Bacterial Profile of Urinary Tract Infections: Evaluation of Biofilm Formation and Antibiotic Resistance Pattern of Uropathogenic Escherichia coli

Urinary tract infection (UTI) is one of the most common complaints in the outpatient clinic and a major health problem owing to the emergence of antibiotic resistance and biofilm formation. The objective of this study was to isolate and identify the causative bacterial agent of UTI and detect in vitro biofilm formation by Escherichia coli and investigate its correlation with antibiotic resistance. Urine samples from 519 patients with suspected UTIs were collected and processed by conventional microbiological procedures. Antimicrobial susceptibility testing for E. coli isolates was performed on Mueller Hinton agar (MHA) plates using the Kirby-Bauer disk diffusion method. Biofilm production was evaluated using the tissue culture plate method. Of 519 urine samples, 115 (22.1%) showed significant bacteriuria. The most common isolate was E. coli (n=57, 49.6%), followed by Klebsiella spp. (n=23, 20%). All E. coli isolates were evaluated for their ability to form biofilms in vitro. Of 57 isolates, 50 (87.7%) were biofilm producers and 7 (12.3%) were non-biofilm producers. Antibiogram of E. coli isolates revealed the highest resistance to ampicillin (96.5%) and nitrofurantoin (91.2%), followed by amoxyclav (82.5%), ceftazidime (73.7%), cefepime (71.9%), and tetracycline (71.9%). A significant association (p<0.05) was observed between biofilm formation and resistance to amoxyclav, ceftazidime, cefepime, imipenem, and nitrofurantoin. A significant correlation was noted between biofilm production and antibiotic resistance. Hence, screening of all isolates of uropathogenic E. coli for biofilm production and studying their antibiogram would allow appropriate choice of antibiotic therapy.


INTRODUCTION
Urinary tract infection (UTI) is considered as the microbial invasion of any tissues extending from the renal cortex to the urethral meatus. The urinary system includes organs that collect, store, and release urine from the body. Accordingly, UTI is classified based on the site of infection as follows: bladder (cystitis), kidney (pyelonephritis), and urethra (bacteriuria) 1 .
UTI is one of the most frequently presented complaints in outpatient clinics, and most patients are in their reproductive age (18-37 years). UTI is one of the most common hospitalacquired infections, representing as high as 35% of nosocomial infections, and accounts for the second most common cause of bacteremia in patients admitted to hospitals 2, 3 . It has been estimated that about 6 million patients have UTI per year worldwide, of which around 30,000 are treated in the wards. In India, UTI is the third most common cause of hospital admission, and its prevalence varies from 21.8 to 31.3 in different parts of the country 4,5 . Uropathogenic Escherichia coli (UPEC) is the most common cause of UTI, accounting for approximately 90% and 50% of communityacquired and hospital-acquired UTIs, respectively. E. coli is as an endogenous microorganism in the human bowel and is deemed harmless under natural conditions. E. coli from the intestine is present in the fecal matter. The passage of trace amounts of fecal matter through the urethral opening allows entry of the microorganism into the urinary tract, wherein it thrives, multiplies, and eventually causes an infection. Some common ways involved in the migration of E. coli through the urethral opening are as follows:

Sexual contact
A woman's urethra is located next to the vagina and anus, making it easy for bacteria to move into the urinary tract during sexual intercourse and sexual contact.

Improper cleaning
Wiping from the back to front after excretion can drag E. coli directly into the urethra.

Holding urine
Frequent urination facilitates the continuous flushing of bacteria such as E. coli from the system. This is particularly important before and after intercourse.

Enlarged prostate gland
This exerts extra pressure on the bladder, thereby preventing it from properly emptying and flushing E. coli from the body 6 .
About 60% to 70% of UPEC have the ability to form biofilms 7 . Relapses and chronic infections by UPEC have been associated with the ability of pathogenic strains to form biofilms. Several studies have shown that 50%-90% of isolates collected from patients with relapsed infections were biofilm producers 8 . Drug resistance among bacteria causing UTIs is increasing and considered as a major hurdle in the treatment of UTI. Biofilms protect the bacteria from the host immune response and impede the effects of antibiotics. High antimicrobial concentrations are imperative to inactivate organisms growing in a biofilm, and this may increase antibiotic resistance by 1000-fold 9 . In this context, the present study aimed to determine the correlation between biofilm production and multidrug resistance in UPEC isolates.

MATERIAL AND METHODS
The study was carried out at the Department of Microbiology, Yenepoya Medical College and Hospital, after receiving ethical clearance from the Yenepoya Ethics Committee.

Inclusion criteria
Culture isolates from urine samples of patients from all age groups with a high colony count (>10 5 colony-forming units [CFU]/mL) were included.

Exclusion criteria
Colony count < 10 5 CFU/mL Culture plates with multiple bacterial growth.

Methodology Study design
Descriptive longitudinal study

Sampling technique
Convenience sampling

Sample collection
Freshly voided midstream urine samples were collected from patients with suspected UTI in a sterile, dry, wide-necked, leak-proof universal sterile container under aseptic conditions 10 .

Culture and identification
The well-mixed and non-centrifuged urine samples were inoculated by a wire loop to deliver 0.001 mL of the specimen onto 5% sheep blood agar, cysteine-lactose electrolytedeficient agar, and MacConkey agar plates using the streak plate method following standard microbiological procedures. The plates were aerobically incubated at 37°C for 24 h and examined for the presence or absence of bacterial growth. Cultures that formed >10 5 CFU/mL were considered to have significant bacteriuria. All positive samples showing significant bacteriuria were further tested for physical characteristics such as colony morphology, odor, swarming, and presence of hemolysis on respective media using different biochemical reactions performed as per standard procedures. Thus, gram-negative rods were identified with the help of a series of biochemical tests such as triple-sugar iron agar, indole, Simmons citrate agar, oxidase, urease, and motility tests. Morphologically identical colonies of suspected strains were taken from agar plates, suspended in nutrient broth, and vortexed. The suspensions were inoculated into butts and slants of biochemical testing media. The inoculated media were aerobically incubated at 37°C for overnight, and bacteria were identified following the standard flow chart. Gram-positive cocci were identified based on their reactions in catalase and coagulase tests 10 .

Procedure
Each bacterial sample was emulsified in sterile saline in a test tube (mixed thoroughly so that no solid particles remained in the solution). The turbidity of the solution was evaluated by matching with turbidity standards (0.5 McFarland standard). The sterile swab was dipped into broth culture, and the excess fluid from the swab was removed by gently squeezing the swab against the wall of the test tube. Using the lawn culture method, the swab was streaked onto a sterile MHA plate, which was allowed to dry for a few minutes. Antibiotic disks (6 on each plate) were aseptically placed on MHA plate and the plate was incubated for 18-24 h at 37°C.

Observation and interpretation
The diameter of the zone of inhibition for each antibiotic was recorded using a metric ruler. Results were interpreted as sensitive, moderately sensitive, and resistant, as per the CLSI guidelines 11 . Biofilm production 12 .
Biofilm production was determined using the tissue culture plate (TCP) method.

Procedure
A colony from an overnight grown culture of isolates on MacConkey agar plate was inoculated into a 3 mL brain heart infusion (BHI) broth with  (Fig. 1).

Statistical analysis
Statistical analysis was carried out using InStat software. A chi-square (χ 2 ) test was performed and a value of p<0.05 was considered statistically significant.

RESULTS
A total of 519 urine samples were processed during the study period of 1 year, of which 115 (22.1%) samples showed significant bacterial growth (>10 5 CFU/mL). There were more female patients (n=75; 65.2%) than male patients (n=40; 34.7%). Patients were divided into nine age groups. The incidence of UTI was the highest among women from the 21-30 year age group followed by women from the 31-40 year age group and was the lowest for women 70 years or older. Among men, the incidence of UTI was the highest among those from the 41-50 year age group, followed by men from the 51-60 year age group ( Table 2).

Association between antimicrobial resistance and biofilm formation
In comparison with non-biofilm producers, biofilm-producing isolates showed stronger resistance to antibiotics. The highest level of resistance was reported for ampicillin (82%) followed by nitrofurantoin (78%) and amoxyclav (72%), while the least resistance was conferred toward norfloxacin (6%) ( Table 5). There was a significant association between resistance to amoxyclav, ceftazidime, cefepime, imipenem, and nitrofurantoin and biofilm formation (p<0.05). E. coli isolates resistant to three or more classes of antibiotics were categorized as multidrug-resistant (MDR) strains.

DISCUSSION
UTI is one of the most common health problems affecting millions of people worldwide and is a leading cause of morbidity and high healthcare expenditures in people of all ages 5 . In the present study, the incidence of UTI was higher in female patients than in male patients. Our results are in line with those by Momoh et al. 13 and Ahmed et al, 14 . who reported UTIs in 60.2% and 73% women and 39.8% and 23% men, respectively. The difference in the female:male ratio may be related to different clinical components. Women remain at a much higher risk of UTI (compared to men) owing to shorter urethra, which permits bacterial entry and infection in the bladder. In addition, hormonal changes may influence the beneficial bacteria that are responsible for competing with harmful microorganisms in the urinary tract.
In our study, the frequency of UTI was the highest in women from 21 to 30 years of age and men between 41 and 50 years of age, consistent with the results of Santosh John thattil et al 15 . and Fatima S. et al. 16 that reported the highest incidence of UTI in women from 26 to 35 years and men from 46 to 60 years of age.
In our study, the most common isolate was E. coli (49.5%), consistent with the observation reported by Kaur et al. 17 (71.7%) and George et al. 18 (69.8%). Thus, the host fecal flora may be a source of E. coli that spreads via the perineal, vaginal, and periurethral areas to the lower urinary tract, wherein it is established. Some common ways for the migration of E. coli include sexual contact, improper cleaning, holding urine (especially before and after intercourse), and enlarged prostate gland 6 .
We investigated the biofilm formation ability of UPEC. Among 57 isolates, 87.7% were positive for biofilm formation in vitro, which is in line with the results of Suman et al. 19 , Poursina F et al. 20 and Yadav et al. 21 , showing 92, 80, and 76% E. coli isolates to be biofilm producers, respectively.
The correlation between biofilm production and resistance to amoxyclav, ceftazidime, cefepime, imipenem, and nitrofurantoin was found to be statistically significant (p<0.05); no significant correlation was observed (p>0.05) with amikacin, ampicillin, tetracycline, cotrimoxazole, piperacillin/ tazobactam, gentamicin, and norfloxacin. The antibiotics found to be effective against biofilmproducing E. coli isolates were norfloxacin, amikacin, imipenem, and piperacillin/tazobactam. A significant correlation was observed between multidrug resistance and biofilm formation. Approximately 90% biofilm producers were resistant to more than three classes of antibiotics. The results of our study are in agreement with those reported by Deotale et al. 22 and Sevanan et al. 23 . where in biofilm-producing organisms were more resistant to antibiotics than non-biofilmproducing isolates. The correlation between antibiotic resistance and biofilm formation may be associated with multiple factors such as restricted penetration of drugs through the biofilm matrix or longer time needed to penetrate the biofilm than treatment duration. The expression of efflux pumps is considered as a mechanism underlying antimicrobial resistance not only in planktonic cells but also in biofilm structures 21 . It has been demonstrated that biofilm-producing microorganisms can tolerate up to 100-1000 times higher concentrations of antibiotics and disinfectants than planktonic cells, and biofilmproducing isolates showed increased resistance against phagocytosis and other host defense mechanisms 9 .

Limitations of the Study
In this study, other virulence factors such as hemagglutination, gelatinase production, and extended-spectrum beta-lactamase Amp C were not evaluated. The biofilm-producing capability of UPEC may differ in vivo. Further studies regarding the in vivo biofilm-forming capacity of uropathogens are warranted in case of treatment failure.

CONCLUSION
UTI was found to be more common in women than in men. The most common isolate was E. coli. Biofilm producers showed higher resistance to antibiotics than non-biofilm producers. Biofilm formation by UPEC may pose a health problem, as these bacteria are difficult to treat and increase the chances of chronic UTI. Norfloxacin, amikacin, imipenem, piperacillin, and tazobactam antimicrobials are particularly effective against biofilm-producing E. coli. These antibiotics may be used in the empirical therapy of UTI caused by biofilm-producing UPEC. A significant correlation was observed between biofilm production and antibiotic resistance in our study. Hence, screening of all isolates of UPEC for biofilm production and studying their antibiogram may help in providing an appropriate antibiotic therapy.

ACKNOwLEDgMENTS
We are thankful to all the faculty a n d t e c h n i c i a n s o f D e p a r t m e n t o f Microbiology-Yenepoya Medical College and hospital for their support.