Alpha Diversity Measures Research Articles

Microbiome composition recovery after liver transplantation correlates with initial liver disease severity and antibiotics treatment

Liver transplantation (LT) is crucial for end-stage liver disease, but it is linked to infection risks. Pathobionts, microorganisms potentially harmful under specific conditions, can cause complications posttransplant. Monitoring such pathogens in fecal samples can be challenging and therefore remains underexplored post-LT. This study aimed to analyze the gut microbiome before and after LT, tracking pathobionts and correlating clinical data. The study involved 17 liver transplant recipients, 17 healthy relatives (spouses), and 13 donors. Gut samples collected pretranplantation and posttransplantation underwent bacterial and fungal profiling through DNA sequencing. Quantitative polymerase chain reaction was used to assess microbial load. Statistical analyses included alpha and beta diversity measures, differential abundance analysis, and correlation tests between microbiome and clinical parameters. Microbiome analysis revealed dynamic changes in diversity posttransplant. Notably, high-severity patients showed persistent and greater dysbiosis during the first months post-LT compared with low-severity patients, partly due to an antibiotic treatment pre-LT. The analysis identified a higher proportion of pathogens such as Escherichia coli/Shigella flexneri in high-severity cases posttransplant. Furthermore, butyrate producers including Roseburia intestinalis, Anaerostipes hadrus, and Eubacterium coprostanoligenes were positively correlated with levels of albumin. This study offers valuable insights into post-LT microbiome changes, shedding light on the need for tailored prophylactic treatment post-LT.

Abstract 6204: Differential gut microbiome alpha diversity in endometrial and ovarian carcinoma patients

Abstract Background: Emerging research underscores the gut microbiome's critical role in cancer development, progression, and response to treatment. As a part of the “ASAP” study, which focuses on the implementation of comprehensive molecular profiling and deep clinical annotation of electronic health records in participants diagnosed with or at risk of developing cancer, we have established a microbiome-associated biobank and corresponding clinical dataset to systematically explore the dynamics of the gut microbiome as patients progress through cancer treatment. We describe a pilot project where we explore the baseline microbiome samples collected from ASAP participants, particularly from those with endometrial and ovarian carcinomas. Methods: Stool samples from patients were collected at the time of enrollment in the ASAP study and sampling is repeated every three months to align with the additional biosampling procedures. Microbial DNA was extracted from the baseline stool samples and subjected to sequencing of the V4 region of the 16S rRNA gene. After generating species level operational taxonomic units (OTUs), alpha diversity measures were generated in the form of the inverse Simpson index. In order to determine whether there was a difference in alpha diversity between individuals with ovarian (N = 37) and endometrial (N = 35) carcinomas, we employed a t-test with 100,000 permutations to derive an empirical p-value. Results: We present sequencing results from the initial n=297 samples. Our comparison of the alpha diversity between ASAP study participants with ovarian and endometrial carcinomas showed a significant difference between the two groups, with the ovarian cancer patients having a lower alpha diversity (empirical p-value = 0.03915). Due to the uniform sampling, biobanking, and analytical procedures, our study allows the ability to explore microbiome features amongst various cancer diagnoses while minimizing laboratory and analytical differences that plague microbiome reproducibility. Conclusions: We observed a significant difference in alpha diversity between patients with endometrial and ovarian carcinomas. Although the current results are preliminary, the wealth of information available in the ASAP protocol will allow us to continue exploring the association of the gut microbiome with cancer and the associated treatment. Citation Format: Casey T. Finnicum, Tobias Meissner, Christel Davis, Sarah Viet, Jason L. Petersen, McKenna Deaton, Benjamin Solomon, William Spanos, David Starks, Rachel Elsey, Erik A. Ehli, Casey Williams. Differential gut microbiome alpha diversity in endometrial and ovarian carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6204.

16s RNA-based metagenomics reveal previously unreported gut microbiota associated with reactive arthritis and undifferentiated peripheral spondyloarthritis.

Reactive arthritis (ReA) provides a unique opportunity to comprehend how a mucosal infection leads to inflammatory arthritis at a distant site without the apparent invasion of the pathogen. Unfortunately, conventional stool cultures after ReA provide limited information, and there is a dearth of metagenomic studies in ReA. The objective of this study was to identify gut microbiota associated with the development of ReA. Patients with ReA or undifferentiated peripheral spondyloarthritis (UpSpA) were included if they presented within 4 weeks of the onset of the current episode of arthritis. Metagenomic DNA was extracted from the stools of these patients and of 36 age- and sex-similar controls. Sequencing and analysis were done using a standard 16S ribosomal pipeline. Of 55 patients, there was no difference between the gut microbiota of postdiarrheal ReA(n = 20) and of upSpA (n = 35). Comparing the gut microbiota of patients vs healthy controls, the patients had significantly higher alpha and beta diversity measures. After stringency filters, Proteobacteria had high abundance while Firmicutes had lesser as compared with the controls. Six families were overexpressed in patients, while another five were overexpressed in controls. Sixteen genera and 18 species were significantly different between patients and controls. At the species level there was strong association of Staphylococcus aureus, Clostridium septicum Klebsiella pneumoniae, Escherichia coli, Empedobacter brevis, Roseburia hominis, Bacillus velezensis, and Crassaminicella with ReA. The microbiota of classical gut-associated ReA and upSpA is similar. Patients have higher diversities in their gut microbiota compared with healthy controls. Both known and previously unreported species associated with ReA/upSpA were identified.

Premature Infant Gut Microbiome relationships with childhood behavioral scales: preliminary insights.

Very Low Birth Weight (VLBW) infants, born weighing less than 1,500 grams, are at risk for both gut dysbiosis and later neuropsychological developmental deficits. Behavioral effects, while related to neurodevelopment, are often more subtle and difficult to measure. The extent of later neurobehavioral consequences associated with such microbial dysbiosis has yet to be determined. We explored associations between the infants' gut microbiome and early childhood behavior at 4 years of age and identified the bacterial taxa through a multivariate analysis by linear models. Parents completed the Child Behavior Checklist (CBCL) focused on different DSM diagnostic categories: affective, anxiety, pervasive developmental, attention deficit/hyperactivity, and oppositional defiant. All the CBCL scores were corrected for gender, delivery method, gestational age, infant birth weight, occurrence of sepsis, and days on antibiotics prior statistical analyses. Canonical correlation analysis (CCA) was performed to determine the relationship between early life gut microbiome and the adjusted CBCL scores. The association of bacterial Amplicon sequence Variants (ASVs) to the CBCL scores were tested with multivariate analysis by linear models (MaAsLin). Nineteen children who were previously born with very low birth weight and studied while hospitalized in the Neonatal Intensive Care Unit (NICU) were included in this study. Statistically significant associations were observed between early life gut bacteria such as Veillonella dispar, Enterococcus, Escherichia coli, and Rumincococcus to later behavior at 4 years. No significant association could be observed with early-life gut microbiome alpha diversity and behavioral measures at 4 years. These preliminary observational data provide insight into the relationships between VLBW gut microbiome dysbiosis and childhood behavior. This study contributes to the literature on gut microbiome analysis by examining various behavioral domains using a standardized tool linked to the Diagnostic and Statistical Manual of Mental Disorders (DSM).

Effect of no-till followed by crop diversification on the soil microbiome in a boreal short cereal rotation

Diversification of agricultural practices, including changes in crop rotation, intercropping or cover cropping, influence the soil microbiome. Here the impact of tillage and crop diversification on the soil microbiome is reported, being one of the few boreal studies. The field experiment consisted of four treatments with four replications all having a short cereal rotation practice namely an oat (Avena sativa) – spring barley (Hordeum vulgare) – wheat (Triticum aestivum) rotation for the past 10 years until spring 2018. During that period two of the treatments were conventionally tilled with moldboard ploughing whereas the other two were no-tillage treatments. From the growing season 2018 until fall 2020 the main crop in all treatments was spring barley. The first conventional tillage treatment was diversified with English ryegrass (Lolium perenne) as an undersown cover crop for the next three growing seasons. The first no-tillage treatment continued with spring barley only. The second conventional tillage and no-tillage treatment had winter rapeseed in rotation in 2019. Bulk soils were sampled in May 2018 before diversification and then in October 2018, 2019, and 2020. The results showed a clear effect of tillage on the beta-diversity of the soil microbiome and an increase in fungal richness. Barley monoculture interrupted with winter rapeseed resulted in a minor change of the fungal and bacterial community composition. Other fungal and bacterial alpha diversity measures did not react to tillage or diversification nor did the gene copy abundances involved in the N cycle. In conclusion tillage had a profound effect on the soil microbiome hindering impact of the diversification.

Ant handling changes myrmecochore seed coat microbiomes and alters diversity of seed‐borne plant pathogenic fungi

Abstract The putative benefits to seeds in myrmecochory (ant‐mediated seed dispersal) are often cast in a reward context. However, microbes have been mostly overlooked as seed mortality agents in myrmecochory, as have potential treatments provided by ant‐handling. We investigated the effects of ant handling on the diversity of seed coat fungal communities of three myrmecochorous plant species. Ant‐handling altered measures of both alpha and beta diversity of fungal communities. Ant‐handled seeds harboured different overall fungal communities and plant pathogen communities than non‐ant‐handled seeds. The myrmecochore pathogenic fungal community showed high dissimilarity (high pairwise community turnover) between ant‐handled and control seeds, while beta diversity measures for ant‐handled seeds and seeds with manually removed elaiosomes were less dissimilar. Ant handling may offer an additional benefit to myrmecochorous seeds via the reduction in the seed coat pathogenic community, which may be driven by elaiosome removal or as a by‐product of ant cleaning behaviours and chemical secretions. Read the free Plain Language Summary for this article on the Journal blog.

DECODING MICROBIOME DYSBIOSIS THROUGH METAGENOMIC ALPHA DIVERSITY

Background: Sarcoidosis is a chronic inflammatory disease that can affect multiple organs. The aetiology of sarcoidosis is not fully understood, but there is increasing evidence that the microbiome may play a role. The blood microbiome is a collection of microorganisms that live in the bloodstream. It is a complex and dynamic community that is influenced by a variety of factors, including the host’s lifestyle and pathology. Recent studies have shown that people with sarcoidosis have alterations in their blood microbiome. These alterations include changes in the diversity, richness, and evenness of the microbial community. The abundance measures by which the blood microbiome diversity may detect instances of dysbiosis related to sarcoidosis aetiology. It should be clearly distinguished from microbiome changes related to unspecific inflammation or sepsis. However, the available evidence suggests that the microbiome may be a promising target for therapeutic interventions. Aim: The primary goal of this review was to assess and compare the existing metrics of microbiome composition and diversity as established by metagenomic analyses. Additionally, we aim to elucidate the potential causal relationship between these measures, the phenomenon of blood microbiome dysbiosis and the pathogenesis of sarcoidosis. Conclusion: In the present review, we investigated alpha diversity measures as characteristics of microbiome communities, examining their potential as indicators of dysbiosis, and the probablemechanisms of microbiome participation. A descriptive qualitative comparison was conducted between lung microbiome data of sarcoidosis patients and blood microbiome data of healthy adults. This comparison elucidates common taxa between the two microbiomes and identifies taxa potentially involved in sarcoidosis.

Effects of diets supplemented with bioactive peptides on nutrient digestibility, immune cell responsiveness, and fecal characteristics, microbiota, and metabolites of adult cats.

Bioactive peptides (BP) are recognized for their ability to function as antioxidants and maintain lipid stability. They may have positive health effects, including antihypertensive, anti-inflammatory, antimicrobial, osteoprotective, gut health, and immunomodulatory properties, but are poorly tested in cats. Our primary objective was to determine the apparent total tract digestibility (ATTD) of BP-containing kibble diets and assess how the fecal characteristics, metabolites, and microbiota were affected in adult cats. Our secondary objective was to test whether BP could impact blood oxidative stress markers and cytokine concentrations following transport stress. Twelve adult cats (4.83±0.37 yr; 4.76±0.14kg) were used in a replicated 4×4 Latin square design to test four extruded kibble diets: Control (no BP), Chicken (4% chicken BP), Marine1 (2% marine BP), and Marine2 (4% marine BP). Each experimental period lasted 28 d, with a 20-d adaptation phase, 5 d for fecal collection, 2 d for blood collection, and 1 d for transport stress testing (driven in vehicle in individual carriers for 45min). Salivary cortisol and blood oxidative stress markers and cytokines were measured after transport. Fecal microbiota data were evaluated using 16S rRNA gene amplicon sequencing and QIIME2. All other data were analyzed using the Mixed Models procedure of SAS, with P<0.05 being considered significant and P<0.10 considered trends. No differences were observed in animal health outcomes, with all cats remaining healthy and serum metabolites remaining within reference ranges. Cats fed the Marine2 diet had higher (P<0.05) ATTD of dry matter (84.5% vs. 80.9%) and organic matter (88.3% vs. 85.8%) than those fed the control diet. The ATTD of protein and energy tended to be higher (P<0.10) for cats fed the Marine2 diet. Fecal characteristics, metabolites, and bacterial alpha and beta diversity measures were not affected by treatment. However, the relative abundances of six bacterial genera were different (P<0.05) and two bacterial genera tended to be different (P<0.10) across treatments. Treatment did not alter salivary cortisol, blood oxidative stress markers, or blood cytokines after transport stress. Our data suggest that BP inclusion may increase nutrient digestibility and modify fecal microbiota and immune measures. More testing is required, however, to determine whether BP may provide additional benefits to cats.

Dietary isoacids effects on growth, nitrogen, and energy digestibility, and fecal volatile fatty acids and microbial ecology in finishing pigs.

Isoacids are branched ketoacids which when fed to ruminants have been shown to enhance the growth of fiber-digesting organisms. Ninety finishing gilts were individually fed dietary treatments consisting of diet type: corn-soybean meal (CSBM), a diet containing 40% distillers dried grains with solubles (DDGS), or a diet containing 40% sugar beet pulp (SBP); in combination with either no feed additive (CNT), the addition of 0.50% isobutyrate (IB), or the addition of a 0.88% mix of isobutyrate, isovalerate, and 2-methylbutyrate (MX). Gilts consumed an average of 2.171kg/d over the 28-d trial. On d 26, fresh fecal samples were collected for determination of apparent total tract digestibility (ATTD) of gross energy (GE) and nitrogen (N), determination of fecal volatile fatty acids (VFA), and evaluation of microbial ecology. There was no interaction between diet type and isoacid addition, and no main effect of isoacid or diet type on alpha or Shannon microbial diversity measures (P > 0.05). There was no interaction between isoacid addition and diet type, and no main effect of isoacid addition on microbial beta diversity (P > 0.05), but differences were observed in microbial beta diversity due to diet type (P ≤ 0.05). There was no interaction between diet type and isoacid addition observed in fecal VFA concentrations (P > 0.05), with only minor differences in fecal VFA concentrations noted due to isoacid addition (P ≤ 0.05). The interaction between diet type and isoacid addition on ATTD of dietary GE and N (P ≤ 0.01) was large because the addition of IB did not affect the ATTD of GE or N in pigs fed the CSBM diet, but increased ATTD of GE and N in pigs fed diets containing DDGS and decreased the ATTD of GE and N in pigs fed diets containing SBP. In contrast, adding a blend of isoacids (i.e., MX) reduced the ATTD of GE and N, regardless of diet type. There was no interaction between diet type and isoacid addition, and no effect of isoacid addition was observed on pig performance (P > 0.05). Diet type did not affect average daily gain (P > 0.05), but pigs fed diets containing DDGS or SBP consumed less feed (P = 0.01) and exhibited greater GF ratios compared to pigs fed the low-fiber CSBM diet (P ≤ 0.05). In conclusion, there was little to no effect of isoacid addition on microbial ecology, fecal VFA concentrations, ATTD of GE or N, or pig performance, but the improvement in ATTD of GE and N in pigs fed diets containing DDGS when IB was added warrants further investigation.

Effects of a veterinary gastrointestinal diet on fecal characteristics, metabolites, and microbiota concentrations of adult cats treated with metronidazole.

Antibiotics are used to treat gastrointestinal diseases or infections but are known to negatively affect stool quality and gut microbiota in cats and dogs. Therefore, identifying dietary strategies that may aid in antibiotic recovery is of interest. The objective of this study was to determine how a veterinary gastrointestinal diet affected the fecal characteristics, microbiota, and metabolite and bile acid (BA) concentrations of cats recovering from metronidazole administration. Twenty-four healthy adult cats were used in an 8-wk completely randomized design study. During a 2-wk baseline, all cats consumed a leading grocery brand diet (GBD). Over the next 2wk, cats consumed GBD and received metronidazole (20mg/kg body weight twice daily). At week 4, cats were randomly allotted to one of 2 treatments [GBD; BLUE Natural Veterinary Diet GI Gastrointestinal Support (BB)] and fed for 4wk. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter (DM) %, metabolites, and microbiota. Microbiota was analyzed by 16S rRNA gene sequencing and qPCR, which was used to calculate dysbiosis index. Data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, testing for effects of diet, time and diet*time. Metronidazole had dramatic effects on all outcomes, including increased fecal scores (looser stools), reduced fecal pH and DM%, reduced fecal short-chain fatty acid, branched-chain fatty acid, ammonia, phenol, and indole concentrations, and altered fecal BA concentrations (increased primary BA; reduced secondary BA). Metronidazole reduced fecal bacterial alpha diversity, increased dysbiosis index, and altered the relative abundance of 78 bacterial genera. Fecal outcomes partially recovered over the next 4wk, with some being impacted by diet. Fecal acetate concentrations were higher after metronidazole in cats fed BB. Dysbiosis index and alpha diversity measures slowly recovered over 4wk, without diet differences. Recovery of 16 bacterial genera was impacted by diet. Fecal BA profiles demonstrated a prolonged impairment of primary to secondary BA conversion, with cholic acid being lower after metronidazole in cats fed BB. In conclusion, our data demonstrate that metronidazole is a powerful antibiotic that has long-lasting effects on the fecal microbiota and metabolites of cats. Outcome variables slowly recovered over time, but a gastrointestinal diet may aid in recovery.

Age and sex-related variations in murine laryngeal microbiota.

The larynx undergoes significant age and sex-related changes in structure and function across the lifespan. Emerging evidence suggests that laryngeal microbiota influences immunological processes. Thus, there is a critical need to delineate microbial mechanisms that may underlie laryngeal physiological and immunological changes. As a first step, the present study explored potential age and sex-related changes in the laryngeal microbiota across the lifespan in a murine model. We compared laryngeal microbial profiles of mice across the lifespan (adolescents, young adults, older adults and elderly) to determine age and sex-related microbial variation on 16s rRNA gene sequencing. Measures of alpha diversity and beta diversity were obtained, along with differentially abundant taxa across age groups and biological sexes. There was relative stability of the laryngeal microbiota within each age group and no significant bacterial compositional shift in the laryngeal microbiome across the lifespan. There was an abundance of short-chain fatty acid producing bacteria in the adolescent group, unique to the laryngeal microbiota; taxonomic changes in the elderly resembled that of the aged gut microbiome. There were no significant changes in the laryngeal microbiota relating to biological sex. This is the first study to report age and sex-related variation in laryngeal microbiota. This data lays the groundwork for defining how age-related microbial mechanisms may govern laryngeal health and disease. Bacterial compositional changes, as a result of environmental or systemic stimuli, may not only be indicative of laryngeal-specific metabolic and immunoregulatory processes, but may precede structural and functional age-related changes in laryngeal physiology.

Validation of method for faecal sampling in cats and dogs for faecal microbiome analysis

BackgroundReproducible and reliable studies of cat and dog faecal microbiomes are dependent on many methodology-based variables including how the faecal stools are sampled and stored prior to processing. The current study aimed to establish an appropriate method for sampling and storing faecal stools from cats and dogs which may also be applied to privately-owned pets. The approach investigated the effects of storing faeces for up to 12 h at room temperature and sampling from various locations within the stool in terms of microbial diversity, relative taxa abundances and DNA yield. Faeces were collected from 10 healthy cats and 10 healthy dogs and stored at room temperature (20 °C). Samples were taken from various locations within the stool (the first emitted part (i), the middle (ii) and the last emitted end (iii), at either surface or core) at 0, 0.5, 1, 2, 3, 6 and 12 h, stabilised and stored at -80 °C. DNA was extracted from all samples, using Illumina NovaSeq.ResultsFaecal bacterial composition of dogs and cats shown no statistically significant differences in alpha diversity. Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria were the most prevalent phyla. Cat and dog samples were characterized by a dominance of Prevotella, and a lack of Fusobacterium in feline stools. Room temperature storage of cat and dog faecal samples generally had no significant effect on alpha diversity, relative taxa abundance or DNA yield for up to 12 h. Sampling from regions i, ii or iii of the stool at the surface or core did not significantly influence the outcome. However, surface cat faecal samples stored at room temperature for 12 h showed a significant increase in two measures of alpha diversity and there was a tendency for a similar effect in dogs. When comparing samples with beta diversity measures, it appeared that for dog and cat samples, individual effect has the strongest impact on the observed microbial diversity (R2 0.64 and 0.88), whereas sampling time, depth and horizontal locations significantly affected the microbial diversity but with less impact.ConclusionCat and dog faeces were stable at room temperature for up to 12 h, with no significant changes in alpha diversity, relative taxa abundance and DNA concentration. Beta diversity analysis demonstrated that despite an impact of the sampling storing time and the surface of the sampling, we preserved the identity of the microbial structure linked to the individual. Finally, the data suggest that faecal stools stored for > 6 h at room temperature should be sampled at the core, not the surface.

PSXI-30 Ruminal Bacterial Diversity Indexes of Grazing Beef Cattle Receiving Different Supplements Plus Phytogenic Additives

Abstract The addition of phytogenic compounds that have antimicrobial activity in cattle supplements can promote changes in ruminal bacterial diversity. This study aimed to evaluate the effect of two levels of supplementation plus a blend of phytogenic additives (essential oils, saponins, and spices) on the measures of alpha and beta diversity in the rumen of beef cattle grazing tropical pasture. The experiment was carried out in the Beef Cattle Sector of UNESP, Jaboticabal-BR. The trial lasted 84 days and nine castrated Nellore steers (body weight of 262 ± 31.2 kg) cannulated in the rumen were assigned in 3 simultaneous 3 × 3 Latin square designs. Animals were kept on the pasture of Urochloa brizantha cv. Marandu in the rainy season. The treatments were: ad libitum mineral supplementation (MS) as control group; added mineral supplementation, 0.1% of body weight per day plus Cargill phytogenic blend (AMSP); and energetic-protein supplementation, 0.3% of body weight per day plus Cargill phytogenic blend (EPSP). Ruminal samples were collected on day 26 of each experimental period. A mix of liquid and solid rumen content (6 mL) was taken before supplementation, placed in cryogenic tubes, and immediately frozen in liquid nitrogen. All samples were stored at −80 °C until DNA extraction and analysis. Total genomic DNA was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep kit. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTWTCTAAT). The PCR fragments were submitted to sequencing on an Illumina NovaSeq 6000 PE 250 platform. The sequences were processed using mothur, grouped into operational taxonomic units (OTUs) using a similarity cut-off value of 97%, and the bacterial OTUs were classified using the SILVA database. Alpha diversity indices including Shannon, Simpson, Fisher, Chao1, and ACE, were calculated using the phyloseq package, while beta diversity was determined using the vegan package, both in Rstudio. Alpha diversity indices were compared between treatments using a non-parametric Kruskal–Wallis test and Wilcoxon’s post-hoc test. Beta diversity was tested using permutational multivariate analysis of variance (PERMANOVA) based on the Bray-Curtis similarity index. The AMSP and EPSP did not affect measures of alpha diversity (Figure 1), such as Shannon and Fisher indices (P = 0.219) as well as the richness index measured using Chao1 and ACE (P ≥ 0.363). A negative effect of supplementation on alpha diversity (P = 0.004) was only observed with Simpson’s Index of Diversity. Additionally, the supplementation did not change the beta-diversity of the ruminal bacterial community based on Bray–Curtis similarity index (P = 0.510; Figure 2). In conclusion, the addition of phytogenic compounds in the two levels of supplementation showed only a minor impact on the alpha diversity in the rumen of grazing beef cattle.

PSIX-4 A Veterinary Gastrointestinal Diet Affects Fecal Characteristics, Metabolites, Bile Acids, and Microbiota Concentrations of Antibiotic-Treated Cats

Abstract Antibiotics are often used to treat animals with gastrointestinal diseases or infections, but their use is known to have negative effects on the intestinal microbiota community. Identifying dietary strategies that may aid in the recovery from antibiotic use is of interest. The objective of this study was to determine how a veterinary gastrointestinal diet affected the fecal characteristics and metabolite, bile acid (BA), and microbiota concentrations of cats recovering from metronidazole administration. Twenty-four healthy adult cats were used in an 8-wk completely randomized design study. The study began with a 2-wk baseline phase where all cats consumed a leading grocery brand dry diet (GBD; wk 0 to wk 2). Over the next 2 wk, cats continued to consume GBD and received metronidazole (20 mg/kg BW; wk 2 to wk 4). At wk 4, cats were randomly allotted to one of two treatments [GBD; Blue Buffalo Gastrointestinal Support (BB)] for the remaining 4 wk (wk 5 to wk 8). Fecal scores were recorded daily, and fresh fecal samples were collected at wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter, and metabolite and BA concentrations. Fecal microbiota were analyzed by 16S rRNA sequencing and qPCR, which was used to calculate dysbiosis index. All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, with treatment treated as a fixed effect, cat as a random effect, and significance set at P&amp;lt; 0.05 and trends at P &amp;lt; 0.10. Metronidazole administration had dramatic effects on the outcomes measured, including increased fecal scores (looser stools), reduced fecal pH and dry matter %, reduced fecal short-chain fatty acid (SCFA), branched-chain fatty acid, ammonia, phenol, and indole concentrations, and altered fecal BA concentrations (increased primary BA; reduced secondary BA). Furthermore, fecal bacterial alpha diversity was reduced, dysbiosis index was increased, and the abundance of several individual bacterial taxa were altered. Fecal outcome variables partially recovered over the next 4 wk, with some being impacted by diet. The change in fecal acetate concentrations were greater and the change in total SCFA concentrations tended to be greater in cats fed BB. Dysbiosis index and alpha diversity measures slowly recovered over 4 wk, without differences due to diet. Recovery of about 20 bacterial genera were impacted by diet. Primary and secondary BA concentrations and ratios demonstrated a prolonged impairment of primary to secondary BA conversion. Most of these changes were an effect of time, but the change of fecal cholic acid concentrations were less in cats fed BB. In conclusion, our data demonstrate that metronidazole is a powerful antibiotic that has long-lasting effects on the fecal microbiota and metabolites of cats. The veterinary gastrointestinal diet tested aided in the recovery of some variables measured.

257 Effects of Prebiotic Galactooligosaccharide (GOS) on Hindgut Microbial Composition of Pigs During the Peri-Weaning Period

Abstract Galactooligosaccharide (GOS) is a prebiotic containing beta-linked galactose oligomers of 2 to 8 units. Previously, GOS positively impacted post-weaning growth performance and altered jejunal morphology. In this study, GOS-enriched whey permeate (Milk Specialties Global, Eden Prairie MN) was supplemented to piglets in farrowing and nursery phases. To maximize pre-weaning GOS intake, novel gruel creep feeders were used. Piglets from 24 litters received either no creep feed (NC), creep without GOS (CG-) or creep with 5.0% GOS (CG+) followed by a phase 1 nursery diet without (NG-) or with 3.8% GOS (NG+). At d 22 (pre-weaning) and d 31 (post-weaning), 6 pigs per treatment were euthanized for sample collection. DNA was extracted from cecal and rectal swabs for downstream 16S rRNA sequencing via Illumina MiSeq (Zymo Research Corp, Irvine CA). An amplicon sequence variant (ASV) table was created using the DADA2 pipeline in R and taxonomy assignment was performed using the SILVA database. Average sequencing depth after filtering was 138,323 and low sequencing counts were discarded based on 20% prevalence. Data were analyzed using the lmer and adonis procedures in RStudio ver. 4.2.2. Analysis of 16S rRNA genes did not detect pre- or post-weaning GOS effects on Chao1, Simpson, or Shannon alpha diversity measures (P &amp;gt; 0.1). Similarly, no pre-weaning GOS effects were detected in Principal Coordinates Analysis (PCoA) beta diversity plots (P &amp;gt; 0.1); however, pigs fed GOS post-weaning segregated from controls (P &amp;lt; 0.01). The relative abundance of several genera including Fusicatenbacter and Collinsella increased by 2.0 and 1.3-fold, respectively, in the cecum of NG+ pigs, whereas, Bacteroides decreased by 3.8-fold (FDR, P &amp;lt; 0.05). In the rectum, relative abundance of Corprococcus decreased by 2.0-fold and Fusicatenbacter increased by 1.5-fold in NG+ pigs compared with NG- pigs (FDR, P &amp;lt; 0.05). Collectively, these genera in the cecum and rectum comprise of less than 3% of the total community. In contrast, lactobacillus in the cecum (33%) and rectum (21%) had a large relative abundance but was unaffected by treatment. We conclude that supplementation of GOS in the post-weaning phase 1 nursery diet alters the hindgut microbial community and may contribute to improvements in growth performance.

PSIX-3 A Veterinary Gastrointestinal Low-Fat Diet Affects Fecal Characteristics, Metabolites, Bile Acids, and Microbiota Concentrations of Antibiotic-Treated Dogs

Abstract In a recent dog study, the antibiotic metronidazole proved to be an effective disruptor of the fecal microbiota and resulted in looser stools and altered fecal bile acid (BA) profiles. Recovery took several weeks and was aided by a dietary supplement. The objective of this study was to determine how a veterinary gastrointestinal low-fat diet affected the fecal characteristics and metabolite, BA, and microbiota concentrations of dogs recovering from metronidazole administration. Twenty-four healthy adult dogs were used in an 8-wk completely randomized design study. The study began with a 2-wk baseline phase where all dogs consumed a leading grocery brand dry kibble diet (GBD; wk 0 to wk 2). Over the next 2 wk, dogs continued to consume GBD and received metronidazole (20 mg/kg BW; wk 2 to wk 4). At wk 4, dogs were randomly allotted to one of two treatments [GBD; Blue Buffalo Gastrointestinal Support Low-Fat (BB)] for the remaining 4 wk (wk 5 to wk 8). Fecal scores were recorded daily, and fresh fecal samples were collected at wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter, and metabolite and bile acid concentrations. Fecal microbiota populations were also analyzed by 16S rRNA sequencing and qPCR, which was used to calculate dysbiosis index. All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, with treatment treated as a fixed effect, dog as a random effect, and significance set at P &amp;lt; 0.05. As anticipated, metronidazole administration increased (P &amp;lt; 0.0001) fecal scores (looser stools) and drastically altered (P &amp;lt; 0.05) fecal metabolite concentrations, reducing fecal short-chain fatty acid (SCFA), branched-chain fatty acid (BCFA), phenol, and indole concentrations, increasing primary BA concentrations, and decreasing secondary bile acid concentrations. Metronidazole dramatically impacted fecal microbiota populations by reducing alpha diversity, altering the abundance of multiple bacterial taxa, and increasing dysbiosis index. Fecal outcome variables recovered over the next 4 wk, with some being impacted by diet. The change in fecal SCFA and immunoglobulin A concentrations were greater (P &amp;lt; 0.01), while the change in fecal BCFA, phenol, and indole concentrations were decreased (P &amp;lt; 0.05) in dogs fed BB. Dysbiosis index and alpha diversity measures recovered quickly for all dogs and without dietary effects. Recovery of over 25 bacterial genera were impacted by diet (P &amp;lt; 0.05). While many bacterial taxa returned to baseline levels after 4 wk, others had still not fully recovered. Fecal BA concentrations recovered quickly for all dogs. The change in lithocholic acid concentration was less (P &amp;lt; 0.0001) in dogs fed BB, but other BA concentrations were not affected by diet. In conclusion, metronidazole drastically alters the fecal microbiota and metabolites of dogs. While most variables return to normal over time, a veterinary gastrointestinal diet may aid in the recovery.

Urinary microbiome community types associated with urinary incontinence severity in women

BackgroundUrinary microbiome (urobiome) studies have previously reported on specific taxa and community differences in women with mixed urinary incontinence compared to controls. Therefore, we hypothesized that higher urinary and vaginal microbiome diversity would be associated with increased urinary incontinence severity. ObjectivesTo test if specific urinary or vaginal microbiome community-types are associated with urinary incontinence severity in a population of women with mixed urinary incontinence. Study DesignThis planned secondary, cross-sectional analysis evaluated associations between the urinary/vaginal microbiomes and urinary incontinence severity in a subset of ESTEEM trial participants with urinary incontinence. Incontinence severity was measured using bladder diaries and Urinary Distress Inventory (UDI) questionnaires collected at baseline. Catheterized urine samples and vaginal swabs were concurrently collected prior to treatment at baseline to assess the urinary and vaginal microbiomes.16S rRNA V4-V6 variable regions were sequenced, characterizing bacterial taxa to the genus level using the DADA2 pipeline and SILVA database. Using Dirichlet multinomial mixture methods, samples were clustered into community-types based on core taxa. Associations between community-types and severity measures (UDI total, UDI subscale scores, and the number of urinary incontinence episodes (total, urgency, and stress) from bladder diary) were evaluated using linear regression models adjusted for age and body mass index. Alpha diversity measures for richness (total taxa numbers) and evenness (proportional distribution of taxa abundance) were also analyzed for associations with urinary incontinence episodes and community-type. ResultsSix urinary microbiome community-types were identified, characterized by varying levels of common genera (Lactobacillus, Gardnerella, Prevotella, Tepidomonas, Acidovorax, Escherichia, and others). Analysis of urinary incontinence severity in 126 participants with MUI identified a Lactobacillus-dominated reference group with the highest abundance of Lactobacillus (76% mean relative abundance). A community characterized by fewer Lactobacilli (19% mean relative abundance) and greater alpha diversity, was associated with higher total urinary incontinence episodes (2.67 daily leaks, 95% CI: 0.76 - 4.59, p=0.007) and urgency urinary incontinence episodes (1.75 daily leaks, 95% CI: 0.24 - 3.27, p=0.02) compared to the reference group. No significant association was observed between community-type and stress urinary incontinence episodes or UDI total or subscores. The composition of vaginal community-types and urinary community-types were similar but comprised of slightly different bacterial taxa. Vaginal community-types were not associated with urinary incontinence severity, as measured by bladder diary or UDI total and subscale scores. Alpha diversity indicated that greater sample richness was associated with more incontinence episodes (observed genera p=0.01) in urine. Measures of evenness (Shannon/Pielou) were not associated with incontinence severity in the urinary or vaginal microbiomes. ConclusionsIn the urobiome of women with mixed urinary incontinence, a community-type with fewer Lactobacilli and more diverse bacteria was associated with more severe urinary incontinence episodes (total and urgency) compared to a community-type with high predominance of a single genus, Lactobacillus. Whether mixed urinary incontinence severity is due to lesser predominance of Lactobacillus, greater presence of other non-Lactobacillus genera, or the complement of bacteria comprising urobiome community-types, remains to be determined.

Land use change rather than surrounding vegetation affects fungal endophyte assemblages in the African wild olive

ContextLand use change can significantly affect plant-fungal interactions.ObjectivesWe assessed how fungal endophytes within African wild olive (Olea europaea subsp. cuspidata) twigs are influenced by different levels of land use change and differences in surrounding vegetation types.MethodsTwigs were sampled in the Western Cape Province (South Africa) and their fungal endophyte assemblages were characterised using culture-independent DNA metabarcoding. We assessed the effects of land use change (natural, semi-natural and planted (completely transformed)) and differences in surrounding vegetation types (grasses/low-growing plants versus shrubs/trees versus other olives) using fungal endophyte alpha and beta diversity measures. Co-occurrence networks were constructed to assess assemblage connectivity under different scenarios and to identify OTUs of potential ecological significance.ResultsOTU richness, but not abundance, was significantly influenced by both land use change and differences in the surrounding vegetation types. Planted African olives and those surrounded by heterospecific trees harboured the highest OTU richness. Only levels of land use change significantly influenced fungal endophyte assemblage composition. Specifically, fungal assemblages from natural habitats were distinct from those in planted and semi-natural habitats, which were similar to each other. Co-occurrence network analyses revealed that cohesive and species rich networks could only be maintained within the natural habitats.ConclusionThese findings suggest that although the African olive is widespread, the identity and composition of their associated fungal assemblages are particularly sensitive to land use change. This study highlights the importance of conserving natural habitats, not just for the plants, but also for the maintenance of their associated fungal endophytes.

Dietary Inclusion of Yellow Mealworms (T. molitor) and Lesser Mealworms (A. diaperinus) Modifies Intestinal Microbiota Populations of Diet-Induced Obesity Mice

BackgroundInsect-based proteins are high-quality alternatives to support the shift toward more sustainable and healthy diets. Additionally, insects contain chitin and have unique fatty acid profiles. Studies have shown that mealworms may beneficially affect metabolism, but limited information is known regarding their effects on gut microbiota. ObjectivesWe determined the effects of defatted yellow mealworm (Tenebrio molitor) and whole lesser mealworm (Alphitobius diaperinus) meals on the intestinal microbiota of diet-induced obesity mice. MethodsMale C57BL/6J mice were fed a high-fat diet (HFD; 46% kcal) to induce obesity. Obese mice were then randomly assigned to treatments (n = 10/group) and fed for 8 wk: HFD, HFD with casein protein; B50, HFD with 50% protein from whole lesser mealworm; B100, HFD with 100% protein from whole lesser mealworm; Y50, HFD with 50% protein from defatted yellow mealworm; Y100, HFD with 100% protein from defatted yellow mealworm. Lean mice (n = 10) fed a low-fat-diet (10% kcal) were included. Fresh feces were collected at baseline and every 2 wk, with cecal digesta collected at kill. Fecal and cecal DNA was analyzed for microbiota using 16S rRNA MiSeq Illumina sequencing. ResultsIn feces and cecal digesta, mice fed mealworms had greater (P < 0.05) bacterial alpha diversity, with changes occurring in a time-dependent manner (P < 0.05). Beta diversity analyses of cecal samples showed a clear separation of treatments, with a time-based separation shown in fecal samples. Widespread microbial differences were observed, with over 45 genera altered (P < 0.05) by diet in cecal digesta. In feces, over 50 genera and 40 genera were altered (P < 0.05) by diet and time, respectively. ConclusionMealworm consumption changes the intestinal microbiota of obese mice, increasing alpha diversity measures and shifting bacterial taxa. More investigation is required to determine what mealworm components are responsible and how they may be linked with the metabolic benefits observed in mealworm-fed mice.

Gut microbiome in men with chronic prostatitis/chronic pelvic pain syndrome: profiling and its predictive significance.

To investigate the difference in gut microbiome composition between patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and healthy controls, and to assess the potential of gut microbiota as predictive markers for CP/CPPS risk. The present study included 41 CP/CPPS patients and 43 healthy controls in China. Fecal specimen data were obtained and analysed using 16S rRNA gene sequencing. Alpha and beta-diversity indices, relative microbiome abundances, cluster analysis, and linear discriminant analysis effect size (LEfSe) were employed. Microbial biomarkers were selected for the development of a diagnostic classification model, and the functional prediction was conducted using PICRUSt2. Alpha-diversity measures revealed no statistically significant difference in bacterial community structure between CP/CPPS patients and controls. However, significant differences were observed in the relative abundances of several bacterial genera. Beta-diversity analysis revealed a distinct separation between the two groups. Significant inter-group differences were noted at various taxonomic levels, with specific bacterial genera being significantly different in abundance. The LEfSe analysis indicated that three bacterial species were highly representative and seven bacterial species were low in CP/CPPS patients as compared to the control group. A diagnostic model for CP/CPPS based on microbial biomarkers exhibited good performance. PICRUSt2 functional profiling indicated significant differences in the development and regeneration pathway. Significant differences in the gut microbiome composition were found between groups. The study provided a novel diagnostic model for CP/CPPS based on microbiota, presenting promising potential for future therapeutic targets and non-invasive diagnostic biomarkers for CP/CPPS patients.