Wickens, Hayley Jayne; (2001) Post antibiotic effects and repair-recovery in Escherichia coli exposed to ciprofloxacin. Doctoral thesis (Ph.D.), University College London.

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Abstract

Quinolone antibacterials interact primarily with the DNA-DNA gyrase complex, but the subsequent events that lead to loss of viability are still subject to debate. This thesis investigates the lethal mechanisms of ciprofloxacin in Escherichia coli, with particular reference to cellular repair and recovery during the period of post antibiotic effect (PAE). Three distinct mechanisms for quinolone lethality may be identified: Mechanism A, which requires RNA and protein synthesis, and cell division for expression; Mechanism B, which remains active under conditions where RNA or protein synthesis are precluded; and Mechanism C, which is active on non-dividing cells. This work shows that the length of the PAE is altered in cells exposed to ciprofloxacin under conditions that inhibit protein or RNA synthesis, or cell division, and that the classic filamentation response of these DNA- damaged cells is delayed. It is suggested that altered PAEs result from delay of DNA repair initiation, and this is investigated using studies of radiolabel incorporation into DNA, RNA and protein of cells previously exposed to ciprofloxacin. Filamentation is shown to be weak or absent at sub-minimum inhibitory concentrations (MIC), maximal at concentrations around the MIC, and to decline at concentrations above the maximal bactericidal concentration. Flow cytometry (FCM), a technique which permits the simultaneous measurement of several single cell parameters, is used to analyse individual filaments during the PAE in an attempt to determine the fate and function of these abnormal forms. The chromosome content of filaments is also analysed by FCM. PAEs of ciprofloxacin on DNA repair proficient and DNA repair deficient strains of E. coli are compared, and recovering cells are investigated using FCM and traditional methods. This provides insight into the controversial roles of intracellular DNA repair in quinolone lethality and recovery.

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