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Year : 2011  |  Volume : 2  |  Issue : 1  |  Page : 20-23  

Insignificant difference seen on biofilm production among indwelling medical device associated bacterial isolates

1 Department of Microbiology, Government Medical College, New Rander Road, Surat, India
2 Department of Microbiology, Government Medical College, Bhatar Road, Surat, India

Date of Web Publication15-Jul-2011

Correspondence Address:
Sangita B Revdiwala
28, Parikshit Society, opp Gajjar Park apartment, Besides Rupali canal, Bhatar road, Surat - 395 001
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0976-9234.82988

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Aim of study : Biofilm formation is a developmental process with intercellular signals that regulate growth. Biofilms contaminate catheters, ventilators and medical implants; they act as a source of disease for humans, animals and plants. In this study we have done quantitative assessment of biofilm formation in device-associated clinical bacterial isolates in response to various concentrations of glucose in tryptic soya broth and with different incubation time. Materials and Methods: The study was carried out on 100 positive bacteriological cultures of medical devices which were inserted in hospitalized patients. The bacterial isolates were processed as per microtitre plate method with tryptic soya broth alone and with varying concentrations of glucose and were observed in response to time. Results: The majority of catheter cultures were positive. Out of the total 100 bacterial isolates tested, 88 were biofilm formers. Biofilm production was more in 0.25% than in 0.5% glucose concentration in tryptic soya broth. After 16-h incubation no significant difference was seen in biofilm production. Conclusions: Availability of nutrition in the form of glucose enhances the biofilm formation by bacteria, but further increase in glucose concentration could not enhance biofilm production. Biofilm formation depends on the adherence of bacteria to various surfaces.

Keywords: Biofilm, glucose, incubation time, microtitre plate method, tryptic soya broth

How to cite this article:
Mulla SA, Revdiwala SB. Insignificant difference seen on biofilm production among indwelling medical device associated bacterial isolates. J Pharm Negative Results 2011;2:20-3

How to cite this URL:
Mulla SA, Revdiwala SB. Insignificant difference seen on biofilm production among indwelling medical device associated bacterial isolates. J Pharm Negative Results [serial online] 2011 [cited 2020 Aug 10];2:20-3. Available from:

   Introduction Top

Microorganisms universally attach to surfaces and produce extracellular polysaccharides, resulting in the formation of a biofilm. Biofilms pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices. An appreciation of the role of biofilms in infection should enhance the clinical decision-making process. Many bloodstream infections and urinary tract infections are associated with indwelling medical devices and, therefore, (in most cases) biofilm-associated. The most effective strategy for treating these infections may be removal of the biofilm-contaminated device. [1]

When an indwelling medical device is contaminated with microorganisms, several variables determine whether a biofilm develops. First the microorganisms must adhere to the exposed surfaces of the device long enough to become irreversibly attached. The rate of cell attachment depends on the number and types of cells in the liquid to which the device is exposed, the flow rate of liquid through the device, and the physicochemical characteristics of the surface. Components in the liquid may alter the surface properties and also affect rate of attachment. Once these cells irreversibly attach and produce extracellular polysaccharides to develop a biofilm, the rate of growth is influenced by flow rate, nutrient composition of the medium, antimicrobial drug concentration, and ambient temperature. [2]

There are many works that discuss some features of biofilm-positive bacteria, but there is no consistency in the culture conditions which are feasible for biofilm formation among authors. [3],[4],[5],[6],[7] The only agreement is in the culture temperature, 37°C seems to be appropriate. Other conditions, e.g. presence of nutrition and time of cultivation, vary in many publications. In our study we paid attention to those culture conditions that differ in most authors. We investigated the potential relationship between colonization of different medical devices by various clinical bacterial isolates to determine the differences in the biofilm formation under different culture conditions necessary for the development of a homogenous and mature biofilm layer. [3]

   Materials and Methods Top

Approval was obtained from our institutional review board. The study was carried out on 100 positive bacteriological cultures of medical devices which were inserted in hospitalized patients.

Catheter culture technique

All catheters/ devices submitted to the clinical laboratory for culture during a three-year period were studied. Each catheter coming to the clinical laboratory for culture was directly cultured by roll plate method, then placed in 10 ml of tryptic soy broth ( himedia), incubated for 2 h at 37°C and then vortexed for 15 sec. Broth was then surface-plated by using a wire loop on blood agar, chocolate agar and MacConkey agar (himedia). [8]

Isolates derived later from the clinical laboratory for the purpose of our study were frozen in nutrient broth with 15% glycerol at -20°C. Samples retrieved for the study were grown on blood agar plates and were processed as described below.

Cultures retrieved from the frozen material retained the same biochemical reactions, confirming that no alteration had occurred in the bacterial isolate because of storage and processing.

Biofilm formation and quantification of activity against biofilms

Preparation of inoculum

Three different media were taken; tryptic soya broth, tryptic soya broth with 0.25% glucose and tryptic soya broth with 0.5% glucose for culture. Isolated colonies were inoculated and incubated for 24h in these media, then cultures were diluted 1:200 with respective fresh media.


Biofilm-producing reference strains of Acinetobacter baumanni (ATCC 19606) and Pseudomonas aeruginosa (ATCC 27853) and non-biofilm-forming reference strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were evaluated. [9]

Microtitre plate assay

Biofilm formation was induced in 96-well flat-bottomed polystyrene microtitre plates. An aliquot of 200 μl of diluted bacterial suspension was added to each well and incubated for 16 h, 20 h and 24 h at 37°C. At the end of incubation period, the wells were carefully aspirated, washed twice with 300 μl of phosphate-buffered saline (PBS, pH, 7.2) to remove planktonic bacteria. Wells were emptied and dried before biomass quantification of the biofilms was performed by staining. The staining was done with 200 μl of 0.1% Safranine and 0.1% Crystal Violet (CV) for 45 min. After that, the wells were carefully washed twice with distilled water to remove excess stain. After staining, 200 μl ethanol/acetone (90:10) was added to each well to dissolve remaining stain from the wells. The optical density was then recorded at 492 nm with 630 nm reference filter using an ELISA reader. [3],[10],[11],[12],[13]

Wells originally containing uninoculated medium, non-biofilm-producing bacteria and known biofilm-producing bacteria were used as controls for cutoff, negative controls and positive controls respectively. The test was carried out in quadruplicate, results were averaged and standard deviations were calculated.

The cutoff was defined as three standard deviations above the mean optical density control (ODc). [14] Each isolate was classified as follows: weak biofilm producer OD= 2xODc, moderate biofilm producer 2xODc < OD = 4xODc, or strong biofilm producer OD > 4xODc. [9],[15]

   Results Top

Fifty-nine endotracheal tubes, 11 central venous catheter (CVC) tips, 10 Foley's catheter tips, seven abdominal drain tubes, five nephrostomy tubes, four tracheostomy tubes, three.Double J (D.J) stent tips, and one supra pubic catheter (SPC) tip were included in the study.

Out of total 100 bacterial isolates 23 Acinetobacter baumanni, 23 Pseudomonas aeruginosa, 20 Klebsiella pneumoniae sub spp. pneumoniae, 16 Escherichia coli, nine coagulase-negative Staphylococci, four Enterobacter cloacae, three Enterococci and two Staphylococcus aureus were isolated as stated in [Table 1]. Out of 100 clinical isolates tested 88 were found to be biofilm formers by micro titre plate method. Out of two different staining methods; 0.1% Safranine detected 88 biofilm producers while 0.1% crystal violet detected 87 biofilm producers.

Biofilm formation in response to different concentrations of glucose was tested. Biofilm production in tryptic soya broth without glucose was noted in 75 (85%) isolates . Out of 75, two (40%) were strong and 28 (64%) were moderate biofilm formers. In tryptic soya broth with 0.25% glucose, 81 (92%) were found positive, out of which three (60%) were strong and 30 (70%) were moderate biofilm formers. In tryptic soya broth with 0.5% glucose, 67 (76%) were found positive, out of which four (80%) were strong and 28 (63%) were moderate biofilm formers.
Table 1: Clinical bacterial isolates in association with type of indwelling medical device

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Biofilm formation at different incubation time is done .It is evident from [Table 2] that After 16-h incubation period, 88 (100%) isolates were found positive, out of which three (3%) were strong and 28 (32%) were moderate biofilm formers. After 20-h incubation period, 81 (92%) isolates were found positive, out of which two (2%) were strong and 36 (44%) were moderate biofilm formers. After 24-h incubation period, 76 (86%) isolates were found positive, four (5%) were strong and 29 (38%) were moderate biofilm formers.
Table 2: Screening of 100 bacterial isolates for biofilm formation by tissue culture plate method in different media and at 16, 20 and 24 h incubation period

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   Conclusions Top

Two different staining methods, Safranine 0.1% and Crystal violet 0.1%, both gave equal positive, stable and accurate results in terms of reproducibility. Sixteen-hour incubation time was optimum for detection of biofilms produced by bacteria. Further increasing incubation time has no positive effect on biofilm production. Moderate to weak biofilm-producing bacteria attach to the surfaces and detachment occurs early because of weak binding. Strong biofilm producers can be detected even at 24 h of incubation period. Availability of nutrition favors biofilm formation by bacteria so glucose enhances the biofilm-forming ability of bacteria but the effect of osmolarity and pH on biofilm formation cannot be ruled out.

   References Top

1.Donlan RM. Biofilm formation: A clinically relevant microbiological process. Clin Infect Dis 2001;33:1387-92.  Back to cited text no. 1
2.Donlan RM. Biofilms and device-associated infections. Emerg Infect Dis 2001;7:277-81.  Back to cited text no. 2
3.Hola V, Ruzicka F, Votava M. The dynamics of Staphylococcus epidermis biofilm formation in relation to nutrition, temperature and time. Scr Med (Brno) 2006;79:169-74.  Back to cited text no. 3
4.Stepanovic S, Vukovic D, Jezek P, Pavlovic M, Svabic-Vlahovic M. Influence of dynamic conditions on biofilm formation by staphylococci. Eur J Clin Microbiol Infect Dis 2001;20:502-4.  Back to cited text no. 4
5.Deighton MA, Balkau B. Adherence measured by microtiter assay as a virulence marker for Staphylococcus epidermidis infections. J Clin Microbiol 1990;28: 2442-7.  Back to cited text no. 5
6.Gelosia A, Baldassarri L, Deighton M, van Nguyen T. Phenotypic and genotypic markers of Staphylococcus epidermidis virulence. Clin Microbiol Infect 2001;7:193-9.  Back to cited text no. 6
7.Dunne WM Jr, Mason EO Jr, Kaplan SL. Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm. Antimicrob Agents Chemother 1993;37:2522-6.  Back to cited text no. 7
8.Raad II, Sabbagh MF, Rand KH, Sherertz RJ. Quantitative tip culture methods and the diagnosis of central venous catheter-related infections. Diagn Microbiol Infect Dis 1992;15:13-20.  Back to cited text no. 8
9.Rao RS, Karthika RU, Singh SP, Shashikala P, Kanungo R, Jayachandran S, et al. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Indian J Med Microbiol 2008;26:333-7.  Back to cited text no. 9
10.Rossi BP, Calenda M, Vay C, Franco M. Biofilm formation by Stenotrophomonas maltophilia isolates from device-associated nosocomial infections. Rev Argent Microbiol 2007;39:204-12.  Back to cited text no. 10
11.Jayanthi M, Ananthasubramanian M, Appalaraju B. Assessment of Pheromone response in biofilm forming clinical isolates of high level gentamicin resistant Enterococcus fecalis. Indian J Med Microbiol 2008;26:248-51.  Back to cited text no. 11
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12.Eftekhar F, Mirmohamadi Z. Evaluation of biofilm production by Staphylococcus epidermidis isolates from nosocomial infections and skin of healthy volunteers. Int J of Med and Med Sci 2009;1:438-41.  Back to cited text no. 12
13.Christensen G, Simpson W, Younger J, Baddour L, Barret F, Melton D, 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. 13
14.Stepanovic S, Cirkovic I, Ranin L, Svabic-Vlahovic M. Biofilm formation by Salmonella spp. and listeria monocytogenes on plastic surface. Lett Appl Microbiol 2004;38:428-32.  Back to cited text no. 14
15.Tenorio E, Saeki T, Fujita K, Kitakawa M, Baba T, Mori H, et al. Systematic characterization of Escherichia coli Genes/orfs affecting biofilm formation. FEMS Microbiol Lett 2003;225:107-14.  Back to cited text no. 15


  [Table 1], [Table 2]


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