الفهرس 
Title pageOnly 14 pages are availabe for public view
 |  | from | 114 |  |  |
| | | from | 114 | | |
Abstract
Biofilm associated wounds infections remain a major health and financial burden, despite of the incredible advancements in wound management in the last decade. The competitive and cooperative interaction of microbial community within biofilms markedly altered the antimicrobial susceptibility of the interacting species, with significant increase in patient morbidity and mortality.
The present study was concerned with determining the invitro impact of P. aeruginosa on a mature S. aureus biofilm cell viability, as well as susceptibility to vancomycin. Both bacteria were isolated from chronic wound swab specimens submitted to the Microbiology laboratory of the Medical Research Institute, during the period of December 2019 till February 2020.
Five strains for each of P. aeruginosa, MRSA, & MSSA clinical isolates were biochemically identified and tested for antimicrobial susceptibility using BD Phoenix™ M50 Automated Microbiology System. All of our S. aureus isolates were vancomycin sensitive.
Qualitative detection of biofilms formed in MTP by S. aureus and P. aeruginosa clinical isolates using spectrophotometric measurement reviled that, all MRSA strains were moderate biofilm-forming. Sixty percentages of MSSA clinical isolates were strong biofilm forming and 40 % were moderate biofilm forming. Among P. aeruginosa, 60% of the isolates showed strong biofilm formation, 20% were moderate biofilm forming and 20% were weak biofilm forming.
The cell free culture supernatant of P. aeruginosa clinical isolates was prepared using 0.2 μm bacterial filters and stored at minus 20°C. P. aeruginosa cell suspension was freshly prepared in the same experimental setting.
The viable cell count for all of our S. aureus clinical isolates in the sessile phase of biofilms was performed using spread plate count technique, to obtain a baseline data for mature S. aureus biofilm cells viability. Using an invitro model of biofilm disruption assay in MTP followed by colony count enumeration with spread plate count technique, we tested the effect of one of our P. aeruginosa clinical isolates on the viability of the ten clinical isolates of S. aureus sessile cells in mature biofilms.
We found impaired viability of S. aureus mature biofilm cells, which was statistically significant for both MRSA & MSSA strains after disruption with P. aeruginosa cell suspension. Although, the cell-free culture supernatant of the same P. aeruginosa clinical isolate did not significantly change the count of MRSA viable cells in the sessile phase of biofilms, but it showed a border line significant reduction in MSSA biofilm cells count.
To investigate the effect of our five P. aeruginosa clinical isolates on the susceptibility of mature S. aureus (3 MRSA, & 3 MSSA) cells in the planktonic and sessile phase of biofilms to vancomycin, we followed the same biofilm disruption assay used in testing S. aureus cell viability. Although, the impact of P. aeruginosa cell suspensions and cell free culture supernatants, was determined using the DROP plate technique. We determined the MBEC of vancomycin on the mature biofilms developed by each of the for mentioned S. aureus cells in the planktonic and sessile phase, and compared it with the MBEC of vancomycin obtained after disrupting S. aureus mature biofilms with the cell suspension and cell free culture supernatant of our five P. aeruginosa clinical isolates individually.
A detectable effect was found on the planktonic cells of S. aureus cells in the form of decreased MBEC of vancomycin (increased susceptibility) in combination with P. aeruginosa cell suspension, and an increased MBEC of vancomycin (decreased susceptibility) in combination with P. aeruginosa cell free culture supernatant. On the contrary, no detectable effect was found for P. aeruginosa cell suspensions or cell free culture supernatants on the MBEC of vancomycin on S. aureus cells in the sessile phase of biofilms.
In the present study, we investigated the relative expression of two genes associated in the fermentation pathway (ldh & adh), as well as one of the VraSR two component system associated genes (vraR) responsible for increasing S. aureus cell wall thickness, in one of our MRSA clinical isolates sessile cells, that displayed the three effects of interaction; an increase, decrease and no change in the MBEC of vancomycin in the planktonic phase of biofilm.
Expression analysis using qPCR was done for this MRSA cells in the sessile phase of mature biofilm that was considered the control group, for assessment of the expression changes resulted in the target genes after interaction of the selected MRSA biofilm cells with the cell free culture supernatants of three of our P. aeruginosa clinical isolates individually using biofilm disruption assay, both in the presence and absence of vancomycin. In addition, we assessed the relative expression of our target genes in the same MRSA mature biofilm cells after disruption with vancomycin alone, in relation to the same control group cells.
Our results suggested that each of vancomycin and P. aeruginosa cell free culture supernatants can be considered inhibitors to the synthesis of S. aureus cell wall, which was represented by down regulation in the expression of vraR gene. Although, the combination of these two interacting agents resulted in the same downregulating effect with no marked variation.
In our study, P. aeruginosa cell free culture supernatants as well as vancomycin halt the established mature S. aureus biofilm cells from shifting their state of respiration from the aerobic to the anaerobic mode, through down regulating the expression of ldh & adh genes. Combining these two interacting agents together resulted in the same downregulating effect with no marked variation