QUANTITATIVE AND QUALITATIVE ASSAYS OF BACTERIAL BIOFILM PRODUCED BY Pseudomonas aeruginosa AND Klebsiella spp.

The aim of this study was to detect biofilm formation by study isolates of Pseudomonas aeruginosa and Klebsiella spp. qualitatively and quantitatively. Twenty-five isolates were taken from patients admitted to Ramadi General Hospital were included in this study. Qualitative biofilm formation assays (tube method and Foley-catheter assay) and quantitative assay by spectrophotometric method with ELISA reader were achieved under two experimental conditions. In tube method, the result showed that out of 8 (32%) isolates of Pseudomonas aeruginosa and 17 (68%) isolates of Klebsiella species, the biofilm were produced in 7 (87.5%), and 14(82.35%) respectively. while, in foley catheter method, the biofilm were produced on the surfaces of the catheters in all pseudomonal isolates (100%), and 15(88.23%)of klebsiella isolates respectively. In the spectrophotometric method, the results showed that out of 17 isolates of Klebsiella spp., all isolates were produced biofilm strongly in the glucose supplemented media while 15 (88.23%) of them were produced biofilm strongly in the glucose non-supplemented media and 2 (11.76%) isolates were weak biofilm producers in the glucose non-supplemented media. With regard to Pseudomonas aeruginosa , the results showed that all pseudomonal isolates, which submitted to this study, were produced biofilm strongly in the glucose supplemented and non-supplemented brain heart infusion broth. The study concluded that all isolates of Pseudomonas aeruginosa and Klebsiella spp. were produced biofilm qualitatively by both techniques. Further, the spectrophotometric method was an accurate method for detection the bacterial adherence to the surface of Microtiter plates. Further more, biofilm production was not affected by this factor in both of Pseudomonas aeruginosa and Klebsiella spp .

The aim of this study was to detect biofilm formation by study isolates of Pseudomonas aeruginosa and Klebsiella spp.qualitatively and quantitatively.Twenty-five isolates were taken from patients admitted to Ramadi General Hospital were included in this study.Qualitative biofilm formation assays (tube method and Foley-catheter assay) and quantitative assay by spectrophotometric method with ELISA reader were achieved under two experimental conditions.
In tube method, the result showed that out of 8 (32%) isolates of Pseudomonas aeruginosa and 17 (68%) isolates of Klebsiella species, the biofilm were produced in 7 (87.5%),and 14(82.35%)respectively.while, in foley catheter method, the biofilm were produced on the surfaces of the catheters in all pseudomonal isolates (100%), and 15(88.23%)ofklebsiella isolates respectively.In the spectrophotometric method, the results showed that out of 17 isolates of Klebsiella spp., all isolates were produced biofilm strongly in the glucose supplemented media while 15 (88.23%) of them were produced biofilm strongly in the glucose non-supplemented media and 2 (11.76%) isolates were weak biofilm producers in the glucose non-supplemented media.With regard to Pseudomonas aeruginosa, the results showed that all pseudomonal isolates, which submitted to this study, were produced biofilm strongly in the glucose supplemented and non-supplemented brain heart infusion broth.The study concluded that all isolates of Pseudomonas aeruginosa and Klebsiella spp.were produced biofilm qualitatively by both techniques.Further, the spectrophotometric method was an accurate method for detection the bacterial adherence to the surface of Microtiter plates.Further more, biofilm production was not affected by this factor in both of Pseudomonas aeruginosa and Klebsiella spp.

Introduction:-
It is now well documented that microbial biofilm is defined as structured communities of microbial species embedded in a biopolymer matrix on either biotic or a biotic substrate.
Studies have revealed that the adherent bacteria, growing in consortia known as biofilms, are present in virtually all natural and pathogenic ecosystems (1,2) .Most importantly, the biofilm is characterized by its resistance to biocides, antibiotic chemotherapy, and clearance by humoral or cellular host defense mechanisms (3,4,5) .Twenty-five isolates were taken from patients admitted to Ramadi General Hospital, in Ramadi during the period from April to August 2006.Out of 25 patients, 17 (68%) were male and 8(32%) were female with male to female ratio 1: 2.1.The age of the patients was varying between 13 years and 60 years old with mean (31.3 Y).The clinical data regarding the distribution of isolates, type of specimens and type of infection are presented in table 1.All study isolates were well bacteriologically identified and confirmed by biochemical tests 7 .Bacteria were stored in brain heart infusion broth (BHI) medium containing 20% glycerol.Before each experiment, one aliquot was thawed quickly at 37 Cº and subcultured on blood agar plate's at37Cº for 24 hr 7 .

Qualitative biofilm formation assays:
Adhesion assay: Tube method Briefly, two to three colonies of isolates were inoculated into 5 ml of BHI broth in plastic conical tubes in duplicate.Saccharide free basal medium (BHI broth) without glucose that lacks the substrate for polysaccharides was used as a control.Cultures were incubated at 37 Cº for 18-20 hr. the contents were aspirated, one tube was examined unstained and one each stained with crystal violate and safranin.Slime positivity was judged by the presence of visible unstained or stained film lining the wall of the tube 8 .
Adhesion assay: Formation of biofilm on catheter: An overnight culture of tested bacteria (10µl) in brain heart infusion broth was inoculated into 500µl of the same medium and injected into clear Foley catheters.The catheters were capped at both ends and incubated at 37ºC overnight.Then catheters were rinsed with phosphate buffer saline.After drying at room temperature for 15 min, 700 µl crystal violet (1%) was added to the catheters for 20 min, then the stained biofilm rinsed several times with BPS and allowed to dry at room temperature before examination (9,10).

Spectrophotometric method
Working cultures were prepared by inoculation on Columbia agar supplemented with 5% blood and incubated aerobically at 37 Cº for 24 hr.The (OD) of each well was measured at 570 nm (8,11) by ElISA reader.

Results:
Under the field of biofilm production, particularly qualitative biofilm assay by tube method, the results showed that out of 8 (32%)isolates of Pseudomonas aeruginosa, 17 (68%)isolates of Klebsiella species, the biofilm were produced on the inner lining of the tubes in 7 (87.5%),and 14(82.35%)respectively.while this phenomenon was not observed in 1 (12.5%), and 3(17.64%)isolates of Pseudomonas aeruginosa and Klebsiella spp.respectively (table 2 Further, in the other qualitative assay (foley catheter method) the study revealed that out of 8 (32%) isolates of Pseudomonas aeruginosa, 17 (68%) isolates of Klebsiella species, the biofilm were produced on the surfaces of the catheters in all pseudomonal isolates (100%), and 15(88.23%) of klebsiella species respectively.On the other hand this phenomenon was not observed in Two (11.76%) of

Klebsiella spp. (table 2-B).
In the quantitative biofilm formation assay, spectrophotometric method was achieved under two set of experimental conditions (with and without glucose).The results showed that out of 17 isolates of Klebsiella spp., all isolates were produced biofilm strongly (OD was more than 0.25) in the glucose supplemented media while 15 (88.23%) of them were produced biofilm strongly in the glucose nonsupplemented media and 2 (11.76%) isolates were weak biofilm producers in the glucose nonsupplemented media.On the other hand no significant differences observed in the readings of optical density at 570 nm with the presence and absence of glucose among isolates of Klebsiella (0.712 ±0.30) and (0.54 ±0.12) respectively (P value = 0.07) (table 3).
With regard to Pseudomonas aeruginosa, the results showed that all pseudomonal isolates, which submitted to this study, were produced biofilm strongly in the glucose supplemented and nonsupplemented brain heart infusion broth.On the other hand no significant differences observed in the readings of optical density at 570 nm with the presence and absence of glucose among isolates of Pseudomonas aeruginosa (1.11 ±0.68) and (0.58 ±0.02) respectively (P value = 0.12) (table 4).
The isolates were classified according to biofilm production according to the criteria laid down by Christensen 12 as following: non-biofilm producers less than 0.125, weak biofilm producer between 0.125-0.25 and strong biofilm producers more than 0.25

Discussion:
It is well realized that bacterial adhesion to biomaterial surfaces is the essential step in the pathogenesis of prosthetic device related infections 13 .Microorganisms attach to surfaces and develop into biofilms.Biofilm-associated cells can be differentiated from their suspended counterparts by generation of an extracellular polymeric substance (EPS) matrix, reduced growth rates, and the regulation of specific genes.Cells may also communicate via quorum assay regarding strongly biofilm producing isolates.
The same authors concluded that it was difficult to discriminate between weak and biofilm negative isolates due to the variability in observed results by different observers.Consequently, high variability was observed and classification in biofilm positive and negative was difficult by tube method.Thus, tube test cannot be recommended as general screening test to identify biofilm-producing isolate (12,16) .
In order to enable easier study of bacterial a gram-negative bacterium, opportunistic human pathogen that causes chronic infections in immunocompromised individuals.These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the P-ISSN 1991-8941 E-ISSN 2706-6703 Journal of University of Anbar for Pure Science (JUAPS) Open Access 2008,(2), (3 ) :06-13 7 formation of antibiotic-tolerant biofilms.The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems.Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase 6 .This study has been undertaken in vitro, to detect biofilm formation by locally isolates of Pseudomonas aeruginosa and Klebsiella spp.qualitatively by tube adhesion method and foley catheter assay.Further, Quantitative determination of bacterial adhesion on the surfaces of microtiter plate by spectrophotometric assay with ELISA reader under two experimental conditions Patients and Methods: cultures were used to prepare bacterial suspension in sterile distilled water adjusted to a 0.5 McFarland standard.The suspensions obtained were inoculated into a brain-heart infusion broth.After that, poured into the wells of plastic microplates 11 .The Wells of sterile 96-well flat-bottomed plastic micro plates were filled with 250µL of the BHI broth.Negative control wells contained the broth only.Twenty µL of bacterial suspension was then added to each well.The plates were incubated at 37 Cº for 24hr.following incubation, the content of each well was P-ISSN 1991-8941 E-ISSN 2706-6703 Journal of University of Anbar for Pure Science (JUAPS) Open Access 2008,(2), (3 ) :06-13 8 aspirated, and each well was washed three times with 300µL of sterile distilled water.The remaining attached bacteria were fixed with 200 µL of methanol per well, and after 15 min the plates were emptied and left to air dry.Afterthat the plates were stained for 5 min with 160 µL per well of crystal violet used for gram stain.Excess stain was rinsed off by placing the microplates under running tap water.After the plates were air dried, the dye which was bound to the adherent cells was resolublized with 160 µL of 33 % (v/v) glacial acetic acid per well.The optical density attachment and colonization, a variety of experimental, direct and indirect, observation methods have been developed.Microtiter plate assay (spectrophotometric method) is the most frequently used techniques for quantifying biofilm formation 17 .Microtiter plate procedure is an indirect method for estimation of bacteria in situ, it has the advantage of enabling researchers to rapidly analyze adhesion of multiple bacterial strains or growth conditions within each experiment, easy technique and used widely for antimicrobial agents susceptibility of biofilm.In the quantitative biofilm formation assay, spectrophotometric method was achieved under two set of experimental conditions (with and without glucose).the results showed that out of 17 isolates of Klebsiella spp., all isolates were produced biofilm strongly (OD was more than 0.25) in the glucose supplemented media while 15 (88.23%) of them were produced biofilm strongly in the glucose supplemented media and 2 (11.76%) isolates were weak biofilm producers in the glucose non-supplemented media.On the other hand no significant differences observed in the readings of optical density at 570 nm with the presence and absence of glucose among isolates of Klebsiella (0.712 ±0.30) and (0.54 ±0.12) respectively (P value = 0.07).With regard to Pseudomonas P-ISSN 1991-8941 E-ISSN 2706-6703 Journal of University of Anbar for Pure Science (JUAPS) Open Access 2008,(2), (3 ) :06-13 10 aeruginosa, Our results showed that all pseudomonal isolates which were submitted to this study were produced biofilm strongly in the glucose supplemented and non-supplemented media.On the other hand no significant differences observed in the readings of optical density at 570 nm with the presence and absence of glucose among isolates of Pseudomonas aeruginosa (1.11 ±0.68) and (0.58 ±0.02) respectively (P value = 0.12).Conclusions:The study concluded that all studied isolates of Pseudomonas aeruginosa and Klebsiella spp.were produced biofilm qualitatively by both of tube methods and Foley catheter methods.Further, the quantitative spectrophotometric method with ELISA reader was an accurate method for detection the bacterial adherence to the surface of Microtiter plates.Further more, under two set of experimental conditions, in the glucose supplemented and non-supplemented media, biofilm production was not affected by this factor in both of Pseudomonas aeruginosa and Klebsiella spp.11-Stepanovic, S., Djukic, V., Djordjevic, V. and Djukic, S. (2003).Influence of the incubation atmosphere on the production of biofilm by staphylococci.Clin Microbiol.Infect, 9: 955-959.12-Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M., and Beachey, E.H. (1985).Adherence of Coagulase negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices.J. Clin.Microbiol., 22: 996-1006.13-Katsikogianni, M. and Missirlis, Y.F.(2004).Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions.Europ.Cell Mat., 8: 37-57.14-Donlan, R.M. (2002).Biofilms: Microbial Life on Surfaces.Emerg.Infect.Dis.8: 881-890.15-Jones, B.V., Mahenthiralingam, E., Sabbuba, N.A., Stickler, D.J. (2005).Role of swarming in the formation of crystalline Proteus mirabilis biofilms on urinary catheters.J. Med.Microbiol., 54: 807-813.16-Mathur, T., Singhal, S., Khan, S., Upadhyay, D.J., Fatma, T. and Rattan, A. (2006).Detection of biofilm formation among the clinical isolates of Staphylococci: An evaluation of three different screening methods.Ind.J. Med.Microbiol., 24: 25-29.17-Stepanovic, S., Vukovic, D., Dakic, I., Savic, B., Svabic-Vlahovic, M. (2000).A modified microtiter plate test for quantification of staphylococcal biofilm formation.J. Microbiol.Meth., 40: 175-179.

Table 1 .
Distribution of clinical isolates of Klebsiella spp.and Pseudomonas spp.according to the type of specimens and infection.

Table 2 .
Qualitative biofilm formation by tube and foly -catheter method among isolates of Pseudomonas aeruginosa and Klebsiella spp.