Overall, these studies revealed presence of two clonal
groups among biovar 1A strains. These studies also showed that clinical and non-clinical serotype O:6,30-6,31 (biovar 1A) strains clustered into two separate groups but failed to reveal any unequivocal associations between genotypes and the source of isolation. Multilocus enzyme electrophoresis (MLEE) is an important tool used to study genetic relationships where allelic variations in housekeeping genes are indexed using electrophoretic mobilities of corresponding enzymes [20, 21]. The technique has been used to study epidemiology of several pathogenic bacteria [22–26]. Multilocus restriction typing (MLRT), a recently developed tool, analyses restriction fragment length polymorphism of several housekeeping Selleck Belinostat genes [27–29]. The objective of this study was to use MLEE and MLRT to gain further insight
into the genetic heterogeneity and relationships among clinical and non-clinical strains of Y. enterocolitica biovar 1A. Methods Bacterial strains Eighty one strains of Y. enterocolitica biovar 1A were examined in this study. Of these, sixty-five were isolated from clinical and non-clinical sources in India viz. diarrheic human patients (35), wastewater (18), swine (7) and pork (5) [30–32]. All isolates have been authenticated, and deposited with Yersinia National Reference Laboratory Semaxanib and WHO Collaborating Centre, Institut Pasteur, Paris (France). Of the remaining 16 isolates, ten were obtained from Elisabeth Carniel (Yersinia National Reference Laboratory and WHO Collaborating Centre, Institut Pasteur, Paris, France) and six from Jürgen Heesemann (Max von Pattenkofer Institute, Munich, Germany). Y. enterocolitica 8081 (biovar 1B, serotype O:8), kindly provided by Mikael Skurnik (Haartman Institute, Finland) was used
as the reference strain for both MLEE and MLRT. The serotypes, sources of isolation, country of origin and reference laboratory accession numbers of these strains have been Mizoribine reported previously . All strains were maintained as glycerol stocks at -40°C. Multilocus enzyme electrophoresis (MLEE) The enzyme Edoxaban extracts were prepared as per the method described by Selander et al . Briefly, cultures grown overnight in tryptone soy broth (TSB) were harvested by centrifugation at 10,000 g for 10 min at 4°C. The cells were washed twice in potassium phosphate buffer (0.15 M, pH 7.0) and the pellet was resuspended in 2 ml of buffer (10 mM Tris-HCl, 1 mM EDTA and 0.5 mM NADP, pH 6.8). The bacteria were lysed by sonication (Sonics) on ice and centrifuged at 13,000 g for 30 min at 4°C to obtain the supernatant (enzyme extract), which was stored in aliquots of 200 μl each at -40°C until use. The enzyme extracts were subjected to horizontal gel electrophoresis in 0.