Involvement of pmrAB and phoPQ in Polymyxin B Adaptation and Inducible Resistance in Non-Cystic Fibrosis Clinical Isolates of Pseudomonas aeruginosa

Author:

Schurek Kristen N.1,Sampaio Jorge L. M.2,Kiffer Carlos R. V.2,Sinto Sumiko2,Mendes Caio M. F.2,Hancock Robert E. W.1

Affiliation:

1. Centre for Microbial Diseases & Immunity Research, University of British Columbia, 2259 Lower Mall, Vancouver, British Columbia, Canada

2. Fleury Medicina e Saúde, Setor de Microbiologia, Av. General Valdomiro de Lima 508, São Paulo, Brazil

Abstract

ABSTRACT During investigation of susceptibility testing methods for polymyxins, 24 multidrug-resistant clinical isolates of Pseudomonas aeruginosa were observed to have a distinct, reproducible phenotype in which skipped wells were observed during broth microdilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated. The effects of various concentrations of polymyxin B on growth, the expression of resistance genes, and outer-membrane permeability were observed. Real-time PCR was performed to compare the expression, in response to selected concentrations of polymyxin B, of genes related to the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems in polymyxin B-susceptible isolate PAO1, polymyxin B-resistant isolate 9BR, and two isolates (19BR and 213BR) exhibiting the skipped-well phenotype. 19BR and 213BR appeared to have similar basal levels of expression compared to that of PAO1 for phoQ , arnB , and PA4773 (from the pmrAB operon), and in contrast, 9BR had 52- and 280-fold higher expression of arnB and PA4773, respectively. The expression of arnB and PA4773 increased in response to polymyxin B in a concentration-dependent manner for 9BR but not for 19BR and 213BR. For these isolates, expression was significantly increased for arnB and PA4773, as well as phoQ , only upon exposure to 2 μg/ml polymyxin B but not at a lower concentration of 0.125 μg/ml. The sequencing of the pmrAB and phoPQ operons for all three isolates revealed a number of unique mutations compared to that for PAO1. 1- N -phenylnaphthylamine (NPN) was used to study the effect of preincubation with polymyxin B on the self-promoted uptake of polymyxin B across the outer membrane. The preincubation of cells with 2 μg/ml polymyxin B affected baseline membrane permeability in 19BR and 213BR and also resulted in a reduced rate of NPN uptake in these isolates and in PAO1 but not in 9BR. The results presented here suggest that the skipped-well isolates have the ability to adapt to specific concentrations of polymyxin B, inducing known polymyxin B resistance genes involved in generating alterations in the outer membrane.

Publisher

American Society for Microbiology

Subject

Infectious Diseases,Pharmacology (medical),Pharmacology

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