Multilocus Sequence Typing Of Streptococcus Thermophilus From Traditional Fermented Dairy Products In China, Russia And Mongolia

Streptococcus thermophilus is a major dairy starter used for the manufacture of dairyproducts. It is massively used for the manufacture of all kinds of dairy products. In thisstudy, S. thermophilus were isolated using pure culture method from traditionalfermented dairy products collected from Yili Kazakh Autonomous Prefecture in Xinjiangand Damxung County in Tibet of China, and Respublika Kalmykiya and Buryatiya ofRussia. Those isolates were identified by morphology and molecular biology. Meanwhile,multilocus sequence typing (MLST) was used to study the genetic polymorphism andevolutionary relationship of260isolates of S. thermophilus from fermented dairyproducts in Russia,12different regions of Mongolia, and6minority areas of China.Streptococcus thermophilus were isolated by M17agar from223traditional fermenteddairy products colleced from Yili Kazakh Autonomous Prefecture in Xinjiang andDamxung County in Tibet of China, and Respublika Kalmykiya and Buryatiya of Russia.Those traditional fermented dairy products including fermented butter, cheese, mare milk,cow milk, yak milk and camel milk. Gram-positive and catalase-negative cocci wereidentified by16S rRNA gene sequencing. The16S rRNA gene of all the tested strains weredetermined by the BLAST program. According to the16S rRNA gene sequences,52strains were identified as S. thermophilus. Fourty S. thermophilus were isolated from YiliKazakh Autonomous Prefecture of Xinjiang,5strains were isolated from Damxung Countyof Tibet,5strains were isolated from Respublika Kalmykiya, and2strains were isolatedfrom Respublika Buryatiya of Russia.Thirty nine representative isolates form Xinjiang and Tibet of China, and RespublikaKalmykiya and Buryatiya of Russia, and221S. thermophilus isolated from traditionalfermented dairy products in previous study were analyzed by MLST. Based on thegeneome of S. thermophilus ND03,10housekeeping genes, carB, clpX, dnaA, murC,murE, pepN, pepX, pyrG, recA and rpoB, were chose and used for MLST analysis. Thesequences of10housekeeping genes were acquired by bidirectional sequencing technology,and forward and reverse sequences were trimmed, aligned, and analyzed. For each MLST locus, an allele number was given to each distinct combination of alleles of the ten genes,and each allele number was assigned a sequence type (ST). Exclusion of generecombination, the genetic polymorphism and evolution of S. thermophilus were analyzedbased on STs and composite sequences by bioinformatics software, such as BioNumericsv6.0, START2.0, eBURST v3.0, DnaSP5.0and SplitsTree4.0, and so on. The results wereshowed as follow:The number of10loci ranged from7(recA and dnaA) to17(pepN and clpX), and8(recA) to22(murC) polymorphic sites in each gene, indicating intragenic diversity exist inS. thermophilus. The DNA G+C%content observed for all alleles of the ten genes rangedfrom36.94%to44.20%, which were similar to the G+C%content of the S. thermophilusND03. The dS/dNratio ranged from0.0414(pyrG) to0.2624(murC), and it is less than1,suggesting potential negative selection in these genes. The split decomposition graphs ofall ten loci by SplitsTree4.0suggesting no recombination occurred during the evolution ofthese ten loci.The260S. thermophilus isolates by MLST identified as119STs. The mostrepresented STs were ST-29, which comprised16strains. Strains from different regionswere assigned to different STs. SIAof0.1043was significantly greater than the value of0expected for a population at linkage disequilibrium. One hundred and seventeen STs weregrouped by eBURST into58groups,31STs were assigned to singleton. Among58groups,there are5clonal complexes (CCs), CC5, CC29, CC54, CC70and CC81, representing44.6%of the strains in total. The same geographical isolates belong to same CC,suggesting S. thermophilus isolates in different environments with different evolutionaryprocesses. The main factors were location, environment and climate, but not milk type.The minimum evolution tree showed isolates of Xinjiang, Tibet in China and Russiawere closely related to Qinghai, Inner Mogulian, and Gannan region strains, but far fromMongolia strains. Strains from12different regions in Mongolia showed near relationship.Partly strains from the central region of Mongolia were close to isolates of China. Clusteranalysis of the concatenated sequences (5718bp) by using UPGMA revealed that differentgeographical isolates clustered into different clusters, and those strains evoluted towardtheir own direction.In the present study, MLST was used to study the genetic polymorphism andevolutionary relationship of260isolates of S. thermophilus from different regions. On theone hand, an MLST scheme was developed for the study on S. thermophilus, and provideddata for MLST database. On the other hand, analysis of geographically diverse and representative collection of isolates by MLST can provide a better understanding of S.thermophilus genome evolution, and some useful information for further studies ofworldwide S. thermophilus structure and genetic evolution.