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Year : 2016  |  Volume : 59  |  Issue : 2  |  Page : 177-179
Comparison of four different methods for detection of biofilm formation by uropathogens

1 Department of Microbiology, Pandit Bhagwat Dayal Sharma Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India
2 Department of Neuroanaesthesiology, All Institute of Medical Sciences, New Delhi, India

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Date of Web Publication9-May-2016


Context: Urinary tract infection (UTI) is one of the most common infectious diseases encountered in clinical practice. Emerging resistance of the uropathogens to the antimicrobial agents due to biofilm formation is a matter of concern while treating symptomatic UTI. However, studies comparing different methods for detection of biofilm by uropathogens are scarce. Aims: To compare four different methods for detection of biofilm formation by uropathogens. Settings and Design: Prospective observational study conducted in a tertiary care hospital. Materials and Methods: Totally 300 isolates from urinary samples were analyzed for biofilm formation by four methods, that is, tissue culture plate (TCP) method, tube method (TM), Congo Red Agar (CRA) method and modified CRA (MCRA) method. Statistical Analysis: Chi-square test was applied when two or more set of variables were compared. P < 0.05 considered as statistically significant. Considering TCP to be a gold standard method for our study we calculated other statistical parameters. Results: The rate of biofilm detection was 45.6%, 39.3% and 11% each by TCP, TM, CRA and MCRA methods, respectively. The difference between TCP and only CRA/MCRA was significant, but not that between TCP and TM. There was no difference in the rate of biofilm detection between CRA and MCRA in other isolates, but MCRA is superior to CRA for detection of the staphylococcal biofilm formation. Conclusions: TCP method is the ideal method for detection of bacterial biofilm formation by uropathogens. MCRA method is superior only to CRA for detection of staphylococcal biofilm formation.

Keywords: Biofilm, detection, four, methods, uropathogen

How to cite this article:
Panda PS, Chaudhary U, Dube SK. Comparison of four different methods for detection of biofilm formation by uropathogens. Indian J Pathol Microbiol 2016;59:177-9

How to cite this URL:
Panda PS, Chaudhary U, Dube SK. Comparison of four different methods for detection of biofilm formation by uropathogens. Indian J Pathol Microbiol [serial online] 2016 [cited 2023 Oct 2];59:177-9. Available from:

   Introduction Top

Now-a-days, urinary tract infection (UTI) is one of the most common infectious diseases encountered in clinical practice, and antimicrobial resistance is a serious concern in treatment of symptomatic UTI.[1] Bacteria attach to surface aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms.[2] Biofilm forming bacteria are more resistant to antimicrobial agents leading to the limited effectiveness of current antibiotic therapies. Hence, detection of biofilm production by uropathogens is important, and it can help in initiating appropriate intervention in cases of symtomatic UTI.

There are various methods described in current literature to detect biofilm production like tissue culture plate (TCP), tube method (TM), Congo Red Agar method (CRA), modified CRA method (MCRA), bioluminescent assay, piezoelectric sensors, and fluorescent microscopic examination.[3] However, not all of these methods can be used in routine clinical laboratories and studies comparing different methods to detect biofilm formation in urinary samples are scarce. The primary objective of this study was to compare four methods (i.e., TCP, TM, CRA, and MCRA), which can be used in a routine clinical laboratory to detect biofilm formation by uropathogens.

   Materials and Methods Top

The current study was conducted in the tertiary care institution over a period of 1-year. The urine samples received in the laboratory during this period were examined initially by standard conventional microbiological techniques. A total of 300 isolates (100 Escherichia coli, 50 Klebsiella spp., 50 Pseudomonas spp., 30 Staphyloccocus spp., 20 Proteus spp., 20 Enterobacter spp., 15 Citrobacter spp., 10 Acinetobacter spp., and 5 enterococcus spp.) obtained from those samples were analyzed further for biofilm production by four methods (described below) that is, TCP, TM, CRA and MCRA. Biofilm production by each of the four described methods was graded as high, moderate, and weak. For all practical purpose high and moderate biofilm production by each method was considered positive and weak/no biofilm production by each method was considered negative.

Tissue culture plate method

Isolates from fresh agar plates were inoculated in brain heart infusion (BHI) broth with 2% sucrose and incubated for 18–24 h at 37°C in a stationary condition. The broth with visible turbidity was diluted to 1 in 100 with fresh medium. Individual wells of flat bottom polystyrene plates were filled with 0.2 ml of the diluted cultures, and only broth served as a control to check sterility and nonspecific binding of the medium. These plates were incubated for 24 h at 37°C. After incubation, the content of the well was gently removed and then were washed 4 times with 0.2 ml of phosphate buffer saline (PBS pH 7.2) to remove free-floating “planktonic” bacteria. Biofilms formed by adherent “sessile” organisms in plate were fixed with sodium acetate (2%) for half an hour and stained with crystal violet (0.1% w/v) for half an hour. Excess stain was rinsed off by thorough washing with deionized water and plates were kept for drying. Adherent bacterial cells usually formed a biofilm on all side wells and were uniformly stained with crystal violet. Optical densities (OD) of stained adherent bacteria were determined with a micro Enzyme-Linked Immunosorbent Assay auto reader at wavelength of 570 nm (OD 570 nm) and were graded as per Christensen et al. [Table 1]. These OD values were considered as an index of bacteria adhering to the surface and forming biofilms. The experiment was performed in triplicate.[4]
Table 1: Classification of bacterial adherence by TCP method

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Tube method

BHI broth with 2% sucrose (10 ml) was inoculated with loopful of microorganism from overnight culture plates and incubated for 24 h at 37°C. The tubes were then decanted and washed with PBS (pH 7.3) and dried. Dried tubes were then stained with crystal violet (0.1%) for half an hour. Excess stain was removed, tubes were then dried and observed for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Tubes were examined, and the amount of biofilm formation was scored as absent, moderate or strong. The experiment was performed in triplicate.[5]

Congo Red Agar method

This requires the use of a specially prepared solid medium BHI broth supplemented with 5% sucrose and Congo red. The CRA plate was inoculated with the microorganism from an overnight culture plate and incubated at 37°C for 24–48 h. Positive result was indicated by black colonies with a dry crystalline consistency. The experiment was performed in triplicate.[6]

Modified Congo Red Agar method

In the MCRA the CRA is modified in the form of changing the concentration of Congo red dye and sucrose, omission of glucose, replacement of BHI Agar by an alternative agar, that is, Blood Base Agar. The MCRA plate was inoculated with organisms from a fresh plate with overnight growth, and then it was incubated for 48 h at 37°C and subsequently 2–4 days at room temperature. Positive result was indicated by black colonies with a dry crystalline consistency. The experiment was performed in triplicate.[7]

The statistical analysis was performed using SPSS software version 16.0. Data were presented as percentages 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. Based on the available literature, we considered TCP to be gold standard method of biofilm detection amongst the four mentioned methods for our study and calculated the other statistical parameters accordingly.

   Results Top

Out of the 300 isolates TCP method detected biofilm production in 137/300 (45.6%) cases. The rate of biofilm detection by TM (39.3%) was not significantly different from that by TCP (P = 0.14). However, in CRA and MCRA the rate of detection was 11% each which is significantly lower than that in TCP (P = 0.0001). Except for staphylococcus aureus we could not find any difference in the rate of biofilm detection between CRA and MCRA. However, MCRA detected biofilm production in 38.4% of staphylococcus isolates as compared to 23% by CRA. TCP method detected more strong (11%) and moderate (34.7%) biofilm producers when compared to other methods [Table 2]. TM was 81% sensitive, 95.1% specific, with positive and negative predictive value, and accuracy of 93.3%, 85.6% and 88.7% respectively as compared to CRA and MCRA methods [Table 3].
Table 2: Grading of biofilm formation in various isolates by the four different methods

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Table 3: Statistical evaluation of TM, CRA, modified CRA methods for detection of biofilm formation

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   Discussion Top

In our study, we studied urinary samples exclusively and compared four methods of biofilm detection that can be used in routine clinical laboratories. There are very few similar studies available in the current literature. We evaluated 300 urinary isolates by four methods (that can be used in routine clinical laboratories) for their ability to form biofilms. Out of the 300 isolates, the TCP method detected biofilm in 137 isolates (45.6%), TM detected biofilm in 118 isolates (39.3%), CRA and MCRA detected biofilm in 33 isolates (11%). The TCP was found to be most sensitive followed by the TM, CRA and the MCRA method.

In their study on 110 isolates Hassan et al.,[3] reported that the TCP method detected biofilm in 70 isolates (63.6%), TM in 54 (49%) and CRA in 11 (10%) isolates. In another study by Mathur et al.,[8] out of the total 152 isolates tested for biofilm formation, 47.3%, 41.4% and 5.2% isolates were biofilm producers as detected by TCP, TM and CRA respectively. Baqai et al.,[9] reported biofilm production in 75% of the isolates as detected by TM while with CRA detected it only in 10% isolates. Murugan et al.,[10] in their study found 81 out of 96 (84.3%) isolates of E. coli formed biofilm as detected by TM while only 33 out of 96 (34.3%) produced biofilm when CRA method was used for detection. Knobloch et al.,[11] in their study found that out of 128 isolates of S. aureus, CRA detected only 3.8% of isolates as compared to TCP (57.1%) as biofilm producers. They have not recommended the CRA method for biofilm detection.

Comparison of the grading of the biofilm detected by different methods in various studies is shown in [Table 4]. Our study results correlated well with those study results. All the studies suggested that the though TM correlated well with the TCP method for strong biofilm detection, it was difficult to discriminate moderate and weak/none biofilm production by TM. This difference could be attributed to subjective observer's assessment used in TM as compared to the more accurate objective assessment in TCP. In all the studies, it was found that the CRA could not differentiate between strong, moderate, and weak biofilm producers. We also found CRA and MCRA not useful for detection biofilm by uropathogens. There was no difference between CRA and MCRA in terms of rate of biofilm detection except in cases of staphylococcal biofilm where MCRA is a better method than CRA for the same.
Table 4: Comparison of grading of biofilm detection by different methods used in previous studies

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   Conclusion Top

TCP method is the ideal method for detection of bacterial biofilm formation by uropathogens. MCRA method is superior to CRA and not to TCP or TM for detection of the staphylococcal biofilm formation.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.[13]

   References Top

Abdallah NM, Elsayed SB, Mostafa MM, El-Gohary GM. Biofilm forming bacteria isolated from urinary tract infection, relation to catheterization and susceptibility to antibiotics. Int J Biotechnol Mol Biol Res 2011;2:172-8.  Back to cited text no. 1
Aparna MS, Yadav S. Biofilms: microbes and disease. Braz J Infect Dis 2008;12:526-30.  Back to cited text no. 2
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 Infect Dis 2011;15:305-11.  Back to cited text no. 3
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. 4
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.  Back to cited text no. 5
Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 1989;42:872-4.  Back to cited text no. 6
Mariana NS, Salman SA, Neela V, Zamberi S. Evaluation of modified Congo Red Agar for detection of biofilm produced by clinical isolates of methicillin-resistance Staphylococcus aureus. Afr J Microbiol Res 2009;3:330-8.  Back to cited text no. 7
Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods. Indian J Med Microbiol 2006;24:25-9.  Back to cited text no. 8
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Baqai R, Aziz M, Rasool G. Urinary tract infection in diabetic patients and biofilm formation of uropathogens. Infect Dis Pak 2008;17:7-9.  Back to cited text no. 9
Murugan S, Devi PV, John PN. Antimicrobial susceptibility pattern of biofilm producing Escherichia coli of urinary tract infections. Curr Res Bacteriol 2011;4:73-80.  Back to cited text no. 10
Knobloch JK, Horstkotte MA, Rohde H, Mack D. Evaluation of different detection methods of biofilm formation in Staphylococcus aureus. Med Microbiol Immunol 2002;191:101-6.  Back to cited text no. 11
Nagaveni S, Rajeshwari H, Oli AK, Patil SA, Chandrakanth RK. Evaluation of biofilm forming ability of the multidrug resistant Pseudomonas aeruginosa. Bioscan 2010;5:563-6.  Back to cited text no. 12
Vishnuprasad S, Ballal M, Shivananda PG. Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: A comparative study of two methods. Indian J Pathol Microbiol 2009;52:191-3.  Back to cited text no. 13

Correspondence Address:
Pragyan Swagatika Panda
Department of Microbiology, Pandit Bhagwat Dayal Sharma Post Graduate Institute of Medical Sciences, Rohtak, Haryana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.182013

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

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