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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 20  |  Issue : 2  |  Page : 88-91

Study of biofilm production in Escherichia coli causing urinary tract infection and its correlation with antimicrobial resistance


1 Department of Microbiology, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
2 Department of Microbiology, S.C.B. Medical College and Hospital, Cuttack, Odisha, India

Date of Web Publication7-Jan-2019

Correspondence Address:
Dr. Debabrata Dash
Department of Microbiology, All India Institute of Medical Sciences, Raipur - 492 099, Chhattisgarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_35_17

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  Abstract 


INTRODUCTION: Urinary tract infections (UTIs) are among the most common bacterial infections. Escherichia coli remains the most frequent cause of UTIs. More important is the increase in resistance to some antimicrobial agents. Furthermore, bacterial species are capable of living in a biofilm. There is increasing evidence for the role of bacterial biofilm in causing recurrent UTIs.
AIM: The present study aimed to evaluate the ability of E. coli, isolated from UTIs to form a biofilm, and its association with catheterisation and to correlate the role of biofilms with their antimicrobial resistance.
MATERIALS AND METHODS: A total of 403 urine samples were processed. All the isolated E. coli strains (226) were grown in Luria broth and were incubated overnight in high-glucose Dulbecco's modified Eagle's medium using a microtitre plate. The plate was stained with crystal violet, and the biofilm was quantified using an enzyme-linked immunosorbent assay plate reader at 570 nm. An optical density value more than that of the mean negative control plus three standard deviations is taken as positive for biofilm production. The antibiogram was done using the Kirby–Bauer disk diffusion method.
RESULTS AND DISCUSSION: Of 226 strains, 54.4% were found to produce biofilms. Of them, 81.3% of patients were catheterised. Most of them were found to be resistant to commonly used antibiotics such as Cephalosporins, Quinolones and Aminoglycosides. Imipenem and Nitrofurantoin are the most effective antibacterial agents, showing 77.3% and 73.2% sensitivity, respectively.
CONCLUSION: The biofilm assay using a microtitre plate is convenient and useful in screening the biofilm producers. Catheterisation is a risk factor for biofilm production, and catheter care is of paramount importance to prevent catheter-associated UTI.

Keywords: Antimicrobial resistance, biofilm, Escherichia coli


How to cite this article:
Dash D, Sarangi G, Patro P, Chayani N. Study of biofilm production in Escherichia coli causing urinary tract infection and its correlation with antimicrobial resistance. J Acad Clin Microbiol 2018;20:88-91

How to cite this URL:
Dash D, Sarangi G, Patro P, Chayani N. Study of biofilm production in Escherichia coli causing urinary tract infection and its correlation with antimicrobial resistance. J Acad Clin Microbiol [serial online] 2018 [cited 2019 Jul 17];20:88-91. Available from: http://www.jacmjournal.org/text.asp?2018/20/2/88/249572




  Introduction Top


One of the most common infections encountered in clinical practice is urinary tract infection (UTI). Escherichia coli is the most common causative agent for UTI.[1] In recent time, a great concern expressed regarding symptomatic UTI due to increased antimicrobial resistance to commonly used antimicrobials and its chronicity. This chronicity and increase in antimicrobial resistance is attributed to biofilm formation.[2]

The three essential components that constitute a biofilm are adherence of microorganism, a change in gene expression resulting in a different phenotype from the planktonic state and an extracellular matrix composed of host components and secreted bacterial products.[3],[4]

Thus, the objective of this study is to detect the biofilm production by E. coli isolated from urine samples and also to study the antimicrobial susceptibility pattern of these biofilm-producing E. coli.


  Materials and Methods Top


This prospective study was conducted in the Department of Microbiology, S.C.B. Medical College and Hospital, Cuttack, for three months.

Inclusion criteria

Admitted patients of all age groups and both sexes who had symptoms of UTI (fever, urgency, frequency, dysuria or suprapubic tenderness in case of non-catheterised patients and new onset or worsening of fever, altered mental status, flank pain, costovertebral angle tenderness, rigors, pelvic discomfort and new or worsening malaise or lethargy in catheterised patients) were included in the study.

Sample collection and processing

The samples were collected under complete aseptic conditions in sterile containers. Ten microlitres of urine samples was inoculated onto cystine lactose electrolyte-deficient medium (HiMedia Laboratories, Mumbai) and incubated at 37°C overnight. The identification of isolates was done on the basis of the colony morphology, Gram-staining and the standard biochemical tests.

Detection of biofilm production

The method was adopted as described by Wakimoto et al.[5] Briefly, the isolates were inoculated into Luria broth (LB) (HiMedia Laboratories, Mumbai) and incubated at 37°C. In a flat bottom microtitre plate, 200 μl of Dulbecco's modified Eagle's medium (HiMedia Laboratories, Mumbai) was taken and 5 μl of inoculated LB was added and again incubated overnight at 37°C. Then, it was discarded, washed, heat fixed and stained at 0.5% crystal violet for 5 min. The biofilm was quantified in duplicate, after adding 200 μL of 95% ethanol, by an enzyme-linked immunosorbent assay plate reader (Bio-Rad Laboratories, USA) at 570 nm. The procedure was repeated three times, and the mean optical density (OD) value was calculated. E. coli ATCC 25922 procured from Microbiologics, USA, was taken as positive control while uninoculated LB was taken as negative control. If the OD of the medium exceeded 0.125, then it was classified the strain as biofilm producers. The number 0.125 was chosen as cut-off because it was three standard deviations (0.013) above the mean OD (0.086) of a clean microtitre plate stained by the method cited above.[6]

Antibiotic susceptibility test

Antibiotic susceptibility test was done using the Kirby–Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines using Ampicillin, Amoxicillin/Clavulanate, Cefazolin, Amikacin, Ceftriaxone, Co-trimoxazole, Gentamicin, Norfloxacin, Nitrofurantoin, Piperacillin/Tazobactam and Imipenem which were also procured from HiMedia laboratories, Mumbai, India.[7]

Statistical analysis was done using IBM Statistical Package for the Social Sciences for Windows, version 21 (IBM Corp., Armonk, NY, USA). The data were presented as percentage and proportions. The Chi-square test was applied when two or more set of variables were compared. The critical value of P indicating the probability of significant difference was taken as <0.05.


  Results Top


A total of 403 consecutive samples (254 from catheterised patients and 149 from non-catheterised patients) were processed for culture. A total of 293 (72%) samples were found to be culture positive, of which 226 (77.13%) were E. coli and others being Pseudomonas aeruginosa (7.5%), Klebsiella pneumoniae (4.1%), acinetobacter spp. (3.4%), enterococcus spp. (3.1%), enterobacter spp. (2.3%), candida spp.(1.7%) and Staphylococcus aureus (0.77%). All the isolated E. coli (226) were evaluated for biofilm production. A total of 123 (54.42%) samples were biofilm producers. Of 226 isolates, 100 (67.11%) were from catheterised patients while 23 (29.87%) were from non-catheterised patients (P = 0.03). The susceptibility pattern showed highest susceptibility to Imipenem (77.3%) followed by Nitrofurantoin (73.2%) among the biofilm producers, while the susceptibility of Imipenem was 100% in case of biofilm non-producing E. coli (P < 0.001 for Imipenem susceptibility).


  Discussion Top


UTIs are gradually becoming a major threat to the society due to increasing antimicrobial resistance. The situation is more complicated in catheterised patients due to biofilm production. This results in limited treatment options, complicate medical management and prolonged hospital stays.[8]

In the current study, 226 (77.13%) were E. coli of the 293 culture-positive cases. In most of the other studies also, E. coli was found to be the most common isolated organism.[1],[9],[10],[11]

Of 226 E. coli, 123 (54.42%) were biofilm producers. A similar study by Abdaagire et al. from India showed 44.85% of biofilm production by uropathogens.[9] It is estimated that more than 90% of microorganisms live in a structured community of cells, i.e. biofilms, since they constitute a more efficient way of surviving in hostile environments.[12]

Among the isolated biofilm producing E. coli, 67.11% were from the catheterised patients. Studies done by Sayal et al. in Punjab, India,[13] Subramanian et al.[14] and Amuthamani et al. in Puducherry, India,[10] on catheterised patients had similar results of 71%, 60% and 71% biofilm production, respectively. Organisms got access to the urinary tract during catheterisation because of poor hygienic practices followed during the procedure. The daily risk of acquisition of bacteriuria varies from 3% to 7% when an indwelling urethral catheter remains in situ. Formation of biofilm along the catheter surface is a contributing factor for chronic, indolent infection in catheter-associated UTI (CAUTI).[15] Hence, the implementation of 'care bundle approaches' such as insertion of catheter by strict aseptic non-touch technique, catheter care by proper hand hygiene, mental care, perineal care and closed drainage system through the entire period of catheter stay has been advocated for prevention of CAUTI.

Biofilm-producing strains showed relatively high drug resistance against all antibiotics tested as compared to non-biofilm producing counterparts. This may be because bacterial biofilms are often associated with long-term persistence of organism in various environments, decreased bacterial growth rate in a biofilm, expression of resistance genes and restricted penetration of antibiotics into biofilm. Furthermore, proximity of cells within a biofilm can facilitate a plasmid exchange and hence enhance the spread of antimicrobial resistance.[1],[16]

The susceptibility pattern showed the highest susceptibility to Imipenem (77.3%) followed by Nitrofurantoin (73.2%) among the biofilm producers, while the susceptibility of Imipenem was 100% in case of biofilm non-producing E. coli. This finding was in close association with a study done by Abdaagire et al. on uropathogens who found that the most effective antibiotics against Gram-negative bacteria were Imipenem and Meropenem.[9] Furthermore, Rewatkar and Wadher in their study on various clinical isolates found that the most effective antibiotics against Gram-negative bacteria were Imipenem and Colistin.[16] From North India,Panda et al. in uropathogens found that Imipenem was the most effective drugs against both biofilm producer and non-biofilm producers.[2] Sharma et al. have shown that the susceptibility pattern of the biofilm-producing isolates of E. coli ranged from 16% to 57% while that of the non-film-producing isolates ranged from 38 to 76%.[17] Hence, it can be said that the role of biofilm is definite in case of increased antimicrobial resistance. Biofilm production is affected by the type of medical devices, duration of stay of medical devices, extracellular production of polysaccharide by bacteria and bacterial quorum sensing. Some of the effective ways of preventing biofilm formations are closed drainage system and the removal of catheter when not necessary, intermittent catheterisation and changing catheter material while antimicrobial prophylaxis, antimicrobial drainage bag solutions and antimicrobial bladder washes were found to be ineffective.[18]

This study is, however, has its limitations as the duration of the study is short. The isolates were not tested for Extended-spectrum β-lactamase, AmpC or Carbapenemase production. Furthermore, the blood cultures of the patients were not compared to know the extent of bacteraemia.


  Conclusion Top


Biofilm production by uropathogens is not an uncommon phenomenon nowadays and more so in catheterised patients. In the present study, antibiotic resistance was significantly higher in biofilm-producing organism, highlighting the role of biofilm production in spreading drug resistance among the uropathogens.

Imipenem and Nitrofurantoin are the few antimicrobial agents that are effective against both biofilm-producing and non-biofilm-producing organisms. The biofilm mode of living is a highly advantageous response of the microorganisms to the environmental stresses of the urinary tract environment. Whether or not we human beings can overcome or subvert this ancient survival mechanism is an open question as few therapeutic options exist for treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Niveditha S, Pramodhini S, Umadevi S, Kumar S, Stephen S. The isolation and the biofilm formation of uropathogens in the patients with catheter associated urinary tract infections (UTIs). J Clin Diagn Res 2012;6:1478-82.  Back to cited text no. 1
    
2.
Panda PS, Chaudhary U, Dube SK. Study of biofilm production and antimicrobial sensitivity pattern of uropathogens in a tertiary care hospital in North India. Int J Community Med Public Health 2016;3:2421-6.  Back to cited text no. 2
    
3.
Gristina AG. Biomaterial-centered infection: Microbial adhesion versus tissue integration. Science 1987;237:1588-95.  Back to cited text no. 3
    
4.
Denstedt JD, Wollin TA, Reid G. Biomaterials used in urology: Current issues of biocompatibility, infection, and encrustation. J Endourol 1998;12:493-500.  Back to cited text no. 4
    
5.
Wakimoto N, Nishi J, Sheikh J, Nataro JP, Sarantuya J, Iwashita M, et al. Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli. Am J Trop Med Hyg 2004;71:687-90.  Back to cited text no. 5
    
6.
Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985;22:996-1006.  Back to cited text no. 6
    
7.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: 24th Informational Supplement. Clinical and Laboratory Standards Institute; 2014.  Back to cited text no. 7
    
8.
Mohamad EA, Shalakan AH. Detection of biofilm formation in uropathogenic bacteria. Egypt J Med Microbiol 2015;24:49-58.  Back to cited text no. 8
    
9.
Abdagire NV, Chincholkar VV, Kulkarni DM, Nilekar SL, Birajdar SV. Biofilm production among uropathogenes and their antibiogram. Int J Pharm Bio Sci 2014;5:261-6.  Back to cited text no. 9
    
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Amuthamani R, Subramaniyan A, Kanungo R. Biofilm producing uropathogens and drug resistance: Dual foe for patients on urinary catheter. Int J Curr Microbiol Appl Sci 2017;6:326-30.  Back to cited text no. 10
    
11.
Momtaz H, Karimian A, Madani M, Safarpoor Dehkordi F, Ranjbar R, Sarshar M, et al. Uropathogenic Escherichia coli in Iran: Serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob 2013;12:8.  Back to cited text no. 11
    
12.
Simões M, Simões LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT Food Sci Technol 2010;43:573-83.  Back to cited text no. 12
    
13.
Sayal P, Singh K, Devi P. Detection of bacterial biofilm in patients with indwelling urinary catheters. CIB Tech J Microbiol 2014;3:9-16.  Back to cited text no. 13
    
14.
Subramanian P, Shanmugam N, Sivaraman U, Kumar S, Selvaraj S. Antiobiotic resistance pattern of biofilm-forming uropathogens isolated from catheterised patients in Pondicherry, India. Australas Med J 2012;5:344-8.  Back to cited text no. 14
    
15.
Lynch AS, Robertson GT. Bacterial and fungal biofilm infections. Annu Rev Med 2008;59:415-28.  Back to cited text no. 15
    
16.
Rewatkar AR, Wadher BJ. Staphylococcus aureus and Pseudomonas aeruginosa-biofilm formation methods. J Pharm Biol Sci 2013;8:36-40.  Back to cited text no. 16
    
17.
Sharma M, Aparna, Yadav S, Chaudhary U. Biofilm production in uropathogenic Escherichia coli. Indian J Pathol Microbiol 2009;52:294. DOI: 10.4103/0377-4929.48960  Back to cited text no. 17
    
18.
Trautner BW, Darouiche RO. Role of biofilm in catheter-associated urinary tract infection. Am J Infect Control 2004;32:177-83.  Back to cited text no. 18
    




 

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