The identification of bacteria was carried out using matrix-assisted-laser-desorption-ionization–time-of-flight mass spectrometry (MALDI Biotyper, Bruker Daltonics). Antimicrobial susceptibility testing was performed using Phoenix, and MERLIN Diagnostika panels. Rectal swabs were plated onto ChromID Carba Smart plates (bioMérieux). The RESIST-3 O.K.N. immunochromatographic assay (Coris BioConcept), or a molecular test, was performed to establish the type of bacterial resistance. The molecular characterization of carbapenemase genes was conducted on the total amount of positive swabs, using either the Xpert CarbaR real-time PCR test (Cepheid) or the Allplex Entero-DR Assay (Seegene).
Chromid carba smart plates
Manufactured by bioMérieux
Sourced in Germany, France
ChromID Carba Smart plates are a type of microbiology culture medium used for the detection and identification of carbapenemase-producing organisms. They provide a rapid and specific method for the detection of these clinically important bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Allplex entero dr assay, by Seegene (2 mentions)
Xpert carbar real time pcr test, by Cepheid (2 mentions)
Maldi biotyper, by Bruker (2 mentions)
Resist 3 o k n immunochromatographic assay, by BioConcept (2 mentions)
Fecalswab system, by Copan (2 mentions)
Trypcase soy agar plates, by bioMérieux (1 mentions)
Antibiotic gradient strips, by Liofilchem (1 mentions)
Vitek ms, by bioMérieux (1 mentions)
Chemiluminescent enzyme immunoassay, by Fujirebio (1 mentions)
Bactec fx instrument, by BD (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
6 protocols using chromid carba smart plates
1
Bacterial Identification and Resistance Profiling
2
Klebsiella pneumoniae ST258 Isolate Characterization
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Klebsiella pneumoniae strains used in this study are listed in Table S1. ST258 isolates were kindly provided by C. Mammina (Italy)24,25 (link) S. Opal (USA), M. Assous (Israel)26 (link) and M. Gniadkowski (Poland).27 (link)
K. pneumoniae O1:K2 strain was obtained from ATCC (43816), and E. coli strains 536 and MG1655 originated from the laboratory of J. Hacker (Germany). Bacteria were inoculated routinely from chromID™ CARBA SMART plates (BioMérieux) into Luria-Bertani (LB) broth to ensure carriage of the KPC encoding plasmid. For CFU enumeration, bacteria were grown on Trypcase Soy Agar plates (BioMérieux).
K. pneumoniae O1:K2 strain was obtained from ATCC (43816), and E. coli strains 536 and MG1655 originated from the laboratory of J. Hacker (Germany). Bacteria were inoculated routinely from chromID™ CARBA SMART plates (BioMérieux) into Luria-Bertani (LB) broth to ensure carriage of the KPC encoding plasmid. For CFU enumeration, bacteria were grown on Trypcase Soy Agar plates (BioMérieux).
3
Surveillance of Carbapenem-Resistant Enterobacterales
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Bacterial strains investigated in this work included all the available CRE isolates (n=38) obtained from surveillance cultures (rectal swabs) of colonized patients from the SBI ward of the Don Carlo Gnocchi Foundation LTCRF of Florence (Italy), during 2016. Each isolate was from a different patient. The studied isolates represented approximately half of the total cases of carriage (n=74) observed in the SBI ward during 2016, and had been isolated throughout the whole study period (1–5 per month).
Rectal swabs were collected using the FecalSwab system (Copan). The specimens (in 10 µl medium) were cultured on ChromID CARBA SMART plates (bioMérieux) to screen for CRE, within 48 h of collection. Plates were inspected for growth after 18–24 h incubation at 35±2 °C. Colonies grown on the selective medium were identified using the MALDI-TOF MS Vitek 2 system (bioMérieux). Suspect CRE isolates with a meropenem minimum inhibitory concentration >0.125 mg l−1 were included in the study.
Rectal swabs were collected using the FecalSwab system (Copan). The specimens (in 10 µl medium) were cultured on ChromID CARBA SMART plates (bioMérieux) to screen for CRE, within 48 h of collection. Plates were inspected for growth after 18–24 h incubation at 35±2 °C. Colonies grown on the selective medium were identified using the MALDI-TOF MS Vitek 2 system (bioMérieux). Suspect CRE isolates with a meropenem minimum inhibitory concentration >0.125 mg l−1 were included in the study.
4
Carbapenem-Resistant Enterobacterales Harboring OXA-48-Like
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Enterobacterales clinical isolates carrying the plasmid-borne OXA-48-like carbapenemases OXA-48, OXA-181, OXA-232, OXA-244, and OXA-484 were recovered from patients of the University Hospital Frankfurt, Germany (Table 1 ). All isolates were phenotypically characterized and tested for blaOXA–48–like by PCR and Sanger sequencing as previously described (Göttig et al., 2015 (link)). Antimicrobial susceptibility was determined using antibiotic gradient strips (Liofilchem, Roseto degli Abruzzi, Italy), broth micro dilution for colistin and agar dilution for fosfomycin as recommended by EUCAST. Minimal inhibitory concentrations (MIC) were interpreted according to EUCAST guidelines v10.0. For detection of carbapenemases, chromID® CARBA SMART plates (bioMérieux, Nürtingen, Germany) and the immunochromatographic CARBA 5 lateral flow test (NG-Biotech, Guipry, France) were used (Baeza et al., 2019 (link); Göttig et al., 2020 (link)).
5
Rapid Detection of Antimicrobial Resistance
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Bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI Biotyper, Bruker Daltonics or VITEK MS, bioMérieux). Antimicrobial susceptibility testing was performed by Vitek2, Phoenix and MERLIN Diagnostika panels and interpreted according to the EUCAST clinical breakpoints [14 14 . European Committee on Antimicrobial Susceptibility Testing (EUCAST) Breakpoint tables for interpretation of MICs and zone diameters Version 9.0-10.0 2020 Available at: https://www.eucast.org/ast_of_bacteria/previous_versions_of_documents/ Google Scholar ]. Molecular characterization of carbapenemase genes was performed by either the Xpert CarbaR real-time PCR test (Cepheid) or the Allplex Entero-DR Assay (Seegene). Rectal swabs were plated on to ChromID Carba Smart plates (bioMérieux), suspected colonies were identified as described above. The RESIST-3 O.K.N. immunochromatographic assay (Coris BioConcept) or a molecular test was performed to establish the type of bacterial resistance. SARS-CoV-2 infection was determined by analysing nasopharyngeal swabs for the presence of the RNA genome or nucleocapsid antigen using a real-time reverse-transcriptase-polymerase-chain-reaction assay (Abbott, Arrow-Seegene, Hologic, and Roche) or a chemiluminescent enzyme immunoassay (Fujirebio), respectively.
6
Rectal Swabs for Carbapenem-Resistant Enterobacteriaceae
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Rectal swabs were collected using the FecalSwab system (Copan, Brescia, Italy) containing 2 mL of modified Cary-Blair medium, and processed by an external laboratory (Synlab Toscana, Sesto Fiorentino, Italy). The specimens (10 mL of medium) were cultured on chromID CARBA SMART plates (bio-Me ´rieux, Marcy L'Etoile, France) to screen for CRE, and on McConkey agar (as a quality control of the rectal swab), within 48 h of collection. Plates were inspected for growth after 18e24 h of incubation at 35AE2 C. Colonies representative of different morphologies grown on the selective medium were identified using the Vitek 2 system (bioMe ´rieux). Phenotypic characterization of carbapenemase production was investigated according to the EUCAST guidelines for detection of mechanisms [24] . In accordance with routine laboratory protocol, blood cultures were performed at the same external laboratory as rectal swab cultures using the BACTEC FX instrument (Becton Dickinson, Franklin Lakes, NJ, USA), and bacterial identification and susceptibility testing were performed using the Vitek 2 system (bioMe ´rieux).
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