Dataset Information

Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

ABSTRACT: "Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n = 9), the Czech Republic (n = 2), and Germany (n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

SUBMITTER: Grankvist A

PROVIDER: S-EPMC4572549 | biostudies-other | 2015 Oct

REPOSITORIES: biostudies-other

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Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

Grankvist Anna A Moore Edward R B ER Svensson Stadler Liselott L Pekova Sona S Bogdan Christian C Geißdörfer Walter W Grip-Lindén Jenny J Brandström Kenny K Marsal Jan J Andréasson Kristofer K Lewerin Catharina C Welinder-Olsson Christina C Wennerås Christine C

Journal of clinical microbiology 20150708 10

"Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species wi ...[more]

PMID: 26157152

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Project description:BackgroundThe oral spirochete bacterium Treponema denticola is associated with both the incidence and severity of periodontal disease. Although the biological or phenotypic properties of a significant number of T. denticola isolates have been reported in the literature, their genetic diversity or phylogeny has never been systematically investigated. Here, we describe a multilocus sequence analysis (MLSA) of 20 of the most highly studied reference strains and clinical isolates of T. denticola; which were originally isolated from subgingival plaque samples taken from subjects from China, Japan, the Netherlands, Canada and the USA.ResultsThe sequences of the 16S ribosomal RNA gene, and 7 conserved protein-encoding genes (flaA, recA, pyrH, ppnK, dnaN, era and radC) were successfully determined for each strain. Sequence data was analyzed using a variety of bioinformatic and phylogenetic software tools. We found no evidence of positive selection or DNA recombination within the protein-encoding genes, where levels of intraspecific sequence polymorphism varied from 18.8% (flaA) to 8.9% (dnaN). Phylogenetic analysis of the concatenated protein-encoding gene sequence data (ca. 6,513 nucleotides for each strain) using Bayesian and maximum likelihood approaches indicated that the T. denticola strains were monophyletic, and formed 6 well-defined clades. All analyzed T. denticola strains appeared to have a genetic origin distinct from that of 'Treponema vincentii' or Treponema pallidum. No specific geographical relationships could be established; but several strains isolated from different continents appear to be closely related at the genetic level.ConclusionsOur analyses indicate that previous biological and biophysical investigations have predominantly focused on a subset of T. denticola strains with a relatively narrow range of genetic diversity. Our methodology and results establish a genetic framework for the discrimination and phylogenetic analysis of T. denticola isolates, which will greatly assist future biological and epidemiological investigations involving this putative 'periodontopathogen'.

Project description:More than 75 "species-level" phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n = 71) of diverse geographical origins. This comprises phylogroup 1 (n = 23) and phylogroup 2 (n = 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n = 41) and Treponema putidum (n = 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct "species-level" phylotypes. These respectively corresponded to "Treponema vincentii" (n = 11), Treponema medium (n = 1), "Treponema sinensis" (Treponema sp. IA; n = 4), Treponema sp. IB (n = 3), and Treponema sp. IC (n = 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE:Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

Project description:Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Dataset Information

Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

Publications

Multilocus Sequence Analysis of Clinical "Candidatus Neoehrlichia mikurensis" Strains from Europe.

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