The structure of sojourners gut microbiota was analyzed at different heights, measuring the abundance of bacteria by WGS, representing known sequences at phyla level more than 1% using Wilcoxon rank test (odds ratio at 95% confidence intervals). There were eight phyla present in the fecal samples at different heights (Figure 3). Phyla Bacteriodetes was predominantly present, accounting for 80% of the identified taxa at H1 time point. The second richest phyla was Fermicutes with 18% presence. The next detectable phyla was 2% Proteobacteria (Figure 3 A). After ascending to and six months stay at H2, there was a significant decrease in Bacteriodetes to 64%, though still remaining the richest phyla, increase in Fermicutes contributing 35% with Proteobacteria remaining the same. The microbial composition varied immensely, depicting an interesting phenomenon when sojourners descended to H3 and stayed for two months. There was a sudden 5% appearance of Fusobacteria and increase in Proteobacteria to 6%. Moreover, the predominance of Bacteriodetes was reversed and increased to 76%, whereas Fermicutes reduced to 8%. At the end when sojourners ascended to H4 and stayed for four months, the newly appeared phyla Fusobacteria at H3, suddenly disappeared remaining with Bacteriodetes, Fermicutes and Proteobacteria 77%, 13% and 3% respectively. Other bacterial members from Actinobacteria, Verrucomicrobia, Lentisphaereae, Euryarchaeota were also detected with lower abundance.
Further, confirmation of the variation in the composition of fecal microbiota of sojourners at different heights was achieved by linear discriminant analyses (LEfSe) using the Greengene software (http://greengenes.lbl.gov). FDR correction of the p value was to determine statistical significance (p < 0.05/number of tests) and considered p < 0.05 statistically significant for reproducibility. Figure 3D indicates the association between the relative abundance of presence or absence of phyla and the significant change in the microbiota mainly with abundance of Bacteriodetes (p= 0.05), Fermicutes (p= 0.05) and Proteobacteria (p=0.05). LEfse analysis also showed concordant result with Wilcoxon rank test (Supplementary FigureS3). The results thus indicate the most important contributing factor to the diversity could be altitude or hypoxia as diet and ethnicity of all the subjects were the same.
The presence of eight phyla by WGS analysis in limited three subjects were reproduced by 16s RNA analysis in another 16 subjects from the same cohort at H1 and H2 heights (p<0.05 Wilcoxon rank test) with odds ratio of 95% (Figure 3B). The second method reconfirmed the significant changes in the abundance of phyla Bacteriodetes and Fermicutes (Figure3C).