Viviana Cafiso

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.

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.

DNA microarray-based characterisation of Panton–Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from Italy

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to collect and characterise the CA-MRSA isolates circulating in Italy, since only some case reports have been published. Eighteen Panton–Valentine-positive CA-MRSA isolates were collected from different Italian hospitals during the period 2005–2009 from severe infections (skin and soft tissue infections, n = 10; necrotising pneumonia, n = 7; and sepsis, n = 1). Accessory gene regulator (agr) typing, staphylococcal cassette chromosome (SCC) mec typing, spa typing, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and DNA microarray were applied to categorise isolates into clones and to compare the relevant genetic features of each clone. Six different clones were identified, the most common (7 out of 18 isolates, 38.8%) being agrI/ST8/SCCmecIV, corresponding to the USA300 clone. Six out of the seven USA300 isolates did not harbour the arginine catabolic mobile element (ACME). Four strains (22.2%) were agrIII/ST80/SCCmecIV, corresponding to the European clone. Two of the other clones, namely, agrIII/ST88/SCCmecV and agrIII/ST772/SCCmecV, corresponded to CA-MRSA clones rarely found in other countries and probably originating from Africa or the Indian subcontinent. The results of microarray hybridisations showed that the distribution of resistance genes and other virulence factors was specific to each clone. Some characteristics could be exploited as specific markers for a clone or a group of isolates, e.g. the mer operon, recovered only in ACME-negative USA300 strains. DNA microarray contributed to a more complete description of the variety of different CA-MRSA clones circulating in Italy.

Pathotype and susceptibility profile of a community-acquired methicillin-resistant Staphylococcus aureus strain responsible for a case of severe pneumonia

Recent articles have described an increasing number of infections due to Panton-Valentine leukocidin PVL-positive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) worldwide. We report a case of necrotizing pneumonia successfully treated with levofloxacin in Italy, sustained by a PVL-positive CA-MRSA, belonging to ST88 and carrying a staphylococcal chromosomal cassette mec type V. Further molecular characterization of isolates revealed that they were PVL positive, belonged to the agr IV allele, possessed a capsular type 8, and had an almost complete pathotype composed of leukocidins and numerous adhesins. In addition, the strains were strong biofilm producers.

Infections with VIM-1 Metallo-β-Lactamase-Producing Enterobacter cloacae and Their Correlation with Clinical Outcome

ABSTRACT The aim of this study was to ascertain the incidence and clinical significance of metallo-β-lactamases among Enterobacter strains isolated from patients with nosocomial infections. We prospectively collected data on patients with Enterobacter infection during a 13-month period. All of the strains were investigated for antibiotic susceptibility, the presence and expression of metallo-β-lactamases, and clonality. Of 29 infections (11 involving the urinary tract, 7 pneumonias, 3 skin/soft tissue infections, 3 intra-abdominal infections, 3 bacteremias, and 2 other infections), 7 (24%) were caused by Enterobacter cloacae strains harboring a blaVIM-1 gene associated or not with a blaSHV12 gene. Infections caused by VIM-1-producing strains were more frequently associated with a recent prior hospitalization (P = 0.006), cirrhosis (P = 0.03), relapse of infection (P < 0.001), and more prolonged duration of antibiotic therapy (P = 0.01) than were other infections. All of the isolates were susceptible to imipenem and meropenem and had blaVIM-1 preceded by a weak P1 promoter and inactivated P2 promoters. Most VIM-1-producing Enterobacter isolates belonged to a main clone, but four different clones were found. Multiclonal VIM-1-producing E. cloacae infections are difficult to diagnose due to an apparent susceptibility to various beta-lactams, including carbapenems, and are associated with a high relapse rate and a more prolonged duration of antibiotic therapy.

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.

Insights and clinical perspectives of daptomycin resistance in Staphylococcus aureus: A review of the available evidence

The emergence of glycopeptide reduced susceptibility and resistance in Staphylococcus aureus strains is a growing clinical problem that poses significant clinical challenges in treatment. Its development is a complex and novel process involving many subtle physiological changes in the micro-organism. Daptomycin is the first cyclic lipopeptide approved for clinical use. Unlike most other antimicrobials, a trend towards increased daptomycin resistance has not been reported, although several cases of daptomycin non-susceptibility have been reported. The present review will present the available evidence on daptomycin resistance of S. aureus, with particular attention to its development. In addition to a literature overview, we have compiled the reported cases of daptomycin non-susceptibility to shed light on possible clinical mechanisms of resistance. In the 36 reports describing 62 clinical cases, infections caused by meticillin-resistant S. aureus (MRSA) strains with a vancomycin minimum inhibitory concentration (MIC) between 1mg/L and 2mg/L often led to vancomycin treatment failure, which may be associated with the development of non-susceptibility to daptomycin. Additional evidence suggests that underdosage of daptomycin is an important clinical aspect that merits further study. The current analysis highlights the importance of determining the MIC when using vancomycin to treat patients with severe S. aureus infections and that when failure is suspected, testing for heterogeneous vancomycin-intermediate S. aureus (hVISA) may also be necessary. Whilst further investigation is needed, it can be hypothesised that MRSA strains become hVISA during prolonged bacteraemia, which may predispose to the development of daptomycin resistance.

Methicillin resistance and vancomycin heteroresistance in Staphylococcus aureus in cystic fibrosis patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are increasingly being reported among cystic fibrosis (CF) populations worldwide. In this paper, we sought to examine at the epidemiology, the molecular characterisation and the antibiotic resistance of MRSA isolates in our cohort of CF patients. All MRSA strains were collected prospectively at the University Hospital of Catania, Italy, during a two-year study between mid 2005 to mid 2007 and underwent molecular, pathotype and susceptibility characterisations. Our study demonstrates persisting infections with both hospital-associated (HA-) and community-associated (CA-)MRSA, including Panton–Valentine leukocidin (PVL)-positive strains, in our CF population with an overall prevalence of 7.8%. We demonstrated that, in these patients, persistence was sustained by either identical clones that underwent subtle changes in their toxin content or by different clones over time. The isolation of MRSA in our CF population aged 7–24 years was associated with an increased severity of the disease even if, due to the small sample of patients included and the paucity of data on the clinical outcome, these results cannot be conclusive. Furthermore, three strains were heteroresistant vancomycin-intermediate S. aureus (hVISA), questioning the use of glycopeptides in the treatment of MRSA infections in these patients.

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.

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.