Journal of Applied Bacteriology zyxwvutsrq 1994, 76, 576-582 zyxwvutsr Resistance of Pseudomonas aeruginosa to isothiazolone V.S. Brozel and T.E. Cloete Environmental Biotechnology Laboratory, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, South Africa 4605/06/93: zyxwvutsrqpo accepted 18 November 1993 V.S.BROZEL AND T.E. CLOETE. 1994. zyxwvutsr This investigation was to determine whether zyxw Pseudomonas aeruginosa could acquire resistance to the bactericide isothiazolone, and what the nature of such a resistance mechanism would be. The Pseudomonas was cultured in nutrient-limited broth in the presence of sub-inhibitory concentrations of isothiazolone (a mixture of 1.1 5% 5-chloro-N-methylisothiazolone (CMIT) and zyxwvu 0-35 % N-methylisothiazolone (MIT)). Three cultures tested in parallel adapted gradually during exposure for 15 d from an initial minimum inhibitory concentration (MIC) of 300 pll-' to 607 pl 1-'. The three parallel cultures adapted at similar rates, so the adaptation was not ascribed to mutation but to a specific mechanism. Resistant cells did not produce any extracellular isothiazolone-quenching compounds nor undergo detectable alterations in their lipopolysaccharide layer. In wild cells, a 35 kDa outer membrane protein (protein T ) was detectable, whereas resistant cells lacked this protein. Production of protein T was suppressed within 24 h of exposure to isothiazolone. It was still suppressed after 72 h of growth in isothiazolone-free medium. It is proposed that Ps. aeruginosa acquires resistance to isothiazolone by a process of adaptation where the outer membrane protein T is suppressed. INTRODUCTION Industrial water systems (e.g. cooling water systems in power plants and mines) contain a variety of bacteria, most of which are Gram-negative aerobic rods, e.g. Pseudomonas stutzeri, Ps. jluorescens and zyxwvuts Ps. aeruginosa (Escher and Characklis 1990; Cloete et zyxwvutsrqpo al. 1992). Many such systems are treated with bactericides to eliminate or reduce micro- bial growth and concomitant microbially-induced corrosion (Cloete et al. 1992). The bacterial species present differ in their susceptibility to various bactericides, some having a higher degree of inherent resistance than others (Brozel and Cloete 1991a). It was observed that some species (e.g. Ps. stutzeri and Bacillus cereus) show a low degree of resistance under pure culture conditions (Brozel and Cloete 1991a) but are the dominant planktonic survivors in cooling water systems 36 h after treatment with bactericides, indicating adaptation to the bactericide (Brozel and Cloete 1992). Various isolates from such a bactericide-treated system, i.e. Ps. stutzeri, Ps. cepacia, B. cereus and Bacillus subtilis acquired a higher degree of resistance to a range of bacteri- cides after extended exposure to sub-inhibitory concentra- tions of bactericide (Brozel and Cloete 1991b). Correspondence to : V.S. Brozel, Environmental Biotechnology Laboratory, Department of Microbiology and Plant Pathology, University of Pretoria, zyxwvutsrq 0002, Pretoria, South Africa. Bacteria develop resistance to various antiseptics (Kolawole 1984; Heinzel 1989; Jones et al. 1989; Sakagami et al. 1989). Such resistance may be due to a mechanism of adaptation of the cell envelope (Heinzel 1989), or to plasmid transfer encoding a bactericide-degrading enzyme (Eagon and Barnes 1986). Bactericides attack specific com- ponents of bacterial cells, usually at the cytoplasmic mem- brane or in the cytoplasm (Gilbert and Wright 1987). Direct action at the outermost surface is rare. For bacteri- cides to be effective they must be able to penetrate the cell envelope and attain a sufficiently high concentration at the target site in order to exert their antibacterial action. Hydrophilic antibacterial agents enter through the outer membrane via membrane proteins (Nikaido and Vaara 1987). In Escherichia coli the uptake of hydrophilic drugs of Mr less than 600 is by solute diffusion through general porins. Pseudomonas aeruginosa has a wide array of specific permeation channels and only one porin (Opr F) which permits a low rate of solute diffusion (Nikaido 1992). This property affords Ps. aeruginosa its high degree of inherent drug resistance. Altered cell envelope structure can contrib- ute decisively towards accessibility of antimicrobial agents to their periplasmic or cytoplasmic targets (Nikaido 1992). Certain resistant isolates either lack or overexpress certain outer membrane proteins. An example is an isolate of Ps. aeruginosa resistant to the antibiotic imipenem. It lacks Opr