Article

Genomic and clinical evidence uncovers the enterohepatic species Helicobacter valdiviensis as a potential human intestinal pathogen

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Abstract

Background: Helicobacter valdiviensis is a recently described enterohepatic species isolated from wild bird's fecal samples. Currently, its pathogenic potential and clinical significance are unknown mainly due to the lack of whole-genome sequences to compare with other helicobacters and the absence of specific screenings to determine its prevalence in humans. Materials and methods: The species type strain (WBE14(T) ) was whole-genome-sequenced, and comparative analyses were carried out including the genomes from other Helicobacter species to determine the exact phylogenetic position of H. valdiviensis and to study the presence and evolution of virulence determinants. In parallel, stools from diarrheic patients and healthy individuals were screened by PCR to assess the clinical incidence of H. valdiviensis. Results: Helicobacter valdiviensis belongs to a monophyletic clade conformed by H. canadensis, H. pullorum, H. winghamensis, H. rodentium, and H. apodemus. Its predicted genome size is 2 176 246 bp., with 30% of G+C content and 2064 annotated protein-coding genes. The patterns of virulence factors in H. valdiviensis were similar to other enterohepatic species, but evidence of horizontal gene transfer from Campylobacter species was detected for key genes like those coding for the CDT subunits. Positive PCR results confirmed the presence of H. valdiviensis in 2 of 254 (0.78%) stools of patients with acute diarrhea while not a single sample was positive in healthy individuals. Conclusions: Horizontal gene transfer has contributed to shape the gene repertory of H. valdiviensis, which codes for virulence factors conserved in other pathogens that are well-known human pathogens. Additionally, the detection of H. valdiviensisDNA in diarrheic patients supports its role as a potential emergent intestinal pathogen. Further, sampling efforts are needed to uncover the clinical relevance of this species, which should be accomplished by the isolation of H. valdiviensis from ill humans and the obtention of whole genomes from clinical isolates.

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... Enterohepatic species of the genus Helicobacter (EHH) have become increasingly important as emerging pathogens (Collado et al., 2014;Fresia et al., 2017) due to their association with acute gastroenteritis, inflammatory bowel disease (Crohn's disease and ulcerative colitis) and liver, gallbladder and bile ducts diseases (Man et al., 2008;Martel et al., 2009;Bascuñana et al., 2011;Thomson et al., 2011). ...
... Although EHH are frequently detected in clinical samples by PCR (Fox, 2002, Mateos-Muñoz et al., 2013Fresia et al., 2017), only occasionally they are isolated using medium and atmospheric conditions designed for Campylobacter spp. According to the Bergey's Manual, there is no ideal method for the isolation of EHH and the recovery of these species is often optimised by the simultaneous use of selective and nonselective procedures (On et al., 2005). ...
... Gastric Helicobacter species such as H. pylori, H. mustelae, H. nemestrinae and H. acinonychis do not require atmospheric hydrogen for growth, while the remaining species have commonly been isolated in microaerobic conditions containing hydrogen, and as stated in the last edition of Bergey´s Manual it is unknown whether it is an essential component, as in the case of some Campylobacter species, or simply enhances Helicobacter growth on culture media (On et al., 2005;2017). Therefore, all cultures were carried out under microaerobic conditions with and without hydrogen supplement. ...
Article
This research aims to compare the culturing conditions for enterohepatic Helicobacter, evaluating culture media, incubation atmosphere and susceptibility to antimicrobials used to generate selective conditions. Four common media for the closely related genus Campylobacter (Columbia, Bolton, Brucella, and CCDA agar), as well as the need for hydrogen in the microaerobic incubation atmosphere, were evaluated. Serial dilutions of 13 strains belonging to six species (H. apodemus, H. bilis, H. canicola, H. canis, H. equorum, and Helicobacter sp.) were inoculated in each media and incubated at 37 °C for 48 to 96 h using CampyGen (OXOID) and gaseous exchange (including hydrogen) in parallel. Columbia or Brucella agars were the most appropriate for culturing EHH (P<0·05). However, there was no significant difference between the atmospheres evaluated (P=0·13). In addition, minimal inhibitory concentration for six antibiotics showed that all isolates were resistant to trimethoprim, while for the rest of the antibiotics (cephalothin, cefoperazone, cefsulodin, teicoplanin, and vancomycin) the inhibition range was between 8 and 64 μg/ml. Our findings suggest that Columbia or Brucella media, regardless of the use of hydrogen, can be used for the EHH isolation. In addition, the concentration of antibiotics included in commercial campylobacteria supplements is suitable for EHH species recovery. This article is protected by copyright. All rights reserved.
... Although the pathogenicity to humans of many species found in the genus is still unknown, Helicobacter pylori is considered the most important species of the genus Helicobacter, which is associated with human gastric lesions and malignancy. In addition, various enterohepatic species of the Helicobacter genus such as H. cinaedi [5,6], H. canis [7], H. pullorum [8][9][10][11], H. valdiviensis [12], H. winghamensis [13] and H. canicola [12] are associated with gastroenteritis in human beings [14]. Also, some Helicobacter species in animals may cause gastritis, typhlocolitis, and hepatitis [15][16][17][18][19][20][21]. ...
... Although the pathogenicity to humans of many species found in the genus is still unknown, Helicobacter pylori is considered the most important species of the genus Helicobacter, which is associated with human gastric lesions and malignancy. In addition, various enterohepatic species of the Helicobacter genus such as H. cinaedi [5,6], H. canis [7], H. pullorum [8][9][10][11], H. valdiviensis [12], H. winghamensis [13] and H. canicola [12] are associated with gastroenteritis in human beings [14]. Also, some Helicobacter species in animals may cause gastritis, typhlocolitis, and hepatitis [15][16][17][18][19][20][21]. ...
Article
Eleven Gram-negative, curved and S-shaped, oxidase activity positive, catalase activity negative bacterial isolates recovered from faeces of Anatolian ground squirrel ( Spermophilus xanthoprymnus ) in the city of Kayseri, Turkey, were subjected to a polyphasic taxonomic study. Results of a genus-specific PCR revealed that these isolates belonged to the genus Helicobacter . The 16S rRNA gene sequence analysis revealed that the 11 isolates had over 99 % sequence identity with each other and were most closely related to Helicobacter ganmani CMRI H02 T with 97.0–97.1 % identity levels and they formed a novel phylogenetic line within the genus Helicobacter . Faydin-H64 and Faydin-H70 T strains were subjected to gyr A and atp A gene and whole genome sequence analyses. These two Helicobacter strains formed separate phylogenetic clades, divergent from other known Helicobacter species. The DNA G+C content and genome size of the strain Faydin-H70 T were 35.3 mol% and 1.7 Mb, respectively. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain Faydin-H70 T and its close phylogenetic neighbour H. winghamensis ATCC BAA-430 T were determined as 81.7 and 34.9 %, respectively. Pairwise sequence comparison showed that it was closely related to H. ganmani CMRI H02 T however it shared the highest ANI and dDDH values with H. winghamensis ATCC BAA-430 T . The data obtained from the polyphasic taxonomy approach, including phenotypic characterization and whole-genome sequences, revealed that these strains represent a novel species within the genus Helicobacter , for which the name Helicobacter turcicus sp. nov., is proposed with Faydin-H70 T as the type strain (=DSM 112556 T =LMG 32335 T ).
... After describing this species, we found a 16S rRNA gene sequence deposited in the GenBank database belonging to H. valdiviensis that correspond to a case of bacteraemia in Thailand (KX503247). A case-control clinical survey carried out in the city of Valdivia and the description of the whole genome of the type strain of H. valdiviensis were recently reported (Fresia et al. 2017). The main results of these studies have demonstrated that horizontal gene transfer has contributed to shaping the gene repertory of H. valdiviensis, which codes for virulence factors (such as the genotoxin CDT) conserved in other pathogens that are well-known human pathogens. ...
... The main results of these studies have demonstrated that horizontal gene transfer has contributed to shaping the gene repertory of H. valdiviensis, which codes for virulence factors (such as the genotoxin CDT) conserved in other pathogens that are well-known human pathogens. Additionally, the detection of H. valdiviensis DNA in diarrhoeic patients (but absent in healthy controls) supports its role as a potential emergent intestinal pathogen (Fresia et al. 2017). ...
Article
The genus Helicobacter defined just over 30 years ago, is a highly diverse and fast-growing group of bacteria that are able to persistently colonize a wide range of animals. The members of this genus are subdivided into two groups with different ecological niches, associated pathologies, and phylogenetic relationships: the gastric Helicobacter (GH) and the enterohepatic Helicobacter (EHH) species. Although GH have been mostly studied, EHH species have become increasingly important as emerging human pathogens and potential zoonotic agents in the last years. This group of bacteria has been associated with the development of several diseases in humans from acute pathologies like gastroenteritis to chronic pathologies that include inflammatory bowel disease, and liver and gallbladder diseases. However, their reservoirs, as well as their routes of transmission, have not been well established yet. Therefore, this review summarizes the current knowledge of taxonomy, epidemiology, and clinical role of the EHH group.
... Both of them are unable to colonize the gastric mucosa, because they do not express a urease to overcome the acidic environment in the stomach. These helicobacters with the term "enterohepatic helicobacters (EHH)" colonize the intestinal mucosa or the liver, and are associated with chronic liver or intestinal in ammation [26]. Recently, many cases of EHH infection in humans were reported, mainly in immunocompromised patients [3]. ...
Preprint
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Background: Helicobacter himalayensis was isolated from Marmota himalayana in the Qinghai-Tibet Plateau, China, and is a new non-H. pylori species, with unclear taxonomy, phylogeny, and pathogenicity. Results: A comparative genomic analysis was performed between the H. himalayensis type strain 80(YS1) and other the genomes of Helicobacter species present in the National Center for Biotechnology Information (NCBI) database to explore the molecular evolution and potential pathogenicity of H. himalayensis. H. himalayensis 80(YS1)T formed a clade with H. cinaedi and H. hepaticus that was phylogenetically distant from H. pylori. The H. himalayensis genome showed extensive collinearity with H. hepaticus and H. cinaedi. However, it also revealed a low degree of genome collinearity with H. pylori. The genome of 80(YS1)T comprised 1,829,936 bp, with a 39.89% GC content, a predicted genomic island, and 1,769 genes. Comparatively, H. himalayensis has more genes for functions in “cell wall/membrane/envelope biogenesis” and “coenzyme transport and metabolism” sub-branches than the other compared helicobacters, and its genome contained 42 virulence factors genes, including that encoding cytolethal distending toxin (CDT). Conclusions: We characterized the H. himalayensis 80(YS1)T genome, its phylogenetic position, and its potential pathogenicity. However, further understanding of the pathogenesis of this potentially pathogenic bacterium is required, which might help to manage H. himalayensis-induced diseases.
... Both of them are unable to colonize the gastric mucosa, because they do not express a urease to overcome the acidic environment in the stomach. These helicobacters with the term "enterohepatic helicobacters (EHH)" colonize the intestinal mucosa or the liver, and are associated with chronic liver or intestinal in ammation [25]. Recently, many cases of EHH infection in humans were reported, mainly in immunocompromised patients [3]. ...
Preprint
Full-text available
Background: Helicobacter himalayensis was isolated from Marmota himalayana in the Qinghai-Tibet Plateau, China, and is a new non-H. pylori species, with unclear taxonomy, phylogeny, and pathogenicity. Results: A comparative genomic analysis was performed between the H. himalayensis type strain 80(YS1)T and other the genomes of Helicobacter species present in the National Center for Biotechnology Information (NCBI) database to explore the molecular evolution and potential pathogenicity of H. himalayensis. H. himalayensis 80(YS1)T formed a clade with H. cinaedi and H. hepaticus that was phylogenetically distant from H. pylori. The H. himalayensis genome showed extensive collinearity with H. hepaticus and H. cinaedi. However, it also revealed a low degree of genome collinearity with H. pylori. The genome of 80(YS1)T comprised 1,829,936 bp, with a 39.89% GC content, a predicted genomic island, and 1,769 genes. Comparatively, H. himalayensis has more genes for functions in “cell wall/membrane/envelope biogenesis” and “coenzyme transport and metabolism” sub-branches than the other compared helicobacters, and its genome contained 42 virulence factors genes, including that encoding cytolethal distending toxin (CDT). Conclusions: We characterized the H. himalayensis 80(YS1)T genome, its phylogenetic position, and its potential pathogenicity. However, further understanding of the pathogenesis of this potentially pathogenic bacterium is required, which might help to manage H. himalayensis-induced diseases.
... Both of them are unable to colonize the gastric mucosa, because they do not express a urease to overcome the acidic environment in the stomach. These helicobacters with the term "enterohepatic helicobacters (EHH)" colonize the intestinal mucosa or the liver, and are associated with chronic liver or intestinal inflammation [25]. Recently, many cases of EHH infection in humans were reported, mainly in immunocompromised patients [3]. ...
Article
Full-text available
Background Helicobacter himalayensis was isolated from Marmota himalayana in the Qinghai-Tibet Plateau, China, and is a new non-H. pylori species, with unclear taxonomy, phylogeny, and pathogenicity. Results A comparative genomic analysis was performed between the H. himalayensis type strain 80(YS1)T and other the genomes of Helicobacter species present in the National Center for Biotechnology Information (NCBI) database to explore the molecular evolution and potential pathogenicity of H. himalayensis. H. himalayensis 80(YS1)T formed a clade with H. cinaedi and H. hepaticus that was phylogenetically distant from H. pylori. The H. himalayensis genome showed extensive collinearity with H. hepaticus and H. cinaedi. However, it also revealed a low degree of genome collinearity with H. pylori. The genome of 80(YS1)T comprised 1,829,936 bp, with a 39.89% GC content, a predicted genomic island, and 1769 genes. Comparatively, H. himalayensis has more genes for functions in “cell wall/membrane/envelope biogenesis” and “coenzyme transport and metabolism” sub-branches than the other compared helicobacters, and its genome contained 42 virulence factors genes, including that encoding cytolethal distending toxin (CDT). Conclusions We characterized the H. himalayensis 80(YS1)T genome, its phylogenetic position, and its potential pathogenicity. However, further understanding of the pathogenesis of this potentially pathogenic bacterium is required, which might help to manage H. himalayensis-induced diseases.
... The study showed that H. valdiviensis belongs to a monophyletic clade formed by Helicobacter canadensis, Helicobacter pullorum, Helicobacter winghamensis, Helicobacter rodentium, and Helicobacter apodemus. 3 Additional PCR analyses revealed the presence of H. valdiviensis in some of stools samples of patients with acute diarrhea suggesting a potential role as an intestinal pathogen. Another interesting study looked at the microbiome composition in coelomic fluid of marine invertebrates. ...
Article
The current article is a review of the most important, accessible, and relevant literature published between April 2017 and March 2018 on other Helicobacters and the gastric microbiome. The first part of the review focuses on literature describing non‐Helicobacter pylori‐Helicobacter (NHPH) infections in humans and animals whilst the subsequent section focuses specifically on the human gastric microbiome. Novel diagnostic methods as well as new NHPHs species have been identified in recent studies. Furthermore, our knowledge about the pathogenesis of NHPH infections has been further enhanced by important fundamental studies in cell lines and animal models. Over the last year, additional insights over the prevalence and potential prevention strategies of NHPHs have also been reported. With regard to understanding the gastric microbiome, new information detailing the structure of the gastric microbiota at different stages of H. pylori infection, within different patient geographical locations, was documented. There was also a study detailing the impact of proton‐pump inhibitor usage and the effect on the gastric microbiome. Newer analysis approaches including defining the active microbiome through analysis of RNA rather than DNA‐based sequencing were also published allowing the first assessments of the functional capabilities of the gastric microbiome.
... Phylen has been already used by our group for building the Helicobacter genus phylogeny (Fresia et al. 2017) from a set of 40 universal marker genes (Mende et al. 2013), and to reconstruct core genome phylogenies of Leptospira genus (Puche et al. 2017;Thibeaux et al. 2018) from orthologous groups defined in the EggNOG database (spiNOG) (Powell et al. 2014). Additionaly, here we screened 93 Epsilonproteobacteria genomes against 4513 orthologous groups from the EggNOG database (eproNOG) to obtain the phylogenetic tree shown in Fig. 1B. ...
Article
Full-text available
Phylen is an R package that performs automatic phylogenetic reconstruction given a set of Hidden Markov Models (HMMs). Genomes are screened against these HMMs, genes found in all genomes ("core genes") are aligned individually, those alignments are concatenated into a single supergene alignment, and a phylogenetic reconstruction is performed and returned as an object of class "phylo" so it can be further analysed using ape/phangorn framework in R. Functions to download well curated HMMs from clade-specific orthologous sets from the EggNOG database are provided although any custom set of HMMs can be used as well.
... Phylen has been already used by our group for building the Helicobacter genus phylogeny (Fresia et al. 2017) from a set of 40 universal marker genes (Mende et al. 2013), and to reconstruct core genome phylogenies of Leptospira genus (Puche et al. 2017;Thibeaux et al. 2018) from orthologous groups defined in the EggNOG database (spiNOG) (Powell et al. 2014). Additionaly, here we screened 93 Epsilonproteobacteria genomes against 4513 orthologous groups from the EggNOG database (eproNOG) to obtain the phylogenetic tree shown in Fig. 1B. ...
Preprint
Full-text available
Phylen is an R package that performs automatic phylogenetic reconstruction given a set of Hidden Markov Models (HMMs). Genomes are screened against these HMMs, genes found in all genomes ("core genes") are aligned individually, those alignments are concatenated into a single supergene alignment, and a phylogenetic reconstruction is performed and returned as an object of class "phylo" so it can be further analysed using ape/phangorn framework in R. Functions to download well curated HMMs from clade-specific orthologous sets from the EggNOG database are provided although any custom set of HMMs can be used as well.
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Enterohepatic Helicobacter (EHH) species have been increasingly associated with acute gastroenteritis, inflammatory bowel disease and hepatobiliary diseases in humans. However, their host range and transmission routes are poorly understood. Therefore, the aim of this study was to determine the presence of EHH in healthy dogs using both cultivation‐dependent and ‐independent methods. Three hundred and ninety faecal samples from domestic dogs without gastrointestinal symptoms were analysed between June 2018 and July 2019 in Valdivia (South of Chile). Samples were inoculated on selective medium and in parallel were filtrated over an antibiotic‐free blood agar. Both media were incubated in a microaerobic atmosphere at 37°C for 7 days. Colonies were identified by PCR and phylogenetic analysis. A subset of 50 samples (half of them positive for EHH by cultivation and the remaining half negative) was analysed by PCR‐Denaturing Gradient Gel Electrophoresis (PCR‐DGGE) for direct detection. Cultivation method detected EHH in 15.4% (60/390) of the samples, being the most prevalent species H. canis (5.8%, 23/390) and H. canicola (5.1%, 20/390), followed by H. bilis (3.6%, 14/390) and ‘H. winghamensis’ (1.3%, 5/390). In contrast, PCR‐DGGE method detected Helicobacter DNA in almost all (96%, 48/50) tested samples. On the other hand, the method used also allowed to isolate other Campylobacterales, in fact 44.3% (173/390) of the samples were positive for Campylobacter upsaliensis (43.3%, 169/390) followed by C. jejuni (2.0%, 8/390). Moreover, two strains that presented Campylobacter‐like morphology were finally identified as Anaerobiospirillum succiniciproducens. Our results indicate that healthy domestic dogs commonly carry EHH and other Campylobacter species. However, further studies are needed to determine whether and how these Helicobacter and Campylobacter species can be transmitted to humans.
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Chapter
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The ankyrin repeat (ANK) is the most common protein-protein interaction motif in nature, and is predominantly found in eukaryotic proteins. Genome sequencing of various pathogenic or symbiotic bacteria and eukaryotic viruses has identified numerous genes encoding ANK-containing proteins that are proposed to have been acquired from eukaryotes by horizontal gene transfer. However, the recent discovery of additional ANK-containing proteins encoded in the genomes of archaea and free-living bacteria suggests either a more ancient origin of the ANK motif or multiple convergent evolution events. Many bacterial pathogens employ various types of secretion systems to deliver ANK-containing proteins into eukaryotic cells, where they mimic or manipulate various host functions. Studying the molecular and biochemical functions of this family of proteins will enhance our understanding of important host-microbe interactions.
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Urease activity is vital for gastric colonization by Helicobacter species, such as the animal pathogen Helicobacter felis. Here it is demonstrated that H. felis expresses two independent, and distinct urease systems. H. felis isolate CS1 expressed two proteins of 67 and 70 kDa reacting with antibodies to H. pylori urease. The 67-kDa protein was identified as the UreB urease subunit, whereas the N-terminal amino acid sequence of the 70-kDa protein displayed 58% identity with the UreB protein and was tentatively named UreB2. The gene encoding the UreB2 protein was identified and located in a gene cluster named ureA2B2. Inactivation of ureB led to complete absence of urease activity, whereas inactivation of ureB2 resulted in decreased urease activity. Although the exact function of the UreA2B2 system is still unknown, it is conceivable that UreA2B2 may contribute to pathogenesis of H. felis infection through a yet unknown mechanism.
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