Different Modulatory Effects of Four Methicillin-Resistant Staphylococcus aureus Clones on MG-63 Osteoblast-Like Cells.

Staphylococcus aureus is a Gram-positive bacterium responsible for a variety of mild to life-threatening infections including bone infections such as osteomyelitis. This bacterium is able to invade and persist within non-professional phagocytic cells such as osteoblasts. In the present study, four different S. aureus strains, namely, 2SA-ST239-III (ST239), 5SA-ST5-II (ST5), 10SA-ST228-I (ST228), and 14SA-ST22-IVh (ST22), were tested for their ability to modulate cell viability in MG-63 osteoblast-like cells following successful invasion and persistence. Methicillin-sensitive S. aureus (MSSA) ATCC-12598-ST30 (ST30) was used as control strain. Despite being proven that ST30, ST239, and ST22 have a similar ability to internalize and persist in MG-63 osteoblast-like cells under our experimental conditions, we demonstrated that the observed decrease in cell viability was due to the different behavior of the considered strains, rather than the number of intracellular bacteria. We focused our attention on different biochemical cell functions related to inflammation, cell metabolism, and oxidative stress during osteoblast infections. We were able to show the following: (1) ST30 and ST239 were the only two clones able to persist and maintain their number in the hostile environment of the cell during the entire period of infection; (2) ST239 was the only clone able to significantly increase gene expression (3 and 24 h post-infection (p.i.)) and protein secretion (24 h p.i.) of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in MG-63 osteoblast-like cells; (3) the same clone determined a significant up-regulation of the transforming growth factorbeta 1 (TGF-ß1) and of the metabolic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs at 24 h p.i.; and (4) neither the MSSA nor the four methicillin-resistant S. aureus (MRSA) strains induced oxidative stress phenomena in MG-63 cells, although a high degree of variability was observed for the different clones with regard to the expression pattern of nuclear factor E2-related factor 2 (Nrf2) and its downstream gene heme oxygenase 1 (HO-1) activation. Our results may pave the way for an approach to S. aureus-induced damage, moving towards individualized therapeutic strategies that take into account the differences between MSSA and MRSA as well as the distinctive features of the different clones. This approach is based on a change of paradigm in antibiotic therapy involving a case-based use of molecules able to counteract pro-inflammatory cytokines activity such as selective cytokine signaling inhibitors (IL-6, TNF-α).

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.

Staphylococcus haemolyticus endocarditis: clinical and microbiologic analysis of 4 cases.

Only 3 cases of infective endocarditis (IE) due to methicillin-resistant Staphylococcus haemolyticus (MRSH) have been reported in English literature. Here we report 4 cases of IE due to MRSH encountered in a single university hospital. Population analysis of the strains was performed to assess the presence of vancomycin/teicoplanin heteroresistant subpopulations. Pulsed-field gel electrophoresis was used for molecular typing of isolates. IE was defined in 3 cases as health care associated, and in 1 case, as community acquired. A causative strain was lost. Two strains were heteroresistant to teicoplanin, and 1 also to vancomycin. Genome macrorestriction profile studies demonstrated that 2 MRSH isolates belonged to clones A and E, possessing a class C1 mecDNA, whereas 1 clone was sporadic. All patients were treated with vancomycin plus rifampin. Two patients were cured with antibiotic therapy alone, 1 patient needed surgery, and 1 patient died. Methicillin-resistant multiresistant S. haemolyticus may represent a difficult-to-treat cause of both community and nosocomially acquired IE.

Identification of a variant "Rome clone" of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit.

We describe the identification of a variant of the "Rome clone" of methicillin-resistant Staphylococcus aureus (MRSA), responsible for an outbreak involving 5 patients in a Cardiac Surgery Intensive Care Unit (CS-ICU) of a tertiary-care University Hospital in Rome. All strains isolated from patients and from nasal swabs obtained from four members of the CS-ICU personnel, belonged to the same identified clone. The characteristics of this clone were: (1) resistance to ampicillin, oxacillin, gentamicin, ciprofloxacin, erythromycin, clindamycin, rifampin, spectinomycin, and tetracycline; (2) vancomycin and teicoplanin MICs respectively of 2 and 4 mg/L; (3) heteroresistant subpopulations in the presence of 4 and 6 mg/L of vancomycin (10(-3) and 10(-5), respectively); (4) clonal type I::J::C determined following an established protocol (mec A::Tn 554 ::PFGE); (5) sequence type ST247 (3-3-1-12-4-4-16), obtained by multilocus sequence typing (MLST); and (6) the staphylococcal cassette chromosome mec (SCC) IA, obtained by multiplex PCR method. This new strain had different characteristics from the epidemic clone circulating in the same hospital from 1997 and designed "Rome clone," which was susceptible to erythromycin, clindamycin, and spectinomycin and belonged to the II::NH::C genetic background. A high genetic similarity between this Rome clone and the previously classified Archaic and Iberian clones was found, because they shared the same allelic profile (ST247), probably originating from the same S. aureus ancestor of the Iberian MRSA strains. Therefore, the strains responsible for the outbreak, with vancomycin MICs 2-4 mg/L, are variant clones, showing the genotype of the "Rome clone," the ST247 in association with SCC mec type IA (ST247-MRSA-IA), and are characterized by a uniform susceptibility to fosfomycin.