The representative and most abundant phyla included Firmicutes, Bacteroidetes, Verrucomicrobia, Proteobacteria, Cyanobacteria, Tenericutes, and Deferribacteres in all samples, irrespective of the time after injury (Fig. 5A). The dominant phyla in both the pre-TBI and the post-TBI groups were Firmicutes and Bacteroidetes. These two phyla comprised 89.8% of the bacteria in the pre-TBI group, and total levels of these two phyla decreased by about 10% by day 3 post-TBI, even though there was an overall increase in Bacteroidetes. Conversely, the phylum Proteobacteria, containing many pathogenic bacteria, more than doubled during the same time post-TBI. The ratio of Firmicutes to Bacteroidetes decreased significantly with the nadir occurring on day 1 post-TBI (2.4 for pre-TBI vs 1.1 for 3 days post-TBI; P = 0.012). Similar data for sham animals are shown in Figure 5B. There was no difference in the GI microbiome between sham and TBI animals prior to craniotomy/TBI. Additionally, in sham animals, the microbiome phyla remained relatively unchanged over time, with the lone exception of a small decrease in the phylum Bacteroidetes on day 3 post-craniotomy compared to pre-craniotomy levels. This finding is the opposite of what is observed in TBI animals, and otherwise there was no change in the microbiome in sham animals (Fig. 5B).
The data in Table 2 shows specific changes in the families of each phylum after TBI. There were significant decreases in relative abundance at the phylum level seen in Firmicutes at days 1 and 3 and Deferribacteres at day 1 following TBI (P < 0.05; Table 2 and Fig. 5A). Conversely, an increased relative abundance in the phyla Bacteroidetes and Deferribacteres was observed at day 1 following TBI (P < 0.05; Table 2 and Fig. 5A). Within the phylum Firmicutes, there was an initial decrease of ∼50% in proportion of sequences in the family Lachnospiraceae at 2 h after TBI, which remains suppressed through 3 days and returned to baseline by 7 days. The Mogibacteriaceae family was also significantly decreased (P < 0.05) at 1 and 3 days, and returned to baseline levels by 7 days. Ruminococcaceae levels decreased at 1 day after TBI only. Lactobacilliaceae levels were not significantly different after TBI. Levels of bacteria from the family Anaeroplasmataceae, in the phylum Tenericutes decreased by ∼60% at 2 h after TBI and remained markedly decreased over time. Deferribacteraceae (a member of the Deferribacteria phylum) levels), were reduced ∼75% at 1 and 3 days and approached baseline by 7 days after TBI). The Bacteroidete family Bacteroidaceae demonstrated a 50% increase in the proportion of sequences by 1 day after TBI with a return to baseline by 7 days). Verrucomicrobia, from the phylum Verrucomicrobia, demonstrated a similar trend with a decrease seen at 1 day and return to baseline levels by 7 days). Enterobacteriaceae and Pseudomonadaceae, members of the Proteobacteria phylum, all demonstrated markedly increased levels by 3 days post-TBI with all levels in these families returning to baseline by 7 days (P < 0.05).
Correlative analysis of MRI lesion volume, behavioral assessments, and the GI microbiome
To directly compare the magnitude of functional and structural changes to the microbiome alterations, linear regression was performed. Linear regression revealed that changes in both Firmicutes (y = −7.46x+110.7, r2 = 0.914, P < 0.0001) and Proteobacteria (y = 3.29x−15.61, r2 = 0.805, P = 0.001; Fig. 6A) were strongly correlated to MRI lesion volume, with a weaker but still significant relationship to the phylum Verrucomicrobia (y = 0.96x+0.88, r2 = 0.55, P = 0.02). Moreover, regression analysis also revealed that larger brain lesion volume resulted in a significantly greater reduction in α-diversity by all indices measured (Fig. 6B). Although not quite as strongly, changes in gut microbial phyla and diversity were also associated with behavioral/functional outcomes as measured by the foot fault test. Specifically, the foot fault scores also tended to positively correlate to Proteobacteria, although this was not quite significant (P = 0.081). However, there was a significant correlation to the phylum Verrucomicrobia (y = 1.25x−0.615, r2 = 0.773, P = 0.0018, Fig. 6C), as well as significant negative correlations to α-diversity measures (Fig. 6D).
The current study was designed to detect changes in the GI microbiome due to a moderate-TBI injury over time. Significant changes in the GI microbiome were evident as early as 2 h after TBI as compared with pre-injured samples and sham rats, with varying trends among the phylogenetic families. This indicates that TBI alters the gastrointestinal microbiome and creates a dysbiosis in the absence of other injury patterns, resuscitation, antibiotics, and analgesics. Peak MRI lesion volume, functional deficits, microbial composition alterations, and the greatest reduction in α-diversity occurred in a similar timeline (i.e., between 2 and 3 days). Perhaps the most salient finding is that the evolution of the lesion directly correlated with changes in the GI microbiome. Specifically, a larger brain lesion was associated with greater decreases in levels of Firmicutes (traditionally beneficial bacteria) and an exacerbated increase in Proteobacteria (with many pathogenic bacteria). Furthermore, a larger brain lesion size was associated with a significant reduction in α-diversity. To our knowledge, this is the first such finding directly correlating GI microbiome changes to lesion size due to TBI.
A decrease in traditionally beneficial bacteria at the phylum and family levels was observed after TBI with parallel increases in families that contain opportunistic pathogens including Bacteroidaceae, Enterobacteriaceae and Pseudomonadaceae. These findings herein are consistent with previous research in human burn victims, which showed a decreased overall diversity of the microbiome and an associated increase in gut permeability (11). This group also found an increase in gram-negative bacteria, particularly in the family Enterobacteraceae. Krezalek et al. found that the intestinal microbiota was disrupted within hours of injury, and that these disturbances were predictive of sepsis-associated mortality in this patient population. Moreover, in patients who developed sepsis, the composition of the gut microbiome dramatically changed such that pathogen communities of extremely low diversity emerged and triggered further virulence in the host leading to the development of a pathobiome (18). In a model of middle cerebral artery stroke, Singh et al. (9) similarly observed reduced diversity of the gut microbiome, with particular decreases in the phylum Bacteroidetes.
The normal microbiome is dominated by the Bacteroidetes and Firmicutes phyla, with more than 90% of species falling within these groups (3, 4, 6). Other representative phyla in the gut microbiome include Actinobacteria, Fusobacteria, Proteobacteria, Verrucomicrobia, and Cyanobacteria(6). Native gut bacteria provide many benefits in that they metabolize indigestible polysaccharides, produce essential vitamins, maintain tissue homeostasis, and protect against invasion of pathogens (19). In Clostridium difficile colitis, there is an increase in Proteobacteria and decreases in the phyla Bacteroidetes and Firmicutes(20). Moreover, an increased ratio of Firmicutes to Bacteroidetes is observed in obesity that is thought to be related to the promotion of Firmicutes from a high fat diet leading to increased caloric intake (21). Alternatively, we found a reduced ratio of Firmicutes to Bacteroidetes, which may be a result of the stress response following TBI.
Reductions in beneficial commensal bacterial populations have been associated with acute and chronic disease states (3). Similarly, we observed decreases in families within the Firmicutes phylum including Lachnospiraceae, Mogibacteriaceae, and Ruminococcaceae, traditionally believed beneficial families of bacteria. Bacteria in the gram-positive family Lachnospiraceae, which includes Clostridia species, generate butyrate by fermenting carbohydrates. Lachnospiraceae have been shown to prevent inflammation in colitis models, and decreased levels are found in inflammatory bowel disease (5, 22). Other beneficial families including Anaeroplasmataceae and Verrucomicrobiaceae were also reduced in animals sustaining TBI in our study. Recent research has shown a correlation between changes in Verrucomicrobia and glucose tolerance, obesity, and diabetes (23). Our data suggest that similar changes observed in our model may potentially contribute to secondary brain injury following TBI.
The Bacteroidaceae family typically acts as one of the dominant families endemic to the human microbiome but also includes the pathogenic bacteria Bacteroides fragilis, whose enterotoxigenic strain is commonly associated with diarrhea in inflammatory bowel disease and colorectal cancer (24). Members of the phylum Proteobacteria, particularly those within the family Enterobacteriaceae, contain many opportunistic pathogenic bacteria, including those from the genera Escherichia, Klebsiella, Proteus, and Citrobacter, which are commonly seen in sepsis (24). Proteus mirabilis and Klebsiella pneumonia, bacteria in the family Enterobacteriaceae, are associated with colitis and can elicit inflammation and spontaneous colitis when transferred to wild-type mice (25). In burn patients, there is an overgrowth of Enterobacteriaceae and Bacteroidaceae(11). Increased abundance of traditionally opportunistic bacteria was also observed in this study, with increases of Bacteroidaceae, Enterobacteriaceae, and Pseudomonadaceae. Moreover, the extent of the brain lesion in our model positively correlated with the increase in Proteobacteria, which includes two of those families suggesting that increased pathogenic flora may contribute to inflammatory and infectious complications. A feedback loop involving the virulent bacterial species within the GI microbiome and the brain-gut axis may also potentiate a neuroinflammatory cascade, leading to secondary brain injury and thus influencing functional outcome.
Specific to the brain-gut axis, increases of Enterobacteriaceae in cirrhotic patients are associated with hyperammonemia-astrocytic changes, hepatic encephalopathy, higher model end stage liver disease (MELD) scores, increased brain magnetic resonance spectroscopy manifestations, and increased systemic and local inflammatory effects (26). The ability of these bacteria to produce toxins that increase levels of gut ammonia and potentiate other metabolic and systemic effects may also play a key role in negative sequelae in patients with TBI via the brain-gut axis. Furthermore, in patients with Parkinson the abundance of Enterobacteriaceae is positively associated with postural instability and gait disturbance severity (27). Opportunistic bacteria such as Pseudomonas aeruginosa play a key role in a number of nosocomial infectious complications such as urinary tract infections and surgical site infections (28). We also found increases in the opportunistic families Pseudomonadaceae and Enterobacteriaceae suggesting that they may also contribute to negative secondary sequelae and outcomes in TBI patients.
Preclinical models have demonstrated that the gastrointestinal microbiota may influence the brain-gut axis via immunological, neuroendocrine, and direct neural mechanisms (7, 29). Afferent and efferent connections from vagal nuclei compose the enteric nervous system and directly connect the brain to the gut (29). Furthermore, neuroendocrine and immunologic communication from areas of the brain such as the hypothalamus, insular cortex, and the cingulate is integrated with visceral signaling via the vagus complex and the pontine nucleus tractus solitaries (29). TBI can disrupt the corticopontine communication between the intestine and the vagal complex causing dysautonomia, which is characterized by tachycardia, hyperthermia, hypertension, increased sweating, muscle tone, and posturing (30). The release of stress hormones in the hypothalamic–pituitary–adrenal axis following injury can also result in intestinal changes in permeability, motility, and secretion (31). Future investigation will help determine whether these phenomena are involved in our model.
Improvements in emergency response times and care have increased survivability and have brought to light an increasing need for developing reliable methods to identify patients at risk of developing secondary complication pathologies. Objective diagnosis and classification of TBI can be challenging with the current clinical modalities. While head computed tomography (CT) and MRI are excellent at determining the presence and extent of hemorrhage, edema, and other physical changes in the brain, they have limited ability to predict adverse secondary events or to detect subtle damage. Moreover, current methods of classification for TBI include the Glasgow Coma Scale, Abbreviated Injury Scale, and the Abbreviated Trauma Scale, all of which struggle to clearly define the severity of TBI due to the heterogeneous presentations. Recent studies have identified several protein molecules that may serve as candidate biomarkers in TBI (32, 33). Recent publications from the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Study have demonstrated the utility of sampling plasma for glial, axonal, and neuronal breakdown molecules following TBI as biomarkers (34, 35). To this end, changes in the microbiome may represent a novel biomarker to stage TBI severity and predict functional outcome, especially when viewed in the context of the brain-gut axis. By characterizing the changes in bacterial families and degree of loss of diversity, it may be possible to correlate TBI severity to the specific changes in the microbiome. Therefore, future studies will focus on determining the impact of varying the severity of TBI on changes in the microbiome.
While the findings of our study support a relationship between TBI and the gut microbiome, there are limitations. This is a preclinical model of TBI in rodents, so conclusions drawn may not translate to patients with TBI. Further clinical studies are needed to better assess the effects on TBI and the microbiome. In addition, gnotobiotic rodents were not utilized in these experiments, but all comparisons were made between rats with TBI and shams with all reported changes reaching significance. As mentioned, PERMANOVA demonstrated that β-diversity was significantly altered within individual animals over time, but failed to reach significance when time points were compared. Given the trend, significance would likely be reached at days 1 and 3 with a larger sample size. In addition, this model utilized a moderate TBI, and given the important relationships with lesion volume, a staged model incorporating mild and severe TBI would likely prove revealing. While the effects of the craniotomy cannot be entirely excluded, only decreased levels of Bacteroidetes were noted in the sham animals, which was the opposite of findings in TBI animals. This was the only change seen in any of the phyla or families in the entire GI microbiota and may be accounted for by only having four sham animals. Finally, since the fecal samples were from excreted pellets, they only represent the microbiota of the large intestine and changes in the small intestinal microbiome cannot be inferred. Similarly, anatomic variation of luminal versus mucosal-associated microbes was not explored.
In summary, we observed changes in the gastrointestinal microbiome as early as 2 h post-TBI, which, in some families, persisted through 7 days in the absence of therapeutic intervention. Many of the alterations at both the phylum and family levels were noted by 3 days and correlated with peak lesion volume on MRI and loss of behavioral function. Many of these changes were directly related to the size of the lesion volumes measured via MRI scanning. The physiologic and clinical implications of these changes to the microbiota in the setting of TBI remain unseen. Further evaluation of the gut microbiome following TBI has the potential to improve clinical detection of TBI and outcome, serve as a potential therapeutic target, and enhance quality-of-life for patients with TBI.
The authors thank the following individuals for their support: Basil A. Pruitt, Jr., Dawn Garcia for 16S sequencing sample processing, Yidong Chen, PhD for bioinformatics support.
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Keywords:© 2019 by the Shock Society
Brain-gut axis; gastrointestinal (GI); gut; commensals; microbiome; traumatic brain injury (TBI)