Establishment and application of multilocus sequence typing for Vibrio metschnikovii

ObjectiveTo establish a multilocus sequence typing (MLST) assay for Vibrio metschnikovii, evaluate the molecular typing effect of the MLST assay, and perform MLST analysis on the collected V. metschnikovii strains.

MethodsSeven housekeeping genes (ftsZ, gapA, mreB, gyrB, purM, recA, ropA) sequences in 17 V. metschnikovii strains were amplified with PCR by using self-designed specific primers. The gene polymorphism, neutral evolution and gene recombination were evaluated by using software DnaSP 6.12.03 and software Split tree 4.0, and corresponding sequence types (ST) were obtained based on the sequence differences among 7 housekeeping genes. The minimum spanning tree and clustering map were constructed for different ST types by using software BioNumerics 7.1, and clonal complexes (CCs) were calculated by using software eBURST 3.0.

ResultsThe selected 7 housekeeping genes were suitable for MLST with sufficient allele loci, sequence differences and high resolving power. The split tree clustering of 7 housekeeping genes was consistent with all of V. metschnikovii strains, and the neutrality tests showed that 7 housekeeping genes were less subject to genetic drift during evolution. MLST divided 17 V. metschnikovii strains into 14 STs, and most of them (64.70 %) formed a single ST respectively, of which ST 007 was probably a potential predominant clonal complex. There was a relatively long genetic distance between these strains, and may have specific geographical distribution in vertical transmission.

ConclusionThe MLST assay established in this study has great capability in analyzing the population structure and evolution relationship of V. metschnikovii.