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The composition of the gut microbiota is associated with various disease states, most notably inflammatory bowel disease, obesity and malnutrition. This underlines that analysis of intestinal microbiota is potentially an interesting target for clinical diagnostics. Currently, the most commonly used sample types are feces and mucosal biopsy specimens. Because sampling method, storage and processing of samples impact microbiota analysis, each sample type has its own limitations. An ideal sample type for use in routine diagnostics should be easy to obtain in a standardized fashion without perturbation of the microbiota. Rectal swabs may satisfy these criteria, but little is known about microbiota analysis on these sample types. In this study we investigated the characteristics and applicability of rectal swabs for gut microbiota profiling in a clinical routine setting in patients presenting with various gastro-intestinal disorders. We found that rectal swabs appeared to be a convenient means of sampling the human gut microbiota. Swabs can be performed on demand, whenever a patient presents; swab-derived microbiota profiles are reproducible, whether they are gathered at home by patients or by medical professionals in an outpatient setting and may be ideally suited for clinical diagnostics and large-scale studies.

Disruption of the intestinal microbiota is considered an etiological factor in pediatric functional constipation. Scientifically based selection of potential beneficial probiotic strains in functional constipation therapy is not feasible due to insufficient knowledge of microbiota composition in affected subjects. The aim of this study was to describe microbial composition and diversity in children with functional constipation, compared to healthy controls. Fecal samples from 76 children diagnosed with functional constipation according to the Rome III criteria (median age 8.0 years; range 4.2-17.8) were analyzed by IS-pro, a PCR-based microbiota profiling method. Outcome was compared with intestinal microbiota profiles of 61 healthy children (median 8.6 years; range 4.1-17.9). Microbiota dissimilarity was depicted by principal coordinate analysis (PCoA), diversity was calculated by Shannon diversity index. To determine the most discriminative species, cross validated logistic ridge regression was performed. Applying total microbiota profiles (all phyla together) or per phylum analysis, no disease-specific separation was observed by PCoA and by calculation of diversity indices. By ridge regression, however, functional constipation and controls could be discriminated with 82% accuracy. Most discriminative species were Bacteroides fragilis, Bacteroides ovatus, Bifidobacterium longum, Parabacteroides species (increased in functional constipation) and Alistipes finegoldii (decreased in functional constipation). None of the commonly used unsupervised statistical methods allowed for microbiota-based discrimination of children with functional constipation and controls. By ridge regression, however, both groups could be discriminated with 82% accuracy. Optimization of microbiota-based interventions in constipated children warrants further characterization of microbial signatures linked to clinical subgroups of functional constipation.

The neonatal period, during which the initial gut microbiota is acquired, is a critical phase. The healthy development of the infant's microbiome can be interrupted by external perturbations, like antibiotics, which are associated with profound effects on the gut microbiome and various disorders later in life. The aim of this study was to investigate the development of intestinal microbiota and the effect of antibiotic exposure during the first three months of life in term infants. Fecal samples were collected from healthy infants and infants who received antibiotics in the first week of life at one week, one month, and three months after birth. Microbial composition was analyzed using IS-pro and compared between antibiotics-treated and untreated infants. In total, 98 infants, divided into four groups based on feeding type and delivery mode, were analyzed. At one week of age, samples clustered into two distinct groups, which were termed \"settler types\", based on their Bacteroidetes abundance. Caesarean-born infants belonged to the low-Bacteroidetes settler type, but vaginally-born infants were divided between the two groups. The antibiotics effect was assessed within a subgroup of 45 infants, vaginally-born and exclusively breastfed, to minimize the effect of other confounders. Antibiotics administration resulted in lower Bacteroidetes diversity and/or a delay in Bacteroidetes colonization, which persisted for three months, and in a differential development of the microbiota. Antibiotics resulted in pronounced effects on the Bacteroidetes composition and dynamics. Finally, we hypothesize that stratification of children's cohorts based on settler types may reveal group effects that might otherwise be masked.

The etiology of diverticulosis is still poorly understood. However, in patients with diverticulitis, markers of mucosal inflammation and microbiota alterations have been found. The aim of this study was to evaluate potential differences of the gut microbiota composition and mucosal immunity between patients with asymptomatic diverticulosis and controls. We performed a prospective study on patients who underwent routine colonoscopy for causes not related to diverticular disease or inflammatory bowel disease. Participants were grouped based on the presence or absence of diverticula. Mucosal biopsies were obtained from the sigmoid and transverse colon. Microbiota composition was analyzed with IS-pro, a 16S-23S based bacterial profiling technique. To predict if patients belonged to the asymptomatic diverticulosis or control group a partial least squares discriminant analysis (PLS-DA) regression model was used. Inflammation was assessed by neutrophil and lymphocyte counts within the taken biopsies. Forty-three patients were enrolled. Intestinal microbiota profiles were highly similar within individuals for all phyla. Between individuals, microbiota profiles differed substantially but regardless of the presence (n = 19) of absence (n = 24) of diverticula. Microbiota diversity in both sigmoid and transverse colon was similar in all participants. We were not able to differentiate between diverticulosis patients and controls with a PLS-DA model. Mucosal lymphocyte counts were comparable among both groups; no neutrophils were detected in any of the studied biopsies. Microbiota composition and inflammatory markers were comparable among asymptomatic diverticulosis patients and controls. This suggests that the gut microbiota and mucosal inflammation do not play a major role in the pathogenesis of diverticula formation.

Green tea catechins may play a role in body weight regulation through interactions with the gut microbiota. We examined whether green tea supplementation for 12 weeks induces changes in composition of the human gut microbiota. 58 Caucasian men and women were included in a randomized, placebo-controlled design. For 12 weeks, subjects consumed either green tea (>0.56 g/d epigallocatechin-gallate + 0.28 ∼ 0.45 g/d caffeine) or placebo capsules. Fecal samples were collected twice (baseline, vs. week 12) for analyses of total bacterial profiles by means of IS-profiling, a 16S-23S interspacer region-based profiling method. No significant changes between baseline and week 12 in subjects receiving green tea or placebo capsules, and no significant interactions between treatment (green tea or placebo) and time (baseline and week 12) were observed for body composition. Analysis of the fecal samples in subjects receiving green tea and placebo showed similar bacterial diversity and community structures, indicating there were no significant changes in bacterial diversity between baseline and week 12 in subjects receiving green tea capsules or in subjects receiving placebo capsules. No significant interactions were observed between treatment (green tea or placebo) and time (baseline and week 12) for the gut microbial diversity. Although, there were no significant differences between normal weight and overweight subjects in response to green tea, we did observe a reduced bacterial alpha diversity in overweight as compared to normal weight subjects (p = 0.002). Green tea supplementation for 12 weeks did not have a significant effect on composition of the gut microbiota. ClinicalTrials.gov NCT01556321.

This study examined the correlation between pancreatic microbiome and patients characteristics. Furthermore, we compared different duodenal materials to examine their reflection of the pancreatic microbiome. Patients undergoing pancreatic surgery were included in the study. Characteristics of those patients were prospectively registered and sterile pancreatic biopsies were collected during surgery. After completion of the resection, duodenal fluid, -tissue and -swab were collected. Bacterial DNA was extracted and analyzed with IS-pro assay. Paired samples of 51 patients were available for evaluation, including pancreatic biopsies from all patients, 22 duodenal fluids, 21 duodenal swabs and 11 duodenal tissues. The pancreatic microbiome consisted mostly of Proteobacteria followed by Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia (FAFV) and Bacteroidetes. On species level, Enterococcus faecalis, Escherichia coli, and Enterobacter-Klebsiella were most abundant. In pancreatic biopsies, the total bacterial load and Proteobacteria load were significantly higher in patients with biliary drainage (54618.0 vs 5623.5; 9119.0 vs 2067.1). Patients who used proton pump inhibitors had a significantly higher total bacterial load (115964.7 vs 8495.8), more FAFV (66862.9 vs 1890.1), more Proteobacteria (24245.9 vs 2951.4) and more Bacteroidetes (542.5 vs 25.8). The head of the pancreas contained significantly more bacteria (21193.4 vs 2096.8) and more FAFV (5225.7 vs 19.0) compared to the tail, regardless of biliary drainage. Furthermore, the microbiome of all duodenal materials showed a weak correlation with the pancreatic microbiome. Biliary drainage, use of proton pump inhibitors, and anatomic location of the pancreatic biopsy influence the pancreatic microbiome. Furthermore, the duodenal microbiome does not suffice as a surrogate for the pancreatic microbiome.

ObjectiveBacterial peritonitis (BP) is a serious complication commonly associated with cirrhosis and ascites, often leading to high mortality rates. Although these effects could be reduced with timely and appropriate antibiotics, traditional BP diagnosis relies on culture, often delaying targeted treatment. Therefore, the use of fast molecular assays holds the potential to enhance laboratory diagnosis. In this study, we assessed the diagnostic performaance of Molecular Culture ID, a broad PCR-based assay targeting the 16S-23S interspace rDNA region in the scope of BP diagnosis.MethodsThe residual material from 247 peritoneal fluid samples submitted for routine diagnostics was analyzed using Molecular Culture ID and compared alongside the standard of care (SOC) results.ResultsSample positivity and species identification outcomes of Molecular Culture ID were compared to those of SOC. Molecular Culture ID yielded 1.6x more positive samples than SOC. Percent positive agreement (PPA) between Molecular Culture ID and SOC at the sample level was 90.1% (IC 95%, 81.0% to 95.1%), and negative percent agreement (NPA) was 70.5% (IC 95%, 63.3% to 76.7%). At the species level, the PPA was 75.2% (95% CI 67.2% to 81.8%). Molecular Culture ID yielded 289 extra bacterial identifications, mainly anaerobic species. High leukocyte counts, indicative of infection, were concordant with Molecular Culture ID positivity.ConclusionMolecular Culture ID demonstrated enhanced BP diagnostic capabilities compared to SOC, with higher positivity rates, more comprehensive species identification for difficult to culture species and a high correlation with leukocyte counts.

[This corrects the article DOI: 10.3389/fcimb.2025.1645965.].

Introduction Clostridioides difficile infection (CDI) is the most common cause of antibiotic-associated diarrhoea. Fidaxomicin and fecal microbiota transplantation (FMT) are effective, but expensive therapies to treat recurrent CDI (reCDI). Our objective was to develop a prediction model for reCDI based on the gut microbiota composition and clinical characteristics, to identify patients who could benefit from early treatment with fidaxomicin or FMT. Methods Multicentre, prospective, observational study in adult patients diagnosed with a primary episode of CDI. Fecal samples and clinical data were collected prior to, and after 5 days of CDI treatment. Follow-up duration was 8 weeks. Microbiota composition was analysed by IS-pro, a bacterial profiling technique based on phylum- and species-specific differences in the 16–23 S interspace regions of ribosomal DNA. Bayesian additive regression trees (BART) and adaptive group-regularized logistic ridge regression (AGRR) were used to construct prediction models for reCDI. Results 209 patients were included, of which 25% developed reCDI. Variables related to microbiota composition provided better prediction of reCDI and were preferentially selected over clinical factors in joint prediction models. Bacteroidetes abundance and diversity after start of CDI treatment, and the increase in Proteobacteria diversity relative to baseline, were the most robust predictors of reCDI. The sensitivity and specificity of a BART model including these factors were 95% and 78%, but these dropped to 67% and 62% in out-of-sample prediction. Conclusion Early microbiota response to CDI treatment is a better predictor of reCDI than clinical prognostic factors, but not yet sufficient enough to predict reCDI in daily practice.

Cladribine tablets are an effective treatment for relapsing remitting multiple sclerosis (RRMS). However, almost half of the treated patients are not free of disease activity after two years. The aim of this study was to describe the changes that cladribine tablets effectuate in the gut and oral microbiota and the peripheral immunological profile between responders and non-responders. In this pilot study of the multicenter, prospective, observational BIA (Brain-Immune-Intestine Axis) study, we included patients aged 18 to 55 years with RRMS who were scheduled to start treatment with cladribine tablets. We assessed the clinical status and the immunological and microbiological profile prior to the start of the treatment and after three and twelve months. At twelve months, we assessed the response status, based on clinical relapses, radiological activity and disability progression on the Expanded Disability Status Scale. The first twenty-five patients of the BIA study were included in this analysis. Ten patients (40%) were responders twelve months after treatment. Three months after treatment we found a significant decline of naïve and transitional B cells and memory B cells, and of CD57 CD56 NK cells. After twelve months the values recovered to baseline levels, except for the memory B cells. We did not find significant changes of the microbiological profile over time, except for a decline of the phylum in the oral samples twelve months after treatment. Baseline values and changes over time did not significantly differ between responders and non-responders. However, several phyla, genera or species ( showed a higher relative abundance, and several phyla, genera or species ( had a lower relative abundance in responders compared to non-responders. After treatment with cladribine tablets, we found significant changes in the immunological landscape. Also, the microbiological profile showed several differences in microbes with known anti- or pro-inflammatory properties between responders and non-responders. Overall, we showed that we can measure a treatment effect from cladribine tablets with our analyses. Future research on data from the BIA study, with a larger sample size and extended follow-up, can possibly confirm the reliability of our findings.