Elzbieta Huggler

Evidence of an Intracellular Reservoir in the Nasal Mucosa of Patients with Recurrent Staphylococcus aureus Rhinosinusitis

Severe infections due to Staphylococcus aureus require prolonged therapy for cure, and relapse may occur even years after the first episode. Persistence of S. aureus may be explained, in part, by nasal carriage of S. aureus, which occurs in a large percentage of healthy humans and represents a major source of systemic infection. However, the persistence of internalized S. aureus within mucosal cells has not been evaluated in humans. Here, we provide the first in vivo evidence of intracellular reservoirs of S. aureus in humans, which were assessed in endonasal mucosa specimens from patients suffering from recurrent S. aureus rhinosinusitis due to unique, patient-specific bacterial clonotypes. Heavily infected foci of intracellular bacteria located in nasal epithelium, glandular, and myofibroblastic cells were revealed by inverted confocal laser scan fluorescence and electron microscopic examination of posttherapy intranasal biopsy specimens from symptom-free patients undergoing surgery on the sinuses. Intracellular residence may provide a sanctuary for pathogenic bacteria by protecting them from host defense mechanisms and antibiotic treatment during acute, recurrent S. aureus rhinosinusitis.

Role of host and bacterial factors in modulating staphylococcal adhesion to implanted polymer surfaces

The aim of this paper is to describe some of the major interactions between host and bacterial factors, as promoters of adhesion and colonization of polymeric implants/devices by staphylococci

Transcriptomic and Functional Analysis of an Autolysis-Deficient, Teicoplanin-Resistant Derivative of Methicillin-Resistant Staphylococcus aureus

ABSTRACT The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

Underestimation of Vancomycin and Teicoplanin MICs by Broth Microdilution Leads to Underdetection of Glycopeptide-Intermediate Isolates of Staphylococcus aureus

ABSTRACT Broth microdilution was compared with tube macrodilution and a simplified population analysis agar method for evaluating vancomycin and teicoplanin MICs and detecting glycopeptide-intermediate isolates of Staphylococcus aureus. Modal vancomycin and teicoplanin MICs recorded by tube macrodilution and the agar plate assay, which both used inocula of 106 CFU, were significantly higher (2 μg/ml) against a panel of borderline glycopeptide-susceptible and glycopeptide-intermediate methicillin-resistant S. aureus (MRSA) bloodstream isolates compared to broth microdilution (1 μg/ml). Vancomycin and teicoplanin MIC distributions by tube macrodilution and agar testing were also markedly different from those evaluated by broth microdilution. The 20-fold-lower inoculum size used for broth microdilution compared to macrodilution and agar MIC assays explained in part, but not entirely, the systematic trend toward lower vancomycin and teicoplanin MICs by microdilution compared to other methods. Broth microdilution assay led to underdetection of the vancomycin-intermediate S. aureus (VISA) phenotype, yielding only three VISA isolates, for which vancomycin MICs were 4 μg/ml compared to 8 and 19 VISA isolates detected by macrodilution and agar testing, respectively. While macrodilution and agar testing detected 7 and 22 isolates with elevated teicoplanin MICs (8 μg/ml), respectively, broth microdilution failed to detect such isolates. Detection rates of isolates with elevated vancomycin and teicoplanin MICs by macrodilution and agar testing assays were higher at 48 h than at 24 h. In conclusion, the sensitivity of broth microdilution MIC testing is questionable for reliable detection and epidemiological surveys of glycopeptide-intermediate resistance in S. aureus isolates.

Identification by Genomic and Genetic Analysis of Two New Genes Playing a Key Role in Intermediate Glycopeptide Resistance in Staphylococcus aureus

ABSTRACT 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.

High Prevalence of Isolates with Reduced Glycopeptide Susceptibility in Persistent or Recurrent Bloodstream Infections Due to Methicillin-Resistant Staphylococcus aureus

ABSTRACT Reduced susceptibility to glycopeptides in methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates is considered a risk factor for failure of glycopeptide therapy. We compared the prevalences of MRSA isolates with reduced glycopeptide susceptibility in patients with versus without persistent or recurrent MRSA bloodstream infections. A retrospective cohort study at the University Hospital of Geneva identified 27 patients with persistent or recurrent clonally related MRSA bacteremic episodes over an 8-year period, which included 208 consecutive nosocomial MRSA bacteremic episodes. Vancomycin and teicoplanin MICs were determined by a modified macrodilution assay allowing improved detection of glycopeptide-intermediate MRSA isolates (GISA), characterized by elevated teicoplanin or/and vancomycin MICs (≥4 μg/ml). For 16 patients (59%), their pretherapy and/or posttherapy MRSA isolates showed elevated teicoplanin MICs, among which 10 (37%) concomitantly displayed elevated vancomycin MICs. In contrast, 11 other patients (41%) were persistently or recurrently infected with non-GISA isolates. In comparison, only 39 (22%) of 181 single isolates from patients with no microbiological evidence of persistent or recurrent infections showed elevated teicoplanin MICs, among which 14 (8%) concomitantly displayed elevated vancomycin MICs. Clinical, microbiological, and pharmacokinetic variables for patients persistently or recurrently infected with GISA or non-GISA isolates were similar. Bacteremic patients with a poor response to glycopeptide therapy had a 2.8-fold- and 4.8-fold-higher rates of MRSA isolates displaying elevated teicoplanin and vancomycin MICs, respectively, than patients with single isolates (P < 0.0001). Detection of elevated teicoplanin MICs may help to predict a poor response to glycopeptide therapy in MRSA bacteremic patients.

Impact of Ciprofloxacin Exposure on Staphylococcus aureus Genomic Alterations Linked with Emergence of Rifampin Resistance

ABSTRACT Intensive use of antimicrobial agents in health care settings not only leads to the selection of multiresistant nosocomial isolates of Staphylococcus aureus but may also promote endogenous, resistance-conferring mutations in bacterial genes that encode drug targets. We evaluated the spectrum of rifampin resistance-conferring mutations in cultures of methicillin-susceptible S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA) strains exposed in vitro to sub-MICs of ciprofloxacin. Growth of ciprofloxacin-susceptible MRSA strain MRGR3 and ciprofloxacin-resistant MSSA strain RA1 (a NCTC 8325 derivative) in the presence of 1/2× or 1/4× MIC of ciprofloxacin led to higher frequencies of rifampin-resistant mutants on agar supplemented with rifampin (0.25 mg/liter) than under ciprofloxacin-free conditions. While rifampin-resistant mutants from ciprofloxacin-free cultures essentially showed single-amino-acid substitutions, a significant proportion of rifampin-resistant mutants from ciprofloxacin-exposed cultures displayed in-frame deletions or insertions in the rpoB gene at several positions of the rifampin resistance cluster I. In-frame deletions or insertions were also recorded in rpoB cluster I of rifampin-resistant mutants from ciprofloxacin-exposed cultures of mutS and mutL DNA repair mutants of ciprofloxacin-resistant S. aureus strain RA1. Frequencies of rifampin-resistant mutants grown under ciprofloxacin-free medium were higher for mutant strains RA1 mutS2 and RA1 mutL, but not RA1 recA, than for their parent RA1. In conclusion, ciprofloxacin-mediated DNA damage in S. aureus, as exemplified by the wide diversity of deletions or insertions in rpoB, suggests the occurrence of major, quinolone-mediated disturbances in DNA fork progression and replication repair. Besides promoting antibiotic resistance, accumulation of unrepaired DNA replication errors, including insertions and deletions, may also contribute to potentially lethal mutations.

Screening for Staphylococcal Superantigen Genes Shows No Correlation with the Presence or the Severity of Chronic Rhinosinusitis and Nasal Polyposis

Background Staphylococcus aureus secretes numerous exotoxins which may exhibit superantigenic properties. Whereas the virulence of several of them is well documented, their exact biological effects are not fully understood. Exotoxins may influence the immune and inflammatory state of various organs, including the sinonasal mucosa: their possible involvement in chronic rhinosinusitis has been suggested and is one of the main trends in current research. The aim of this study was to investigate whether the presence of any of the 22 currently known staphylococcal exotoxin genes could be correlated with chronic rhinosinusitis. Methodology/Principal Findings We conducted a prospective, multi-centred European study, analysing 93 Staphylococcus aureus positive swabs taken from the middle meatus of patients suffering from chronic rhinosinusitis, with or without nasal polyposis, and controls. Strains were systematically tested for the presence of the 22 currently known exotoxin genes and genotyped according to their agr groups. No direct correlation was observed between chronic rhinosinusitis, with or without nasal polyposis, and either agr groups or the presence of the most studied exotoxins genes (egc, sea, seb, pvl, exfoliatins or tsst-1). However, genes for enterotoxins P and Q were frequently observed in nasal polyposis for the first time, but absent in the control group. The number of exotoxin genes detected was not statistically different among the 3 patient groups. Conclusions/Significance Unlike many previous studies have been suggesting, we did not find any evident correlation between staphylococcal exotoxin genes and the presence or severity of chronic rhinosinusitis with or without nasal polyposis.

Differential effects on neutrophil activation of staurosporin and its protein kinase C-selective derivative cgp 41231

The relative insensitivity of the chemoattractant-induced respiratory burst to non-specific kinase inhibitors, such as staurosporin, is widely considered as evidence against the involvement of protein kinase C in signal transduction by chemoattractants. In this study we compared the effect on neutrophil activation of the non-specific kinase inhibitor staurosporin with the effect of its protein kinase C-selective derivative cgp 41251. Staurosporin activates secondary granule release by itself and enhances chemoattractant-induced primary granule release; it inhibits superoxide production in response to phorbol esters at low concentrations, but superoxide production in response to chemoattractants only at considerably higher concentrations. In contrast, cgp 41251 did not interfere with granule release, but inhibited phorbol ester- and chemoattractant-induced superoxide production with similar potency. These results suggest that many of the staurosporin effects, including its low potency to inhibit chemoattractant-induced superoxide production, are due to protein kinase C-independent effects. The results obtained with cgp 41251 are compatible with a role of protein kinase C in the mediation of the chemoattractant-induced respiratory burst of human neutrophils.

Successful single-dose prophylaxis of Staphylococcus aureus foreign body infections in guinea pigs by fleroxacin.

Single-dose administration of fleroxacin was evaluated as a means of preventing foreign body infection due to staphylococci. Tissue cages were implanted into guinea pigs and subsequently infected (100% rate) with 10(2) or more CFU of Staphylococcus aureus Wood 46. When a single dose of 30 mg of fleroxacin or vancomycin per kg of body weight was administered intraperitoneally, bactericidal levels of the antimicrobial agent were found in the tissue cage fluid after 3 h (when guinea pigs were inoculated with S. aureus) and during the next 24 h. Either fleroxacin or vancomycin successfully prevented experimental infection in all tissue cages challenged by 10(2) CFU of S. aureus Wood 46. When tissue cages were challenged with 10(4) CFU of S. aureus Wood 46, however, fleroxacin was more effective than vancomycin (P less than 0.05) in reducing colony counts below the detection limit of 10 CFU/ml in the inflammatory fluid of all tissue cages during the initial 48 h. In contrast to their initially different actions, the effects of the antibiotics were similar after 7 days, mostly because bacterial regrowth occurred more frequently in the fleroxacin-treated than in the vancomycin-treated tissue cages. These data show that experimental infections of subcutaneous tissue cages are a useful model for studying the prophylaxis of foreign body infections with antimicrobial agents. Images