Comparative genomic analyses of Lactobacillus rhamnosus isolated from Chinese subjects
Introduction
Lactobacillus is the most diverse genus of lactic acid bacteria (LAB), and is usually found in fermented foods, feeds, oral and gastrointestinal tracts (Barrons & Tassone, 2008). Lactobacillus in the gut have an important role, for example, immunity regulation, cholesterol control, and gastrointestinal function improvement, on the intestinal micro-ecological balance and health of hosts (Damodharan et al., 2016; Reuman et al., 1986). In addition, Lactobacillus have an important role in food production and processing, such as fermented dairy products (Todorov, 2010).
L. rhamnosus is a Gram-positive bacterium that exists in a variety of niches, such as the human intestine and vagina (Pascual et al., 2008). L. rhamnosus GG is one of the best commercialized strain among L. rhamnosus. It has a strong resistance to gastrointestinal digestion and potential probiotic characteristics. Previous studies on L. rhamnosus focused on its functional benefits including various diarrhea treatments, immunity improvement, and respiratory infections prevention (Barbieri et al., 2017; Evans et al., 2016). Phenotype studies were carried out, such as acid tolerance, bile salt tolerance, carbohydrate utilization and adherence to epithelial cells (Ceapa et al., 2016, 2015; Succi et al., 2005; Tripathi et al., 2013).
To explore the metabolism, evolution and speciation, high-throughput methods (e.g., site sequence typing (MLST) and OmniLog (Biolog) phenotyping (Di Cagno et al., 2010) have been used to analyze genotype and phenotype (Bao et al., 2016). Comparative genomic analysis is a common tool in bioinformatics (Kant et al., 2011) that is able to identify the association between strains and their origins as well as to evaluate the gene distribution of specific species (Kelleher et al., 2017). It is significant for strain characteristics such as combining phenotypic profiles with strain-specific genetic diversity allows the assignment of unknown functions to specific genetic loci and to determine interactions with the hosts (Siezen et al., 2010). It also provides a new approach to effectively assess the diversity of strains. Genetic diversity of several species among the Lactobacillus genus had been carried out, such as L. plantarum (Duar et al., 2017; Martino et al., 2016), L. casei (Broadbent, 2012), L. reuteri (Zheng et al., 2015) and L. salivarius (Raftis et al., 2011). Current research on the L. rhamnosus genome was primarily concerned with the use of carbohydrates. Ceapa et al. (2015; 2016) predicted that L. rhamnosus had a number of carbohydrate transport and utilization genes, combined with phenotypic analysis, with which the adhesion-related functional genes were predicted. To investigate the genomic diversity of L. rhamnosus, Douillard et al. (2013b) sequenced and compared the genome and phenotype validation of 100 L. rhamnosus strains from different niches.
To further learn about the genetic diversity of L. rhamnosus from Chinese subjects, 70 L. rhamnosus strains were isolated, draft-genome sequenced and analyzed with comparative genomics approaches as well as gene-trait matching analysis for both carbohydrate utilization and antibiotic resistance based on the genotype-phenotype combination.
Section snippets
Isolation of strains
One hundred and twenty fecal samples were collected from different regions of China. The fecal samples were mixed with 30% sterile glycerin solution (China National Medicines Corp. Ltd., Beijing, China), stored temporarily at 4 °C, and stored at −80 °C within 48 h for a maximum of 8 wk. One g of each stool sample was blended with 9 ml sterile physiological saline (China National Medicines Corp. Ltd.) (Ingham, 1999). Serial dilution and plating were done in an anaerobic workstation (AW400TG,
Isolation of strains
The donors came from 18 different regions in China and their ages varied from 2 d to 102 yr (35 samples of infants < 1 yr; 9 samples from 2 to 16 yr; 10 samples from 27 to 54 yr; 10 samples from 60 to 79 yr; 7 samples from 82 to 102 yr). Most of donors were located in three cities (Wuhu, Anhui Province; Wuxi, Jiangsu Province; Wusu, Xinjiang Uygur Autonomous Region). A total of 600 isolates were isolated from the LBS agar, and all of them were species identified with 16S rRNA sequencing. Among
Discussion
With sequencing technology improvements, more researches involving the genome of the strain. L. rhamnosus contained a 3.0 Mb genome, which was one of the largest for LAB (Lebeer et al., 2008). Several genomic sequences of L. rhamnosus have been identified to date, including human intestinal derived L. rhamnosus GG (ATCC 53103) (Hidetoshi et al., 2009), cheese-derived L. rhamnosus LC705 (Prisciandaro et al., 2011) and soil derived L. rhamnosus CASL (Yu et al., 2011). In the current study, 70
Conclusion
Seventy strains of L. rhamnosus were isolated from Chinese infants and adults and their draft-genome sequenced. Combined with LGG, the genome and biological properties of all the strains were analyzed. The results showed that the pan-genome of L. rhamnosus was 8395 and core genome was 1835. All the strains could utilize d-galactose, sucrose, d-trehalose and l-fucose and only a few strains could use XOS and d-xylose, consistent with their genotype. For antibiotics resistance, most genotypic
CRediT authorship contribution statement
Dan Huang: Data curation, Formal analysis, Methodology, Software, Writing - original draft. Bo Yang: Conceptualization, Formal analysis, Resources, Validation, Visualization, Writing - original draft. Yang Chen: Data curation, Methodology. Catherine Stanton: Investigation, Writing - review & editing. R. Paul Ross: Investigation, Writing - review & editing. Jianxin Zhao: Formal analysis, Methodology. Hao Zhang: Project administration, Resources, Software, Supervision. Wei Chen:
Declaration of Competing interest
All authors declared no conflict of interest.
Acknowledgement
This research was supported by the National Natural Science Foundation of China (Nos. 31771953, 31820103010), the National First-Class Discipline Program of Food Science and Technology (JUFSTR20180102), and the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province.
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