Gut microbiota and metabolomic profile changes play critical roles in tacrolimus-induced diabetes in rats.

Aims: Hyperglycemia is one of the adverse effects of tacrolimus (TAC), but the underlying mechanism is not fully identified. We used multi-omics analysis to evaluate the changes in the gut microbiota and metabolic profile of rats with TAC-induced diabetes. Methods: To establish a diabetic animal model, Sprague Dawley rats were divided randomly into two groups. Those in the TAC group received intraperitoneal injections of TAC (3 mg/kg) for 8 weeks, and those in the CON group served as the control. 16S rRNA sequencing was used to analyze fecal microbiota. The metabolites of the two groups were detected and analyzed by nontargeted and targeted metabolomics, including amino acids (AAs), bile acids (BAs), and short-chain fatty acids (SCFAs). Results: The rats treated with TAC exhibited hyperglycemia as well as changes in the gut microbiota and metabolites. Specifically, their gut microbiota had significantly higher abundances of Escherichia-Shigella, Enterococcus, and Allobaculum, and significantly lower abundances of Ruminococcus, Akkermansia, and Roseburia. In addition, they had significantly reduced serum levels of AAs including asparagine, aspartic acid, glutamic acid, and methionine. With respect to BAs, they had significantly higher serum levels of taurocholic acid (TCA), and glycochenodeoxycholic acid (GCDCA), but significantly lower levels of taurodeoxycholic acid (TDCA) and tauroursodeoxycholic acid (TUDCA). There were no differences in the levels of SCFAs between the two groups. Correlations existed among glucose metabolism indexes (fasting blood glucose and fasting insulin), gut microbiota (Ruminococcus and Akkermansia), and metabolites (glutamic acid, hydroxyproline, GCDCA, TDCA, and TUDCA). Conclusions: Both AAs and BAs may play crucial roles as signaling molecules in the regulation of TAC-induced diabetes.

Intratumoral and fecal microbiota reveals microbial markers associated with gastric carcinogenesis.

Background: The relationship between dysbiosis of the gastrointestinal microbiota and gastric cancer (GC) has been extensively studied. However, microbiota alterations in GC patients vary widely across studies, and reproducible diagnostic biomarkers for early GC are still lacking in multiple populations. Thus, this study aimed to characterize the gastrointestinal microbial communities involved in gastric carcinogenesis through a meta-analysis of multiple published and open datasets. Methods: We analyzed 16S rRNA sequencing data from 1,642 gastric biopsy samples and 394 stool samples across 11 independent studies. VSEARCH, QIIME and R packages such as vegan, phyloseq, cooccur, and random forest were used for data processing and analysis. PICRUSt software was employed to predict functions. Results: The α-diversity results indicated significant differences in the intratumoral microbiota of cancer patients compared to non-cancer patients, while no significant differences were observed in the fecal microbiota. Network analysis showed that the positive correlation with GC-enriched bacteria increased, and the positive correlation with GC-depleted bacteria decreased compared to healthy individuals. Functional analyses indicated that pathways related to carbohydrate metabolism were significantly enriched in GC, while biosynthesis of unsaturated fatty acids was diminished. Additionally, we investigated non-Helicobacter pylori (HP) commensals, which are crucial in both HP-negative and HP-positive GC. Random forest models, constructed using specific taxa associated with GC identified from the LEfSe analysis, revealed that the combination of Lactobacillus and Streptococcus included alone could effectively discriminate between GC patients and healthy individuals in fecal samples (area under the curve (AUC) = 0.7949). This finding was also validated in an independent cohort (AUC = 0.7712). Conclusions: This study examined the intratumoral and fecal microbiota of GC patients from a dual microecological perspective and identified Lactobacillus, Streptococcus, Roseburia, Faecalibacterium and Phascolarctobacterium as intratumoral and intestinal-specific co-differential bacteria. Furthermore, it confirmed the validity of the combination of Lactobacillus and Streptococcus as GC-specific microbial markers across multiple populations, which may aid in the early non-invasive diagnosis of GC.

Sex-specific post-inflammatory dysbiosis mediates chronic visceral pain in colitis.

BACKGROUND: Despite achieving endoscopic remission, over 20% of inflammatory bowel disease (IBD) patients experience chronic abdominal pain. Visceral pain and the microbiome exhibit sex-dependent interactions, while visceral pain in IBD shows a sex bias. Our aim was to evaluate whether post-inflammatory microbial perturbations contribute to visceral hypersensitivity in a sex-dependent manner. METHODS: Males, cycling females, ovariectomized, and sham-operated females were given dextran sodium sulfate to induce colitis and allowed to recover. Germ-free recipients received sex-appropriate and cross-sex fecal microbial transplants (FMT) from post-inflammatory donor mice. Visceral sensitivity was assessed by recording visceromotor responses to colorectal distention. The composition of the microbiota was evaluated via 16S rRNA gene V4 amplicon sequencing, while the metabolome was assessed using targeted (short chain fatty acids - SCFA) and semi-targeted mass spectrometry. RESULTS: Post-inflammatory cycling females developed visceral hyperalgesia when compared to males. This effect was reversed by ovariectomy. Both post-inflammatory males and females exhibited increased SCFA-producing species, but only males had elevated fecal SCFA content. FMT from post-inflammatory females transferred visceral hyperalgesia to both males and females, while FMT from post-inflammatory males could only transfer visceral hyperalgesia to males. CONCLUSIONS: Female sex, hormonal status as well as the gut microbiota play a role in pain modulation. Our data highlight the importance of considering biological sex in the evaluation of visceral pain.

DNA methylation analysis of the SDC2, SEPT9 and VIM genes in fecal DNA for colorectal cancer diagnosis.

BACKGROUND: Colorectal cancer is one of the most common cancers worldwide. DNA methylation sites may serve as a new gene signature for colorectal cancer diagnosis. The search for representative DNA methylation sites is urgently needed. This study aimed to systematically identify a methylation gene panel for colorectal cancer diagnosis via tissue and fecal samples. METHODS: A total of 181 fecal and 50 tumor tissue samples were collected. They were obtained from 83 colorectal cancer patients and 98 healthy subjects. These samples were evaluated for DNA methylation of 9 target genes via quantitative bisulfite next-generation sequencing. We employed the rank-sum test to screen the colorectal cancer-specific methylation sites in the tissue and fecal cohorts. A data model was subsequently constructed and validated via the dedicated validation dataset. RESULTS: Compared with the fecal and negative control samples, the colorectal cancer tissue samples presented significantly higher methylation rates for all the selected gene sites. The methylation rates of the tissue and preoperative fecal samples showed the same high and low rates at the same sites. After screening, a panel of 29 loci in the SDC2, SEPT9, and VIM genes proved to be reliable biomarkers for colorectal cancer diagnosis in fecal samples. Logistic regression models were then constructed and validated using this panel. The sensitivity of the model was 91.43% (95% CI = [89.69, 93.17]), the specificity was 100% (95% CI = [100,100]), and the AUC value is 99.31% (95% CI = [99,99.62]). The diagnostic accuracy of the model for stage I and stage II colorectal cancer was 100% (11/11) and 91.3% (21/23), respectively. Overall, this study confirms that the gene locus panel and the model can be used to diagnose colorectal cancer effectively through feces. CONCLUSIONS: Our study identified a set of key methylation sites for colorectal cancer diagnosis from fecal samples, highlighting the importance of using tissue and fecal samples to accurately assess DNA methylation levels to screen for methylation sites, and developing an effective diagnostic model for colorectal cancer.

Frequency and genotyping of group A rotavirus among Egyptian children with acute gastroenteritis: a hospital-based cross-sectional study.

BACKGROUND: This hospital-based cross-sectional study aims to investigate the epidemiologic and clinical characteristics of rotavirus group A (RVA) infection among children with acute gastroenteritis and to detect the most common G and P genotypes in Egypt. METHODS: A total of 92 stool samples were collected from children under five who were diagnosed with acute gastroenteritis. RVA in stool samples was identified using ELISA and nested RT-PCR. Common G and P genotypes were identified utilizing multiplex nested RT-PCR assays. RESULTS: RVA was detected at a rate of 24% (22 /92) using ELISA and 26.1% (24 /92) using VP6 nested RT-PCR. The ELISA test demonstrated diagnostic sensitivity, specificity, and accuracy of 91.7%, 100%, and 97.8%, respectively. G3 was the most prevalent G type (37.5%), followed by G1 (12.5%), whereas the most commonly detected P type were P[8] (41.7%) and P[6] (8.2%). RVA-positive samples were significantly associated with younger aged children (p = 0.026), and bottle-fed (p = 0.033) children. In addition, RVA-positive samples were more common during cooler seasons (p = 0.0001). Children with rotaviral gastroenteritis had significantly more frequent episodes of diarrhea (10.87 ± 3.63 times/day) and vomiting (8.79 ± 3.57 times/day) per day (p = 0.013 and p = 0.011, respectively). Moreover, they had a more severe Vesikari clinical score (p = 0.049). CONCLUSION: RVA is a prevalent cause of acute gastroenteritis among Egyptian children in our locality. The discovery of various RVA genotypes in the local population, as well as the identification of common G and P untypeable strains, highlights the significance of implementing the rotavirus vaccine in Egyptian national immunization programs accompanied by continuous monitoring of strains.

Stratification of human gut microbiomes by succinotype is associated with inflammatory bowel disease status.

BACKGROUND: The human gut microbiome produces and consumes a variety of compounds that interact with the host and impact health. Succinate is of particular interest as it intersects with both host and microbiome metabolism. However, which gut bacteria are most responsible for the consumption of intestinal succinate is poorly understood. RESULTS: We build upon an enrichment-based whole fecal sample culturing approach and identify two main bacterial taxa that are responsible for succinate consumption in the human intestinal microbiome, Phascolarctobacterium and Dialister. These two taxa have the hallmark of a functional guild and are strongly mutual exclusive across 21,459 fecal samples in 94 cohorts and can thus be used to assign a robust "succinotype" to an individual. We show that they differ with respect to their rate of succinate consumption in vitro and that this is associated with higher concentrations of fecal succinate. Finally, individuals suffering from inflammatory bowel disease (IBD) are more likely to have the Dialister succinotype compared to healthy subjects. CONCLUSIONS: We identified that only two bacterial genera are the key succinate consumers in human gut microbiome, despite the fact that many more intestinal bacteria encode for the succinate pathway. This highlights the importance of phenotypic assays in functionally profiling intestinal microbiota. A stratification based on "succinotype" is to our knowledge the first function-based classification of human intestinal microbiota. The association of succinotype with IBD thus builds a bridge between microbiome function and IBD pathophysiology related to succinate homeostasis. Video Abstract.

Dual-targeted treatment for inflammatory bowel disease: Whether fecal microbiota transplantation can be an important part of it.

Inflammatory bowel disease (IBD) is a chronic gastrointestinal inflammatory disease. With the emergence of biologics and other therapeutic methods, two biologics or one biologic combined with a novel small-molecule drug has been proposed in recent years to treat IBD. Although treatment strategies for IBD are being optimized, their efficacy and risks still warrant further consideration. This editorial explores the current risks associated with dual-targeted treatment for IBD and the great potential that fecal microbiota transplantation (FMT) may have for use in combination therapy for IBD. We are focused on addressing refractory IBD or biologically resistant IBD based on currently available dual-targeted treatment by incorporating FMT as part of this dual-targeted treatment. In this new therapy regimen, FMT represents a promising combination therapy.

Early diagnostic strategies for colorectal cancer.

At present, cancer is still an important factor threatening human health. Colorectal cancer (CRC) is one of the top three most common cancers worldwide and one of the deadliest malignancies in humans. The latest data showed that CRC incidence and mortality rank third and second, respectively, among global malignancies. Early and accurate diagnosis is crucial to reduce the morbidity, mortality and improve survival of patients with CRC, but the current early diagnostic methods have limitations. The effectiveness and compliance of diagnostic methods have a certain impact on whether people choose screening. In this editorial, we explore strategies for the early diagnosis of CRC, including stool-based, blood-based, direct visualization, and imaging examinations.

The effects of AG1® supplementation on the gut microbiome of healthy adults: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: This study aimed to examine the effect of a commercially available multi-ingredient powder (AG1Ⓡ) on the gut microbiome and assess the impact of AG1Ⓡ on GI tolerability and other clinical safety markers in healthy men and women. METHODS: Using a double-blind, randomized, two-arm, placebo-controlled, parallel design, we examined a 4-week daily supplementation regimen of AG1Ⓡ vs. placebo (PL). Fifteen men and 15 women provided stool samples for microbiome analysis, questionnaires for digestive quality of life (DQLQ), and completed visual analog scales (VAS) and Bristol stool charts to assess stool consistency and bowel frequency before and after the 4-week intervention. Participant's blood work (CBC, CMP, and lipid panel) was also assessed before and after the 4-week intervention. Alpha diversity was determined by Shannon and Chao1 index scores and evaluated by a two-way ANOVA, beta diversity in taxonomic abundances and functional pathways was visualized using partial least squares-discriminant analyses and statistically evaluated by PERMANOVA. To identify key biomarkers, specific feature differences in taxonomic relative abundance and normalized functional pathway counts were analyzed by linear discriminant analysis (LDA) effect size (LEfSe). Questionnaires, clinical safety markers, and hemodynamics were evaluated by mixed factorial ANOVAs with repeated measures. This study was registered on clinicaltrials.gov (NCT06181214). RESULTS: AG1Ⓡ supplementation enriched two probiotic taxa (Lactobacillus acidophilus and Bifidobacterium bifidum) that likely stem from the probiotics species that exist in the product, as well as L. lactis CH_LC01 and Acetatifactor sp900066565 ASM1486575v1 while reducing Clostridium sp000435835. Regarding community function, AG1Ⓡ showed an enrichment of two functional pathways while diminishing none. Alternatively, the PL enriched six, but diminished five functional pathways. Neither treatment negatively impacted the digestive quality of life via DQLQ, bowel frequency via VAS, or stool consistency via VAS and Bristol. However, there may have been a greater improvement in the DQLQ score (+62.5%, p = 0.058, d = 0.73) after four weeks of AG1Ⓡ supplementation compared to a reduction (-50%) in PL. Furthermore, AG1Ⓡ did not significantly alter clinical safety markers following supplementation providing evidence for its safety profile. CONCLUSIONS: AG1Ⓡ can be consumed safely by healthy adults over four weeks with a potential beneficial impact in their digestive symptom quality of life.

Intake and digestibility of four rations with different fiber levels in alpacas (Vicugna pacos).

The aim of this study was to evaluated the impact of different fibre levels in alpaca diet on voluntary feed intake and apparent digestibility, and to estimate the digestibility of organic matter (OMD) from the content of crude protein (CP) in feces. The study was carried out with twelve alpacas (36.7 ± 6.4 kg body weight- BW), which were offered 4 treatments with different neutral detergent fiber content (NDF. T1: 40.3%; T2: 62%; T3: 68%; T4: 72%) under a switch back design. Absolute daily dry matter intake (DMI) was higher for T1 (678 g/d) than T4 (312 g/d) (p ≥ 005). NDF intake was similar between treatments when related to BW or MW (on average 1% BW and 22 g/kg MW. p ≥ 0.05). Water intake (L/kg DMI) was higher in T1 compared to the other treatments, with values ranging from 2.9 L/kg DMI(T1) to 2.8 L/kg DMI(T4), respectively (p ≤ 0.05). Digestibility of dry matter, organic matter and CP was higher in T1 than in the other treatments, with average values ranging from 72% for T1 to 32% for T4 (p ≤ 0.05). NDF digestibility was similar among treatments (p ≥ 0.05). The regression equation generated to predict OMD (y) was as follows: y = 0.360 + 0.08294*fecal CP (g/kg OM). Further studies will indicate whether faecal nitrogen can be used to estimate digestibility and hence diet quality in South American camelids.

Molecular characterization of common zoonotic protozoan parasites and bacteria causing diarrhea in dairy calves in Ningxia Hui Autonomous Region, China.

Diarrhea caused by zoonotic pathogens is one of the most common diseases in dairy calves, threatening the health of young animals. Humans are also at risk, in particular children. To explore the pathogens causing diarrhea in dairy calves, the present study applied PCR-based sequencing tools to investigate the occurrence and molecular characteristics of three parasites (Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi) and three bacterial pathogens (Escherichia coli, Clostridium perfringens, and Salmonella spp.) in 343 fecal samples of diarrheic dairy calves from five farms in Lingwu County, Ningxia Hui Autonomous Region, China. The total positive rate of these pathogens in diarrheic dairy calves was 91.0% (312/343; 95% CI, 87.9-94.0), with C. perfringens (61.5%, 211/343; 95% CI, 56.3-66.7) being the dominant one. Co-infection with two to five pathogens was found in 67.3% (231/343; 95% CI, 62.4-72.3) of investigated samples. There were significant differences (p < 0.05) in the positive rates of Cryptosporidium spp. and diarrheagenic E. coli among farms, age groups, and seasons. Two Cryptosporidium species (C. parvum and C. bovis) and five gp60 subtypes of C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1, and a novel IIdA13G1) were identified. Two assemblages (assemblage E and zoonotic assemblage A) of G. duodenalis and six ITS genotypes of E. bieneusi (J, Henan-IV, EbpC, I, EbpA, and ESH-01) were observed. Four virulence genes (eaeA, stx1, stx2, and st) of diarrheagenic E. coli and one toxin type (type A) of C. perfringens were detected. Our study enriches our knowledge on the characteristics and zoonotic potential of diarrhea-related pathogens in dairy calves.

Title: Caractérisation moléculaire des protozoaires parasites zoonotiques courants et des bactéries responsables de diarrhée chez les veaux laitiers dans la région autonome Hui du Ningxia, en Chine. Abstract: La diarrhée causée par des agents pathogènes zoonotiques est l'une des maladies les plus courantes chez les veaux laitiers, menaçant la santé des jeunes animaux. Ceci est également un risque pour la santé humaine, en particulier les enfants. Pour explorer les agents pathogènes responsables de la diarrhée chez les veaux laitiers, cette étude a utilisé des outils de séquençage basés sur la PCR pour étudier l'occurrence et les caractères moléculaires de trois parasites (Cryptosporidium spp., Giardia duodenalis et Enterocytozoon bieneusi) et de trois agents pathogènes bactériens (Escherichia coli, Clostridium perfringens et Salmonella spp.) dans 343 échantillons fécaux de veaux laitiers diarrhéiques provenant de cinq fermes du comté de Lingwu, région autonome Hui du Ningxia, en Chine. Le taux total positif de ces pathogènes chez les veaux laitiers diarrhéiques était de 91,0 % (312/343; IC à 95 %, 87,9­94,0), et C. perfringens (61,5 %, 211/343; IC à 95 %, 56,3­66,7) était le plus répandu. Une co-infection avec deux à cinq pathogènes a été trouvée dans 67,3 % (231/343; IC à 95 %, 62,4­72,3) des échantillons étudiés. Il y avait des différences significatives (p < 0,05) dans les taux positifs de Cryptosporidium spp. et d'E. coli diarrhéogènes entre les fermes, les groupes d'âge et les saisons. Deux espèces de Cryptosporidium (C. parvum et C. bovis) et cinq sous-types de gp60 de C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1 et un nouveau, IIdA13G1) ont été identifiés. Deux assemblages (assemblage E et assemblage zoonotique A) de G. duodenalis et six génotypes ITS d'E. bieneusi (J, Henan-IV, EbpC, I, EbpA et ESH-01) ont été observés. Quatre gènes de virulence (eaeA, stx1, stx2 et st) d'E. coli diarrhéogènes et un type de toxine (type A) de C. perfringens ont été détectés. Notre étude enrichit les connaissances sur les caractères et le potentiel zoonotique des agents pathogènes liés à la diarrhée chez les veaux laitiers.

Clinical Significance of Faecal Calprotectin in Differentiating Inflammatory Bowel Disease from Irritable Bowel Syndrome.

Inflammatory bowel disease (IBD) is a chronic idiopathic inflammatory disorder of the gastrointestinal tract with relapsing and remitting course. Recurrent abdominal pain and discomfort in association with abnormal defecation in the absence of structural abnormality of the gut is the key feature of irritable bowel syndrome (IBS). Faecal biomarker may be used a precise tool in the differentiation of IBD and IBS. The aim of this study was to measure faecal calprotectin (FC) in patients with IBD and IBS and compare between them. This was a cross-sectional study done in the department of Gastroenterology, BSMMU, Bangladesh from May 2017 to August 2018. IBD patients were diagnosed on the basis of compatible history, clinical examination, laboratory, radiological and endoscopic findings. IBS patients were selected by using the Rome IV criteria. Quantitative faecal calprotectin ELISA (BUHLMANN Quantum Blue) test was done and compared between IBD and IBS patients. In this study, ninety (90) patients were enrolled, 45 patients with IBD and 45 patients with IBS. Mean age of the IBD patients was 32.24±9.76 years and IBS patients was 33.80±9.70 years. There were 28(62.2%) male and 17(37.8%) female patients with IBD and 30(66.7%) male and 15(33.3%) female patients with IBS. We found faecal calprotectin (FC) level was 445.68±237.35µg/gm in IBD patients and 39.16±17.31µg/gm in IBS patients. There was a significant difference of faecal calprotectin level between IBD and IBS patients (p-value <0.001). The sensitivity and specificity of faecal calprotectin to differentiate IBD from IBS was 91.1% and 86.7% respectively. The test accuracy was 88.9%. Area under ROC was 0.959 (95% CI, 0.909 to 1.0). This study showed that faecal calprotectin appears to be clinically useful, non-invasive, rapid and reliable marker to differentiate IBD from IBS.

Performance, ruminal and fecal microbiome of lambs fed diets supplemented with probiotics.

The present study aimed to investigate the impact of adding two doses of a commercial probiotic on productive performance, ruminal and fecal microbiome in growing lambs. Forty-two Texel or Ile de France crossbred lambs aged 86.9 ± 8.0 days (body weight: 27.4 ± 3.7 kg) were distributed into three groups: basal diet without probiotic supplementation (CG); basal diet + 1 g/animal/day of probiotic (GP1) and basal diet + 5 g/animal/day of probiotic (GP5). The experimental period was 84 days. The weight was evaluated weekly and dry matter intake (DMI) and leftovers were measured daily. At the end of the experiment, lambs were slaughtered. Feces and rumen fluid were collected for microbiome analysis and rumen fragments for histological evaluation. The use of probiotics did not affect weight gain, but GP1 showed a higher silage and DMI intake than CG (p < 0.001). The CG had a greater thickness of keratinized epithelium and stratum corneum (< 0.001) than GP1 and GP5, and greater total papilla width (p = 0.039) than GP1. There was no difference in the general abundance in the rumen and fecal microbiomes. GP5 had a higher proportion of Azoarcus and Dialister taxa in the rumen fluid (p = 0.012 and p = 0.017, respectively) and higher proportion of Treponema and Fibrobacter taxa in the fecal microbiome (p = 0.015 and p = 0.026, respectively), whereas CG had a higher proportion of Anaeroplasma than the other groups (p = 0.032). These results demonstrated the benefits of probiotics for ruminal epithelium protection and microbial diversity. However, there was no effect on performance parameters.

Enhancing diagnostic accuracy: Direct immunofluorescence assay as the gold standard for detecting Giardia duodenalis and Cryptosporidium spp. in canine and feline fecal samples.

The enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. are common cause of diarrhea in pet dogs and cats, affecting primarily young animals. This comparative study evaluates the diagnostic performance of conventional and molecular methods for the detection of G. duodenalis and Cryptosporidium spp. infection in dogs and cats.The compared diagnostic assays included merthiolate-iodine-formalin (MIF) method, lateral flow immunochromatography rapid test (ICT) and real-time PCR; using direct immunofluorescence assay (DFA) as golden standard. The study included the analysis of 328 fecal samples from different dog (n = 225) and cat (n = 103) populations.According to DFA, the overall prevalence of G. duodenalis was 24.4% (80/328, 95% CI: 19.8-29.4), varying from 11.6% (12/103, 95% CI: 6.2-19.5) in cats to 30.2% (68/225, 95% CI: 24.3-36.7) in dogs. The overall prevalence of Cryptosporidium spp. was 4.0% (13/328, 95% CI: 2.1-6.7), varying from 2.9% (3/103, 95% CI: 0.6-8.3) in cats to 4.4% (10/225, 95% CI: 2.1-8.0) in dogs. MIF was only used for the detection of G. duodenalis, which was identified by this method in 22.7% of dogs and 7.8% of cats, respectively. DFA was the most sensitive technique for detecting G. duodenalis in samples from dogs and cats (p-value: < 0.001), followed by real-time PCR. Identification of Cryptosporidium infections was most effectively accomplished by the combination of DFA and PCR technique (p-value: < 0.001). In addition, epidemiological (sex, age, origin) and clinical (fecal consistency) variables were collected to assess their potential associations with an increased likelihood of infection by G. duodenalis and/or Cryptosporidium spp. Breeder dogs were more likely to harbor G. duodenalis infection (p-value: 0.004), whereas female cats were significantly more infected with Cryptosporidium (p-value: 0.003).In conclusion, DFA (alone or in combination with PCR) has been identified as the most accurate and cost-effective method for detecting G. duodenalis and Cryptosporidium spp. in fecal samples from pet dogs and cats. This highlights their importance in both veterinary and clinical settings for enabling prompt treatment and preventing potential transmission to humans.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Delivery mode and maternal gestational diabetes are important factors in shaping the neonatal initial gut microbiota.

Background: The infant gut microbiome's establishment is pivotal for health and immune development. Understanding it unveils insights into growth, development, and maternal microbial interactions. Research often emphasizes gut bacteria, neglecting the phageome. Methods: To investigate the influence of geographic or maternal factors (mode of delivery, mode of breastfeeding, gestational diabetes mellitus) on the gut microbiota and phages of newborns, we collected fecal samples from 34 pairs of mothers and their infants within 24 hours of delivery from three regions (9 pairs from Enshi, 7 pairs from Hohhot, and 18 pairs from Hulunbuir) using sterile containers. Gut microbiota analysis by Shotgun sequencing was subsequently performed. Results: Our results showed that geographic location affects maternal gut microbiology (P < 0.05), while the effect on infant gut microbiology was not significant (P = 0.184). Among the maternal factors, mode of delivery had a significant (P < 0.05) effect on the newborn. Specific bacteria (e.g., Bacteroides, Escherichia spp., Phocaeicola vulgatus, Escherichia coli, Staphylococcus hominis, Veillonella spp.), predicted active metabolites, and bacteriophage vOTUs varied with delivery mode. Phocaeicola vulgatus significantly correlated with some metabolites and bacteriophages in the early infant gut (P < 0.05). In the GD group, a strong negative correlation of phage diversity between mother and infants was observed (R = -0.58, P=0.04). Conclusion: In conclusion, neonatal early gut microbiome (including bacteria and bacteriophages) colonization is profoundly affected by the mode of delivery, and maternal gestational diabetes mellitus. The key bacteria may interact with bacteriophages to influence the levels of specific metabolites. Our study provides new evidence for the study of the infant microbiome, fills a gap in the analysis of the infant gut microbiota regarding the virome, and emphasizes the importance of maternal health for the infant initial gut virome.

Effect of oral metformin on gut microbiota characteristics and metabolite fractions in normal-weight type 2 diabetic mellitus patients.

Background and aims: To analyze the effect of oral metformin on changes in gut microbiota characteristics and metabolite composition in normal weight type 2 diabetic patients. Methods: T2DM patients in the cross-sectional study were given metformin for 12 weeks. Patients with unmedicated T2DM were used as a control group to observe the metrics of T2DM patients treated with metformin regimen. 16S rDNA high-throughput gene sequencing of fecal gut microbiota of the study subjects was performed by llumina NovaSeq6000 platform. Targeted macro-metabolomics was performed on 14 cases of each of the gut microbiota metabolites of the study subjects using UPLC-MS/MS technology. Correlations between the characteristics of the gut microbiota and its metabolites, basic human parameters, glycolipid metabolism indicators, and inflammatory factors were analyzed using spearman analysis. Results: Glycolipid metabolism indexes and inflammatory factors were higher in normal-weight T2DM patients than in the healthy population (P<0.05), but body weight, BMI, waist circumference, and inflammatory factor concentrations were lower in normal-weight T2DM patients than in obese T2DM patients (P<0.05). Treatment with metformin in T2DM patients improved glycolipid metabolism, but the recovery of glycolipid metabolism was more pronounced in obese T2DM patients. None of the differences in α-diversity indexes were statistically significant (P>0.05), and the differences in ß-diversity were statistically significant (P <0.05). Community diversity and species richness recovered after metformin intervention compared to before, and were closer to the healthy population. We found that Anaerostipes/Xylose/Ribulose/Xylulose may play an important role in the treatment of normal-weight T2DM with metformin by improving glycemic lipids and reducing inflammation. And Metformin may play a role in obese T2DM through Romboutsia, medium-chain fatty acids (octanoic acid, decanoic acid, and dodecanoic acid). Conclusion: Gut microbial dysbiosis and metabolic disorders were closely related to glucose-lipid metabolism and systemic inflammatory response in normal-weight T2DM patients. Metformin treatment improved glucose metabolism levels, systemic inflammation levels in T2DM patients, closer to the state of healthy population. This effect may be mediated by influencing the gut microbiota and microbial host co-metabolites, mainly associated with Anaerostipes and xylose/Ribulose/Xylulose. Metformin may exert its effects through different pathways in normal-weight versus obese T2DM patients.

Identification of insect body fragments found in fecal samples of Sapajus nigritus (Primates: Cebidae) from five Conservation Units in Rio de Janeiro state, Brazil.

Sapajus nigritus (Primates: Cebidae) is endemic of the Atlantic Forest, occurring from the Southeastern and Southern regions of Brazil to Northeastern Argentina. This species plays a role in two ecosystem services: seed dispersal through endozoochory and insect population control in agricultural plantations and forest environments. Fruits and invertebrates represent approximately 90% of their diet, and there is a large number of insects in the diet of S. nigritus in urban and conserved areas. However, it is known that insect diversity decreases in anthropized environments. Our objective was to identify the insects present in the fecal samples of S. nigritus from five Conservation Units in Rio de Janeiro state, Brazil. We aim to estimate the percentage of each taxon of insects found in feces either, hypothesizing that there are a greater variety of insect species in the diet of S. nigritus that inhabit preserved forested areas. A fecal screening was conducted using a light microscopy and the insects were identified based on their external morphology. Insect fragments were found in eight out of ten fecal samples of S. nigritus, revealing that they belonged to insects from five orders: Hymenoptera, Hemiptera, Orthoptera, Coleoptera and Blattodea, suggesting a good conservation status of the sampling areas.

Low-cost biosecurity measures are associated with reduced detection of non-Typhoidal Salmonella in Nigerian poultry while inappropriate antibiotic use is widespread.

Large-scale poultry production in low- and middle-income countries may be a source of adulterated products (e.g., Salmonella contamination, antibiotic residues) that can be disseminated over wide areas. We employed a cross-sectional survey of 199 randomly selected poultry farms in Lagos State, Nigeria, to estimate the prevalence of non-typhoidal Salmonella (NTS), and biosecurity and antibiotic use practices. Pooled fecal samples were collected from laying chickens and from poultry handlers. Selective culture, biochemical assays, and PCR (invA) were used to isolate and confirm NTS isolates. NTS was detected at 14% of farms (28/199) and from 10% of farm workers (6/60). Multivariate logistic regression analysis indicated that antiseptic foot dips reduced the odds ratio (OR) for detecting NTS in chicken feces [OR: 0.55; 95% confidence interval (CI) 0.07-0.58]. Most farms (94.5%, 188/199) used antibiotics for treatment and prophylaxis, but no farms (0/199) exercised withdrawal before sale of products. Most farms (86.4%, 172/199) reported using antibiotic cocktails that included medically important colistin, ciprofloxacin, chloramphenicol, and gentamicin. Egg production in Lagos State relies heavily on antibiotics and antibiotic residues are likely passed to consumers through poultry products, but there is evidence that low-cost biosecurity controls are effective for limiting the presence of NTS on farms.

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.