Identification And Typing Of Vibrio Vulnificus Based On MAIDI-TOF MS And MLST

Objective: 1.To generate and expand MALDI-TOF MS spectral database of Vibrio vulnificus so as to improve its accuracy in Vibrio vulnificus' s indentification and explore the extended application between MALDI-TOF MS and Vibrio vulnificus.2.To identify whether a strain suspected of Vibrio vulnificus is a new species.3.To evaluate the genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the regions of our country like Beijng,Zhejiang,Guangdong and Taiwan by multilocus sequence typing(MLST).Methods: 1.The MALDI-TOF MS protein fingerprint spectra database of Vibrio vulnificus was established by Microflex(Bruker Daltonik,Germany)and the cluster analysis of protein fingerprint was obtained by Biotyper 4.0.2.Phylogenetic analysis was constructed based on 16 S r RNA gene sequences and multilocus sequence analysis(MLSA)to infer the evolutionary history of vibrios using eight housekeeping genes(fts Z,gap A,gyr B,mre B,pyr H,rec A,rop A and top A)to find the closely related strains.Furthermore,In silico DNA–DNA hybridization(is DDH)and average nucleotide identity(ANI)were estimated for the novel strain.3.A strain's allelic profile and its sequence type were obtained by PCR amplification of Vibrio vulnificus' ten housekeeping genes.Nucleotide and allelic diversity were evaluated with software Dna SP 6.12 and e BURST Version 3.0,and the population structure were analysed with Splits Tree4 and STRUCTURE.Results: 1.Established a protein fingerprint database composed of 107 Vibrio vulnificus including both clinical and environmental isolates originating from four regions of our country.2.The cluster analysis of protein fingerprint do not show a clear distinction between stains from different regions,and between clinical and environmental strains.3.The 16 S r RNA gene sequence analysis showed that the strain belongs to the Vibrio vulnificus clade.Multilocus sequence analysis showed that the strain was closely related to Vibrio vulnificus and Vibrio litoralis,followed by Vibrio navarrensis and Vibrio proteolyticus.In silico DNA-DNA hybridization(is DDH)indicated that the strain showed66.1%,13.7%,28.3%,and 16.3% in DDH values with the above four species respectively,lower than the proposed cutoff level(70%)for species delineation.Moreover,the ANI results ranged from 70.8 to 92.3%,values that were lower than the 95% threshold previously recommended for novel species.4.86 STs were identified among all of the107 strains,and 67 of them were new STs which were discovered in our study.24 STs of34 Beijing isolates were identified and there were 21 new STs among them,for Zhejiang strains,there were24 STs while 34 of them were new STs,for Guangzhou collections,7 STs were all new STs,and for Taiwan collections,14 out of 23 STs were new STs.5.There were significant recombinations within and across Vibrio vulnificus isolated from four regions,the population structure analysis showed that 86 STs of 107 strains were divided into two subgroups by using Splits Tree4 software,while divided into three 3 subgroups with STRUCTURE among 86 STs.Though the MLST clusters I and II do not show a clear distinction between clinical and environmental strains,it was found that in cluster I,clinical strains are over represented.Conclusion: 1.By using different number of strains to construct the protein fingerprint spectra database,the database identified the best matching score of Vibrio vulnificus as the number of strains increased,indicating the necessity of expanding the library capacity.2.The resolution of MALDI-TOF MS cluster analysis is not sufficient to distinguish strains from different regions,and it is also impossible to distinguish between clinical and environmental strains.3.Based on the polyphasic evidence presented here,it can be concluded that the strain belongs to the novel species of the genus Vibrio.4.The MLST results showed high level diversity of 107 strains,and the nucleotide and alleles were also represent high diversity.There was significant recombination within and across regions.5.The population structure analysis of 107 Vibrio vulnificus were divided into two clusters,one of which may be associated with clinical strains.