The Effect of Urinary Catheters on Microbial Biofilms and Catheter Associated Urinary Tract Infections Sahra Kırmusaoğlu1*, Seyhun Yurdugül2, Ahmet Metin3, Suphi Vehid4 Purpose: The aims of this study were to determine relationship between biofilm producer microorganisms attached to urinary catheters (UCs) and urinary catheter-associated urinary tract infections (CAUTIs), to determine the rate of CAUTI development and the relationship between CAUTI and catheterization period in catheterized patients. Materials and Methods: Urinary catheters from 143 inpatients who were hospitalized in Abant Izzet Baysal University Hospital Urinary Service, and urine samples of these patients before and after catheterization of urinary catheter were collected. Culture-based microbiological evaluation of urinary catheters removed from inpatient and urine samples collected from inpatients were performed before and after catheterization of urinary catheter to identify various organisms and determine biofilm production by them. Results: The incidence of CAUTIs was 13% (18/143) in catheterized inpatients. Biofilm producer microorganisms such as Escherichia coli (E. coli ), Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis that were isolated from UCs removed from inpatients were found to cause CAUTI (P < .001). Conclusion: Incidence of CAUTIs is increased by the usage of UCs and prolonged catheterization period. Keywords: urinary catheter; biofilm; catheter-associated urinary tract infection; Escherichia coli; Klebsiella pneu- monia; Pseudomonas aeroginosa; Proteus mirabilis. INTRODUCTION Biofilm infections cause problems in hospitalized and immunocompressed patients.(1) Indwelling de- vice related urinary tract infections are one of the most common biofilm infections of the urinal system.(2,3) In Europe, the mortality rate of nosocomial infections is 10%, 97% of which are related with catheters.(4) Ap- proximately 80% of nosocomial urinary tract infections are associated with indwelling urinary catheters.(5) Urinary bladder infection that is associated with biofilm causes failure in the drainage of urine due to congestion of catheter lumen that can be caused by crystalline de- bris of biofilms.(4) Biofilm embedded bacterial commu- nities can be made up of heterogeneous cells that can resist immune defence and antibiotics because of their low metabolic activity caused by nutrient and oxygen limitations at the lower parts of the biofilm, decreased penetration of antibiotics through biofilm caused by binding of antibiotics to the structural contents of the biofilm matrix.(1) Biofilms have an important role in the pathogenesis of bacteria in indwelling device related infections. Biofilms are formed by bacteria, which at- tach to biotics such as, tissues, or abiotic surfaces such as, medical devices and are slime-like glycocalyx. Af- ter colonization of bacteria, mature biofilms disperse 1 Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, T.C. Haliç University, Sütlüce-Beyoğlu/Istanbul 34445, Turkey. 2 Department of Biology, Faculty of Arts and Sciences, Abant Izzet Baysal University, Bolu 14030, Turkey. 3 Department of Urology, Faculty of Medicine, Abant Izzet Baysal University, Bolu 14030, Turkey. 4 Department of Public Health, Cerrahpaşa Faculty of Medicine, Istanbul University Istanbul 34098, Turkey. *Correspondence: Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, T.C. Haliç University, Sütlüce-Beyoğlu/Istanbul 34445, Turkey. Tel: +90 212 924 24 44-1148. Fax: +90 212 999 78 52. E-mail: kirmusaoglu_sahra@hotmail.com. Received October 2016 & Accepted March 2017 which leads to bacterial spread to the whole body.(6, 7,8) Antimicrobial resistant indwelling device related infections can cause chronic and recurrent infections. (1) Untreated urinary tract infection (UTI) can lead to acute pyelonephritis, chronic renal infection, bacterial vaginosis, chronic bacterial prostatitis, bacteraemia and death.(3) Enterococcus spp. especially Enterococcus faecalis, Methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus epider- midis, Providencia stuartii and Morganella morganii are the main urinary pathogens that cause biofilm relat- ed urinary tract infections.(3,9) The aims of this study were to determine relationship between biofilm producer microorganisms attached to urinary catheters (UCs) and urinary catheter associated urinary tract infections (CAUTIs), to determine the rate of CAUTI development and the relationship between CAUTI and catheterization period in catheterized pa- tients. MATERIALS AND METHODS Study population All patients who had been hospitalized in Urology MISCELLANEOUS Miscellaneous 3028 Clinics of Abant Izzet Baysal University Faculty of Medicine Hospital due to health problems which did not include urinary tract infections (UTI) in a period of 6 months, were included in study. Study participants were inpatients who used urinary catheter, did not have UTI, were not immunosuppressed, had no other diseas- es, and did not take antibiotic prophylaxis before taking catheter out of the body. Inclusion criteria was presence of urinary catheter. Exclusion criteria were UTI, being immunosuppressed, having other diseases, and antibi- otic prophylaxis before taking catheter out of the body. Informed consent was obtained from all the inpatients participated in the study. This study was approved by Ethics Committee of Clinical Studies of T.C. Haliç Uni- versity, Institute of Health Sciences. Urine samples of those patients were collected before and after catheterization of urinary catheter to evaluate urine analysis and urine cultures. Approximately 4-5 cm long tips of Foley urinary catheters were cut by ster- ile scalpel and transferred to the sterile urine container to culture and detect whether biofilms were formed by microorganisms grew. Urine samples taken from the patients before and after the catheterization of urinary catheter were cultured. These samples were processed and evaluated microbiologically and biochemically in Abant Izzet Baysal University Department of Biology Biochemistry Laboratory.(Table 1) Study design and Evaluation This study which was performed in prospective single center and on random inpatients was conducted in Urol- ogy Clinics of Abant Izzet Baysal University Faculty of Medicine Hospital in Bolu, Turkey. Inpatients who had urinary catheters participated in this study in the period of 6 months. After informed consent was obtained from all the inpatients participated in the study, and being approved by ethics committee, the study proceeded as explained below. Not only development of biofilm producer uropatho- gens on UCs, but also positivities of urinalysis test that contains leukocyte, nitrite and microorganism, and de- velopment of biofilm producer uropathogens in urine were defined as CAUTIs. Procedures Analysis of urinary catheters and urine Urinary catheters were transferred into tryptic soy broth (TSB) (Merck TM) and incubated for 24 hours at 37o C. Then, after observing microbial growth, a subsequent transfer was done into the blood and EMB agars and incubated for 24 hours at 37o C.(10) Urines were also in- oculated into the blood and EMB agar and incubated for 24 hours at 37o C. Then, the microorganisms isolated from urinary catheters and urines were identified. The capability of biofilm production of microorganisms was determined by Congo red agar method, tube method and microtiter plate assay.(11,12) Since not only microbiological growth and develop- ment of bacteriuria (higher than 105 cfu/mL of microor- ganisms grown in urine cultures)(5,13), but also the posi- The effect of UCs on microbial biofilms and CAUTIs- Kırmusaoğlu et al. Table 1. All processes of methods Urine Sample Before Catheterization Urine Sample After Catheterization Urinary Catheter Culturation of urines Culturation of urines Culturation of tips of catheters Identification of isolates Identification of isolates Identification of isolates Antibiotic susceptibility tests Antibiotic susceptibility tests Antibiotic susceptibility tests Assessment of biofilm Production Assessment of biofilm Production Assessment of biofilm production a) Qualitative Determination of Biofilm a) Qualitative Determination of Biofilm a) Qualitative Determination of Biofilm Congo red agar method (CRA) Congo red agar method (CRA) Congo red agar method (CRA) Tube method (TM) Tube method (TM) Tube method (TM) b) Quantitative Determination of Biofilm b) Quantitative Determination of Biofilm b) Quantitative Determination of Biofilm Microtiter plate assay Microtiter plate assay Microtiter plate assay Urinalysis test Urinalysis test Number Percentage % Urinary Catheter / Patient 143 100 Catheter Colonized 75 52 Catheter uncolonized 68 48 Total Microorganisms Isolated from Catheters 88 100 The Incidence of Biofilm Producer Microorganisms in Whole Microorganisms 18 21 (18/88) The Incidence of Biofilm Related UTI 18 13 (18/143) The Incidence of Biofilm Producer Microorganisms in Catheters Colonized 18 24 (18/75) Table 2. Status of catheters and incidences Vol 14 No 02 March-April 2017 3029 tivities of leukocyte (higher than 10 leukocytes per mm3 of urine(14)) and nitrite, and the observation of micro- organism in urine microscopy(15) were criterias for the definition of CAUTI, the complete urinalysis including pH, nitrite, and microscopic (leukocyte, bacteria, crys- tals) were also performed. Identification of Microorganisms After incubation of urines and urinary catheters, mi- croorganisms that grow on blood and EMB agar me- dia were determined whether they are gram positive or negative according to gram staining. Identification of S. aureus was based upon colony morphology on blood and mannitol-salt agar, catalase and coagulase tests. Identifications of Gram negative bacteria was based upon colony morphology on EMB agar, IMVIC test, and API systems.(10) Assessment of MRSA and MRSE Methicillin resistance of S. aureus and S. epidermidis is determined by cefoxitin by Kirby Bauer disk diffusion method and broth microdilution method according to the Clinical Laboratory Standards Institute criteria 2013 (CLSI). Bacterial suspensions of Staphylococcal strains were prepared in Tryptic soy broth (TSB), and adjusted to 0.5 McFarland (1.108 cfu/mL). The staphylococcal strains from bacterial suspensions were inoculated by the spread plate method to Mueller Hinton agar, and 30 µg cefoxitin disks were put on the inoculated plate. Zone diameters of cefoxitin were measured after incu- bation in 24 hours at 37°C. The zone measurements were categorized into sensitive (≥ 22 mm), or resistant (≤ 21 mm for cefoxitin) categories.(16) Assessment of Biofilm Production a) Qualitative Determination of Biofilm Congo red agar method (CRA). The strains isolated from urinary catheters and urines were inoculated to Congo red agar media (CRA) (Merck TM) as described by Freeman et al. (1989) to identify whether strains were biofilm producer or not.(11) The CRA medium was constructed by mixing 0.8 g of Congo red and 36 g of sucrose (Sigma, Missouri, EUA) to 37g/L of brain heart infusion (BHI) agar (Oxoid, Basingstoke, Hampshire, England). After an incubation period of 24 hours at 37°C, morphology of colonies that undergone to differ- ent colours were differentiated as biofilm producers or not. Black colonies with a dry crystalline consistency indicated biofilm producers, whereas colonies that re- mained pink were non-biofilm producers. Tube method (TM). The biofilm formation of strains that were isolated from urinary catheters and urines was also detected by tube method described by Christensen et al. (1985). The strains were inoculated in polystyrene test tube which contained TSB and incubated for 24 h at 37°C.(12) The sessile strains of which biofilms ad- hered on the walls of polystyrene test tube were stained with saphranin for 1 hour, after planktonic cells were discharged by washing twice with phosphate buffered saline (PBS). Then, saphranin stained polystyrene test tube was washed twice with PBS to discharge saphranin stain. After air drying of the test tube, the occurence of visible film lining the walls and the bottom of the tube indicates biofilm production.(12) b) Quantitative Determination of Biofilm Preparation of Bacterial Suspension Bacterial suspensions of strains that were isolated from urinary catheters and urines were prepared and adjusted to 0.5 McFarland (1.108 cfu/mL). This bacterial sus- pensions were twenty fold (1/20) diluted to gain 5.106 cfu/mL. Bacterial suspension was adjusted by ten fold dilution (1/10) in such a way as the final concentration become 5.105 cfu/mL. Microtiter Plate Assay 180 µl of TSB and 10 µl of bacterial suspensions were inoculated into 96-well flat-bottomed sterile poly- Table 3. Microorganisms isolated in urinary catheters and urine samples Urinary Catheter Urine (After Catheterization) CAUTI Microorganisms Microbial Growth Biofilm Producer Microbial Growth Biofilm Producer No % No % No % No % No % S. epidermidis MRSE 19 22 14 26 0 0 0 0 0 0 MSSE 2 2 1 2 0 0 0 0 0 0 S. aureus MRSA 18 20 9 17 0 0 0 0 0 0 MSSA 2 2 2 4 0 0 0 0 0 0 E. coli 29 33 18 34 14* 78 14* 78 14* 78 Klebsiella pneumonia 4 5 1 2 1* 6 1* 6 1* 6 Candida albicans 8 9 4 8 0 0 0 0 0 0 Streptococcus spp. 3 3 1 2 0 0 0 0 0 0 Pseudomonas aeroginosa 2 2 2 4 2* 11 2* 11 2* 11 Proteus mirabilis 1 1 1 2 1* 6 1* 6 1* 6 Total 88 100 53 100 18* 100 18* 100 18* 100 Abbreviations: %, percentage; no, number. * Parameters which defines CAUTI were compared by Pearson χ2 test (χ2: 49.685, P < .001). The effect of UCs on microbial biofilms and CAUTIs- Kırmusaoğlu et al. Miscellaneous 3030 styrene microplate (LP Italiana SPA TM) to obtain 5.105 cfu/mL as a final concentration (ten fold dilution (1/10)). Uninoculated wells containing sterile TSB were used as negative controls. Microplates incubated at 24 h at 37°C. The sessile isolates of which biofilms formed on the walls of wells of microplate were stained with saphranin for 1 hour, after planktonic cells in wells of microplate had discharged by washing twice with phosphate-buffered saline (PBS) (pH 7.2) and wells had dried at 60 °C for 1 h.(12) Then, saphranin stained wells of microplates were washed twice with PBS to discharge saphranin stain. After air drying process of wells of microplate, biofilms lined the walls of the mi- croplate were measured spectrophotometrically at 595 nm by a microplate reader (Thermo Instruments TM). The studies were repeated in triplicates. Uninoculated Table 4. The catheterization periods and microorganisms that caused catheter-associated UTI Microorganisms Isolated in Urinary Catheters Microorganisms Isolated in Urines Urinalysis Cath Sex Per. M.o.s Biofilm M.o.s Biofilm M.o.s in Urine (cfu/mL) Leuko. (per mm3 of urine) pH Nitrite 1 F P. aeroginosa Positive P. aeroginosa Positive > 105 > 10 6.5 + 1 F E. coli Positive E. coli Positive > 105 > 10 5 + (ESBL +) 1 M E. coli Positive E. coli Positive > 105 > 10 5 + (ESBL +) 1 M E. coli Positive E. coli Positive > 105 > 10 8 + (ESBL +) 2 F E. coli Positive E. coli Positive > 105 > 10 6.5 + 2 M E. coli Positive E. coli Positive > 105 > 10 6.5 + 2 F P. Positive P. aeroginosa aeroginosa Positive > 105 > 10 6.5 + 2 M K. pneumonia Positive K. Positive > 105 > 10 5 + pneumonia 3 F E. coli Positive E. coli Positive > 105 > 10 6.5 + 4 M E. coli Positive E. coli Positive > 105 > 10 6.5 + (ESBL +) 4 F E. coli Positive E. coli Positive > 105 > 10 6.5 + 4 F E. coli Positive E. coli Positive > 105 > 10 6.5 + 7 F E. coli Positive E. coli Positive > 105 > 10 7.5 + (ESBL +) 7 M E. coli Positive E. coli Positive > 105 > 10 6.5 + 7 F E. coli Positive E. coli Positive > 105 > 10 6.5 + 7 M E. coli Positive E. coli Positive > 105 > 10 5 + (ESBL +) 8 M Proteus Positive Proteus Positive > 105 > 10 6.5 + mirabilis mirabilis 21 M E. coli Positive E. coli Positive > 105 > 10 5.5 + Abbreviations: Cath. per., Catheterization periods; M.o.s, Microorganisms; Leuko, Leukocytes; F, Female; M, Male. The effect of UCs on microbial biofilms and CAUTIs- Kırmusaoğlu et al. Vol 14 No 02 March-April 2017 3031 wells containing sterile TSB that were considered to be the negative controls used as blanks. The blank ab- sorbance values were used to identify whether biofilm formation of isolates exist or not. The wells of isolates of which OD values are higher than blank well are con- sidered to be biofilm producers. The Statistical Analysis The data were analyzed by the SPSS software version 21 that is licensed to Istanbul University. Pearson χ2 test was used to detect existence of significance between the cultures of urinary catheter and urine, and between urine samples that were taken before and after catheter- ization. As a result of which existence of significance between the urinary catheter and biofilm related urinary tract infection were detected. All results were consid- ered statistically significant if the p-value was equal to or less than 0.05. RESULTS 143 urinary catheter samples were collected from inpa- tients (age ranges from 20 to 75, 33 female, 110 male) who had been hospitalized in Urology Clinics of Abant Izzet Baysal University Faculty of Medicine Hospital due to health problems which did not include urinary tract infections (UTI) in a period of 6 months. 88 strains of microorganisms were isolated from urinary catheters of 68 patients among 143 (Table 2). 18 strains of microorganisms were isolated from the urines that were taken after catheterization of inpatients, but, on the other hand, no microorganisms were found in any urine samples taken before catheterization. The strains of E. coli, Klebsiella pneumonia, Pseudomonas aeroginosa and Proteus mirabilis were isolated from the urines that were taken after catheterization of inpatients (Table 3). Not only growth of definite species in biofilms of uri- nary catheters were observed, but also heterogeneous microorganisms grew in biofilms of urinary catheters. Heterogeneous microorganisms that grew in biofilms of urinary catheters were, mostly, Candida albicans, MRSA and E. coli. Some of the isolates from the urinary catheters includ- ing E. coli, Klebsiella pneumonia, Candida albicans, Streptococcus spp., MRSA, MSSA, MRSE and MSSE were found to be biofilm producers only; on the other hand, some of the isolates of E. coli, Klebsiella pneu- monia, Pseudomonas aeroginosa and Proteus mirabi- lis were found to be both biofilm producers and cause catheter-associated UTIs (CAUTIs) (Tables 3 and 4) (P < .001). Six of E. coli strains were found to be the extended spectrum beta-lactamase producers (ESBL). The values of complete urinalysis of catheterized in- patients such as positivity of leukocyte and nitrite, and bacteria seen in urine microscopy supported CAUTIs of catheterized inpatients (Table 4). The incidences of E. coli, Pseudomonas aeroginosa, Klebsiella pneumonia and Proteus mirabilis that caused CAUTIs were 78%, 11%, 6% and 6%, respectively (Table 3). The incidences of CAUTIs were 27% (9/33) and 8% (9/110) among female and male, respectively. The inci- dences of CAUTIs caused by E. coli and Pseudomonas aeroginosa were 21% (7/33) and 6% (6/33) among fe- male, respectively. The incidences of CAUTI caused by E. coli, Klebsiella pneumonia and Proteus mirabilis were 6% (7/110), 1% (1/110) and 1% (1/110) among male, respectively (Table 4). Although, four strains of E. coli, four strains of Candida albicans, one strain of Streptococcus spp. and 26 strains of S. aureus and S. epidermidis isolated from urinary catheter of patients were biofilm producers, they were not found to cause UTIs (Table 3). 18 strains of microorganisms isolated from urinary catheters of patients were found to be biofilm produc- ers and caused biofilm or CAUTIs. The incidence of biofilm related UTI was 13% (18/143) in catheterized inpatients. The incidence of biofilm producer micro- organism was 21% (18/88) among all microorganisms that were isolated from colonized urinary catheters (Ta- ble 2). Leukocytes and microorganisms were observed in urine microscopy, nitrite were positive, and at least 104 cfu/mL of microorganisms grow in urine cultures of these 18 catheterized inpatients who also showed clini- cal symptoms of UTI. These data show that biofilm pro- ducer microorganisms that can adhere to urinary cathe- ters facilitate adhesion, colonization of microorganisms and cause UTI in catheterized patients (P < .001). The incidence of CAUTI in patients who were cathe- terized four days and below, and above four days were 9% (12/127) and 38% (6/16), respectively (Table 5). 91 percent of inpatients who were catheterized four days and below and 62 percent of inpatients who were cathe- terized four days and above did not have CAUTI. DISCUSSION In our study, Escherichia coli, Klebsiella pneumoni- ae, Pseudomonas aeruginosa, Proteus mirabilis were found to be the main urinary pathogens that cause cath- eter-associated urinary tract infection (CAUTIs). Alves et al., as well as Kucheria et al. also concluded that these pathogens were the main urinary pathogens(3,17) (Table 3). Urinary tract infection is caused by bacteria that colo- nize urinary catheters produce biofilm and disperse to the bladder. Diagnosis of symptomatic CAUTI varies. CAUTI is defined based on microbiological growth, de- velopment of bacteriuria and UTI symptoms during and after catheterisation period.(5) Stenzelius defined CAU- TI as bacteriuria higher than 105 cfu/mL of microorgan- isms grown in urine cultures and urinary symptoms dur- ing and after catheterization period.(18) Thibon defined Table 5. The percentages of CAUTIs according to catheterization periods Catheterization days Inpatient with CAUTI Inpatient without CAUTI Total Inpatient ≤ 4 9% (12/127)* 91% (115/127)* 127 > 4 38% (6/16)* 62% (10/16)* 16 Abbreviations: CAUTI, catheter-associated urinary tract infection * Parameters which defines CAUTI were compared by Pearson χ2 test (χ2: 20.232, P < .001). The effect of UCs on microbial biofilms and CAUTIs- Kırmusaoğlu et al. Miscellaneous 3032 UTI as bacteriuria (higher than 105 cfu/mL of micro- organisms grown in urine cultures) with higher than 10 leukocytes per mm3 of urine.(14) Karchmer defined UTI as bacteriuria equal and higher than 105 cfu/mL of mi- croorganisms grown in urine cultures.(13) According to the Center for Disease Control and Prevention (CDC), the positivities of leukocyte, and nitrite, the observation of microorganism in urine microscopy, and at least 104 cfu/mL of microorganisms grown in urine cultures of catheterized patients indicates CAUTIs.(15) In our study, these parameters were positive in inpatients who had CAUTIs, and they also showed clinical symptoms of UTI. However, high and rising of urine pH leads crys- tallization in urine that promotes biofilm formation.(19) In our study, urine pHs of inpatients who had CAUTI ranged 5-8. Urine pHs of two inpatients who had CAU- TI elavated from 5 and 5.5 to 8 and 7.5, respectively. Rising of pH in urine can be due to the ability of urease production of bacteria that colonize urinary catheter.(19) Although four strains of E. coli, four strains of Can- dida albicans, one strain of Streptococcus spp. and 26 strains of S. aureus and S. epidermidis that were iso- lated from urinary catheters of inpatients and found to be biofilm producers, these isolates were not present in urine samples of patients, and inpatients did not show clinical symptoms of UTI. So, these isolates did not cause urinary tract infection. The reason for this may be due to undetachment of biofilm, so sessile microorgan- ism did not disperse from catheter to urine and did not cause UTI till that time. When the biofilm embedded microorganism are detached and dispersed, they cause UTI. It is hard to identify microorganism and biofilm in urine before UTI due to the down-regulation of phenol soluble modulins (PSMs) since microorganisms are just identified in the dispersal stage of biofilm that is caused by PSMs.(20,21,22) Another reason might be due to pH of urines that are not optimum for microbial growth. Gen- erally, pH of the urine ranges between 5 to 8.5. Above pH 7.5, and below pH 6.5 bacteria can not grow effec- tively. The optimum pH for bacteria and yeast growth ranges from 6.5 to 7.5 and from 5 to 6, respectively.(23) This result can also be explained by short catheteriza- tion period. Decreased catheterization period of patient reduces the risk of CAUTI. Prolonged catheterization period of patient increased the incidence of CAUTI.(24) If catheterization period of these inpatients were pro- longed, risk of CAUTI would be increased. In our study, six patients that had CAUTI were catheter- ized more than 4 days. One patient that had CAUTI was catheterized for a period of 21 days. Twelve patients that had CAUTI were catheterized below 4 days (Table 4). The incidence of CAUTI in patients who were cath- eterized four days and below, and above four days were 9% (12/127) and 38% (6/16), respectively (Table 5). According to our study, prolonged catheterization pe- riod increases the risk of CAUTI (p < 0.05). Incidence of bacteriuria development in patients who has urinary catheter is 5%. When catheterization period prolonges to more than 7 and 14 days, incidence of bacteriuria de- velopment rises to 35% and 70 %, respectively.(5) Crouzet et al. reported that termination of catheteriza- tion at the fourth day decreased the incidence rate of CAUTI from 10.6 to 1.1.(24) Dohnt et al. found that inci- dence rate of CAUTI of short term (to 7 days) and long term catheterized patients (28 days) were approximate- ly 50% and 100%, respectively.(25) In our study, biofilm The effect of UCs on microbial biofilms and CAUTIs- Kırmusaoğlu et al. producer Proteus mirabilis that was isolated in urinary catheter was also isolated in urine of an inpatient who was catheterized for 8 days (Table 4). Proteus mirabilis that does not cause UTI in short catheterization period, causes UTI in prolonged catheterization.(4) In addition to prolonged catheterization period, the risks of CAUTI of inpatients, may be enhanced with older age, female sex and immunosuppression due to other diseases.(26) In our study, patients who had CAUTI were not immunosuppressed, had no other diseases, and half of the patients were female. Another study revealed that, before taking catheter out of the body, antibiotic prophylaxis decreased the incidence of UTI (27, 28), while antibiotic resistance can be emerged by prophylaxis.(29) In our study, antibiotic prophylaxis was not given to catheterized inpatients be- fore taking catheter out of the body. CONCLUSIONS According to this study, incidence of CAUTIs is in- creased by the usage of urinary catheters and prolonged catheterization period. 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