| Abstract|| |
Background: Microorganisms develop biofilm on various medical devices. The process is particularly relevant in public health since biofilm associated organisms are much more resistant to antibiotics and have a potential to cause infections in patients with indwelling medical devices. Materials and Methods: To determine the efficiency of an antibiotic against the biofilm it is inappropriate to use traditional technique of determining Minimum Inhibitory Concentration (MIC) on the free floating laboratory phenotype. Thus we have induced formation of biofilm in two strains (Pseudomonas aeruginosa and Staphylococcus aureus, which showed heavy growth of biofilm in screening by Tube method) in a flow cell system and determined their antibiotic susceptibility against ciprofloxacin by agar dilution method in the range (0.25 mg/ml to 8 mg/ml). The MIC value of ciprofloxacin for the biofilm produced organism was compared with its free form and a standard strain as control on the same plates. Observations: Both the biofilm produced strains showed a higher resistance (MIC > 8 mg/ml) than its free form, which were 2 μg/ml for Pseudomonas aeruginosa and 4 mg/ml for Staphylococcus aureus. Thus biofilm can pose a threat in the patient treatment.
Keywords: Antibiotic resistance, biofilm, ciprofloxacin, flow cell system, minimum inhibitory concentration, Pseudomonas aeruginosa, Staphylococcus aureus, tube method
|How to cite this article:|
Gupta S, Agarwal S, Sahoo DR, Muralidharan S. In vitro production of biofilm in a flow cell system in a strain of Pseudomonas aeruginosa and Staphylococcus aureus and determination of efficiency of ciprofloxacin against them. Indian J Pathol Microbiol 2011;54:569-71
|How to cite this URL:|
Gupta S, Agarwal S, Sahoo DR, Muralidharan S. In vitro production of biofilm in a flow cell system in a strain of Pseudomonas aeruginosa and Staphylococcus aureus and determination of efficiency of ciprofloxacin against them. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Mar 25];54:569-71. Available from: http://www.ijpmonline.org/text.asp?2011/54/3/569/85095
| Introduction|| |
Biofilm is an accumulation of microorganisms and their extracellular products forming a structured community on a surface.  Biofilms are difficult to eradicate with therapeutic doses of antibiotics and are always a source of many chronic infections.  It is known that bacterial biofilm colonizing on indwelling/implanted medical device can be a source of many blood stream and urinary infections. ,, The hallmark of biofilm microbes is their resistance to antimicrobial agents and host immune defenses, thus have a great significance for public health.  It is being observed that planktonic bacteria are more susceptible to the effects of antibiotics converse to their sessile counterpart. Traditionally, efficiency of an antibiotic is measured by its minimum inhibitory concentration (MIC) on a planktonic free floating laboratory phenotype. However this technique may be inappropriate for the sessile form, which shows more resistance. ,,, Thus to test antimicrobial agents against biofilm, various devices had been made like the Calgary biofilm device by Ceri et al.  and modified Robbin's device by Domingue et al. 
In the present study we have determined biofilm formation among 50 clinical isolates by tube method.  Among the 50 isolates we have taken a gram positive (Staphylococcus aureus) and a gram negative (Pseudomonas aeruginosa) bacteria, which showed heavy growth of biofilm in tube method. In these two isolates biofilm production was induced in vitro in a flow cell system.  The MIC of ciprofloxacin was determined and compared on both the biofilm state and the planktonic state of the two test organisms to see whether biofilm makes an organism more resistant to antibiotics.
| Materials and Methods|| |
A total of 50 non-repetitive clinical isolates, namely Escherichia More Details coli (n = 28), Klebsiella pneumoniae (n = 4), Pseudomonas aeruginosa (n = 3), non fermenting gram negative bacilli (NFGNB) (n = 3), Coagulase negative Staphyloocci (CoNS) (n = 8), Staphylococcus aureus (n = 3) and Enterococcus faecalis (n = 1) were isolated from catheter tip (n = 3), stent tip (n = 2) central line tip (n = 5) and urine of catheterized patients (n = 40). These 50 isolates were screened for biofilm production by using tube method.
Detection of Biofilm Formation by Tube Method
Ten ml of Brain Heart Infusion Broth (BHIB) with 2% sucrose in unsensitised polysterene tubes were inoculated with loop full of microorganisms from overnight culture plates and incubated for 24 hours at 37°C. The tubes were decanted and washed with PBS (pH 7.2) and allowed to dry. Dried test tubes were stained with 1% crystal violet. Excess stain was removed and test tubes were washed with deionised water. Test tubes were then dried in inverted position and observed for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and the bottom. Ring formation at the interface was not indicative of biofilm formation. Test tubes were scored as heavy, moderate and none or weak biofilm growth. 
In vitro Formation of Biofilm
Flow system-The continuous flow system consisted of a nutrient reservoir containing BHIB, a single channel flow cell, temperature controller, a flow regulator with a drip set maintaining the desired flow and a waste vessel. All parts were connected with silicon tubing.
The flow cell was 12 cm long, 10 cm wide and 1 cm deep, and is built of poly-acrylic materials with a poly urethane base. A rectangular glass cover slip was used which covers the flow system. It was sealed by using a rubber gasket lining. The flow cell system consists of an inlet through which the entry of nutrient is regulated. It was guided by a three way stop-flow valve. The flow outlet was as well regulated by a three way stop flow valve.
Six circular recesses were provided in the poly-urethane base for the placement of the ceramic discs, serving as the substrate for the adhesion of microorganism. Each recess was 1 mm deep and 6.35 mm in diameter. The discs were made of ceramic having a smooth bottom surface and a rough top surface. The discs measured 6 mm in diameter and 0.75 mm thick. The flow cell with discs in place was sterilized by ethylene oxide treatment.
The flow cell containing the uninoculated discs was filled with sterile BHIB media from the reservoir and a continuous flow was maintained. Overnight inoculum was introduced into the bioreactor cell with a syringe injection, upstream of the discs. The culture volume used covered all of the discs and was maintained in the flow cell for 30 minutes for allowing the bacteria to attach on the discs.
The flow cell containing the inoculum was left undisturbed for 24 hours at a minimal flow rate of 20 ml/hour, at a temperature of 37°C maintained by the temperature controller. The biofilm was allowed to grow and the discs were removed, detected for presence of biofilm by observation of slime growth and used in the antibiotic susceptibility assay  .
From the 50 isolates screened for biofilm production by tube method, we have chosen one gram positive (Staphylococcus aureus) and one gram negative (Pseudomonas aeruginosa) bacteria, which showed formation of heavy growth of biofilm. In these two organisms the biofilm was produced in vitro on the ceramic discs, in the continuous flow system. MIC of ciprofloxacin was detected against the biofilm producing organism on ceramic discs and the freshly sub-cultured organism by agar dilution method. On agar plates containing the known dilutions of antibiotic, the biofilm containing ceramic disc was placed with the biofilm produced surface on the agar along with a loopful of free form of the same organism from broth and a standard strain. The MIC range of ciprofloxacin was taken between 0.25 μg/ml to 8 μg/ml. Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were used as the standard control for the determination of MIC.
| Results|| |
Fifty different isolates which constituted of Escherichia coli (n = 28), Klebsiella pneumoniae (n = 4), P. aeruginosa (n = 3), NFGNB (n = 3), Staphylococcus aureus (n = 3), CoNS (n = 8) and Enterococcus faecalis (n = 1) from urine and catheter tips were screened for biofilm formation by tube method. Results of the screening for biofilm by the tube method is shown in the [Table 1].
|Table 1: Results of biofi lm screening by tube method in 50 clinical isolates|
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Among the 50 isolates 20% showed heavy growth (n = 10), 22% showed moderate growth (n = 11) and 58% showed no growth or weak growth (n = 29). Among the 10 isolates showing heavy growth, one isolate of Pseudomonas aeruginosa and one of Staphylococcus aureus was taken as the test organism for in vitro formation of biofilm and detection of MIC for ciprofloxacin against it.
The MIC of ciprofloxacin for the biofilm forming isolate and young isolate for both the test organisms (Pseudomonas aeruginosa and Staphylococcus aureus) along with their standard strains were determined by the agar dilution method at various concentration range (0.25 mg/ml to 8 mg/ml), is given in the following [Table 2].
|Table 2: MIC of ciprofloxacin against standard strains, biofi lm formed strains and young isolates of Staphylococcus aureus and Pseudomonas aeruginosa various MIC ranges|
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It was observed that the MIC value of Staphylococcus aureus young isolate is 4 μg/ml whereas the MIC value of Staphylococcus aureus with biofilm is more than 8μg/ml and MIC value of Pseudomonas aeruginosa young isolate is 2 μg/ml, whereas the MIC value of Pseudomonas aeruginosa with biofilm is more than 8 μg/ml. Thus from the experimental result we can conclude that the MIC value of an organism is increased in presence of biofilm and it has higher antibiotic resistance.
| Discussion|| |
Bacterial biofilm can colonize both the surfaces of tissues and implanted medical devices, which may often lead to infection. , A biofilm develops when the attached cells excrete polymers that facilitate adhesion, matrix formation and alteration of the organisms phenotype with respect to the growth rate and gene transcription.  Biofilms poses public health problem for persons requiring indwelling medical devices. Tendency of microorganisms to develop biofilms has been well documented for a number of medical devices and it is being found to be difficult to eradicate. ,,, This process is particularly relevant for clinicians, because biofilm associated microorganisms show decreased susceptibility to antimicrobial agents than are planktonic organisms and also because colonization of a medical device with biofilm may be associated with infection. ,,
Biofilms which are most often resistant to systemic antibiotic therapy are responsible for 65% of infection treated in the developed world.  The antibiotic resistance showed can be attributed to the restricted penetration of antibiotics, nutrient limitations, altered environment and slow growth. , A study conducted by Ceri et al.  showed that biofilm associated with Escherichia coli required 1500 times the MIC of ampicillin to provide 3 log reduction and Williams et al.  showed that Staphylococcus aureus biofilms required 110 times the MBC of vancomycin to provide 3 log reduction.
In diagnostic laboratories, antibiotic efficiency is determined by MIC on free floating laboratory phenotypes. However this method is not appropriate for biofilm produced organism. Thus we have devised a flow cell system for in vitro production of biofilm and determined the MIC levels of ciprofloxacin for the biofilm producing organism against its planktonic type by agar dilution method. The test organisms namely Pseudomonas aeruginosa and Staphylococcus aureus, which showed a heavy growth of biofilm in screening, needed a higher concentration of MIC (>8 mg/ml) than its free form. This study shows that biofilm makes bacteria more resistant to an antibiotic, which in this case was ciprofloxacin, posing a threat in the patient treatment.
In a study by Duguid et al.  showed that ciprofloxacin activity was influenced by the cell cycle; newly formed daughter cells were more susceptible than other population in the biofilm.
Biofilms are emerging problem. A clear understanding of the role of biofilms in infection should guide the clinical decision making and also in proper use of therapeutics. More studies need to be performed to understand the structure, mechanism and their resistance to antibiotics.
| References|| |
|1.||Tenke P, Riedl CR, Jones GJ, Stickler D, Nagy E. Bacterial biofilm formation on urologic devices and heparin coating as preventive strategy. Int J Antimicrob Agents 2004;23suppl1: s67-74. |
|2.||Lewis K. Riddle of Biofilm resistance. Antimicrob Agents Chemother 2001;45:999-1007. |
|3.||Donlan RM. Biofilm formation: A clinically relevant microbiological process. Clin Infect Dis 2001;33:1387-92. |
|4.||Donlan RM. Biofilms and device associated infections. Emerg Infect Dis 2001;7:277-81. |
|5.||Ryder MA. Catheter related infections: It's all about biofilm. Topics in Advanced Practice Nursing eJournal 2005; 5 ©2005 Medscape posted. Available from http://www.medscape.com/view article/508109. [Last cited on 2005 Aug 08]. |
|6.||Costerton W, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G. The application of biofilm science to the study and control of chronic bacterial infections. J Clin Invest 2003;112:1466-77. |
|7.||Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999;284:1318-22. |
|8.||Prosser BT, Taylor D, Dix BA, Cleeland R. Methods for evaluating antibiotics on bacterial biofilms. Antimicrob Agents Chemother 1987;31:1502-6. |
|9.||Ceri h, Olson ME, Stermick C, Read RR, Morck D, Buret A. The Calgary biofilm device: New technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 1997;37:1771-6. |
|10.||Domingue G, Ellis B, Dasgupta M, Costerton JW. Testing antimicrobial susceptibilities of adherent bacteria by a method that incorporates guidelines of the national committee for clinical laboratory standards. J Clin Microbiol 1994;32:2564-8. |
|11.||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. Ind J Med Microbiol 2006;24:25-9. |
|12.||Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative evaluation of endodontic irrigants against Enterococcus faecalis biofilms. J Endod 2006;32:527-31. |
|13.||Williams I, Venables WA, Lloyd D, Paul F, Critchley I. The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus. Microbiology 1997;143:2407-13. |
|14.||Duguid IG, Evans E, Brown MR, Gilbert P. Growth rate independent killing by ciprofloxacin of biofilm derived Staphylococcus epidermidis: Evidence for cell cycle dependency. J Antimicrob Chemother 1992;30:791-802. |
Department of Microbiology, St. Johns Medical College, Bangalore - 560 034, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]