The tongue microbiome of young patients with chronic kidney disease and their healthy mothers.

OBJECTIVES: Oral microbiome plays a crucial role in the incidence and development of oral diseases. An altered intestinal microbiome has been reported in adults with chronic kidney disease (CKD). This study aimed to characterize the tongue microbiome of young patients with CKD compared to their healthy mothers to identify the influence of CKD-associated factors on resilient tongue ecosystem. MATERIAL AND METHODS: Thirty patients with CKD (mean age, 14.2 years; 16 males and 14 females) and generalized gingivitis were included in the study. Swabs of the posterior tongue were collected from the patients and 21 mothers (mean age 40.8 years). Next-generation sequencing of 16S rDNA genes was employed to quantitatively characterize microbial communities. RESULTS: The bacterial communities were similar in terms of richness and diversity between patients and mothers (p > 0.05). In patients with CKD, 5 core phyla, 20 core genera, and 12 core species were identified. CONCLUSIONS: The tongue microbiome of the study participants showed no relevant CKD-associated differences compared to their mothers and appears to be a highly preserved niche in the oral cavity. Differences observed in the abundance of individual species in this study could be attributed to the age rather than CKD, even after a mean disease duration of 11 years. CLINICAL RELEVANCE: CKD and its associated metabolic changes appear to have no detectable impact on the resilient tongue microbiome observed in young patients.

Dietary omega-6/omega-3 ratio is not associated with gut microbiota composition and disease severity in patients with nonalcoholic fatty liver disease.

In this cross-sectional study, we hypothesized that a high dietary ratio of omega-6 (n-6) to omega-3 (n-3) fatty acids could be associated with an altered gut bacterial composition and with the disease severity in patients with nonalcoholic fatty liver disease (NAFLD). A total of 101 NAFLD patients were included in the study, of which 63 underwent a liver biopsy. All 101 patients completed a 14-day food and activity record. Ebispro 2016 professional software was used to calculate individual macronutrients and micronutrients consumed. Patients were grouped into 3 quantiles (Q) according to a low (Q1: <6.1, n = 34), moderate (Q2: 6.1-7.8, n = 33), or high (Q3: >7.8, n = 34) dietary n-6/n-3 ratio. Stool samples were analyzed using 16S rRNA gene sequencing. Spearman correlation coefficients and principal coordinate analysis were used to detect differences in the bacterial composition of the gut microbiota. The median dietary n-6/n-3 ratio of all patients was 6.7 (range, 3.1-14.9). No significant associations between the dietary n-6/n-3 ratio and the gut microbiota composition or disease severity were observed. However, the abundance of specific bacteria such as Catenibacterium or Lactobacillus ruminis were found to be positively correlated and the abundance of Clostridium were negatively correlated with dietary n-6 fatty acid intake. The results indicate that a high dietary n-6/n-3 ratio is probably not a highly relevant factor in the pathogenesis of human NAFLD. Further studies are needed to clarify the importance of interactions between gut bacterial taxa and n-6 fatty acids in the pathophysiology of NAFLD.

Longitudinal variability in the urinary microbiota of healthy premenopausal women and the relation to neighboring microbial communities: A pilot study.

BACKGROUND: The understanding of longitudinal changes in the urinary microbiota of healthy women and its relation to intestinal microbiota is limited. METHODS: From a cohort of 15 premenopausal women without known urogenital disease or current symptoms, we collected catheter urine (CU), vaginal and periurethral swabs, and fecal samples on four visits over six months. Additionally, ten participants provided CU and midstream urine (MU) to assess comparability. Urine was subjected to expanded culture. 16S rRNA gene sequencing was performed on all urine, fecal, and selected vaginal and periurethral samples. Sequence reads were processed (DADA2 pipeline) and analyzed using QIIME 2 and R. RESULTS: Relative abundances of urinary microbiota were variable over 6-18 months. The degree of intraindividual variability of urinary microbiota was higher than that found in fecal samples. Still, nearly half of the observed beta diversity of all urine samples could be attributed to differences between volunteers (R2 = 0.48, p = 0.001). After stratification by volunteer, time since last sexual intercourse was shown to be a factor significantly contributing to beta diversity (R2 = 0.14, p = 0.001). We observed a close relatedness of urogenital microbial habitats and a clear distinction from intestinal microbiota in the overall betadiversity analysis. Microbiota compositions derived from MU differed only slightly from CU compositions. Within this analysis of low-biomass samples, we identified contaminating sequences potentially stemming from sequencing reagents. CONCLUSIONS: Results from our longitudinal cohort study confirmed the presence of a rather variable individual urinary microbiota in premenopausal women. These findings from catheter urine complement previous observations on temporal dynamics in voided urine. The higher intraindividual variability of urinary microbiota as compared to fecal microbiota will be a challenge for future studies investigating associations with urogenital diseases and aiming at identifying pathogenic microbiota signatures.

T-cells of invasive candidiasis patients show patterns of T-cell-exhaustion suggesting checkpoint blockade as treatment option.

OBJECTIVE: Recent data imply that strengthening host immunity by checkpoint inhibition improves outcome in invasive fungal infections (IFI), particularly in candidiasis. METHODS: To assess T-cell exhaustion in this context, we compared peripheral blood mononuclear cells (PBMCs) and serum samples of patients with invasive Candida albicans infection (IC, n = 21) to PBMCs or tumor-infiltrating lymphocytes (TILs) from cancer patients (n = 14) and PBMCs of healthy controls (n = 20). Type and differentiation of lymphocytes and expression of 29 immune-regulatory molecules were analyzed by flow cytometry. C. albicans specific responses were assessed by FluoroSpot (n = 8) and antibody measurement (n = 14). RESULTS: Fractions and phenotypes of lymphocyte subsets in PBMCs of IC patients were similar compared to PBMCs of controls, while they were different in TILs. PBMCs of patients with IC showed increased expression of immune-checkpoint molecules. The pattern of upregulated molecules was similar to TILs, but not present in PBMCs of control cancer patients. Fractions of T-cells expressing PD-1 and TIGIT were higher in IC patients that died. FluoroSpot analysis showed a Candida-specific IFN-y or IL-2 response in 5/8 patients, enhanced by addition of nivolumab in vitro. CONCLUSIONS: Together with preclinical data and preliminary evidence of clinical efficacy in mucormycosis, our results support clinical evaluation of immune-checkpoint inhibition in IFI treatment. TRIAL REGISTRATION: NCT04533087; retrospectively registered on August 31, 2020.

Combined analysis of gut microbiota, diet and PNPLA3 polymorphism in biopsy-proven non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is a global health burden. Risk factors for disease severity include older age, increased body mass index (BMI), diabetes, genetic variants, dietary factors and gut microbiota alterations. However, the interdependence of these factors and their individual impact on disease severity remain unknown. METHODS: In this cross-sectional study, we performed 16S gene sequencing using fecal samples, collected dietary intake, PNPLA3 gene variants and clinical and liver histology parameters in a well-described cohort of 180 NAFLD patients. Principal component analyses were used for dimensionality reduction of dietary and microbiota data. Simple and multiple stepwise ordinal regression analyses were performed. RESULTS: Complete data were available for 57 NAFLD patients. In the simple regression analysis, features associated with the metabolic syndrome had the highest importance regarding liver disease severity. In the multiple regression analysis, BMI was the most important factor associated with the fibrosis stage (OR per kg/m2 : 1.23, 95% CI 1.10-1.37, P < .001). The PNPLA3 risk allele had the strongest association with the histological grade of steatosis (OR 5.32, 95% CI 1.56-18.11, P = .007), followed by specific dietary patterns. Low abundances of Faecalibacterium, Bacteroides and Prevotella and high abundances of Gemmiger were associated with the degree of inflammation, ballooning and stages of fibrosis, even after taking other cofactors into account. CONCLUSIONS: BMI had the strongest association with histological fibrosis, but PNPLA3 gene variants, gut bacterial features and dietary factors were all associated with different histology features, which underscore the multifactorial pathogenesis of NAFLD.

Prediction of advanced fibrosis in non-alcoholic fatty liver disease using gut microbiota-based approaches compared with simple non-invasive tools.

Liver fibrosis is the major determinant of liver related complications in patients with non-alcoholic fatty liver disease (NAFLD). A gut microbiota signature has been explored to predict advanced fibrosis in NAFLD patients. The aim of this study was to validate and compare the diagnostic performance of gut microbiota-based approaches to simple non-invasive tools for the prediction of advanced fibrosis in NAFLD. 16S rRNA gene sequencing was performed in a cohort of 83 biopsy-proven NAFLD patients and 13 patients with non-invasively diagnosed NAFLD-cirrhosis. Random Forest models based on clinical data and sequencing results were compared with transient elastography, the NAFLD fibrosis score (NFS) and FIB-4 index. A Random Forest model containing clinical features and bacterial taxa achieved an area under the curve (AUC) of 0.87 which was only marginally superior to a model without microbiota features (AUC 0.85). The model that aimed to validate a published algorithm achieved an AUC of 0.71. AUC's for NFS and FIB-4 index were 0.86 and 0.85. Transient elastography performed best with an AUC of 0.93. Gut microbiota signatures might help to predict advanced fibrosis in NAFLD. However, transient elastography achieved the best diagnostic performance for the detection of NAFLD patients at risk for disease progression.

High Protein Intake Is Associated With Histological Disease Activity in Patients With NAFLD.

Overconsumption of carbohydrates and lipids are well known to cause nonalcoholic fatty liver disease (NAFLD), while the role of nutritional protein intake is less clear. In Western diet, meat and other animal products are the main protein source, with varying concentrations of specific amino acids. Whether the amount or composition of protein intake is associated with a higher risk for disease severity has not yet been examined. In this study, we investigated associations of dietary components with histological disease activity by analyzing detailed 14-day food records in a cohort of 61 patients with biopsy-proven NAFLD. Furthermore, we used 16S ribosomal RNA gene sequencing to detect associations with different abundances of the gut microbiota with dietary patterns. Patients with definite nonalcoholic steatohepatitis (NAFLD activity score of 5-8 on liver biopsy) had a significantly higher daily relative intake of protein compared with patients with a NAFLD activity score of 0-4 (18.0% vs. 15.8% of daily protein-based calories, P = 0.018). After adjustment for several potentially confounding factors, a higher protein intake (≥17.3% of daily protein-based calories) remained associated with definite nonalcoholic steatohepatitis, with an odds ratio of 5.09 (95% confidence interval 1.22-21.25, P = 0.026). This association was driven primarily by serine, glycine, arginine, proline, phenylalanine, and methionine. A higher protein intake correlated with a lower Bacteroides abundance and an altered abundance of several other bacterial taxa. Conclusion: A high protein intake was independently associated with more active and severe histological disease activity in patients with NAFLD. Further studies are needed to investigate the potential harmful role of dietary amino acids on NAFLD, with special attention to meat as their major source.

Phenotyping non-alcoholic fatty liver disease by the gut microbiota: Ready for prime time?

BACKGROUND AND AIM: Several studies observed alterations in the gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD). However, analyzed patient populations and methods strongly differ among these studies. The aim of this study was to prove the reproducibility of published results and to provide a detailed overview of all findings in our NAFLD cohort using next generation sequencing methods. METHODS: The individual taxonomic microbiota composition of fecal samples from 90 NAFLD patients and 21 healthy controls was analyzed using 16S rRNA gene sequencing. Study participants were grouped according to their disease stage and compared regarding their gut microbiota composition. Studies were identified from PubMed listed publications, and the results were compared with the findings in our cohort. RESULTS: Results from 13 identified studies were compared with our data. A decreased abundance of the Bacteroidetes and Ruminococcaceae as well as an increased abundance of Lactobacillaceae and Veillonellaceae and Dorea were the most frequently reported changes among NAFLD patients in 4/13, 5/13, 4/13, 2/13, and 3/13 studies, respectively. Even though these alterations in the gut microbiota composition were also observed in our patient cohort, the majority of published differences could not be reproduced, neither in our own nor in other NAFLD cohort studies. CONCLUSION: Despite repeatedly reproduced abundance patterns of specific bacteria, the heterogeneous study results did not reveal a consistent disease specific gut microbiota signature. Further prospective studies with homogenous patient cohorts and standardized methods are necessary to phenotype NAFLD by the gut microbiota.

Fecal microbiota transplantation in a kidney transplant recipient with recurrent urinary tract infection.

PURPOSE: We report on a kidney transplant recipient treated with fecal microbiota transplantation (FMT) for recurrent urinary tract infections. METHODS: FMT was administered via frozen capsulized microbiota. Before and after FMT, urinary, fecal and vaginal microbiota compositions were analyzed. RESULTS: The patient remained without symptoms after FMT. CONCLUSIONS: Underlying mechanisms of action need to be addressed in depth by future research.

Incorporation of antigens into viral capsids augments immunogenicity of adeno-associated virus vector-based vaccines.

Genetic modification of adeno-associated virus (AAV) capsids has previously been exploited to redirect viral tropism. Here we demonstrate that engineering of AAV capsids as scaffolds for antigen display augments antigen-specific immunogenicity. Combining antigen display with vector-mediated overexpression resulted in a single-shot prime-boost vaccine. This new class of vaccines induced immune responses significantly faster and an IgG antibody pool of higher avidity than conventional vectors, highlighting the potency of capsid modification in vaccine development.

Insights into the function of the WhiB-like protein of mycobacteriophage TM4--a transcriptional inhibitor of WhiB2.

WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.