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ABSTRACTThe arnlocus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-l-arabinose modification of the lipid A moiety of lipopolysaccharide. In Pseudomonas aeruginosa, several two-component regulatory systems (TCSs) control the arnlocus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of the P. aeruginosaPAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety of P. aeruginosastrains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such as P. aeruginosaPAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designated cprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. The cprAgene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutated cprAallele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion of cprAdid not affect the arn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance.

ABSTRACTPseudomonas aeruginosais a major pathogen in cystic fibrosis (CF) lung disease. Children with CF are routinely exposed to P. aeruginosafrom the natural environment, and by adulthood, 80% of patients are chronically infected. P. aeruginosain the CF airway exhibits a unique biofilm-like structure, where it grows in small clusters or aggregates of bacteria in association with abundant polymers of neutrophil-derived components F-actin and DNA, among other components. These aggregates differ substantially in size and appearance compared to surface-attached in vitrobiofilm models classically utilized for studies but are believed to share properties of surface-attached biofilms, including antibiotic resistance. However, little is known about the formation and function of surface-independent modes of biofilm growth, how they might be eradicated, and quorum sensing communication. To address these issues, we developed a novel in vitromodel of P. aeruginosaaggregates incorporating human neutrophil-derived products. Aggregates grown in vitroand those found in CF patients' sputum samples were morphologically similar; viable bacteria were distributed in small pockets throughout the aggregate. The lasAquorum sensing gene was differentially expressed in the presence of neutrophil products. Importantly, aggregates formed in the presence of neutrophils acquired resistance to tobramycin, which was lost when the aggregates were dispersed with DNase, and antagonism of tobramycin and azithromycin was observed. This novel yet simple in vitrosystem advances our ability to model infection of the CF airway and will be an important tool to study virulence and test alternative eradication strategies against P. aeruginosa.

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQmutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRSgenes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQmutant, as did individual or tandem deletion of cprRS. Individual deletion of colRor colSin a ΔphoQmutant also suppressed 4-amino-l-arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRSor cprRSin the ΔphoQmutant or individual deletion of cprRor cprSfailed to suppress 4-amino-l-arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in P. aeruginosa. Episomal expression of colRSor cprRSin tandem or of cprRindividually complemented the Pm resistance phenotype in the ΔphoQmutant, while episomal expression of colR, colS, or cprSindividually did not. Highly polymyxin-resistant phoQmutants of P. aeruginosaisolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRSand cprS; when expressed in a ΔphoQbackground, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in P. aeruginosa, indicate that addition of 4-amino-l-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRSand cprSmutations can contribute to high-level clinical resistance.

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance, P. aeruginosaisolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-function pmrBalleles that conferred polymyxin resistance to strains with a wild-type or pmrABdeletion background. Double mutant pmrBalleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrBalleles induced transcription from the promoter of the arnBoperon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate that pmrBgain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of polymyxin resistance (MICs of 8 to 64 mg/liter) in laboratory and clinical strains of this organism. To explore the role of PhoPQ in high-level clinical polymyxin resistance, P. aeruginosastrains with colistin MICs > 512 mg/liter that had been isolated from cystic fibrosis patients treated with inhaled colistin (polymyxin E) were analyzed. Probable loss-of-function phoQalleles found in these cystic fibrosis strains conferred resistance to polymyxin. Partial and complete suppressor mutations in phoPwere identified in some cystic fibrosis strains with resistance-conferring phoQmutations, suggesting that additional loci can be involved in polymyxin resistance in P. aeruginosa. Disruption of chromosomal phoQin the presence of an intact phoPallele stimulated 4-amino-l-arabinose addition to lipid A and induced transcription from the promoter of the pmrH(arnB) operon, consistent with the known role of this lipid A modification in polymyxin resistance. These results indicate that phoQloss-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.