EUROMEDICA  Hanover 6-7  Juni 2008	Advanced methods of diagnosis, treatment and prophylactics
	European Academy of Natural Sciences, Hanover European Scientific Society, Hanover Russian Academy of Natural Sciences, Moscow

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The purpose of this work is to study the cultures of Lactobacillus spp, circulating in different geographical regions of Kazakhstan with the use of molecular biological techniques.

Based on the analysis morphological-cultural and physiological-biochemical parameters, 74 Lactobacillus spp isolates were divided into 3 main groups: Lb.acidophilus - 16 isolates, Lb.fermentum - 21 isolate, Lb.casei - 37 iolyatov. All three groups are evenly distributed across all regions of Kazakhstan.

Analysis of restriction fragment length polymorphism of 16S rDNA and interspacer region of 16 - 23 S rDNA revealed: 25 cultures showed a picture of hydrolysis, typical for Lb.casei, 12 isolates showed similar results for Lb.casei, Lb.rhamnosus, Lb.plantarum, 8 isolates - similar to the species of Lactobacillus acidophilus and Lactobacillus delbrueckii, 18 cultures similar to Lb.fermentum. A direct nucleotide sequence analysis of 16S rDNA was carried out. The result was confirmed with 27 cultures of Lactobacillus casei/Lactobacillus paracasei, 10 cultures of Lb.acidophilus, 23 cultures of Lb.fermentum. remaining cultures were attributed to the species of Lactobacillus plantarum/Lactobacillus pentosus and Lactobacillus delbrueckii. Correlation analysis of phenotypic and phylogenetic identification of 16S rDNA revealed incorrect identification of 14 isolates.

Random amplification of polymorphic DNA (RAPD PCR) using two primers allowed dividing a group of Lactobacillus casei / Lactobacillus paracasei to 8 groups, differing from one another by patterns.

For further analysis MLST typing for 18 isolates of L. casei was conducted. Under investigation were sets of alleles of elongation factor EF-2 (fusA), isoleucyl tRNA synthetase (ileS), GTP-binding protein LepA (lepA), leucyl-tRNA synthetase (leuS), CTP synthetase (pyrG), recombinase A (recA), ATP-dependent DNA helicase (recG). In the course of this work there were found new alleles fusA (option 1), ileS - Two new allele, lepA - Two new allele, leuS - one new allele, recG - one new allele. The combination of new alleles allowed dividing isolates of L. casei on five sequence-type: first group(I) form the sequence type of isolates submitted from all regions of Kazakhstan, the second group (II) consists of isolates Akmola and southern regions of Kazakhstan; III, IV and V form the isolates from northern regions of Kazakhstan.