|Year : 2018 | Volume
| Issue : 2 | Page : 175-179
Analysis of antibiotic sensitivity profile of biofilm-forming uropathogenic Escherichia coli
Kulkarni Ramesh Sudheendra1, Peerapur V Basavaraj2
1 Department of Microbiology, BLDEA's Shri B M Patil Medical College, Bijapur, India
2 Department of Microbiology, Raichur Institute of Medical Sciences, Raichur, Karnataka, India
|Date of Web Publication||20-Jun-2018|
Kulkarni Ramesh Sudheendra
Department of Microbiology, BLDEA's Shri B M Patil Medical College, Bijapur, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Biofilms are group of microorganisms which are embedded within a self-produced matrix of extracellular polymeric substance which adhere to each other. They are found to be involved in a wide range of infections in the body like urinary tract infections (UTIs). Biofilms are considered to be highly resistant to antimicrobial agents. Escherichia coli (E. coli) is the most common organism causing both community as well as hospital acquired UTI leading to serious health issues. Objectives: This study was conducted to analyse the antibiotic sensitivity profile of biofilm forming Escherichia coli (E. coli) isolated from patients with suspected UTI attending a Teaching hospital of North Karnataka. Materials And Methods: 388 E. coli isolates recovered from1000 suspected cases of UTI were tested for susceptibility to fourteen different antibiotics. In vitro biofilm formation was detected by Tube adherence method, Congo red agar method and Tissue culture plate method. Results: 277 isolates (71.39%) produced biofilm in-vitro by all the three methods. Biofilm forming E. coli developed significantly higher degree of resistance towards antimicrobial drugs Ampicillin (87.36%), Cefuroxime (81.58%), Amoxicillin clavulanic acid (77.61%), Ciprofloxacin (71.48%) and Ceftriaxone (71.48%). They were sensitive to higher antibiotics like Imipenem, Piperacillin-tazobactam, Nitrofurantoin, and Amikacin. Conclusion: Detection of biofilm in E. coli and its resistance to commonly prescribed antibiotics in the clinical practice is essential in improving the efficacy of empirical treatment. This study revealed the prevalence and antimicrobial susceptibility pattern of biofilm forming E. coli which helps clinicians to treat UTI effectively.
Keywords: Antibiotic resistance, biofilm, Escherichia coli, urinary tract infection
|How to cite this article:|
Sudheendra KR, Basavaraj PV. Analysis of antibiotic sensitivity profile of biofilm-forming uropathogenic Escherichia coli. J Nat Sc Biol Med 2018;9:175-9
|How to cite this URL:|
Sudheendra KR, Basavaraj PV. Analysis of antibiotic sensitivity profile of biofilm-forming uropathogenic Escherichia coli. J Nat Sc Biol Med [serial online] 2018 [cited 2018 Dec 12];9:175-9. Available from: http://www.jnsbm.org/text.asp?2018/9/2/175/234710
| Introduction|| |
Urinary tract infections (UTIs) are the major and most important cause of serious health problems and morbidity. UTIs account for more than 7 million visits to physicians per year ,, affecting persons of all ages including children, women, and elderly but most predominant in women, especially in developing countries such as India., Approximately 40% of women have had a UTI in their lifetime and over 20% of young sexually active women who had previous UTIs have recurrent UTIs.
Escherichia coli is the most common organism causing both community and hospital-acquired UTIs, leading to serious secondary health issues worldwide., Currently, recurrent UTI is a serious health problem for many women despite our broad array of very successful antimicrobial agents. Recurrent and relapse UTIs may be due to bacterial virulence factors exhibited by uropathogenic E. coli (UPEC) which enable colonization of the bacteria and help the organism overcome host defenses and invade the urinary tract.
Biofilm formation is one of the most important virulence factors exhibited by E. coli among other virulence factors. Microbial biofilms are community of bacteria and other microorganisms that are irreversibly attached to self-produced extracellular polymeric substances and adhere to a surface or each other. Biofilms are ubiquitous and can be found in a variety of niches or sites or devices. They play an important role in medicine and have been proven to cause a wide range of microbial infections in the human body such as UTIs, catheter-associated infections, or dental plaques.
Biofilms decrease the susceptibility of organism to antimicrobial agents by enclosing them in an extracellular matrix. A high content of polysaccharides in biofilm prevents the access of antimicrobial agents. Limited penetration of antimicrobial agents into the biofilm and slow rate of cell multiplication of organisms in the biofilm may contribute to the development of chronic infections. Biofilm-forming bacteria exhibit higher resistance to antimicrobial drugs used for the treatment of UTIs, which also lead to recurrent infections.
Our study aimed to unveil the association of biofilm-forming E. coli and their antimicrobial susceptibility pattern. This study would help the clinicians in choosing suitable antibiotics for effective treatment of UTI.
| Materials and Methods|| |
Sample collection and processing
This study was conducted in the Department of Microbiology, Bidar Institute of Medical Sciences (BRIMS), Bidar, after getting approval from the Institutional Ethical Committee. One thousand patients of all age groups and both sexes complaining of burning micturition and other associated illness attending the outpatient department of BRIMS teaching hospital were included in this study. Informed consent was obtained from all the patients. Clean-catch midstream urine samples were collected in a sterile widemouthed container along with information about their age, sex, and brief clinical history. Samples were transported to the laboratory immediately and processed for culture and antimicrobial drug susceptibility testing as per the routine microbiological techniques and recommendations of Kass. Further, the isolates were identified by standard biochemical tests, and a diagnosis of UTI was made when pathogens were grown at least 105 colony forming unit/ml of urine. Only E. coli isolates were included in this study.
Antibiotic sensitivity testing
Antibiotics (obtained from HiMedia Laboratories, Mumbai, Maharashtra, India) such as ampicillin (AMP 10 μg), amikacin (AK 30 μg), amoxicillin-clavulanic acid (AMC 30 μg), aztreonam (AT 30 μg), ceftriaxone (CTR 30 μg), cefuroxime (CXM 30 μg), cefepime (CPM 30 μg), ciprofloxacin (CIP 5 μg), chloramphenicol (C 30 μg), gentamicin (GEN 10 μg), imipenem (IPM 10 μg), nitrofurantoin (NIT 300 μg), norfloxacin (NX 10 μg), and piperacillin-tazobactam (PIT 100/10 μg) were tested according to Kirby–Bauer's disc diffusion method  as per the Clinical and Laboratory Standards Institute's (CLSI) guidelines.
The CLSI control strain of E. coli ATCC 25922 was used as a control for antimicrobial susceptibility testing.
| In Vitro Biofilm Detection|| |
In vitro detection of biofilm was done by three different methods as follows: tube adherence method, Congo red agar method (CRA), and tissue culture plate method (TCP).
Tube adherence method
This test described by Christensen et al. is a qualitative method for biofilm detection. A loopful of test organisms was inoculated in 10 ml of trypticase soy broth with 1% glucose in test tubes. The tubes were incubated at 37°C for 24 h. After incubation, tubes were decanted and washed with phosphate-buffered saline (pH 7.3) and dried. Tubes were then stained with crystal violet (0.1%). Excess stain was washed with deionized water and dried. The scoring for tube method was done according to the results of the control strains. Biofilm formation was considered positive when a visible film lined the wall and the bottom of the tube.
Congo red agar method
Freeman et al. have described a simple qualitative method to detect biofilm production using CRA medium. CRA medium was prepared with brain–heart infusion broth 37 g/L, sucrose 50 g/L, agar no. 1 10 g/L (HiMedia Laboratories, Mumbai, Maharashtra, India), and Congo red indicator 8 g/L (Nice chemicals, Cochin). Initially, Congo red stain was prepared as a concentrated aqueous solution and autoclaved (121°C for 15 min) separately from the other medium constituents. It was then added to the autoclaved brain–heart infusion agar with sucrose at 55°C. CRA plates were inoculated with test organisms and incubated at 37°C for 24 h aerobically. Black colonies with a dry crystalline consistency indicated biofilm production.
Tissue culture plate method
This quantitative test described by Christensen et al. is considered the gold standard method for biofilm detection. Isolates were inoculated in 10 ml of trypticase soy broth with 1% glucose and incubated at 37°C for 24 h. The cultures were then diluted 1:100 with fresh medium. Individual wells of sterile TCPs were filled with 200 μL of the diluted cultures including control strains. Plates were incubated at 37°C for 24 h. After incubation, contents of each well were removed by gentle tapping. The wells were washed with 0.2 mL of phosphate-buffered saline (pH 7.2) four times. Biofilms formed by bacteria adherent to the wells were fixed by 2% sodium acetate and stained by crystal violet (0.1%). Excess stain was removed by using deionized water and plates were dried. Optical density of stained adherent biofilms was obtained using micro ELISA autoreader (model 680, Biorad, UK) at a wavelength of 570 nm. The interpretation of biofilm production was done according to the criteria of Stepanovic et al.
The biofilm producers such as Staphylococcus epidermidis ATCC 35984 (positive control) and the nonbiofilm producers such as S. epidermidis ATCC 12228 (negative control) were used as standard control strains.
Statistical software package SPSS version 22 (IBM SPSS Statistics for Windows, IBM Corp., Released 2013, Armonk, NY, USA) was used to analyze the data. Chi-square test was applied. P < 0.05 was considered statistically significant.
| Results|| |
Of 1000 urine specimens processed from patients of suspected UTI, 388 E. coli were isolated (38.8%). Infection was predominant in females with a rate of 80.92% between the age group of 20 and 29 years (39%). Among males, the infection rate was 19.07%.
Among 388 E. coli strains subjected to in vitro biofilm production, 277 isolates (71.39%) produced biofilm by all the three methods.In vitro biofilm formation by different methods was as follows: 40 (10.3%) strains showed highly positive, 35 strains (9%) showed moderately positive, and 91 strains (23.5%) showed weakly positive by tube method [Figure 1]. Similarly, in CRA method, 254 strains (65.5%) showed highly positive [Figure 2], whereas in TCP method, 284 (73.2%) strains showed strongly positive, 23 strains (5.9%) showed moderately positive, and 81 strains (20.9%) showed weakly positive [Figure 3] and [Table 1].
|Figure 1: Strong biofilm formation of Escherichia coli by tube adherence method|
Click here to view
|Figure 3: Positive biofilm formation of Escherichia coli by tissue culture plate method|
Click here to view
|Table 1: Screening of the Escherichia coli isolates for biofilm formation by tube adherence method, Congo Red Agar method, and tissue culture plate method|
Click here to view
Biofilm-producing isolates showed the highest resistance to the antibiotics compared to nonbiofilm-producing isolates. Biofilm producers demonstrated resistance to AMP (87.36%) followed by CXM (81.58%), AMC (77.61%), CIP (71.48%), CTR (54.6%), and CPM (64.98%) [Table 2]. Significant association was observed between biofilm formation and multidrug resistance which was proved to be statistically significant regarding antibiotics such as AK, AMC, AT, CTR, CXM, CPM, CIP, and C [Table 3]. Isolates were sensitive to antibiotics such as PIT (97.83%), IPM (97.14%), and NIT (92.41%).
|Table 2: Antibiotic sensitivity profile of biofilm-forming and nonbiofilm-producing Escherichia coli isolates|
Click here to view
|Table 3: Association between antimicrobial resistance and biofilm-forming uropathogenic Escherichia coli isolates|
Click here to view
| Discussion|| |
E. coli is the most prominent causative agent of both symptomatic and asymptomatic UTIs, which accounts for more than 80% of the infections., In our study, we found that the frequency of UTI was higher in females compared to males, which concord with other studies conducted., This difference in frequency may be due to several clinical factors, including anatomic differences and hormonal effects. UTI is associated with an expression of different virulence factors including biofilm formation. Biofilm formation is closely related to the susceptibility pattern of E. coli toward the antimicrobial drugs which are commonly used to treat UTIs. The resistance pattern in UTI patients of this region is not known. Understanding the resistance pattern will be helpful for treatment. Resistance to antibiotics by biofilm-producing E. coli increases the chronicity and recurrence of UTI as bacteria are enclosed within the biofilm and do not allow the antibiotic access to the bacteria.
In this study, the incidence of in vitro biofilm formation by UPEC was 71.39%, which was similar to the studies conducted by Subramanian et al., Sharma et al., and Suman et al. who reported biofilm formation at the rates of 63%, 67.5%, and 92.0%, respectively. In our study, we analyzed the in vitro biofilm formation by three different methods. Nearly 42.78% of isolates were found positive by tube adherence method, and we classified them as highly positive (10.3%), moderately positive (9%), and weakly positive (23.5%). In CRA method, 65.5% isolates were found positive. In TCP method, again it was classified as strongly positive (73.2%), moderately positive (5.9%), and weakly positive (20.9%). These findings were much closer to the study results reported by Tabasi et al. In our study, we detected biofilm formation in all the 388 E. coli isolates (100%) by TCP method which was similar to the findings reported by Fattahi et al. who outlined biofilm formation in 100% of isolates by TCP method.
We studied antibiotic susceptibility pattern for all UPEC isolates. We analyzed the antibiotic resistance pattern of biofilm- and nonbiofilm-forming E. coli isolates. Biofilm-forming isolates demonstrated increased resistance to the commonly used antibiotics to treat UTI compared to nonbiofilm producers. Our study results revealed significant correlation between biofilm formation and multidrug resistance. There was an increase in resistance pattern of the drugs such as AK, AMC, AT, CTR, CXM, CPM, CIP, and C which were routinely used to treat UTIs from a long time. This pattern of resistance coincides with the study findings reported by Mittal et al. and Ponnusamy et al.,
Bacterial biofilms are associated with long-term persistence of the organisms in various environments. Biofilms make the organisms impermeable to antibiotics and bind the agents at the outer surface of the matrix layer which protects the bacteria from penetration of the antibiotics. This causes recurrent infection and results in the organism developing multidrug resistance. These strains respond poorly or not respond at all to conventional and routine antimicrobial therapies.
In the present study, the drugs PIT, IPM, and NIT were effective against biofilm-producing UPEC isolates and these drugs can serve as useful reserved drugs for the treatment of UTI. Understanding biofilms in UTIs will help clinicians in decision-making toward effective treatment guidelines for recurrent UTI in this geographical region.
| Conclusion|| |
Conclusively, we have noticed significant association between biofilm production and multidrug resistance. We believe that the detection of biofilm formation might be worth in the management of UTI therapy. Therefore, the knowledge of biofilm formation by E. coli and their antibiotic susceptibility pattern will help in deciding on an appropriate antibiotic treatment for UTI. It also helps restrain the emergence of drug-resistant strains.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Patton JP, Nash DB, Abrutyn E. Urinary tract infection: Economic considerations. Med Clin North Am 1991;75:495-513.
Hooton TM, Stamm WE. Diagnosis and treatment of uncomplicated urinary tract infection. Infect Dis Clin North Am 1997;11:551-81.
Schappert SM, Burt CW. Ambulatory care visits to physician offices, hospital outpatient departments, and emergency departments: United states, 2001-02. Vital Health Stat 13 2006;(159):1-66.
Al-Jiffri O, El-Sayed ZM, Al-Sharif FM. Urinary tract infection with Escherichia coli
and antibacterial activity of some plants extracts. Int J Microbiol Res 2011;2:01-7.
Williams NS, Bulstrode CJ, O'Connell PR, editors. Bailey & Love's Short Practice of Surgery. 25th
ed. London, United Kingdom: Edward Arnold Publishers; 2008. p. 1329-30.
Kunin CM. Urinary tract infections in females. Clin Infect Dis 1994;18:1-10.
Schito GC. Why fosfomycin trometamol as first line therapy for uncomplicated UTI? Int J Antimicrob Agents 2003;22 Suppl 2:79-83.
Rodríguez-Baño J, Navarro MD, Romero L, Muniain MA, Perea EJ, Pérez-Cano R, et al.
Clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli
as a cause of nosocomial infection or colonization: Implications for control. Clin Infect Dis 2006;42:37-45.
Wang X, Lünsdorf H, Ehrén I, Brauner A, Römling U. Characteristics of biofilms from urinary tract catheters and presence of biofilm-related components in Escherichia coli.
Curr Microbiol 2010;60:446-53.
Tambyah PA. Catheter-associated urinary tract infections: Diagnosis and prophylaxis. Int J Antimicrob Agents 2004;24 Suppl 1:S44-8.
Tajbakhsh E, Ahmadi P, Abedpour-Dehkordi E, Arbab-Soleimani N, Khamesipour F. Biofilm formation, antimicrobial susceptibility, serogroups and virulence genes of uropathogenic E. coli
isolated from clinical samples in Iran. Antimicrob Resist Infect Control 2016;5:11.
Tayal RA, Baveja SM, De Anuradha S. Analysis of biofilm formation and antibiotic susceptibility pattern of uropathogens in patients admitted in a tertiary care hospital in India. Int J Health Allied Sci 2015;4:247. [Full text]
Kass EH. Pyelonephritis and bacteriuria. A major problem in preventive medicine. Ann Intern Med 1962;56:46-53.
Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.
Clinical and Laboratory Standards Institute (CLSI) Approved standard M2-A10. Performance Standards for Antimicrobial Disk. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2007.
Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime-producing strains of Staphylococcus epidermidis
to smooth surfaces. Infect Immun 1982;37:318-26.
Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 1989;42:872-4.
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.
Stepanović S, Vuković D, Hola V, Di Bonaventura G, Djukić S, Cirković I, et al.
Quantification of biofilm in microtiter plates: Overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS 2007;115:891-9.
Hedlund M, Duan RD, Nilsson A, Svensson M, Karpman D, Svanborg C, et al.
Fimbriae, transmembrane signaling, and cell activation. J Infect Dis 2001;183 Suppl 1:S47-50.
Svanborg C, Godaly G. Bacterial virulence in urinary tract infection. Infect Dis Clin North Am 1997;11:513-29.
Miller O, Hemphill RR. Urinary tract infection and pyelonephritis. Emerg Med Clin North Am 2001;19:655-74.
Foxman B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Am J Med 2002;113:5-13.
Subramanian P, Shanmugam N, Sivaraman U, Kumar S, Selvaraj S. Antibiotic resistance pattern of biofilm-forming uropathogens isolated from catheterised patients in Pondicherry, India. Australas Med J 2012;5:344-8.
Sharma M, Yadav S, Chaudhary U. Biofilm production in uropathogenic Escherichia coli.
Indian J Pathol Microbiol 2009;52:294.
Suman E, Jose J, Varghese S, Kotian MS. Study of biofilm production in Escherichia coli
causing urinary tract infection. Indian J Med Microbiol 2007;25:305-6.
] [Full text]
Tabasi M, Asadi Karam MR, Habibi M, Yekaninejad MS, Bouzari S. Phenotypic assays to determine virulence factors of uropathogenic Escherichia coli
(UPEC) isolates and their correlation with antibiotic resistance pattern. Osong Public Health Res Perspect 2015;6:261-8.
Fattahi S, Kafil HS, Nahai MR, Asgharzadeh M, Nori R, Aghazadeh M, et al.
Relationship of biofilm formation and different virulence genes in uropathogenic Escherichia coli
isolates from Northwest Iran. GMS Hyg Infect Control 2015;10:Doc11.
Mittal S, Sharma M, Chaudhary U. Biofilm and multidrug resistance in uropathogenic Escherichia coli.
Pathog Glob Health 2015;109:26-9.
Ponnusamy P, Natarajan V, Sevanan M.In vitro
biofilm formation by uropathogenic Escherichia coli
and their antimicrobial susceptibility pattern. Asian Pac J Trop Med 2012;5:210-3.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]