Helicobacter Himalayana Sp.nov. Isolated From Gastric Mucosa Of Marmota Himalayana

In this study, we used 16S rDNA amplification, cloning and sequence analysis methods to study the gastric flora of 10 marmota himalayanafrom Qinghai-Tibet Plateau of China.Predominant bacterium crowd were carried out isolation and culture. Then, the implementation of acidic tolerance test on them wasconducted out.16S clone analysis showed that Lactobacillus flora accounting for 37%, followed by Streptococcus bovis, Escherichia coli and clostridium respectively;according to 16S cloning, showing positive clone for Helicobacter spp. and it provided strong clues for the discovery ofHelicobacter himalayana sp.nov. by the separation and culture ofpredominant bacterium flora of 100 gastric mucosa tissue, we found the results are consistent with the 16S cloning results.A total of 200 M. himalayana were captured and sampled in Qinghuai-Tibet Marmota himalayana natural foci of Yersinia pestis by the national annual plague surveillance program 2012 to monitor activity of plague in animals. The animals tested negative for Y.pestis were screened for Helicobacter by genus specific PCR. Positive rates of gastric specimens and feces for Helicobacter were 5.5% and 19% based on Helicobacter genus-specific PCR. Bacterial cultivation was conducted for PCR positive specimens. Thirty mg mucus specimen was scraped using a glass slide, then mixed with 1 ml BHI broth supplemented with 20% fetal bovine serum. Approximately 500 ?l of the mixture was filtrated through a 0.45-?m-pore-size filter on BHI agar containing 8%(v/v) horse blood, 5 mg ml-1 amphotericin B (Fungizone; Fluka), Campylobacter-selective supplement (Oxoid) containing 10 mg 1-1 vancomycin,5 mg 1-1 trimethoprim lactate and 2500 U 1-1 polymyxin B),0.1% activated charcoal. Plates were incubated in miroaerobic atmosphere(5% CO2,5% H2,0.5% O2 and 89.5% N2) at 35? under 95% humidity. We successfully isolated 3 strains of Helicobacter from Marmota himalayana in Qinghai-Tibet plateau China. Two from gastric mucosa another one from faeces of marmot himalayana. Based on the data from biochemical reactions, morphology and phylogenetic analysis with four conserved gene revealed that the strain YS1T represents a new species of the genus Helicobacter, named as Helicobacter himalayana sp.nov. the Homology of these three Helicobacter himalayana sp.nov. strains is very poor by PFGE method.The whole genome sequencing and analysis of YS1T showed that the total length ofHelicobacter himalayana sp.nov genomeDNA was 1829936bp, with the content of G+C 39.89%. Virulence gene analysis showed that this strain genome contains no urease gene, but carrying cytolethal distending toxin gene (cdtB), neuB gene,hemolysin gene, flagella and other virulence genes. Phylogenetic analysis with 16S rRNA, 23 S rRNA,gyrB and hsp60 sequences indicated that YS1 is distantly related with all recognized Helicobacter species. The virulence gene cdtB was common virulence gene among H.hepaticus, H.cinaedi and H.himalayana.sp.nov. Further studies are required to define the natural habitat and host range of to determine its clinical significance and zoonotic potential.