Discussion
The present study was performed to analyze the gut microbiota of 19 Indian sojourners on an extreme altitude expedition. We compared two sequencing methods, WGS and 16srRNA to latch on the complete picture of the gut microbial diversity and functional networks involved at extreme altitudes under low oxygen pressure. By doing so, we uncovered both known and novel microbial components, upto the species and strain levels, including a highly altered network of various functions indicative of leading to pathological changes. The two analytical methods employing various state of art pipelines presently available for gut meta genomes, verified our meta-analysis framework to be robust and appropriate. To the best of our knowledge there has been so far no study on microbiome -wide expression changes in Indians high and extreme altitude responses and that ours is the first one and is therefore imperative in shedding light on some prospective cohort studies and pooling data at various altitudes with large sample size, that should be performed in order to study the effect on the human gut microbiome in identifying important associations that may be involved with detection and prevention of some pathological diseases.
The study represents significant microbial changes in individuals exposed to various altitudes which could be attributed to environment while with similar diet and genetic patterns. The encouraging part of the study was, samples were collected longitudinally making it possible to determine microbial changes associated with the induction to different altitudes and easily compared with their own baseline profile. However, the study was not without limitations. It had relatively small sample size, therefore, we were underpowered to detect many statistically significant details, which were nevertheless circumvented by using more than one methods and latest pipelines, providing additional information for down-stream data pooling and meta-analysis50 . We were able to use 16S rRNA gene sequencing to reproduce significant bacterial abundance in the same population that was assessed using WGS. We chose to collect fecal samples mainly because of its easy accessibility, which is extremely important if changes in the microbiome revealed were to be used as indicators of the entire human health status.
Human gut is extraordinary dense with microbes, facing the greatest challenge of number & diversity of intestinal species29,51 . There are many factors having impact on the composition of gut microbiota. However, so far, the statistical analysis indicated that the three most important contributing factors are environment, genetics and diet29 . Hence, we chose to focus on examining the impact of altitude and related environmental conditions on the differences in the gut microbiota of Indian male sojourners of same age range, same ethnicity, altitude (210m H1) and similar dietary habits, visiting different altitudes (3500, 4420 to 5800m) for short time periods.
The gut microbiota of Indian sojourners was compositionally distinct at high and low altitudes. Three samples for WGS had similar diversity measures compared to another sixteen sequenced by 16s rRNA method. Overall Bacteriodetes and Firmicutes revealed their marked presence. A relatively lower abundance of Proteobacteria is apparently a positive state of gut health, indicating no epithelial dysfunction52 in the recruited cohort. At sea level H1, the relative abundance of phyla Bacteriodetes was much more than Firmicutes, which is in line with the previous studies on Indians19,20,21,30 .
The same subjects after staying at H2 for six months had altered pattern with decreased Bacteriodetes, though still remaining the maximum. Whereas after descending to H3 and staying for two months, resulted in reversal of pattern by increase in Bacteriodetes & Proteobacteria, similar to the findings of Das et al at 3500m30 and sudden appearance of Fusobacteria. Again, after ascending to H4 and stay for four months, Fusobacteria disappeared with increase in Fermicutes and decrease in Proteobacteria whereas Bacteriodetes remaining almost the same and maximum. All the study subjects had normal BMI as per the standards53 and similar diet. Bacteriodetes have been reported to make distinctive lipids associated with atheroma formation and contribute to heart disease54 . Therefore, to investigate the effect of different altitudes on gut microbiota of Indian subjects of same ethnicity with high abundance of Bacteriodetes, consuming similar diet and visiting greater altitudes becomes very important. On the other hand, it could also be possible under altered circumstances, body actively adapted to the required bacterial flora as it is very common during pregnancy55 . It is quite conceivable that living at HA under multiple harsh conditions like cold, hypoxia, radiations etc., requires well balanced and strong gut microbiota to support the nutrients availability for metabolism. Nature plays its role by altering the microbial diversity which helps in building an adaptive response to the extreme conditions.
Comparison of genera at different altitudes, revealed the changes within Bacteriodetes, majorly due to significant alteration in the genera Bacteroides & Prevotella. Human gut has been classified into three enterotypes and each one is predominantly occupied by Prevotella, Bacteroides and Ruminococcus because they explain the most human taxonomic variations56,57,58 . In the present study, the analysis of microbiota at different altitudes revealed Bacteroides enterotypes significantly higher at sea level H1 and moderate at altitude H3 whereas Prevotella enterotypes prevailed at H2, H3 and H4. Our findings are in line with the previous reports of negatively correlated relative abundance of Bacteroides and Prevotella59 , suggesting their antagonistic nature, that needs to be ascertained.
It is believed that dietary factor has a dominant role in different gut microbiota23,60 . In the present study the general diet of these sojourners at sea level was low fiber, high protein and high fat, whereas at HA from 3500m onwards the diet was composed of high fiber and low protein. The present study indicates that the anti-correlation may not exclusively be due to diet, because the diet of sojourners was same at H2, H3, and H4, yet the abundance of Bacteroides at H3 was close to that of at H1 than at H2 and H4. Interestingly, at H4 the abundance of Bacteroides was significantly higher than at H2. However the prevalence and abundance of Prevotella was higher at H2, H3 and H4. Our findings on Indian population are not in agreement with the analysis of Li et al29reporting that most Han population at 3600 m belonged to the Bacteroides enterotype, rather in agreement with most Tibetans staying at HA at 4800m who are of the Prevotella enterotype. Das et al have also reported Prevotella abundance at 3500m, with highest percentage of individuals consuming non-vegetarian diet30 . In the present study out of 19 subjects only four were vegetarians, therefore higher abundance of Prevotella in 15 non-vegetarian individuals consuming similar diet at different altitudes was quite interesting. These findings are in contrast to the studies indicating association of Prevotella with high levels of carbohydrates and fruits & vegetables intake61,62 , whereas in line with other studies confirming with highest percentage of individuals consuming non- fiber diet63 . Interesting to note that western Indian population at 53m above sea level with highest percentage of individuals consuming carbohydrate and fibre­rich components31 , and north Indian population at 197m with majority of non-vegetarians30 , have predominance of Prevotella. It is also indicated that omnivorous oligotypes of Prevotella are unique and significantly high, specifically associated with animal origin diet in Indian population30,23 . Our analysis at four different altitudes revealed association of Prevotella with non-vegetarian dietary pattern which was further confirmed on sub-typing and revealing higher abundance of P11 and P12 oligotypes. The association of these oligotypes has been established with animal origin diet and tri-methyl amine oxide concentration, a carnitine and choline catabolism derived metabolite related to development of CVD23 . These evidences of different responses of Bacteroides and Prevotella in Indian population clearly indicate the involvement of factors other than diet pointing towards the dominance of Prevotella at sea level20,30,31 , which increased with the increment in the altitude. Therefore, alteration in the genera Bacteroides and Prevotella cannot be attributed to the dietary or genetic factors only but could be the altitude or hypoxia or other environmental factors which needs to be further looked into.