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Antimicrobial Agents and Chemotherapy, December 2005, p. 5149-5152, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5149-5152.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Variants of the Klebsiella pneumoniae OKP Chromosomal Beta-Lactamase Are Divided into Two Main Groups, OKP-A and OKP-B

Cindy Fevre, Virginie Passet, François-Xavier Weill, Patrick A. D. Grimont, and Sylvain Brisse*

Unité Biodiversité des Bactéries Pathogènes Emergentes (U389 INSERM), Institut Pasteur, 25-28 rue du Dr. Roux, 75 724 Paris Cedex 15, France

Received 29 July 2005/ Returned for modification 16 September 2005/ Accepted 22 September 2005


   ABSTRACT
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Two blaOKP subgroups were found, diverging by 4.2%. Subgroups blaOKP-A (10 enzyme variants, pIs from 7.1 to 8.3) and blaOKP-B (11 variants, pI 7.1) showed similar antibiotic susceptibilities. Sequencing of rpoB, gyrA, and mdh demonstrated a concordant subdivision of Klebsiella pneumoniae phylogenetic group KpII into two subgroups, KpII-A and KpII-B.


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Klebsiella pneumoniae clinical isolates are naturally resistant to ampicillin, amoxicillin, carbenicillin, and ticarcillin, but not to extended-spectrum beta-lactams, due to the constitutive expression of a chromosomally encoded beta-lactamase. Three families of the chromosomal beta-lactamase gene, blaSHV, blaOKP, and blaLEN, evolved from a common ancestor over millions of years, in parallel with the diversification of the three K. pneumoniae phylogenetic groups KpI, KpII, and KpIII (3, 6). The blaOKP family was found in group KpII (6) and was more heterogeneous than the SHV and LEN families, with four OKP enzyme variants. These variants appeared to belong to two OKP subgroups, but a higher number of variants was judged necessary to firmly demonstrate the phylogenetic distinctness of these subgroups (6). In addition, because homologous recombination is more frequent among closely related strains than between distant phylogenetic groups (8, 10), the correspondence observed at the level of the three phylogenetic groups between beta-lactamase and housekeeping gene classifications (5, 6) could be disrupted by homologous gene transfer within KpII.

Precise identification of bla variants can be critical for epidemiological purposes and infection control. The objectives of the present study were to discover additional OKP enzyme variants, to determine whether strains harboring distinct OKP variants differ in their antibiotic susceptibility, and to determine whether blaOKP gene variants are subdivided into groups that correspond to housekeeping gene sequence groups.

In order to discover new OKP variants, we selected 24 KpII strains (Table 1), including the three reference strains SB18, SB59, and SB30 (6), 14 clinical isolates, 4 fecal isolates, and 3 environmental isolates. The clinical strains were identified in a previous study (2), in which 6.9% of K. pneumoniae clinical isolates belonged to KpII. The fecal isolates were identified by gyrA PCR restriction fragment length polymorphism screening (1, 2) of 137 K. pneumoniae fecal carriage isolates (14). The entire coding region (861 bp) was amplified by PCR using primers blaU5F (AAA GAG GAA TTG TGA ATC AGC) and blaU4R (GTT CAC CAC CAT CAT TAC CG). The annealing temperature was 50°C. For sequencing, internal primers blaU6F (TGC GCC AGA TCG GYG ACA A) and blaU7R (CGT CGC CGG GAA GCG C) were used in addition to the PCR primers. Sequencing was performed as described previously (4). Isoelectric point determination and antibiotic susceptibility testing were performed as described previously (5). In order to investigate the phylogenetic structure of the KpII group, an internal portion of the three housekeeping genes rpoB, gyrA, and mdh was amplified and sequenced with primers Vic2 and Vic3 (5), gyr-A and gyr-C (5), and mdh130-F and mdh867-R (4).


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  		TABLE 1. Klebsiella pneumoniae strains studied and their characteristics

 
Alignment of the 24 blaOKP sequences and 2 additional blaOKP sequences (13) showed no insertion-deletion events and no missing or ambiguous data. Alignment of the 26 nucleotide sequences showed 82 (8.9%) polymorphic sites, corresponding to 41 synonymous substitutions, 38 nonsynonymous substitutions, and 3 complex sites. Twenty-one alleles were distinguished. Phylogenetic analysis revealed an obvious subdivision of the blaOKP sequences into two subgroups, named blaOKP-A and blaOKP-B (Fig. 1A). The nucleotide sequence divergence was lower between blaOKP-A and blaOKP-B subgroups than between any of these subgroups and blaSHV or blaLEN (Table 2).



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  		FIG. 1. Phylogeny of the chromosomal beta-lactamase gene (A) and of the rpoB gene (B) of K. pneumoniae phylogenetic group KpII strains. Trees were obtained with PAUP* version 4.0b10, using the neighbor-joining method with Kimura's two-parameter distance. The tree was rooted at the midpoint of the branch leading from strains SB132 (K. pneumoniae group KpI) and SB1 (K. pneumoniae group KpIII) to the other groups. The values at the nodes correspond to the bootstrap values obtained after 1,000 replicates. Trees obtained with gyrA and mdh gene sequences (not shown) were similar to the rpoB phylogeny, except that the branch leading to strain SB2728 was shorter.

 

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  		TABLE 2. Sequence divergence of the four genes among Klebsiella pneumoniae phylogenetic groups

 
Twenty-one amino acid sequences, OKP-A-1 to OKP-A-10 and OKP-B-1 to OKP-B-11, were found (Table 1). The proposed nomenclature of OKP enzyme variants is available at http://www.pasteur.fr/recherche/genopole/PF8/betalact_en.html. The mean amino acid sequence divergence within OKP-A and OKP-B was 2.21% and 1.15%, respectively, but it was 5.45% between them.

Isoelectrofocusing of the beta-lactamases revealed only one band per isolate (Table 1). All isolates presented similar MICs. The 24 isolates showed resistance against amoxicillin (range of MICs, 128 to 256 µg/ml) and ticarcillin (64 to 128 µg/ml). Resistance to these two drugs fell in the presence of clavulanic acid (4 to 8 µg/ml and 1 to 8 µg/ml, respectively). Resistance to piperacillin (4 to 16 µg/ml) was intermediate and fell in the presence of tazobactam (1 to 8 µg/ml). All isolates were susceptible to cephalothin (1 to 8 µg/ml), cefoxitin (2 to 8 µg/ml), cefotaxime (<0.06 to 0.125 µg/ml), ceftazidime (<0.06 to 0.25 µg/ml), cefepime (<0.06 to 0.125 µg/ml), aztreonam (<0.06 µg/ml), and imipenem (0.25 to 1 µg/ml). No difference was found between isolates harboring blaOKP-A and blaOKP-B genes.

In order to investigate the phylogenetic structure of group KpII, an internal portion of genes rpoB, gyrA, and mdh was sequenced. The proportion of polymorphic sites was 3.9% (37/940) for rpoB, 1.56% (6/383) for gyrA, and 5.4% (37/688) for mdh. The lower amount of variable sites in the housekeeping genes compared to that in the blaOKP gene (8.9%) was mainly due to the rarity of nonsynonymous substitutions (none in rpoB and gyrA and only five in mdh), indicative of a stronger selective pressure against amino acid changes, relative to the beta-lactamase gene.

Phylogenetic analysis of the housekeeping gene sequences demonstrated the subdivision of KpII into two subgroups, KpII-A and KpII-B, which corresponded totally to blaOKP-A and blaOKP-B subgroups (Fig. 1B and Table 2).

Under the molecular-clock hypothesis, sequence variation at synonymous sites, Ks, can be used to estimate the time since divergence of the last common ancestor (7, 9). For the mdh, rpoB, and gyrA genes, the Ks values for the pairwise comparisons of KpII-A and KpII-B phylogenetic groups were 0.072, 0.074, and 0.028, respectively (12). The Ks values of genes mdh, rpoB, and gyrA between Escherichia coli and Salmonella enterica, which diverged 140 million years ago (11), were 0.875, 0.223, and 0.367, respectively, indicating that KpII-A and KpII-B would have diverged 11 to 46 million years ago.

In conclusion, we showed that the chromosomal beta-lactamase blaOKP gene diversified into many variants which can be classified into two major subgroups. The diversity of OKP enzymes occurred in parallel to the phylogenetic diversification of KpII-A and KpII-B subgroups but without concomitant phenotypic changes in beta-lactam susceptibility. This result is totally similar to the situation found at higher levels of phylogenetic divergence (5, 6). Therefore, even at this lower level of nucleotide divergence, horizontal transfer of the chromosomal beta-lactamase gene does not appear to occur frequently between strains, and this gene may therefore be used as a phylogenetic marker. It is interesting to note that the beta-lactamase gene has a faster evolutionary rate than those of housekeeping genes and will thus give better resolution in distinguishing strains, as was observed in K. oxytoca (5).

Nucleotide sequence accession numbers. Nucleotide sequences were submitted to EMBL/GenBank databases under accession numbers AM051091 to AM051186.


   ACKNOWLEDGMENTS
 
We thank P. Toivanen and D. Hansen-Schröder for providing fecal isolates.


   FOOTNOTES
 
* Corresponding author. Mailing address: Unité Biodiversité des Bactéries Pathogènes Emergentes (U389 INSERM), Institut Pasteur, 25-28 rue du Dr. Roux, 75 724 Paris Cedex 15, France. Phone: 33 (0) 1 40 61 33 57. Fax: 33 (0) 1 45 68 88 37. E-mail: sbrisse{at}pasteur.fr. Back


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Antimicrobial Agents and Chemotherapy, December 2005, p. 5149-5152, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5149-5152.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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