الفهرس 
Discovery of persistenceOnly 14 pages are availabe for public view
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Abstract
A total of 40 clinical bacterial isolates were obtained from different specimens. The isolates were identified using the classical phenotypic characteristic tests including API 20E identification kit. The results showed that Proteus mirabilis (55%), E.coli (17%), P. aeruginosa (12%), Klebsiella pneumoniae (10%), Enterobacter cloacae (3%), Acinetobacter baumannii (3%) comprised the different bacterial species of the collected isolates. The agar dilution technique was used to determine the minimum inhibitory concentration of the collected isolates against ciprofloxacin. The results showed that sensitive isolates constituted (60%), resistant isolates (32%) and intermediate isolates (8%) of the collected isolates. Two isolates were selected for completing the study and the criteria of the selection depended on choosing most sensitive isolates as well as two different bacterial species not commonly reported in literature for persistence phenomenon study. A Klebsiella pneumoniae isolate (MIC = 0.5 μg/ml) and A Proteus mirabilis isolate (MIC= 0.031 μg/ml) fulfilled the selection criteria and they were of codes 33 and 17, respectively.
Persister cell isolation was done by dose dependent killing experiment of ciprofloxacin for 24h; the resultant dose kill curve was biphasic leaving a plateau of survivors. from the obtained plateau, the most suitable concentration of ciprofloxacin was selected to be used for persister cells isolation in subsequent experiments. About 2.3% persisters was obtained when using, 500 μg/ml ciprofloxacin for Klebsiella pneumoniae, whereas about 1.3% survivors was obtained by using 30 μg/ml ciprofloxacin for P. mirabilis. To ensure that these cells were not drug resistant mutants rather tolerant cells, the MIC of the survivors were determined against ciprofloxacin for both isolates and the results showed no change in their values as compared to those of their corresponding wild type cell populations. Additionally, when LB agar plates with ciprofloxacin added at concentrations equal to that used for persister cell isolation of both test isolates were spreaded with the corresponding recovered survivors, no growth was detected while the corresponding plates without added ciprofloxacin showed growth.
Different environmental factors were investigated for their effects on persister cell recovery of both tested isolates. All used environmental stressors used for 3 h exposure period decreased the percentage of survivors in K. pneumoniae as compared to ciprofloxacin exposure alone for 24 h. While for P. mirabilis most of environmental stressors (3 h) gave the same percentage of survivors as ciprofloxacin exposure (24 h). The least percentage of K. pneumoniae survivors (0.000005%) was obtained upon using different concentrations of hydrogen peroxide. While for P. mirabilis, the least percentage of survivors was observed with heat shock at 40 oC (0.33%) and 45oC (0.32%). The effect of ciprofloxacin on the pre-stressed cells showed that it decreased the percentage of pre stressed recovered survivors in case of K. pneumoniae. While for P. mirabilis, the percentage of survivors recovered from stressors pre exposure were un effected upon post exposure to ciprofloxacin. Therefore,environmental stressors rendered persisters of K. pneumoniae to be more sensitive to ciprofloxacin the case that was unattained in case of P. mirabilis. Therefore, P. mirabilis persisters were more tolerant than those of K. pneumoniae persisters for ciprofloxacin killing effect.
For both test isolates, slow rate of growth and cell elongation were the characters observed in persisters when resuscitated in rich nutrient media. Persister revival assay was used to prove the slow rate of growth. The results revealed that after 5 h incubation period, K. pneumoniae persisters optical density recorded about 65% that of the wild type cell population whereas P. mirabilis persisters optical density reached about 30% that of wild type cell population. Scanning electron microscopy was used to detect the elongation of cell length. About 5 times, increase in cell length for resuscitated of K. pneumoniae persisters and about 7 times increase in cell length of P. mirabilis persisters were recorded.
Inhibition of persisters was attempted using three approaches. First, clearance was tested by priming with different sugars before ciprofloxacin addition. The results showed that priming with each of the tested sugars decreased percentage of survivors as compared to the control (ciprofloxacin alone). Priming with glucose (0.4%) recorded the least percentage of survivors for P. mirabilis isolate whereas priming with sucrose (0.0008%) recorded the least persister percentage in case of K. pneumoniae.
Secondly, persisters clearance was tested using combination of silver nitrate with ciprofloxacin. A complete eradication of persisters was obtained for both tested isolates at all concentrations used of silver nitrate (10 to 120 μM).
Finally, persister cell clearance by antimicrobial agent in the presence of sodium salicylate was examined. For K. pneumoniae, a complete eradication was observed. In contrast the percentage of survivors reached 0.4% for P. mirabilis at 50 mM sodium salicylate.
Thus, targeting persisters in the treatment of any sensitive population reduces recurrence of bacterial infections as well as emergence of chronic infections, which in turn will decrease the development of multi drug resistant bacteria.