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

Plasmid encoded (SHV)-S-bla 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 (LEN)-L-bla and (OKP)-O-bla 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 (SHV)-S-bla-like genotype was used in a universal SHV PCR assay using SP6- (forward) and T7- (reverse) sequence-tagged primers. Identification and differentiation of (SHV)-S-bla, (LEN)-L-bla and (OKP)-O-bla 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 similar to 800bp (similar to 93%) of the (SHV)-S-bla, (LEN)-L-bla and (OKP)-O-bla enzyme encoding sequences, allowed for an unequivocal discrimination of SHV, LEN and OKP genes. Moreover, sequencing revealed the presence of (SHV)-S-bla 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 (SHV)-S-bla, (OKP)-O-bla and (LEN)-L-bla genes. (C) 2009 Elsevier Ltd. All rights reserved.