Multilocus sequence typing of Streptococcus thermophilus from naturally fermented dairy foods in China and Mongolia
- Jie Yu†1,
- Zhihong Sun†1,
- Wenjun Liu1,
- Xiaoxia Xi1,
- Yuqin Song1,
- Haiyan Xu1,
- Qiang Lv1,
- Qiuhua Bao1,
- Bilige Menghe1 and
- Tiansong Sun1Email author
© Yu et al. 2015
Received: 26 November 2014
Accepted: 7 October 2015
Published: 26 October 2015
Streptococcus thermophilus is a major dairy starter used for manufacturing of dairy products. In the present study, we developed a multilocus sequence typing (MLST) scheme for this important food bacterium. Sequences of 10 housekeeping genes (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, and rpoB) were obtained for 239 S. thermophilus strains, which were isolated from home-made fermented dairy foods in 18 different regions of Mongolia and China.
All 10 genes of S. thermophilus were sequenced, aligned, and defined sequence types (STs) using the BioNumerics Software. The nucleotide diversity was calculated by START v2.0. The population structure, phylogenetic relationships and the role of recombination were inferred using ClonalFrame v1.2, SplitsTree 4.0 and Structure v2.3.
The 239 S. thermophilus isolates and 18 reference strains could be assigned into 119 different STs, which could be further separated into 16 clonal complexes (CCs) and 38 singletons. Among the 10 loci, a total of 132 polymorphic sites were detected. The standardized index of association (I A S = 0.0916), split-decomposition and ρ/θ (relative frequency of occurrence of recombination and mutation) and r/m value (relative impact of recombination and mutation in the diversification) confirms that recombination may have occurred, but it occurred at a low frequency in these 10 loci. Phylogenetic trees indicated that there were five lineages in the S. thermophilus isolates used in our study. MSTree and ClonalFrame tree analyses suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality, but revealed no association with the types of fermented dairy product. Phylogenetic analysis of 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) indicated that our MLST scheme could clearly separate three closely related species within the salivarius group and is suitable for analyzing the population structure of the other two species in the salivarius group.
Our newly developed MLST scheme improved the understanding on the genetic diversity and population structure of the S. thermophilus, as well as provided useful information for further studies on the genotyping and evolutionary research for S. thermophilus strains with global diversity.
KeywordsHome-made fermented dairy foods Streptococcus thermophilus Multilocus sequence typing Population genetics Food microbiology
Streptococcus thermophilus is a lactic acid bacteria species that is essential for the manufacturing of many types of fermented dairy products, occurring naturally as well as in commercial starter cultures . Because S. thermophilus can produce large amounts of lactic acid, exopolysaccharides, and flavor compounds in fermentation processes, it is considered the second most important industrial dairy starter after Lactococcus lactis [2, 3]. Therefore, analyses of the genetic diversity, population structure, and phylogenetic relationships of S. thermophilus are important for understanding the evolution of ecological or biological properties of bacterial strains and for optimizing their industrial exploitation. Especially, S. thermophilus is genetically similar to S. vestibularis and S. salivarius in S. salivarius group. Several restriction- or PCR-based typing methods have been applied to the typing of S. thermophilus [4–8]. More recently, a powerful method for population genetic studies is multilocus sequence typing (MLST), which is based on sequencing of conserved housekeeping genes. The method provides faster and less expensive nucleotide sequence determination, and has become the gold standard for studying the evolution and population genetics of pathogenic microbes .
MLST involves the sequencing of a small number (at least seven) of housekeeping genes for each strain. The sequences of each fragment are assigned allele numbers for each locus. All allele numbers are combined to define the allelic profile of the strain and each different allelic profile is assigned as a sequence type (ST), which is used to describe the strain . Because the typing is based on nucleotide sequence, MLST is primarily used for the exchange of electronic data between laboratories via the internet and provides accurate information on strain evolution [11, 12]. MLST has been used successfully in molecular epidemiology studies [13–15], and for identifying and typing lactic acid bacteria of various species of Lactobacillus [16–18], Pediococcus , Enterococcus , Oenococcus , and Lactococcus [22, 23]. The MLST scheme also reported for S. thermophilus analyzed the nucleotide variability at eight genetic loci of 27 S. thermophilus and the phylogenic relationship of two oral streptococci S. salivarius and S. vestibularis in the salivarius group .
In this study, 239 S. thermophilus isolated from different ecological sources and geographical areas and 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) were used to: (i) developed an effective MLST scheme for characterization of S. thermophilus and distinguish three closely related species in S. salivarius group; (ii) apply MLST to assess phylogenetic relationship and evolutionary characteristics of these strains; (iii) compare the genetic diversity of S. thermophilus from different type of dairy foods and geographic origin.
Bacterial isolates and DNA extraction
36 whole genome strains belonging to the salivarius group used in this study
GenBank Accession no.
Oral swab from healthy volunteer
Saliva from healthy child
Milk from a healthy woman
Fermented milk product (curd)
Fermented milk product (curd)
Curd of “Grana Padano” cheese
Artisanal goat cheese from raw milk
Naturally fermented yak milk
Buffalo mozzarella whey
Buffalo moarella curd
Starter for yogurt and mozzarella cheese
Artisanal goat cheese from raw milk
Commercial starter culture
All S. thermophilus strains were grown in 5.0 mL M17-broth (OXOID, CM0817B, Germany) supplemented with 5.0 g/L of lactose at 30 °C for 18–22 h, and then strains were harvested by centrifugation and cell pellets were used for DNA extraction. Total genomic DNA was extracted from cultures by using a previously reported method . Purified DNA was diluted to a final concentration of 100 ng/μL for application.
MLST loci selection
Genes and primers used for Multilocus sequence typing
Amplicon size (bp)
Application size (bp)
carBamoyl phosphate synthetase
ATP-dependent Clp protease subunit X
chromosomal replication initiator protein dnaA
UDP-N-acetyl muramate-alanine ligase
UDP-N-acetylmuramyl tripeptide synthase
lysyl-aminopeptidase, aminopeptidase N
X-prolyl dipeptidyl aminopeptidase
RNA polymerase beta subunit
PCR amplification and DNA sequencing
For each strain, the genomic DNA was used as a template for PCR amplification of MLST loci on the automatic thermal cycler (PTC-200, MJ Research, Waltham, MA). Thermal cycling conditions for PCR were: 94 °C for 5 min; 30 cycles of 94 °C for 1 min, corresponding temperature of each locus for 1 min, and 72 °C for 2 min; a final elongation step of 72 °C for 10 min. For each target, PCR mixture (50 μL) containing 150 ng of genomic DNA, 10 mM of each dNTP, 10 pmol of each primer, 2.5 U Taq polymerase in 1x PCR buffer (with 2.5 mmol/L Mg2+). PCR products were electrophoresed in a 1.2 % agarose gel. Sequencing of the PCR products was performed in Shanghai Majorbio Bio-pharm Technology Corporation. The same primers were used for PCR and sequencing on both DNA strands.
MLST data analysis
For MLST analysis, forward and reverse sequences were trimmed, aligned, and analyzed using MEGA 6.0 software package (version 6.0, www.megasoftware.net) . Definition of alleles using a nonredundant dataset and minimum spanning tree analysis were preformed using the BioNumerics Software (version 6.6, Applied-Maths, Sint Maartens-Latem, Belgium). Different allelic sequences (with at least one nucleotide difference) were assigned arbitrary numbers. For each of the ten MLST loci, a unique nucleotide sequence defined an allele. Unique allelic profiles, consisting of the allele numbers at each of the ten MLST loci, defined STs. The same ST was used for several strains when they shared the same allelic profiles. Groups of isolates with closely related allelic profiles have been called clonal complexes (CCs). STs were grouped into CC with the eBURST program  and located in the BioNumerics program.
START version 2.0 program  was used for calculating the number of polymorphic sites, mol% G + C content, d N /d S , and I A and I A S value for linkage analysis of the population as well as recombination testing. The nucleotide diversity (π) per site was calculated using Dnasp version 5.0 . The split decomposition method was used to assess the degree of tree-like structure for alleles of each locus and all STs using SplitsTree 4.0 . The phi test for recombination based on individual loci of the whole strain collection was also done with SplitsTree. The phylogenetic trees from the concatenated sequences (5718 bp) were constructed by the neighbor-joining method with a Kimura two-parameter distance model using MEGA 6.0. Bootstrap analysis with 1,000 replicates was performed.
The software Structure v2. 3 with linkage model  was used to identify the ancestral subpopulations and assign ancestry proportions for each isolate. The K value that generated the highest posterior probability was used as the probable number of ancestral populations. Three independent runs were performed for each value of the number of populations K ranging from 3 to 15. Each run consisted of 100,000 Markov Chain Monte Carlo (MCMC) iterations, of which the first 20,000 iterations were discarded as burn-in. The K value that generated the highest median posterior probability was used as the probable number of ancestral populations.
The evolutionary relationships amongst of S. thermophilus strains were inferred using ClonalFrame v1.2 . Three independent runs of ClonalFrame were performed each consisting of 200,000 MCMC iterations, and a posterior sampling of 300,000 iterations. The prior iterations were discarded and model parameters were sampled in the second period of the run every 100th iteration thereafter, resulting in 3,000 samples from the posterior. The genealogy of the population was summarized and the robustness of the tree topology was evaluated by concatenating the posterior samples of the 3 runs to built-up a 50 % majority rule consensus tree using the ClonalFrame GUI. The ρ/θ and r/m was also accounted by ClonalFrame.
Nucleotide sequence accession numbers
All MLST data of S. thermophilus in this study are available at http://pubmlst.org/sthermophilus/ . Allele sequences of the ten MLST loci have been deposited in the GenBank database under accession numbers KF794203 to KF796592.
Allelic profiles and sequence types
The 10 genes were successfully amplified for all strains and sequenced by bidirectional sequencing technology, and forward and reverse sequences were trimmed, aligned, and analyzed. Nucleotide positions of the sequences showing ambiguities were excluded from the analysis. Ultimately, the sequences of 10 housekeeping genes, ranging in size from 451 to 630 bp (Table 2), were used for MLST analysis.
Additional file 2 summarizes the allelic profiles of the 239 isolates and 18 S. thermophilus reference strains. A total of 119 different STs (ST1–ST119) were obtained; 83 STs corresponded to single isolates, 24 STs included 2–4 isolates, 8 STs included 5–9 isolates, and 4 STs included 11–15 isolates. The most represented STs were ST5 composed of 15 isolates (5.8 % of all isolates), ST2 (14 isolates, 5.4 %), ST79 (13 isolates, 5.1 %), and ST39 (11 isolates, 4.3 %). The breakdown by region was as follows: 140 strains from 12 different regions of Mongolia were identified as 67 STs, and 99 strains from 6 different provinces of China were assigned to 43 STs. Among the 119 STs identified in our collection, 14 STs were attributed to strains isolated in more than two provinces. The widest type was ST5, which was identified in strains isolated in as many as five different provinces across China and Mongolia. A similar distribution was observed for ST65, ST66, and ST91, which were identified in strains isolated from two provinces of China and Mongolia, respectively; the remaining ST strains were isolated in a specific province. Moreover, almost all genome strains were identified as a single ST, the exceptions being ND03, MN-ZLW-002, ASCC 1275, DGCC 7710, and LMD-9. Strain ND03 belonged to ST2, MN-ZLW-002 belonged to ST65, ASCC 1275 and DGCC 7710 belonged to ST66, and strain LMD-9 belonged to ST91.
Nucleotide sequence variation at each MLST locus
Descriptive analysis of MLST data genetic variability at Streptococcus thermophilus loci
G + C content (mol %)
d N /d S b
Evidence for recombination in S. thermophilus
The multilocus linkage disequilibrium between MLST loci was measured using the index of association (I A)  and standardized index of association (I A S ) . To minimize linkage disequilibrium introduced by sampling bias or recent expansion of adaptive genotypes, only one strain from each ST was analyzed . An I A of 0.8242 (P = 0.000) and an I A S of 0.0916 (P =0.000) for the 10 loci were obtained, which were greater than the value of 0 expected for a population at linkage equilibrium. This also shows the formation of an underlying clonal population structure of all the studied isolates. However, the low I A S value confirms that recombination played a role in the evolution of the analyzed genes, and the split graphs of all ten loci and the concatenated sequences corroborated this. From the split graphs (Additional files 3 and 4), we can see that some parallelogram structures were formed for the clpX, pepN, pepX, pyrG, and carB genes indicating that these genes have undergone a little intergenic recombination during evolution (Additional file 3). In addition, the concatenated sequences of the 10 loci displayed a complex network-like structure (Additional file 4), suggesting that there have been several recombination events for these 10 loci based on the phi test (P = 2.108E-9). It is deduced that the selection of 5 genes with a more recombination history maybe contribute to the recombination events.
The linkage disequilibrium analysis and split graphs proved that recombination are existent in the evolution of 257 S. thermophilus strains. Then ClonalFrame was used to further assess the recombination or mutation play a leading role in the evolution of S. thermophilus. The r/m value (relative impact of recombination and mutation in diversification) and ρ/θ value (relative frequency of occurrence of recombination and mutation) were 0.0036 (95 % Credibility Interval 0.001 - 0.034) and 0.0034 (95 % Credibility Interval 0.001 - 0.031), respectively. These low values indicate that recombination occurred less frequently than mutation at these loci in the evolution of 257 S. thermophilus.
Clonal complexes and minimum-spanning tree analysis
To analyze the relationship between the clusters, geographic origin, and the source dairy product among the 257 S. thermophilus isolates, allelic profile-based phylogenetic analysis was performed using a Minimum-Spanning Tree (MSTree) analysis with the BioNumerics v5.10 software. To facilitate analysis, Gansu and Sichuan province of China were grouped into Gannan region, and 11 provinces and one city in Mongolia were grouped into three larger regions (central, northwestern, and northeastern Mongolia) based on geographic position and environment. As shown in Fig. 1, strains of the same allelic profile were in the same circle, the size of which was proportional to the number of strains of that particular profile, and the different colors of the circles indicate different strain regions. The MSTree revealed four major branches and three minor branches. Branch 1 contained 39 strains from CC1, CC7 and 5 single STs. All strains were isolated from Mongolia except ST 92. Branch 2 contained three CCs (CC5, CC12, and CC16) and strain F8CT. The isolates in these branches were from Qinghai in China (19 strains) and central Mongolia (9 strains). Branch 3 contained three CCs (CC3, CC8, and CC15), with isolates from Xinjiang (6 strains), Gannan (13 strains), Qinghai (11 strains), Inner Mongolia (11 strains), central Mongolia (9 strains), and 13 reference strains. Regarding CC2 as the center, Branch 4 was divided into three minor branches (4a-c). Branch 4a included 38 strains of CC2, mainly from Mongolia (32 strains). Branch 4b, containing CC11 and CC13, contained 28 strains isolated from Mongolia. Branch 4c contained 55 strains of CC4, CC6, CC9, CC10, and CC14, most isolated from Xinjiang (20 strains), Tibet (3 strains), Gannan (9 strains), and central Mongolia (15 strains). Moreover, ST11, ST70, ST102, and ST122 do not belong to these four branches and are very different from ST 49. It is suggested that the phylogenetic relationships are more distant between these isolates compared to the others.
Population structure and phylogenetic relationships
The phylogeny of the 119 STs was analyzed by constructing a NJ tree from the concatenated sequences of 10 loci. As shown in Fig. 2a, the NJ tree revealed four major branches. The colored branches of the NJ tree are in accordance with the color of the groups identified in STRUCTURE. As shown in Fig. 2, we found that the STs of subpopulations 2 (yellow) and 4 (blue), 6 (orange) were distributed in differenced branches of the NJ tree. To examine any possible influences of recombination on tree topology, we inferred a clonal genealogy from our data using ClonalFrame, which takes recombination into account during tree building. The ClonalFrame tree (Additional file 5) showed all isolates fell into five lineages, A-E. Five subpopulations (1–5) identified in STRUCTURE (Fig. 2a) corresponded to the subpopulations shown in Additional file 5 and are represented by corresponding colors. However, subpopulation 6, found in STRUCTURE, did not constitute a lineage in the NJ analysis and ST114 (MTH17CL396) fell into lineage C. The other STs formed small clades at this level of analysis based on the ClonalFrame results. Based on the combined evidence from the STRUCTURE and ClonalFrame analyses, the 257 S. thermophilus strains formed five lineages during evolution.
Phylogenetic analysis of S. thermophilus within the salivarius group
S. thermophilus is a key species involved in the acidification of milk and the development of texture in various fermented dairy products. However, few studies have characterized the diversity of the genetic population and the evolutionary scheme of this food bacterium from different ecological origins. To contribute to the characterization of the population structure of S. thermophilus, we utilized the MLST method to examine 239 strains isolated from different ecological sources and geographical areas.
MLST was first used for pathogenic microbes . This technique is useful for elucidating the phylogenetic relationships and evolution of isolates. Traditional MLST schemes generally include only five to seven gene loci, the lengths of which range from 400 to 500 bp. This small number of gene loci and short length may lead to a decrease in information and unreliable results. Based on previous MLST studies of LAB and the S. thermophilus ND03 genome, 12 gene targets (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, groEL, uvrC, and rpoB gene) were first selected for MLST analysis in this study. Two loci (groEL and uvrC) could not be amplified from all examined strains and were removed. From these 10 gene loci, 239 S. thermophilus isolates and 18 genome strains were identified as 119 STs. The number of alleles per locus ranged from 7 to 18. When any gene locus with the lowest number of alleles was removed from the analysis, the number of STs decreased. Finally, an optimization of the MLST scheme based on these 10 housekeeping genes was chosen for analyzing S. thermophilus. Compared to previous MLST schemes , an average of 12 distinguishable alleles per locus among the 119 STs were identified, which is more than the average of 6–7 alleles observed from 27 S. thermophilus isolates in a previous study. Phylogenetic analysis of whole genome strains within the salivarius group showed that our method could clearly separate three closely related species and also confirmed the existence of an additional cluster. Our MLST scheme has strong typing discrimination for S. thermophilus and could be applied to the other two species in the salivarius group.
Analysis of synonymous and non-synonymous changes in the allele sequences of a locus can be used to determine whether it is subject to selection pressure. In our analysis, all loci displayed a d N /d S ratio lower than 1, indicating a strong purifying selection preventing amino acid changes, which is a typical phenomenon for housekeeping genes and is desired in MLST schemes. The π of the 10 genes varied from 0.0040 in rpoB to 0.0056 in pepN. Previous reports on other lactic acid bacteria have ranged from 0.0004 to 0.0072 for L. plantarum , 0.0051 to 0.0096 for L. delbruecki , and 0.0038 to 0.0119 for Leuconostoc mesenteroides . These results suggest that the examined S. thermophilus housekeeping genes are relatively conserved. Loss of gene function, recombination, and horizontal gene transfer (HGT) were proposed to contribute to the plasticity of the S. thermophilus genome [48, 49]. The low I A S value (0.0916) and network structure of the split-graph in this study confirms that recombination may have occurred, while the low ρ/θ and r/m value indicate that recombination has not played a major role in the evolution of these loci. We infer that the discrepancy between our data and the literature may be related to the selected loci and S. thermophilus isolates.
The population structure within the sequences was analyzed using the linkage model in Structure. It is suggested that the six ancestral subpopulations in our isolates and some STs contain a high degree of admixture (Fig. 2). To examine any possible influences of recombination on tree topology, we inferred a phylogenetic tree using ClonalFrame. The ClonalFrame tree (Additional file 5) revealed that subpopulation 6 identified in STRUCTURE did not represent a true lineage, because this subpopulation is, in fact, a random mixture of strains that did not fall into one of the five true lineages and does not represent a real evolutionary lineage. However, the NJ tree contains four major branches, but they are not well supported because of low bootstrap values. The low bootstrap values are representative of the inherently incongruent phylogenetic signals in the NJ topology. It is suggested that the admixture of STs possibly contributed to the overall poor support values for the NJ topology . Compared with the analytical tools in ClonalFrame and STRUCTURE, our data demonstrate that NJ analysis is not necessarily a suitable method for analyzing phylogenetic relationships between bacteria using MLST data.
One interesting application of the MLST technique was the identification of an association between strains and their origin. Previous studies on 40 Lactobacillus casei isolates using MLST demonstrated specificity to particular ecological niches . However, the MSTree (Fig. 1) and ClonalFrame tree (Additional file 5) analyses in this study suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality. Often, most strains from the same location clustered together, while the rest were dispersed across other clusters. The isolates from the same region are likely to have been exposed to similar environment selective pressures. For instance, Tibet and Xinjiang are located in western China, and have similar climate characteristics of drought and extensive sunshine, thus most isolates from those regions belong to the same lineage. Reference strain ND03 was isolated from Qinghai province in China, so it belongs to ST2 along with the 13 Qinghai isolates. In addition, it is interesting that the central Mongolian strains are distributed in several lineages. Central Mongolia includes the city of UlaanBaatar, the capital of Mongolia. Because of the traffic network in UlaanBaatar, trade contacts were more frequent with the other provinces of Mongolia and China. We deduced that fermented food exchange was a factor that may have contributed to the distribution of the central Mongolian strains, but the reference strains, especially the 12 European strains, have no strong relationship to the lineages and origins of the examined strains that could be identified, based either on the isolation of dairy sources or the sampling location. Furthermore, no significant associations between lineages and the type of dairy products were found in our collection of S. thermophilus (data not shown), which was probably due to an unequal number of isolates from different fermented products. A total of 182 S. thermophilus were isolated from fermented cow milk, whereas only 57 strains were from other fermented dairy products (3 isolates from fermented mare milk, 11 isolates from fermented goat milk, 5 isolates from Qula, and 38 isolates from fermented yak milk). As more isolates are collected from different countries and types of fermented products, further evidence may become available for the grouping of S. thermophilus compared to worldwide isolates, which would provide a strong indication of the factors that have affected its evolutionary history.
MLST was used to study the genetic polymorphisms and evolutionary relationships of 239 isolates of S. thermophilus from different origins. Analysis of a geographically diverse and representative collection of isolates using MLST can provide a better understanding of S. thermophilus genome evolution, and provide information for future studies on the structure and genetic evolution of S. thermophilus globally.
This research was supported by the National Natural Science Foundation of China (Grant No. 31471711), National Key Technology R&D Program (2013BAD18B01), Program for International S&T Cooperation Projects of China (ISTCP, 2014DFR31150), the Hi-Tech Research and Development Program of China (863 Planning, Grant No. 2011AA100902), the China Agriculture Research System (Grant No. CARS-37), the Natural Science Foundation of Inner Mongolia (No. 2013MS1205) and the Open Projects of Inner Mongolia Natural Science Foundation (No. 20102010). This publication made use of the Streptococcus thermophilus MLST website (http://pubmlst.org/sthermophilus/) sited at the University of Oxford, U.K. We would like to thank Professor Jolley KA and Maiden MCJ for kind help with MLST data submission.
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