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.

dltA overexpression: A strain-independent keystone of daptomycin resistance in methicillin-resistant Staphylococcus aureus.

The mechanisms leading to reduced susceptibility to daptomycin (DAP) are multifactorial and have not been fully elucidated. We analysed, by sequencing and expression studies, the role of the major molecular targets (cell-envelope charge genes, dltA, mprF, cls2; cell-wall turnover and autolysis genes, sceD, atl) involved in the emergence of DAP resistance in three series of isogenic clinical methicillin-resistant Staphylococcus aureus (MRSA) in which DAP resistance emerged after a heterogeneous glycopeptide-intermediate S. aureus (hGISA) step under teicoplanin and DAP therapy. All of the isolates had different genotypes and were δ-haemolysin negative, reflecting a strain proclivity to acquire DAP/glycopeptide non-susceptibility under antibiotic pressure. DAP exposure led to the emergence of DAP resistance after an hGISA step probably in parallel with the timing of the two antimicrobial administrations and, in two of three cases, in conditions of DAP underdosage. Real-time qPCR data revealed that all DAP-resistant (DAP-R) isolates had dltA overexpression, whereas mprF upregulation was found only in DAP-R strains with the S295L and T345I amino acid substitutions. Strains that were heteroresistant to DAP did not possess DAP-R-like characteristics. DAP-R strains presented high cls2 expression and no known cls2 mutations, and moreover exhibited sceD and atl upregulation. In conclusion, these findings highlight that dltA overexpression is the common pathway of resistance among genotypically different series of isolates and may represent the keystone of DAP resistance in MRSA, leading to electrostatic repulsion and, indirectly, to a reduction of autolysin activity. mprF mutations related to increased transcription may play a role in this complex phenomenon.

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

INTRODUCTION: Staphylococcus aureus 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 S.aureus infections were traditionally limited to hospitals, community-associated cases of methicillin-resistant S.aureus infections have been reported. In our case, we observed an unexpected event during treatment. CASE PRESENTATION: A 60-year-old Caucasian man developed fever and multiple muscle and brain abscesses caused by Panton-Valentine leukocidin-negative community-associated methicillin-resistant S. aureus. CONCLUSION: Although our patient was given antimicrobials active against the isolated methicillin-resistant S. aureus strain, it was only after the introduction of daptomycin that his skin, soft tissue and muscle lesions and also brain manifestations improved.

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.

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

The prevalence and molecular characterisation of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) strains were determined in a large group of Italian strains isolated between 2005 and mid 2007. Amongst the 1284 strains isolated from documented infections in hospitalised patients (bloodstream infection, pneumonia, and skin and skin-structure infections), 139 S. aureus with vancomycin minimum inhibitory concentrations (MICs) between 1 mg/L and 2 mg/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. Amongst the three screening methods used, the macro Etest (MET) demonstrated 100% specificity and 75% sensitivity. hVISA strains were accessory gene regulator (agr) 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 amongst MRSA isolates with MICs between 1 mg/L and 2 mg/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.

Tigecycline inhibition of a mature biofilm in clinical isolates of Staphylococcus aureus : comparison with other drugs.

The purpose of our study was to evaluate the anti-staphylococcal biofilm activity of tigecycline, compared with a group of recently developed or commonly used antimicrobials such as linezolid, daptomycin, levofloxacin, tobramycin and rifampin, all possessing putative antibiofilm properties, on a sample of multi-drug-resistant methicillin-resistant Staphylococcus aureus grown as a planktonic and mature biofilm. We determined conventional minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for the planktonic forms, MICs of adherent cells and finally, minimum biofilm eradication concentrations (MBECs). No drug was able to inhibit adherent bacteria at the same concentration necessary for eradicating a mature biofilm; the latter concentrations varied from three to seven times higher than the ones inhibiting adhesion. The concentrations eradicating biofilm were reached by rifampin and daptomycin at lower concentrations with respect to the other antibiotics tested; tigecycline was able to inhibit mature biofilms at higher concentrations, while all the other antibiotics were only able to inhibit adhering cells.

The apoptotic machinery as a biological complex system: analysis of its omics and evolution, identification of candidate genes for fourteen major types of cancer, and experimental validation in CML and neuroblastoma.

BACKGROUND: Apoptosis is a critical biological phenomenon, executed under the guidance of the Apoptotic Machinery (AM), which allows the physiologic elimination of terminally differentiated, senescent or diseased cells. Because of its relevance to BioMedicine, we have sought to obtain a detailed characterization of AM Omics in Homo sapiens, namely its Genomics and Evolution, Transcriptomics, Proteomics, Interactomics, Oncogenomics, and Pharmacogenomics. METHODS: This project exploited the methodology commonly used in Computational Biology (i.e., mining of many omics databases of the web) as well as the High Throughput biomolecular analytical techniques. RESULTS: In Homo sapiens AM is comprised of 342 protein-encoding genes (possessing either anti- or pro-apoptotic activity, or a regulatory function) and 110 MIR-encoding genes targeting them: some have a critical role within the system (core AM nodes), others perform tissue-, pathway-, or disease-specific functions (peripheral AM nodes). By overlapping the cancer type-specific AM mutation map in the fourteen most frequent cancers in western societies (breast, colon, kidney, leukaemia, liver, lung, neuroblastoma, ovary, pancreas, prostate, skin, stomach, thyroid, and uterus) to their transcriptome, proteome and interactome in the same tumour type, we have identified the most prominent AM molecular alterations within each class. The comparison of the fourteen mutated AM networks (both protein- as MIR-based) has allowed us to pinpoint the hubs with a general and critical role in tumour development and, conversely, in cell physiology: in particular, we found that some of these had already been used as targets for pharmacological anticancer therapy. For a better understanding of the relationship between AM molecular alterations and pharmacological induction of apoptosis in cancer, we examined the expression of AM genes in K562 and SH-SY5Y after anticancer treatment. CONCLUSION: We believe that our data on the Apoptotic Machinery will lead to the identification of new cancer genes and to the discovery of new biomarkers, which could then be used to profile cancers for diagnostic purposes and to pinpoint new targets for pharmacological therapy. This approach could pave the way for future studies and applications in molecular and clinical Medicine with important perspectives both for Oncology as for Regenerative Medicine.

agr-Genotyping and transcriptional analysis of biofilm-producing Staphylococcus aureus.

We investigated the correlation between biofilm production and the accessory-gene-regulator (agr) in 29 strains isolated from catheter-associated infections compared to a control group (30 isolates). All strains were tested for their ability to produce biofilm in a static system, and their agr genotype was determined. ScaI-restriction fragment length polymorphism for agr-typing showed that strong biofilm-producing strains belong to agr-type II. We found two new agr-variants, and sequence analysis of the three PCR products revealed the insertion of IS256 within the agr-locus. Biofilm production was assessed and correlated with agr functionality, with the expression of the ica-operon and of two transcriptional regulators, sarA and rsbU. Our data show that agr-II strains produce large amounts of biofilm, possess a defective agr-system show early transcription of icaA and are defective in haemolysin activity, icaR transcription, and in the expression of the sigma(B) activator rsbU. Strains with agrIII are medium biofilm producers, have an inactive agr-system, but express icaAR and rsbU in the late- and postexponential growth phases. In agrI-IV- and -IA-variants, medium or weak biofilm production was found. In these strains, the agr-locus was fully functional, rsbU-icaR and icaA were found in the late- and/or postexponential phases. Biofilm production was not affected by sarA.