Karley K. Mahalak

A stable gut microbial community (SGMC) created by the culture of fecal samples in a chemostat-based gut model is a physiologically relevant, reproducible tool for ecological research on the gut microbiome. It is also a subject of continuous improvement for the model to better mimic the in vivo system. In this research, compositions of fecal homogenates and the derived SGMC were analyzed based on shotgun metagenomic sequencing. It was found that taxa in the fecal inocula could be divided into resilient or sensitive types according to their response to environmental factors; taxa were sorted into three subsets—shared, emerged, and lost—by how they were represented in the established communities. The first two subsets consisted of resilient species, constituting an SGMC, which was suitable for long-term, ecological research. In contrast, species of the lost subset were sensitive to the new environmental conditions and were missing from the SGMC. However, these lost species may carry host-dependent information that is useful or even critical for drug and functional foods development or nutrition research. The differentiation between the three subsets reveal the community shift and metabolic profile along with SGMC formation and could serve as a diagnosis for understanding the extent to which the SGMC mimics, or differs from, the actual ecosystem in vivo or the donor’s fecal samples, which may be useful in work to upgrade in vitro models.

Perfluorooctanoic acid (PFOA) is an environmentally persistent chemical that enters the gastrointestinal tract (GIT) via the food chain, posing a harmful, long-term threat to human health. In response to this challenge, research on the PFOA-GIT interaction is thriving. Currently, studies on the effect of PFOA on the epithelial cells of the GIT and those on its influence on the microbial community are often implemented separately, and less attention has been paid to the combinational effects of the chemical, the gut microbiome and metabolome. In the present study, we co-cultured fecal samples from healthy adults aged 25–70 in the ex vivo SIFR^® simulator, adding PFOA at 10 mg/L to represent the accumulated effects of long-term exposure. The results obtained from bacterial cell counting by flow cytometry and shotgun metagenomic sequencing revealed that PFOA was broadly disruptive to the microbiome and that Pseudomonadota emerged as the dominant phylum by replacing Bacteriodota and Bacillota, including key members of short-chain fatty acid-producing groups. Bacterial culture media with and without PFOA were collected and used in human colonoid cell culture for TEER and transcription measurement. It was shown that the PFOA-impacted microbial culture had stronger effects on the cell’s protective functions, in terms of tissue junction tightening, mucin biosynthesis, and immune response, than either untreated bacterial culture or PFOA alone. The results point out the possibility that the combination of PFOA and PFOA-impacted bacterial metabolites more strongly induces a change in epithelial cells’ protective function than either one alone.

Ginger ( Zingiber officinale ) has a long history of use in traditional medicine and the modern world to alleviate health conditions, particularly those related to indigestion and nausea. Gingerols are phenolic bioactive compounds found in ginger. It has been suggested that health benefits associated with gingerols may be due to modification of the gut microbiome, especially in disease models. However, the impact of gingerol on a healthy human gut microbiome, and whether age affects gingerol activity, is not well understood. To address this, the impact of 6-gingerol, the most abundant polyphenol found in ginger, on the gut microbiomes of four age groups (infants, children, adults (22–40), and adults (60+)) was determined using SIFR® technology. Following a 24-h incubation with 6-gingerol, microbial community genomic analysis was performed together with metabolic analysis to determine the impact of 6-gingerol on the gut microbiota ex vivo . Using this method, 6-gingerol was determined to have no significant impact on the gut microbiota in terms of community density, community diversity, or short-chain fatty acid production. This study found that, in healthy gut microbiota, 6-gingerol did not have a strong effect within a 24-h period of treatment.

The food additive sodium benzoate (SB) has been used for decades as an antimicrobial to prevent food spoilage. SB has been deemed to pose no risk to human health when consumed at levels under 5 mg/kg body weight per day; however, when many of the supporting studies were conducted, the importance of the gut microbiome to human health was not yet appreciated. Given SB’s known antimicrobial qualities, it is important to assess the effect of this food additive on the human gut microbiome. The ex vivo SIFR^® (Systemic Intestinal Fermentation Research) technology was used to test the effect of SB on microbial communities from 24 donors, aged infants to older adults. A dose of 3.5 g/L SB elicited a drop in the Pseudomonadota phylum for multiple age groups but did not alter the alpha or beta diversity within any of these groups. This was accompanied by changes in the functional outputs that included an overall rise in butyrate and a drop in propionate production. This higher butyrate correlates with an increase in the abundance of several known butyrate producers in the presence of SB, although the genetic potential for its production in the community did not change. Overall, despite using a dose ten times higher than the accepted daily intake limit, the effect on the gut microbiome was minimal.

Hydrogen sulfide (H₂S) can be produced from the metabolism of foods containing sulfur in the gastrointestinal tract (GIT). At low doses, H₂S regulates the gut microbial community and supports GIT health, but depending on dose, age, and individual health conditions, it may also contribute to inflammatory responses and gut barrier dysfunction. Controlling H₂S production in the GIT is important for maintaining a healthy gut microbiome. However, research on this subject is limited due to the gaseous nature of the chemical and the difficulty of accessing the GIT in situ. In the present ex vivo experiment, we used a single-dose sodium sulfide preparation (SSP) as a H₂S precursor to test the effect of H₂S on the human gut microbiome across different age groups, including breastfed infants, toddlers, adults, and older adults. Metagenomic sequencing and metabolite measurements revealed that the development of the gut microbial community and the production of short-chain fatty-acids (SCFAs) were age-dependent; that the infant and the older adult groups were more sensitive to SSP exposure; that exogeneous SSP suppressed SCFA production across all age groups, except for butyrate in the older adult group, suggesting that H₂S selectively favors specific gut microbial processes.

Background: The human gut microbiota develops in concordance with its host over a lifetime, resulting in age-related shifts in community structure and metabolic function. Little is known about whether these changes impact the community’s response to microbiome-targeted therapeutics. Providing critical information on this subject, faecal microbiomes of subjects from six age groups, spanning from infancy to 70-year-old adults (n = six per age group) were harvested. The responses of these divergent communities to treatment with the human milk oligosaccharide 2’-fucosyllactose (2’FL), fructo-oligosaccharides (FOS), and lactose was investigated using the Ex vivo SIFR^® technology that employs bioreactor fermentation and is validated to be predictive of clinical findings. Additionally, it was evaluated whether combining faecal microbiomes of a given age group into a single pooled microbiome produced similar results as the individual microbiomes. Results: First, marked age-dependent changes in community structure were identified. Bifidobacterium levels strongly declined as age increased, and Bifidobacterium species composition was age-dependent: B. longum, B. catenulatum/pseudocatenulatum, and B. adolescentis were most prevalent for breastfed infants, toddlers/children, and adults, respectively. Metabolomic analyses (LA-REIMS) demonstrated that these age-dependent differences particularly impacted treatment effects of 2’FL (more than FOS/lactose). Further analysis revealed that while 2’FL enhanced production of short-chain fatty acids (SCFAs) and exerted potent bifidogenic effects, regardless of age, the specific Bifidobacterium species enhanced by 2’FL, as well as subsequent cross-feeding interactions, were highly age-dependent. Furthermore, single-pooled microbiomes produced results that were indicative of the average treatment response for each age group. Nevertheless, pooled microbiomes had an artificially high diversity, thus overestimating treatment responses (especially for infants), did not recapitulate interindividual variation, and disallowed for the correlative analysis required to unravel mechanistic actions. Conclusions: Age is an important factor in shaping the gut microbiome, with the dominant taxa and their metabolites changing over a lifetime. This divergence affects the response of the microbiota to therapeutics, demonstrated in this study using 2’FL. These results evidence the importance of screening across multiple age groups separately to provide granularity of how therapeutics impact the microbiome and, consequently, human health.

Introduction: Studies have shown that a diet high in fiber and prebiotics has a positive impact on human health due largely to the fermentation of these compounds by the gut microbiota. One underutilized source of fiber may be rice bran, a waste product of rice processing that is used most frequently as an additive to livestock feed but may be a good source of fibers and other phenolic compounds as a human diet supplement. Previous studies focused on specific compounds extracted from rice bran showed that soluble fibers extracted from rice bran can improve glucose response and reduce weight gain in mouse models. However, less is known about changes in the human gut microbiota in response to regular rice bran consumption.Methods: In this study, we used a Simulator of the Human Intestinal Microbial Ecology (SHIME®) to cultivate the human gut microbiota of 3 different donors in conditions containing either soluble or insoluble fiber fractions from rice bran. Using 16S rRNA amplicon sequencing and targeted metabolomics via Gas Chromatography-Mass Spectrometry, we explored how gut microbial communities developed provided different supplemental fiber sources.Results: We found that insoluble and soluble fiber fractions increased short-chain fatty acid production, indicating that both fractions were fermented. However, there were differences in response between donors, for example the gut microbiota from donor 1 increased acetic acid production with both fiber types compared with control; whereas for donors 2 and 3, butanoic acid production increased with ISF and SF supplementation. Both soluble and insoluble rice bran fractions increased the abundance of Bifidobacterium and Lachnospiraceae taxa.Discussion: Overall, analysis of the effect of soluble and insoluble rice bran fractions on the human in vitro gut microbiota and the metabolites produced revealed individually variant responses to these prebiotics.

Ivermectin is a an anti-helminthic that is critical globally for both human and veterinary care. To the best of our knowledge, information available regarding the influence of ivermectin (IVM) on the gut microbiota has only been collected from diseased donors, who were treated with IVM alone or in combination with other medicines. Results thus obtained were influenced by multiple elements beyond IVM, such as disease, and other medical treatments. The research presented here investigated the impact of IVM on the gut microbial structure established in a Triple-SHIME^® (simulator of the human intestinal microbial ecosystem), using fecal material from three healthy adults. The microbial communities were grown using three different culture media: standard SHIME media and SHIME media with either soluble or insoluble fiber added (control, SF, ISF). IVM introduced minor and temporary changes to the gut microbial community in terms of composition and metabolite production, as revealed by 16S rRNA amplicon sequencing analysis, flow cytometry, and GC-MS. Thus, it was concluded that IVM is not expected to induce dysbiosis or yield adverse effects if administered to healthy adults. In addition, the donor’s starting community influences the relationship between IVM and the gut microbiome, and the soluble fiber component in feed could protect the gut microbiota from IVM; an increase in short-chain fatty acid production was predicted by PICRUSt2 and detected with IVM treatment.

Tomatoes are a perishable and seasonal fruit with a high economic impact. Carbon dioxide (CO₂), among several other reagents, is used to extend the shelf-life and preserve the quality of tomatoes during refrigeration or packaging. To obtain insight into CO₂ stress during tomato ripening, tomatoes at the late green mature stage were conditioned with one of two CO₂ delivery methods: 5% CO₂ for 14 days (T1) or 100% CO₂ for 3 h (T2). Conventional physical and chemical characterization found that CO₂ induced by either T1 or T2 delayed tomato ripening in terms of color change, firmness, and carbohydrate dissolution. However, T1 had longer-lasting effects. Furthermore, ethylene production was suppressed by CO₂ in T1, and promoted in T2. These physical observations were further evaluated via RNA-Seq analysis at the whole-genome level, including genes involved in ethylene synthesis, signal transduction, and carotenoid biosynthesis. Transcriptomics analysis revealed that the introduction of CO₂ via the T1 method downregulated genes related to fruit ripening; in contrast, T2 upregulated the gene encoding for ACS6, the enzyme responsible for S1 ethylene synthesis, even though there was a large amount of ethylene present, indicating that T1 and T2 regulate tomato ripening via different mechanisms. Quantitative real-time PCR assays (qRT-PCR) were used for validation, which substantiated the RNA-Seq data. The results of the present research provide insight into gene regulation by CO₂ during tomato ripening at the whole-genome level.

In the present research, we investigated changes in the gut metabolome that occurred in response to the administration of the Laticaseibacillus rhamnosus strain GG (LGG). The probiotics were added to the ascending colon region of mature microbial communities established in a human intestinal microbial ecosystem simulator. Shotgun metagenomic sequencing and metabolome analysis suggested that the changes in microbial community composition corresponded with changes to metabolic output, and we can infer linkages between some metabolites and microorganisms. The in vitro method permits a spatially-resolved view of metabolic transformations under human physiological conditions. By this method, we found that tryptophan and tyrosine were mainly produced in the ascending colon region, while their derivatives were detected in the transverse and descending regions, revealing sequential amino acid metabolic pathways along with the colonic tract. The addition of LGG appeared to promote the production of indole propionic acid, which is positively associated with human health. Furthermore, the microbial community responsible for the production of indole propionic acid may be broader than is currently known.