Comprehensive Screening of Klebsiella pneumoniae Virulence Genes

A total of 41 primer sets were used to screen and detect K. pneumoniae virulence genes which included PKS colibactin gene, K. pneumoniae serotypes (K1(K1-magA), K2 (K2wzy), K5 (K5wzx), K20 (wzyK20), K54 (wzxK54), K57 (wzyK57)) as well as Carbapenem genes (OXA-48 (blaOXA-48), NDM-1 (blaNDM-1), KPC (blaKPC), IMP (blaIMP), and VIM (blaVIM)). Previously published primers that were used in this study along with the length of the expected PCR product are listed in Supplementary S1. Briefly, the counterpart virulence genes that were screened are Adhesins (mrkD1, mrkD2, fimH, kpn), Enterobactin siderophores (entB, entB1), Ferric aerobactin receptor (iutA, iutA1,iucB), Aerobactin siderophore, Yersiniabactin siderophore (ybtA, ybtS), Salmochelin catecholate receptor (iroN, iroNB), Klebsiella iron uptake systems (kfu, kfuBC), Genotoxins(Colibactin (clbB), Haemolysin (hlyA), Cytotoxic necrotizing factor (cnf)), Capsule synthesis (K2 serotype capsule synthesis (k2A), Fucose capsule synthesis (wcaG), Lipid polysaccharide (LPS) synthesis (wabG), outer membrane lipoprotein (ycfM), Uridine diphosphategalacturonate 4-epimerase (uge), Regulator of mucoid phenotype A (rmpA1, rmpA2), urease synthesis (ureA), and Allantoin metabolism (allS, allS2). The cycling conditions for the detection of PKS colibactin gene were initial denaturation at 95 °C for 10 min, denaturation at 94 °C for 45 s, annealing at 54 °C for 45 s, and extension at 72 °C for 1 min. This was repeated for 30 cycles. The amplified PCR products were separated and visualized through gel electrophoresis at 120 V for 2 h in 1.8% (wt/vol) agarose gel containing SYBR Safe DNA Gel Stain (Invitrogen). Cycling conditions used for the multiplex PCR along with the primer grouping will be made available upon request.

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Kaur C.P., Iyadorai T., Sears C., Roslani A.C., Vadivelu J, & Samudi C. (2023). Presence of Polyketide Synthase (PKS) Gene and Counterpart Virulence Determinants in Klebsiella pneumoniae Strains Enhances Colorectal Cancer Progression In-Vitro. Microorganisms, 11(2), 443.

Publication 2023

Adhesins Aerobactin Agarose Allantoin Capsule Carbapenem Colibactin Electrophoresis Enterobactin Epimerase Ferric aerobactin receptor Fucose Gene conditions Genes Genotoxins Haemolysin Iron K pneumoniae Klebsiella Lipid synthesis Lipoprotein Membrane Metabolism Multiplex pcr Phenotype Polysaccharide Primer Salmochelin Siderophore Stain Synthesis Urea Urease Uridine Virulence Yersiniabactin

Corresponding Organization : University of Malaya

Other organizations : Johns Hopkins University, Johns Hopkins Medicine, Sidney Kimmel Comprehensive Cancer Center

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Variable analysis

independent variables

Primer sets used to screen and detect K. pneumoniae virulence genes
Cycling conditions for the detection of PKS colibactin gene

dependent variables

Detection of K. pneumoniae virulence genes, including PKS colibactin gene, K. pneumoniae serotypes, and Carbapenem genes

control variables

Previously published primers used in the study
Length of the expected PCR product

controls

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