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

Genomic perspective on the bacillus causing paratyphoid B fever

Paratyphoid B fever (PTB) is caused by an invasive lineage (phylogroup 1, PG1) of Salmonella enterica serotype Paratyphi B (SPB). However, little was known about the global population structure, geographic distribution, and evolution of this pathogen. Here, we report a whole-genome analysis of 568 historical and contemporary SPB PG1 isolates, obtained globally, between 1898 and 2021. We show that this pathogen existed in the 13th century, subsequently diversifying into 11 lineages and 38 genotypes with strong phylogeographic patterns. Following its discovery in 1896, it circulated across Europe until the 1970s, after which it was mostly reimported into Europe from South America, the Middle East, South Asia, and North Africa. Antimicrobial resistance recently emerged in various genotypes of SPB PG1, mostly through mutations of the quinolone-resistance-determining regions of gyrA and gyrB. This study provides an unprecedented insight into SPB PG1 and essential genomic tools for identifying and tracking this pathogen, thereby facilitating the global genomic surveillance of PTB

Acetylation of surface carbohydrates in bacterial pathogens requires coordinated action of a two-domain membrane-bound acyltransferase

Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an "AT3-SGNH fused" domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in O-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates

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

Gel electrophoresis image of the multiplex PCR used for the screening of <i>S. enterica</i> serotypes Typhi and Paratyphi A.

<p>M: 100-bp DNA ladder (Invitrogen, Carlsbad, CA, USA); lane 1, <i>S. enterica</i> serotype Typhi isolate 09-2213, lane 2, <i>S. enterica</i> serotype Typhi isolate 09-6791; lane 3, <i>S. enterica</i> serotype Paratyphi A isolate 09-2344; lane 4, <i>S. enterica</i> serotype Paratyphi A isolate 09-9426; lane 5, <i>S. enterica</i> serotype Typhimurium strain LT2; lane 6: <i>S. enterica</i> serotype Emek strain 297K; lane 7, <i>Escherichia coli</i> isolate 10-7043; lane 8: <i>Shigella flexneri</i> isolate 11-1445; lane 9, <i>S. enterica</i> serotypes Typhi and Paratyphi A reference strains Ty2 and 1K, respectively; lane 10, negative control (sterile water). We checked that the template DNA was of sufficiently high quality, by amplifying the housekeeping genes <i>aroC</i> for <i>Salmonella</i> and <i>adk</i> for <i>E. coli</i>/<i>Shigella</i> (data not shown), as previously described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Achtman1" target="_blank">[32]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Wirth1" target="_blank">[33]</a>.</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