Didier Hocquet

Are pathogenic bacteria just looking for food? Metabolism and microbial pathogenesis

It is interesting to speculate that the evolutionary drive for microbes to develop pathogenic characteristics was to access the nutrient resources that animals provided. Animal environments that pathogens colonize have likely driven the evolution of new bacterial characteristics to maximize these new nutritional opportunities. This review focuses on genomic and functional aspects of pathogen metabolism that allow efficient utilization of nutrient resources provided by animals. Similar to genes encoding specific virulence traits, genes encoding metabolic functions have been horizontally acquired by pathogens to provide a selective advantage in host tissues. Selective advantage in host tissues can also be gained by loss of function mutations that alter metabolic capabilities. Greater understanding of bacterial metabolism within host tissues should be important for increased understanding of host-pathogen interactions and the development of future therapeutic strategies.

Clinical Strains of Pseudomonas aeruginosa Overproducing MexAB-OprM and MexXY Efflux Pumps Simultaneously

ABSTRACT Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa. DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon. In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon. Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics. Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants). These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.

Molecular Characterization of an Epidemic Clone of Panantibiotic-Resistant Pseudomonas aeruginosa

ABSTRACT We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymorphism. Resistance mechanisms were characterized, including production of naturally occurring and acquired β-lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen isolates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo-β-lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively controlled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.

MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides

ABSTRACT Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, “adaptive” resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.

Role of the Multidrug Efflux System MexXY in the Emergence of Moderate Resistance to Aminoglycosides among Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis

ABSTRACT This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa. Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs. As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria. This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin. Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain. Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients. In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs. These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P. aeruginosa.

Involvement of the MexXY-OprM Efflux System in Emergence of Cefepime Resistance in Clinical Strains of Pseudomonas aeruginosa

ABSTRACT Cefepime (FEP) and ceftazidime (CAZ) are potent β-lactam antibiotics with similar MICs (1 to 2 μg/ml) for wild-type strains of Pseudomonas aeruginosa. However, recent epidemiological studies have highlighted the occurrence of isolates more resistant to FEP than to CAZ (FEPr/CAZs profile). We thus investigated the mechanisms conferring such a phenotype in 38 clonally unrelated strains collected in two French teaching hospitals. Most of the bacteria (n = 32; 84%) appeared to stably overexpress the mexY gene, which codes for the RND transporter of the multidrug efflux system MexXY-OprM. MexXY up-regulation was the sole FEP resistance mechanism identified (n = 12) or was associated with increased levels of pump MexAB-OprM (n = 5) or MexJK (n = 2), synthesis of secondary β-lactamase PSE-1 (n = 10), derepression of cephalosporinase AmpC (n = 1), coexpression of both OXA-35 and MexJK (n = 1), or production of both PSE-1 and MexAB-OprM (n = 1). Down-regulation of the mexXY operon in seven selected strains by the plasmid-borne repressor gene mexZ decreased FEP resistance from two- to eightfold, thereby demonstrating the significant contribution of MexXY-OprM to the FEPr/CAZs phenotype. The six isolates of this series that exhibited wild-type levels of the mexY gene were found to produce β-lactamase PSE-1 (n = 1), OXA-35 (n = 4), or both PSE-1 and OXA-35 (n = 1). Altogether, these data provide evidence that MexXY-OprM plays a major role in the development of FEP resistance among clinical strains of P. aeruginosa.

Pseudomonas aeruginosa May Accumulate Drug Resistance Mechanisms without Losing Its Ability To Cause Bloodstream Infections

ABSTRACT In this study, we systematically investigated the resistance mechanisms to β-lactams, aminoglycosides, and fluoroquinolones of 120 bacteremic strains of Pseudomonas aeruginosa. Pulsed-field gel electrophoresis genotyping showed that 97 of these strains were represented by a single isolate, 10 by 2 and 1 by 3 clonally related isolates, respectively. Seventy-five percent (90 out of 120) of the bacteremic P. aeruginosa strains displayed a significant resistance to one or more of the tested antimicrobials (up to 11 for 1 strain). These strains were found to harbor a great diversity of resistance mechanisms (up to 7 in 1 strain), leading to various levels of drug resistance. Interestingly, 11 and 36% of the isolates appeared to overproduce the MexAB-OprM and MexXY-OprM efflux systems, respectively. Altogether, our results show that P. aeruginosa may accumulate intrinsic (overproduction of cephalosporinase AmpC, increased drug efflux, fluoroquinolone target mutations, and deficient production of porin OprD) and exogenous (production of secondary β-lactamases and aminoglycoside-modifying enzymes) resistance mechanisms without losing its ability to generate severe bloodstream infections. Consequently, clinicians should be aware that multidrug-resistant P. aeruginosa may remain fully pathogenic.

Genetic and Phenotypic Variations of a Resistant Pseudomonas aeruginosa Epidemic Clone

ABSTRACT From May 1997 to December 2001, a serotype O:6 multidrug-resistant strain of Pseudomonas aeruginosa colonized or infected 201 patients in the University Hospital of Besançon (France). The susceptibility profile of this epidemic clone to fluoroquinolones and aminoglycosides was relatively stable during the outbreak but showed important isolate-to-isolate variations (up to 64-fold) in the MICs of β-lactams. Analysis of 18 genotypically related isolates selected on a quaterly basis demonstrated alterations in the two DNA topoisomerases II and IV (Thr83→Ile in GyrA and Ser87→Leu in ParC) and production of an ANT(2)-I enzyme. Although constitutively overproduced in these bacteria, the MexXY efflux system did not appear to contribute significantly to aminoglycoside resistance. β-Lactam resistance was associated with derepression of intrinsic AmpC β-lactamase (with isolate-to-isolate variations of up to 58-fold) and sporadic deficiency in a 46-kDa protein identified as the carbapenem-selective porin OprD. Of the 18 isolates, 14 were also found to overproduce the efflux system MexAB-OprM as a result of alteration of the repressor protein MexR (His107→Pro). However, complementation experiments with the cloned mexR gene demonstrated that MexAB-OprM contributed only marginally to β-lactam and fluoroquinolone resistance. Of the four isolates exhibiting wild-type MexAB-OprM expression despite the MexR alteration, two appeared to harbor secondary mutations in the mexA-mexR intergenic region and one harbored secondary mutations in the putative ribosome binding site located upstream of the mexAB oprM operon. In conclusion, this study shows that many mechanisms were involved in the multiresistance phenotype of this highly epidemic strain of P. aeruginosa. Our results also demonstrate that the clone sporadically underwent substantial genetic and phenotypic variations during the course of the outbreak, perhaps in relation to local or individual selective drug pressures.

Cumulative Effects of Several Nonenzymatic Mechanisms on the Resistance of Pseudomonas aeruginosa to Aminoglycosides

ABSTRACT Screening of a Tn5-Hg insertional library (12,000 clones) constructed in wild-type Pseudomonas aeruginosa strain PAO1 identified four genes (namely, galU, nuoG, mexZ, and rplY) whose disruption individually led to increased resistance to aminoglycosides (means of twofold). Inactivation of these genes was associated with (i) impaired outer membrane uptake, (ii) reduced active transport, (iii) increased MexXY-OprM-mediated active efflux, and (iv) alteration of target of aminoglycosides, respectively. In addition, suppression of the gene rplY, which codes for ribosomal protein L25, was found to result in both moderate upregulation of the efflux system MexXY-OprM and hypersusceptibility to β-lactam antibiotics. Construction of double, triple, and quadruple mutants demonstrated cumulative effects of the different mechanisms on aminoglycoside resistance, with MICs increasing from 16- to 64-fold in the quadruple mutant compared to the wild-type strain PAO1. Altogether, these results illustrate how P. aeruginosa may gradually develop high resistance to these antibiotics via intrinsic (i.e., nonenzymatic) mechanisms, as in cystic fibrosis patients.

Mutations in PA3574 (nalD) Lead to Increased MexAB-OprM Expression and Multidrug Resistance in Laboratory and Clinical Isolates of Pseudomonas aeruginosa

ABSTRACT Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-à-vis MexAB-OprM overexpression in these isolates.