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In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC beta-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L(-1) tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled beta-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginosa.

In this study, we assessed by broth microdilution the in vitro activity of oritavancin, a semisynthetic lipoglycopeptide currently under development, against selected meticillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates (n=56) from Geneva University Hospital, Switzerland, displaying a wide range of vancomycin minimum inhibitory concentrations (MICs) (0.25-4 microg/mL). The MRSA resistance phenotype was confirmed by broth microdilution (oxacillin MIC > or = 4 microg/mL) for all isolates; 89% and 100% of the tested isolates were also resistant to erythromycin and ciprofloxacin, respectively. For 53 MRSA isolates for which vancomycin MICs were in the susceptible range (0.5-2.0 microg/mL), the oritavancin MICs ranged from 0.03 microg/mL to 0.5 microg/mL. For these 53 vancomycin-susceptible isolates, the cumulative distribution of oritavancin MICs was markedly different from those of vancomycin, teicoplanin, daptomycin and linezolid MICs, yielding an oritavancin MIC for 90% of the organisms (MIC(90)) (0.25 microg/mL) that was four times lower than the MIC(90) values (1 microg/mL) of comparators. For three MRSA isolates with a vancomycin-intermediate phenotype (vancomycin MIC=4 microg/mL), oritavancin MICs (0.5-1.0 microg/mL) were 2-4-fold lower than vancomycin, teicoplanin or daptomycin MICs, but were equivalent to linezolid MICs. Pairwise comparison for each bloodstream isolate showed that oritavancin was > or =4-fold more active than vancomycin, teicoplanin and daptomycin, against 86%, 75% and 59% of all MRSA isolates, respectively.

Endogenous, low-level glycopeptide resistance in Staphylococcus aureus results from multifactorial genetic changes. Comparative genomic hybridization analysis revealed the specific deletion of a 1.8-kb segment encompassing two adjacent open reading frames (ORFs) of unknown function in a teicoplanin-susceptible revertant (strain 14-4rev) compared to the sequence of its isogenic, teicoplanin-resistant parental strain, strain 14-4. This provocative finding prompted us to perform a detailed genetic analysis of the contribution of this genomic segment to glycopeptide resistance. Despite repeated efforts in our laboratory, 14-4 and 14-4rev have proven refractory to most genetic manipulations. To circumvent this difficulty, we evaluated the contribution of both putative ORFs (designated teicoplanin resistance factors trfA and trfB ) on teicoplanin resistance in a different, genetically tractable background. Genetic analysis showed that single or double trfA and/or trfB mutations abolished teicoplanin resistance in two independent teicoplanin-resistant derivatives of NCTC8325 strain ISP794 generated by two-step passages with the drug. The frequency of teicoplanin-resistant mutants was markedly decreased by the absence of trfAB in the teicoplanin-susceptible ISP794 background. Nevertheless, a low rate of teicoplanin-resistant mutants was selected from ISP794 trfAB , thus indicating an additional contribution of trfAB -independent pathways in the emergence of low-level glycopeptide resistance. Further experiments performed with clinical glycopeptide-intermediate S. aureus isolate NRS3 indicated that the trfAB mutation could affect not only teicoplanin resistance but also vancomycin and oxacillin resistance. In conclusion, our study demonstrates the key role of two novel loci in endogenous, low-level glycopeptide resistance in S. aureus whose precise molecular functions warrant further investigation.

Several reports indicate the emergence of subpopulations resistant to glycopeptides in some clinical isolates of Staphylococcus aureus. While the development of glycopeptide resistance in S. aureus is easily observed in vitro, the in vivo conditions promoting emergence of glycopeptide-resistant subpopulations are unknown. Using a rat model, subcutaneous implants were chronically infected with a methicillin-resistant strain of S. aureus, MRGR3, devoid of a significant (>10(-7)) glycopeptide-resistant subpopulation at 2 mg/L of either teicoplanin or vancomycin. After 3 weeks of infection in antibiotic-untreated animals, subpopulations emerged, growing on agar containing 10 mg/L of either glycopeptide. These subpopulations were detected in all tissue cage fluids containing >7 log cfu/mL at average frequencies of 4 x 10(-5) and 2 x 10(-5) on teicoplanin- and vancomycin-containing agar, respectively. While teicoplanin MICs increased two- to 16-fold, vancomycin MICs increased by less than two-fold. Population analysis and survival kinetic studies of three teicoplanin-selected subclones indicated that transfer from solid to liquid medium conditions decreased expression of teicoplanin resistance in the bacterial population. In Mueller-Hinton broth, >90% of cells remained fully resistant to antibiotic, but did not grow in the presence of teicoplanin for an initial period of at least 6 h. All three teicoplanin-resistant subclones expressed stable teicoplanin resistance with slight cross-resistance to vancomycin after a few transfers on teicoplanin-supplemented agar. These data suggest that some in vivo conditions may lead to selection of S. aureus subpopulations exhibiting decreased glycopeptide susceptibility and growing in the presence of otherwise inhibitory concentrations of these antimicrobial agents.

We used a well-characterized isogenic set of clinical bloodstream Staphylococcus aureus strains to study (i) regulation of mprF -mediated phosphatidylglycerol lysinylation in the contexts of in vitro daptomycin (DAP) nonsuceptibility and (ii) the role of mprF mutation in endovascular virulence. We observed a correlation between increased expression of a mutant mprF gene and reduced in vitro DAP susceptibility. There were no detectable fitness differences between strains in experimental infective endocarditis.

Background/Aims: Endogenous development of glycopeptide-intermediate resistance is linked to multiple genetic and phenotypic changes in clinical and laboratory isolates of Staphylococcus aureus. This study evaluated endocytic uptake and intracellular survival of a teicoplanin-resistant derivative of S. aureus in a human epithelial cell line, and compared these to the isogenic teicoplanin-susceptible parent or a spontaneously derived, susceptible revertant. Methods: Endocytic uptake of teicoplanin-resistant and teicoplanin-susceptible strains by human embryonic kidney 293 cells was estimated by a lysostaphin protection assay. Differential intracellular survival of all S. aureus strains from 2 to 24 h was evaluated by colony-forming unit counts of Triton X-100-lysed 293 cells, following lysostaphin inactivation. Results: Endocytic uptake of the teicoplanin-resistant strain increased by approximately 4-fold over its teicoplanin-susceptible counterparts. Furthermore, the teicoplanin-resistant strain showed an 11-fold increase in intracellular colony-forming unit counts from 2 to 24 h, compared to its teicoplanin-susceptible counterparts that showed marginal (Conclusions: Intracellular location might confer a significant fitness benefit to glycopeptide-intermediate isolates of methicillin-resistant S. aureus and further protect them from cell wall-active antibiotics whose intracellular activity is limited.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting intermediate susceptibility to glycopeptides (GISA) is challenging for clinical microbiology laboratories. We compared three different screening assays for evaluating trends in decreased glycopeptide susceptibility during two periods. Ninety four and ninety five consecutive MRSA blood isolates from 189 bacteremic patients collected during periods A (1989-1994) and B (1999-2001), respectively, were screened in parallel for vancomycin or teicoplanin susceptibility by glycopeptide-containing brain-heart infusion agar (BHIA) tests, Etest MICs performed at a standard (0.5 McFarland) or high (2.0 McFarland) inoculum on Mueller-Hinton (MHA) or BHIA, respectively. Any MRSA isolate yielding 50 CFU on either V2-BHIA or T5-BHIA significantly (P

Alcohol-based gels have been introduced recently in many hospitals worldwide for hand antisepsis. We investigated the antimicrobial efficacy of ten gels and four rinses according to European standards (EN 1500). No gel met the EN 1500 requirements within 30 s of application, whereas all hand rinses did. The tested hand gels should be considered a retrograde step for hand hygiene because the application time in clinical practice is often shorter than 30 s; they should not replace alcohol-based liquid hand disinfectants currently used in hospitals or be implemented as first choice agents.

Teicoplanin resistance was transformed from a teicoplanin-resistant Staphylococcus aureus into the susceptible strain BB255 to give strain BB938. The cell wall composition, amidation of the iD-glutamate, and peptide crosslinking were identical in BB938 as in BB255 except for a 60% increased length of the glycan chain. Transductional crosses revealed that at least two distinct loci contributed in a cumulative fashion to teicoplanin resistance. One of these loci correlated with a mutation inactivating the anti-sigma factor RsbW. This mutation must have occurred during transformation and selection for teicoplanin resistance in BB938. Genetic manipulations involving the sigB operon showed that transcription factor SigB contributed to decreased teicoplanin susceptibility.

In order to characterize the impact and pattern of Gram-negative bacteraemia (GNB) at a Swiss University hospital and to assess the effect of multi-resistance on mortality, we conducted a 6-y retrospective cohort study using linear regression and multivariate Cox-proportional hazard analysis. 1766 patients had 1835 episodes of GNB; 61% were community-acquired. The incidence of GNB increased linearly (r2 = 0.90, p = 0.014) from 7.07 episodes to 8.32 episodes per 1000 admissions, but this trend was no longer significant after adjustment for the number of blood cultures drawn/y. The in-hospital mortality for patients with GNB decreased from 20% in 1989 to 16% in 1994 (r2 = 0.94, p = 0.005). The risk ratio for death remained unchanged over the study period and was 7-fold higher for patients with GNB than for patients without GNB. Factors independently associated with an increased hazard of death after GNB were: severity of illness as measured by exposure to intensive care (hazard ratio [HR], 1.5); age = 66-79 y (HR 1.8); GNB due to Klebsiella spp. (HR 1.7) or Pseudomonas aeruginosa (HR 1.6); and polymicrobial infection (HR 1.6). Multi-resistance was not associated with an increased risk of death (HR 1.0). Although the crude mortality of GNB decreased, the population-attributable risk ratio for death remained significant. These data suggest the absence of a major impact of multi-resistant GNB on patient mortality.

The influence of calcium and magnesium in free form on the sensitivity of Pseudomonas aeruginosa to aminoglycoside antibiotics was evaluated on 42 clinical isolates. When compared to the minimal inhibitory concentrations (MIC) measured in Mueller-Hinton broth - a medium almost devoid of Ca and Mg - the MIC obtained in the same medium, supplemented with calcium and magnesium to achieve physiological concentrations of the free divalent ions (50 micrograms/ml and 10 micrograms/ml respectively), increased by a mean factor of 8 for gentamicin. Consequently, the percentage of gentamicin resistant strains increased from 2 to 78% when tested in the supplemented medium containing physiological concentrations of free calcium and magnesium. Similar increases in MIC were observed for tobramycin and amikacin. These results cast doubt on the clinical relevance of antibiotic disc sensitivities performed on Mueller-Hinton agar by the Kirby-Bauer technique. Moreover, they could explain some of the therapeutic failures observed with Pseudomonas aeruginosa strains falsely reported as sensitive to aminoglycosidic antibiotics.