Permeability of Pseudomonas Aeruginosa

Abstract

P. aeruginosa PAOL was chosen as the organism for investigation. β-lactamase production was influenced by induction of the chromosomally mediated class Id enzyme by growth of the organism in the presence of a sub-MIC concentration of a β-lactam or by plasmid mediation. Outer membrane permeability was measured by adding a solution of a chromogenic β-lactam, nitrocefin, of known concentration (S₀) to whole cells. The rate of hydrolysis of nitrocefin by whole cells was calculated (vi). β-lactamase kinetic parameters, V₀ and Kₘ, were determined from a sonicated cell suspension. Reproducibility was assessed by repetition of the outer membrane permeability assay and calculation of the accumulating averages for each parameter (V₀, Kₘ, and vi) as the number of repeats increased. The averages converged to a single value for each parameter after six repeats. V₀, Kₘ, and vi allowed the calculation of the periplasmic nitrocefin concentration (Sₚ) and a permeability coefficient (C). The influence of assay conditions on measured permeability was investigated. A harvest temperature of 16-20°C and a magnesium/phosphate-based nitrocefin solvent were chosen. This minimized damage to cells while measuring hydrolysis rates of nitrocefin by whole cells (vi), thus influencing C. C also varied with S₀ and method of β-lactamase production, plasmid mediation, and inducer concentration. Outer membrane permeability should therefore be defined with respect to S₀, β-lactamase level (and/or method of production), and harvesting conditions. The influence of growth rate on measured permeability was investigated in PAQ1 grown in batch culture. By both criteria of measured permeability (Sₚ/S₀ and Cc), logarithmically growing cells were more permeable than stationary-phase cells—a difference that could not be accounted for by differences in β-lactamase level. In vivo-grown cells had a measured permeability approximately the same as in vitro-grown stationary-phase cells. In vivo-grown cells had a much lower measured permeability than in vitro logarithmically growing cells. Investigations into the influence of incubation of PAQ1 with pre-immune and immune serum on measured permeability suggest that this difference may be accounted for in part by clumping of cells in vivo.