Objective To investigate the influences of exposure to different environmental microbes on early-life gut microbiota colonization in mice.Methods Male (n=8) and female (n=16) adult specific pathogen free (SPF) BALB/c mice were caged together at a ratio of 2:l.After conception,the mice were divided into four groups according to the environments where the offsprings were reared at three different periods (fetal period,breastfeeding period and childhood).Group A:Offsprings were kept in a SPF environment throughout the study;group B:SPF environment during fetal and breastfeeding periods,and then ordinary environment during childhood;group C:SPF environment during fetal period,and then ordinary environment during breastfeeding period and childhood;group D:ordinary environment all the time.Fecal samples were collected at the end of week 3 and 5.Total bacterial DNA was extracted from each sample and analyzed by high throughput analysis.Kruskal-Wallis and Dunn-Bonferroni test were applied for statistical anaysis.Results 1.At the end of three weeks:(1) Diversity:① Phylum level:There were significant differences in the abundance of Firmicutes,Verrucomicrobia,Proteobacteria and Actinobacteria among the four group (all P＜0.01).Compared with group C and D,group A and B showed significantly decreased abundance of Firmicutes [30.876(23.448-41.218)× 10-2,3.317(1.116-4.641) 10-2 vs 71.936(53.587-86.713)× 10-2,79.105(56.305-82.736)× 10-2],but increased abundance of Verrucomicrobia and Proteobacteria [Verrucomicrobia:17.249(9.748-35.106)× 10-2,58.883(0.017-6.047)× 10-2 vs 0.152(0.066-1.890)× 10-2,0.003(0.000-0.016)× 10-2;Proteobacteria:12.640(0.336-15.070)× 10-2,3.653(3.362-4.5955)× 10-2 vs 0.219(0.134-0.325)× 10-2,0.124(0.116-0.165) × 10-2,all P＜0.05 or 0.01].② Genus level:There were significant differences in the abundance of Lactobacillus,Akkermansia and Bacteroides among the four groups (all P＜0.01).Compared with group C and D,group A and B showed significantly decreased abundance of Lactobacillus [19.283(8.618-31.541)× 10-2,0.339(0.264-22.278) × 10-2 vs 58.414(34.874-71.942)× 10-2,66.007(55.141-76.940)× 10-2],but increased abundance of Akkermansia,Bacteroides and Klebsiella [Akkermansia:17.247(9.748-35.106)× 10-2,58.883(0.017-60.475)× 10-2 vs 0.152(0.066-1.890)× 10-2,0.003(0.000-0.017)× 10-2;Bacteroides:3.978(0.683-25.171)× 10-2,8.216(6.023-9.946)× 10-2 vs 0.141(0.061-0.281)× 10-2,0.568(0.149-1.455)× 10-2;Klebsiella:0.209(0.050-8.888)× 10-2,1.402(0.865-1.692)× 10-2 vs 0.003(0.000-0.039) 10-2,0.000(0.000 0.001)× 10-2,all P＜0.05 or 0.01].(2) Alpha diversity:Significant differences were found in operational taxonomic unit (OTU) and Chaol index (P＜0.05),but not in Shannon index among the four groups (P＞0.05).The OTUs of group A and B were significantly lower than that of group D [246(221-348),257(209-280) vs 387(324-478),P=0.045 and 0.008,respectively].2.At the end of five weeks:(1) Diversity:① Phylum level:There were significant differences in the abundance of Firmicutes,Verrucomicrobia and Proteobacteria among the four groups (P＜0.05 or 0.01).The abundance of Firmicutes in gut microbiota in group A was lower than that in group B,C and D [13.765(64.181-24.238)× 10-2 vs 48.912(37.280-59.466)× 10-2,86.065(50.149-89.856) × 10-2,53.847(31.946-72.936) × 10-2],while that of Verrucomicrobia was higher [58.089(22.459-61.285)× 10-2 vs 0.001(0.000-0.005)× 10-2,0.000(0.000-0.001)× 10-2,0.003(0.000-0.006)× 10-2],all P＜0.05 or 0.01.② Genus level:There were significant differences in the abundance of Lactobacillus and Akkermansia among the four groups (P＜0.01).The abundance of Lactobacillus in gut microbiota in group A was lower than that in group B,C and D[1.755(0.805-8.833)× 10-2 vs 26.391(17.550-37.265)× 10-2,70.688(45.713-77.953) × 10-2,28.675 (15.660-57.224) × 10-2],while that of Akkermansia was higher [58.089(22.460-61.285)× 10-2 vs 0.000(0.000-0.006)× 10-2,0.000(0.000-0.001)× 10-2,0.003(0.000-0.006)× 10-2,all P＜0.05 or 0.01].(2) Alpha diversity:There were significant differences in OTU,Chaol and Shannon index among the four groups (P＜0.05 or 0.01).The OTU of group A was lower than that of group B,C and D [268(241-410) vs 438(380-516),562(533-588),546(473-599)],and the OTU,Chaol and Shannon index of group B were all lower than those of group C and D [OTU:438(380-516) vs 562(533-588),546(473-599);Chaol index:1 033(883-1 181) vs 1 285(1 220-1 338),1 328(1 155-1 516);Shannon index:3.85(3.25-4.50) vs 4.28(3.30-5.11),4.17(3.62-4.38),all P＜0.05 or 0.01].Conclusions Early-life exposure to different environments has an obvious impact on the diversity and composition of intestinal microbiota in mice.The less clean the living environment is,the more diverse the gut microflora will be.Furthermore,the window of opportunity for gut microbiota colonization seems to be related to breastfeeding period.