الفهرس 
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Abstract
Biofilm formation is considered as an essential virulence factor in the pathogenesis of various opportunistic bacteria. Biofilm has been described as aggregates of microorganisms in which cells are attached firmly and embedded in a self-secreted matrix of extracellular polymeric substances (EPS) which enable the bacteria to stick together and adhere to any surface included. It is composed of a sessile community characterized by cells that are irreversibly attached to an interface or to each other and free-floating populations which include bacterial colonies growing as planktonic cells on a surface and exhibit none of the inherent resistance characteristics of true biofilms
It is obvious by evidence that biofilms have a vital role in infectious diseases, in both: specific conditions such as cystic fibrosis and periodontitis and in bloodstream and urinary tract infections as a result of indwelling medical devices as: prosthetic heart valves, central venous, catheters, urinary catheters, intrauterine devices, and dental unit water. Many bloodstream and urinary tract infections progressed after the detachment of cell aggregates that are formed on the indwelling medical device biofilms.
The exposure of bacteria to low levels of antimicrobial concentrations may happen for different reasons such as the low antimicrobial bioavailability in systemic, systemic absorption of topically applied antimicrobials. All that may lead to find Sub-MIC concentrations of these antimicrobials. In many cases, the existence of the biofilm may lower the penetration of antimicrobials to the internal environment of the biofilms and as a result they are exposed to the lower levels of antimicrobials due to diffusion gradients.
This study investigated the effect of Sub-MIC concentrations of 5 different classes of antimicrobials (AZM, GEN, CIP, DOX, IMP) on the biofilm formation of 4 different bacterial species (S. aureus, P. aeruginosa, E. coli, K. pneumoniae). The MIC of each antimicrobial on each bacterial strain was determined. Microorganisms are then allowed to form biofilms in presence of different sub-MIC concentrations (12.5%, 25% and 50% of MIC) of the antimicrobial agents. Biofilm was evaluated using two methods which are the CV staining method for total biofilm biomass quantification and the qPCR quantitation method to assess the total number of cells either dead or live according to the nucleic acid present using specific gene primers, except for S. aureus, where 16s rRNA was used and a third method was applied to detect only the live cells involved within the biofilm formed.
In case of S. aureus, Azithromycin and Gentamicin showed no effect on biofilm formation by both detection methods. Both Imipenem and Ciprofloxacin showed a significant stimulatory potential in biofilm formation by CV staining, but this stimulatory effect was not significant in molecular quantification method. However, doxycycline showed a slight stimulatory effect on biofilm formation in CV staining however no significant effect was found by qPCR method. The spread method showed that the exposure to sub-MIC doses of all antimicrobials had no statistical effect on viable staph cell count.
In case of P. aeruginosa, Azithromycin sub-MIC concentrations had significant stimulatory effect in the CV staining method and in the qPCR method. Imipenem showed a significant decreasing effect in CV staining method, while its effect was not significant in the qPCR method. On the other hand, different response for Doxycycline was detected as it resulted in slight increase in CV staining method among the isolates and slight decrease in total DNA count by qPCR and this was not significant.
In case of E. coli, different responses for Azithromycin were detected as it resulted in slight decrease in CV staining method among the isolates and slight increase in total DNA count by qPCR and this was not significant. Imipenem slightly increases in biomass CV staining method, while in qPCR it showed no overall statistical significance effect on total DNA. Gentamicin showed significant decrease of total DNA in qPCR quantitation method only and no significant effect was detected by CV staining method. Doxycycline showed no significant effect by both tested methods.
In K. pneumoniae, all antimicrobials showed no significant effect on klebsiella isolates by both CV staining and qPCR method.