Slidex staph plus

Nasal swabs were incubated in brain heart infusion broth (BHI) with 7% NaCl for 18–24 h at 37 °C. Then, 10 µL of this broth was plated onto selective chromID^®MRSA agar culture (bioMérieux^®, Marcy l’Etoile, France), and incubated for 48 h at 37 °C. MRSA colonies were preliminarily identified as characteristic green malachite colored round colonies and purified on Columbia agar plates with 5% sheep blood (bioMérieux^®). Isolates were confirmed as S. aureus by Gram stain appearance catalase test and coagulase test agglutination (Slidex^® Staph Plus; bioMérieux^®). Species’ identifications were confirmed by Vitek^®2 Automated Microbiology System with a Gram positive identification card (bioMérieux^®). Methicillin resistance was confirmed by testing the presence of penicillin-binding protein A (PBP2a) (MRSA-screen; Denka Seiken Co™, Tokyo, Japan) and detecting the presence of the mecA gene by Real Time PCR (IQ™5; Bio-Rad, Hercules, CA, USA). The mecA gene (496bp) was amplified with the following primers: mecA- 1: 5′-GGG GTG GTT ACA ACG TTA CAA G-3′ mecA- 2: 5′-AGT TCT GCA GTA CCG GAT TTG C-3′. S. aureus ATCC 29213 was used as the reference strain. The primers were marked with IQ SYBR Green Supermix (BioRad^®, Madrid, Spain).

The nasopharyngeal samples were thawed at room temperature and 10 μl of each sample were inoculated on Chapman medium (Oxoid Ltd) and incubated for 24 hours at 37 degrees Celsius. Presumed colonies of S. aureus, indicated by the presence of a golden yellow pigment, were purified by cultivation on fresh agar containing 5% fresh sheep’s blood and incubated under the same conditions. The coagulase agglutination test (Slidex Staph plus, BioMérieux^®SA) was carried out on well-characterized colonies to confirm their identification as S. aureus. Standard 0.5 McFarland S. aureus colony suspensions were seeded in Mueller Hinton agar for the following antibiotic sensitivity tests: azithromycin (15 μg), penicillin (10 IU) and cefoxitin (30 μg) (Oxoid Ltd). Susceptibility results were interpreted in accordance with Clinical and Laboratory Standards Institute [18 ]. azithromycin resistance (AzmR) was defined by an inhibition diameter of 13 mm or less around the antibiotic disk, penicillin by an inhibition diameter of 28 mm or less, and that of cefoxitin by a diameter of 21 mm or less.

Staphylococci were identified to species level based on colony morphology, Gram staining, catalase production, coagulase testing (Slidex Staph Plus; bioMérieux, Marcy l’ Etoile, France) and by the Vitek2 System (GP card, bioMerieux). Susceptibility to cefoxitin (FOX), penicillin (PEN), erythromycin (ER), clindamycin (CLI), tobramycin (TOB), gentamicin (GM), ciprofloxacin (CIP), fusidic acid (FA), rifampicin (RIF), tetracycline (TET) and sulfamethoxazole/ trimethoprim (SXT), was tested by the disk diffusion method according to EUCAST guidelines [36 ]. Testing for inducible and constitutive lincosamide resistance was performed with the D-test. MICs of mupirocin (MUP) and oxacillin (OX) were determined by E-test® (bioMerieux). Isolates resistant to at least three classes of antimicrobials were considered multidrug resistant. Phenotypic determination of MSSA was based on cefoxitin disk susceptibility [36 ]. Isolates with mupirocin MIC > 1 mg/L were further analysed.

Blood from neonates was cultured in BACTEC plus PEDS aerobic bottles and incubated in the Bactec FX (BD, Heidelberg, Germany). In case of positive blood cultures, plates were inoculated and, after 16–24 h of incubation at 37 °C, screened for S. aureus based on colony morphology. Identification was performed by means of a latex agglutination test (Slidex Staph Plus, bioMérieux, Marcy-l’Etoile, France) and/or via matrix-assisted laser desorption/ionisation, time-of-flight, mass spectrometry (MALDI-TOF MS system, Bruker). S. aureus isolates were stored at − 20 °C or – 80 °C until use. The VITEK 2 system (bioMérieux) was used for antimicrobial susceptibility testing (AST).

Specimens were collected from infection sites of every patient enrolled and cultured on blood agar. Preliminary identification was performed based on bacterial morphology, Gram staining, hemolysis, and catalase tests at the central laboratory. Then, Slidex Staph Plus (bioMérieux, Marcy I'Etoile, France) latex agglutination was performed for the rapid detection of S. aureus. To avoid overrepresentation, we included only the first isolate from each patient. MRSA isolates were initially identified using the oxacillin minimum inhibitory concentration method and confirmed for the presence of the mecA gene by PCR as previously described.^{5 (link)}

All swabs were placed into the corresponding transport tube and were subjected to microbiological analysis one hour after swabbing the nose models to simulate optimum transport conditions. CFU were determined by streaking swabs in a standardized fashion onto Columbia agar supplemented with 5% sheep blood in 5 streaks with a length of 5 cm while constantly rotating the swab shaft in an angle of 45° to the plate and exerting gentle pressure (swab shaft was slightly bended).
For the elution of Copan FLOQSwabs according to manufacturer's instructions, the swabs were rotated (10 turns) in 1 ml eSwab liquid Amies preservation medium, Copan, Brescia, Italy, ref. 490CE.A. 100 µl aliquots were plated onto Columbia agar supplemented with 5% sheep blood.
Agar plates were subsequently cultured at 37°C under ambient atmosphere for 48 h.
CFU were then counted by macroscopic inspection. Staphylococcus aureus was distinguished from Staphylococcus epidermidis by hemolysis (β-hemolysis vs. no hemolysis) and colony color (golden yellow vs. white), if necessary by agglutination assay (Slidex Staph Plus, bioMérieux, Marcy l'Etoile, France).