Nasal and groin samples collected were cultured on MRSA CHROMagar (CHROMID® MRSA, bioMérieux) and incubated at 37°C for 24 hours. Plates were examined for green MRSA colonies; other colours were disregarded. In addition, the latex agglutination test was performed to identify penicillin-binding protein, PBP2A. The isolates were confirmed as MRSA by disc diffusion test using 30 μg cefoxitin disc on Mueller-Hinton agar according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Quality control was done by using Staphylococcus aureus ATCC 25923.
Chromid mrsa
Manufactured by bioMérieux
Sourced in France
ChromID MRSA is a chromogenic media used for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical specimens. The product allows for the rapid and selective identification of MRSA colonies based on their characteristic color.
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Lab products found in correlation
Vitek 2 system, by bioMérieux (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Columbia agar, by bioMérieux (1 mentions)
Maldi tof, by Bruker (1 mentions)
Mueller hinton agar, by Bio-Rad (1 mentions)
Axima assurance maldi tof mass spectrometer, by Shimadzu (1 mentions)
Sheep blood, by BD (1 mentions)
Columbia agar, by BD (1 mentions)
Slidex staph plus, by bioMérieux (1 mentions)
Chromid cps elite, by bioMérieux (1 mentions)
Vitek 2 cards, by bioMérieux (1 mentions)
Chromid candida, by bioMérieux (1 mentions)
9 protocols using chromid mrsa
1
Rapid MRSA Identification Protocol
2
Surveillance of MRSA in Wound Infections
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Fecal specimens, nostril- and wound swabs were initially cultured on Columbia agar with 5% sheep blood (bioMérieux, Germany), and chromID® MRSA (bioMérieux, Germany) agar plates. Identification of S. aureus was determined by MALDI-TOF MS (Bruker, Germany). Wound swabs were taken immediately upon admission from the “open wound” patients within the reception area. Further information on the complete sampling procedure and local setting has been published elsewhere (Walther et al., 2018 (link)). Antimicrobial susceptibility testing (AST) using the VITEK®2 system (BioMérieux, Germany) was performed according to the standards given by the CLSI VET01-A4 and M100-S21 (Clinical Laboratory Standards Institute, 2011 , 2013 ).
3
MRSA Isolation and Identification
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The MRSA isolate was recovered by culture on a selective medium (Chrom ID MRSA, bioMérieux Marcy I’Etoile). Identification was achieved by linear matrix-assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF) (Bruker; Billerica, MA, United States). The isolate was stored frozen for subsequent genotyping.
4
MRSA Surveillance in Neonatal Unit
5
S. aureus Detection from Nasal and Endotracheal Samples
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Nasal and endotracheal samples were cultured in the microbiology laboratory. S. aureus was detected in both samples using molecular methods (Xpert SA Nasal Complete assay). Samples were processed using PCR according to the manufacturer's instructions, as detailed elsewhere [20 (link)]. Xpert is a qualitative in vitro diagnostic test designed for rapid detection of MSSA and MRSA from nasal swabs. The test utilizes automated real-time PCR to detect nucleic acid sequences of the staphylococcal protein A (spa), the gene for methicillin/oxacillin resistance (mecA), and staphylococcal cassette chromosome (SCCmec).
Samples were cultured on a mannitol-salt agar plate and on a chromogenic medium for the isolation of MRSA (chromID MRSA, bioMérieux, Craponne, France) and processed for a semiquantitative count.
Plates were incubated for 48 hours at room temperature.
Samples were cultured on a mannitol-salt agar plate and on a chromogenic medium for the isolation of MRSA (chromID MRSA, bioMérieux, Craponne, France) and processed for a semiquantitative count.
Plates were incubated for 48 hours at room temperature.
6
Validation of CHROMagar LIN-R for Bacterial Identification
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For the validation on pure bacterial colonies, Bacterial cultures were performed in 4 mL of Brain Heart Infusion for 2 h at 37 °C until reaching an OD600nm of 0.1. A 1/10 dilution was performed in sterile water and 10 µL of this solution was plated on CHROMagar™ LIN-R. Regarding the validation on the blood cultures, 2 drops (ca. 80 µL) of positive blood cultures were directly spread on CHROMagar™ LIN-R. Blood agar was also inoculated to further perform antimicrobial susceptibility testing on Mueller-Hinton (MH) agar (Biorad, Marnes-la-Coquette, France). Nasal swabs were directly spread on CHROMagar™ LIN-R. As a growth control, the nasal swabs were also inoculated on MH agar and on ChromID® MRSA (bioMérieux, La Balmes les Grottes, France). Growth, colony size and color were determined after 24 and 48 h at 37 °C.
7
Microbiological Analysis of Nasal Swabs
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All swabs were subjected to microbiological analysis immediately after swabbing the noses or the nose models. The swab-NaCl-combination was vortexed for 5 seconds. CFU were determined by plating 100 μl of 1:10 serial dilutions onto Columbia agar supplemented with 5% sheep blood (BD, Heidelberg, Germany), resulting in a detection threshold of 10 CFU. Bacterial amounts below this limit could not be detected and swabs were regarded as MRSA negative. In parallel, a chromogenic MRSA medium (chromID MRSA, bioMérieux, Marcy l’Etoile, France) was inoculated with 100 μl of the undiluted suspension. Agar plates were subsequently cultured at 37°C under ambient atmosphere for 48 h. CFU were then counted by macroscopic inspection. Staphylococcus aureus was identified by β-hemolysis and colony color (golden yellow on Columbia agar, green color on chromID MRSA agar), if necessary by agglutination assay (Slidex Staph Plus, bioMérieux, Marcy l’Etoile, France), or by matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using a Shimadzu “AXIMA Assurance” MALDI-TOF mass spectrometer (Shimadzu Germany Ltd., Duisburg, Germany) as described elsewhere [31 (link)].
8
Oral Tumor Microbiome Profiling
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Oral swabs were done from the surface of the tumor. Bacterial cultures were performed in the Laboratory of Bacteriology of Medical Diagnostic Laboratory of Fryderyk Chopin Provincial Specialist Hospital No. 1 in Rzeszów, Poland. Columbia Agar with 5% sheep blood, CHROMID® CPS® Elite, CHROMID® MRSA, and CHROMID® Candida (all of them bioMérieux SA, France) solid media were used for identification of aerobic bacteria and candida. Cultures for identification of bacteria were incubated for 24 h at 37°C and for candida culture for 48 h at 35°C.For isolation of anaerobic bacteria Schaedler agar +5% sheep blood (bioMérieux SA, France) were used and was incubated for 48 h in GENBAG ANAEROBIC sachets (bioMérieux SA, France) in an incubator at 37°C. Bacteria and yeasts were identified with the use of VITEK® 2 cards (bioMérieux SA, France) according to the manufacturer's protocol.
9
Characterization of MRSA Isolates
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For the strain selection, isolation, and characterization, we followed the methods of Kotnik-Kevorkijan et al. [22] . S. aureus cultivated from clinically relevant samples were recognized as MRSA because of the resistance to cefoxitin, and were subsequently confirmed with mecA testing. For surveillance samples, the conventional culture media, including MRSA-screening plates (CHROMID ® MRSA, bioMerieux, Marcy-l'Etoile, France) and trypticase soy broth containing NaCl, were used. MRSA strains were confirmed by PCR amplification of the mecA gene by a modification of previously published methods [23, 24] . An in-house method is established and is based on amplification of three different PCR products; two are S. aureus-specific and one targets mecA gene. The mecC testing was introduced in the second half of the year 2013 for strains that were resistant to cefoxitine and negative for mecA. No strains fulfilling these criteria were detected within the study interval. All isolates were frozen at -70 °C until further characterization. Only the first isolate of each patient was stored.
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