Different analysis strategies of 16S rRNA gene data from rodent studies generate contrasting views of gut bacterial communities associated with diet, health and obesity

Background One of the main functions of diet is to nurture the gut microbiota and this relationship affects the health of the host. However, different analysis strategies can generate different views on the relative abundance of each microbial taxon, which can affect our conclusions about the significance of diet to gut health in lean and obese subjects. Here we explored the impact of using different analysis strategies to study the gut microbiota in a context of diet, health and obesity. Methods Over 15 million 16S rRNA gene sequences from published studies involving dietary interventions in obese laboratory rodents were analyzed. Three strategies were used to assign the 16S sequences to Operational Taxonomic Units (OTUs) based on the GreenGenes reference OTU sequence files clustered at 97% and 99% similarity. Results Different strategies to select OTUs influenced the relative abundance of all bacterial taxa, but the magnitude of this phenomenon showed a strong study effect. Different taxa showed up to 20% difference in relative abundance within the same study, depending on the analysis strategy. Very few OTUs were shared among the samples. ANOSIM test on unweighted UniFrac distances showed that study, sequencing technique, animal model, and dietary treatment (in that order) were the most important factors explaining the differences in bacterial communities. Except for obesity status, the contribution of diet and other factors to explain the variability in bacterial communities was lower when using weighted UniFrac distances. Predicted functional profile and high-level phenotypes of the microbiota showed that each study was associated with unique features and patterns. Conclusions The results confirm previous findings showing a strong study effect on gut microbial composition and raise concerns about the impact of analysis strategies on the membership and composition of the gut microbiota. This study may be helpful to guide future research aiming to investigate the relationship between diet, health, and the gut microbiota

Commentary on key aspects of fecal microbiota transplantation in small animal practice

The gastrointestinal tract of dogs, cats, and other mammals including humans harbors millions of beneficial microorganisms that regulate and maintain health. Fecal microbiota transplantation (FMT) is a procedure involving the administration of a fecal infusion from a healthy individual (donor) to a patient with disease to help improve health. Despite the effectiveness of FMT to treat intestinal disorders in humans, in particular recurrent Clostridium difficile infection, there is a paucity of scientific data regarding the application of FMT in veterinary patients. Here, we outline key aspects of FMT in small animal practice

Evaluation of aflatoxin and fumonisin co-exposure in urine samples from healthy volunteers in northern Mexico

Aflatoxins (AF) and fumonisins (FB) are common contaminants of maize and have been associated with cancer, immune suppression, and growth stunting. In this work, AFM(1) and FB(1) were measured in urine samples of healthy volunteers from the metropolitan area of Monterrey, Mexico, while AF and FB were detected in foods collected near the sampling zone. Urine samples from 106 adults were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry and toxins in foods were measured by fluorometry. The mean value of AFM(1) and FB(1) was 4.3 pg/mg creatinine from 76 samples (72 %), and 50 pg/mg creatinine from 75 samples (71 %), respectively. More than half of the samples (n = 56, 53 %) had detectable levels of both AFM(1) and FB(1). No differences in toxin levels were found between males and females or between age groups, but AFM(1) and FB(1) levels were higher (p < 0.01) when detected as a single exposure compared to co-exposed. Some significant results were found when comparing AFM(1) and FB(1) levels among groups of people assigned to levels of food consumption. Food samples had average concentrations of 5.3 μg/kg for AF and 800 μg/kg for FB. The results showed that co-exposure to AF and FB is common in the metropolitan area of Monterrey

Randomized clinical trial to evaluate the effect of fecal microbiota transplant for initial Clostridium difficile infection in intestinal microbiome

Objective The aim of this study was to evaluate the impact of fecal donor-unrelated donor mix (FMT-FURM) transplantation as first-line therapy for C. difficile infection (CDI) in intestinal microbiome. Methods We designed an open, two-arm pilot study with oral vancomycin (250mg every 6 h for 10–14 days) or FMT-FURM as treatments for the first CDI episode in hospitalized adult patients in Hospital Universitario “Dr. Jose Eleuterio Gonzalez”. Patients were randomized by a closed envelope method in a 1: 1 ratio to either oral vancomycin or FMT-FURM. CDI resolution was considered when there was a reduction on the Bristol scale of at least 2 points, a reduction of at least 50% in the number of bowel movements, absence of fever, and resolution of abdominal pain (at least two criteria). From each patient, a fecal sample was obtained at days 0, 3, and 7 after treatment. Specimens were cultured to isolate C. difficile, and isolates were characterized by PCR. Susceptibility testing of isolates was performed using the agar dilution method. Fecal samples and FMT-FURM were analyzed by 16S rRNA sequencing. Results We included 19 patients; 10 in the vancomycin arm and 9 in the FMT-FURM arm. However, one of the patients in the vancomycin arm and two patients in the FMT-FURM arm were eliminated. Symptoms resolved in 8/9 patients (88.9%) in the vancomycin group, while symptoms resolved in 4/7 patients (57.1%) after the first FMT-FURM dose (P = 0.26) and in 5/7 patients (71.4%) after the second dose (P = 0.55). During the study, no adverse effects attributable to FMT-FURM were observed in patients. Twelve isolates were recovered, most isolates carried tcdB, tcdA, cdtA, and cdtB, with an 18-bp deletion in tcdC. All isolates were resistant to ciprofloxacin and moxifloxacin but susceptible to metronidazole, linezolid, fidaxomicin, and tetracycline. In the FMT-FURM group, the bacterial composition was dominated by Firmicutes, Bacteroidetes, and Proteobacteria at all-time points and the microbiota were remarkably stable over time. The vancomycin group showed a very different pattern of the microbial composition when comparing to the FMT-FURM group over time. Conclusion The results of this preliminary study showed that FMT-FURM for initial CDI is associated with specific bacterial communities that do not resemble the donors’ sample.Peer reviewedFinal Published versio

The Effect of Gluten-Free Diet on Health and the Gut Microbiota Cannot Be Extrapolated from One Population to Others

Gluten-related disorders (GRD) affect millions of people worldwide and have been related to the composition and metabolism of the gut microbiota. These disorders present differently in each patient and the only treatment available is a strict life-long gluten-free diet (GFD). Several studies have investigated the effect of a GFD on the gut microbiota of patients afflicted with GRD as well as healthy people. The purpose of this review is to persuade the biomedical community to think that, while useful, the results from the effect of GFD on health and the gut microbiota cannot be extrapolated from one population to others. This argument is primarily based on the highly individualized pattern of gut microbial composition and metabolic activity in each person, the variability of the gut microbiota over time and the plethora of factors associated with this variation. In addition, there is wide variation in the composition, economic viability, and possible deleterious effects to health among different GFD, both within and among countries. Overall, this paper encourages the conception of more collaborative efforts to study local populations in an effort to reach biologically and medically useful conclusions that truly contribute to improve health in patients afflicted with GRD

The Effect of Gluten-Free Diet on Health and the Gut Microbiota Cannot Be Extrapolated from One Population to Others

Gluten-related disorders (GRD) affect millions of people worldwide and have been related to the composition and metabolism of the gut microbiota. These disorders present differently in each patient and the only treatment available is a strict life-long gluten-free diet (GFD). Several studies have investigated the effect of a GFD on the gut microbiota of patients afflicted with GRD as well as healthy people. The purpose of this review is to persuade the biomedical community to think that, while useful, the results from the effect of GFD on health and the gut microbiota cannot be extrapolated from one population to others. This argument is primarily based on the highly individualized pattern of gut microbial composition and metabolic activity in each person, the variability of the gut microbiota over time and the plethora of factors associated with this variation. In addition, there is wide variation in the composition, economic viability, and possible deleterious effects to health among different GFD, both within and among countries. Overall, this paper encourages the conception of more collaborative efforts to study local populations in an effort to reach biologically and medically useful conclusions that truly contribute to improve health in patients afflicted with GRD

Descriptive Bacterial and Fungal Characterization of Propolis Using Ultra-High-Throughput Marker Gene Sequencing

Bees harbor microorganisms that are important for host health, physiology, and survival. Propolis helps modulate the immune system and health of the colony, but little information is available about its microbial constituents. Total genomic DNA from samples of natural propolis from Apis mellifera production hives from four locations in Mexico were used to amplify a region of the 16S rRNA gene (bacteria) and the internal transcriber spacer (fungi), using PCR. The Illumina MiSeq platform was used to sequence PCR amplicons. Extensive variation in microbial composition was observed between the propolis samples. The most abundant bacterial group was Rhodopila spp. (median: 14%; range: 0.1%&ndash;27%), a group with one of the highest redox potential in the microbial world. Other high abundant groups include Corynebacterium spp. (median: 8.4%; 1.6%&ndash;19.5%) and Sphingomonas spp. (median: 5.9%; 0.03%&ndash;14.3%), a group that has been used for numerous biotechnological applications because of its biodegradative capabilities. Bacillus and Prevotella spp. alone comprised as much as 88% (53% and 35%, respectively) of all bacterial microbiota in one sample. Candida (2%&ndash;43%), Acremonium (0.03%&ndash;25.2%), and Aspergillus (0.1%&ndash;43%) were among the most abundant fungi. The results contribute to a better understanding of the factors associated with the health of Apis mellifera production hives

Akkermansia and Microbial Degradation of Mucus in Cats and Dogs : Implications to the Growing Worldwide Epidemic of Pet Obesity

Akkermansia muciniphila is a mucin-degrading bacterium that has shown the potential to provide anti-inflammatory and anti-obesity effects in mouse and man. We here focus on companion animals, specifically cats and dogs, and evaluate the microbial degradation of mucus and its health impact in the context of the worldwide epidemic of pet obesity. A literature survey revealed that the two presently known Akkermansia spp., A. muciniphila and A. glycaniphila, as well as other members of the phylum of Verrucomicrobia seem to be neither very prevalent nor abundant in the digestive tract of cats and dog. While this may be due to methodological aspects, it suggests that bacteria related to Akkermansia are not the major mucus degraders in these pets and hence other mucus-utilizing taxa may deserve attention. Hence, we will discuss the potential of these endogenous mucus utilizers and dietary interventions to boost these as well as the use of Akkermansia spp. related bacteria or their components as strategies to target feline and canine obesity.Peer reviewe

Molecular assessment of the fecal microbiota in healthy cats and dogs before and during supplementation with fructo-oligosaccharides (FOS) and inulin using high-throughput 454-pyrosequencing

Prebiotics are selectively fermentable dietary compounds that result in changes in the composition and/or activity of the intestinal microbiota, thus conferring benefits upon host health. In veterinary medicine, commercially available products containing prebiotics have not been well studied with regard to the changes they trigger on the composition of the gut microbiota. This study evaluated the effect of a commercially available nutraceutical containing fructo-oligosaccharides (FOS) and inulin on the fecal microbiota of healthy cats and dogs when administered for 16 days. Fecal samples were collected at two time points before and at two time points during prebiotic administration. Total genomic DNA was obtained from fecal samples and 454-pyrosequencing was used for 16S rRNA gene bacterial profiling. The linear discriminant analysis (LDA) effect size (LEfSe) method was used for detecting bacterial taxa that may respond (i.e., increase or decrease in its relative abundance) to prebiotic administration. Prebiotic administration was associated with a good acceptance and no side effects (e.g., diarrhea) were reported by the owners. A low dose of prebiotics (50 mL total regardless of body weight with the end product containing 0.45% of prebiotics) revealed a lower abundance of Gammaproteobacteria and a higher abundance of Veillonellaceae during prebiotic administration in cats, while Staphylococcaceae showed a higher abundance during prebiotic administration in dogs. These differences were not sufficient to separate bacterial communities as shown by analysis of weighted UniFrac distance metrics. A predictive approach of the fecal bacterial metagenome using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) also did not reveal differences between the period before and during prebiotic administration. A second trial using a higher dose of prebiotics (3.2 mL/kg body weight with the end product containing 3.1% of prebiotics) was tested in dogs and revealed a lower abundance of Dorea (family Clostridiaceae) and a higher abundance of Megamonas and other (unknown) members of Veillonellaceae during prebiotic administration. Again, these changes were not sufficient to separate bacterial communities or predicted metabolic profiles according to treatment. A closer analysis of bacterial communities at all time-points revealed highly individualized patterns of variation. This study shows a high interindividual variation of fecal bacterial communities from pet cats and dogs, that these communities are relatively stable over time, and that some of this variation can be attributable to prebiotic administration, a phenomenon that may be affected by the amount of the prebiotic administered in the formulation. This study also provides insights into the response of gut bacterial communities in pet cats and dogs during administration of commercially available products containing prebiotics. More studies are needed to explore potentially beneficial effects on host health beyond changes in bacterial communities

Influence of whole-wheat consumption on fecal microbial community structure of obese diabetic mice

The digestive tract of mammals and other animals is colonized by trillions of metabolically-active microorganisms. Changes in the gut microbiota have been associated with obesity in both humans and laboratory animals. Dietary modifications can often modulate the obese gut microbial ecosystem towards a more healthy state. This phenomenon should preferably be studied using dietary ingredients that are relevant to human nutrition. This study was designed to evaluate the influence of whole-wheat, a food ingredient with several beneficial properties, on gut microorganisms of obese diabetic mice. Diabetic (db/db) mice were fed standard (obese-control) or whole-wheat isocaloric diets (WW group) for eight weeks; non-obese mice were used as control (lean-control). High-throughput sequencing using the MiSeq platform coupled with freely-available computational tools and quantitative real-time PCR were used to analyze fecal bacterial 16S rRNA gene sequences. Short-chain fatty acids were measured in caecal contents using quantitative high-performance liquid chromatography photo-diode array analysis. Results showed no statistical difference in final body weights between the obese-control and the WW group. The bacterial richness (number of Operational Taxonomic Units) did not differ among the treatment groups. The abundance of Ruminococcaceae, a family containing several butyrate-producing bacteria, was found to be higher in obese (median: 6.9%) and WW-supplemented mice (5.6%) compared to lean (2.7%, p = 0.02, Kruskal-Wallis test). Caecal concentrations of butyrate were higher in obese (average: 2.91 mmol/mg of feces) but especially in WW-supplemented mice (4.27 mmol/mg) compared to lean controls (0.97 mmol/mg), while caecal succinic acid was lower in the WW group compared to obese but especially to the lean group. WW consumption was associated with ∼3 times higher abundances of Lactobacillus spp. compared to both obese and lean control mice. Analysis of weighted UniFrac distances revealed a distinctive clustering of lean microbial communities separately from both obese and WW-supplemented mice (p = 0.001, ANOSIM test). Predictive metagenome analysis revealed significant differences in several metabolic features of the microbiota among the treatment groups, including carbohydrate, amino acids and vitamin metabolism (p < 0.01, Kruskal-Wallis test). However, obese and WW groups tended to share more similar abundances of gene families compared to lean mice. Using an in vivo model of obesity and diabetes, this study suggests that daily WW supplementation for eight weeks may not be enough to influence body weight or to output a lean-like microbiome, both taxonomically and metabolically. However, WW-supplementation was associated with several statistically significant differences in the gut microbiome compared to obese controls that deserve further investigation