The infra-specific phylogenetic diversity and genetic structure of both Klebsiella pneumoniae and Klebsiella oxytoca was investigated using a combination of randomly amplified polymorphic DNA (RAPD) analysis, sequencing of gyrA and parC genes, and automated ribotyping. After RAPD analysis with four independent primers of 120 clinical isolates collected from 22 European hospitals in 13 countries, K. pneumoniae isolates fell into three clusters and K. oxytoca isolates fell into two clusters, while Klebsiella planticola isolates formed a sixth cluster. Each cluster was geographically widespread. K. pneumoniae cluster I (KpI) accounted for 80% of the isolates of this species and included reference strains of the three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. Clusters KpII and KpIII were equally represented, as were the two K. oxytoca clusters. Individualization of each cluster was fully confirmed by phylogenetic analysis of gyrA and parC gene sequences. In addition, sequence data supported the evolutionary separation of K. pneumoniae from a phylogenetic group including K. oxytoca, Klebsiella terrigena, K. planticola and Klebsiella ornithinolytica. Automated ribotyping using Mlu I appeared suitable for identification of each Klebsiella cluster. The adonitol fermentation test was found to be useful for cluster identification in K. pneumoniae, since it was negative in all strains of clusters KpIII and in some KpII strains, but always positive in cluster KpI. The usefulness of gyrA and parC sequence data for population genetics and cluster identification in bacteria was demonstrated, even for the phylogenetic positioning of quinolone-resistant isolates.

On the basis of the idea that DNA sequences encoding cell surface-exposed regions of outer membrane proteins are genus or species specific, two oligonucleotide probes which were based on the PhoE protein of Klebsiella pneumoniae were evaluated. In slot blot hybridizations and in polymerase chain reactions, no cross-hybridizations were observed with non-Klebsiella strains. When the probes were tested on 75 different K-antigen reference Klebsiella strains, 16 strains were not recognized although they did produce PhoE protein under phosphate starvation. To determine whether these 16 strains belong to (a) different species, the reference strains were also tested for the ability to produce indole and to grow at 10 degrees C and their whole-cell fatty acid patterns were analyzed by gas chromatography. A strong correlation was observed among (i) reaction with the probes, (ii) the inability to produce indole, (iii) the inability to grow at 10 degrees C, and (iv) the presence of the hydroxylated fatty acid C14:0-2OH. From these results we conclude that the two oligonucleotides are specific for the species K. pneumoniae. Furthermore, analysis of fatty acid patterns appears to be a useful tool to distinguish K. pneumoniae from other Klebsiella species.