May-August 2023 UNIVERSA MEDICINA Vol.42- No.2 pISSN: 1907-3062 / eISSN: 2407-2230 Repurposing of fluoxetine for antibacterial activities in catheter-associated urinary tract biofilm infections: an in vitro analysis Muhammad Musthafa Poyil1* , Rameesha Shafqat2 , and Mamoun A. Alfaki1 ABSTRACT BACKGROUND Urinary tract infections are often initiated by indwelling catheters and bring about serious consequences, especially when they are caused by multidrug-resistant bacterial pathogens. The biofilms of uropathogens such as Enterococcus faecalis and Escherichia coli pose serious challenges. Therefore the scientific world is trying to experiment with alternative drugs to replace conventional antibiotics as the latter are more prone to cause the development of antibacterial resistance. Here, we evaluate the repurposing of the antidepressant fluoxetine as an antibacterial agent against the mentioned pathogens. METHODS To repurpose fluoxetine for its antibacterial activity against Enterococcus faecalis and Escherichia coli, the agar diffusion method was used. The minimal inhibitory concentration was found by the microdilution method. The drug was also analyzed as a coating on catheters to evaluate its efficiency against biofilm formation by pathogens. RESULTS The drug fluoxetine showed potential antibacterial and anti-biofilm activities. Its minimum inhibitory concentration was found to be 18.75 µg/ mL and 37.5 µg/mL against Enterococcus faecalis and Escherichia coli respectively. The antibiofilm activity on polystyrene surfaces was also remarkable as it reduced the formation of Enterococcus faecalis and Escherichia coli biofilms by 70% and 74%, after being treated with 1x MICs and 2x MICs respectively. CONCLUSIONS Fluoxetine - one of the drugs of choice in treating depression, when repurposed, has shown considerable antibacterial and antibiofilm effects against two of the major catheter-associated urinary tract infection-causing bacteria - viz. Enterococcus faecalis and Escherichia coli. Therefore, further studies are needed to understand its applicability as an antibacterial agent. Keywords: Biofilms, catheter-associated urinary tract infections, fluoxetine, repurposing 1Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University, Al-Kharj - 11942, Saudi Arabia 2Dow University of Health Sciences, Karachi - 74200, Pakistan *Correspondence: Muhammad Musthafa Poyil Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University, Al-Kharj - 11942, Saudi Arabia Email: m.poyil@psau.edu.sa ORCID: 0000-0003-4826-3603 Date of first submission, January 17, 2023 Date of final revised submission, June 16, 2023 Date of acceptance, June 23, 2023 This open access article is distributed under a Creative Commons Attribution- Non Commercial-Share Alike 4.0 International License ORIGINAL ARTICLE 128 DOI: http://dx.doi.org/10.18051/UnivMed.2023.v42:128-136 Copyright@Author(s) - https://univmed.org/ejurnal/index.php/medicina/article/view/1421 Cite this article as: Poyil MM, Shafqat R, Alfaki MA. Repurposing of fluoxetine for antibacterial activities in catheter- associated urinary tract biofilm infections: an in vitro analysis. Univ Med 2023;42:128-36. doi: 10.18051/ UnivMed.2023.v42:128-136 mailto:m.poyil@psau.edu.sa https://orcid.org/0000-0002-8415-3893 https://orcid.org/0000-0002-4022-1524 https://orcid.org/0000-0002-1283-8375 https://univmed.org/ejurnal/index.php/medicina/article/view/1421 129 Univ Med Vol. 42 No. 2 INTRODUCTION In modern medical science, indwelling medical devices have been playing a revolutionary role in the management of various diseases, and urinary catheters which relieve the discomforts of patients with urinary bladder complications are the most significant among them. Unfortunately, in hospitalized patients these indwelling catheters significantly increase the risk for iatrogenic infections that are common when the patient is immunocompromised. In the modern age, medical device-associated infections contribute to a major part of nosocomial infections and among these, urinary tract infections (UTIs) are one of the most significant hospital-acquired infections associated with catheters.(1,2) Catheter-associated urinary tract infections (CAUTIs) occur when the presence of uropathogenic bacteria is manifested in the urine of hospitalized patients with catheter usage and are the most common healthcare- associated infections that affect millions of patients globally.(3-5) The use of catheters facilitates the entry of microbial pathogens to the urinary tract and their colonization leads to various complications such as sepsis and bacteriuria, resulting in long hospital stays and in extreme cases, higher mortality rates causing socio- economic burdens.(6,7) Prolonged use of urinary catheters favours polymicrobial infections including those by bacteria and fungi.(8) Reports suggest that Escherichia coli and Enterococcus faecalis are the most prevalent bacterial pathogens that cause CAUTIs.(9,10) These prevalent bacteria form biofilms by adhering to the catheter surfaces and establishing communications among them making their eradication complicated.(11) The biofilm- forming ability enables the microorganisms to resist several antibiotics through various resistance mechanisms, such as the physical protective effects of the extracellular polymeric substances, efflux pumps, and the transfer of antibiotic resistance genes between bacterial cells.(12-14) As a result, in most cases, the treatment of CAUTIs has been practically difficult.(15,16) Hence, there is an immediate need for alternative therapies that enable the prevention of colonization and biofilm formation on urinary catheters by bacterial pathogens such as Enterococcus faecalis and Escherich ia coli and their eradication. However, the search for new drugs from the available sources is a time-consuming and expensive process as these drugs under development have to undergo many clinical trials before entering the market. Among the sever al ways of dr ug development, repurposing already existing drugs for a new and novel application is one approach that has gained much attention because of the unique nature of this drug development process. The quest of repurposing the existing drug is significant as the drug has already gone through many safety, pharmacological efficacy, and human trials which reduces the cost, time, and risks associated with anti biotic innovation. (1 7) Considering all these facts, in the present study, an effort was made to evaluate the antibacterial activity of the antidepressant drug fluoxetine against two of the major CAUTI biofilm-forming bacterial pathogens, viz, Enterococcus faecalis and Escherichia coli. METHODS Research design An in vitro analysis was carried out to screen the anti-depression drug fluoxetine against two of the major catheter-associated urinary tract infection-causing bacterial pathogens viz., Enterococcus faecalis and Escherichia coli. The experiments and the following analyses were conducted a t the Ba sic Medical Science Labor atory, Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia, and at Biotech Research Centre, Salem- 636010, Tamil Nadu, India, during the period between November 4th, 2022 and January 2nd, 2023. Reagents and materials For the study, Mueller Hinton Broth, ampicillin, and rifampicin were procured from Hi 130 Media (USA) and fluoxetine from Sigma Aldrich. Fluoxetine (1 mg/ mL) was prepared in sterile double distilled water. The strains Enterococcus faecalis (ATCC 29212) and Escherichia coli (ATCC 25922) were obtained from American Type Culture Collection (ATCC). The antibiotics rifampicin (5 µg) and ampicillin (5 µg) were used as controls. Determination of antibacterial activity The antibacterial activity of fluoxetine against Enterococcus faecalis and Escherichia coli was investigated using the well diffusion method as per standard protocols. (18) In brief, overnight cultures of Enterococcus faecalis and Escherichia coli were grown in Mueller-Hinton Broth (MHB) at densities of 0.5 MacFarland units and were used for determining the antibacterial activity of fluoxetine against Enterococcus faecalis and Escherichia coli. The mentioned cultures were swabbed over the surface of sterile Mueller-Hinton Agar (MHA) plates, in which the wells of 6 mm diameters were made to allow the different concentrations of fluoxetine, and incubated at standard conditions overnight. Then, the plates were observed for zones of inhibition, which were measured in millimetres (mm), to determine the antibacterial activity of fluoxetine. The exper iments were done in duplicate. Ampicillin and rifampicin were used as positive control s for Enterococcus faecalis an d Escherichia coli respectively. Minimum inhibito ry concentr ation determination The microdilution method was adopted, as described by Meiyazhagan et al.,(19) to find out the minimum inhibitory concentrations (MICs) of fluoxetine against Enterococcus faecalis and Escherichia coli. Here, in 96 well plates, 300 µg/mL of fluoxetine was serially diluted using 200 µl of MHB to achieve a final concentration of 2.3 µg/mL. Later, the plates were incubated under standard conditions after adding the overnight cultures of the above-indicated organisms. After incubation, the plates were observed for turbidity, and the optical density was read at 600 nm using a spectrophotometer. The experiments were done in triplicate. Effect of fluoxetine on bacterial colony formation To find out the effect of fluoxetine on Enterococcus faecalis and Escherichia coli colonization, the drug was serially diluted in 96- well plates using MHB, then the overnight cultures were added and ncubated for 96 hours. Then, the phosphate-buffered saline (PBS) wash was done to each well for the removal of unattached cells. The adherent cells were fixed with methanol and stained with crystal violet for several minutes. The ethanol and acetone (1:9) mixture was added to the stained cells and the plate was r ead a t 570 nm using a spectrophotometer.(19) The experiments were done in triplicate. Cells without treatment acted as a negative control. Biofilm inhibition efficiency of fluoxetine The biofilm formation assay was performed in polystyrene microlitre plates as illustrated by Gowri et al.,(20) to ascertain the effect of fluoxeti ne on Enterococcus faecalis an d Escherichia coli biofilms. Briefly, the biofilm formation was achieved after incubating the Enterococcus faecalis and Escherichia coli cultures in a 12-well plate for 96 hours. After the incubation, 1x MIC and 2x MIC concentrations of fluoxetine were added and the plate again incubated for 24 hours. The non-adherent cells were then removed by PBS wash, followed by methanol fixation and crystal violet staining. Then, the excess stain was removed by washing followed by air drying. The ethanol and acetone (1:9) mixture was added to the stained cells and the pla te wa s read at 570 nm using a spectrophotometer. Cells without treatment were considered negative controls. Ampicillin and rifampicin were the positive controls for Enterococcus faecalis and Escherichia coli respectively. The experiments were done in triplicate. Poyil, Shafqat, Alfaki Catheter-associated urinary tract infections 131 Univ Med Vol. 42 No. 2 Antibacterial activity of catheter coated with fluoxetine To determine the antibacterial activity of a cathe ter c oated with fluoxetine against Enterococcus faecalis and Escherichia coli, the in vitro catheter model was adopted as described by Goda et al.(21) Briefly, small pieces of sterile catheter tubes were dipped into 25 mg/mL of fluoxetine solution for 30 mins followed by air drying. For the assay, the air-dried fluoxetine- coated tubes were placed on the sterile MHA plates which were swabbed with Enterococcus faecalis and Escherichia coli followed by 24 hours of incubation. After incubation, the plates were observed for zones of inhibition. The experiments were done in duplicate. Statistical analysis Means and standard deviations were calculated for MIC determination, colony formation, and biofilm formation assays to analyze the statistical significance. RESULTS Determination of antibacterial activity of fluoxetine The antibacterial activities of various concentrations of fluoxetine were investigated against Enterococcus faecalis and Escherichia coli and the zones of inhibition exhibited by different concentrations of fluoxetine against prevalent microbes involved in CAUTI are presented in Figure 1. As seen in the Figure, the antibacterial activity was achieved with 75 µg of fluoxetine against Enterococcus faecalis and 125 µg of fluoxetine against Escherichia coli. It is noted that, when the concentrations increased, the activity of fluoxetine was also higher against both of the microbes. MIC determination The MICs of fluoxetine were evaluated against Enterococcus faecalis and Escherichia coli involved in CAUTIs and the lowest concentration of fluoxetine which inhibited the growth of the respective bacteria was calculated, as presented in Figur e 2. As shown, the calculated MICs of fluoxetine was 18.75 µg/mL against Enterococcus faecalis and 37.5 µg/mL against Escherichia coli. Effect of fluoxetine on bacterial colonization The activities of fluoxetine against colony formation by Enterococcus faecalis a nd Escherichia coli wer e studi ed and are represented in Figure 3. As shown, the presence of fluoxetine up to its MIC level in polystyrene Figure 1. Fluoxetine exhibited zone of inhibition against A) Enterococcus faecalis B) Escherichia coli 132 plates did not allow any bacterial growth on the surface. Surprisingly, even traces of fluoxetine present in the wells were able to decrease the colony-forming ability of Enterococcus faecalis and Escherichia coli on the surface of the plate when compared with untreated wells which is reflected in the results. Effect of fluoxetine on biofilm formation The effects of fluoxetine on Enterococcus faecalis and Escherichia coli biofilm formation, after treatment with various concentrations of the drug, have been quantified and are shown in Figure 4. As seen in the Figure, the percentage of effects of fluoxetine on Enterococcus faecalis biofilm formation was calculated as 64.4 and 70 for 1x MICs and 2x MICs respectively. In contrast, Escherichia coli biofilm formation was reduced by 69% and 74% after being treated with 1x MICs and 2x MICs respectively. Act ivity of f luoxetine coated cathet ers against the bacteria Antibacterial activity was investigated for the fluoxetine-coate d catheter s against Enterococcus faecalis and Escherichia coli under suitable in vitro conditions as presented in Figure 5. As seen in the Figure, the drug-coated Figure 2. MICs of fluoxetine against Enterococcus faecalis and Escherichia coli Values are for mean ± standard deviation (error bar) Figure 3. Graphical representation of fluoxetine effect on Enterococcus faecalis and Escherichia coli colony formation Poyil, Shafqat, Alfaki Catheter-associated urinary tract infections 133 Univ Med Vol. 42 No. 2 catheter tube exhibited a clear zone of inhibition around the silicone tube which represents the antibacterial activity of fluoxetine. DISCUSSION Catheter-associated urinary tract infection is one of the most significant hospital-associated infections causing serious clinical complications leading to high morbidity and mortality owing to their polymicrobial nature, which creates treatment challenges because of the antibiotic resistance and biofilm-forming ability of the prevalent organisms.(22,23) Considering these facts, there have been novel ways of searching for new antibacterial agents, and one of them is the repurposing of existing drugs for a different scope that has achieved much attention owing to their known pharmaceutical profiles. Here, the anti- depression drug fluoxetine was repurposed for its antibacterial activity against E. faecalis and E. coli which are prevalent in CAUTI. The fluoxetine antibacterial activity was explored and the lowest inhibitor y concentr ation was determined. Recently, duloxetine had been evaluated against multi-drug resistant E. coli and Figure 4. Percentage of biofilm inhibition of fluoxetine against A) Enterococcus faecalis B) Escherichia coli Note: PC-Positive control Figure 5. Antibacterial activity of fluoxetine coated catheter tube against A) Escherichia coli B) Enterococcus faecalis. Note: UN- Uncoated, C- coated with drug fluoxetine 134 found to have excellent activity when combined with chloramphenicol.(24) Correspondingly, the antibacterial and anti-biofilm activity of the repurposed drug auranofin was investigated and the drug was recorded to possess potential activity against Bacteroides fragilis.(25) In the same way, the anti-depression dr ug sertraline was investigated for its antibacterial activity against E. faecalis, Acine tobacte r baumanii, Pseudomonas aeruginosa, and E. coli. (26) Various repurposed drugs such as amodiaquine, curcumin, ibuprofen, ellagic acid, and quercetin were evaluated against two important ESKAPE pathogens and the evaluation revealed the antibacterial activity of curcumin and ellagic acid against Staphylococcus aureus a nd Pseudomonas aeruginsa.(27) The antiamoebic drug diiodohydroxyquinoline was repurposed for its antibacterial activity against Clostridioides difficile. (2 8) Likewise , mitomycin-c was repurposed for the antibacterial activity against Klebsiella pneumoniae and found to have enhanced antibacterial activity when combined with imipenem.(29) In the present study, fluoxetine was studied for its effect on E. faecalis and E. coli colony formation which is the most important stage in biofilm formation. The insertion of a catheter may provide an entry for bacteria to initiate infection in the urinary tract by adhering to the catheter surface. (30) Once the bacteria are attached to the surface area, they can form a complex structure that protects the bacteria from external sources, such as host defence mechanisms resulting in the development of antibiotic resistance, making the management of CAUTI critical.(31) Therefore, the quest is to concentrate on each stage of biofilm formation, from adhesion to mature biofilm, while studying biofilm prevention.(32,33) Therefore, the effect of fluoxetine was studied on E. faecalis and E. coli colony formation and it was found that the drug was able to prevent colony formation of E. faecalis and E. coli on surfaces, and thereby inhibiting biofilm formation. Similarly, etoposide- A was repurposed for its anti-biofilm activity against S. aureus and has shown excellent activity in removing the biofilm which formed on hydroxyapatite.(34) Also, the drug penfluridol was repurposed for its antibacterial and anti- biofilm activities against E. faecalis,(35)with promising results. Besides the antibacterial and antibiofilm activity, fluoxetine was studied for the effect of a coating on the silicone catheter tube which was evaluated against E. faecalis and E. coli. The coating of the catheter tube is an excellent approach to prevent the microbial colonization of uropathogens in the inner and outer surfaces of the tube. Hence, our study showed the efficacy of fluoxetine-coated silicone catheter tubes against E. faecalis and E. coli in the in- vitro bladder model which imitates the general process. The drug fluoxetine was able to suppress the growth of E. faecalis and E. coli which was represented by the clear zone of inhibition. Similarly, the antibacterial activity of silver nanocomposite-coated catheter tubes was s t ud i e d a ga i n s t E. co l i a n d S . a u re u s representing biofilm inhibition.(36) In another report by Abbott et al.(37) on repurposing fosfomycin, it was said that the drug showed excellent activity against E. faecalis in bladder infection in-vitro model. . In short, fluoxetine can be used as an alternative for CAUTI when the mechanisms of action will be explored and in vivo efficacy of the drug is analyzed. CONCLUSION This study demon strated tha t the antidepressant drug fluoxetine can make an excellent antibacterial drug and an anti-biofilm coating component. Further, in vivo analysis of fluoxetine is needed to determine the efficacy against CAUTI. CONFLICT OF INTEREST The authors declare that the present study was performed in the absence of any conflict of interest. Poyil, Shafqat, Alfaki Catheter-associated urinary tract infections 135 Univ Med Vol. 42 No. 2 ACKNOWLEDGEMENT The authors are grateful to the Deanship of Scientific Research, Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia, for its support and encouragement in conducting the research and publishing this report. AUTHOR CONTRIBUTIONS Conceptualization MMP; methodology and experiments MMP, MAA; result analysis and interpretation MMP, RS, MAA; writing of original draft RS, MAA; review and editing MMP. All authors have read and agreed to the published version of the manuscript. FUNDING None. REFERENCES 1. Skelton-Dudley F, Doan J, Suda K, Holmes SA, Evans C, Trautner B. Spinal cord injury creates unique challenges in diagnosis and management of catheter-associated urinary tract infection. Top Spinal Cord Inj Rehabil 2019;25:331-9. doi: 10.1310/sci2504-331. 2. Medina M, Castillo-Pino E. An introduction to the epidemiology and burden of urinary tract infections. Ther Adv Urol 2019;11:3-7. doi: 10.1177/1756287219832172. 3. Papanikolopoulou A, Maltezou HC, Stoupis A, et al. Catheter-associated urinary tract infections, bacteremia, and infection control interventions in a hospital: a six-year time-series study. J Clin Med 2022;11:5418. doi: 10.3390/jcm11185418. 4. Wooller KR, Backman C, Gupta S, Jennings A, Hasimja-Saraqini D, Forster AJ. A pre and post intervention study to reduce unnecessary urinary catheter use on general internal medicine wards of a large academic health science center. BMC Health Serv Res 2018;18:1-9. https://doi.org/ 10.1186/s12913-018-3421-2. 5. Saint S, Greene MT, Krein SL, et al. A program to prevent catheter-associated urinary tract infection in acute care. N Engl J Med 2016;374:2111-9. https://doi.org/10.1056/NEJMoa1504906. 6. Flores-Mireles A, Hreha TN, Hunstad DA. Pathophysiology, treatment, and prevention of catheter-associated urinary tract infection. Top Spinal Cord Inj Rehabil 2019 Summer;25:228-40. doi: 10.1310/sci2503-228. 7. Magill SS, O’Leary E, Janelle SJ, et al. Changes in prevalence of health care–associated infections in US hospitals. N Engl J Med 2018;379:1732-44. https://doi.org/10.1056/NEJMoa1801550. 8. Kim B, Pai H, Choi WS, et al. Current status of indwelling urinary catheter utilization and catheter-associated urinary tract infection throughout hospital wards in Korea: a multicenter prospective observational study. PLOS ONE 2017; 12:e0185369. https://doi.org/10.1371/journal. pone.0185369. 9. Bjarnsholt T. The role of bacterial biofilms in chronic infections. APMIS Suppl 2013 ;136:1-51. doi: 10.1111/apm.12099. 10. Sharma G, Sharma S, Sharma P, et al. Escherichia coli biofilm: development and therapeutic strategies. J Appl Microbiol 2016;121:309-19. https://doi.org/10.1111/jam.13078. 11. Sandhu R, Sayal P, Jakkhar R, Sharma G. Catheterization-associated urinary tract infections: epidemiology and incidence from tertiary care hospital in Haryana. J Health Res Rev 2018;1;5:135-41. 12. Walker JN, Flores-Mireles AL, Lynch AJ, et al. High-resolution imaging reveals microbial biofilms on patient urinary catheters despite antibiotic administration. World J Urol 2020;38: 2237-45. doi: 10.1007/s00345-019-03027-8. 13. Olivares E, Badel-Berchoux S, Provot C, Prévost G, Bernardi T, Jehl F. Clinical impact of antibiotics for the treatment of Pseudomonas aeruginosa biofilm infections. Front Microbiol 2020;10:2894. https://doi.org/10.3389/fmicb.2019.02894. 14. Hrvatin V. Combating antibiotic resistance: new drugs or alternative therapies? CMAJ 2017;189: E1199. doi: 10.1503/cmaj.109-5469. 15. Kamali E, Jamali A, Ardebili A, Ezadi F, Mohebbi A. Evaluation of antimicrobial resistance, biofilm forming potential, and the presence of biofilm- related genes among clinical isolates of Pseudomonas aeruginosa. BMC Res Notes 2020; 13:1-6. https://doi.org/10.1186/s13104-020-4890-z. 16. Karigoudar RM, Karigoudar MH, Wavare SM, Mangalgi SS. Detection of biofilm among uropathogenic Escherichia coli and its correlation with antibiotic resistance pattern. J Lab Physicians 2019;11:017-22. doi: 10.4103/ JLP.JLP_98_18. 17. T hangamani S, Younis W, Seleem MN. Repurposing ebselen for treatment of multidrug- resistant staphylococcal infections. Sci Rep 2015; 5:11596. doi: 10.1038/srep11596. 136 18. Gowri M, Sofi Beaula W, Biswal J, et al. β-lactam substituted polycyclic fused pyrrolidine/ pyrrolizidine derivatives eradicate C. albicans in an ex vivo human dentinal tubule model by inhibiting sterol 14-α demethylase and cAMP pathway. Biochim Biophys Acta 2016;1860:636- 47. doi: 10.1016/j.bbagen.2015.12.020. 19. Meiyazhagan G, Raju R, Winfred SB, et al. Bioactivity studies of β-lactam derived polycyclic fused pyrroli-dine/pyrrolizidine derivatives in dentistry: in vitro, in vivo and in silico studies. PLoS ONE 2015;10:e0131433. doi: 10.1371/ journal.pone.0131433. 20. Gowri M, Jayashree B, Jeyakanthan J, Girija EK. Sertraline as a promising antifungal agent: inhibition of growth and biofilm of Candida auris with special focus on the mechanism of action in vitro. J Appl Microbiol 2020;128:426-37. https:// doi.org/10.1111/jam.14490. 21. Goda RM, El-Baz AM, Khalaf EM, Elkhooly TA, Shohayeb MM. Combating bacterial biofilm formation in urinary catheter by green silver nanoparticle. Antibiotics (Basel) 2022;11:495. https://doi.org/10.3390/antibiotics11040495. 22. Jordan RP, Malic S, Waters MG, DJ Stickler, DW Williams. Development of an antimicrobial urinary catheter to inhibit urinary catheter encrustation. Microbiol Discov 2015; 3:1. http://dx.doi.org/ 10.7243/2052-6180-3-1. 23. Ansari MA, Albetran HM, Alheshibri MH, et al. Synthesis of electrospun TiO2 nanofibers and characterization of their antibacterial and antibiofilm potential against gram-positive and gram-negative bacteria. Antibiotics (Basel) 2020;9:572. doi: 10.3390/antibiotics9090572. 24. Shi D, Hao H, Wei Z, et al. Combined exposure to non-antibiotic pharmaceutics and antibiotics in the gut synergistically promote the development of multi-drug-resistance in Escherichia coli. Gut Microbes 2022;14:2018901. https://doi.org/ 10.1080/19490976.2021.2018901. 25. Jang HI, Eom YB. Antibiofilm and antibacterial activities of repurposing auranofin against Bacteroides fragilis. Arch Microbiol 2020;202: 473-82. https://doi.org/10.1007/s00203-019-01764- 3. 26. Ayaz M, Subhan F, Ahmed J, et al. Sertraline enhances the activity of antimicrobial agents against pathogens of clinical relevance. J Biol Res (Thessalon) 2015;22:4. doi: 10.1186/s40709-015- 0028-1. 27. Kamurai B, Mombeshora M, Mukanganyama S. Repurposing of drugs for antibacterial activities on selected ESKAPE bacteria Staphylococcus aureus and Pseudomonas aeruginosa. Int J Microbiol 2020;2020. https://doi.org/10.1155/ 2020/8885338. 28. Abutaleb NS, Seleem MN. Repurposing the antiamoebic drug diiodohydroxyquinoline for treatment of Clostridioides difficile infections. Antimicrob Agents Chemother 2020;64e02115- 19. doi: 10.1128/AAC.02115-19. 29. Pacios O, Fernández-García L, Bleriot I, et al. Enhanced antibacterial activity of repurposed mitomycin C and imipenem in combination with the lytic phage vB_KpnM-VAC13 against clinical isolates of Klebsiella pneumoniae. Antimicrob Agents Chemother 2021;65:e0090021. doi: 10.1128/AAC.00900-21. 30. Zhu Z, Wang Z, Li S, et al. Antimicrobial strategies for urinary catheters. J Biomed Mater Res A 2019; 107:445-67. doi: 10.1002/jbm.a.36561. 31. Pelling H, Nzakizwanayo J, Milo S, et al. Bacterial biofilm formation on indwelling urethral catheters. Lett Appl Microbiol 2019;68:277-93. doi: 10.1111/ lam.13144. 32. Ghosh A, Jayaraman N, Chatterji D. Small- molecule inhibition of bacterial biofilm. ACS Omega 2020;5:3108-15. doi: 10.1021/acsomega. 9b03695. 33. Muhammad MH, Idris AL, Fan X, et al. Beyond risk: bacterial biofilms and their regulating approaches. Front Microbiol 2020;11:928. doi: 10.3389/fmicb.2020.00928. 34. Ganesan V, Meiyazhagan G, Devaraj M, et al. Repurposing the antibacterial activity of etoposide% a chemotherapeutic drug in combination with eggshell-derived hydroxyapatite. ACS Biomater Sci Eng 2022;8: 682-93. doi: 10.1021/acsbiomaterials.1c01481. 35. Zeng X, She P, Zhou L, et al. Drug repurposing: antimicrobial and antibiofilm effects of penfluridol against Enterococcus faecalis. Microbiologyopen 2021;10:e1148. doi: 10.1002/ mbo3.1148. 36. Rahuman HBH, Dhandapani R, Palanivel V, Thangavelu S, Paramasivam R, Muthupandian S. Bioengineered phytomolecules-capped silver nanoparticles using Carissa carandas leaf extract to embed on to urinary catheter to combat UTI pathogens. PloS One.2021;16:e0256748. doi: 10.1371/journal.pone.0256748. 37. Abbott IJ, Van Gorp E, van der Meijden A, et al. Oral fosfomycin treatment for enterococcal urinary tract infections in a dynamic in vitro model. Antimicrob Agents Chemother 2020;64:e00342- 20. doi: 10.1128/AAC.00342-20. Poyil, Shafqat, Alfaki Catheter-associated urinary tract infections