During the baseline period, no rapid diagnostic methods were used for positive blood cultures. Gram stain was performed immediately when a blood culture signaled positive, and results were exported into the electronic information system. In addition, the positive blood culture gram stain result was called to the treating physician within 1 h of the positive BACTEC alert. After biochemical identification, Staphylococcus aureus was sub-cultured onto solid media and early growth tested for the presence of PBP2a using the PBP2a SA Culture Colony Test (Alere™, Waltham, MA).
Pbp2a sa culture colony test
Manufactured by Abbott
Sourced in United States
The PBP2a SA Culture Colony Test is a laboratory equipment used for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from bacterial cultures. It is designed to identify the presence of the PBP2a protein, which is a specific marker for MRSA strains.
Automatically generated - may contain errors
Lab products found in correlation
Chromid, by bioMérieux (2 mentions)
Amoxicillin, by bioMérieux (1 mentions)
Oxacillin, by BD (1 mentions)
Maldi tof ms biotyper, by Bruker (1 mentions)
Amoxicillin clavulanic acid, by bioMérieux (1 mentions)
Oxacillin, by bioMérieux (1 mentions)
Pastorex, by Bio-Rad (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
Matrix assisted laser desorption ionization time of flight mass spectrometry, by Bruker (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Maldi tof mass spectrometry, by Bruker (1 mentions)
Biotyper microflex, by Bruker (1 mentions)
6 protocols using pbp2a sa culture colony test
1
Rapid Identification of Staphylococcus aureus
2
Microbial Identification and MRSA Characterization
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Microbial identification was confirmed by Bruker Biotyper MALDI-TOF MS (Bruker, Billerica, MA). For MRSA designation, a PBP2a SA culture colony test (Alere) was performed according to the manufacturer’s instructions using 18- to 24-h subculture growth. The presence of mecA and the homolog mecC were determined by in-house PCRs (22 (link), 23 (link)). Susceptibility testing included that for cefoxitin by disk diffusion and for oxacillin by disk diffusion and gradient diffusion. Methods followed the procedural guidelines outlined by the Clinical and Laboratory Standards Institute (documents M02 and M23) (55 , 56 ). Disk diffusion testing of cefoxitin (Hardy Diagnostics) and oxacillin (BD) was performed on conventional Mueller-Hinton agar (MHA) (Hardy Diagnostics). Gradient diffusion testing of oxacillin (bioMérieux) was performed on MHA with 2% NaCl agar (Remel). Detection of beta-lactamase production was assessed by the disk diffusion penicillin zone edge test (Hardy Diagnostics) and nitrocefin-based Cefinase disk test (Hardy Diagnostics). Beta-lactamase inhibitor rescue phenotype was determined by assessing the fold change in MIC from amoxicillin (bioMérieux) and amoxicillin-clavulanic acid (bioMérieux) gradient diffusion testing.
3
Methicillin-Resistant Staphylococcus aureus Characterization
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Swabs were cultured on Columbia blood agar for 24 to 48 hours at 37 ± 1 C. Suspected colonies were tested for a positive catalase, coagulase and latex agglutination test (Pastorex; Bio-Rad, Marnesla-Coquette, France). Species confirmation was done in Germany using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Daltonics, Leipzig, Germany). Antimicrobial susceptibility was tested with the VITEK 2 (bioM erieux, Marcy l'Etoile, France) automated systems using the AST632 card. Susceptibility to trimethoprim and chloramphenicol was tested using disk diffusion. Etests were used to measure the minimum inhibitory concentration (MIC) of cefoxitin and oxacillin. All susceptibility tests were performed and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST; clinical breakpoints version 6.0).
Methicillin resistance was confirmed using a PBP2a detection test (PBP2a SA Culture Colony test; Alere, Scarborough, ME, USA).
OS-MRSA were passaged on oxacillin gradient agar (0e2 mg/L) which consisted of a bottom layer (Müller-Hinton agar, 2 mg/L oxacillin) and an upper layer (Müller-Hinton agar, 5% sheep's blood) [10] . The colonies which grew at the highest oxacillin concentration were used for passaging. The oxacillin MIC of these colonies was measured after each passage using the Etest following EUCAST recommendations.
Methicillin resistance was confirmed using a PBP2a detection test (PBP2a SA Culture Colony test; Alere, Scarborough, ME, USA).
OS-MRSA were passaged on oxacillin gradient agar (0e2 mg/L) which consisted of a bottom layer (Müller-Hinton agar, 2 mg/L oxacillin) and an upper layer (Müller-Hinton agar, 5% sheep's blood) [10] . The colonies which grew at the highest oxacillin concentration were used for passaging. The oxacillin MIC of these colonies was measured after each passage using the Etest following EUCAST recommendations.
4
MRSA Screening by Nasopharyngeal Samples
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MRSA screening by nasopharyngeal samples was performed using Polywipe™ (Medical Wire & Equipment, Wiltshire, United Kingdom) pre-moistened sponge swabs. Detection of MRSA was performed by using selective agar plates (chromID®, bioMérieux, Marcy l’Étoile, France) and incubation at 35 ± 1 °C for 24–48 h both with and without enrichment (Dextrose Bouillon) at 36 °C for 24 h. Suspicious colonies were confirmed via MALDI-TOF–MS (Bruker Corporation, Bremen, Germany). Susceptibility testing was performed and interpreted in accordance with the current European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards for clinical breakpoints [13 ]. Methicillin resistance was confirmed by the PBP2a latex agglutination test (PBP2a SA Culture Colony Test®, Abbott, Illinois, US) and genotypically by detecting the mecA, mecC resistance, and the Panton-Valentine leukocidin (PVL) virulence genes (Geno-Type MRSA®, HAIN, Nehren, Germany) [14 (link), 15 (link)].
5
Identification and Susceptibility Testing of MRSA
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Detection of MRSA was performed by using selective agar plates (chromID, bioMérieux, Marcy l’Étoile, France, 36 ± 1 °C for 24 h). For species identification, we used MALDI-TOF mass spectrometry (Bruker Daltonics, Bremen, Germany). Susceptibility testing (see also Table 2 ) was performed using the VITEK-2 system (bioMérieux) and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards for clinical breakpoints (version 11.0). Isolates were further screened by PBP2a (PBP2a SA Culture Colony Test, Abbott, Scarborough, Maine, USA) and, in case of inconsistent results, for mecA, mecC and panton-valentine leukocidin (PVL) (eazyplex® MRSAplus, amplex, Gars-Bahnhof, Germany).
6
Bacterial Identification and Antibiotic Resistance
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The identification of bacteria was performed by MALDI-TOF MS (Biotyper Microflex, Bruker Daltonics, Bremen, Germany). The susceptibility/resistance to antibiotics was tested by the broth microdilution method according to EUCAST [16 ]. The following bacterial strains were used as references for the quality control: Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212. The production of broad-spectrum beta-lactamases, such as ESBL and AmpC, was detected by phenotypic tests [17 (link)]. Positive results were verified by PCR detection of genes for the respective type of beta-lactamase. All strains of S. aureus were tested for the resistance to methicillin using selective diagnostic chromogenic media (Colorex/TM/MRSA, TRIOS, Prague, Czech Republic) and immunochromatographic assays for the detection of PBP2a (PBP2a SA Culture Colony Test, AlereTM, Abbott, Prague, Czech Republic). Positive results were confirmed by the detection of the mecA gene. The resistance to vancomycin in VRE was also confirmed by the detection of the vanA and vanB genes.
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