BIOLOGICAL CHARACTERISTICS OF SOME MICROBES CONTRIBUTING IN BIOFILM FORMATION

The objectives of the present study were determining the microbial population contributing in biofilm formation. In addition to, study the behavior and biological characteristics of some pathogenic bacteria in biofilm state. To achieve the first aim forty eight natural biofilm samples were collected from different types of microhabitats (drinking water distribution system (DWDS), kitchen sink drainage pipes, bathroom sink drainage pipes and lab sink drainage pipes) to determine the characteristics of the predominant microorganisms. Spread plate method was used to determine the pathogenic microbes by using enzymatic cultures. The results of microbiological characterization of natural biofilm collected from three different materials of the DWDS (PP, PVC and iron pipes) showed that, the heterotrophic plate count (TVBC) of biofilm scraped from iron pipe material was higher than PVC and PP pipe materials in DWDS. Moreover, the tested pathogenic bacteria were absent in all biofilm samples in PVC and PP pipe materials, while there were present in iron pipe material. Regarding to the results of the microbiological examination of natural biofilm samples which collected from kitchen sink drainage pipes, bathroom sink drainage pipes and lab sink drainage pipes it can be observed that the average counts of bacterial indicators and tested pathogens except Cl. perfringens were high in natural biofilm samples collected from kitchen sink drainage than others. To achieve the second aim the behaviour of two pathogenic bacteria (L. monocytogenes and S. Typhimurium) for biofilm formation were studied by using desigened DWDS model. The results recorded that, the highest growth curve of L. monocytogenes and S. Typhimurium biofilm were reported in I pipe, while the lowest was recorded in Cu pipe. In the case of plastic-based materials (PVC, PP, PE and R) the results were observed that no differences between them in the growth curve. The amounts of exopolysaccharide were determined in L. monocytogenes and S. Typhimurium biofilm grown on six different domestic plumping materials during experiments periods (10 to 90 days). After estimation the amounts of exopolysaccharide produced by L. monocytogenes and S. Typhimurium biofilm, the results indicated that there was a positive correlation with significant between the quantities of and the biofilm ages (P?0.01). Additionally, the results indicated that, the highest amount of exopolysaccharide of L. monocytogenes biofilm was recorded in iron (I) pipe. Whereas, the lowest amount of exopolysaccharide was observed in Cu pipe material in all biofilm ages from 10 to 90 days. In addition to, the amount of exopolysaccharide (EPS) in biofilm produced by L. monocytogenes (Gram positive bacterium) was greater than S. Typhimurium (Gram negative bacterium). In spite of, the biomass growth rate of S. Typhimurium biofilm was more than L. monocytogenes biofilm grown on all tested plumping materials. Three microscopic examination methods (epi-fluorescence, transmission and scanning electron microscope) were used to investigate biofilm formation of L. monocytogenes and S. Typhimurium biofilm grown on six different domestic plumping materials. When the direct counting of biofilm cells using epi-flourescence microscopy the results demonstrated that, highest total cells counts of either L. monocytogenes or S. Typhimurium biofilm were recorded in I pipe. On the contrary, the lowest total cells count was found in Cu pipe materials. By using TEM for investiagtion of 90 days-old L. monocytogenes biofilm , the photomicrograph results indicated that the biofilm cells were embedded in a polymer matrix and different shapes of exopolysaccharide. Regarding to results of SEM, the structure of 90 days-old S. Typhimurium biofilm grown on PVC, PP, PE, I, Cu and R observed the thick biofilm wrapped by EPS and the highest thickness was shown in I pipe materials. While, the lowest thickness in Cu pipe material was showed. To control and prevent of biofilm formation by using different antimicrobial agents against planktoinc and biofilm cells of L. monocytogenes and S. Typhimurium, the results concluded that L. monocytogenes and S. Typhimurium biofilm grown on all tested pipe materials was more resistant to chlorine, Ag ions, AgNPs and antibiotics than planktonic cells. Also younger biofilm age (10 days) was more sensitive to these antimicrobial agents than older ages (40 and 90 days-old). The findings of this study, it can be summerized, the L. monocytogenes biofilm was more resistant to chlorine, Ag ions and AgNPs than S. Typhimurium biofilm. While in case of antibiotic, results showed S. Typhimurium biofilm was more resistant than L. monocytogenes biofilm. From the previous results of the present study cleared that 90 days-old of biofilm was more resistant to antimicrobial agents and environmental factors. Thus, the survival and persistence of 90 days-old L. monocytogenes and S. Typhimurium biofilm were determined in two types of water (tap and ground water). The results showed that, the longest survival time in groundwater of L. monocytogenes and S. Typhimurium biofilm cells scratched from I pipe material was observed. In contrast, the shortest survival time of biofilm cells scratched from Cu pipe material was recorded. Finally, the toxicity test was carried out using Microtox 500 analyser to evaluate all tested plumping materials. The results found that all tested materials were nontoxic. Abstract The aims of this study were to determine microbial population contributing in natural biofilm. In addition to, study the behavior and biological characteristics of two pathogenic bacteria including L. monocytogenes and S. Typhimurium in biofilm state. To achieve first aim, forty eight natural biofilm samples were collected from different types of microhabitats to determine the characteristics of the predominant microorganisms. The results of natural biofilm samples showed that the most frequent pathogens were Staph. aureus, L. monocytogenes, Salmonella spp and Candida albicans in all tested samples. While, to achieve second aim, water distribution system model was designed for testing of these bacteria. Six samples of synthetic biofilm were collected at three different biofilm ages (10, 40 and 90 days-old). The collected biofilm samples were exposed to different antimicrobial agents. Results cleared that the highest growth of L. monocytogenes and S. Typhimurium biofilm was reported in I pipe material. Results showed the log reduction of 90 days-old L. monocytogenes biofilm formation, when exposed to 3.0 mg/l of chlorine dose, on six pipe materials; PVC, PP, PE, I, Cu and R been 3.96, 4.16, 4.21, 4.17, 4.32 and 4.03 CFU/cm2, respectively. While, the effective MICs of Ag ions were 500 mg at 5 min against 90 days-old L. monocytogenes and 300 at 15 min for S. Typhimurium biofilm. Additionally, the effective MICs of AgNPs were recorded 500 mg at 15 min for 90 days-old L. monocytogenes and 500 mg at 10 min for S. Typhimurium. On the other hand, the results of antibiotic susceptibility indicated the planktonic cells were more sensitive than the biofilm cells. Also, the longest survival time in tested water of L. monocytogenes and S. Typhimurium biofilm cells scraped from I pipe material was observed. In contrast, the shortest survival time of biofilm cells scraped from Cu pipe material was recorded.