Amino Acid Substitutions Account for Most MexS Alterations in Clinical nfxC Mutants of Pseudomonas aeruginosa.

Multidrug-resistant mutants ofPseudomonas aeruginosathat overproduce the active efflux system MexEF-OprN (callednfxCmutants) have rarely been characterized in the hospital setting. Screening of 221 clinical strains exhibiting a reduced susceptibility to ciprofloxacin (a substrate of MexEF-OprN) and imipenem (a substrate of the negatively coregulated porin OprD) led to the identification of 43 (19.5%)nfxCmutants. Subsequent analysis of 22 nonredundant mutants showed that, in contrast to theirin vitro-selected counterparts, only 3 of them (13.6%) harbored a disruptedmexSgene, which codes for the oxidoreductase MexS, whose inactivation is known to activate themexEF-oprNoperon through a LysR-type regulator, MexT. Nine (40.9%) of the clinicalnfxCmutants contained single amino acid mutations in MexS, and these were associated with moderate effects on resistance and virulence factor production in 8/9 strains. Finally, the remaining 10 (45.5%)nfxCmutants did not display mutations in any of the regulators known to controlmexEF-oprNexpression (themexS,mexT,mvaT, andampRgenes), confirming that other loci are responsible for pump upregulation in patients. Collectively, these data demonstrate thatnfxCmutants are probably more frequent in the hospital than previously thought and have genetic and phenotypic features somewhat different from those ofin vitro-selected mutants.

Carbapenem resistance in cystic fibrosis strains of Pseudomonas aeruginosa as a result of amino acid substitutions in porin OprD.

The aim of this work was to investigate the impact of single amino acid substitutions occurring in specific porin OprD on carbapenem resistance of cystic fibrosis (CF) strains of Pseudomonas aeruginosa. A PAO1ΔoprD mutant was complemented with the oprD genes from five carbapenem-resistant CF strains exhibiting very low amounts of mutated OprD porins in their outer membrane despite wild-type levels of oprD transcripts. Compared with wild-type porin from strain PAO1, single amino acid substitutions S403P (in periplasmic loop 8), Y242H, S278P and L345P (in ß-sheets 10, 12 and 14, respectively) were found to result in reduced amounts of OprD in the outer membrane, increased carbapenem resistance, and slower growth in minimal medium containing gluconate, an OprD substrate, as the sole source of carbon and energy. This indicates that in CF strains of P. aeruginosa, loss of porin OprD may not only result from mutations downregulating the expression of or disrupting the oprD gene, but also from mutations generating deleterious amino acid substitutions in the porin structure.

Diversity of ß-lactam resistance mechanisms in cystic fibrosis isolates of Pseudomonas aeruginosa: a French multicentre study.

OBJECTIVES: To investigate the resistance mechanisms of ß-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France. METHODS: Two-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to ß-lactams were investigated. Their ß-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic ß-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations. RESULTS: MLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to ß-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to ß-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary ß-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene. CONCLUSIONS: ß-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of ß-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.

Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa can become resistant to carbapenems by both intrinsic (mutation-driven) and transferable (ß-lactamase-based) mechanisms. Knowledge of the prevalence of these various mechanisms is important in intensive care units (ICUs) in order to define optimal prevention and therapeutic strategies. METHODS: A total of 109 imipenem-non-susceptible (MIC >4 mg/L) strains of P. aeruginosa were collected in June 2010 from the ICUs of 26 French public hospitals. Their resistance mechanisms were characterized by phenotypic, enzymatic, western blotting and molecular methods. RESULTS: Single or associated imipenem resistance mechanisms were identified among the 109 strains. Seven isolates (6.4%) were found to produce a metallo-ß-lactamase (one VIM-1, four VIM-2, one VIM-4 and one IMP-29). Porin OprD was lost in 94 (86.2%) strains as a result of mutations or gene disruption by various insertion sequences (ISPa1635, ISPa1328, IS911, ISPs1, IS51, IS222 and ISPa41). Thirteen other strains were shown to be regulatory mutants in which down-regulation of oprD was coupled with overexpressed efflux pumps CzcCBA (n = 1), MexXY (n = 9) and MexEF-OprN (n = 3). The lack of OprD was due to disruption of the oprD promoter by ISPsy2 in one strain and alteration of the porin signal sequence in another. CONCLUSIONS: Imipenem resistance in ICU P. aeruginosa strains may result from multiple mechanisms involving metallo-ß-lactamase gene acquisition and genetic events (mutations and ISs) inactivating oprD, turning down its expression while increasing efflux activities or preventing insertion of porin OprD in the outer membrane. This diversity of mechanisms allows P. aeruginosa, more than any other nosocomial pathogen, to rapidly adapt to carbapenems in ICUs.