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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