RESUMO
Pseudomonas aeruginosa can 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 phoQ mutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRS genes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQ mutant, as did individual or tandem deletion of cprRS. Individual deletion of colR or colS in a ΔphoQ mutant 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 colRS or cprRS in the ΔphoQ mutant or individual deletion of cprR or cprS failed 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 colRS or cprRS in tandem or of cprR individually complemented the Pm resistance phenotype in the ΔphoQ mutant, while episomal expression of colR, colS, or cprS individually did not. Highly polymyxin-resistant phoQ mutants of P. aeruginosa isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRS and cprS; when expressed in a ΔphoQ background, 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 colRS and cprS mutations can contribute to high-level clinical resistance.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/efeitos dos fármacos , Polimixinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Arabinose/análogos & derivados , Arabinose/metabolismo , Proteínas de Bactérias/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana/genética , Deleção de Genes , Teste de Complementação Genética , Loci Gênicos , Humanos , Lipídeo A/metabolismo , Mutação , Plasmídeos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismoRESUMO
Pseudomonas aeruginosa can 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. aeruginosa isolates 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 pmrB alleles that conferred polymyxin resistance to strains with a wild-type or pmrAB deletion background. Double mutant pmrB alleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrB alleles induced transcription from the promoter of the arnB operon 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 pmrB gain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana/genética , Mutação , Polimixinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Colistina/farmacologia , Colistina/uso terapêutico , Fibrose Cística/tratamento farmacológico , Regulação Bacteriana da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificaçãoRESUMO
Moxifloxacin is under development for expanded use against Mycobacterium tuberculosis. Rifampin is a mainstay of therapy. We examined the interaction of moxifloxacin plus rifampin for log-phase and nonreplicating persister (NRP) organisms. For this evaluation, we employed our hollow-fiber infection model, in which organisms are exposed to clinically relevant drug concentration-time profiles and the impact on bacterial cell kill and resistant subpopulation amplification is determined. In log phase, resistance emergence was observed in all monotherapy regimens and in no combination therapy regimen. No difference was seen in time to a 3-log reduction in the bacterial burden; there was a significant difference in time to resistance emergence (P = 0.0006). In the NRP experiment, no resistance emergence was seen. There was a significant difference between the monotherapy and combination therapy regimens in time to a 3-log reduction in the bacterial burden (P = 0.042). The combination is efficacious for suppressing resistant organisms but is antagonistic for cell kill.