ID 247
Viducic, Darija Department of Microbiology, Graduate School of Dentistry, The University of Tokushima
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Departmental Bulletin Paper
The capacity of bacteria to survive, but not grow during antibiotic treatment may be defined as antibiotic tolerance. In bacteria, significant physiological changes occur depending on various environmental conditions ; low metabolic activity, slow growth, iron limitation and different stress conditions may be considered as factors playing a role in the tolerance to antimicrobial agents in Pseudomonas aeruginosa. One of the factors underlying these conditions is an alarmone, ppGpp that accumulates in the cells during transient starvation and nutritional deficiency. In order to investigate the possible role of ppGpp in the antibiotic tolerance in P. aeruginosa, the knockout mutants in the genes involved in the stringent response such as relA, spoT and dksA were constructed and investigated for their antibiotic susceptibility to quinolones. The susceptibility of these mutants to quinolone was determined using minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) testing and colony forming units per ml (CFU/ml). MIC/MBC ratios for ofloxacin in the dksA and the spoT knockout mutant were 1/4 and 0.5/2, respectively, in the comparison with the wild type strain, 1/1. The survival rate of the dksA and spoT knockout mutant in the presence of 8μg/ml of ofloxacin showed to be approximately 100 times higher than the same for the wild type strain. In the presence of 1μg/ml of ciprofloxacin, the survival rate for dksA and spoT mutant was 10-50 times higer than seen in the wild type strain. In comparison with the wild type, the relA and the relA spoT mutant displayed increased sensitivity to quinolones. The intracellular levels of ppGpp determined by high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. In conclusion, the data indicate that significantly elevated levels of ppGpp might be responsible for rendering these mutants tolerant to quinolones. The data presented in this study not only furnish some additional insights into the pleiotropic effect of ppGpp but also expand the importance of ppGpp as an antimicrobial target in P. aeruginosa. Moreover, given the facts that RpoN, an alternative sigma factor activates the expression of wide variety of environmentally regulated genes and is required for virulence in a variety of pathogens, we hypothesized that it might additionally be a target for interaction with antimicrobial agents. Two knockout mutants in the rpoN gene were constructed and investigated for their response to quinolone treatment. Using different construction approach, we obtained rpoN::Tc^r which displayed tolerance to quinolone and responded to quinolone addition by increased production of siderophore ; in addition, rpoN::Gm^r showed increased susceptibility to quinolone treatment. Results obtained imply that RpoN exerts its response to antibiotics through different pathways of regulation, and one of them might be due to the production of siderophore.
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