Computational protocol: Results from the Canadian Nosocomial Infection Surveillance Program on Carbapenemase-Producing Enterobacteriaceae, 2010 to 2014

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Protocol publication

[…] Detection of the genes for the following carbapenemases by PCR was conducted as previously described (): NDM, KPC, IMP, VIM, GES, and SME. Detection further included genes for OXA-48-type () and NMC/IMI-type (NMC-1 5′-TGGTGTCTACGCTTTAGAC-3′ NMC-2 5′-ACCATGTCTGATAGGTTTCC-3′) enzymes. PCR mapping of the Tn4401 element was conducted using previously described primers (). Multilocus sequence typing (MLST) (http://bigsdb.pasteur.fr/klebsiella/ and http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) and macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) as previously described () were conducted on all CPE. BioNumerics software (version 3.5; Applied Maths, Saint Lartens-Latem, Belgium) was used to analyze fingerprints. Plasmid restriction fragment length polymorphism (pRFLP) analysis was conducted as previously described using electroporation to transfer a carbapenemase gene-harboring plasmid to Escherichia coli DH10B () in which NDM-type plasmids were digested with BglII and all other carbapenemase gene-containing plasmids with EcoRI. Plasmid-based replicon typing (PBRT) was conducted as previously described (, ). Antimicrobial susceptibility testing was performed using Vitek2 (AST-GN25 or AST-N219; bioMérieux, St. Laurent, Canada) using 2014 CLSI breakpoints (). Tigecycline breakpoints were based on FDA breakpoints for Enterobacteriaceae (susceptible [S], ≤2 mg/liter; intermediate, 4 mg/liter; resistant [R], ≥8 mg/liter). Etest (bioMérieux) was used to determine colistin MICs with EUCAST breakpoints for Enterobacteriaceae (S ≤ 2 mg/liter; R > 2 mg/liter). Antimicrobial testing on plasmids was done on representatives from large pRFLP clusters along with all plasmids with unique pRFLP profiles. […]

Pipeline specifications

Software tools BIGSdb, BioNumerics
Application De novo sequencing analysis
Organisms Homo sapiens