Phylogenetic Analysis Of Housekeeping Genes Research Articles

Genome-Based Reclassification of Strain KIST612, Previously Classified as Eubacterium limosum, into a New Strain of Eubacterium callanderi.

The strain KIST612, initially identified as E. limosum, was a suspected member of E. callanderi due to differences in phenotype, genotype, and average nucleotide identity (ANI). Here, we found that E. limosum ATCC 8486T and KIST612 are genetically different in their central metabolic pathways, such as that of carbon metabolism. Although 16S rDNA sequencing of KIST612 revealed high identity with E. limosum ATCC 8486T (99.2%) and E. callanderi DSM 3662T (99.8%), phylogenetic analysis of housekeeping genes and genome metrics clearly indicated that KIST612 belongs to E. callanderi. The phylogenies showed that KIST612 is closer to E. callanderi DSM 3662T than to E. limosum ATCC 8486T. The ANI between KIST612 and E. callanderi DSM 3662T was 99.8%, which was above the species cut-off of 96%, Meanwhile, the ANI value with E. limosum ATCC 8486T was not significant, showing only 94.6%. The digital DNA-DNA hybridization (dDDH) results also supported the ANI values. The dDDH between KIST612 and E. callanderi DSM 3662T was 98.4%, whereas between KIST612 and E. limosum ATCC 8486T, it was 57.8%, which is lower than the species cut-off of 70%. Based on these findings, we propose the reclassification of E. limosum KIST612 as E. callanderi KIST612.

MALDI-TOF MS database expansion for identification of Bacillus and related genera isolated from a pharmaceutical facility

Bacillus and related genera are among the main bacterial groups isolated from pharmaceutical production areas. The identification of Bacillus species and related genera by classical methods is particularly difficult, due to similarities between closely related species. The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is one of the most promising techniques for chemotaxonomic characterization of microorganisms, being an alternative to genotypic methods. This study aimed to identify Bacillus strains and related genera isolated from immunobiological production areas by phylogenetic analysis of housekeeping genes and expand the database associated with MALDI-TOF MS to improve their identification. In a previous study, 97 aerobic endospore-forming bacteria isolated from a pharmaceutical facility were analyzed by MALDI-TOF MS and 16S rRNA gene full-length sequencing. All strains were identified as Bacillus and related genera by the latest methodology. Among the 97 strains, 22 were unidentified and 2 strains were misidentified by MALDI-TOF MS. In the present study, these 24 strains were subjected to 16S rRNA gene phylogenetic analysis. Strains not identified at species level by this methodology were submitted to rpoB gene phylogenetic analysis. After identifying the strains, 19 of the 24 strains were incubated for 24, 48, and 72 h on Tryptic Soy Agar and Sheep Blood Agar and subjected to analysis by MALDI-TOF MS. A SuperSpectrum for each strain was created and entered into the equipment database. Finally, the 24 strains were again submitted to proteomic analysis by MALDI-TOF MS, and, at this time, all were correctly identified. The genotypic identification of in-house isolated strains and the introduction of these spectra in MALDI-TOF MS, in order to obtain a customized database, proved to be an extremely effective tool in the identification of Bacillus and related genera from pharmaceutical industry origin.

Validation of Species-Specific PCR Assays for the Detection of Pantoea ananatis, P. agglomerans, P. allii, and P. stewartii.

Species of Pantoea represent a group of plant pathogenic bacteria that infect a variety of agro-economically important plant species. Among these, a complex of P. ananatis, P. allii, P. agglomerans, and P. stewartii subsp. indologenes cause center rot in onion, resulting in significant economic losses. As species of Pantoea are phenotypically closely related, identification of Pantoea species relies on the sequencing and phylogenetic analysis of housekeeping genes. To aid in rapid identification of Pantoea species, efforts have been made in developing species-specific primers to be used in PCR assays. In the current study, two P. ananatis, one P. allii, one P. agglomerans, and three P. stewartii published primers as well as newly developed P. agglomerans PagR primers were evaluated for their specificity against 79 Pantoea strains, belonging to 15 different species. To ensure that selected primers were evaluated against accurately identified species, sequencing and phylogenetic analysis of housekeeping gene infB were conducted. Thereafter, PCR assays using selected species-specific primers were performed. The results showed that previously described P. ananatis-specific PANA_1008; P. allii-specific allii-leuS; P. stewartii-specific PANST_rpoB, 3614galE, and DC283galE primers; and one newly designed P. agglomerans-specific PagR primer pair were highly specific for their target Pantoea species. They accurately identified these strains into their species and, in some cases, their subspecies level. The findings of the current study will facilitate rapid and reliable identification of P. ananatis, P. agglomerans, P. allii, and P. stewartii.

Multiphase characterization of wild Vigna associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant Bradyrhizobium strains having high symbiotic compatibility with soybean and mungbean

ABSTRACT Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with V. riukiuensis grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to Bradyrhizobium japonicum, B. elkanii and B. jicamae family. However, analysis of symbiotic nifH and nodD1 genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with B. elkanii, growth was observed at 40°C, which exceed the highest record for B. elkanii to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with B. diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V. riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than B. diazoefficiens USDA110. In addition, six isolates attained higher soybean biomass production compared with B. diazoefficiens USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29% higher soybean biomass production than B. diazoefficiens USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics. Abbreviations ARA: acetylene reduction assay, ISR: intergenic spacer regions, ITS: internal transcribed spacer

Mesorhizobium jarvisii sv. astragali as predominant microsymbiont for Astragalus sinicus L. in acidic soils, Xinyang, China

To identify the rhizobia nodulating Astragalus sinicus in the acid soils at Xinyang, China and to estimate the effects of fertilization strategies on the rhizobial community. Soil samples were collected from treatments submitted to different fertilization strategies (chemical fertilizers, A. sinicus green manure, various input levels). Nodules obtained from the A. sinicus trapping plants grown in the soil samples were used for rhizobial isolation. The isolates were grouped into PCR-RFLP genotypes based on analyses of 16S rRNA gene and IGS, and representatives for each genotype were further subjected to phylogenetic analyses of housekeeping genes (16S rRNA gene, atpD, glnII, and rpoB), as well as symbiotic genes (nodC and nifH). Soil physicochemical characteristics were also estimated and redundancy analysis was performed to examine the relations between soil features and distribution of rhizobial genotypes across different fertilization treatments. A total of 257 rhizobial isolates were obtained and discriminated into 12 IGS types in a single 16S rRNA type by RFLP analyses. Further 16S rRNA gene sequence-based phylogeny and multilocus sequence analysis (MLSA) distinguished a minor group (10 strains, 4 IGS types) affiliated to Mesorhizobium huakuii and a main group (247 strains, 8 IGS types) to Mesorhizobium jarvisii, for which a novel symbiovar, sv. astragali, was described. The long-term input of A. sinicus green manure increased rhizobial diversity and modified rhizobial community structures, via the increase of organic matter and decrease of soil pH, available phosphate, available potassium, and total salt. M. jarvisii sv. nov. astragali was the predominant microsymbiont of A. sinicus in the tested acidic soils. The long-term input of green manure modified the soil features, enhancing the genetic diversity of rhizobia associated with A. sinicus and alternating the rhizobial community structure.

Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates.

Municipal wastewater treatment facilities are considered to be “hotspots” for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7–9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A) qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B) that small qnrS-harboring pGNB2-like plasmids are ubiquitous in wastewater treatment facilities and are most likely environmental in origin.

Symbiotic efficiency and genetic diversity of soybean bradyrhizobia in Brazilian soils

The symbiotic N2-fixing genus Bradyrhizobium includes 29 species distributed throughout different geographic regions. Only five species have recently been described based on isolates from tropical soils, three species from Brazil (B. manausense, B. ingae, B. neotropicale) and two species from Peru (B. paxllaere e B. icense), although tropical region is considered to be the origin of legume rhizobia symbiosis. Besides, some authors suggested that Bradyrhizobium was introduced in Brazil with first soybeans inoculants from USA. In this work, 46 Bradyrhizobium strains were isolated from soils collected in different regions of Brazil (Midwest, Northeast, Southeast, and South), using soybean as a trap plant. These strains were characterized genetically by analyzing the 16S rRNA gene and five housekeeping genes (atpD, gyrB, dnaK, recA, and rpoB). They were also characterized in terms of their symbiotic efficiency with soybean plants grown under axenic conditions in Leonard jars. The phylogenetic analysis of housekeeping genes revealed the possible presence of novel species in the Northeast and Southeast soils, some of which exhibited high symbiotic efficiency with soybean plants. These results emphasize the great diversity among native strains belonging to Bradyrhizobium genus in Brazilian soils as well as potential ones to be used as inoculants. They also indicate that symbiotically efficient native bradyrhizobia occur in Brazilian soils and are independent of strains introduced as soybean inoculant.

Insights into the environmental reservoir of pathogenic Vibrio parahaemolyticus using comparative genomics.

Vibrio parahaemolyticus is an aquatic halophilic bacterium that occupies estuarine and coastal marine environments, and is a leading cause of seafood-borne food poisoning cases. To investigate the environmental reservoir and potential gene flow that occurs among V. parahaemolyticus isolates, the virulence-associated gene content and genome diversity of a collection of 133 V. parahaemolyticus isolates were analyzed. Phylogenetic analysis of housekeeping genes, and pulsed-field gel electrophoresis, demonstrated that there is genetic similarity among V. parahaemolyticus clinical and environmental isolates. Whole-genome sequencing and comparative analysis of six representative V. parahaemolyticus isolates was used to identify genes that are unique to the clinical and environmental isolates examined. Comparative genomics demonstrated an O3:K6 environmental isolate, AF91, which was cultured from sediment collected in Florida in 2006, has significant genomic similarity to the post-1995 O3:K6 isolates. However, AF91 lacks the majority of the virulence-associated genes and genomic islands associated with these highly virulent post-1995 O3:K6 genomes. These findings demonstrate that although they do not contain most of the known virulence-associated regions, some V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates. This highlights the dynamic nature of the V. parahaemolyticus genome allowing them to transition between aquatic and host-pathogen states.

The capsule biosynthesis locus of Haemophilus influenzae shows conspicuous similarity to the corresponding locus in Haemophilus sputorum and may have been recruited from this species by horizontal gene transfer.

The newly described species Haemophilus sputorum has been cultured from the upper respiratory tract of humans and appears to have little pathogenic potential. The species encodes a capsular biosynthesis locus of approximately 12 kb composed of three distinct regions. Region I and III genes, involved in export and processing of the capsular material, show high similarity to the corresponding genes in capsulate lineages of the pathogenic species Haemophilus influenzae; indeed, standard bexA and bexB PCRs for detection of capsulated strains of H. influenzae give positive results with strains of H. sputorum. Three ORFs are present in region II of the sequenced strain of H. sputorum, of which a putative phosphotransferase showed homology with corresponding genes from H. influenzae serotype c and f. Phylogenetic analysis of housekeeping genes from 24 Pasteurellaceae species showed that H. sputorum was only distantly related to H. influenzae. In contrast to H. influenzae, the capsule locus in H. sputorum is not associated with transposases or other transposable elements. Our data suggest that the capsule locus of capsulate lineages of H. influenzae may have been recruited relatively recently from the commensal species H. sputorum by horizontal gene transfer.

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.

Comparative polyphasic characterization of Streptococcus phocae strains with different host origin and description of the subspecies Streptococcus phocae subsp. salmonis subsp. nov.

A polyphasic study was undertaken to clarify the taxonomic position of Streptococcus phocae strains isolated from Atlantic salmon (Salmo salar) cage-farmed in Chile. Four salmon and three seal isolates showed minor differences in the SDS-PAGE protein analysis. Thus, a major protein band present in the salmon isolates, of approximately 22.4 kDa, was absent in the pinniped strains, regardless of the growth media employed. In addition, the pinniped strains showed protein bands with molecular masses of 71.5 and 14.2 kDa, when grown on trypticase soy agar supplemented with 1% NaCl, or 25.6 kDa, when grown on Columbia blood agar, not present in the Atlantic salmon strains. A high similarity in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS spectra of the strains was observed, although some minor peaks were absent in the fish isolates. Fatty acid methyl esters from isolates with different host origin significantly (P<0.05) differed in the content of C16:0, C17:0, C18:1ω9c, C20:4ω6,9,12,15c and summed features 3, 5 and 8. The salmon isolates formed a separate cluster in the phylogenetic analysis of housekeeping genes, separately or as concatenated sequences. Sequence divergences among salmon and seal strains were in the range of inter-subspecies differentiation for groEL (2.5%), gyrB (1.8%), recN (2.1%), rpoB (1.7%) and sodA (2.0%) genes. DNA-DNA hybridization results confirmed those of sequencing, showing reassociation values between seal and salmon strains close to the borderline of species definition. Differences in growth at low temperatures and in the haemolytic capacities were also observed between both groups of isolates. On the basis of all these results, the salmon isolates represent a novel subspecies of S. phocae, for which the name Streptococcus phocae subsp. salmonis subsp. nov. is proposed. The type strain is C-4T (=CECT 7921T=DSM 24768T). The subspecies Streptococcus phocae subsp. phocae subsp. nov. is automatically created. An emended description of S. phocae is also provided.

Replicon-Dependent Differentiation of Symbiosis-Related Genes in Sinorhizobium Strains Nodulating Glycine max

In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

Genetic diversity of rhizobia nodulating lentil (Lens culinaris) in Bangladesh

In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1–94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5–10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli–R. leguminosarum group and may correspond to novel species in the genus Rhizobium.

Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E).

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.

Rhizobium helanshanense sp. nov., a bacterium that nodulates Sphaerophysa salsula (Pall.) DC. in China

Studying rhizobia in the root nodules of Sphaerophysa salsula (Pall.) DC in the northwest of China, we obtained five strains classified as genus Rhizobium on the basis of their 16S rRNA gene sequences. The sequence similarity of strain CCNWQTX14(T) with the most related species was 99.0%. Further phylogenetic analysis of housekeeping genes (recA and atpD) suggested the five strains comprised a novel lineage within Rhizobium. The nifH and nodD gene sequences of CCNWQTX14(T) were phylogenetically closely related with those of Sinorhizobium kummerowiae and R. sphaerophysae, respectively. The five strains isolated from different places were also distinct from related Rhizobium species using ERIC fingerprint profiles. The DNA-DNA hybridization value was 41.8% between CCNWQTX14(T) and Rhizobium sphaerophysae CCNWGS0238(T). Our novel strains were only able to form effective nodules on its original host Sphaerophysa salsula. Our data showed that the five Rhizobium strains formed a unique genomic species, for which a novel species Rhizobium helanshanense sp. nov. is proposed. The type strain is CCNWQTX14(T) (=ACCC 16237(T) =HAMBI 3083(T)).

Virulent Clones of Klebsiella pneumoniae: Identification and Evolutionary Scenario Based on Genomic and Phenotypic Characterization

Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländer's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23K1 and clone CC82K1, were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82K1 indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23K1 remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent clones associated with specific infections and provide an evolutionary framework for research into the links between clones, virulence and other genomic features in K. pneumoniae.