Modha, Jayshree L.; (1991) Sensitivity to Antibacterial Agents Associated with Envelope Changes in Escherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Gram-negative bacteria are resistant to many hydrophobic antibacterial agents due mainly to the fact that their outer membrane acts as a very efficient permeability barrier against the entry of such compounds. Sensitivity to hydrophobic, cationic and 4-quinolone antibacterial agents was investigated using strains altered in outer membrane permeability due to the effects of cationic permeabilisers, Col V plasmids and mutations affecting outer membrane proteins. The cationic agents N-(2-pyrimidinyl)piperazine, 4,5,6-triamino pyrimidine, 1,4-diaminopiperazine, methylglyoxyl bis(guanyl-hydrazone), spermidine, agmatine sulfate, 1,3-diaminoacetone,3,3’-diaminobenzidine and 1,4-diaminobutanone were found to considerably enhance the growth inhibitory effects of novobiocin. In constrast, the agents 1,3-diaminoguanidine, triethylenetetramine, tetraethylene pentamine, N,N,N’ ,N’ -tetramethy 1-p-phenylenediamine and formamidine disulfide had no effect on novobiocin activity. Diaminoacetone also enhanced the activity of erythromycin, rifampicin, nalidixic acid, bacitracin, serum, polymyxin B, hydrogen peroxide and hexanoic acid (at pH 4), but failed to have any significant effect on the inhibitory activities of rifamycin, fiisidic acid, vancomycin, oxacillin, nafcillin, nitrofurantoin and deoxycholate. The growth inhibitory activity of EDTA and hexanoic acid were partially reversed by diaminoacetone. Magnesium ions were found to reverse novobiocin acitivity and also to compete with the enhancers of novobiocin activity. Inhibitory effects of the DNA gyrase inhibitors norfloxacin and ofloxacin were unaffected by the cationic agents diaminoacetone and methylglyoxyl bis(guanyl-hydrazone), but that of flumequin was significantly increased by both the cationic agents. Norfloxacin and ofloxacin are hydrophilic and amphoteric compounds respectively and hence use the porin pathway for entry into the Gram-negative cell. Flumequin, on the other hand is the most hydrophobic of these three DNA gyrase inhibitors tested, and may enter the cell via one of the other two pathways. It seems that the cationic agents act as permeabilisers of hydrophobic agents and therefore act at the surface of the cell ie. at the outer membrane; the possible mechanism of this effect is discussed. The effects of the ColV plasmids ColV,Ia-K94, ColV,Ia-K94 TnlO, ColV-K30, ColV-8 and ColV-41 were investigated on sensitivity of their E.coli K12 host strain to various antibacterial agents. Plasmids ColVJa-K94, its derivative ColV,Ia-K94 TnlO and ColV-41 were found to considerably increase the sensitivity of their host strain to erythromycin, rifampicin, gentamicin, novobiocin, but only to flumequin (and some to ofloxacin) out of the five 4- quinolones tested. Plasmid ColV-8 sensitised its host to most of the quinolones tested whereas plasmid ColV-41 conferred no effect on the sensitivity to these agents. With ColV,Ia-K94, colicin V and its immunity component, transfer components and an unidentified factor were found to contribute towards the observed increased antibiotic sensitising effect. I propose that the increased antibacterial agent sensitivity effect associated with the presence of most of the Col V plasmids is due to a change in the proportion of the outer membrane components and possibly also due to changes in LPS structure, but not due to any specific uptake system encoded by these plasmids; ColV-8 may however provide an additional route of entry for the quinolones. Novobiocin was found to be very active at low pH and this change in activity may be related to an observed change in its conformation at pH 5. On the other hand, novobiocin may be using a different pathway of entry into the cell at pH 5. This latter concept was investigated using strains containing mutations in the various major outer membrane proteins eg. porin proteins and those involved in the iron-assimilation system.

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