II clade, RD23 was not deleted, thus showing that deletion of RD23 is not correlated with sensitivity to erythromycin. The molecular mechanisms of resistance to erythromycin have not been functionally established, but mutations identified in domain V of the 23S rRNA of biovar II strains, could provide a likely explanation . Although 25 VNTR markers have been described for the typing of Francisella, it is pragmatic PD173074 nmr to investigate only loci of interest depending on the prevalent subspecies of F. tularensis, the efficiency of PCR assays for single loci, and
existing data [1, 13, 34]. Sequence analysis of the locus Ft-M3 resulted in two different repeats denominated here as Ft-M3a corresponding with SSTR9E and Ft-M3b corresponding with SSTR9A as described previously by Johansson et al. . Johansson et al. and Byström et al. also found that locus Ft-M3 is the most variable marker [1, 13]. In the Francisella genome variations of DNA sequences in spite of identical repeat length have been described for short-sequence tandem repeats [35, 36]. Locus Ft-M6 showed less variability with only three PCR fragment sizes being observed
among the strains. We obtained the same amplicon sizes that were described in previous studies for locus Ft-M3 (Additional file 1: Table S2) [14, 37] and for locus Ft-M6 (Additional file 1: Table S2) [14, 37]. Svensson et al. developed a sophisticated real-time PCR array for hierarchical identification of Francisella isolates . Only three (Ftind33, Ftind38, Ftind49) Talazoparib ic50 out of five INDEL loci were
discriminatory among our set of F. tularensis subsp. holarctica isolates. Ftind48 is a marker for B.I to B.IV clades (non-japonica/non-california) and is not expected to vary for these isolates, and Ftind50 is targeting a specific deletion that so far only has been found in LVS. It was possible to simplify these assays to conventional PCR assays that allowed a simple read out based on gel electrophoresis. Bcl-w We identified clusters of strains that had the same INDELs and SNPs as strains described by Svensson et al. . In our study the analysis of VNTR and INDELs of two F. tularensis subsp. holarctica strains (06T0001, 10T0191) that were passaged twenty times in Ma-104 cells showed that these genomic elements were stable. Johansson et al. demonstrated for two VNTR loci (SSTR9 and SSTR16) that they were actually stable over 55 passages . The VNTR NVP-BSK805 ic50 pattern for strains belonging to clade B.I was more variable compared with the pattern obtained for clade B. IV (Additional file 1: Table S2), as was observed previously [21, 23–25]. This might indicate that clade B.IV is more recently introduced in Germany than clade B.I. We have applied several typing tools in a polyphasic approach in order to determine their value for identifying groups of Francisella strains in Germany. We found strains belonging to biovars I and II of F.