AbstractPseudomonas aeruginosa displays high levels of tolerance and resistance to many antimicrobial agents. Much of this tolerance is related to the nature of the Gram-negative cell envelope and in particular, the outer membrane. The outer membrane plays an important role in excluding harmful molecules from the cell, whilst being selectively permeable to other solutes via its implanted proteins (outer membrane proteins or OMPs). In order to exert their antibacterial action, antimicrobial agents must enter the cell and attain sufficiently high concentrations at their target site(s). The OMPs are highly sensitive to environmental changes and have a physiological ability to respond to such changes. It is thought that the altered cell envelope structure contributes to the accessibility of antimicrobial agents into the cell interior and resistance to such agents is related to over expression or loss of certain OMPs.
Brozel and Cloete (1994) observed a gradual increase in tolerance to increasing concentrations of biocide upon exposure of P. aeruginosa to Kathon™, a commercial biocide containing 1.15% v/v 5-chloro-N-methylisothiazolone (CMIT) and 0.35% v/v N-methylisothiazolone (MIT). This adaptation was associated with the concurrent disappearance of a 35kDa OMP, designated T-OMP. Therefore, they concluded that the biocide entered the sensitive cells via the T-OMP and that the observed resistance was the result of the absence of this OMP. The aim of this investigation was to induce tolerance in cultures of P. aeruginosa PAOl towards the pure active forms of the three isothiazolone biocides 1,2-benzisothiazolone (BIT), MIT, CMIT and the thiol-interactive agent thiomersal (used as a positive control).
An increase was observed in the minimum inhibitory concentrations (MIC) of all four biocides by at least 58% between the sensitive and resistant cultures. In some cases the percentage increase in MIC was in excess of 150%. However, when the tolerant cells were removed from the presence of the biocide, the MIC began to decrease, indicating a loss in tolerance. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) analysis of the OMP profiles from the tolerant-induced cultures illustrated the loss of T-OMP in all cases. Analysis of the sensitive and resistant cultures using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) indicated that the T-OMP disappeared in the tolerant cultures. However, these observations also suggested that other outer membrane alterations occur concurrently in T-OMP depleted tolerant cells. Investigations into the cross-resistance of the resistant cultures towards the other test biocides, indicated that resistance was, to some extent, transferable, once it had been developed towards one member of the biocide group. Following routine passaging of the resistant cultures on gradient plates two distinct colonial morphologies were observed, mucoid and non-mucoid. An increase in the cell surface hydrophobicity was noted between the mucoid and non-mucoid cultures, which indicated a loss or reduction in the B-band OPolysaccharide. However, there were no observable differences in the lipopolysaccharide
banding patterns between the mucoid and non-mucoid cells. These observations suggested that other alterations were occurring in the tolerant cells upon exposure to biocide, over and above the simple disappearance of T-OMP. Therefore, it is suggested that the observed tolerant development in biocide exposed cells, was not solely due to the loss of T-OMP. Investigations into Gram-negative bacteria isolated from contaminated industrial samples preserved with isothiazolone compounds exhibited higher MICs towards the preservative biocides than would normally be expected in the species of bacteria isolated and identified. However, there were no observable alterations in their OMP profiles.
|Date of Award||Sep 2000|
|Supervisor||Phillip J. Collier (Supervisor)|