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 Table of Contents  
ORIGINAL ARTICLES
Year : 2021  |  Volume : 9  |  Issue : 3  |  Page : 103-106

Relationship of biofilm formation and antibiotic susceptibility profile of uropathogenic Escherichia coli


Department of Microbiology, Dr. Harvansh Singh Judge Institute of Dental Sciences & Hospital, Panjab University, Chandigarh, India

Date of Submission18-Sep-2021
Date of Acceptance28-Oct-2021
Date of Web Publication11-May-2022

Correspondence Address:
Jyoti Sharma
Department of Microbiology, Dr. Harvansh Singh Judge Institute of Dental Sciences & Hospital, Panjab University, Panjab University South Campus, Vidya Path, Near UIET, Sector 25, Chandigarh 160014
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/dypj.dypj_54_21

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  Abstract 

Introduction: Escherichia coli is the most frequent etiological agent isolated from urinary tract infections. Ability of these bacteria to form biofilm is one of its important virulence factors. Moreover in the past decades, there has been a growing increase in the prevalence of antibiotic resistance against commonly prescribed antibiotics. Bacteria in biofilm are considered to be highly resistant to antimicrobial agents. Materials and Methods: All the urine isolates were cultured to isolate the etiological agent of the infection and were tested for their susceptibility against various antibiotics recommended for their treatment. Of these isolates, only E. coli were subjected for biofilm formation detection. The resistance pattern obtained after antibiotic susceptibility testing was correlated with the results obtained from biofilm detection. All the results were statistically analyzed. Results: Of all the isolated obtained after culturing urine samples, 90% of them turned out to be E. coli only. When these isolates were subjected to biofilm detection, 35% of them showed positive results. Further, their resistance pattern revealed that bacteria-forming biofilms were more resistant toward penicillins, cephalosporins, and fluoroquinolones where very little resistance was reported for aminoglycosides, carbapenems, and nitrofurantoins. Conclusion: As this study concludes a positive association between biofilm formation and antibiotic resistance, it is strongly suggested that detection of biofilm and awareness about the susceptibility pattern of the bacteria toward locally prescribed antibiotics can be helpful in improving the efficacy of the empirical treatment of types of infections.

Keywords: Antibiotic resistance, biofilm, uropathogenic


How to cite this article:
Mehta M, Sharma J, Bhardwaj SB, Ghai A, Chawla A. Relationship of biofilm formation and antibiotic susceptibility profile of uropathogenic Escherichia coli. D Y Patil J Health Sci 2021;9:103-6

How to cite this URL:
Mehta M, Sharma J, Bhardwaj SB, Ghai A, Chawla A. Relationship of biofilm formation and antibiotic susceptibility profile of uropathogenic Escherichia coli. D Y Patil J Health Sci [serial online] 2021 [cited 2022 May 27];9:103-6. Available from: http://www.dypatiljhs.com/text.asp?2021/9/3/103/345107




  Introduction Top


Escherichia coli is a gram-negative, facultatively anaerobic, rod-shaped coliform bacterium.[1] This is a fabulously divergent bacterial species, which has the competence to colonize and prevail in plentiful niches both in the environment and within animal hosts. Majority of the E. coli strains do not cause disease, as they exist normally in the gut but virulent strains can cause gastroenteritis, urinary tract infections (UTIs), neonatal meningitis, hemorrhagic colitis, and Crohn’s disease. Escherichia coli is the most prevalent organism causing both community and hospital-acquired UTIs, leading to severe secondary health issues worldwide.[2]

The idiosyncratic aspect of urinary pathogenic E. coli (UPEC) is biofilm formation. It forms intracellular bacterial associations with biofilm-like properties within bladder epithelium.[3] Also, it can cause cystitis; the organism is also competent to maneuver further through the ureters to kidneys and cause pyelonephritis.[4] Biofilms are the bacterial accumulation securely embedded in the extracellular matrices of polysaccharides, proteins, enzymes, and nucleic acid, so as to expedite anchorage to any surface inevitably.[5] Biofilm producers display a transformed phenotype corresponding to growth rate and gene transcription.[6] Higher resistance to antibiotics is proclaimed in bacteria confined in biofilm comparative to planktonic cells. The resistance mechanism in biofilm implicates the decelerated infiltration of antibiotics through polysaccharide matrix of the biofilm, resistance gene transfer within the biofilm, which is higher in planktonic cells, the higher expression of antibiotic efflux pumps in biofilm, the existence of perpetual cells that are not mutants, but resist the antibiotic, the slow growth rate, and nutrient deficiency that cut-back antibiotic susceptibility.[7] Biofilm promotes the endurance of these pathogenic isolates in the urinary tract and hampers with bacterial elimination. Biofilm infections are strenuous to uproot with antibiotic treatment.[8] As reported by the National Institute of Health, more than 60% of all microbial infections are owed to biofilms.[3] The prevalence of biofilm among UPEC ranges from 60% to 70%.[9] Regression by UPEC has also been pertinent to the capability of this bacterium to form biofilm.

This study aimed to perform the in vitro detection of biofilm formation and antibiotic resistance pattern of UPEC isolates and also to find out the correlation between them.


  Materials and Methods Top


Samples

This study was conducted in the Department of Microbiology, Dr. Harvansh Singh Judge Institute of Dental Sciences & Hospital, Panjab University, Chandigarh. The midstream urine samples were collected from the outpatients suspecting UTIs. Patients from all age groups and both sexes were included in the study. The samples were subjected to macroscopic as well as microscopic examination and culture. Macroscopic examination was done to document any change in color or presence of turbidity or deposits in the urine samples. Microscopic examination was done with the sediment harvested after the samples were centrifuged at 2000 rpm for 15 min, to check for the presence of pus cells, red blood cells, bacteria, yeast, epithelial cells, or any kind of cast and crystals. The samples were cultured by semiquantative culture technique.[10] All the samples were inoculated on cysteine lactose-deficient (CLED) agar. The plates were incubated at 37°C for 24–48 h. Urine culture which yielded colony counts of >105 organism/mL of a single type along with >10 pus cells/high-power field (HPF) of the centrifuged urine samples was interpreted as diagnostic bacteriuria. Bacteria were identified based on colony morphology, gram-staining, and conventional biochemical methods.[11] Of all the uropathogens isolated, only E. coli isolates were included in the study. A total of 150 urine samples were screened to get the desired E. coli isolates. All the isolates were stored in brain heart infusion broth at –20°C for further studies.

Antibiotic sensitivity testing

Antibiotic sensitivity was performed on Mueller–Hinton agar using Kirby–Bauer disk diffusion susceptibility test technique following Clinical & Laboratory Standard Institute (CLSI) recommendations.[12] The antibiotics tested in this study were ampicillin, amoxicillin-clavulanic acid, ciprofloxacin, nitrofurantoin, cefuroxime, ceftriaxone, chloramphenicol, gentamicin, imipenem, cefoperazone-sulbactum, and piperacillin-tazobactum. The CLSI control strain of E. coli ATCC 25922 was used as a control for antibiotic susceptibility testing.

Detection of biofilm formation

The formation of biofilm was detected by using the previously described tissue culture plate method.[13] For this overnight growth of E. coli in Trypticase soya broth (TSB) was diluted with fresh TSB to 1:100 dilution. Individual wells of sterile tissue culture plates were filled with 200 µL of the diluted cultures including controls. Plates were incubated for 24 h at 37°C overnight. After incubation, the contents of the plates were removed by gentle tapping. Then the wells were washed four times with 0.2 µL of phosphate-buffered saline (pH 7.2) to remove planktonic cells. Wells were stained with crystal violet (0.1%). Excess stain was removed by washing with deionized water and plates were kept for drying. Optical density of the adherent bacteria was detected with the help of enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm.

Statistical analysis

The results of the study were statistically determined by using κ2 test. The significance of the difference was evaluated at P < 0.05.


  Results Top


Of 150 urine samples, 90 samples showed significant growth. Escherichia coli was isolated from 80 of 90 samples.

Antimicrobial susceptibility testing

Overall resistance pattern of all the E. coli isolates is shown in [Table 1]. Escherichia coli isolates showed higher resistance toward ampicillin (76.20%), cefuroxime (66.25%), amoxicillin-clavulanic acid (56.25%), ciprofloxacin (56.25%), ceftazidime (50%), cefoperazone (48.75%), cefotaxime (43.75%), and cotrimoxazole (40%). The strains were comparatively sensitive to gentamicin (95%), nitrofurantoin (98.75%), imipenem (98.75%), and piperacillin tazobactum (98.75%). For gentamicin, only four strains showed resistance, whereas only one strain showed resistance toward nitrofurantoin, imipenem, and pipracillin tazobactum.
Table 1: Resistance pattern of Escherichia coli isolates

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Biofilm formation

Of all the E. coli isolates which were subjected to biofilm production, 35% (28) strains turned out to be biofilm former by tissue culture plate method of biofilm detection.

Biofilm formation and antibiotic resistance

[Figure 1] shows the resistance pattern of biofilm producer and nonproducer UPEC. Among biofilm former UPEC 86% strains showed resistance toward ampicillin as compared to nonbiofilm producers (71%). Similarly, biofilm producer strains showed higher resistance toward cefuroxime (82%), amoxicillin-clavulanic acid (82%), ciprofloxacin (68%), ceftazidime (57%), cefoperzone (60%), cefotaxime (64%), and cotrimoxazole (57%) as compared to nonbiofilm producer strains of UPEC, cefuroxime (48%), amoxicillin-clavulanic acid (33%), ciprofloxacin (50%), ceftazidime (52%), cefoperzone (42%), cefotaxime (42%), and cotrimoxazole (42%), respectively. Only four isolates from biofilm producers showed resistance toward gentamicin and one only was resistant to nitrofurantoin, imipenem, and piperacillin tazobactum, whereas none from nonbiofilm producers was resistant to these antibiotics. The results of comparison of resistance pattern of biofilm producers and nonbiofilm producers using κ2 test were found to be statistically significant for ampicillin, cefuroxime, amoxicillin-clavulanic acid, ciprofloxacin, ceftazidime, cefoperzone, cefotaxime, and cotrimoxazole.
Figure 1: Resistance pattern of biofilm former vs. nonbiofilm former urinary pathogenic Escherichia coli (UPEC)

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  Discussion Top


UTI speaks for a large-scale health hazard as a result of escalation in the prevalence of antibiotic resistance. Escherichia coli is the most frequent cause of UTIs, accounting for more than 80% of the infections.[14] In our study, 35% of the isolates were biofilm producers. The majority of the UPEC strains were resistant toward penicillins, cephalosporins, and quinolones. However, biofilm former strains showed a higher degree of resistance toward these antimicrobials as compared to nonbiofilm producing isolates. This difference is susceptibility came out to be statistically significant. Similar findings have been shown by the study conducted by Sevanan et al.,[3] who also reported biofilm-forming strains to be more resistant than nonbiofilm producers. However, no statistically significant difference in the resistance pattern of biofilm vs. nonbiofilm UPEC was observed for amikacin, nitrofurantoin, imipenem, and pipracillin-tazobactum. Many studies have documented a considerable proportion of antibiotic resistance in biofilm producers in comparison to nonbiofilm producers.[15],[16] Biofilm formation by UPEC is a leading determinant in the establishment of UTIs. Biofilm-forming bacteria are a thousand times more resistant to antimicrobial agents and host immune attacks.[17] This may be because of the inadequate concentration of the antibiotics encompassing some areas of the biofilms and the metabolic dormancy of the bacteria placed at the base of the biofilm.[7]

We have found a significant association between biofilm formation and antibiotic resistance. Therefore, the awareness of antibiotic sensitivity profile and biofilm formation by UPEC will help in concluding on a relevant treatment of patients with antibiotic resistance UTIs.

Acknowledgement

The technical help offered by our technician Mrs. Arti is duly acknowledged. The authors also express their sincere gratitude to Mrs. Promila for her assistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Tenaillon O, Skurnik D, Picard B, Denamur E The population genetics of commensal Escherichia coli. Nat Rev Microbiol 2010;8:207-17.  Back to cited text no. 1
    
2.
Schito GC Why fosfomycin trometamol as first line therapy for uncomplicated UTI? Int J Antimicrob Agents 2003;22:79-83.  Back to cited text no. 2
    
3.
Sevanan M, Pongiya U, Peedikavil NJ Antimicrobial susceptibility pattern of biofilm producing Escherichia coli of urinary tract infections. Curr Res Bacteriol 2011;4:73-80.  Back to cited text no. 3
    
4.
Marrs CF, Zhang L, Foxman B Escherichia coli mediated urinary tract infections: Are there distinct uropathogenic E. coli (UPEC) pathotypes? FEMS Microbiol Lett 2005;252:183-90.  Back to cited text no. 4
    
5.
Macià MD, Rojo-Molinero E, Oliver A Antimicrobial susceptibility testing in biofilm-growing bacteria. Clin Microbiol Infect 2014;20:981-90.  Back to cited text no. 5
    
6.
Donlan RM, Costerton JW Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167-93.  Back to cited text no. 6
    
7.
Soto SM Importance of biofilms in urinary tract infections: New therapeutic approaches. Adv Biol 2014;2014:1-13.  Back to cited text no. 7
    
8.
Costerton JW Introduction to biofilm. Int J Antimicrob Agents 1999;11:217-21; discussion 237-9.  Back to cited text no. 8
    
9.
Saroj G, Vivek H, Sujata KK, Reddy M Correlation between biofilm formation of uropathogenic Escherichia coli and its antibiotic resistance pattern. J Evol Med Dent Sci 2012;1:166-75.  Back to cited text no. 9
    
10.
Cheesbrough M District Laboratory Practice in Tropical Countries. Part II. 2nd ed. New York: Cambridge University Press; 2006. p. 112-3.  Back to cited text no. 10
    
11.
Washington CW, Jr, Stephen DA, William MJ, Elmer WK, Gray WP, Paul CSGL Koneman’s Colour Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006.  Back to cited text no. 11
    
12.
Clinical Laboratory Standards Institute (CLSI). CLSI Document M100S-S22. Performance Standards for Antimicrobial Susceptibility Testing. Twenty Second Informational Supplement edition. Wayne, PA: CLSI; 2012.  Back to cited text no. 12
    
13.
Das S, Dash HR Microbial Biotechnology: A Laboratory Manual for Bacterial Systems. India: Springer; 2015.  Back to cited text no. 13
    
14.
Svanborg C, Godaly G Bacterial virulence in urinary tract infection. Infect Dis Clin North Am 1997;11:513-29.  Back to cited text no. 14
    
15.
Punia P, Goel N, Asati S, Phogat R, Chaudhary U Biofilm detection amongst extended spectrum beta lactamase (ESBL) and metallo beta lactamase (MBL) producing clinical isolates of Acinetobacter baumannii. Int J Enchanced Res Med Dent Care 2016;3:10.  Back to cited text no. 15
    
16.
Shahidul KM, Farahnaaz F, Sunjukta A Determination of antibiotic resistance pattern of biofilm producing pathogenic bacteria associated with UTI. Int J Drug Dev Res 2013;5:312-9.  Back to cited text no. 16
    
17.
Anderson GG, O’Toole GA Innate and induced resistance mechanisms of bacterial biofilms. Curr Top Microbiol Immunol 2008;322:85-105.  Back to cited text no. 17
    


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