Molecular identification of (bla)SHV, (bla)LEN and (bla)OKP beta-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged (bla)SHV-PCR amplicons

Abstract

Plasmid encoded (bla)SHV enzymes represent an important sub-group of class A beta-lactamases causing an ESBL-phenotype which is increasingly found in Enterobacteriaceae including Klebsiella pneumoniae. The chromosomally encoded beta-lactamase (bla)LEN and (bla)OKP enzymes, which so far only have been found in K. pneumoniae, do not hydrolyse extended-spectrum cephalosporins. In the present study, multiple displacement amplified DNA derived from 20 K. pneumoniae clinical isolates with a (bla)SHV-like genotype was used in a universal SHV PCR assay using SP6- (forward) and T7- (reverse) sequence-tagged primers. Identification and differentiation of (bla)SHV, (bla)LEN and (bla)OKP genes was obtained by bi-directional amplicon sequencing using SP6- and T7-specific primers. Three well characterised K. pneumoniae strains having a SHV-genotype were included in the study. The bi-directional amplicon sequencing, covering approximately 800 bp (approximately 93%) of the (bla)SHV, (bla)LEN and (bla)OKP enzyme encoding sequences, allowed for an unequivocal discrimination of SHV, LEN and OKP genes. Moreover, sequencing revealed the presence of (bla)SHV allelic variants in six K. pneumoniae isolates in which the amplicons had to be cloned accordingly. Based on deduced amino-acid sequences, a dendrogram was constructed. Seventeen out of 20 K. pneumoniae isolates with an ESBL-phenotype formed a SHV-like cluster, two were LEN-like, and one isolate was OKP-like. The PCR-based molecular typing method described here enables a rapid, reliable and cost-effective identification and differentiation of (bla)SHV, (bla)OKP and (bla)LEN genes.