MS clonal complexes were named MSCC followed by the ST number of the central ST in the tree. eBurst clonal complexes were named eBCC followed by the number of the predicted founder ST. When the founder is unpredicted or when the complex contained only 2 STs, the complex was named by the most represented ST or by default by the ST with the lower numbering. In both MS and eBURST analyses, the singleton (S) STs corresponded to STs differing
from every other ST at 3 or more of the 7 loci. A distance matrix in nexus format was generated from the set of allelic profiles and then used for decomposition analyses with SplitsTree 4.0 software [30]. Program LIAN 3.1 [35] was used to calculate the standardized IA (sIA) and to test the null hypothesis of linkage disequilibrium Cl-amidine mw as well as to determine mean genetic Dasatinib cell line diversity (H) and genetic diversity at each locus (h). The number of synonymous (dS) AZD0156 purchase and non-synonymous
(dN) substitutions per site was determined on codon-aligned sequences using SNAP software [36]. Results Development of a MLST scheme for O. anthropi typing Since MLST approaches have never been performed for bacteria of the genus Ochrobactrum, we developed an original MLST scheme in this study. The choice of the seven loci was done on the basis of the complete genome sequence of O. anthropi ATCC 49188T (accession number: CP000758). Amplification primers (Table 3) were designed using the alignment of genes from O. anthropi ATCC 49188T and its closest totally sequenced relatives Brucella suis 1330T, Brucella melitensis 16M and Brucella abortus 2308. We selected 6 genes encoding housekeeping products involved in transcription (rpoB), DNA repair (recA), stress response (dnaK), amino-acid biosynthesis (aroC and trpE) and the glycolytic pathway (gap) (Table 3). They were frequently used in MLST because mutations occurred slowly and were believed to be mostly neutral [37]. The seventh gene, omp25, encoding an outer membrane protein, was supposed to be a more variable marker. The selected loci were distributed as much as possible across the large chromosome
of the bipartite genome of O. anthropi to ensure the absence of physical links between loci (Table 3). Rapamycin The MLST scheme showed between 4.5% to 13.7% of polymorphic sites among genes and a total of 235 single nucleotide polymorphisms (SNPs) in the 7 loci (Table 4). The mean genetic diversity (H) among strains was 0.7083 +/- 0.0506 and the genetic diversity at each locus (h) is given in Table 4. H in the clinical strains population (0.5959 +/- 0.0572) did not differ significantly from H in the environmental population (0.7301 +/- 0.0286), p = 0.11. Table 4 Sequence analysis of the seven loci. Locus Number of alleles Number of polymorphic sites (%) Genetic diversity (h) Number of non-synonymous codon dN dS dN/dS dnaK 6 24 (4.5%) 0.6625 3 0.0037 0.0811 0.0456 recA 6 32 (6.5%) 0.4286 0 0.000 – - rpoB 12 38 (7.6%) 0.7648 4 0.0036 0.1038 0.
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