الفهرس 
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Abstract
In the present study, a total of 1000 samples including 600 clinical samples compromising urine (58), blood (166), sputum (247), stool (36), and wound pus (93) plus 400 environmental samples were collected from different departments of Tanta university hospitals throughout the period from January to July 2017. All samples were cultured on nutrient agar, MacConkey and mannitol salt agar then stained by Gram stain and examined microscopically. Gram negative (n=485) and Gram positive bacteria (n=115) were subjected to biochemical identification and then confirmed by culture on Hi chrome agar medium. The susceptibility of all the recovered bacterial isolates (n=600) to different antibiotics was performed using agar dilution method. Also, the susceptibility of all bacterial isolates (n=600) to different biocides (BAC, TCS, phenol, hydrogen peroxide, chlorohexidine and citric acid) was performed using broth dilution method. Both biocide MIC and MBC were determined then, adaptation of each isolate to each biocide was performed. After adaptation to different biocides, MIC increase factor (fold increase) for each antibiotic was determined for each adapted isolate as a ratio between antibiotic MIC after adaptation and antibiotic MIC before adaptation. It was observed that the values of the MIC increase factor of the tested antibiotics were relatively higher in E.coli (n=78), K.pneumoniae (n=50), P.mirabilis (n=31) and P.aeruginosa (n=43) isolates after adaptation to BAC and TCS, relative to the remaining tested isolates, where 4-32 fold increase in antibiotic MICs was recorded in the tested isolates after adaptation. Moreover, higher numbers of antibiotics (up to 10) showed such increase in MIC values. Therefore, these isolates were selected for further studies. In order to examine the effect of adaptation to biocides on the growth process of the tested isolates, growth curves were generated for the tested cells before and after adaptation. They were constructed by plotting log OD versus time (hr) before and after adaptation. It was found that growth was retarded in 43% and 66% in E. coli and p. aeruginosa isolates after adaptation to BAC with non-significant change (p>0.05) was found in the remaining tested isolates. To elucidate the potential role of efflux pumps in adaptation of the tested isolates to biocides, the efflux system of the original and adapted cells was examined by EtBr cartwheel method. It was noted that BAC and TCS significantly increased (p˂ 0.05) efflux activity in E.coli and K.pneumoniae isolates, respectively, after adaptation in comparison with their original cells. Detection of 4 efflux genes (acrB, mdfA, norE and yihV) was examined in the isolates showing increase in fluorometric efflux with EtBr in the cartwheel method after biocide adaptation. With the aim of better understanding of the influence of adaptation to BAC and TCS on efflux systems, the expression of the efflux pump genes in 10 K. pneumoniae isolates and 10 E. coli isolates (showing the highest increase in fluorometric efflux with EtBr in the cartwheel method after adaptation) before and after repeated exposure to sub lethal concentrations of BAC and TCS, respectively was measured. It was observed that there was a marked increase (> 2 fold) in the expression of the efflux pump genes in 50% of the adapted isolates for acrB and yihV genes, 60% of the adapted isolates for mdfA gene and 70% of the adapted isolates for norE gene in the adapted K. pneumoniae isolates. Also, an increase in the expression of the efflux pump genes in 50% of the adapted isolates for acrB gene and 60% of the adapted isolates for either mdfA, norE or yihV genes in the adapted E. coli isolates was recorded. The production of β-lactamases was detected in the tested isolates using iodometric overlay method before and after adaptation. It was found that there was non-significant (p>0.05) change in β-lactamase production in the adapted bacterial isolates in comparison with their original non adapted counterparts. Interactions of bacterial membranes with biocides cause fundamental changes in the membrane structure and function. In this study, effects of adaptation to biocides on outer membrane permeability, cell membrane integrity, permeability, zeta potential and depolarization were examined. A significant decrease (p˂0.05) in outer membrane permeability was detected in 43% and 37% of TCS adapted E.coli, and K.pneumoniae isolates, respectively. There was non-significant change (p>0.05) in the membrane integrity in the TCS adapted E.coli, or K.pneumoniae isolates in comparison with their original non-adapted counterparts. On the other hand, there was a significant decrease (p˂ 0.05) in the membrane integrity with variable percentages in TCS adapted P.mirabilis and P.aeruginosa isolates as well as in the BAC adapted E.coli, K.pneumoniae, P.mirabilis and P.aeruginosa isolates. In addition, a significant decrease (p˂0.05) in the cell membrane permeability was detected in 55% and 50% of the TCS adapted E.coli, and K.pneumoniae isolates, respectively. Furthermore, it was found that 50% and 28% of the TCS adapted E.coli and K.pneumoniae isolates, respectively, showed a significant increase (p˂ 0.05) in the zeta potential values of their membranes. It was observed that 75.6% of TCS adapted E.coli and 66% of BAC adapted K. pneumoniae isolates showed a significant increase (p˂ 0.05) in membrane depolarization after adaptation. The impact of biocide adaptation on bacterial cell morphology was analyzed using SEM and TEM. The SEM analysis revealed considerable morphological modifications were exhibited in K.pneumoniae isolates adapted to BAC that ranged from deformed cells, with individual bumps, grooves, ridges, and cavities, to overall cell surface wrinkling. Cell wall disruption was observed in the form of cracks, holes, or even cell lysis. Also, clusters of some lysed and fused cells were observed. Moreover, the cells appeared thinner and longer than the non-adapted cells. On the other hand, the TEM examination of the adapted cells showed electron dense regions, the cytoplasm was disorganized and spacing between inner and outer membrane occurred. The remaining tested isolates didn’t show observable changes after adaptation. Effect of adaptation to biocides on some virulence factors of the tested isolates was investigated. This included adherence, invasion, cell surface hydrophobicity, biofilm formation, lipase production, swarming, urease activity and pyocyanin production. Adherence of tested isolates was investigated using congo red agar test and positive adherence results were confirmed by adhesion to the human CaCO-2 cell line assay before and after adaptation. It was observed that the average adhesion index of the tested P. aeruginasa isolates increased from the range of (1.1-1.6) bacteria per cell to (6.43-7.2) bacteria per cell after adaptation to BAC. Additionally, it was noted that there was non-significant change (p>0.05) in the invasion of the adapted isolates in comparison with their original counterparts. It was found that 63% and 16% of the tested P. aeruginasa and K. pneumoniae isolates showed a significant increase (p˂ 0.05) in their HI after adaptation to BAC and TCS, respectively. Semi-quantitative measurement of the mature biofilms formed by the tested isolates using crystal violet assay revealed non-significant (p>0.05) change in biofilm formation except for P.aeruginosa isolates. It was found that the percentage of strong and moderate biofilm producing P.aeruginosa isolates has increased from 39% to 62.5% after adaptation to BAC. There was non-significant (p>0.05) change in the viability of P.aeruginosa isolates in the formed biofilm after biocide adaptation as determined by MTT assay. In order to further understand the impact of adaptation to BAC on biofilm formation by P. aeruginosa isolates, the relative expression of the biofilm gene (ndvB) was detected using RT-PCR in 10 P. aeruginosa isolates before and after adaptation to BAC. It was found that 60% of the selected isolates showed overexpression of ndvB biofilm gene after adaptation to BAC (> 2-fold increase in gene expression). After adaptation to BAC and TCS, there was a significant decrease (p˂ 0.05) in lipase production by E.coli, K.pneumoniae and P.aeruginosa isolates with variable percentages. Also, adaptation to either BAC or TCS significantly decreased (p˂0.05) the swarming motility of P. mirabilis after adaptation with percentage of 29% and 42% of isolates, respectively. In addition, it was found that there was non-significant (p>0.05) change in urease production by the tested P.mirabilis isolates after adaptation. Moreover, adaptation to BAC and TCS significantly (p˂ 0.05) decreased quantity of pyocyanin produced by the adapted P. aeroginosa isolates with percentages of 21% and 30.2%, respectively. A variable increase in the quantity of the total proteins among the adapted isolates was observed. Moreover, adaptation to BAC and TCS resulted in a decrease the total lipids of some of the adapted isolates. The examination of the lipid profiles of the tested bacterial isolates before and after adaptation to biocides demonstrated non observable changes in the lipid profiles of the tested isolates after adaptation. In the current study, intI and intII genes were detected in the tested isolates using PCR technique to detect co-resistance between antibiotics and biocides. Additionally, presence of efflux genes in the tested isolates might indicate the occurrence of cross resistance.