Unexplored microbial diversity from 2,500 food metagenomes and links with the human microbiome.

Complex microbiomes are part of the food we eat and influence our own microbiome, but their diversity remains largely unexplored. Here, we generated the open access curatedFoodMetagenomicData (cFMD) resource by integrating 1,950 newly sequenced and 583 public food metagenomes. We produced 10,899 metagenome-assembled genomes spanning 1,036 prokaryotic and 108 eukaryotic species-level genome bins (SGBs), including 320 previously undescribed taxa. Food SGBs displayed significant microbial diversity within and between food categories. Extension to >20,000 human metagenomes revealed that food SGBs accounted on average for 3% of the adult gut microbiome. Strain-level analysis highlighted potential instances of food-to-gut transmission and intestinal colonization (e.g., Lacticaseibacillus paracasei) as well as SGBs with divergent genomic structures in food and humans (e.g., Streptococcus gallolyticus and Limosilactobabillus mucosae). The cFMD expands our knowledge on food microbiomes, their role in shaping the human microbiome, and supports future uses of metagenomics for food quality, safety, and authentication.

Discovering and exploring the hidden diversity of human gut viruses using highly enriched virome samples.

Viruses are an abundant and crucial component of the human microbiome, but accurately discovering them via metagenomics is still challenging. Currently, the available viral reference genomes poorly represent the diversity in microbiome samples, and expanding such a set of viral references is difficult. As a result, many viruses are still undetectable through metagenomics even when considering the power of de novo metagenomic assembly and binning, as viruses lack universal markers. Here, we describe a novel approach to catalog new viral members of the human gut microbiome and show how the resulting resource improves metagenomic analyses. We retrieved >3,000 viral-like particles (VLP) enriched metagenomic samples (viromes), evaluated the efficiency of the enrichment in each sample to leverage the viromes of highest purity, and applied multiple analysis steps involving assembly and comparison with hundreds of thousands of metagenome-assembled genomes to discover new viral genomes. We reported over 162,000 viral sequences passing quality control from thousands of gut metagenomes and viromes. The great majority of the retrieved viral sequences (~94.4%) were of unknown origin, most had a CRISPR spacer matching host bacteria, and four of them could be detected in >50% of a set of 18,756 gut metagenomes we surveyed. We included the obtained collection of sequences in a new MetaPhlAn 4.1 release, which can quantify reads within a metagenome matching the known and newly uncovered viral diversity. Additionally, we released the viral database for further virome and metagenomic studies of the human microbiome.

Favorable subgingival plaque microbiome shifts are associated with clinical treatment for peri-implant diseases.

We performed a longitudinal shotgun metagenomic investigation of the plaque microbiome associated with peri-implant diseases in a cohort of 91 subjects with 320 quality-controlled metagenomes. Through recently improved taxonomic profiling methods, we identified the most discriminative species between healthy and diseased subjects at baseline, evaluated their change over time, and provided evidence that clinical treatment had a positive effect on plaque microbiome composition in patients affected by mucositis and peri-implantitis.

Urinary microbiota is associated to clinicopathological features in benign prostatic hyperplasia.

BACKGROUND: The urinary microbiota of patients with benign prostatic hyperplasia (BPH) has been associated with lower urinary tract symptoms (LUTS), however, little is known about urinary microbiota correlations with clinicopathological parameters associated with BPH. Here, we investigate associations between the urinary microbiota and clinical parameters of patients with BPH undergoing surgery. METHODS: Forty-one patients with BPH undergoing surgery were recruited from two medical centers. Catheterized urine specimens were collected and the microbiota was characterized by 16S rRNA gene sequencing. Patients were segregated into two groups according to each clinical parameter and differences in urinary microbiota diversity and composition were evaluated. RESULTS: Higher prostate weight and prostate-specific antigen (PSA) levels were associated with higher alpha diversity in the urinary microbiota of BPH patients. At the specific microbe level, we found that the greater the prostatic weight, the lower the relative abundance of Streptococcus, while the greater the PSA levels, the higher the abundance of Lactobacillus. Treatment with 5-α-reductase inhibitor was associated with overall urinary microbiota composition, in part due to a higher abundance of Corynebacterium and Anaerococcus in this group. CONCLUSIONS: We demonstrated that the urinary microbiota of BPH patients is associated with clinicopathological features, paving the way for larger studies in which causality between urinary microbiota and BPH can be appropriately explored.

The bladder microbiota is not significantly altered by intravesical BCG therapy.

INTRODUCTION: Bladder microbiota dysbiosis has been associated with several urological disorders. However, dysbiosis markers in bladder cancer have not been identified and little is known about the effect of Bacillus Calmette-Guérin (BCG) intravesical therapy on the bladder microbiota. In this study, we compared the bladder microbiota of patients with non-muscle-invasive bladder cancer (NMIBC) undergoing BCG therapy to nononcological controls. We also longitudinally analyzed the impact of BCG therapy on the bladder microbiota of NMIBC patients and addressed whether bladder microbiota is associated with BCG efficacy. METHODS: We collected catheterized urine samples from males with intermediate/high-risk NMIBC (cancer group, n = 32) or benign prostatic hyperplasia (control group, n = 41). The cancer group also provided urine samples during and after BCG induction. We used 16S rRNA gene sequencing to characterize the bladder microbiota. Bladder microbiota parameters, such as diversity and taxonomic composition, were compared between groups and associated with clinicopathological data and BCG efficacy. RESULTS: We observed no significant differences between the bladder microbiota of NMIBC patients and controls. BCG intravesical instillations did not significantly alter the bladder microbiota of NMIBC patients, and BCG was rarely detected in the bladder during and after BCG therapy. Microbiota diversity and overall composition before BCG induction did not influence disease persistence at 3 months. However, higher abundance of Lactobacillus, Streptococcus, and Cutibacterium in the pre-BCG bladder microbiota was associated with BCG effectiveness. CONCLUSION: We were unable to identify markers of bladder microbiota dysbiosis among male NMIBC patients. Moreover, we demonstrated for the first time using longitudinally collected samples that BCG cannot persist in the bladder microbiota nor significantly alter its diversity and composition. The associations found between bladder microbes and BCG efficacy highlight the potential of microbial-based therapeutic and risk-stratification strategies in the intermediate/high-risk NMIBC setting.

Longitudinal analysis at three oral sites links oral microbiota to clinical outcomes in allogeneic hematopoietic stem-cell transplant.

IMPORTANCE: The oral cavity is the ultimate doorway for microbes entering the human body. We analyzed oral microbiota dynamics in allogeneic hematopoietic stem-cell transplant recipients and showed that microbiota injury and recovery patterns were highly informative on transplant complications and outcomes. Our results highlight the importance of tracking the recipient's microbiota changes during allogeneic hematopoietic stem-cell transplant to improve our understanding of its biology, safety, and efficacy.

Unusual gingival actinomycosis post allogeneic hematopoietic stem-cell transplant: case report.

BACKGROUND: Allogeneic hematopoietic stem cell transplant (allo-HSCT) is used to treat several hematological diseases, but immunosuppression during allo-HSCT facilitates opportunistic microbial growth in tissues, such as actinomycosis. An effective diagnosis of opportunistic diseases is essential for correct management of the disease and preservation of the immunosuppressed patient's life. CASE DESCRIPTION: A 57-year-old female patient was diagnosed with extranodal nasal type NK/T cell lymphoma and underwent curative treatment with allo-HSCT. Twenty-one days after the last clinical follow-up, the patient presented a necrotizing lesion in the papilla region between the first and second molars of the second quadrant. Histopathological analysis showed the presence of a bacterial cluster consistent with Actinomyces infection, and a dense lymphoid infiltrate was also observed. Immunohistochemistry for CD20, CD3, and CD56 was performed to exclude the possibility of the recurrence of extranodal NK/T cell lymphoma. Oral microbiota profiling showed a huge increase in the abundance of Actinomyces bacteria in the subgingival region three weeks prior to appearance of the lesion. CONCLUSIONS: Opportunistic infections with an unusual clinical appearance are confounding factors in therapeutic decision-making. We present for the first time a case of actinomycosis in the gingival papilla region following allo-HSCT. We also highlight how microbiota profiling through next-generation sequencing could be used to anticipate bacterial infection diagnosis.

Dynamics of Gut Microbiota and Clinical Variables after Ketogenic and Mediterranean Diets in Drug-Naïve Patients with Type 2 Diabetes Mellitus and Obesity.

Type 2 diabetes mellitus (T2DM), the most common form of diabetes, is a progressive chronic metabolic disease that has increasingly spread worldwide, enhancing the mortality rate, particularly from cardiovascular diseases (CVD). Lifestyle improvement through diet and physical activity is, together with drug treatment, the cornerstone of T2DM management. The Mediterranean diet (MD), which favors a prevalence of unprocessed vegetable foods and a reduction in red meats and industrial foods, without excluding any food category, is usually recommended. Recently, scientific societies have promoted a very low-calorie ketogenic diet (VLCKD), a multiphasic protocol that limits carbohydrates and then gradually re-introduces them, with a favorable outcome on body weight and metabolic parameters. Indeed, gut microbiota (GM) modifications have been linked to overweight/obesity and metabolic alterations typical of T2DM. Diet is known to affect GM largely, but only a few studies have investigated the effects of VLCKD on GM, especially in T2DM. In this study, we have compared anthropometric, biochemical, lifestyle parameters, the quality of life, and the GM of eleven patients with recently diagnosed T2DM and overweight or obesity, randomly assigned to two groups of six and five patients who followed the VLCKD (KETO) or hypocaloric MD (MEDI) respectively; parameters were recorded at baseline (T0) and after two (T2) and three months (T3). The results showed that VLCKD had more significant beneficial effects than MD on anthropometric parameters, while biochemical improvements did not statistically differ. As for the GM, despite the lack of significant results regarding the alpha and beta diversity, and the Firmicutes/Bacteroidota ratio between the two groups, in the KETO group, a significant increase in beneficial microbial taxa such as Verrucomicrobiota phylum with its members Verrucomicrobiae, Verrucomicrobiales, Akkermansiaceae, and Akkermansia, Christensenellaceae family, Eubacterium spp., and a reduction in microbial taxa previously associated with obesity (Firmicutes and Actinobacteriota) or other diseases (Alistipes) was observed both at T2 and T3. With regards to the MEDI group, variations were limited to a significant increase in Actinobacteroidota phylum at T2 and T3 and Firmicutes phylum at T3. Moreover, a metagenomic alteration linked to some metabolic pathways was found exclusively in the KETO group. In conclusion, both dietary approaches allowed patients to improve their state of health, but VLCKD has shown better results on body composition as well as on GM profile.

Commensal oral microbiota impacts ulcerative oral mucositis clinical course in allogeneic stem cell transplant recipients.

Oral mucositis (OM) is a complex acute cytotoxicity of antineoplastic treatment that affects 40-85% of patients undergoing hematopoietic stem-cell transplantation. OM is associated with prolonged hospitalization, increased extensive pharmacotherapy, need for parenteral nutrition, and elevated treatment costs. As OM onset relates to the mucosal microenvironment status, with a particular role for microbiota-driven inflammation, we aimed to investigate whether the oral mucosa microbiota was associated with the clinical course of OM in patients undergoing allogeneic hematopoietic stem-cell transplantation. We collected oral mucosa samples from 30 patients and analyzed the oral mucosa microbiota by 16S rRNA sequencing. A total of 13 patients (43%) developed ulcerative OM. We observed that specific taxa were associated with oral mucositis grade and time to oral mucositis healing. Porphyromonas relative abundance at preconditioning was positively correlated with ulcerative OM grade (Spearman ρ = 0.61, P = 0.028) and higher Lactobacillus relative abundance at ulcerative OM onset was associated with shortened ulcerative OM duration (P = 0.032). Additionally, we generated a machine-learning-based bacterial signature that uses pre-treatment microbial profiles to predict whether a patient will develop OM during treatment. Our findings suggest that further research should focus on host-microbiome interactions to better prevent and treat OM.

Choice of 16S Ribosomal RNA Primers Impacts Male Urinary Microbiota Profiling.

Accessibility to next-generation sequencing (NGS) technologies has enabled the profiling of microbial communities living in distinct habitats. 16S ribosomal RNA (rRNA) gene sequencing is widely used for microbiota profiling with NGS technologies. Since most used NGS platforms generate short reads, sequencing the full-length 16S rRNA gene is impractical. Therefore, choosing which 16S rRNA hypervariable region to sequence is critical in microbiota profiling studies. All nine 16S rRNA hypervariable regions are taxonomically informative, but due to variability in profiling performance for specific clades, choosing the ideal 16S rRNA hypervariable region will depend on the bacterial composition of the habitat under study. Recently, NGS allowed the identification of microbes in the urinary tract, and urinary microbiota has become an active research area. However, there is no current study evaluating the performance of different 16S rRNA hypervariable regions for male urinary microbiota profiling. We collected urine samples from male volunteers and profiled their urinary microbiota by sequencing a panel of six amplicons encompassing all nine 16S rRNA hypervariable regions. Systematic comparisons of their performance indicate V1V2 hypervariable regions better assess the taxa commonly present in male urine samples, suggesting V1V2 amplicon sequencing is more suitable for male urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future male urinary microbiota studies.

Are short-read amplicons suitable for the prediction of microbiome functional potential? A critical perspective.

Taxonomic marker gene analysis allows uncovering taxonomic profiles of microbial communities at low cost, making it omnipresent in microbiome research. There is an ever-expanding set of tools to extract further biological information from this kind of data. In this perspective, we enunciate several concerns regarding the biological validity of predicting functional potential from taxonomic profiles, especially when they are generated by short-read sequencing. The taxonomic resolution of marker genes, intragenomic variability of marker genes, and the compositional nature of microbiome data are discussed. Combining actual measurements of microbiome functions with predicted functional potentials is proposed as a powerful approach to better understand microbiome functioning. In this context, the significance of predicted functional potentials for generating and testing hypotheses is highlighted. We argue that functions of microbiomes predicted from microbiome DNA read count data generated by short-read amplicon sequencing should not serve as the only basis to draw biological inferences.

Disruption of the oral microbiota is associated with a higher risk of relapse after allogeneic hematopoietic stem cell transplantation.

Intestinal microbiota (IM) diversity and composition regulates host immunity and affects outcomes after allogeneic stem cell transplantation (allo-HSCT). We evaluated if the oral mucosa microbiota (OM) could impact the outcomes in patients who underwent allo-HSCT. Samples from the oral mucosa of 30 patients were collected at three time points: before the conditioning regimen, at aplasia, and at engraftment. We analyzed the associations of OM diversity and composition with allo-HSCT outcomes. Lower OM diversity at preconditioning was associated with a higher risk of relapse at 3 years (68% versus 33%, respectively; P = 0.04). Dominance (relative abundance ≥ 30%) by a single genus at preconditioning was also associated with a higher risk of relapse (63% versus 36% at 3 years, respectively; P = 0.04), as well as worse progression-free survival (PFS; 19% versus 55%, respectively; P = 0.01), and overall survival (OS) at 3 years (38% versus 81%, respectively; P = 0.02). In our study we observed that OM dysbiosis is associated with a higher risk of relapse and worse survival after allo-HSCT.

Dental Biofilm Microbiota Dysbiosis Is Associated With the Risk of Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation.

Acute graft-versus-host disease (aGVHD) is one of the major causes of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recently, aGVHD onset was linked to intestinal microbiota (IM) dysbiosis. However, other bacterial-rich gastrointestinal sites, such as the mouth, which hosts several distinctive microbiotas, may also impact the risk of GVHD. The dental biofilm microbiota (DBM) is highly diverse and, like the IM, interacts with host cells and modulates immune homeostasis. We characterized changes in the DBM of patients during allo-HSCT and evaluated whether the DBM could be associated with the risk of aGVHD. DBM dysbiosis during allo-HSCT was marked by a gradual loss of bacterial diversity and changes in DBM genera composition, with commensal genera reductions and potentially pathogenic bacteria overgrowths. High Streptococcus and high Corynebacterium relative abundance at preconditioning were associated with a higher risk of aGVHD (67% vs. 33%; HR = 2.89, P = 0.04 and 73% vs. 37%; HR = 2.74, P = 0.04, respectively), while high Veillonella relative abundance was associated with a lower risk of aGVHD (27% vs. 73%; HR = 0.24, P < 0.01). Enterococcus faecalis bloom during allo-HSCT was observed in 17% of allo-HSCT recipients and was associated with a higher risk of aGVHD (100% vs. 40%; HR = 4.07, P < 0.001) and severe aGVHD (60% vs. 12%; HR = 6.82, P = 0.01). To the best of our knowledge, this is the first study demonstrating that DBM dysbiosis is associated with the aGVHD risk after allo-HSCT.

Aurora A kinase and its activator TPX2 are potential therapeutic targets in KRAS-induced pancreatic cancer.

PURPOSE: Oncogenic KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs). As yet, however, no effective therapies are available for KRAS-induced malignancies. Therefore, research aimed at the identification of KRAS targets with therapeutic potential is warranted. Our goal was to investigate Aurora A (AURKA) and targeting protein for Xklp2 (TPX2) as potential therapeutic targets in PDAC. METHODS: AURKA and TPX2 expression was assessed using RNAseq and qRT-PCR in PDAC patient samples and matched non-tumor pancreatic tissues. Publicly available PDAC datasets were used to investigate associations of AURKA and TPX2 expression levels with patient survival and the presence of KRAS mutations. Next, we used an Aurora kinase inhibitor, or KRAS, AURKA and TPX2 targeting using RNA interference in KRAS-mutant PDAC cells and, subsequently, analyzed their clonogenic and anchorage-independent growth and migration. RESULTS: We found that relative to matched non-tumor tissues, PDAC tumors displayed significantly higher expression levels of AURKA and TPX2. In addition, we found that AURKA and TPX2 were co-expressed in PDAC datasets, and that high expression levels of AURKA and TPX2 were associated with a shorter patient survival and with the presence of oncogenic KRAS mutations. In addition, we found that siRNA-mediated KRAS targeting in KRAS-mutant PDAC cells reduced AURKA and TPX2 expression. Furthermore, targeting AURKA or TPX2 in KRAS-mutant PDAC cells reduced their clonogenic and anchorage-independent growth, as well their migration. CONCLUSIONS: From our data we conclude that AURKA and TPX2 may act as KRAS biomarkers in PDAC that can predict a worse prognosis, and that AURKA or TPX2 targeting in PDAC cells may reduce their transformed phenotype. These results indicate that AURKA and TPX2 may serve as promising targets to be explored for KRAS-mutant PDAC therapy.

Aurora A kinase and its activator TPX2 are potential therapeutic targets in KRAS-induced pancreatic cancer

Purpose Oncogenic KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs). As yet, however, no effective therapies are available for KRAS-induced malignancies. Therefore, research aimed at the identification of KRAS targets with therapeutic potential is warranted. Our goal was to investigate Aurora A (AURKA) and targeting protein for Xklp2 (TPX2) as potential therapeutic targets in PDAC. Methods AURKA and TPX2 expression was assessed using RNAseq and qRT-PCR in PDAC patient samples and matched nontumor pancreatic tissues. Publicly available PDAC datasets were used to investigate associations of AURKA and TPX2 expression levels with patient survival and the presence of KRAS mutations. Next, we used an Aurora kinase inhibitor, or KRAS, AURKA and TPX2 targeting using RNA interference in KRAS-mutant PDAC cells and, subsequently, analyzed their clonogenic and anchorage-independent growth and migration. Results We found that relative to matched non-tumor tissues, PDAC tumors displayed significantly higher expression levels of AURKA and TPX2. In addition, we found that AURKA and TPX2 were co-expressed in PDAC datasets, and that high expression levels of AURKA and TPX2 were associated with a shorter patient survival and with the presence of oncogenic KRAS mutations. In addition, we found that siRNA-mediated KRAS targeting in KRAS-mutant PDAC cells reduced AURKA and TPX2 expression. Furthermore, targeting AURKA or TPX2 in KRAS-mutant PDAC cells reduced their clonogenic and anchorage-independent growth, as well their migration. Conclusions From our data we conclude that AURKA and TPX2 may act as KRAS biomarkers in PDAC that can predict a worse prognosis, and that AURKA or TPX2 targeting in PDAC cells may reduce their transformed phenotype. These results indicate that AURKA and TPX2 may serve as promising targets to be explored for KRAS-mutant PDAC therapy.

Aurora A kinase and its activator TPX2 are potential therapeutic targets in KRAS-induced pancreatic cancer

Purpose Oncogenic KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs). As yet, however, no effective therapies are available for KRAS-induced malignancies. Therefore, research aimed at the identification of KRAS targets with therapeutic potential is warranted. Our goal was to investigate Aurora A (AURKA) and targeting protein for Xklp2 (TPX2) as potential therapeutic targets in PDAC. Methods AURKA and TPX2 expression was assessed using RNAseq and qRT-PCR in PDAC patient samples and matched nontumor pancreatic tissues. Publicly available PDAC datasets were used to investigate associations of AURKA and TPX2 expression levels with patient survival and the presence of KRAS mutations. Next, we used an Aurora kinase inhibitor, or KRAS, AURKA and TPX2 targeting using RNA interference in KRAS-mutant PDAC cells and, subsequently, analyzed their clonogenic and anchorage-independent growth and migration. Results We found that relative to matched non-tumor tissues, PDAC tumors displayed significantly higher expression levels of AURKA and TPX2. In addition, we found that AURKA and TPX2 were co-expressed in PDAC datasets, and that high expression levels of AURKA and TPX2 were associated with a shorter patient survival and with the presence of oncogenic KRAS mutations. In addition, we found that siRNA-mediated KRAS targeting in KRAS-mutant PDAC cells reduced AURKA and TPX2 expression. Furthermore, targeting AURKA or TPX2 in KRAS-mutant PDAC cells reduced their clonogenic and anchorage-independent growth, as well their migration. Conclusions From our data we conclude that AURKA and TPX2 may act as KRAS biomarkers in PDAC that can predict a worse prognosis, and that AURKA or TPX2 targeting in PDAC cells may reduce their transformed phenotype. These results indicate that AURKA and TPX2 may serve as promising targets to be explored for KRAS-mutant PDAC therapy.