| There are many factors influencing the bacterial colonization in the intestine,which can be divided into environmental and host related factors.Environmental factors include diet,geographic location,and delivery patterns at birth.Host related factors include disease,gender,age and host genotype.The intraspecies differences among strains,especially in the dominant and the most important species of the intestinal tract,attract more and more attention recently.Bifidobacteria,as one of the the representative genus,can reach 10~100 million/g in human intestine,and are critical to the host's health and metabolism.However,as the intraspecies 16S rDNA homology is almost 100%,it is difficult to identify or type Bifidobacterium isolates at strain level.So far,there is a lack of research on the diversity of Bifidobacteria strain types in human intestine,and the diversity among individuals.It is still poorly understood regarded to the relationship between in vivo colonization of strains,host genotypes and the environmental factors.In the present study,a multilocus sequence typing(MLST)scheme for Bifidobacteria typing was established,and the feasibility of using this method for strain level typing of Bifidobacterium isolates was confirmed.Then,child and adult twins,as well as monozygotic(MZ)and dizygotic(DZ)twin pairs were recruited for differenciating environmental and host related factors.B.longum subsp.longum(BL)isolates from the twins'feces were analyzed by MLST to observe the influences of environment and the host genotype.Representative BL strains of each sequence types(STs)were subsequently screened by random amplification polymorphic DNA(RAPD)to search for strain-specific amplification bands and screening of strains with unique biomarker sequences.Then a selective quantitative detection technology at strain level from the complex samples was established by arbitrary primer qRT-PCR targeting the biomarker sequences.Based on the strain-specific quantitative detection method,animal experiment was then introduced to further confirm the model bacterial colonization affected by host genotypes and the environment.A mixture of three BL strains at the same concentration was simultaneously inoculated into the premature mice of two inbred lines C57 and Balb/c.After that the inoculation of BL mixture suspended during the adult period of mice,fed on high fat and normal diets respectively.Comparison of the implantation results at the premature period,as well as the comparison of strain persistence in the intestinal tract of mice during adulthood was conducted.It is revealed that the colonization of BL strain is a result of the combined influences and selection of strains,hosts and environment.This is very important for understanding the interactions between Bifidobacterium and host metabolism,as well as developing or screening more personalized probiotic strains with better host and environment adaptability in the future.In this study,seven housekeeping gene loci dnaJ,purF,rpoB,clpC,fusA,gyrB and ileS were finally selected from 12 gene loci based on the existing 57Bifidobacteria strains in our laboratory.The corresponding primers were used for Bifidobacteria MLST,the length of amplified partial allele gene sequences varied from 387bp(dnaJ)to 1015bp(rpoC),and 57 strains could be classified into 28 STs.The identification result at species level was consistent with 16S rRNA gene analysis conducted previously,and it also showed good polymorphism and resolution at the strain level.Twenty-eight BL strains were classified into 12 STs,indicating that this MLST protocol could be applied for the typing of Bifidobacterium isolates at strain level.Child and adult twins were recruited in this study as the model group in the same environment and in different environments.Both child and adult groups consisted of two MZ twin pairs and one DZ twin pair,representing the same host genotype and different host genotypes.Fecal samples were collected lasting for 8 months at two-month'interval,results showed that the number of Bifidobacteria in the feces of the adults was more stable,the concentration in the samples of children varied greatly with age,and the content of total Bifidobacteria was more similar within twin pairs.A total of 577 Bifidobacterium isolates were obtained from the twins'fecal samples,and345 isolates were identified as Bifidobacterium longum subsp.longum by 16S rRNA gene sequence.These BL strains were then clustered into 35 STs and 82 individual strains by MLST.Results analyzed by e-Burst and START2.0 software indicated that,BL isolates from different individuals were highly diversified and specific,and no isolate with same STs were found among the unrelated individuals(non-twins).However,there are eight STs monophyletic exist within twin pairs.The overlap rates of monophyletic ST isolates in child twins(same environment)and adult twins(different environment)were significantly different.No significant difference of the overlap rates of monophyletic ST isolates in child MZ twins(same environment,same host genotype)was found compared with child DZ twins(same environment,different host genotype).While the overlap rates in the MZ adult twins was significantly higher than that in the DZ adult twins.These results suggest that environmental factors are the major determinant of Bifidobacterium strain types in childhood before stable intestinal flora shaped,and later in adulthood,which specific strains can be colonized in the long term may not only be affected by environmental factors,the host genotype also play a role.Thirty-five above mentioned BL strains from different STs were screened by RAPD analysis of 20 random primers and cloned sequencing of unique amplification bands.Arbitrary primers were designed based on the unique sequences of each strain.Finally three BL strains(N58,L2 and A45)and corresponding strain specific primers for qRT-PCR were obtained.The standard curves of strains(N58,A45 and L2)were prepared on the basis of the fresh feces suspension of mice,and results of qRT-PCR showed that the linearity of the standard curve was satisfied.The strain selective quantitative technology was suitable for quantitative detection of target strains in mice feces.And the detection limit was 1.0 x 10~5 cfu/g to 2.0 x 10~9 cfu/g target strains in the feces.In order to further confirm the results found in the twins study,an animal experiment was introduced to discover the influences of host genotype and diet on the colonization of three different BL strains in the intestines of mice.Premature C57 and Balb/c mice of two inbred lines were inoculated the mixture of three BL strains(N58,A45 and L2)by daily gavage at 2.0×10~9 cfu/day of each strain,lasting from age 3weeks till 8 weeks.Concentrations of each strain in feces,colon contents and cecal contents were determined every week or two weeks respectively.Results showed that the concentration of BL strains increased gradually,reached the highest level and kept stable at the age of 6 weeks(after 28-days'gavage).While strains were simultaneously inoculated at the same concentration,it was observed that the implantation results among each strain significantly differed,and the difference varied in two inbred mice.Results demonstrated that the strain implantation in the intestine was affected by both the characteristics of strains and the host genotype of mice.Subsequently,in order to observe the persistency of these strains in adult mice with different host genotypes or diets,other than the survival ability during continously administration,the gavage suspended at the age of 9 weeks,mice of each inbred line were then divided as high fat diet feeding and normal diet feeding groups respectively.The concentration of each strain in feces,colon contents and cecal contents was quantitatively determined,and results showed that,L2 and N58 strains seemed demonstrated better adaptability to the intestine of C57 mice than that of Balb/c under normal diet,and the adaptability of A45 to the two hosts performed no significant difference.Otherwise,on the high fat diet,L2 strains showed lower adaptability to C57 than that of Balb/c,but the adaptability of other two strains seemed no difference between different hosts.The influence of the host genotype on the persistence of BL strains was relatively weakened under high-fat diet feeding.Compared with the normal diet feeding,the persistent days of three strains both in feces and colon contents in high fat diet feeding groups were significantly shortened,which indicated that the high fat diet significantly affected the colonization of BL strains in the intestine of mice,resulting in a rapid decrease of their concentration.It was also interestingly found that the sensitivity to dietary changes among three BL strains varied.Strain A45 was the most susceptible to high fat diet and performed the shortest persistent time.In addition,when the implanted strain kept stable at the end of the premature period,concentrations of the three strains in the intestinal tract of C57 mice were:N58>A45>L2,in Balb/c mice:A45>N58>L2.However,during the persistent period,strain L2 which had the lowest initial implantation content,demonstrated best persistency in adult mice.On the contrary,A45,which had a high initial implanted concentration,have the weakest persistency,i.e.,the shortest persistent time.This indicated that,strains obtained at a high level in the early life of mice by continuous inoculation,was not necessarily survive and dominate in the long run but determined and affected by the adaptive capacity to particular host genotype and dietary structure.Therefore,in this study,the relationship of colonization ability of Bifidobacterium,the host genotype,and environmental factors such as dietary was discussed at strain level,suggesting that many influencing factors including population characteristics,age or diet habits should be considered during the selection of the next generation probiotic strains,as well as its colonization ability and healthy function assessment. |
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