Illumina MiSeq 16S rRNA Gene Library Preparation for Poultry Processing Microbiome Analyses.

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety.This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay.

BACKGROUND: Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. METHODS: In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. RESULTS: A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/µL, 5.8 × 10-2 ng/µL and 1.4 × 10-2 ng/µL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay's high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. CONCLUSION: The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

Metagenomic analysis of colonic tissue and stool microbiome in patients with colorectal cancer in a South Asian population.

BACKGROUND: The gut microbiome is thought to play an important role in the development of colorectal cancer (CRC). However, as the gut microbiome varies widely based on diet, we sought to investigate the gut microbiome changes in patients with CRC in a South Asian population. METHODS: The gut microbiome was assessed by 16s metagenomic sequencing targeting the V4 hypervariable region of the bacterial 16S rRNA in stool samples (n = 112) and colonic tissue (n = 36) in 112 individuals. The cohort comprised of individuals with CRC (n = 24), premalignant lesions (n = 10), healthy individuals (n = 50) and in those with diabetes (n = 28). RESULTS: Overall, the relative abundances of genus Fusobacterium (p < 0.001), Acinetobacter (p < 0.001), Escherichia-Shigella (p < 0.05) were significantly higher in gut tissue, while Romboutsia (p < 0.01) and Prevotella (p < 0.05) were significantly higher in stool samples. Bacteroides and Fusobacterium were the most abundant genera found in stool samples in patients with CRC. Patients with pre-malignant lesions had significantly high abundances of Christensenellaceae, Enterobacteriaceae, Mollicutes and Ruminococcaceae (p < 0.001) compared to patients with CRC, and healthy individuals. Romboutsia was significantly more abundant (p < 0.01) in stool samples in healthy individuals compared to those with CRC and diabetes. CONCLUSION: Despite marked differences in the Sri Lankan diet compared to the typical Western diet, Bacteroides and Fusobacterium species were the most abundant in those with CRC, with Prevotella species, being most abundant in many individuals. We believe these results pave the way for possible dietary interventions for prevention of CRC in the South Asian population.

Antimicrobial potential of Streptomyces sp. NP73 isolated from the forest soil of Northeast India against multi-drug resistant Escherichia coli.

This study reports the isolation and characterization of a Streptomyces sp. from soil, capable of producing bioactive secondary metabolites active against a variety of bacterial human pathogens. We targeted the antimicrobial activity against Escherichia coli ATCC-BAA 2469, a clinically relevant strain of bacteria harbouring resistance genes for carbapenems, extended spectrum beta-lactams, tetracyclines, fluoroquinones, etc. Preliminary screening using the spot inoculation technique identified Streptomyces sp. NP73 as the potent strain among the 74 isolated Actinomycetia strain. 16S rRNA gene and whole genome sequencing (WGS) confirmed its taxonomical identity and helped in the construction of the phylogenetic tree. WGS revealed the predicted pathways and biosynthetic gene clusters responsible for producing various types of antibiotics including the isolated compound. Bioactivity guided fractionation and chemical characterization of the active fraction, carried out using liquid chromatography, gas chromatography-mass spectrometry, infra-red spectroscopy, and nuclear magnetic resonance spectroscopy, led to the tentative identification of the active compound as Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-, a diketopiperazine molecule. This compound exhibited excellent antimicrobial and anti-biofilm properties against E. coli ATCC-BAA 2469 with an MIC value of 15.64 µg ml-1, and the low cytotoxicity of the compound identified in this study provides hope for future drug development.

Akkermansia muciniphila in the small intestine improves liver fibrosis in a murine liver cirrhosis model.

Recent evidence indicates that liver cirrhosis (LC) is a reversible condition, but there is no established intervention against liver fibrosis. Although the gut microbiota is considered involved in the pathogenesis of LC, the underlying mechanisms remain unclear. Although the antibiotic, rifaximin (RFX), is effective for hepatic encephalopathy (HE) with LC, the impact of RFX on intestinal bacteria is unknown. We investigated the bacterial compositions along the GI tract under RFX treatment using a murine LC model. RFX improved liver fibrosis and hyperammonemia and altered the bacterial composition in the small intestine. The efficacy of RFX was associated with increases in specific bacterial genera, including Akkermansia. Administration of a commensal strain of Akkermansia muciniphila improved liver fibrosis and hyperammonemia with changing bacterial composition in the small intestine. This study proposed a new concept "small intestine-liver axis" in the pathophysiology of LC and oral A. muciniphila administration is a promising microbial intervention.

Analysis of the characteristics of intestinal microbiota in patients with different severity of obstructive sleep apnea.

Intestinal microbiota imbalance plays an important role in the progression of obstructive sleep apnea (OSA), and is considered to be the main mediator that triggers metabolic comorbidities. Here, we analyzed the changes in intestinal microbiota in patients with different severities of OSA based on apnea hypopnea index (AHI) classification, and explored the role of intestinal microbiota in the severity of OSA. This study included 19 healthy volunteers and 45 patients with OSA [5 ≤ AHI < 15 (n = 14), 15 ≤ AHI < 30 (n = 13), AHI ≥ 30 (n = 18)]. Relevant sleep monitoring data and medical history data were collected, and microbial composition was analyzed using 16S rRNA high-throughput sequencing technology. The diversity analysis of intestinal microbiota among different groups of people was conducted, including alpha diversity, beta diversity, species diversity, and marker species as well as differential functional metabolic pathway prediction analysis. With the increase of AHI classification, the alpha diversity in patients with OSA significantly decreased. The results revealed that the severity of OSA is associated with differences in the structure and composition of the intestinal microbiota. The abundance of bacteria producing short-chain fatty acids (such as Bacteroides, Ruminococcacea, and Faecalibacterium) in severe OSA is significantly reduced and a higher ratio of Firmicutes to Bacteroidetes. Random forest analysis showed that Parabacteroides was a biomarker genus with important discriminatory significance. The differential metabolic pathway prediction function shows that the main function of maintaining intestinal microbiota homeostasis is biosynthetic function. Our results show that the differences in the composition of intestinal microbiota in patients with different severities of OSA are mainly related to short-chain fatty acid-producing bacteria. These changes may play a pathological role in OSA combined with metabolic comorbidities.

The effects of a high-flavonoid corn cultivar on the gastrointestinal tract microbiota in chickens undergoing necrotic enteritis.

The search for alternative therapies to antimicrobial growth promoters (AGP) in poultry production has gained momentum in the past years because of consumer preference and government restrictions on the use of AGP in animal production. Flavonoids are plant-derived metabolites that have been studied for their health-promoting properties that could potentially be used as an alternative to AGP in poultry. In a previous study, we showed that the inclusion of a flavonoid-rich corn cultivar (PennHFD1) in the diet improved the health of broilers undergoing necrotic enteritis. However, the mechanisms of action by which the PennHFD1-based diet ameliorated necrotic enteritis are unknown. This study describes the microbial diversity and composition of the jejunum and ileum of chickens co-infected with Eimeria maxima and Clostridium perfringens and treated with a high-flavonoid corn-based diet. Luminal content and mucosal samples from the jejunum and ileum were collected for DNA extraction, 16S rRNA amplicon sequencing and data analyses. The infection model and the dietary treatments significantly changed the alfa diversity indices (Mucosal samples: ASVs, P = 0.04; Luminal content samples: ASVs, P = 0.03), and beta diversities (Mucosal samples: P < 0.01, Luminal content: P < 0.01) of the ileal samples but not those of the jejunal samples. The microbial composition revealed that birds fed the high-flavonoid corn diet had a lower relative abundance of C. perfringens compared to birds fed the commercial corn diet. The treatments also changed the relative abundance of other bacteria that are related to gut health, such as Lactobacillus. We concluded that both the infection model and the dietary high-flavonoid corn changed the broilers' gut microbial diversity and composition. In addition, the decrease in the relative abundance of C. perfringens corroborates with a decrease in mortality and intestinal lesions due to necrotic enteritis. Collecting different segments and sample types provided a broader understanding of the changes in the gut microbiota among treatments.

Fusarium graminearum spores disrupt gut microbiota and metabolome via the lung-gut axis in mice.

Fusarium graminearum, the primary pathogen responsible for wheat Fusarium head blight, can induce pulmonary damage through its spores. However, the detailed mechanism by which these spores cause intestinal injury is not yet fully understood. This study aimed to investigate the impact of exposure to fungal spores on the intestinal microbiota using a mice model that mimics the effects of fusarium graminearum spores on the gut microbiota and its metabolic profile. The study utilized 16S rRNA sequencing and metabolomics methodologies to analyze the contents of the cecum and feces in mice. The results showed that exposure to fungal spores led to significant changes in the composition of the intestinal microbiota in mice, characterized by an increase in Akkermansia and Staphylococcus populations. A non-targeted metabolomics analysis identified 316 metabolites associated with various metabolic pathways, particularly galactose metabolism. Pre-exposure to antibiotics before fungal spore exposure resulted in a decrease in the metabolic capacity of the intestinal microbiota in mice. This research demonstrates that fusarium graminearum spores can disrupt the intestinal microbiota and metabolome via the lung-gut axis. These findings provide valuable insights into the intestinal damage caused by fungal spores and offer important support for the development of therapeutic strategies for intestinal diseases.

Altered blood microbiome in patients with HCV-related Child-Pugh class B cirrhosis.

BACKGROUND: Altered bacterial translocation is associated with changes in hepatic function and the progression from compensated to decompensated cirrhosis. Child-Turcotte-Pugh (CTP) score is an essential indicator of liver severity. Thus, we aimed to study differences in the blood microbiome together with metabolome profile between HCV-infected patients with CTP class B (CTP-B, significant functional compromise) and patients with CTP class A (CTP-A, well-compensated cirrhosis). METHODS: We conducted a cross-sectional study in patients with advanced HCV-related cirrhosis (n = 88) stratified by CTP-B and CTP-A. Bacterial 16S rRNA sequencing was sequenced by MiSeq Illumina technology and non-targeted metabolomics was performed by GC-MS and LC-MS ESI+ and ESI- to complement the analysis. RESULTS: Patients with CTP-B had lower levels of richness (Chao1), and alpha diversity (Shannon and Simpson indexes) at phylum level than patients with CTP-A. Likewise, we observed significant differences in beta diversity between groups at phylum, class, and order levels, showing lower diversity in patients with CTP-B. Higher relative abundance of Proteobacteria (p = 0.012), Alphaproteobacteria (p = 0.005), Sphingomonadales (p = 0.012) and Sphingomonadaceae (p = 0.016) were significantly associated with CTP-B. The phylum Proteobacteria was positively correlated with ethanolamine and oleic acid (p = 0.005 and p = 0.004, respectively) and negatively with p-cresol (p = 0.006). In addition, the order Sphingomonadales and the family Sphingomonadaceae was also negatively correlated with p-cresol (p = 0.001 and p = 0.001). CONCLUSIONS: Blood microbial diversity was significantly decreased in patients with CTP-B, who presented an enrichment of Proteobacteria, Alphaproteobacteria, Sphingomonadales and Sphingomonadaceae compared to patients with CTP-A.

Unveiling the pathogenic and multidrug-resistant profiles of Vibrio alfacsensis: A potential identified threat in turbot (Scophthalmus maximus) aquaculture.

Vibrio alfacsensis is traditionally seen as an environmental symbiont within its genus, with no detailedly documented pathogenicity in marine aquaculture to date. This study delves into the largely unexplored pathogenic potential and emerging antibiotic resistance of V. alfacsensis. The VA-1 strain, isolated from recirculating aquaculture system (RAS) effluent of cultured turbot (Scophthalmus maximus), underwent comprehensive analysis including biochemical identification, antibiotic susceptibility testing and reinfection trials. The results confirmed VA-1's pathogenicity and significant multiple antibiotic resistance. VA-1 could induce systemic infection in turbot, with symptoms like kidney enlargement, exhibiting virulence comparable to known Vibrio pathogens, with an LD50 around 2.36 × 106 CFU/fish. VA-1's remarkable resistance phenotype (14/22) suggested potential for genetic exchange and resistance factor acquisition in aquaculture environments. Phylogenetic analysis based on 16S rDNA sequences and whole-genome sequencing has firmly placed VA-1 within the V. alfacsensis clade, while genome-wide analysis highlights its similarity and diversity in relation to strains from across the globe. VA-1 contained numerous replicons, indicating the possibility for the spread of resistance and virulence genes. This study suggests V. alfacsensis may acquire and transfer pathogenic and resistant traits through horizontal gene transfer, a likelihood intensified by changing environmental and aquaculture conditions, highlighting the need for vigilant pathogen monitoring and new non-antibiotic treatments.

Vertical transfer of gut microbiota from dam to neonate calf in the early of life.

The aim of this study was to investigate the vertical transfer of microbiota from dams to the offspring. We studied a pair of 20 dams and its offspring. Maternal sources (colostrum, feces and vaginal secretion) and newborn fecal samples were analyzed using 16S rDNA amplicon sequencing on days 1, 3, 7, 14 and 28. Overall, newborns were maintained healthy and did not receive antimicrobial therapy. The Source Tracker analysis indicated that the newborn fecal microbiota was similar to colostrum and vaginal secretion from day 1 up to 7. However, an unknown source (probably from the environment) showed a gradual increase in its similarity with fecal samples from calves measured from day 3 to 28. The most abundant bacteria groups on meconium (day 1) and calf fecal samples on day 3 were Escherichia-Shigella and Clostridium, respectively. On day 7, the predominant genus were Bifidobacterium and Lactobacillus, while Fusobacterium was the most abundant genus on day 14, coinciding with the diarrhea peak. Faecalibacterium showed a gradual increase throughout the neonatal period. Maternal sources contribute to the neonatal microbiota, however other unknown sources (probably environment) had a strong influence on development of the gut microbiota later in the neonate period.

Rifaximin ameliorates influenza A virus infection-induced lung barrier damage by regulating gut microbiota.

Prior research has indicated that the gut-lung-axis can be influenced by the intestinal microbiota, thereby impacting lung immunity. Rifaximin is a broad-spectrum antibacterial drug that can maintain the homeostasis of intestinal microflora. In this study, we established an influenza A virus (IAV)-infected mice model with or without rifaximin supplementation to investigate whether rifaximin could ameliorate lung injury induced by IAV and explore the molecular mechanism involved. Our results showed that IAV caused significant weight loss and disrupted the structure of the lung and intestine. The analysis results of 16S rRNA and metabolomics indicated a notable reduction in the levels of probiotics Lachnoclostridium, Ruminococcaceae_UCG-013, and tryptophan metabolites in the fecal samples of mice infected with IAV. In contrast, supplementation with 50 mg/kg rifaximin reversed these changes, including promoting the repair of the lung barrier and increasing the abundance of Muribaculum, Papillibacter and tryptophan-related metabolites content in the feces. Additionally, rifaximin treatment increased ILC3 cell numbers, IL-22 level, and the expression of RORγ and STAT-3 protein in the lung. Furthermore, our findings demonstrated that the administration of rifaximin can mitigate damage to the intestinal barrier while enhancing the expression of AHR, IDO-1, and tight junction proteins in the small intestine. Overall, our results provided that rifaximin alleviated the imbalance in gut microbiota homeostasis induced by IAV infection and promoted the production of tryptophan-related metabolites. Tryptophan functions as a signal to facilitate the activation and movement of ILC3 cells from the intestine to the lung through the AHR/STAT3/IL-22 pathway, thereby aiding in the restoration of the barrier. KEY POINTS: • Rifaximin ameliorated IAV infection-caused lung barrier injury and induced ILC3 cell activation. • Rifaximin alleviated IAV-induced gut dysbiosis and recovered tryptophan metabolism. • Tryptophan mediates rifaximin-induced ILC3 cell activation via the AHR/STAT3/IL-22 pathway.

Gut and respiratory microbiota landscapes in IgA nephropathy: a cross-sectional study.

BACKGROUND: IgA nephropathy (IgAN) is intimately linked to mucosal immune responses, with nasopharyngeal and intestinal lymphoid tissues being crucial for its abnormal mucosal immunity. The specific pathogenic bacteria in these sites associated with IgAN, however, remain elusive. Our study employs 16S rRNA sequencing and machine learning (ML) approaches to identify specific pathogenic bacteria in these locations and to investigate common pathogens that may exacerbate IgAN. METHODS: In this cross-sectional analysis, we collected pharyngeal swabs and stool specimens from IgAN patients and healthy controls. We applied 16SrRNA sequencing to identify differential microbial populations. ML algorithms were then used to classify IgAN based on these microbial differences. Spearman correlation analysis was employed to link key bacteria with clinical parameters. RESULTS: We observed a reduced microbial diversity in IgAN patients compared to healthy controls. In the gut microbiota of IgAN patients, increases in Bacteroides, Escherichia-Shigella, and Parabacteroides, and decreases in Parasutterella, Dialister, Faecalibacterium, and Subdoligranulum were notable. In the respiratory microbiota, increases in Neisseria, Streptococcus, Fusobacterium, Porphyromonas, and Ralstonia, and decreases in Prevotella, Leptotrichia, and Veillonella were observed. Post-immunosuppressive therapy, Oxalobacter and Butyricoccus levels were significantly reduced in the gut, while Neisseria and Actinobacillus levels decreased in the respiratory tract. Veillonella and Fusobacterium appeared to influence IgAN through dual immune loci, with Fusobacterium abundance correlating with IgAN severity. CONCLUSIONS: This study revealing that changes in flora structure could provide important pathological insights for identifying therapeutic targets, and ML could facilitate noninvasive diagnostic methods for IgAN.

Differential growth enhancement followed by notable microbiota modulation in growing-finishing pigs by Bacillus subtilis strains ps4060, ps4100, and a 50:50 strain mixture.

A 50:50 blend of two Bacillus subtilis strains positively impacted the productivity of finishing pigs. Given this observed effect, we hypothesized that each strain has distinct effects on weight gain and their influence on gut microbiota. In a 16-week test, 160 pigs were divided into four groups: basal diet, B. subtilis ps4100, B. subtilis ps4060, and 50:50 mixture supplemented. Subsequently, we compared body weight and fecal microbiota. Among the supplements, ps4100, ps4060, and the 50:50 mix yielded respective average daily weight gains (ADG) of 3.6%, 4.6%, and 3.9% by the 6th week. The weight difference was maintained through the 16th week. At the 11th week, the difference in α-diversity among the fecal microbiota was marginal, and 17 of 229 genera showed differential abundance between the control and either of the treatment groups. A total of 12 of the 17 genera, including Lactobacillus, showed differential abundance between the ps4100 and ps4060-fed groups, and only Eubacterium consistently decreased in abundance in both the ps4100 and ps4060 groups. In comparison, microbial diversity was significantly different at the 16th week (p < 0.05), with 96 out of 229 genera exhibiting differential abundance. A total of 42 of the 96 genera exhibited similar patterns in both the ps4100 and ps4060 groups compared to the control group. Additionally, 236 of 687 microbial enzymes with differential abundance deduced from 16S rRNA reads showed similar differential abundance in both groups compared to the control group. We concluded that the overall microbial balance, rather than the dominance or significant decrease of a few specific genera, likely caused the enhanced ADG until the 11th week. Substantial changes in microbiota manifested at the 16th week did not cause dramatically increased ADG but were a consequence of weight gain and could positively affect animal physiology and health afterward.

Multi-omics study unravels gut microbiota and metabolites alteration in patients with Wilson's disease.

Hepatolenticular degeneration (HLD), also known as Wilson's disease (WD), is a rare autosomal recessive disorder regarding copper metabolism. Whether gut microbiota imbalance is involved in developing HLD remains unknown. A comprehensive 16S rRNA amplicon sequencing, metagenomic sequencing, and metabonomic analysis were undertaken in patients with WD to analyze the composition and function profiles of gut microbiota in patients with WD. The data demonstrated differences in gut microbiota and metabolic pathways between WD patients and normal individuals, significantly decreasing bacterial richness and diversity. The levels of Selenomonaceae and Megamonas in WD patients are significantly higher than those in healthy individuals. The relative abundances of Roseburia inulinivorans in patients with WD are lower than in healthy individuals. Compared with healthy people, the level of metabolites in patients with WD is abnormal. Leucylproline, 5-Phenylvaleric Acid and N-Desmethylclobazam, which have nutritional and protective effects, are significantly reduced fecal metabolites in patients with WD. D-Gluconic acid, which can chelate metal ions, may be a potential treatment for WD. The positive correlation it demonstrates with Alistipes indistinctus and Prevotella stercora indicates potential bacteria able to treat WD. These metabolites are mainly related to the biosynthesis of antibiotics, alpha-linolenic acid metabolism, one carbon pool by folate, nicotinate and nicotinamide metabolism. In conclusion, the data from this study elucidate novel mechanisms describing how abnormal gut miccrobiota contribute to the pathogenesis of WD and outlines new molecules for the treatment of WD.

High-solid digestion - A comparison of completely stirred and plug-flow reactor systems.

High-solid digestion (HSD) for biogas production is a resource-efficient and sustainable method to treat organic wastes with high total solids content and obtain renewable energy and an organic fertiliser, using a lower dilution rate than in the more common wet digestion process. This study examined the effect of reactor type on the performance of an HSD process, comparing plug-flow (PFR) type reactors developed for continuous HSD processes, and completely stirred-tank reactors (CSTRs) commonly used for wet digestion. The HSD process was operated in thermophilic conditions (52 °C), with a mixture of household waste, garden waste and agricultural residues (total solids content 27-28 %). The PFRs showed slightly better performance, with higher specific methane production and nitrogen mineralisation than the CSTRs, while the reduction of volatile solids was the same in both reactor types. Results from 16S rRNA gene sequencing showed a significant difference in the microbial population, potentially related to large differences in stirring speed between the reactor types (1 rpm in PFRs and 70-150 rpm in CSTRs, respectively). The bacterial community was dominated by the genus Defluviitoga in the PFRs and order MBA03 in the CSTRs. For the archaeal community, there was a predominance of the genus Methanoculleus in the PFRs, and of the genera Methanosarcina and Methanothermobacter in the CSTRs. Despite these shifts in microbiology, the results showed that stable digestion of substrates with high total solids content can be achieved in both reactor types, indicating flexibility in the choice of technique for HSD processes.

L. acidophilus/L. johnsonii ratio affects slow transit constipation in rats.

Slow Transit Constipation (STC) is characterized by impaired colonic motility, but its relationship with gut microbiota remains unclear. This study investigated the correlation between specific gut microbial populations and STC, focusing on the Lactobacillus acidophilus to Lactobacillus johnsonii (A/J) ratio. We used four rat groups: Control (CON), Loperamide-induced STC (LOP), antibiotic-treated (ABX), and antibiotic plus Loperamide (ABX + LOP). Fecal samples were analyzed using 16S rRNA gene sequencing, and serum metabolites were examined through LC-MS. The LOP group showed an increased A/J ratio, while ABX and ABX + LOP groups had decreased ratios. Notably, the ABX + LOP group did not develop STC symptoms. Metabolomic analysis revealed alterations in key metabolites across groups, including changes in levels of guanidinoacetate, glycine, L-glutamine, nicotine, and nicotinate D-ribonucleotide in the LOP group, and variations in L-glutamine, L-phenylalanine, L-tyrosine, histamine, D-ornithine, and lecithin in the ABX and ABX + LOP groups. Our findings suggest a correlation between the A/J ratio and STC development, offering insights into STC pathophysiology and potential microbiome-targeted therapies.

Microbial community storm dynamics signal sources of "old" stream water.

Accurate characterization of the movement of water through catchments, particularly during precipitation event response, is critical for hydrological efforts such as contaminant transport modeling or prediction of extreme flows. Abiotic hydrogeochemical tracers are commonly used to track sources and ages of surface waters but provide limited details about transit pathways or the spatial dynamics of water storage and release. Alternatively, biotic material in streams is derived from thousands of taxa originating from a variety of environments within watersheds, including groundwater, sediment, and upslope terrestrial environments, and this material can be characterized with genetic sequencing and bioinformatics. We analyzed the stable water isotopes (δ18O and δ2H) and microbiome composition (16S rRNA gene amplicon sequencing) of the Marys River of western Oregon, USA during an early season storm to describe the processes, storage, and flowpaths that shape surface water hydrology. Stable water isotopes (δ18O and δ2H) typified an event response in which stream water is composed largely of 'old' water introduced to the catchment before the storm, a common though not well understood phenomenon. In contrast, microbial biodiversity spiked during the storm, consisting of early- and late-event communities clearly distinguishable from pre-event communities. We applied concentration-discharge (cQ) analysis to individual microbial taxa and found that most Alphaproteobacteria sequences were positively correlated (i.e., were mobilized) with discharge, whereas most sequences from phyla Gammaproteobacteria and Bacteroidota were negatively correlated with discharge (i.e., were diluted). Source predictions using the prokaryote habitat preference database ProkAtlas found that freshwater-associated microbes composed a smaller fraction of the microbial community during the stream rise and a larger fraction during the recession, while soil and biofilm-associated microbes increased during the storm and remained high during recession. This suggests that the "old" water discharged during the storm was likely stored and released from, or passed through, soil- and biofilm-rich environments, demonstrating that this approach adds new, biologically derived tracer information about the hydrologic pathways active during and after this event. Overall, this study demonstrates an approach for integrating information-rich DNA into water resource investigations, incorporating tools from both hydrology and microbiology to demonstrate that microbial DNA is useful not only as an indicator of biodiversity but also functions as an innovative hydrologic tracer.

A realistic benchmark for differential abundance testing and confounder adjustment in human microbiome studies.

BACKGROUND: In microbiome disease association studies, it is a fundamental task to test which microbes differ in their abundance between groups. Yet, consensus on suitable or optimal statistical methods for differential abundance testing is lacking, and it remains unexplored how these cope with confounding. Previous differential abundance benchmarks relying on simulated datasets did not quantitatively evaluate the similarity to real data, which undermines their recommendations. RESULTS: Our simulation framework implants calibrated signals into real taxonomic profiles, including signals mimicking confounders. Using several whole meta-genome and 16S rRNA gene amplicon datasets, we validate that our simulated data resembles real data from disease association studies much more than in previous benchmarks. With extensively parametrized simulations, we benchmark the performance of nineteen differential abundance methods and further evaluate the best ones on confounded simulations. Only classic statistical methods (linear models, the Wilcoxon test, t-test), limma, and fastANCOM properly control false discoveries at relatively high sensitivity. When additionally considering confounders, these issues are exacerbated, but we find that adjusted differential abundance testing can effectively mitigate them. In a large cardiometabolic disease dataset, we showcase that failure to account for covariates such as medication causes spurious association in real-world applications. CONCLUSIONS: Tight error control is critical for microbiome association studies. The unsatisfactory performance of many differential abundance methods and the persistent danger of unchecked confounding suggest these contribute to a lack of reproducibility among such studies. We have open-sourced our simulation and benchmarking software to foster a much-needed consolidation of statistical methodology for microbiome research.