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

The speciation and hybridization history of the genus Salmonella.

Bacteria and archaea make up most of natural diversity, but the mechanisms that underlie the origin and maintenance of prokaryotic species are poorly understood. We investigated the speciation history of the genus Salmonella, an ecologically diverse bacterial lineage, within which S. enterica subsp. enterica is responsible for important human food-borne infections. We performed a survey of diversity across a large reference collection using multilocus sequence typing, followed by genome sequencing of distinct lineages. We identified 11 distinct phylogroups, 3 of which were previously undescribed. Strains assigned to S. enterica subsp. salamae are polyphyletic, with two distinct lineages that we designate Salamae A and B. Strains of the subspecies houtenae are subdivided into two groups, Houtenae A and B, and are both related to Selander's group VII. A phylogroup we designate VIII was previously unknown. A simple binary fission model of speciation cannot explain observed patterns of sequence diversity. In the recent past, there have been large-scale hybridization events involving an unsampled ancestral lineage and three distantly related lineages of the genus that have given rise to Houtenae A, Houtenae B and VII. We found no evidence for ongoing hybridization in the other eight lineages, but detected subtler signals of ancient recombination events. We are unable to fully resolve the speciation history of the genus, which might have involved additional speciation-by-hybridization or multi-way speciation events. Our results imply that traditional models of speciation by binary fission and divergence are not sufficient to account for Salmonella evolution

CRISPR Typing and Subtyping for Improved Laboratory Surveillance of Salmonella Infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool

CRISPR profiles obtained for 12 strains and two genomes of <i>S. enterica</i> serotype Paratyphi A.

<p>The spacer sequences and direct repeat (DR) sequences are indicated. The set of strains and genomes has been reported elsewhere <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a>. n” is the number of strains harboring each profile. The primers used for serotype Paratyphi A-specific amplification (PA-F and PA-R) and for amplification of the entire CRISPR1 sequence (A1 and A2) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a> are indicated in different colors.</p

CRISPR profiles obtained for the 18 strains and two genomes of <i>S. enterica</i> serotype Typhi.

<p>The spacer sequences and direct repeat (DR) sequences are indicated. The set of strains and genomes has been reported elsewhere <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a>. “n” is the number of strains harboring each profile. Haplotypes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Roumagnac1" target="_blank">[19]</a> are indicated, to illustrate the genetic diversity of the strains studied. ND, not determined. The primers used for serotype Typhi-specific amplification (TY-F and TY-R) and those for amplification of the entire CRISPR2 sequence (B1 and B2) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a> are indicated in different colors.</p

Strategy used to design TY-R, the reverse primer for the serotype Typhi-specific PCR assay.

<p>The DR sequence upstream from EntB0/EntB0var1 is indicated in black letters. The spacers EntB0 and EntB0var1 are indicated in blue letters. The nucleotides belonging to the template for primer TY-R are represented by letters of larger size. The sequence of TY-R is indicated in red, with the deliberate mismatch in green. Mismatch positions between TY-R and the templates of serotypes Emek (EntB0) and Enteritidis (EntB0var1) are underlined.</p

Results of conventional PCR assays targeting <i>S. enterica</i> serotypes Typhi and Paratyphi A.

<p>¹ The details of the strains are provided in Supplemental <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd-0002671-t001" target="_blank">Table 1</a>.</p><p>²+, amplicon of the expected size; −, no amplicon of the expected size.</p

EvaGreen real-time PCR assay.

<p>(A) Amplification and melting curves for 43 serotype Typhi isolates tested in duplicate with the TY-F and TY-R primers, yielding a mean Ct value of 22.6±1.1 and a single melting curve peak at 82.5–83°C. The two negative controls (<i>S. enterica</i> serotype Paratyphi A 1K and sterile water), also tested in duplicate, appear as flat lines. (B) Amplification and melting curves for 37 serotype Paratyphi A isolates tested in duplicate with the PA-F and PA-R primers, yielding a mean Ct value of 23.1±1.1 and a melting curve peak at 85–85.5°C. The two negative controls (<i>S. enterica</i> serotype Typhi Ty2 and sterile water), also tested in duplicate, appear as flat lines.</p

Supplement 2008-2010 (no. 48) to the White-Kauffmann-Le Minor scheme.

International audienceThis supplement (no. 48) of the White-Kauffmann-Le Minor scheme reports on the characterization of 63 new Salmonella serovars and 25 new variants of previously described Salmonella serovars recognized by the WHO Collaborating Centre for Reference and Research on Salmonella between 2008 and 2010. Forty-four new serovars were assigned to Salmonella enterica subspecies enterica, 12 to subspecies salamae, two to subspecies arizonae, two to subspecies diarizonae and three to subspecies houtenae. All these new serovars or new variants are described with their multilocus sequence type