Associations of gut microbiome richness and diversity with objective and subjective sleep measures in a population sample.

STUDY OBJECTIVES: Alterations in gut microbiota composition have been associated with several conditions, and there is emerging evidence that sleep quantity and quality are associated with the composition of the gut microbiome. Therefore, this study aimed to assess the associations between several measures of sleep and the gut microbiome in a large, population based sample. METHODS: Data were collected from participants in the Survey of the Health of Wisconsin from 2016-2017 (N=720). Alpha-diversity was estimated using Chao1 richness, Shannon's diversity, and Inverse Simpson's diversity. Beta-diversity was estimated using Bray-Curtis dissimilarity. Models for each of the alpha-diversity outcomes were calculated using linear mixed effects models. Permutational multivariate analysis of variance tests were performed to test whether gut microbiome composition differed by sleep measures. Negative binomial models were used to assess whether sleep measures were associated with individual taxa relative abundance. RESULTS: Participants were a mean (SD) age of 55 (16) years and 58% were female. The sample was 83% non-Hispanic White, 10.6% non-Hispanic Black, and 3.5% Hispanic. Greater actigraphy-measured night-to-night sleep duration variability, wake after sleep onset (WASO), lower sleep efficiency, and worse self-reported sleep quality were associated with lower microbiome richness and diversity. Sleep variables were associated with beta-diversity, including actigraphy-measured night-to-night sleep duration variability, sleep latency and efficiency, and self-reported sleep quality, sleep apnea, and napping. Relative abundance of several taxa was associated with night-to-night sleep duration variability, average sleep latency and sleep efficiency, and sleep quality. CONCLUSION: This study suggests that sleep may be associated with the composition of the gut microbiome. These results contribute to the body of evidence that modifiable health habits can influence the human gut microbiome.

Listeria monocytogenes infection in pregnant macaques alters the maternal gut microbiome†.

OBJECTIVES: The bacterium Listeria monocytogenes (Lm) is associated with adverse pregnancy outcomes. Infection occurs through consumption of contaminated food that is disseminated to the maternal-fetal interface. The influence on the gastrointestinal microbiome during Lm infection remains unexplored in pregnancy. The objective of this study was to determine the impact of listeriosis on the gut microbiota of pregnant macaques. METHODS: A non-human primate model of listeriosis in pregnancy has been previously described. Both pregnant and non-pregnant cynomolgus macaques were inoculated with Lm and bacteremia and fecal shedding were monitored for 14 days. Non-pregnant animal tissues were collected at necropsy to determine bacterial burden, and fecal samples from both pregnant and non-pregnant animals were evaluated by 16S rRNA next-generation sequencing. RESULTS: Unlike pregnant macaques, non-pregnant macaques did not exhibit bacteremia, fecal shedding, or tissue colonization by Lm. Dispersion of Lm during pregnancy was associated with a significant decrease in alpha diversity of the host gut microbiome, compared to non-pregnant counterparts. The combined effects of pregnancy and listeriosis were associated with a significant loss in microbial richness, although there were increases in some genera and decreases in others. CONCLUSIONS: Although pregnancy alone is not associated with gut microbiome disruption, we observed dysbiosis with listeriosis during pregnancy. The macaque model may provide an understanding of the roles that pregnancy and the gut microbiota play in the ability of Lm to establish intestinal infection and disseminate throughout the host, thereby contributing to adverse pregnancy outcomes and risk to the developing fetus.

The effect of low-abundance OTU filtering methods on the reliability and variability of microbial composition assessed by 16S rRNA amplicon sequencing.

PCR amplicon sequencing may lead to detection of spurious operational taxonomic units (OTUs), inflating estimates of gut microbial diversity. There is no consensus in the analytical approach as to what filtering methods should be applied to remove low-abundance OTUs; moreover, few studies have investigated the reliability of OTU detection within replicates. Here, we investigated the reliability of OTU detection (% agreement in detecting OTU in triplicates) and accuracy of their quantification (assessed by coefficient of variation (CV)) in human stool specimens. Stool samples were collected from 12 participants 22-55 years old. We applied several methods for filtering low-abundance OTUs and determined their impact on alpha-diversity and beta-diversity metrics. The reliability of OTU detection without any filtering was only 44.1% (SE=0.9) but increased after filtering low-abundance OTUs. After filtering OTUs with <0.1% abundance in the dataset, the reliability increased to 87.7% (SE=0.6) but at the expense of removing 6.97% reads from the dataset. When filtering was based on individual sample, the reliability increased to 73.1% after filtering OTUs with <10 copies while removing only 1.12% of reads. High abundance OTUs (>10 copies in sample) had lower CV, indicating better accuracy of quantification than low-abundance OTUs. Excluding very low-abundance OTUs had a significant impact on alpha-diversity metrics sensitive to the presence of rare species (observed OTUs, Chao1) but had little impact on relative abundance of major phyla and families and alpha-diversity metrics accounting for both richness and evenness (Shannon, Inverse Simpson). To increase the reliability of microbial composition, we advise removing OTUs with <10 copies in individual samples, particularly in studies where only one subsample per specimen is available for analysis.

Assessing the relationship between physical activity and the gut microbiome in a large, population-based sample of Wisconsin adults.

The gut microbiome is an important factor in human health and disease. While preliminary studies have found some evidence that physical activity is associated with gut microbiome richness, diversity, and composition, this relationship is not fully understood and has not been previously characterized in a large, population-based cohort. In this study, we estimated the association between several measures of physical activity and the gut microbiota in a cohort of 720 Wisconsin residents. Our sample had a mean age of 55 years (range: 18, 94), was 42% male, and 83% of participants self-identified as White. Gut microbial composition was assessed using gene sequencing of the V3-V4 region of 16S rRNA extracted from stool. We found that an increase of one standard deviation in weekly minutes spent in active transportation was associated with an increase in alpha diversity, particularly in Chao1's richness (7.57, 95% CI: 2.55, 12.59) and Shannon's diversity (0.04, 95% CI: 0.0008, 0.09). We identified interactions in the association between Inverse Simpson's diversity and physical activity, wherein active transportation for individuals living in a rural environment was associated with additional increases in diversity (4.69, 95% CI: 1.64, 7.73). We also conducted several permutational ANOVAs (PERMANOVA) and negative binomial regression analyses to estimate the relationship between physical activity and microbiome composition. We found that being physically active and increased physical activity time were associated with increased abundance of bacteria in the family Erysipelotrichaceae. Active transportation was associated with increased abundance of bacteria in the genus Phascolarctobacterium, and decreased abundance of Clostridium. Minutes in active transportation was associated with a decreased abundance of the family Clostridiaceae.

Examining the association between the gastrointestinal microbiota and Gulf War illness: A prospective cohort study.

Gulf War Illness (GWI) affects 25-35% of the 1991 Gulf War Veteran (GWV) population. Patients with GWI experience pain, fatigue, cognitive impairments, gastrointestinal dysfunction, skin disorders, and respiratory issues. In longitudinal studies, many patients with GWI have shown little to no improvement in symptoms since diagnosis. The gut microbiome and diet play an important role in human health and disease, and preliminary studies suggest it may play a role in GWI. To examine the relationship between the gut microbiota, diet, and GWI, we conducted an eight-week prospective cohort study collecting stool samples, medications, health history, and dietary data. Sixty-nine participants were enrolled into the study, 36 of which met the case definition for GWI. The gut microbiota of participants, determined by 16S rRNA sequencing of stool samples, was stable over the duration of the study and showed no within person (alpha diversity) differences. Between group analyses (beta diversity) identified statistically significant different between those with and without GWI. Several taxonomic lineages were identified as differentially abundant between those with and without GWI (n = 9) including a greater abundance of Lachnospiraceae and Ruminococcaceae in those without GWI. Additionally, there were taxonomic differences between those with high and low healthy eating index (HEI) scores including a greater abundance of Ruminococcaceae in those with higher HEI scores. This longitudinal cohort study of GWVs found that participants with GWI had significantly different microbiomes from those without GWI. Further studies are needed to determine the role these differences may play in the development and treatment of GWI.

Characterization of captive and wild 13-lined ground squirrel cecal microbiotas using Illumina-based sequencing.

BACKGROUND: Hibernating animals experience extreme changes in diet that make them useful systems for understanding host-microbial symbioses. However, most of our current knowledge about the hibernator gut microbiota is derived from studies using captive animals. Given that there are substantial differences between captive and wild environments, conclusions drawn from studies with captive hibernators may not reflect the gut microbiota's role in the physiology of wild animals. To address this, we used Illumina-based sequencing of the 16S rRNA gene to compare the bacterial cecal microbiotas of captive and wild 13-lined ground squirrels (TLGS) in the summer. As the first study to use Illumina-based technology to compare the microbiotas of an obligate rodent hibernator across the year, we also reported changes in captive TLGS microbiotas in summer, winter, and spring. RESULTS: Wild TLGS microbiotas had greater richness and phylogenetic diversity with less variation in beta diversity when compared to captive microbiotas. Taxa identified as core operational taxonomic units (OTUs) and found to significantly contribute to differences in beta diversity were primarily in the families Lachnospiraceae and Ruminococcaceae. Captive TLGS microbiotas shared phyla and core OTUs across the year, but active season (summer and spring) microbiotas had different alpha and beta diversities than winter season microbiotas. CONCLUSIONS: This is the first study to compare the microbiotas of captive and wild rodent hibernators. Our findings suggest that data from captive and wild ground squirrels should be interpreted separately due to their distinct microbiotas. Additionally, as the first study to compare seasonal microbiotas of obligate rodent hibernators using Illumina-based 16S rRNA sequencing, we reported changes in captive TLGS microbiotas that are consistent with previous work. Taken together, this study provides foundational information for improving the reproducibility and experimental design of future hibernation microbiota studies.

Assessing the impact of storage time on the stability of stool microbiota richness, diversity, and composition.

BACKGROUND: New technologies like next-generation sequencing have led to a proliferation of studies investigating the role of the gut microbiome in human health, particularly population-based studies that rely upon participant self-collection of samples. However, the impact of methodological differences in sample shipping, storage, and processing are not well-characterized for these types of studies, especially when transit times may exceed 24 h. The aim of this study was to experimentally assess microbiota stability in stool samples stored at 4 °C for durations of 6, 24, 48, 72, and 96 h with no additives to better understand effects of variable shipping times in population-based studies. These data were compared to a baseline sample that was immediately stored at - 80 °C after stool production. RESULTS: Compared to the baseline sample, we found that the alpha-diversity metrics Shannon's and Inverse Simpson's had excellent intra-class correlations (ICC) for all storage durations. Chao1 richness had good to excellent ICC. We found that the relative abundances of bacteria in the phyla Verrucomicrobia, Actinobacteria, and Proteobacteria had excellent ICC with baseline for all storage durations, while Firmicutes and Bacteroidetes ranged from moderate to good. We interpreted the ICCs as follows: poor: ICC < 0.50, moderate: 0.50 < ICC < 0.75, good: 0.75 < ICC < 0.90, and excellent: ICC > 0.90. Using the Bray-Curtis dissimilarity index, we found that the greatest change in community composition occurred between 0 and 24 h of storage, while community composition remained relatively stable for subsequent storage durations. Samples showed strong clustering by individual, indicating that inter-individual variability was greater than the variability associated with storage time. CONCLUSIONS: The results of this analysis suggest that several measures of alpha diversity, relative abundance, and overall community composition are robust to storage at 4 °C for up to 96 h. We found that the overall community richness was influenced by storage duration in addition to the relative abundances of sequences within the Firmicutes and Bacteroidetes phyla. Finally, we demonstrate that inter-individual variability in microbiota composition was greater than the variability due to changing storage durations.

Assessing the Response of Ruminal Bacterial and Fungal Microbiota to Whole-Rumen Contents Exchange in Dairy Cows.

A major goal for the dairy industry is to improve overall milk production efficiency (MPE). With the advent of next-generation sequencing and advanced methods for characterizing microbial communities, efforts are underway to improve MPE by manipulating the rumen microbiome. Our previous work demonstrated that a near-total exchange of whole rumen contents between pairs of lactating Holstein dairy cows of disparate MPE resulted in a reversal of MPE status for ∼10 days: historically high-efficiency cows decreased in MPE, and historically low-efficiency cows increased in MPE. Importantly, this switch in MPE status was concomitant with a reversal in the ruminal bacterial microbiota, with the newly exchanged bacterial communities reverting to their pre-exchange state. However, this work did not include an in-depth analysis of the microbial community response or an interrogation of specific taxa correlating to production metrics. Here, we sought to better understand the response of rumen communities to this exchange protocol, including consideration of the rumen fungi. Rumen samples were collected from 8 days prior to, and 56 days following the exchange and were subjected to 16S rRNA and ITS amplicon sequencing to assess bacterial and fungal community composition, respectively. Our results show that the ruminal fungal community did not differ significantly between hosts of disparate efficiency prior to the exchange, and no change in community structure was observed over the time course. Correlation of microbial taxa to production metrics identified one fungal operational taxonomic unit (OTU) in the genus Neocallimastix that correlated positively to MPE, and several bacterial OTUs classified to the genus Prevotella. Within the Prevotella, Prevotella_1 was found to be more abundant in high-efficiency cows whereas Prevotella_7 was more abundant in low-efficiency cows. Overall, our results suggest that the rumen bacterial community is a primary microbial driver of host efficiency, that the ruminal fungi may not have as significant a role in MPE as previously thought, and that more work is needed to better understand the functional roles of specific ruminal microbial community members in modulating MPE.

A Sarcina bacterium linked to lethal disease in sanctuary chimpanzees in Sierra Leone.

Human and animal infections with bacteria of the genus Sarcina (family Clostridiaceae) are associated with gastric dilation and emphysematous gastritis. However, the potential roles of sarcinae as commensals or pathogens remain unclear. Here, we investigate a lethal disease of unknown etiology that affects sanctuary chimpanzees (Pan troglodytes verus) in Sierra Leone. The disease, which we have named "epizootic neurologic and gastroenteric syndrome" (ENGS), is characterized by neurologic and gastrointestinal signs and results in death of the animals, even after medical treatment. Using a case-control study design, we show that ENGS is strongly associated with Sarcina infection. The microorganism is distinct from Sarcina ventriculi and other known members of its genus, based on bacterial morphology and growth characteristics. Whole-genome sequencing confirms this distinction and reveals the presence of genetic features that may account for the unusual virulence of the bacterium. Therefore, we propose that this organism be considered the representative of a new species, named "Candidatus Sarcina troglodytae". Our results suggest that a heretofore unrecognized complex of related sarcinae likely exists, some of which may be highly virulent. However, the potential role of "Ca. S. troglodytae" in the etiology of ENGS, alone or in combination with other factors, remains a topic for future research.