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

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 concentrationdependent 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 g/ml polymyxin B but not at a lower concentration of 0.125 g/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 g/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.The emergence of multidrug-resistant gram-negative organisms and the simultaneous lack of new clinically available antimicrobial agents have led to a resurgence of older compounds such as the polymyxins (6). These agents, including polymyxin B and colistin, have highly potent activity against gramnegative organisms, including Pseudomonas aeruginosa, but were previously abandoned due to a reported high incidence of nephrotoxicity and neurotoxicity (12).Resistance to polymyxin B is predominantly associated with decreased uptake into the bacterial cell resulting from a reduced capacity for initial binding (23). Polymyxin B and other polycationic compounds enter the cell via a process known as self-promoted uptake (10, 11). Polymyxin B binds to outer-membrane lipopolysaccharide (LPS) displacing Mg 2ϩ and disrupting the Mg 2ϩ cross bridges between anionic LPS molecules in the outer membrane, thus leading to membrane des...

Irreproducible and Uninterpretable Polymyxin B MICs for Enterobacter cloacae and Enterobacter aerogenes

“…Several two-component regulatory systems that control expression of the arn operon have been identified. Under the pressure of low-Mg conditions (19) or exposure to polymyxins (12,18,20,21), PhoPQ has been demonstrated to regulate expression of the arn operon. Similarly, PmrAB can also affect expression of the arn operon under low-cation conditions (22) or following exposure to polymyxins (12,18,22,23).…”

“…Under the pressure of low-Mg conditions (19) or exposure to polymyxins (12,18,20,21), PhoPQ has been demonstrated to regulate expression of the arn operon. Similarly, PmrAB can also affect expression of the arn operon under low-cation conditions (22) or following exposure to polymyxins (12,18,22,23). Polymyxin resistance has been associated with altered expression of these regulators (12,24), including upregulation of pmrA (18), pmrB (12,23), and phoQ (12) and the disabling of phoQ (21).…”

“…The finding of "cocarde" growth (increased growth near a disk surrounded by a zone of inhibition) has been reported with Serratia marcescens (11). The presence of "skip wells" with polymyxin MIC testing of P. aeruginosa (12) and A. baumannii (13) has also been reported. Heteroresistance, defined by population analysis profiles, has been described with isolates of A. baumannii, especially at concentrations of colistin surrounding the breakpoint of susceptibility (14).…”

Effect of PhoP-PhoQ Activation by Broad Repertoire of Antimicrobial Peptides on Bacterial Resistance

“…Resistance-The likelihood that bacteria will develop resistance to AMPs by activation of PhoP-PhoQ suggests that these bacteria should be more resistant to AMPs upon preincubation with the peptides (22,33,37). We therefore determined the MICs of the peptides against WT bacteria cultivated for 20 h with subinhibitory concentrations of AMPs (0.25 MIC) (Fig.…”

Alternativas terapêuticas para o tratamento de infecções por Pseudomonas aeruginosa multirresistentes endêmicas no Brasil.

“…Um estudo de Rahal et al (1998) Devido à resistência aos carbapenêmicos e a dificuldade no desenvolvimento de novas drogas (Tillotson, 2008), muitos autores têm previsto um retorno à era pré-antibiótica, postulando a volta de antibióticos abdicados atualmente (Levin, Oliveira, 2008;Paterson, Lipman, 2007;Schurek et al, 2009;Theuretzbacher, 2012;Timurkaynak et al, 2006), porém, uma alternativa mais sustentável é o estudo de combinações de antibióticos que alcancem efeitos sinérgicos (Choi et al, 2004;Tripodi et al, 2007;Zhanel et al, 2006 (Bouza, Muñoz, 2000).…”