Standard microbiological methods for microorganism's identification were used [18 (link)]. Then, S. aureus identification was based on Gram staining, morphology, catalase positivity (ID color Catalase; bioMérieux, Marcy l'Etoile, France), agglutination in the Pastorex Staph Plus test (Bio-Rad, Marnes la Coquette, France), and free coagulase production with lyophilized rabbit plasma [19 ]. Finally, the isolates were confirmed by API Staph (bioMérieux, Marcy l'Etoile, France).
Pastorex staph plus test
Manufactured by Bio-Rad
Sourced in France
The Pastorex Staph-Plus test is a rapid latex agglutination test used for the detection and identification of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA), in clinical specimens. The test utilizes antibodies directed against Staphylococcus aureus antigens to detect the presence of the bacteria in a sample.
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3 protocols using pastorex staph plus test
1
Identification of Staphylococcus aureus
2
Identification and Antimicrobial Susceptibility of S. aureus
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Clinical specimens and nasal swabs were cultivated according to standard procedures on Mueller–Hinton broth at 37 °C for 24 h, mannitol salt agar at 37 °C for 48 h, and on Columbia blood agar at 37 °C for 24 h, before molecular studies. S. aureus was presumably identified by catalase slide test, coagulase tube test, and Pastorex Staph-Plus test, performed according to the manufacturer’s protocol (Bio-rad, Marnes-la-Coquette, France) [2 ,52 (link)]. Antimicrobial susceptibility testing was achieved by the VITEK-2 Antimicrobial Susceptibility Testing automated systems, using the AST-P580 card according to the manufacturer’s specifications (bioMérieux, Marcy l’Etoile, France) and EUCAST clinical breakpoints (version 1.3) to define susceptibility. Species confirmation was achieved by Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI Biotyper system, Bruker Daltonics, Bremen, Germany) and, additionally, by PCR targeting the S. aureus nuc gene as described elsewhere [53 (link),54 (link),55 (link)]. DNA was extracted from S. aureus cells using the QIA amp tissue kit (Qiagen, Hilden, Germany) by following the manufacturer’s recommendations. Methicillin resistance was confirmed by PCR targeting mecA and mecC, respectively [56 (link),57 (link)].
3
Comprehensive Microbial Profiling of Food Samples
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Microbial analyses of the 54 samples were performed on selective media in duplicate. The total counts, yeasts and molds, staphylococci, lactic acid bacteria (LAB), Enterobacteriaceae, coagulase positive staphylococci and S. aureus, Listeria monocytogenes and Salmonella were analyzed as described by Lebert et al. (2007) . In particular, the presence of S. aureus isolated from Baird Parker agar supplemented with tellurite yolk egg was confirmed by the Pastorex Staph-Plus test, a latex agglutination test for the identification of S. aureus (Bio-Rad, Marnes La Coquette, France). For LAB, the MRS media was incubated under anaerobic conditions. In addition, Bacillus cereus was numerated on Bacillus cereus Selective Agar (Oxoid, USA), incubated at 30 °C for 18-48 h; Clostridium perfringens on Tryptose Sulfite Agar (Biokar, France), incubated at 37 °C for 20 h and confirmed with Lactose Sulfite medium (Biokar, France) at 46 °C.
A collection of 829 isolates of staphylococci was constituted by selecting for each sample an average of 15 colonies on countable plates of Mannitol Salt Agar (MSA, Bio-Rad, France, Leroy et al., 2010) . These isolates were cultivated on Brain Heart Infusion (BHI, Difco, USA) at 30 °C overnight. They were then stored at -80 °C in BHI broth containing glycerol 20% before identification.
A collection of 829 isolates of staphylococci was constituted by selecting for each sample an average of 15 colonies on countable plates of Mannitol Salt Agar (MSA, Bio-Rad, France, Leroy et al., 2010) . These isolates were cultivated on Brain Heart Infusion (BHI, Difco, USA) at 30 °C overnight. They were then stored at -80 °C in BHI broth containing glycerol 20% before identification.
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