Computational protocol: Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China

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Protocol publication

[…] A total of 64 bacterial strains were used in this work to develop the LAMP assay (Supplemental Materials). K. pneumoniae ATCC BAA-2146 carrying blaNDM−1 and K. pneumoniae ATCC BAA-1705 carrying blaKPC−2 were used as the positive control. Twenty-one non- K. pneumoniae bacterial species maintained in our microorganism center, including common clinical infectious and opportunistic pathogens, were included to evaluate the specificity of the LAMP assay. One hundred and ten clinical sputum samples containing suspected K. pneumoniae strains and multi-resistant infections were collected from ICU hospitalized patients with cough or pneumonia in the Wujing hospital, 307 hospital and 301 hospital in Beijing, China. The species identification was carried out using an automated system (Phoenix and BD systems) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The sequences of 16S ribosomal DNA (rDNA) and rcsA were validated by PCR-based sequencing and showed 100% identity with the sequences of previously reported genes.Seven housekeeper genes including gapA, infB, mdh, pgi, phoE, ropB, and tonB were detected by PCR. The allele number for each gene was assigned to the MLST database (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). A combination of the allelic sequences of the 7 genes yielded the allelic profile. Antimicrobial susceptibility testing was performed by microbroth dilution VITECaccording to the Clinical and Laboratory Standards Institute (CLSI, Performance standards for antimicrobial susceptibility testing; Twenty-third informational supplement CLSI Document M100-S23. Wayne, PA, USA 2013) and Etest strips (bioMérieux) for carbapenems.The strains were screened for the presence of known MBL and other β-lactamase genes (blaNDM−1, blaKPC−2, blaTEM, blaVIM, blaIMP, blaCTX, blaSIM−1, blaAIM−1, and blaOXA−48) by PCR with primers as reported previously (Poirel et al., ; Patzer et al., ).The bacterial strains and clinical samples were cultured in brain heart infusion (BHI) broth at 37°C according to a standard protocol. Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Co. USA). [...] The sequence of the rcsA gene (http://www.ncbi.nlm.nih.gov/gene/7946097) was downloaded from NCBI GenBank database and further analyzed by Primer Explorer (Version 4, http://primerexplorer.jp/elamp4.0.0/index.html). Five primer sets were designed (Table and Table ). To compare the sensitivity of the LAMP and conventional PCR assay, PCR was conducted with the KP-27F3 and KP-27B3 primer pair (Table ), which amplifies a 176-bp fragment. All primers were synthesized commercially (Sangon Biotech Co., Ltd, Shanghai, China). […]

Pipeline specifications

Software tools BIGSdb, PrimerExplorer
Applications De novo sequencing analysis, qPCR
Organisms Klebsiella pneumoniae, Homo sapiens
Diseases Pneumonia