power of each VNTR and all 6 VNTRs combined was measured by Simpson’s Index of Diversity (D). The highest D value was 0.957 and was recorded for learn more vca0283. Except for vca0283 and vca0171, all D values were lower than previously reported. Our focus on 7th pandemic isolates which have been shown to be highly homogeneous may have contributed to these lower D values. VNTR vc1457 had the lowest D value of 0.437, which was lower than previously reported (D value = 0.58) . The combined D value of 7th pandemic isolates for all 6 VNTRs in this study was 0.995. We also calculated D values from previous studies by excluding MLVA data of environmental and non-7th pandemic isolates [19–22] and found that the D values were similar and ranged from 0.962 to 0.990 [19–22], when only 7th pandemic
isolates were analysed. Selleck KPT-330 Analysis using the two most variable VNTRs, vca0171 and vca0283, produced comparable D values, which could potentially reduce the need to use the other markers. This would be particularly useful in outbreak situations where there is limited time and resources available to type isolates. However, typing the isolates in this study using only two loci would not reveal any useful relationships. Phylogenetic analysis using MLVA We analysed the MLVA using eBURST . Using the criteria of 5 out of 6 loci identical as definition of a clonal Phospholipase D1 complex, 26 MLVA profiles were grouped into 7 clonal complexes with 37 singletons. For the 7 clonal complexes, a minimal spanning network (MSN) was constructed to show the relationships of the MLVA profiles (Figure 1 A). Many nodes in the 2 largest clonal complexes showed multiple alternative connections. There were 27 possible nodes differing by 1 locus, 4 nodes were due to the difference in vc0147
and 23 others were due to VNTR loci in chromosome II. Out of the 23 single locus difference in the 2 chromosome II VNTRs, the majority (57%) also differed by gain or loss of a single repeat unit. Thus 1 repeat change was the most frequent for the VNTRs on both chromosomes. It has been shown previously that it is more likely for a VNTR locus to differ by the gain or loss of a single repeat unit as seen in E. coli and we have also found this was the case in V. cholerae. We then used the MLVA data for all 7th pandemic isolates to construct a minimal spanning tree (Additional file 1 Selleckchem AZD8186 Figure S 1A). For nodes where alternative connections of equal minimal distance were present we selected the connection with priority rules in the order of: between nodes within the same SNP group, between nodes differing by 1 repeat difference and between nodes by closest geographical or temporal proximity. The majority of isolates differed by either 1 or 2 loci, which is attributable to vca0171 and vca0283 being the 2 most variable loci.