To be included in the multicenter evaluation study, the commercial selective agar plates had to fulfill the following criteria: i) plates should be available in all countries, ii) they should be available as ready-to-use plates, iii) no bi/dual plates or similar should be included. One exception to the criteria was done for the CHROMagar™ mSuperCARBA™ which is only available as an in-house medium. To avoid any preparation biases of the CHROMagar™ mSuperCARBA™ plates these were prepared for the participating laboratories by the MAST group (MAST DI-AGNOSTICS, Amiens, France). Table 3 lists commercial agar plates included and whether the plate detects OXA-48 CPE, non-OXA-48 CPE or both.
Msupercarba
Manufactured by CHROMagar
Sourced in France
CHROMagar mSuperCARBA is a chromogenic culture medium designed for the detection and differentiation of carbapenemase-producing Gram-negative bacteria. It allows for the direct isolation and presumptive identification of these microorganisms from clinical samples.
Automatically generated - may contain errors
Lab products found in correlation
Chromid mrsa agar, by bioMérieux (1 mentions)
Eswab, by Copan (1 mentions)
Chromid esbl agar, by bioMérieux (1 mentions)
Microscan walkaway, by Beckman Coulter (1 mentions)
Mueller hinton agar (mha), by Avantor (1 mentions)
Bhi broth, by Avantor (1 mentions)
Rapid e coli 2, by Bio-Rad (1 mentions)
Cefotaxime, by Merck Group (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Col apse, by CHROMagar (1 mentions)
Maldi tof, by Bruker (1 mentions)
Maldi tof ms biotyper, by Bruker (1 mentions)
11 protocols using msupercarba
1
Multicenter Evaluation of Selective Agar Plates
2
Tracking Antibiotic-Resistant Bacteria in Newborns
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Cervical, rectal, and amniotic fluid samples were collected from the childbearing women at the admission using cotton swabs (e-swab, Amies Medium, COPAN, Italy, cat.no.480CE) and were immediately transferred to the laboratory. Oral samples were collected from the newborn babies in the first 48 h after birth (e-swab, Amies Medium, COPAN, Italy, cat.no.480CE). Maternal and neonatal samples were first seeded on four different selective chromogenic agar plates—CHROMID ESBL agar (bioMérieux, Marcy l’Etoile, Lion, France), CHROMID MRSA agar (bioMérieux, Marcy l’Etoile, Lion, France), CHROMagar VRE (CHROMagar, Paris, France), and CHROMagar mSuperCARBA (Paris, France), which were incubated for 24–48 h at 37 °C under aerobic conditions.
Clinical and demographic data were gathered from medical records. The standard protocol is that the women gave birth and they and their newborn babies are hospitalized for three days. In the case of the women that presented positive bacterial cultures, the period of hospitalization was extended during their treatment, along with the treatment of their newborn babies, if there was needed, to seven days. After discharge, a follow-up was done for a period of 6 month by phone call once a month to find out if any infection occurred in the newborn babies that were initially infected with antibiotic-resistant bacteria at birth.
Clinical and demographic data were gathered from medical records. The standard protocol is that the women gave birth and they and their newborn babies are hospitalized for three days. In the case of the women that presented positive bacterial cultures, the period of hospitalization was extended during their treatment, along with the treatment of their newborn babies, if there was needed, to seven days. After discharge, a follow-up was done for a period of 6 month by phone call once a month to find out if any infection occurred in the newborn babies that were initially infected with antibiotic-resistant bacteria at birth.
3
Carbapenemase-producing Enterobacterales Isolation Protocol
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Fifty-one strains from 40 patients plus 1 environmental sample were included in this study, for a total of 52 strains. The samples were collected from an Hospital Centre in northern Portugal, August–December 2020, using standard clinical operating procedures. Identification was performed by microbiology laboratories using conventional methods or automated systems such as MicroScan WalkAway (Beckman Coulter, Lisboa, Portugal)). Rectal swabs were seeded in a chromogenic culture medium CHROMagar™ mSuperCARBA™ (CHROMagar, Frilabo, Portugal). For further molecular characterisation, all strains were maintained frozen at −80 °C in BHI broth (VWR Prolabo, Lisboa, Portugal) plus 15% glycerol. For analysis purposes, the strains were grown overnight in BHI broth (18 h at 37 °C) and seeded on Mueller-Hinton agar (VWR, Lisboa, Portugal).
4
Comprehensive Microbiological Screening
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Participant (rectal and vaginal) and environmental swabs were placed in Amies media and stored at 2–8 °C until they could be transported to the lab. All swabs were processed within 24 h of collection. Participant swabs were directly inoculated onto CHROMagar ESBL and CHROMagar mSuperCARBA (CHROMagar, Paris, France). Environmental swab samples were enriched with trypticase soy broth (TSB) with overnight incubation at 37 °C before the samples were inoculated onto the same media as the participant swabs. From the chromogenic agars, bacterial colony growth color and characteristics were recorded after overnight incubation at 37 °C.
5
Isolation of Antibiotic-Resistant Aeromonas
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A series of dilutions of municipal and hospital wastewater samples were prepared in 0.85% NaCl (tenfold dilution up to 1:10,000). The dilutions were filtered in triplicate under vacuum through sterile mixed cellulose ester membrane filters (47 mm diameter, 0.22 µm pore size, Whatman, GE Healthcare, Life Science, Chicago, IL, USA) and the filters were placed on Rapid’ E. coli 2 (BioRad, Hercules, CA, USA) agar plates supplemented with 4 mg/L cefotaxime (CTX) and CHROMagar mSuperCARBA (CHROMagar, Paris, France) agar plates. The plates were incubated at 37 °C for 24 h. Different colonies of Rapid’ E. coli 2 + CTX and CHROMagar mSuperCARBA were selected for isolation of presumptive 3GC- or carbapenem-resistant Aeromonas spp. A total of 200 presumptive isolates were purified on the same medium and stored in a 20% glycerol stock at −80 °C.
6
Isolation of Klebsiella pneumoniae from Pig and Human Fecal Samples
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Pig and human faecal isolates were obtained through a cross-sectional study carried out between September and December 2018 in Khon Kaen, Thailand.9 Samples were cultured for KP isolation using Simmons citrate agar (SCA) containing 1% inositol (SCAI)10 (link) and antibiotic-enriched media including MacConkey agar (Oxoid, Hampshire, England) containing cefotaxime 1 μg/mL (Sigma-Aldrich, Saint Louis, USA), CHROMagar™ mSuperCARBA™, and CHROMagar™ COL-APSE (CHROMagar, Paris, France). Species were identified by MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany) and refined using whole-genome sequences.
7
Comprehensive Bacterial Identification and AMR Profiling
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Clinical and environmental specimens were cultured on CHROMagar™ ESBL or CHROMagar™ mSuperCARBA™ selective media (CHROMagar, France). Single colony picks were identified using matrix-assisted laser desorption-ionisation time-offlight mass spectroscopy machine (MALDI-TOF MS, Brucker Diagnostics, Germany). Phenotypic antimicrobial resistance profiles were determined using VITEK®2 AST N350 card (Biomérieux, Marcy L'Étoile, France) and reported as resistant, intermediate or susceptible based on the Advanced Expert System [10] . Whole genome sequencing (WGS), Quality Control (QC) analysis, phylogenetic inference, comparative genomics and statistical analysis was performed as describe in detail in Supplementary Methods.
8
Screening and Characterization of ESBL and Carbapenemase-Producing Isolates
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Screening rectal swabs were directly inoculated onto two selective chromogenic media (CHROMagar™ ESBL and CHROMagar™ mSuperCARBA, CHROMagar, France) to detect ESBL and carbapenemase producers, respectively. Colonies isolated from any of the screening media and Enterobacteriaceae isolated from clinical specimens, were identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF) (Bruker, Germany) and tested for antimicrobial susceptibility using the BD Phoenix™ automated identification and susceptibility testing system (Becton Dickinson, USA). Minimum inhibitory concentrations (MIC) and breakpoints were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [6] . Isolates with intermediate susceptibility and resistant to each antibiotic tested were grouped in a single non-susceptible category. The CLSI susceptible-dose-dependent category for cefepime was considered as intermediate for the purpose of this study. Nonsusceptible isolates to any of the third-generation cephalosporins or cefepime were considered as potential ESBL producers.
9
Isolation of Carbapenem-Resistant Bacteria
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The influent sample was collected from the Jungnang (JN) WWTP on the Han River, Seoul, South Korea in May of 2018 by using sterile bottles. After collection, the samples were immediately shipped to the laboratory under cool conditions (4°C) and filtered through a 0.22 μm pore size membrane filter (Advantec, Tokyo, Japan). The membranes were suspended in 10 mL of Mueller-Hinton (MH) broth (MBCell, Seoul, South Korea), thoroughly vortexed, and then processed with a serial dilution up to 10–3 times (100, 10–1, 10–2, and 10–3). A 100 μL of sample of the MH broth was spread on mSuperCARBA (CHROMagar, France) agar plates and the plates were incubated at 37°C for 48 h. After incubation, the colonies on the plates were streaked on new MH agar plates containing 8 mg/L of meropenem to obtain a single colony of presumptive carbapenemase-producing bacteria. The isolate grown on the plates were taxonomically identified using 16S rDNA gene sequencing (Macrogen, Seoul, South Korea).
10
Wastewater Analysis for Hospital Superbugs
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Two 1 L grab samples of wastewater from the hospital sewers were collected from two separate locations around the studied hospital. Sampling was performed in July 2021 in concordance with post-cleaning samples of the hospital environment. The samples were transferred to the laboratory in a coolbox and further processed within 12 h from the time of collection. A volume of 100 μl of the wastewater was pipetted and spread on CHROMagar mSuperCARBA and further processed as post-cleaning hospital environment samples without the enrichment step.
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