Evaluation of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay as a method for detection of MRSA isolates, using a large collection of European and North African isolates.

Using a large collection of European and North African methicillin-resistant Staphylococcus aureus (MRSA) isolates with a variety of genetic backgrounds and staphylococcal cassette chromosome mec (SCCmec) types, we evaluated the reliability of the BD GeneOhm MRSA assay. Results revealed high performance of this test for detecting MRSA strains provided from Europe and North Africa (98.3%).

Ward-specific rates of nasal cocolonization with methicillin-susceptible and -resistant Staphylococcus spp. and potential impact on molecular methicillin-resistant Staphylococcus aureus screening tests.

We report that the rates of nasal cocolonization with methicillin-susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci can vary widely between patients admitted to different wards within a single hospital. Such cocolonization can greatly influence the performance of molecular methicillin-resistant S. aureus (MRSA) screening tests depending on the methods used and targets selected.

Evaluation of a commercial immunochromatographic assay for rapid routine identification of PBP2a-positive Staphylococcus aureus and coagulase-negative staphylococci.

We evaluated the performance of an immunochromatographic assay (PBP2a Culture Colony Test - Alere™), detecting protein-binding penicillin 2a on staphylococci primary isolates in only 6minutes. The assay is highly sensitive for the direct detection of MRSA on various culture media whereas it requires cefoxitin induction for methicillin-resistant coagulase-negative staphylococci.

Staphylococcus epidermidis in orthopedic device infections: the role of bacterial internalization in human osteoblasts and biofilm formation.

BACKGROUND: Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms. MATERIALS AND METHODS: Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method. RESULTS: No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/-631 and 347+/-431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups. CONCLUSION: Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.

PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts.

Epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is associated with more severe and acute forms of osteomyelitis than healthcare-associated (HA-) MRSA. Although S. aureus is now recognized as a facultative intracellular pathogen, the contribution of osteoblast invasion by CA-MRSA to the pathogenesis of osteomyelitis is unknown. Using an ex vivo model of intracellular infection of human osteoblasts, we demonstrated that CA-MRSA strains of diverse lineages share an enhanced ability to kill infected osteoblasts compared to HA-MRSA. Cytotoxicity comparisons of CA-MRSA isogenic deletion mutants revealed that phenol-soluble modulins (PSMs), a class of membrane-damaging exoproteins that are expressed at higher levels in CA-MRSA than in HA-MRSA, are involved in this osteoblast killing, whereas other major CA-MRSA virulence determinants, the Panton-Valentine leukocidin and alpha-toxin, are not involved. Similarly, functional agr and sarA regulators, which control the expression of PSMs and alpha-toxin, were required for the expression of the intracellular cytotoxic phenotype by CA-MRSA, whereas the saeRS regulator, which controls the expression of alpha-toxin but not PSMs, had no impact on cytotoxicity. Finally, PSM transcript levels determined by quantitative reverse-transcriptase PCR were significantly higher in CA-MRSA than in HA-MRSA strains and associated with cell damage in MRSA-infected osteoblasts. These findings provide new insights into the pathogenesis of severe CA-MRSA osteomyelitis and unravel a novel virulence strategy of CA-MRSA, based on the invasion and subsequent killing of osteoblasts by PSMs acting as intracellular toxins.

Methicillin-resistant Staphylococcus capitis with reduced vancomycin susceptibility causes late-onset sepsis in intensive care neonates.

BACKGROUND: Coagulase-negative staphylococci, mainly Staphylococcus epidermidis, are the most frequent cause of late-onset sepsis (LOS) in the neonatal intensive care unit (NICU) setting. However, recent reports indicate that methicillin-resistant, vancomycin-heteroresistant Staphylococcus capitis could emerge as a significant pathogen in the NICU. We investigated the prevalence, clonality and vancomycin susceptibility of S. capitis isolated from the blood of NICU infants and compared these data to adult patients. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a retrospective laboratory-based survey of positive blood cultures in NICU infants ≥ 3 days of age (n = 527) and in adult ICU patients ≥ 18 years of age (n = 1473) who were hospitalized from 2004 to 2009 in two hospital centers in Lyon, France. S. capitis was the most frequent pathogen in NICU infants, ahead of S. epidermidis (39.1% vs. 23.5% of positive blood cultures, respectively). Conversely, S. capitis was rarely found in adult ICU patients (1.0%) compared to S. epidermidis (15.3%). S. capitis bloodstream isolates were more frequently resistant to methicillin when collected from NICU infants than from adult patients (95.6% vs. 53.3%, respectively). Furthermore, we collected and characterized 53 S. capitis bloodstream isolates from NICU infants and adult patients from six distant cities. All methicillin-resistant S. capitis isolates from NICU infants were clonally related as determined by pulsed-field gel electrophoresis. These isolates harbored a type V-related staphylococcal chromosomal cassette mec element, and constantly showed either vancomycin resistance (37.5%) or heteroresistance (62.5%). Conversely, the isolates that were collected outside of the NICU were genetically diverse and displayed much lower rates of vancomycin resistance and heteroresistance (7.7% and 23.1%, respectively). CONCLUSIONS/SIGNIFICANCE: A clonal population of methicillin-resistant S. capitis strains has spread into several French NICUs. These isolates exhibit reduced susceptibility to vancomycin, which is the most widely used antimicrobial agent in the NICU setting.

Accuracy of 6 commercial systems for identifying clinical Aeromonas isolates.

We compared the accuracy of 6 commercial systems for Aeromonas identification by testing 87 clinical isolates in routine conditions, using partial rpoB gene sequencing as the reference standard. The systems were API-20E, API-32GN, the ID-GN card with the Vitek2 system (bioMérieux, Marcy l'Etoile, France), the identification portion of the NFC47 panel (MicroScan Walk/Away system; Siemens Healthcare, Sacramento, CA), ID69 (Phoenix system; BD Diagnostic Systems, Sparks, MD), and GN2 microplates (Omnilog system; Biolog, Hayward, CA), for which 67 (77.1%), 80 (91.9%), 72 (82.7%), 70 (80.5%), 64 (73.5%), and 59 (67.8%) isolates, respectively, were correctly identified at the genus and species level. Confusion with Vibrio affected 6.9% and 16.1% of results obtained with NFC47 and API-20E, respectively. Overall, the accuracy of identification for aeromonads was hampered by outdated databases and taxonomy, weak algorithms, and impractical additional tests. Commercial identification systems should be redesigned to make Aeromonas identification algorithms more robust and to cover infrequent clinical species of this genus.