| Abstract|| |
Background: 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. Aim: 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: Majority of catheter cultures were positive. Out of the total 100 bacterial isolates tested, 88 of them were biofilm formers. Incubation period of 16-20 h was found to be optimum for biofilm development. Conclusions: Availability of nutrition in the form of glucose enhances the biofilm formation by bacteria. Biofilm formation depends on adherence of bacteria to various surfaces. Time and availability of glucose are important factors for assessment of biofilm progress.
Keywords: Biofilm, glucose, incubation time, microtitre plate method, tryptic soya broth
|How to cite this article:|
Mulla SA, Revdiwala S. Assessment of biofilm formation in device-associated clinical bacterial isolates in a tertiary level hospital. Indian J Pathol Microbiol 2011;54:561-4
|How to cite this URL:|
Mulla SA, Revdiwala S. Assessment of biofilm formation in device-associated clinical bacterial isolates in a tertiary level hospital. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Jul 22];54:561-4. Available from: http://www.ijpmonline.org/text.asp?2011/54/3/561/85093
| Introduction|| |
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. 
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 the rate of attachment. Once these cells irreversibly attach and produce extracellular polysaccharides to develop a biofilm, rate of growth is influenced by flow rate, nutrient composition of the medium, antimicrobial drug concentration, and ambient temperature. 
There are many works that discuss some features of biofilm-positive bacteria, but there is no consistency in the conditions, which are feasible for biofilm formation among authors. ,,,, The only agreement is in the culture temperature, 37°C seems to be appropriate. Other conditions, for example, 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 and to determine the differences in biofilm formation in different conditions and to determine the minimum time and conditions necessary for the development of a homogenous and mature biofilm layer. 
| Materials and Methods|| |
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 3-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 soya broth (Himedia, Mumbai, India), incubated for 2 hours at 37°C and then vortexed for 15 seconds. Broth was then surface plated by using a wire loop on blood agar, chocolate agar, and Macconkey agar (Himedia, Mumbai, India). 
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 bacterial isolate because of storage and processing.
Biofilm Formation and Quantification of Activity Against Biofilms
Preparation of inoculum
3 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 24 h 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 Eshcherichia coli (ATCC 25922) were used. 
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, 20, and 24 hours at 37°C. At the end of incubation period, the wells were carefully aspirated and 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 into respective wells for 45 min. At the end of time, 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 the remaining stain from the wells. The optical density was then recorded at 492 nm with 630 nm reference filter using an ELISA reader. ,,,,
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 ODc.  Each isolate was classified as follows: Weak biofilm producer OD = 2 x ODc, moderate biofilm producer 2 x ODc < OD = 4 x ODc, or strong biofilm producer OD > 4 x ODc. ,
| Results|| |
Fifty-nine endo-tracheal tubes, 11 CVP tips, 10 Foley's catheter tips, 7 abdominal drain tubes, 5 nephrostomy tubes, 4 Tracheostomy tubes, 3 D.J. stent tips, and 1 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 E. coil, 9 Coagulase negative Staphylococci, 4 Enterobacter cloacae, 3 Enterococci , and 2 Staphylococcus aureus were isolated details of which can be seen in [Table 1].
|Table 1: Relation of clinical bacterial isolates and the type of device inserted|
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Out of 100 clinical isolates tested, 88 were found to be biofilm formers by microtiter plate method. Out of two different staining methods; 0.1% safranine had detected 88 biofilm producers while 0.1% crystal violet had detected 69 biofilm producers as seen in [Figure 1] and [Figure 2] had described ability of two different stains in of detetection biofilms. [Table 2] had shown different bacterial species capable of making biofilms.
|Table 2: Quantitative analysis of biofilm production by clinical bacterial isolates as evaluated by microtitre plate method|
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|Figure 1: Diff erence in the intensity of color between biofi lm positive and biofilm negative bacteria stained with safranine and crystal violet|
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|Figure 2: Ability of safranine and crystal violet staining methods to detect biofilms by microtitre plate assay|
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Biofilm formation in response to different concentrations of glucose was studied. Tryptic soya broth without glucose showed biofilm formation in 75 (85%) isolates. Out of 75, 2 were strong and 28 were moderate biofilm formers as shown in [Table 3]. In tryptic soya broth with 0.25% glucose, 81 (92%) were found positive, of which 3 were strong and 30 were moderate biofilm formers. In tryptic soya broth with 0.5% glucose, 67 (76%) were found positive, out of which 4 were strong and 28 were moderate biofilm formers.
|Table 3: Screening of 100 bacterial isolates for biofilm formation by microtitre plate method in diff erent media and at 16, 20, and 24 h incubation periods|
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Biofilm formation at different incubation time periods was studied. At 16 h incubation period, 88 (100%) were found to be positive, out of it, 3 were strong and 28 were moderate biofilm formers. At 20 h incubation period, 81 (92%) were found positive, 2 were strong and 36 were moderate biofilm formers. At 24 hour incubation period, 76 (86%) found positive, 4 were strong and 29 were moderate biofilm formers.
Experiment was done in quadruplet and repeated two times. All OD 492-630 mm values were expressed as average with standard deviation.
| Discussion|| |
Indwelling medical devices are frequently used in all health setups, whereas critical care units of hospitals use multiple medical devices for treatment and intervention in patient care. Endotracheal tube amounting more than 50% of our specimen; may be due to more specimens are from patients admitted in critical care, which were either incubated or needing ventilator support in multispecialty hospitals. Second most common specimen for investigation was central venous catheters (CVCs) amounting 12% of total specimen volume under study. CVCs pose a greater risk of device-related infection than does any other indwelling medical device, with infection rates of 3-5%. Catheters may be inserted for administration of fluids, blood products, medications, nutritional solutions, and hemodynamic monitoring. Twelve percent of the specimens were of urinary catheter for our study. Urinary catheter was used for many indications in hospital, such as to measure urine output, collect urine during surgery, prevent urinary retention, or control urinary incontinence.
These organisms may originate from the skin of patients or healthcare workers, tap water to which entry ports are exposed, or other sources in the environment.  Acinetobacter, Pseudomonas, Klebsiella, Staphylococcus, Enterobacter, and E. coli are the most common causes of nosocomial infections and that may be common cause of colonization in indwelling medical devices even responsible for biofilm production. These microorganisms survive in hospital environments despite unfavorable conditions, such as desiccation, nutrient starvation, and antimicrobial treatments. It is hypothesized that its ability to persist in these environments, as well as its virulence, is a result of its capacity to colonize medical devices. 
In a study by Feldman et al0.  , it was documented that the interior of the ETT (endo tracheal tube) of patients undergoing mechanical ventilation rapidly became colonized with Gram-negative microorganisms, which commonly appeared to survive within a biofilm While it appears that colonization of the ETT may begin from as early as 12 hours, it is most abundant at 96 hours. Colonization of the ETT with microorganisms commonly causing nosocomial pneumonia appears to persist in many cases despite apparently successful treatment of the previous pneumonia. A study by Donlan et al.  showed that the organisms most commonly isolated from the CVC biofilms are Staphylococcus epidermidis, S. aureus, Candida albicans, P. aeruginosa, K. pneumoniae, and Enterococcus faecalis. ,, Stickler et al0.  showed the organisms commonly contaminating this urinary catheter and developing biofilms are S. epidermidis, Enterococcus faecalis, E. coil, Proteus mirabilis, P. aeruginosa, K. pneumoniae, and other gram-negative organisms. , One study by Rao et al.  showed 30% biofilm-forming bacterial isolates among medical devices, such as endotracheal tubes followed by CVCs, and urinary catheters are third the most common site of biofilm-forming bacterial colonization. 
| Conclusions|| |
Out of the two different staining methods, safranine 0.1% and crystal violet 0.1%; safranine staining gave more positive, stable, and accurate results in terms of reproducibility, for both, gram-positive as well as gram-negative bacteria. Twenty hours incubation time was found to be optimum for detection of biofilms produced by bacteria. Moderate-to-weak-biofilm-producing bacteria although do attach to the surfaces, detachment occurs early because of weak binding. Strong biofilm producers can be detected even at 24 hours of incubation period. Availability of nutrition favors biofilm formation by bacteria so glucose enhances biofilm-forming ability of bacteria but effect of osmolarity and pH cannot be ruled out on biofilm formation.
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Department of Microbiology, 5/1373, 'Jay Ambe Nivas' Kaljug Mahollo, Haripura, Surat - 395 003, Gujarat
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]