Diversity and evolution of the class A chromosomal beta-lactamase gene in Klebsiella 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.

FIG. 1.

Phylogeny of the chromosomal beta-lactamase gene of K. pneumoniae. The tree was obtained by the neighbor-joining method using the HKY85+G model of nucleotide substitution (see Materials and Methods). The root was determined using the sequence of beta-lactamase TEM-1, the closest known relative to the chromosomal beta-lactamase of K. pneumoniae. Because of the long branch leading to the TEM-1 sequence, the root position is represented by a triangle in order to increase the scale of the figure so that the relationships among K. pneumoniae beta-lactamase sequences are clearly visible. The scale indicates the proportion of dissimilar nucleotides among sequences after correction for multiple substitutions. Three major groups of sequences are visible, corresponding to the SHV, LEN, and OKP families of beta-lactamase genes. The values at the nodes correspond to the percent confidence obtained after 1,000 bootstrap replicates. Bootstrap values inside the two groups corresponding to the SHV and LEN families were well below 50% for most nodes and are not given.

FIG. 2.

Phylogeny of the gyrA gene of K. pneumoniae. The tree was obtained by the neighbor-joining method using the Jukes-Cantor model of nucleotide substitution (see Materials and Methods). The tree was rooted at the midpoint of the branch leading from group KpII to the other two groups. The scale indicates the proportion of dissimilar nucleotides among sequences after correction for multiple substitutions. Three major groups of sequences are visible, corresponding to the K. pneumoniae phylogenetic groups KpI, KpII, and KpIII defined previously (11).