These data have similarities to recently published clinical pilot studies, however, our methods and results have some notable differences that provide guidance for developing future studies. Howard et al. found changes in phylogenetic composition and relative abundance within 72 hours of injury in a population of 12 severely injured trauma patients.22 However, they found no difference in composition on admission compared with their controls. Our study had the power to detect a difference; however, it is important to note that we also found a small subpopulation of patients in whom the gut microbiome was not different compared with our healthy controls. Moreover, Howard et al. utilized patients that sustained a trauma but were not found to have injuries as their controls as opposed to our control population of healthy, uninjured volunteers. In this light, it can be speculated that the stress of transport to the hospital has effects on microbial flora in the gut. Future studies might incorporate both this control group and normal healthy individuals to explore this possibility. The findings from our study concerning changes with hospital longevity are also consistent with previous studies performed in critically ill patients with prolonged hospitalization demonstrating dysbiosis and the emergence of low-diversity communities.16–19
Extensive evidence supports the gut as an immune organ, especially given its intimate relationship with the gut microbiota.9,33–35 Trauma-induced injury to the gastrointestinal system can produce profound effects on the gut microbiome and the immunoinflammatory response with resultant consequences on clinical outcome. Increased inflammation at the intestinal level and decreased antimicrobial peptides appear to influence the pathophysiologic processes following injury.24 Damage to the intestinal wall leads to mucosal barrier inflammation, resulting in higher gut nitrate levels and an abnormal mucosal oxygen gradient.36–38 These environmental and metabolic changes lead to proliferation of pathogenic microbes in the Proteobacteria phylum (including Pseudomonas aeruginosa and Escherichia coli), in addition to pathogenic species from the normally health-promoting Firmicutes phylum (including Staphylococcus aureus and Enterococcus spp.).38–40 This new unstable microbiome ecosystem that emerges more closely resembles that of an infectious state with low-diversity microbial communities.38 Targeting this pathobiome with alternatives to antibiotics (e.g., probiotic adjuncts or virulence directed medications) also has the potential to improve outcomes.
Physiologic stressors such as hypoperfusion and vasoconstriction also impair gut motility and alter the intestinal flora. The resulting ischemia reperfusion injury has been shown to induce changes in ileal and colonic microbiota.41–43 Thus, our finding that resuscitation seemingly protects gut microbiota is intriguing. However, the brain-gut axis and central nervous system dysfunction may also impact the gut microbiome through bidirectional vagal pathways between the CNS and the gut, neuroendocrine signaling, immunologic signaling, and the effects of microbe-derived metabolites such as butyrate on the blood brain barrier.27,44–46 In other types of trauma, intestinal permeability allows for translocation of certain types of bacteria, which may be related to ZO-1, occluding, or mucin levels24,47,48 Additional insults during the hospitalization such as subsequent episodes of hypoxia, prolonged exposure to medications (e.g., antibiotics, opiates, vasopressors, steroids, proton pump inhibitors), multiple procedures and trips to the operating room, and periods of inadequate or artificial nutrition can all further disturb the gut microbiota.32 These aberrations may then influence clinical outcomes such as mortality from late onset sepsis and inflammatory disorders, hospital and ICU length of stay, infection rates, and inflammatory disorders.
There are several limitations to our study. While associations can be inferred, the findings do not prove causality. Future preclinical studies could elucidate some of the mechanisms involved that may be causing the changes observed in the gut microbiome following injury. In our study, controls were healthy volunteers with some included from hospital personnel. Future clinical studies could expand the control population to include patients sustaining trauma but found to be uninjured. Also, subsequent samples after admission swabs were taken from stool at the time of defecation; this led to inexact time points, which we subsequently pooled. While rectal swabs may provide consistency in the future, it has been shown that rectal swabs have the same integrity of isolated DNA.49,50 Furthermore, the use of antibiotics was not accounted for in the current study which would almost assuredly influence the gut flora during the hospitalization. The microbiome of antibiotic exposed subjects (trauma patients) is likely different than healthy volunteers not exposed to antibiotics. In addition, massively transfused subjects are known to be depleted of infused drugs, especially antibiotics, which could affect the transfusion-related differences. Of note, no antibiotics were given in the prehospital setting prior to admission to the ED and rectal swabs: therefore, antibiotics likely had little effect admission samples. Future microbial diversity analyses could attempt to account for antibiotic usage.
S.E.N. participated in the study design and idea, literature search, data collection and patient enrollment, data generation, data analysis, data interpretation, article drafting and critical revision, project oversight;.D.M.B. participated in the data and statistical analysis and bioinformatics, data interpretation, critical revision, generation of figures. T.R.J. participated in the literature search, data collection, and article drafting. Y.Z. participated in the data analysis and bioinformatics. Z.L. participated in the metagenomic sequencing and data analysis. S.S. participated in the data collection, sample preparation, laboratory analysis, and data generation. M.D.R. participated in the data collection and patient enrollment, study coordinator for patient enrollment and study completion. R.B.J. participated in the data collection and patient enrollment, study coordinator for patient enrollment and study completion. D.R.M. participated in the data collection and patient enrollment, sample preparation, laboratory analysis. C.Z. participated in the data collection and patient enrollment, literature review, and critical revision. E.S. participated in the data interpretation and critical revision. R.M.S. participated in the critical revision, and mentorship. M.G.S. participated in the data interpretation, critical revision, mentorship. D.H.J. participated in the data interpretation, critical revision, mentorship. B.J.E. participated in the critical revision and mentorship.
The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant KL2 TR001118. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Department of the Army and the Department of Defense. Support was also received by the University of Texas Health San Antonio Military Health Institute and the Bob Kelso Endowment awarded to the University of Texas Health San Antonio Department of Surgery. We would like to thank the following individuals for their support: Basil A. Pruitt, Jr., Dawn Garcia and Korri S. Weldon for 16S sequencing sample processing and data generation, Yidong Chen, PhD for bioinformatics support.
The authors have no conflicts of interest to disclose.
No competing financial interests exist.
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Mitchell J. Cohen, M.D. (Denver, Colorado): That’s the Ernest E. Moore Shock Trauma Center, I might add. Dr. Agarwal and Dr. deMoya, it’s my honor to be asked by the AAST to discuss this excellent work that was nicely presented by Dr. Nicholson.
Indeed, it’s my greatest honor to be part of AAST and the important science our organization supports. I am equally honored to be invited to discuss this particular paper which I should say is very important.
I think this science is, indeed, the future and this work is very good. I am very much an admirer of both this work and the group from San Antonio.
In this work the outstanding and very prolific group from San Antonio is centered around the very important topic of the microbiome after injury, in particular their insight about not only the infectious biology but, more importantly, the mechanistic insights into both what drives the biome and also what the biome drives, which is separate from the commonly discussed infectious disease sequelae.
In this work the group follows on from work from many groups, including ours, which has shown in a few papers that both the lung and the gut biome are affected by injury.
In the lung work our group, when I was in San Francisco, showed that smoking exposure predisposes the biome to make more likely acute injury after trauma. And in the gut work we showed a narrowing and increased virulence very quickly after injury as a result of trauma.
In that work our group struggled with what the control group should be. Indeed, there is a huge body of work in the biome outside of trauma showing vast differences in the biome depending on social and health factors.
Your living situation, your socio-economic status, what you ate, who you live with and your acute and chronic health status all radically change what your biome looks like.
When we looked at uninjured, healthy volunteers and uninjured trauma patients we saw vast differences between these control groups. Right? Those of us just walking around the hospital who were willing to give a sample and the patients who came to our trauma center and weren’t severely injured.
And because of that in our previous paper we used uninjured actual patients because we felt that they more closely represented our trauma cohort than did the healthy volunteers that were walking around the hospital.
And to that end, with that preface, I have some concerns about the methodology in this paper of picking the control group from healthy volunteers. The authors used these healthy controls which I worry don’t reflect an appropriate control to the trauma group. So I have a few questions.
What do we know about these healthy controls? Are they similar in socio-economic or health status to the trauma group? Were they on antibiotics? Did they have any health problems?
And, Number 2, why not use uninjured healthy volunteers? Clearly we all have an over-triage rate, patients that get brought to our trauma center as high-level trauma activations who were totally uninjured. Why not use those? To make that the center of comparison would probably be better.
Number 3. Tell us about the time points. I know that you got these at Time Zero and then at multiple different times but I see from the manuscript that they were convenient samples when patients had bowel movements. That makes the timing very different. Why not use a digital rectal exam to do the rectal swab and then you get really comparable time points?
Number 4. There is a claim that the biome is changed within 30 minutes after injury. This, by my read, is a comparison of those trauma patients to those healthy controls so it very well may be that the trauma patients are just different than the healthy controls. I absolutely believe that the biome changes quickly but may not that quickly.
And so I am wondering do different injury severity types change the alpha diversity? What about the comparison to less-injured or uninjured trauma patients, as I keep harping on? Does that difference persist in your data set?
And, lastly, I have some question about the finding of blood transfusion being protective of beta diversity. While it’s an important finding and it clearly fits my bias, is it controlled for all of the other factors, including injury severity, shock, and individual patient characteristics? This needs to be done in a multivariate way rather than a single variate way.
Overall, I have to say that I congratulate the authors on this very important and well-done study. To be clear that they have my scientific admiration and I think this is really important work. I really do think it is the future and stuff we are going to be talking about for many years to come.
My questions are not meant to suggest otherwise, but only to foster scientific discussion and to get to the bottom of this crucial biology and topic.
I thank the AAST for the privilege of discussing this paper.
Susan Evans, M.D. (Charlotte, North Carolina): You used ISS as your way to determine whether or not you were going to include patients in the study. How did you do ISS?
Most of us I think don’t have access to ISS while in the trauma bay. Can you tell us, do you do that or do you have registrars who are immediately ready for that information? Thank you.
Susannah E. Nicholson, M.D. (San Antonio, Texas): I’d like to thank Dr. Cohen and Dr. Evans for their questions and for the important discussion.
So to answer the question about the controls, certainly that is a very important observation. But we, you know, in this early pilot study we did, we picked healthy volunteers in this study.
And I think certainly worth including uninjured trauma patients is a great suggestion for future studies and would like to incorporate that into future work. That being said, our controls that were used were not on any antibiotics, overall were fairly healthy.
Most of the patients, in reviewing that data – although I would have to go back in and do more of a statistical analysis for my patient data – in terms of health problems I do have some of that but most of the patients were actually fairly healthy, as well. We tried to limit patients that were on antibiotics.
Also, in terms of the fecal samples, again, another great point. You know in designing this study I think if I had to do it over again I would go ahead and try to use the rectal swab on the subsequent days so to take that out of the equation.
That being said, so a rectal swab, there is literature to support the rectal swab as the same as a stool sample but definitely the timing part of the stool sample would be helpful to have that more standardized.
In terms of the 30-minute, that question, again, we certainly looked at ISS and there was no difference in alpha diversity in ISS or however there was a difference in the severely-injured patients compared to the less-severely-injured patients.
There was no difference in beta diversity in blunt versus penetrating trauma and no difference in shock index in beta diversity. However, there was a difference in alpha diversity in patients that had penetrating trauma did have a higher, actually had a higher diversity.
Again, more work is needed to better delineate that time period. Also, you know, this study obviously does not show, does not discuss causality but some more preclinical work is needed to also address this.
In regards to – in regards also to the ISS or in looking at some of the other factors in some of our analysis, so with the beta diversity the analysis for that is ____________ in nature and is not discreet.
So we did – that being said, we did do multiple _______novas for the ISS for shock index for blunt and penetrating trauma in addition to the numbers of blood products and our RVCs first received.
To address your point, we definitely could incorporate in looking at alpha diversity since it incorporates specific numeric data points we could certainly incorporate some of the other factors into an additional analysis.
For the beta diversity, also going back to with the transfusion, there was a significant difference in the beta diversity
And this was in the absence of those healthy controls in the patients that on Day Zero that received more blood versus those that received less blood. In the alpha diversity they had a more diverse microbiome which has traditionally been seen as a more of a beneficial thing.
So to sum all of that up, it is definitely great to, a great area where we want to do more research.
Oh, and, also, to address Dr. Evans’ question, how we determined ISS, our group has developed a table, basically, that helps us define you know based on CT findings. And we estimate to the best of our knowledge the ISS and then can go back and confirm based on the trauma registry.