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Whole-Genome Sequencing and Bioinformatics as Pertinent Tools to Support Helicobacteracae Taxonomy, Based on Three Strains Suspected to Belong to Novel Helicobacter Species

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Whole-Genome Sequencing and Bioinformatics as Pertinent Tools to Support Helicobacteracae Taxonomy, Based on Three Strains Suspected to Belong to Novel Helicobacter Species

Elvire Berthenet et al. Front Microbiol.

Abstract

The present study describes three putative novel species received at the French National Reference Center for Campylobacters & Helicobacters (CNRCH). The CNRCH 2005/566H strain was isolated in 2005 from the feces of a patient with a hepatocellular carcinoma and gastroenteritis. Strain 48519 was isolated in 2017 from the blood of a male patient suffering from a bacteremia. Strain Cn23e was isolated from a gastric biopsy from a dog suffering from chronic gastritis. Biochemical and growth characteristics and electron microscopy for these three strains were studied. Their genomes were also sequenced. gyrA based phylogeny built with 72 nucleotide sequences placed CNRCH 2005/566H among the unsheathed enterohepatic helicobacters, close to Helicobacter valdiviensis; strain 48519 among the sheathed enterohepatic helicobacters, close to Helicobacter cinaedi; and strain Cn23e among gastric helicobacters, close to Helicobacter felis. 16S rRNA gene phylogeny showed similar results, but with weak discriminant strength. Average nucleotide identity and in silico DNA-DNA hybridization analyses revealed that CNRCH 2005/566H and 48519 strains belong to new putative species, but confirmed that Cn23e corresponds to H. felis. Cn23e was able to infect C57BL6 mice and to induce gastric inflammation. The genomics data, together with their different morphological and biochemical characteristics, revealed that these two strains represent novel Helicobacter species. We propose the following names: 'Helicobacter burdigaliensis,' with the type strain CNRCH 2005/566H ( =CECT 8850 =CIP 111660), and 'Helicobacter labetoulli,' with the type strain 48519 ( =CCUG 73475 =CIP 1111659). This study highlights that the diversity of the Helicobacteraceae family remains to be fully explored.

Keywords: Helicobacter genus; gyrA; novel species; taxonomy; whole-genome sequencing.

Figures

FIGURE 1
FIGURE 1
Observation of the three investigated isolates by transmission electron microscopy. (A) Observation of CNRCH 2005/566H revealing an amphitrichous bacterium with unsheathed flagella. Length of the bacteria (flagella excluded) was 2 μm on average. (B) Observation of Cn23e revealing a lophotrichous bacterium with sheathed flagella. Length of the bacteria (flagella excluded) was 6 μm on average. (C) Observation of 48519 revealing an amphitrichous bacterium with sheathed flagella. Length of the bacteria (flagella excluded) was 4 μm on average.
FIGURE 2
FIGURE 2
Genomic tree from gyrA based phylogeny built with 72 nucleotide sequences. The evolutionary history was inferred using the neighbor-joining method. The proportion of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Branches in blue correspond to gastric species, branches in black to enterohepatic species. Helicobacter species named in red are sheathed helicobacters, those named in black are unsheathed species.
FIGURE 3
FIGURE 3
Genomic tree from 16S rRNA gene based phylogeny built with 72 nucleotide sequences. The evolutionary history was inferred using the neighbor-joining method. The proportion of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Branches in blue correspond to gastric species, branches in black to enterohepatic species. Helicobacter species named in red are sheathed helicobacters, those named in black are unsheathed species.
FIGURE 4
FIGURE 4
Histological analyses of Cn23e-infected C56BL6 mice at 6 weeks post-oral gavage. (A) Quantification of the bacterial load in gastric biopsies from infected mice. Results were obtained by quantitative PCR as described in Section “Materials and Methods.” H. felis/1,000 murine cell ratio in non-infected (NI) and Cn23e-infected mice at 6 weeks post-infection (n = 5) versus 18 months post-infection (n = 5 for each group). Graphic representations are box plots, with the box representing 50% of values around the median (horizontal line) and the whiskers representing the minimum and maximum figures of all of the data. ∗∗p < 0.01, ns, non-significant. (B) Inflammation (INF), lymphoid infiltration (IL) scoring: for inflammation, scores range from 0 to 4; for the lymphoid infiltration, scores range from 0 to 3 (see section “Materials and Methods”). Data are plotted as bar graphs displaying the median ± standard deviation for each group. Non-infected (NI) (n = 5), Cn23e-infected (n = 5). For each group, the bar represents the arithmetic median of scores. ∗∗p < 0.01, ns, non-significant for Cn23e-infected mice versus NI. (C) Sections (3 μm thick) from paraffin-embedded tissues were processed for H&E staining. Partial sections of the large curvature of the stomach (fundus and corpus) are presented on these pictures. Representative histopathological features of NI and Cn23e-infected mice: polymorphonuclear infiltrates (black arrow) and lymphoid infiltrates (white arrow) are shown. Scale bars: 200 μm.

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