Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Abstract

Benzisothiazolone (BIT), N‐methylisothiazolone (MIT) and 5‐chloro‐N‐methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub‐minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol‐interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T‐OMP, in SDS‐PAGE preparations. T‐OMP was observed to disappear from the biocide‐exposed preparations and reappear when the resistance‐induced cultures were passaged in the absence of biocide. This reappearance of T‐OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross‐resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T‐OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon™ form (CMIT and MIT).

Pseudomonas aeruginosa PAO1 adapted to 2-phenoxyethanol shows cross-resistance to dissimilar biocides and increased susceptibility to antibiotics

“…BIT (isothiazolone biocide) is often described as thiol-interactive preservative (Bloomfield 1991). The level of BIT induced resistance (Winder 2000) was similar to the level of resistance observed by us. There are no common targets between PE and BIT (Russell 2003) yet three-fold reduction in a susceptibility can only indicate a common penetration site for the two biocides.…”

“…A possible explanation for increased resistance to PE is the physiological adaptation of the envelope to prevent entry of the biocide. Changes in susceptibility towards biocides have been correlated with OM changes (Loughlin et al 2002;Abdel Malek et al 2002;Winder et al 2000;Brozel and Cloete 1994). It is known that a major mechanism of resistance to biocides is reduced uptake due to impermeability or efflux (Russell 2003).…”

“…PE adaptation experiments were performed according to Winder et al (2000) and Abdel Malek et al (2002). Briefly, fresh P.a.…”

“…Pellets were used for OM preparations (Pugsley et al 1986). SDS-PAGE analysis was performed according to Winder et al (2000) and Abdel Malek et al (2002). Briefly, OMs were resuspended in 100 L of Tris-HCl buffer (pH 7.4).…”

“…Adaptation of P.a. to disinfectants by serial passage in increasing concentration of biocides is well documented (Loughlin et al 2002;Abdel Malek et al 2002;Winder et al 2000). The presence of constitutive and inducible efflux mechanisms removing a huge range of antimicrobials from the cell has been detected in P.a.…”