Identibac s aureus genotyping

All MRSA isolates were genotyped by multiple-locus variable number of tandem repeat analysis (MLVA) by the Dutch National Reference Center (RIVM, Bilthoven, the Netherlands) [30] (link). MLVA is known for its higher discriminatory power for LA-MRSA strains as compared to either multilocus sequence typing (MLST) or pulsed-field gel electrophoresis (PFGE) [30] (link). The MLVA profiles were clustered using a categorical clustering coefficient (unweighted-pair group method using arithmetic averages, UPGMA) and a minimum spanning tree was constructed to display the relationships between the various MLVA complexes (MC) and MRSA sources. For this study, we incorporated phiSa3 into the MLVA scheme. Furthermore, tetM was determined by use of DNA microarray (Identibac S. aureus Genotyping, Alere).

A subset of 226 strains from the collection of the Centre National de Référence des Staphylocoques (Lyon, France), composed of 103 strains of the main community and hospital-acquired MRSA clones, and 123 strains of MSSA were used in this study. They were sent to the laboratory for detection of toxin production in the context of nasal colonization, skin and soft tissue infection, pneumonia, bacteremia, or endocarditis. The S. aureus HT20030749 strain belonging to the clone Geraldine was isolated from blood culture of patient with bone–joint infection.
All strains were genotyped as previously described. Briefly, bacterial DNA was extracted according to the manufacturer's recommended protocol using commercial extraction kits (Qiagen). The diagnostic DNA microarrays, identibac S. aureus Genotyping (Alere) used for this study, as well as related procedures and protocols, have been previously described in detail [40 (link)]. This microarray covers 332 different target sequences corresponding to approximately 185 distinct genes and their allelic variants. The assigning of isolates to CCs was determined by the comparison of hybridization profiles with those previously characterized by using multilocus sequence typing reference strains [40 (link)].

Tampons were collected as previously described [9 (link),21 (link)]. Menstrual fluid was extracted from the tampon by soaking it in 15 mL of sterile distilled water and then pressing it. Fifty microliters of menstrual fluid were spread on a SAID chromogenic plate to selectively detect S. aureus (chromID™ S. aureus, Biomérieux, Marcy l’Étoile, France). Plates were incubated at 35 °C for 18–24 h under aerobic conditions. Suspicious colonies (pink to light pink) were identified by matrix-associated laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry [21 (link)]. All S. aureus strains were genotyped using Identibac S. aureus Genotyping^® (Alere) DNA microarrays, as described previously [27 (link)]. Particular attention was paid to the presence of tst, sea, sec, and sed genes encoding TSST-1 and the enterotoxins SEA, SEC and SEC, respectively. A subset of 28 samples were selected from the collection of 737 samples previously described [9 (link),21 (link)], 6 from patients with mTSS and 22 from healthy volunteers (Table 1).