Molecular Typing Research For Bartonella Henselae In China

Bartonella henselae is an important zoonosis pathogen in Bartonella, which can affect the whole body tissues and organs with a wide range of pathogenicity. Cat scratch disease is the main pathogenic species, usually expressed as a benign self-limiting. But once the body's immune function was limited, it would became serious systemic disease, even lead to death. Some studies suggest that geographic distribution of B.henselae is subtype-specific, and some subtypes have a relation to the disease and the intensity. Some attemps of molecular methods have been used to analysis B.henselae subtype in many countries. But in China, there were no reports on intra-species diversity of B.henselae. Also, it is not clear about the pathogen in intra-genotype situation, population structure and geographic distribution. To answer these questions, in this research we used molecular typing analysis such as multilocus sequence typing (MLST), multilocus variable-number tandem repeat analysis (MLVA) and genomeic single nucleotide polymorphism (SNP) analysis for B.henselae intra-species typing.Using MLST method, we divided 80 isolates into three sequence types (ST), in which the total number of ST1 accounted for 90%(72/80), ST9, and ST30, respectively,8.25%(7/80) and 1.25%(1/80). Phylogenetic analysis showed that the three sequence types come from a same clonal complex, suggesting Chinese isolates evovled from a single clonal complex and had little mutation, indicating the established MLST scheme is not suitable for the prevalent investigation of B.henselae in China. And a widespread variation of ST1 strains need to be distinguished using more discrimination approach.At the same time, we established MLVA, a more powerful method in our lab to compare the difference between Chinese and British B.henselae isolates. For the 6 selected VNTR loci, the Nei's index is from 0.54 to 0.90 respectively, and the discrimination index is up to 0.98 while combine them together which could divided 84 isolates into 53 different MLVA types (MTs). Cluster analysis showed that the MLVA method can devided the strains into Group A and Group B.7 isolates contented in Group A which are from the United Kingdom, and all remaining strains belonging to Group B. Group B can be divided into Group B1 and Group B2, and B2 contains 5 small subsets. Different groups and subsets show different geographic distribution. Minimum spanning tree diagram divided 62 ST1 into six groups, suggesting the intra-ST diversity among ST1 isolates.6 of 7 ST9 were from one group while the remaining one have grouped to ST1, suggesting the evolution posibility routine of B.henselae isolates.To further understand the diversity among the ST1 strains, we resequenced four ST1 strains isolated from different loci and time to analysis the genomic SNPs using the next-genaration sequence ehchnology-SOLiDTM sequencer of Apply Biosystem. An additional eight strains from UK were included for sequencing and comparative analysis. Result showed a total 9566 SNPs loci were distributed during 12 isoaltes and for ST1 strain, the SNP range from 374 to 1932. Phylogenetic tree besed on SNP concatenation could separate all ST1 strains but formed two branches, one clustered with reference strain and the other just including Chinese ST1, indicating diversity existed in ST1 strains, and the variant could form another cluster but the diversity is limited, and the established MLST scheme failed to describe it.The MLST scheme has been improved according to genomic SNP data and applied in isolates. In the improved MLST scheme, we identified the subtype hidden in ST1, and make the cluster result consistent with SNP-based phylogenetic tree. Comparing with the other two methods, we found that the improved MLST scheme was partly congruous with MLVA clustering, and completely congruous with the PFGE main groups. Considring all the result in this study, we recommended a combination of MLST and pulsed-field gel electrophoresis (PFGE) method to investigate the epidemiological characteristics of B.henselae. Genome analysis including the protein coding and variation information in the B.henselae would proceed in the futhur research.