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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 1  |  Page : 15-19

Epidemiology of biofilm formation by gram-negative bacilli in patients with urinary tract infection in a tertiary care hospital


1 Department of Microbiology, Govind Ballabh Pant Hospital, New Delhi, India
2 Department of Microbiology, BSA Medical College and Hospital, New Delhi, India

Date of Web Publication15-Feb-2017

Correspondence Address:
Abha Sharma
Department of Microbiology, BSA Medical College and Hospital, Rohini, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.200206

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  Abstract 

INTRODUCTION: Urinary tract infection poses a serious health threat with respect to antibiotic susceptibility and high recurrence rate. Uropathogenic bacteria may produce biofilm, rendering the bacteria very difficult to eradicate with antibiotics. This study was carried out to compare biofilm formation by Gram-negative organisms in catheterized and noncatheterized patients and also to evaluate their antibiotic susceptibility pattern.
MATERIALS AND METHODS: The study comprised fifty consecutive specimens of urine yielding significant colony count of Gram-negative bacteria. Twenty-five specimens were collected from catheterized patients while the rest of the 25 specimens were collected from noncatheterized patients. All the specimens were processed following the standard microbiological guidelines. The isolated organisms were screened for biofilm production by tube method, and antibiotic susceptibility of both biofilm- and nonbiofilm-producing isolates was done by Kirby–Bauer disc diffusion method.
RESULTS: Eighteen (72%) of the 25 organisms isolated from the catheterized patients were found to be biofilm producers, while only 4 (16%) of the rest of the 25 organisms, isolated from the noncatheterized patients were found to produce biofilm. The percentage sensitivity of all the biofilm-producing organisms was found to be lower than the nonbiofilm-producing organisms.
CONCLUSION: The present study shows that urinary catheterization is a major environmental factor that facilitates biofilm formation by uropathogenic Gram-negative bacilli. As biofilm-producing organisms are difficult to treat, proper measures should be taken to prevent its formation.

Keywords: Biofilm, catheterization, Gram-negative bacteria, urinary tract infections


How to cite this article:
Hussain JH, Sharma A, Jaggi T, Mishra B, Thakur A, Dogra V, Loomba PS. Epidemiology of biofilm formation by gram-negative bacilli in patients with urinary tract infection in a tertiary care hospital. Int J Health Allied Sci 2017;6:15-9

How to cite this URL:
Hussain JH, Sharma A, Jaggi T, Mishra B, Thakur A, Dogra V, Loomba PS. Epidemiology of biofilm formation by gram-negative bacilli in patients with urinary tract infection in a tertiary care hospital. Int J Health Allied Sci [serial online] 2017 [cited 2022 Aug 11];6:15-9. Available from: https://www.ijhas.in/text.asp?2017/6/1/15/200206

Urinary tract infection (UTI) is one of the most common human infections in both catheterized and noncatheterized patients. Uropathogenic organisms may produce intracellular bacterial communities called biofilm within the bladder, largely consisting of polysaccharides which prevent the access of antibiotics, antibodies, and white blood cells. Catheterization is supposed to enhance biofilm production by providing a nidus for the bacteria for attachment.

The biofilms have major medical significance as they decrease the susceptibility to the antimicrobial agents. Furthermore, the proximity of cells within a biofilm can facilitate a plasmid exchange and hence enhance the spread of antimicrobial resistance.[1] Environmental changes are also responsible for the transition from planktonic growth to biofilm [2] and cause changes in the expression of surface molecules, virulence factors, and metabolic status, allowing the bacteria to acquire properties that enable their survival in unfavorable conditions.[3],[4]

Several authors around the world have reported that the Gram-negative bacteria including  Escherichia More Details coli and Klebsiella spp. are the most frequent organisms that cause UTIs.[5],[6],[7],[8],[9]

In the present study, the incidence of biofilm formation by Gram-negative organisms in catheterized and noncatheterized patients is compared to evaluate whether catheterization provides a favorable environmental condition for biofilm formation and also the antibiotic susceptibility pattern of the biofilm- and nonbiofilm-producing organisms was evaluated.


  Materials and Methods Top


The study cohort, after obtaining patient consent and Ethical Committee approval, consisted of fifty patients of various age groups having UTI, from which Gram-negative bacteria were isolated. Of which 25 were admitted in the various departments of the hospital and were catheterized for at least 2 days, while the rest of the 25 patients attended the outpatient department of the hospital and were not catheterized.

All the specimens collected were transported following standard microbiological guidelines.[10] Wet mount microscopy and semi-quantitative culture of the specimens were done according to the standard guidelines.[11] Samples showing abundant epithelial cells in wet mount microscopy were not included in the study. Colony counts of 105 colony-forming units/ml were considered as the significant colony count.[11] Specimens yielding significant colony count of Gram-negative bacteria were included in the study.

All the organisms were then further processed for their identification, following standard microbiological procedures,[12] and were tested for antibiotic susceptibility following Kirby–Bauer disc diffusion technique.[13] The zones of inhibition were interpreted following the Clinical and Laboratory Standards Institute 2014 guidelines. For antibiotic susceptibility, discs from HiMedia Pvt. Ltd., Mumbai, were used. Antibiotic susceptibility tests were done for five groups of antibiotics: penicillin with β-lactamase inhibitors (amoxicillin–clavulanic acid and piperacillin–tazobactam), cephalosporins (ceftriaxone), fluoroquinolones (norfloxacin, ciprofloxacin, ofloxacin), aminoglycosides (gentamicin and amikacin), and carbapenems (imipenem, meropenem, and ertapenem).

All isolates were subjected to tube method described by Christensen et al.,[14] for biofilm detection. Pseudomonas aeruginosa ATCC 27853 was used as the positive control for detection of biofilm formation.

Statistical analysis

The statistical analysis was done by Fisher's exact test and the P values were calculated. P ≤ 0.05 was considered to be statistically significant.


  Results Top


Of the total 50 patients, 30 (60%) patients were females while 20 (40%) were males. Thirteen (52%) of the 25 catheterized patients were males while the rest of the 12 (48%) patients were females. Among the 25 patients included in the noncatheterized group, 7 (28%) were males while 18 (72%) patients were females. The difference of occurrence of catheter-associated UTI in male and female patients was not found to be statistically significant in this study (P = 0.148).

E. coli was found to be the most commonly isolated organism followed by Pseudomonas spp., Klebsiella spp., and Proteus spp. [Table 1]. Eighteen (72%) of the 25 organisms isolated from the catheterized patients were found to be biofilm producers, while only 4 (16%) of the rest of the 25 organisms, isolated from the noncatheterized patients, were found to produce biofilm [Table 2]. The P value for biofilm formation in catheterized and noncatheterized patients was found to be 0.0001, which is extremely statistically significant. Thus, in this study, it was found that catheterization is a strong environmental factor that facilitates biofilm formation.
Table 1: Number, percentage, and distribution of organisms

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Table 2: Distribution of biofilm.forming organisms in catheterized and noncatheterized groups

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[Table 3],[Table 4],[Table 5],[Table 6] show the antibiotic susceptibility pattern of E. coli, Pseudomonas spp., Klebsiella spp., and Proteus spp. respectively. The most efficacious antibiotics for biofilm-producing strains of E. coli were piperacillin–tazobactam, amikacin, imipenem, and ertapenem while that for the nonbiofilm-producing strains were piperacillin–tazobactam and ertapenem.
Table 3: Antibiotic susceptibility pattern of Escherichia coli strains

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Table 4: Antibiotic susceptibility pattern of Pseudomonas spp.

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Table 5: Antibiotic susceptibility pattern of Klebsiella spp.

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Table 6: Antibiotic susceptibility pattern of Proteus spp.

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The most efficacious antibiotic for biofilm-producing strains of Pseudomonas spp. was amikacin while the most efficacious antibiotics for the nonbiofilm-producing strains were piperacillin–tazobactam, amikacin, and gentamicin.

The most efficacious antibiotics for biofilm-producing strains of Klebsiella spp. were ofloxacin, amikacin, and imipenem while that for the nonbiofilm-producing strains were ofloxacin and amikacin.

Both biofilm- and nonbiofilm-producing strains of Proteus spp. were sensitive to most of the antibiotics.

The P value for susceptibility to all the five groups of antibiotics for all the strains isolated from catheterized and noncatheterized patients was found to be >0.05, which means the difference in antibiotic susceptibility pattern of biofilm- and nonbiofilm-producing strains of the isolated organisms is not statistically significant.


  Discussion Top


In this study, males were found to be slightly more affected in the catheterized group. This finding coincides with the findings of a study done by Taiwo and Aderounma in Nigeria.[15]

However, in the noncatheterized group, female patients outnumbered the male patients, which is consistent with the proven fact that has been established by various published reports.[10],[15]

However, the association of gender difference with catheterization in the occurrence of UTI was not found to be statistically significant (P = 0.148) in the study.

In the present study, among the fifty strains isolated from fifty specimens of urine, 62% (n = 31) of the strains of E. coli were isolates and were found to be the predominant organism in both catheterized (n = 15) and noncatheterized (n = 16) groups. E. coli was followed by Pseudomonas spp. (16%), Klebsiella spp. (14%), and Proteus spp. (8%). Various previous studies [16],[17],[18] have also reported E. coli to be the most common organism isolated from the specimens of urine from patients suffering from UTIs, which match to the present study.

In our study, we have used the tube method to detect biofilm formation in the uropathogens. Hassan et al.[22] evaluated the different detection methods of biofilm formation and found that the tube method detected 49% of isolates as biofilm producers, with 73% sensitivity and 92.5% specificity. Ruzicka et al.[23] have shown that tube method is better than the Congo red agar method for the detection of biofilm formation.

In this study, of the 25 organisms isolated from the catheterized group, 72% (n = 18) were found to be biofilm producing, while 16% (n = 4) of the 25 organisms isolated from the noncatheterized group were biofilm producers. Another study by Abdallah et al.[21] has reported 43.3% of biofilm production in catheterized patients and 30% of biofilm production in noncatheterized patients. The difference of biofilm-producing organisms in catheterized and noncatheterized groups was found to be statistically significant (P = 0.0001), suggesting a strong association between catheterization and biofilm formation. This result is consistent with a previously reported study,[19] which also states that catheter surface makes it an ideal site for bacterial attachment and biofilm formation.

The antibiotic susceptibility of both biofilm- and nonbiofilm-producing organisms was tested for five groups of antibiotics. The percentage sensitivity of all the biofilm-producing organisms was found to be lower than the nonbiofilm-producing organisms [Table 3],[Table 4],[Table 5],[Table 6]. The reasons for low sensitivity of biofilm-producing organisms maybe due to the fact that the penetration of antimicrobial agent through the biofilm matrix is difficult, the expression of efflux pumps increases in biofilm bacteria, and the growth rate of biofilm bacteria is slow in the center of the biofilm. Similarly, a previously published study [20] has also reported that biofilm facilitates the adherence of these microorganisms to biomedical surfaces and protect them from host immune system and antimicrobial therapy, thereby resulting in greater resistance.

However, the P value for susceptibility to all the five groups of antibiotics for the biofilm- and nonbiofilm-producing strains was found to be >0.05, which means the difference in antibiotic susceptibility pattern of biofilm- and nonbiofilm-producing strains is not statistically significant. This may be attributed to lower sample size in the study.


  Conclusion Top


E. coli is the most common uropathogen with 72% of urinary isolates showing biofilm formation in catheterized patients. Urinary catheterization remains a major risk factor for biofilm formation. It is very difficult to treat biofilm-producing bacteria due to low antimicrobial sensitivity. Therefore, catheterization should be minimized for the shortest duration possible with proper catheter care. Combination antibiotic therapy may be considered for treating UTI in prolonged catheterized patients where biofilm formation is anticipated. Further research is needed to discover new antimicrobial agents that inhibit bacterial virulence and biofilm formation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Taiwo SS, Aderounma AO. Catheter associated urinary tract infection: Aetiologic agents and antimicrobial susceptibility pattern in Ladoke Akintola University Teaching Hospital, Osogbo, Nigeria. Afr J Biomed Res 2006;9:141-8.  Back to cited text no. 15
    
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Abdallah NMA, Elsayed SB, Mostafa MMY, El-gohary GM. Biofilm forming bacteria isolated for UTI, relation to catheterization and susceptibility to antibiotics. Int J Biotechnol Mol Biol Res 2011;2:172-178.  Back to cited text no. 21
    
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Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Inf Dis 2011;15:305-11.  Back to cited text no. 22
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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