Capítulo 6 Estrategia de implementación de la PMO de Go-Labs
6.1 Alcance
The advent of the Human Microbiome Project (HMP) has expanded our knowledge base regarding our microbial companions as well as exposing us to the broadness and complexity of this micro-environment. Studies have shown that the initial paradigm of "one microbe, one disease" is not ubiquitous but instead that there are communities of microbes associated with pathogenesis of disease within a host. Furthermore, studies have assessed that while there is no one sole microbe or microbial profile associated with "health", there are conserved functions associated with healthy gut micro-environment. Along with this knowledge, studies have ascertained that there exists a high level of microbial variation at an individual level that is not driven by host genetics but instead by host diet and environment54,55. While I have made steps in characterizing the gut microbiota and defining the role IBD-associated dysbiosis plays in IBD pathogenesis, much still needs to be learned about the biological mechanism as well as to design studies that take advantage of microbial survey data to investigate both existing hypotheses regarding the disease as well as generate new hypotheses.
In chapter II, I investigated the existence of a common microbial profile associated with post- operative occurrence of CD; identified region-specific alterations in the mucosally-adherent
microbiota; proposed a novel approach to analyzing the gut microbiota through the lens of
aerotolerance; and applied this to examining the data in relation to the "oxygen hypothesis"108. Using 16S rRNA sequencing and predictive functional analysis, I showed that while loss of diversity was universal across intestinal regions in the mucosa in CD, there existed distinct region-specific
alterations as well. Furthermore, I performed an aerotolerance analysis, where we classified taxa as "aerobic", "obligate aerobic", "anaerobic", "obligate anerobic", "facultative
anaerobic/microaerophilic" to determine if there was evidence supporting the oxygen hypothesis which posits that the dysbiosis seen in IBD that changes in oxygen tension leads to the loss of
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obligate anaerobes and the increase in the abundance of facultative anaerobes. My hypothesis was that if there was lower relative abundance of obligate anaerobes and an increase in the relative abundance of facultative species, this would be in silico evidence of the oxygen hypothesis.
Surprisingly, I saw in my patient cohort that (1) the colonic mucosa from CD patients did not undergo a significant loss in the relative abundance of obligate anaerobes compared to nonIBD patients, and (2) the ileal mucosa underwent an increase in the relative abundance of obligate anaerobes in CD in conflict with my hypothesis. I found that within the intestinal mucosa of CD patients, previously identified alterations seen in IBD-associated dysbiosis were not universal across the intestinal regions. For example, increased relative abundance of Klebsiella, a pathogen associated with IBD, was significantly altered in the ileal mucosa but not in the colonic mucosa. Furthermore, considering the high interindividuality of the human gut microbiota, I took an alternative approach to identifying dysbiotic bacteria that were not seen by traditional approaches. To determine whether the high relative abundance of a particular bacteria was unique to that cohort, I compared them to the relative abundance in a related cohort, for example CD vs nonIBD patients. Those that were found to occur at greater than 5% in at least one patient in the initial cohort, at less than 5% relative abundance in all patients in the second cohort, and further that the greatest relative abundance in any one patient in the second cohort was 3-fold less than the greatest relative abundance in the initial cohort, were considered specifically highly abundant in the initial cohort or “dysbiotic”. Traditional approaches to identifying differentially abundant taxa often end up detecting taxa that are differentially abundant but are often in low abundance in patients (<1%) or biased by one or two samples with a high relative abundance (>10%) of the identified taxa with the rest of the patients within the subgroup having low or no presence of the taxa. This alternative approach allows us to identify a broader range of taxa that would be missed by traditional methods. Consequently, in part due to the high interindividuality of the human gut microbiota, I was unable to identify common microbial profile that were associated with post-operative recurrence across cohorts.
In chapter III, I decided to focus on testing my in silico approach for the oxygen hypothesis in the colonic mucosa from nonIBD and IBD patients, considering both UC and CD subtypes. Through
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this analysis, I continued investigating the oxygen hypothesis and noted that: (1) UC exhibited higher variability in the relative abundance of obligate anaerobes than CD; (2) the UC cohort stratified into two subgroups based on the relative abundance of obligate anaerobe. I found they exhibited distinct microbial compositions with the "anaerobe-low" subgroup exhibiting an increased relative abundance
of aerobic-leaning bacteria such as Pseudomonas. This shows that within colonic mucosa of IBD
patients that the shifts in aerotolerance identified in silico are not universal across IBD subtypes, and that within UC, a subset of patients do align from an in silico perspective to the oxygen hypothesis potentially independent of clinical phenotyping. Furthermore, independent of the oxygen hypothesis, this emphasizes the disease-specific nature of aerotolerance between UC and CD within the colonic mucosa.
As an area of research, our understanding of the IBD-associated dysbiosis and the role of the microbiome in IBD is still evolving. While it has been accepted that the environment plays a role in the pathogenesis of IBD, efforts to characterize the dysbiosis seen in IBD through 16S rRNA
sequencing was first seen in 200488,184–186, and one of the first studies surveying the composition and alteration of the mucosally-adherent microbiota in IBD through the use 16S rRNA sequencing was published in 2009.47 It was a twin study that reported that patients with ileal CD had a lower relative abundance of Faecalibacteriumprausnitizii and increased relative abundance of E. coli compared to their healthy twin, and in the Crohn's colitis twin, bacterial shifts seen in patients was strongly correlated with the subtype of CD, ileal or colonic, as opposed to host genetics47. While there has been extensive research to characterize the dysbiosis and identify consistent microbial signals associated with IBD, the extensive heterogeneity of the disease and the interindividual variability seen in the intestinal mucosa as well as knowledge regarding the ileal mucosal microbiota compared to the colon has limited our understanding. My work in chapter II and III contributes to our overall
understanding of the region-specific nature of the intestinal microbiota in both CD and nonIBD, probes the oxygen hypothesis of IBD, and shows a greater need for sensitive approaches to
differential analyses of the microbiome as well as a broader approach to understanding the intestinal micro-environment. In particular, in relation to the oxygen hypothesis, my research has shown that
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from an in silico perspective, the oxygen hypothesis may not be applicable to all of IBD. While a subset of UC patients within my cohort do seem to support the oxygen hypothesis, further in-depth and experimental validation is needed to confirm this.
Looking towards future of research in the intestinal microbiota, there are multiple avenues that need to be explored to gain a clearer picture of the role that the intestinal microbiota plays in health and disease. First, while variable-region specific surveys have been essential in expanding our knowledge of the human microbiome as whole, there is a need to transition to the use of full-length 16S gene (~1500bp) surveys. While previously this approach had been prone to high sequencing error rates, making the benefits of using longer reads inefficient, as of 2019, there have been signs that accuracy of this process has improved making it potentially a viable option for microbial surveys187. The caveat is that the process has been only applied to mock community and fecal samples at this time, but I believe this is a fruitful avenue of research. Many bacterial strains, such as
E. coli, have been shown to differ by a single nucleotide. Longer and full-length 16S sequencing will allow us the ability to obtain finer resolution which will allow us order to more consistently identify bacteria at a species and strain level188. This is essential due to the existing functional diversity seen in bacteria this level. An example of this is the species E. coli. While E.coli is consider a common component of the intestinal microbiota, it is an extremely diverse at a strain level189. The ability to discrimination between bacterial strains is critical to gain a deeper understand of the dysbiosis seen in IBD.
Second, longitudinal studies are crucial to understanding the long-term dynamics of the intestinal microbiota and, in the context of my work, to understand the stability of the obligate
anaerobic levels in IBD over time. While there is high interpersonal variation in the composition of the intestinal microbiota between humans, from a temporal standpoint it is highly stable8,9. Longitudinal studies give the advantage of seeing the temporal dynamics of the intestinal microbiota as well as examining the impact of external confounding factors such as the application of therapy within the intestinal microbiota. This also will allow us to gauge the individualized impact of external factors on
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the intestinal microbiota. While the impact of antibiotics on the intestinal microbiota is well
studied190,191, more data is needed on the impact on common xenobiotics on the temporal dynamics
of the intestinal microbiota. This is need is two-fold, (1) understanding the interplay between xenobiotics and the intestinal microbiota will give us a deeper understanding on understanding functionally what is occurring; and (2) this will allow us to design more personalized approaches to therapies for IBD.
Third, functional studies of the microbiome are a key part for future studies into the interplay of the intestinal microbiota and IBD. Research has shown that while there is no one microbe
associated with a healthy microbiota, there are highly conserved functions associated with health8. The primary approaches to functional analysis are predictive functional analysis and metagenomic sequencing. Metagenomic sequencing gives us the ability to survey the entirety of the microbial community, thus we are able to survey not only the bacterial component but also the fungal, archeal, and viral components which we have limited knowledge on. Furthermore, not all bacterial variation is seen within the 16S gene. While there are multiple benefits to utilizing metagenomic sequencing, in the case of the mucosal microbiome, it is often considered impractical due to the high amounts of human contamination (often > 90%) in those studies192. Thus, the potential insight gained is heavily outweighed by the data that needs to be discarded in these experiments. In contrast, while it is predictive, tools like PICRUSt are decent proxies for investigating alterations in microbial function within the intestinal mucosa in both health and disease.
Finally, in addition to microbial surveys, there is a strong need for not only investigating host- microbe interactions but also microbe-microbe interactions. The majority of research has been focused on identifying and treating identified bacteria as individual members as opposed to treating them instead as a network community structure and the relationship between components of this network. I believe that this is critical for gain more insight into the dynamics seen in the intestinal microbiota and how they related to both diseased and non-diseased states. There have already been examples of co-occurrence networks being used to discriminated between polymicrobial oral
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diseases with similar presentations193,194, and I believe that this approach could be transferred to IBD research as well.
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