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.