Diversity and Evolution of the Class A Chromosomal Beta-Lactamase Gene inKlebsiella pneumoniae

Abstract

We investigated the diversity of the chromosomal class A beta-lactamase gene in Klebsiella pneumoniae in order to study the evolution of the gene. A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified in K. pneumoniae and of different chromosomal beta-lactamase variants previously identified. Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for other K. pneumoniae beta-lactamase). Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups. One new SHV variant, seven new LEN variants, and four OKP variants were identified. The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0. The nucleotide sequences of the housekeeping genes gyrA, coding for subunit A of gyrase, and mdh, coding for malate dehydrogenase, were also determined. Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively. This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families. We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome of K. pneumoniae.

Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons

“…Due to the growing number of Klebsiella species that possess plasmid and chromosomally encoded bla SHV-enzymes, the traditional iso-electrofocusing technique appears not to be the method of choice for discriminating between a K. pneumoniae bla SHV, bla LEN and bla OKP-genotype any longer [9].…”

“…In a previous study, Haeggman and co-workers [9] identified a new, third group of sequences related to the bla SHV and bla LEN families, namely the chromosomally encoded bla OKP family (for other K. pneumoniae β-lactamase). To date, 14 different bla OKP-type β-lactamases have been described [10].…”

“…To date, 14 different bla OKP-type β-lactamases have been described [10]. Phylogenetic analysis of the three genes revealed parallel evolution, corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively [9]. It was proposed that the chromosomally encoded bla LEN and bla OKP enzymes should be grouped in a single new family "K. pneumoniae-specific chromosomal β-lactamases," excluding plasmid encoded bla SHV enzymes from this family [11].…”

“…However, identification of a SHV genotype at the nucleotide level often requires cloning of the amplicons, followed by sequencing. Alternatively, amplicons have been sequenced using a set of gene-specific primers [9,13,19]. These methods are labour intensive and time-consuming.…”

“…Numerous molecular typing methods have been developed for identification of the growing number of bla SHV-genes [4,[14][15][16][17][18], including specific PCR assays of SHV, LEN, and OKP genes [9,19]. However, identification of a SHV genotype at the nucleotide level often requires cloning of the amplicons, followed by sequencing.…”