName":"","lastName":"Thekkekara","suffix":""},{"id":314404218,"identity":"89071903-5092-41de-866a-c94982164fad","order_by":3,"name":"Rima Rafi","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Rima","middleName":"","lastName":"Rafi","suffix":""},{"id":314404219,"identity":"4c780aa4-91c2-4b91-a706-c3089e0233c5","order_by":4,"name":"Michael Magaogao","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Magaogao","suffix":""},{"id":314404220,"identity":"46d3c751-b269-4044-8816-7ecaa2ea077f","order_by":5,"name":"Mohamed Anas Patni","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Anas","lastName":"Patni","suffix":""},{"id":314404222,"identity":"0178e173-0dd6-4142-b314-54bf0bb70a51","order_by":6,"name":"Hafiz Ahmad","email":"","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Hafiz","middleName":"","lastName":"Ahmad","suffix":""},{"id":314404223,"identity":"58fde1cb-4f4d-42b9-8ca1-a49315edfe8b","order_by":7,"name":"Ashfaque Hossain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYFACHoYPIMqAAULKgagDD/BrYZyBrMUYrCWBeC0MDIkNIBKfFv7+swcbfpQdljNnb3/84UfBnfT5YYcfAm2xk9NtwK5F4kZeYmPPucPGlj1nzCR7DJ7lbrydZgDUkmxsdgCHNTd4zB/wth1O3HAjh42Bx+Bw7sbZCSAtBxK34dAif/6MYePftsP1G26kP/74x+BwuuHs9A94tRgcyDFsBtqSYHAjwUAaaEuCvHQOflsMbwC1yJxLN9xw5oyZtIzBYcMN0jkFBxIMcPtFDuSwN2XW8gbH2x9/fPPnsLz87PTNHz5U2Mnh9D4YsKE4FUziU46uRb6BkOpRMApGwSgYaQAAXlVqnQGNYdAAAAAASUVORK5CYII=","orcid":"","institution":"RAK College of Medical Sciences RAK Medical and Health Sciences University","correspondingAuthor":true,"prefix":"","firstName":"Ashfaque","middleName":"","lastName":"Hossain","suffix":""}],"badges":[],"createdAt":"2024-06-05 07:25:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4532112/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4532112/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59390869,"identity":"804e3625-6aee-4ac4-9c77-9d3f200f87bb","added_by":"auto","created_at":"2024-07-01 08:03:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColumn chart for representing the average MIC, MBC and BPC values.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMIC. MBC and BPC values for each strain of UPECcarried out in 3 independent experiments carried out in triplicate. Y-axis represents vitamin C concentration (mg/ml).\u003c/p\u003e","description":"","filename":"Fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/05751c95f09e8eb266758d78.png"},{"id":59390875,"identity":"2e1e2a64-af2d-4ac8-8726-2e353e464e76","added_by":"auto","created_at":"2024-07-01 08:03:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":483672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiofilm production by EPEC strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrystal violet dye binding assay in tubes. Biofilm production potential was graded from 0 - 3+ on an arbitrarily scale in comparison to biofilm forming potential of a known biofilm producer strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (4+ biofilm producer)/ Three UPEC strains were positive for biofilm production, producing different levels of biofilm [Fig.2; Table-1] and one strain, Ec 12 was negative for biofilm production.\u003c/p\u003e","description":"","filename":"Fig.21.png","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/09cabd0319c1749cfae6ee67.png"},{"id":59391281,"identity":"88407931-a2af-4f06-aa53-d8b77a369024","added_by":"auto","created_at":"2024-07-01 08:11:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitation of biofilm on silicone catheter tubes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProduction of biofilm on silicone catheter tubes quantitated using crystal violet dye binding assay by the UPEC strain Ec 5 as influenced by sub-MIC to supra-MIC concentrations of vitamin C.\u003c/p\u003e","description":"","filename":"Fig.31.png","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/b50f40d74b42173415974ff1.png"},{"id":59390871,"identity":"2dfc214b-d10d-49ea-9e95-aa0b59826c28","added_by":"auto","created_at":"2024-07-01 08:03:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":291710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAgarose gel electrophoresis for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003efimH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene amplicons of UPEC\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Fig.41.png","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/f2a6a375555dca2fd005017e.png"},{"id":62886072,"identity":"d829b6f3-69a0-4065-9d66-2888601c9307","added_by":"auto","created_at":"2024-08-20 15:56:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1406406,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/079ec2f1-65f4-4257-9a9e-bc854fb06f07.pdf"},{"id":59390873,"identity":"be0784b5-4a26-4fa8-b841-be7460c5071e","added_by":"auto","created_at":"2024-07-01 08:03:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14509,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/1176d3f920cbd98105d5aba3.docx"},{"id":59390872,"identity":"f38ec045-35b3-44d1-9478-eccc795bec16","added_by":"auto","created_at":"2024-07-01 08:03:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":306809,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4532112/v1/fd9ca92035f4988ae884957d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Vitamin C is an effective biofilm prevention agent against uropathogenic Escherichia coli on urinary catheter tubes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUrinary tract infection (UTI), which involves infection in any part of the urinary system such as urethra, bladder, ureter or kidney is the most common type health-care associated infection (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Urinary catheter is a tube inserted into the bladder to drain urine. Approximately 75%-80% of the hospital acquired UTIs are associated with use of urinary catheter (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe presence of catheter tube offers the uropathogens an opportunity to colonize its surface, creating a pathway for the organism to reach the urinary tract and cause infection. Predominant organisms associated with catheter associated UTI are uropathogenic \u003cem\u003eE. coli\u003c/em\u003e (UPEC) as this pathogen accounts for more than 80% of the cases both in the hospital and community environments (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). UPEC contain different types of fimbriae and adhesins which facilitie its adherence to host cells and cause infection (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent strategies were adopted to reduce the incidence of catheter associated infection, which included frequent changing the catheter tube and following aseptic techniques. But the prevalence of UPEC infection continues to be high. did not been reduced which demands consideration of alternative strategies (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiofilm is a colony of bacteria enclosed in a self-produced extracellular material, consisting of proteins, carbohydrates and nucleic acids, protecting them from biotic and abiotic stressors (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Formation of biofilm by bacteria is induced under stressed conditions as a survival strategy such as nutritional scarcity, exposure to antibiotics and immune challenge posed by the host (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and thus is considered as a virulence attribute of pathogens (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). For a variety of reasons, antibiotics given to patients may not achieve optimal concentration at the site of infection; also administered antibiotics may become sub-MIC after a specific time period (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Sub-MIC dose of antibiotics alter physiology of bacteria, resulting in modulation of gene expression, virulence and sensitivity to antimicrobial agents and are responsible for emerging of new antibiotic resistance. Moreover, exposure of bacteria sub-MIC antimicrobial agents lead to biofilm production (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eType 1 fimbriae is involved in two important virulence attributes of UPEC i.e. adherence to urinary epithelial cells and formation of biofilm (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Cell to cell communication is established in UPEC via quorum sensing in biofilm, enabling the bacteria to further strengthening their resistance against antibiotics (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEradication of UPEC using the conventional antibiotics has dramatically decreased over the past decade (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) which has led researchers to find alternative means for treatment (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Vitamic C has emerged as a promising candidate as an antibacterial agent and has led researchers to explore their antibacterial as well as antibiofilm properties against UPEC, in addition to other pathogens including methicillin-resistant \u003cem\u003eStaphylococcus spp.\u003c/em\u003e (MRSA) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In addition, anti-oxidant property of vitamin C helps in killing the microbes present inside the phagocytic cells by generating reactive oxidative species (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study we evaluated the antibacterial and biofilm prevention property of both vitamin C against UPEC to explore the possibility of repurposing their usage as an alternative and effective approach to reduce the pathogen burden. We explored whether vitamin C can interfere with the process of biofilm formation on urinary catheter tubes, which is a common vehicle of infection in UTI patients. We also determined the association of \u003cem\u003efimH\u003c/em\u003e gene of type 1 fimbriae with biofilm formation in UPEC strains.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and processing\u003c/h2\u003e \u003cp\u003eFully characterized and preserved 4 UPEC strains were used. These strains were isolated from UTI patients attending a local hospital of Ras Al Khaimah, UAE for treatment.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eMIC and MBC estimation\u003c/h2\u003e \u003cp\u003eThe minimal inhibitory concentration (MIC) of vitamin C against all strains was determined by micro-dilution method, according to the clinical and laboratory standard institute (CLSI) guidelines Clinical and Laboratory Standard Institute (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), using 96-well microtiter plates.\u003c/p\u003e \u003cp\u003eThe plates were processed as described earlier (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The first column received 180 ul of 50 mg/ml vitamin C. Then, two-fold serial dilution was prepared in brain heart infusion (BHI) broth, so that concentration of vitamin C ranged from 25 mg/ml to 0.39 mg/ml in 90 ul. Ten microliter of overnight bacterial culture in BHI broth was added to the test wells. Uninoculated BHI broth served as negative control. The microtiter plates was then kept in the incubator at 37\u0026deg;C for 24 h. The microtiter plates were visually inspected to determine the MIC by checking the wells lacking growth. The overall average MIC was calculated by averaging the MICs of all the four strains of UPEC we tested. For MBC estimation, 5 ul of culture from each well of MIC assay plates were spotted on Mueller Hinton agar plates and incubated for at 37\u0026deg;C in a for 24 h. Afterward, MBC was visually interpreted as the vitamin C concentration showing no growth. The overall average MBC was calculated by averaging the MBCs of all the strains tested\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm assay on plastic surfaces\u003c/h2\u003e \u003cp\u003eBiofilm assay was carried as described earlier (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Briefly, BHI broth in microtiter plates (200 \u0026micro;L/well) and in plastic tubes (2 ml/tube) were inoculated with overnight culture of bacterial strains (5 \u0026micro;L/well and 50 \u0026micro;L /tube) adjusted at 0.5 McFarland standard and inoculated at 37\u0026deg;C for 48 hours for biofilm formation. Bacterial cultures were then carefully removed, washed with normal saline (3X) and stained 0.1%. crystal violet solution for 10 min. Biofilm on tube appeared as purple coloured ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The crystal violet dye retained by the purple coloured ring in tubes or microtiter plates were extracted with 95% ethanol (2 ml/tube and 200 \u0026micro;L/well) and absorbance was read at 560 nm using a spectrophotometer or ELISA reader, as needed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation on catheter tubes\u003c/h2\u003e \u003cp\u003eBiofilm production and assay was carried out as described earlier (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), with little modification. Briefly, biofilms were allowed to be formed on the test tubes in 2 ml of growth media containing vitamin C in a series of 2-fold dilutions ranging from 25 mg/ml to 0.39 mg/ml. The silicone catheter tubes were cut into 1 cm long pieces, keeping the package still intact to avoid contamination. Using sterile forceps, the catheter pieces were added to the test tube and the tubes were inoculated UPEC strains (10 \u0026micro;L from overnight culture), adjusted to 0.5 McFarland standard and allowed to grow for 48 hours at 37\u0026deg;C. UPEC strains were inoculated in triplicate. After 48 hours, the catheter tube pieces were taken out of the tubes using the forceps, gently washed three times with normal saline. The biofilm formed on the catheter tube pieces were subjected to crystal violet staining and quantitation as described in the previous section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Biofilm prevention concentration (BPC) on urinary catheter tubes\u003c/h2\u003e \u003cp\u003eBPC is the minimum concentration of vitamin C which can inhibit the formation of biofilm. This is due to sufficient reduction in viable bacteria leading to prevention of biofilm formation. BPC was determined by adding a fixed number of bacteria to a series of concentrations of vitamin C (25 mg/ml to 0.39 mg/ml) which sufficiently represented concentrations of vitamin C both sub-MIC and supra-MIC levels and measuring formation of biofilm at each test point. Quantitation of biofilm formation on catheter tubes was carried as described in the earlier section. The minimum concentration of the vitamin C which reduced the biofilm formation, as measured by OD at 560 nm, to that of uninoculated control tubes is considered as BPC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHemagglutination assay\u003c/h2\u003e \u003cp\u003eHemagglutination assay was carried out on glass slides with human O\u0026thinsp;+\u0026thinsp;ve red blood cell (RBC) with little modification (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Freshly collected blood was washed (3X) with normal saline and made up to a 1% suspension in normal saline. UPEC strains were grown at 37\u0026deg;C for 48 hours for full fimbriation. One drop of RBC suspension was added to a drop of the broth culture on a glass slide and slide was rocked at room temperature for 5 min. Presence of clumping was noted as positive for hemagglutination.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003efimH Gene Detection\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eCommercially designed primers for \u003cem\u003efimH\u003c/em\u003e genes were adapted from a previous study (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The amplification of \u003cem\u003efimH\u003c/em\u003e gene was carried out in a Thermal Cycler (Eppendorf Master Cycler) under the following PCR conditions; denaturation at 94\u0026deg;C for 2 min, followed by 30 cycles of denaturation at 94\u0026deg;C for 60 s, annealing at 63\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 90 s, with a final extension at 72\u0026deg;C for 5 min. The amplified PCR products were visualized by 1.5% ethidium bromide staining after gel electrophoresis; size for \u003cem\u003efimH\u003c/em\u003e gene was 564 bp.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eThe research proposal was approved by the RAK Medical and Health Sciences University Research and Ethics Committee; approval number- RAKMHSU-REC-076-2021/22-UG-M\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eWe used SPSS software (statistical package for the social sciences, version 26, International Business Machines Corporation, Armonk, NY, USA) to carry out the statistical tests, all of which were two-sample t-tests, and the significance level was determined at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. We compared OD at 560 nm as an index of biofilm formation with vitamin C concertation of 0 mg/ml with 3.12 mg/ml, at which concentration biofilm production reached maximum level. We used two two-sample (paired) t-test and estimated the range of difference between their means by measuring the confidence interval; it was found to significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMIC and MBC of vitamin C\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) is defined as the lowest concentration of compound needed to inhibit the growth of bacteria as evidenced by absence of visible growth after an overnight incubation. Minimum bactericidal concentration (MBC) is the lowest concentration of that agent required to kill the bacterial inoculums over a fixed period of time as evidenced by the absence of growth of the microorganism in antibiotic-free media. The overall average of MIC of vitamin C for the 4 strains of UPEC were 3.90 mg/ml and MBC 7.81 mg/ml. The results are presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;1)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTable-1. Profile of the UPEC strains used in this study.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"7\"\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 \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\"\u003e \u003cp\u003eUPEC Strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003cp\u003emg / ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMBC\u003c/p\u003e \u003cp\u003emg / ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBPC\u003c/p\u003e \u003cp\u003emg / ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBiofilm production\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHem-\u003c/p\u003e \u003cp\u003eagglutination\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003efimH\u003c/em\u003e gene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.37\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc 12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+ve\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\u003eEc 45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3.12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e6.25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.68\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+ve\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+ve\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEc 48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3.12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e6.25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.68\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.5+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+ve\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+ve\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAverage\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e3.90\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eAverage\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e7.81\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eAverage\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e6.24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\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 \u003cb\u003eBPC of the strain Ec 12 was not determined as it did not produce any detectable biofilm. Details of each assay is given in the\u003c/b\u003e \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e\u003cb\u003ematerials and methods\u003c/b\u003e\u003c/span\u003e \u003cb\u003esection.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm production by UPEC strains and BPC of vitamin C\u003c/h2\u003e \u003cp\u003eUPEC Strains were screened for biofilm production microtiter plates and plastic tubes. Strain Ec 5 routinely produced higher amounts of biofilm; while strain Ec12 did not produce any detectable biofilm. Strains Ec 45 and Ec 48 produced intermediate levels of biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table-1). Biofilm production potential was graded from 0\u0026ndash;4\u0026thinsp;+\u0026thinsp;on an arbitrarily scale in comparison to biofilm forming potential of a known biofilm producer strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (4\u0026thinsp;+\u0026thinsp;biofilm producer) (Table\u0026nbsp;1). UPEC strain, which was negative for biofilm production. not included in BPC assays.\u003c/p\u003e \u003cp\u003eFor BPC assay on catheter tubes, we quantitated biofilm production as a function of the concentration of vitamin C ranging from sub-MIC to supra-MIC levels. This allowed us to determine at which concentration biofilm production was prevented. It was interesting to note that biofilm production reached maximum level at 3.12 mg/ml; then it sharply dropped to the level of negative control at 6.25 mg/ml, at the next dilution of the 2-fold dilution series. As at 3.12 mg/ml concentration, biofilm formation reached the maximum levels, the dropped to negative level at 6.25 mg/ml, we inferred that BPC is more than \u0026gt;\u0026thinsp;3.12 but \u0026lt;\u0026thinsp;6.25. To arrive at a fixed BPC value, we considered the average of these two values (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From the BPC value of each strains, the average BPC of vitamin C against the strains tested was calculated (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGene detection\u003c/h2\u003e \u003cp\u003eFimbrial gene \u003cem\u003efimH\u003c/em\u003e was present in all 4 UPEC samples tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The primers used to detect the fimbrial genes are presented in supplementary data-1. The amplicon size was 564 bp.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHemagglutination\u003c/h2\u003e \u003cp\u003eThe result of hemagglutination is presented in Table-1 along with biofilm production and the presence of fimbrial gene, \u003cem\u003efimH\u003c/em\u003e. Presence of fimbrial gene correlated with hemagglutination property but not with biofilm production as the strain Ec 12 which was positive for \u003cem\u003efimH\u003c/em\u003e gene and caused hemagglutination but did not produce any detectable biofilm. Microscopic image of hemagglutination is presented in the supplementary data-2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the primary research question was whether vitamin C can inhibit biofilm formation on silicone catheter tube by fresh clinical strains of UPEC. In addressing this question, we carried out experiments for determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm prevention concentration (BPC) of vitamin C. To the best of our knowledge, our study investigated for the first time the effect of vitamin C on biofilm formation by UPEC strains on silicone catheter tubes. In addition, as production of fimbriae by UPEC is critical for biofilm formation, we also explored heamagglutination of human o\u0026thinsp;+\u0026thinsp;ve blood to detect fimbriation and used PCR for detection of fimbrial genes in these test strains.\u003c/p\u003e \u003cp\u003eIt is evident from the results obtained in this study that vitamin C possess antibacterial effect against UPEC. The MIC and MBC data obtained for vitamin C against UPEC strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table-1) in this study was comparable with those obtained in other studies in which other bacteria were tested. Antimicrobial and anti-biofilm effect of vitamin C either singly or in combination with different antibiotics have been demonstrated against different pathogens such as \u003cem\u003ePseudomonas aeruginosa, Streptococcus mutans\u003c/em\u003e, \u003cem\u003eProteus\u003c/em\u003e sp., \u003cem\u003eEnterococcus\u003c/em\u003e sp, and \u003cem\u003eCitrobacter\u003c/em\u003e sp. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor determination of antibiofilm effect of different antimicrobials and non-antimicrobial compounds, determination of BPC is an important assay (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). As pathogenic bacteria commonly exist in biofilm at infection site, BPC is more clinically relevant than MIC. In determining the BPC, we assayed biofilm formation as a function of vitamin C centration ranging from a series of sub- MIC concentration to a series of supra-MIC concentrations. Interestingly, we observed that UPEC produced increasingly higher amounts of biofilm (as determined by the amount of crystal violet dye binding, assayed spectrophotometrically), as the concentration of the vitamin increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It is reported that antimicrobial agents induce biofilm production at sub- MIC concentrations by different bacteria such as \u003cem\u003eE. coli, P. aeruginosa\u003c/em\u003e and XXX (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). It is apparent that from the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e that biofilm production peaked at a highest sub-MIC concentration (3.12 mg/ml), closest to the MIC (3.90 mg/ml) but not exceeding it. Then there was massive drop in biofilm production as the concentration of vitamin C reached near MIC value. This sudden drop can be interpreted with reference to previous studies (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). As biofilm production is considered as a stress response phenotype, it is possible that at sub-MIC concentration of vitamin C, the bacteria are sufficiently stressed (but not inhibited or killed) as the vitamin C concentration increased. However, when the vitamin C concentration reached the critical concentration of BPC, biofilm production dropped to the range of negative control (uninoculated tubes). The drop in biofilm formation was most likely due to sufficient reduction in viable bacteria to form biofilm.\u003c/p\u003e \u003cp\u003eOne important observation is that one strain (Ec 12), although did not produce any detectable biofilm, had identical MIC and MBC as those of two other strains (Ec 45 and Ec 48). Moreover, this strain was genetically and phenotypically positive for the presence of \u003cem\u003efimH\u003c/em\u003e gene and hemagglutination assays (Fig-4, Table-1). Taken together, these findings indicate that multiple mechanisms may be operative linking fimbriation and biofilm production.\u003c/p\u003e \u003cp\u003eLastly, the gene associated with biofilm formation \u003cem\u003efimH\u003c/em\u003e was found to be present in all 4 samples of the clinical strains. This type1 fimbriae are responsible for virulence of UPEC and it is present in more than 95% of \u003cem\u003eE. coli\u003c/em\u003e and it contributes towards adherence bacteria to target cells formation of biofilm (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). How vitamin C exerts its antibacterial effect of UPEC is not known at present. It has been shown that \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e is killed by vitamin C through generation of oxidative radicals and (p)ppGpp mediated stress response (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Similar mechanism may be operative against UPEC; further studies are needed to answer this question. Our findings add UPEC to the growing list of pathogens against which vitamin C possess antimicrobial and antibiofilm activity. Potential of vitamin C as therapeutic adjunct need to be explored.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe antibacterial and antibiofilm properties of vitamin C has been clearly demonstrated against fresh clinical strains of UPEC. The potential of vitamin C to inhibit biofilm formation on catheter tubes at sub-MIC strength exhibits its potential as antibiofilm agent in UTI cases where prolonged use of urinary catheter needed. It is possible that Vitamin C may be routinely prescribed alone or along antimicrobial agents as antibiotic modifier / therapeutic adjunct to treat UTI infections in clinical settings as this combination may shorten the antibiotic course.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLimitation of the study\u003c/h2\u003e \u003cp\u003eLimited number of strains have been used. It needs to be tested with larger number fresh clinical strains to completely realize the applicability of the findings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eUTIs: Urinary tract infections; MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration; BPC: Biofilm prevention concentration; UPEC: Uropathogenic \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the support extended by RAK Medical and Health Sciences University, Ras Al Khaimah, UAE in conducting this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: AH\u003c/p\u003e\n\u003cp\u003eResearch work, Original draft preparation:\u0026nbsp;AP, AR, RR, ST, MM\u003c/p\u003e\n\u003cp\u003eData analysis: HA, MAP, AH\u003c/p\u003e\n\u003cp\u003eReview and editing: HA, MAP, AH\u003c/p\u003e\n\u003cp\u003eThe final version of the manuscript was approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the Department of Medical Microbiology of RAK Medical and Health Sciences University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Additional files available. The datasets used and/or analysed during this study are available from the corresponding author ([email protected]) on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAP, AR, ST, RR, MM, HA and AH: \u0026nbsp; Department of Medical Microbiology and Immunology, RAK Medical College; RAK Medical and Health Sciences University (RAKMHSU). MSP: Department of Public Health, RAK College of Medicine; RAK Medical and Health Sciences University (RAKMHSU), Ras Al Khaimah, UAE.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCDC. Urinary tract infection (catheter-associated urinary tract infection [CAUTI] and non-catheter-associated urinary tract infection [UTI]) events. 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Clin Microbiol Rev. 2002;15:167\u0026ndash;193. doi.org/10.1128/CMR.15.2.167-193.2002\u003c/li\u003e\n\u003cli\u003eHassan, D., Magaogao. M., and Hossain, A. Characterization of small colony variants of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e: Correlation with antibiotic resistance and biofilm formation. Biomed. Biotechnol. Res. J. 2022;26:438-442.doi. 10.4103/bbrj.bbrj_154_22\u003c/li\u003e\n\u003cli\u003eKatongole P, Nalubeg F, Florence NC. Asiimwe, B., and Andia, I. Biofilm formation, antimicrobial susceptibility and virulence genes of uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e isolated from clinical isolates in Uganda. BMC Infect Dis. 2020;0:453. doi.org/10.1186/s12879-020-05186-1\u003c/li\u003e\n\u003cli\u003eSato Y, Unno Y, Ubagai T, Ono Y. Sub-minimum inhibitory concentrations of colistin and polymyxin B promote \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e biofilm formation. 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Adv Expt Med Biol. 2018; 1112: 321\u0026ndash;332. doi.org/10.1007/978-981-13-3065-0_22\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":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":"UPEC, Biofilm, Vitamin C, MIC, MBC, BPC","lastPublishedDoi":"10.21203/rs.3.rs-4532112/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4532112/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eUropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (UPEC) is the major cause of catheter associated urinary tract infections (UTI). As the production of biofilm is usually associated with enhanced antibiotic resistance, formation of biofilm by UPEC poses a challenge against its eradication. Vitamin C is showing great promise as an antimicrobial agent in recent studies. In this study, we explored antibacterial and biofilm prevention property of vitamin C against biofilm produced by fresh UPEC strains on urinary catheter tubes. As expression of fimbriae can enhance biofilm formation by UPEC, the capacity of the strains for heamagglutionation with human O\u0026thinsp;+\u0026thinsp;ve red blood cells and detection of fimbrial gene by PCR were also explored.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe average minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vitamin C for UPEC strains were 3.90 mg/ml and 7.81 mg/ml, respectively. Increased biofilm formation on plastic surfaces and on silicone catheter tubes were noted at sub-MIC concentrations of vitamin C. Biofilm prevention concentration (BPC) of vitamin was calculated to be 6.24 mg/ml. Gene detection by PCR revealed UPEC strains possessed type1-fimbriae (\u003cem\u003efimH\u003c/em\u003e) gene and were also capable of hemagglutinating human O\u0026thinsp;+\u0026thinsp;ve red blood cells, indicating the presence of fimbriae. Taken together, these findings indicate that vitamin C possess antibacterial and biofilm prevention properties against UPEC strains both on plastic surfaces and on silicone catheter tubes.\u003c/p\u003e","manuscriptTitle":"Vitamin C is an effective biofilm prevention agent against uropathogenic Escherichia coli on urinary catheter tubes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-01 08:03:02","doi":"10.21203/rs.3.rs-4532112/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":"84d9f8cd-ec56-456e-983e-a38851d4814a","owner":[],"postedDate":"July 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-20T15:48:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-01 08:03:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4532112","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4532112","identity":"rs-4532112","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}