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.

Date of Award	Sept 1986
Original language	English
Awarding Institution	Aston University