Effects of subinhibitory ofloxacin on selected genetic processes among enterobacteria

The fluorinated 4-quinolone, ofloxacin, interacts with, and inhibits the negative DNA supercoiling activity of the bacterial tetrameric enzyme, DNA gyrase. DNA gyrase is one of several topoisoraerases that homeostatically regulates the tertiary structure and function of both chromosomal and plasmid DNA. Inhibition of this process is recognised as the precursor to a cascade of cytopathic events that leads to bacterial death. It is recognised that in stationary phase, changes in gene expression promote diverse mechanisms in bacteria to maintain their 'living state'. Analysis of changes in stationary phase has become increasingly important, since Enterobacteria in many environments are in a constant state of stationary phase primarily through nutrient competition, and that rapid growth is not possible. The transposition of Tn7 into the IncP plasmid RP4, and the stability and variation of a natural co integrate plasmid, pOG669, were studied in E. coli K12. The reversion of a spontaneous pleiotropic mutant of Serratia marcescens was investigated in relation to both different growth conditions together with pigmentation and antibiotic resistance. In each of the experimental systems the time course was extended to incorporate possible changes that occurred in stationary phase. Restriction endonuclease fragmentation patterns (REFP) techniques were used to identify DNA rearrangements in the co-integrate R-plasmid pOG669 and to analyse the insertion of Tn7 into RP4. Subinhibitory ofloxacin lead to DNA rearrangements, that encompassed alteratioyn the insertion of Tn7, IncX & IncP plasmid maintenance and expression of antibiotic resistance. Subinhibitory ofloxacin significantly altered the distribution of Tn7 insertion in multiple sites within RP4. Most Tn7 insertions were found to be increased in the 22.2kb Kpnl-SmaI fragment and reduced in the 5kb KpnI-SmaI RP4 fragment. Although, fewer insertions were found in the other KpnI-SmaI fragments in RP4, subinhibitory ofloxacin reduced insertions into the 8.5kb and 5.8 fragments. It is proposed this high and low density of Tn7 insertions results from changes in local DNA superhelicity, and led to exposure of insertion sites to Tn7. Preliminary evidence suggests the tra3 and trfA genes may be contiguous in the RP4 molecule, since no Tn7 insertions were detected in these regions. This study has demonstrated that changes in antibiotic expression are induced by subinhibitory ofloxacin, as observed in the plasmids pOG669 and RP4. The mobility of these plasmids are also influenced by subinhibitory ofloxacin through their elimination. The insertion and transposition of Tn7 and therefore mobility and transposon resistance determinants have also been influenced by subinhibitory ofloxacin. Therefore, the results presented in this study describe ofloxacin to act on several genetic processes. (Abstract shortened by ProQuest.)