Modulating Activity of Vancomycin and Daptomycin on the Expression of Autolysis Cell-Wall Turnover and Membrane Charge Genes in hVISA and VISA Strains

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF

Daptomycin efficacy in the central nervous system of a patient with disseminated methicillin-resistant Staphylococcus aureus infection: a case report

<p>Abstract</p> <p>Introduction</p> <p><it>Staphylococcus aureus</it> has emerged as a major nosocomial pathogen in the last decades and also represents the second most common pathogen isolated from patients in outpatient settings. Although methicillin-resistant <it>S.aureus</it> infections were traditionally limited to hospitals, community-associated cases of methicillin-resistant <it>S.aureus</it> infections have been reported. In our case, we observed an unexpected event during treatment.</p> <p>Case presentation</p> <p>A 60-year-old Caucasian man developed fever and multiple muscle and brain abscesses caused by Panton-Valentine leukocidin-negative community-associated methicillin-resistant <it>S. aureus</it>.</p> <p>Conclusion</p> <p>Although our patient was given antimicrobials active against the isolated methicillin-resistant <it>S. aureus</it> strain, it was only after the introduction of daptomycin that his skin, soft tissue and muscle lesions and also brain manifestations improved.</p

Heteroresistance to glycopeptides in Italian meticillin-resistant (MRSA) isolates

International audienceThe prevalence and molecular characterisation of heteroresistant vancomycin-intermediate (hVISA) strains were determined in a large group of Italian strains isolated between 2005 and mid 2007. Among the 1284 strains isolated from documented infections in hospitalised patients (bloodstream infection, pneumonia, and skin and skin-structure infections), 139 with vancomycin minimum inhibitory concentration (MICs) between 1mg/L and 2mg/L were screened for the presence of hVISA using three different methods and were confirmed by population analysis profile (PAP). Thirty-six hVISA strains (25.9%) were detected. Among the three screening methods used, the macro Etest (MET) demonstrated 100% specificity and 75% sensitivity. hVISA strains were accessory gene regulator () types I and II and belonged to the major nosocomial clones circulating in Italy (ST8, ST239, ST247 and ST228). All strains were susceptible to quinupristin/dalfopristin, linezolid, daptomycin, tigecycline and dalbavancin. In conclusion, we have demonstrated that hVISA isolates are common among MRSA isolates with MICs between 1mg/L and 2mg/L in Italy. MET, with its high sensitivity and specificity, should be used for early detection of hVISA, especially in patients with serious or prolonged infections sustained by MRSA. Finally, the most recent anti-Gram-positive drugs maintained their full spectrum of in vitro activity against these strains

Phenotypic and genotypic characterization of daptomycin-resistant methicillin-resistant Staphylococcus aureus strains: relative roles of mprF and dlt operons.

Development of in vivo daptomycin resistance (DAP-R) among Staphylococcus aureus clinical isolates, in association with clinical treatment failures, has become a major therapeutic problem. This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three well-characterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases (i.e., dysregulation of dltA and mprF), (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains (i.e., increased fluidity) may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms

Transcript analysis in drug-free conditions.

<p>(A) Relative quantitative expression of the <i>rna</i>III and <i>wal</i>K regulator genes and (B) of some autolytic (<i>atl</i>, <i>lyt</i>M), cell-wall turnover (<i>sce</i>D) and cell envelope charge genes (<i>mpr</i>F, <i>dlt</i>A) in hVISA and VISA. Statistically significant difference between sample <i>vs.</i> VSSA, p<0.05, are indicated with *.</p

Strains included in the study.

<p>Legend: V = Vancomycin, T = Teicoplanin, D = Daptomycin, S = Susceptible, R = Resistant.</p

δ-hemolysis, Autolysis Ratio, Percentage of Triton X-100 autolysis <i>vs.</i> VSSA.

<p>Legend:</p><p>*(++) large δ-hemolysis zone; (−/+) low δ-hemolysis zone; (−) no δ-hemolysis zone.</p><p>**measured as the T₀/T₅ O.D. ratio ± SD (Standard Deviation).</p

δ-hemolysis assay of prototype microrganisms.

<p>A) NRS149 (VSSA); B) Mu3 (hVISA); C) Mu50 (VISA).</p