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 ).
Chromid s aureus
Manufactured by bioMérieux
Sourced in France
ChromID S. aureus is a chromogenic culture medium used for the identification and detection of Staphylococcus aureus in clinical samples. It enables the rapid and selective growth of S. aureus colonies, which appear mauve in color on the medium.
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Lab products found in correlation
4 protocols using chromid s aureus
1
Menstrual Fluid Analysis for S. aureus
2
Neutralization of Antimicrobial Connectors
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Needle-free connectors were immersed into bijous containing 1 mL of neutralizing solution consisting of 30 g/L Tween 80, 30 g/L saponin, 3 g/L lecithin, 1 g/L L-histidine, 5 g/L sodium thiosulphate in tryptone sodium chloride (all VWR International). Nullification of antimicrobial activity and non-microbial toxicity was verified prior to commencement of the study (unpublished data). The bijous were then sonicated for 10 min at 50 Hz. The entire volume of neutralizing solution was inoculated (in addition to dilutions from positive control connectors) onto chromogenic S. aureus plates (ChromID S. aureus [Biomerieux]) in duplicate.
3
Quantifying Staphylococcus aureus Infection
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To determine the number of CFUs at the site of infection, a second set of mice was inoculated as described above. On days 5 to 9 after infection, mice were euthanized by cervical dislocation; the lesion and the surrounding tissues were removed and transferred in sterile tubes. Tissue samples were weighed, homogenized in 1 ml of PBS (gentleMACS Dissociator, Miltenyi Biotec, Germany), diluted in sterile PBS, and plated on selective agar (ChromID S. aureus, bioMérieux, France). Enumeration of CFUs was performed 24 h later. For determination of cytokine and MPO levels, lesion homogenates were centrifuged and the supernatant removed and immediately stored at -80°C. Cytokine levels were determined using Luminex assays (Bio-Techne) according to the manufacturer’s instructions. The amount of MPO in the skin lesions was quantified using a commercially available ELISA kit (Bio-Techne).
4
Genotyping Staphylococcus aureus Isolates
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Nasal and oropharyngeal samples were separately plated on chromID S. aureus and chromID MRSA agar plates (bioMérieux, La Balme, France), and subsequently placed in two Mueller–Hinton (MH) broth supplemented with 6.5% NaCl. The overnight MH broth were separately subcultured onto both chromID S. aureus and chromID MRSA agar plates. All agar plates were read after 18–24 h incubation at 35–37°C according to manufacturer's instructions [23] (link). All cefoxitin resistant isolates were tested using a PCR for the presence of the mecA and nuc gene [24] (link)–[25] . All S. aureus strains were genotyped by staphylococcal protein A (spa) typing [26] (link) and multiple-locus variable-number tandem repeat analysis (MLVA) as described previously [27] (link). MLVA types (MTs) were clustered using a categorical clustering coefficient and a minimum spanning tree was constructed to display the relationships between the various MTs. MLVA complexes (MC) were assigned if two neighbouring MTs did not differ in more than one variable number tandem repeat (VNTR) locus and if at least five neighbouring MTs fulfilled this criterion.
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