The Impacts Of Intestinal Selective Pressure On The Genetic Stability Of Lactobacillus Plantarum

The host intestinal tract is the main target for beneficial microorganisms to play a probiotic function,but probiotics have a brutal competition instead of coexisting peacefully with the indigenous intestinal microbiota for limited nutrients and intestinal ecological niche after entering the host intestine,which generating the selection pressure will affect the genetic stability of the strain.Therefore,the study on the genetic stability of probiotics in the intestinal environment is not only related to the safety evaluation,but also affected the probiotic performance directly.At the same time,it also provides a new idea for screening the more excellent and efficient colonized mutants.In order to clarify the effect of intestinal selection pressure on the genetic stability of probiotics,from the point of view of intestinal microecosystem,this study took the Lactobacillus plantarum HNU082,which had the potentially probiotic function,isolated from traditional fermented food in Hainan province,to study the effects of intestinal selection pressure on the genetic stability of strain using zebrafish,C57 mouse and human as models.The mutation information of the Lactobacillus plantarum HNU082 genome in the intestinal tract was identified by combining the metagenomic sequencing and genome re-sequencing technology.In addition,the genomic genetic variation map of the strain in the intestinal tract was accurately drawn to find the potential relationship between the mutation and its phenotypic and probiotic characteristics,and to explore the mechanism of concerted evolution of intestinal microbiota.Firstly,based on the unique gene characteristics of Lactobacillus plantarum HNU082,we designed specific primers and established a method for isolation of Lactobacillus plantarum HNU082 from host intestinal tract.The isolates from different model hosts and different time points were screened by traditional culture methods combined with genomic techniques.Then the single-nucleotide polymorphism(SNPs)and the mobile elements in the genome of the isolates of Lactobacillus plantarum HNU082 were annotated,and the adaptive mutation of the strain was analyzed.It was found that the mutation frequency of strain was related to the colonization time,but not to the selection of the host model.The parallel evolution strategy was adopted in the mutation,which was driven by five parallel evolution genes:Gene 1470,Gene 2804,Gene 3003,Gene 3110 and Gene 3111 respectively,which were associated with carbohydrate utilization and transposase.After phenotypic verification,it was showed that the short-term intestinal colonization promoted the adaptive mutation of the strain and improved the adaptability of the strain in the host,which was reflected that the utilization of rhamnose and acid tolerance of the mutants enhanced.Finally,the response and co-evolution of host intestinal microbiota to probiotic intervention were revealed.The intake of probiotics changed the intestinal microbiota of mouse greater than that's of human,and led to the coevolution of intestinal microbiota in the host.The number of mutations of the intestinal microbiota in mouse model was much higher than that of in human model in the experimental group.The main competitors of probiotics in the intestinal tract,including Bacteroides and Bifidobacterium,produced a large number of exclusive mutations,which indicated the species should adapt not only to the environment,but also to other species in the process of evolution.It revealed the "Red Queen Hypothesis" in the evolution of intestinal microbiota.This study revealed the evolution of probiotics after entering the intestinal tract and explored the mechanism of concerted evolution between intestinal microbiota and probiotics,which also provided a new idea for the screening of more excellent mutants targeting the intestinal tract.