Gastric microbiota composition in patients with corpus atrophic gastritis.

BACKGROUND: In corpus atrophic gastritis (CAG), hypochlorhydria makes plausible the overgrowth of intragastric bacteria, whose role in gastric carcinogenesis is under debate. AIMS: To characterize the antrum/corpus composition of the gastric bacterial microbiota in CAG patients compared to controls without CAG. METHODS: A cross-sectional monocentric study on consecutive patients with known histological diagnosis of CAG undergoing gastroscopy for gastric cancer surveillance and patients without CAG undergoing gastroscopy for dyspepsia or anemia (108 biopsies from 55 patients, median age 61.5). Genomic DNA from one antral and one corpus biopsy from each case (n = 23) and control (n = 32) was extracted. Gastric microbiota was assessed by sequencing hypervariable regions of the 16SrRNA gene. RESULTS: Bacterial abundance and diversity were significantly lower in CAG cases than in controls (p < 0.001). Firmicutes were more frequent in cases, Bacteroidetes and Fusobacteria in controls (p < 0.0001). Streptococcaceae were more abundant in cases (p < 0.0001), Prevotellaceae in controls (p < 0.0001). The genus Streptococcus was positively correlated with severe OLGA/OLGIM stages linked to a higher risk of gastric cancer. CONCLUSION: Gastric bacterial microbiota in CAG showed a reduced abundance and complexity but was characterized by higher colonization of Firmicutes, in particular Streptococcus, increased in subjects with severe atrophy/metaplasia stages at higher risk of gastric cancer.

Assessing the Genomic Variability of Gardnerella vaginalis through Comparative Genomic Analyses: Evolutionary and Ecological Implications.

Gardnerella vaginalis is described as a common anaerobic vaginal bacterium whose presence may correlate with vaginal dysbiotic conditions. In the current study, we performed phylogenomic analyses of 72 G. vaginalis genome sequences, revealing noteworthy genome differences underlying a polyphyletic organization of this taxon. Particularly, the genomic survey revealed that this species may actually include nine distinct genotypes (GGtype1 to GGtype9). Furthermore, the observed link between sialidase and phylogenomic grouping provided clues of a connection between virulence potential and the evolutionary history of this microbial taxon. Specifically, based on the outcomes of these in silico analyses, GGtype3, GGtype7, GGtype8, and GGtype9 appear to have virulence potential since they exhibited the sialidase gene in their genomes. Notably, the analysis of 34 publicly available vaginal metagenomic samples allowed us to trace the distribution of the nine G. vaginalis genotypes identified in this study among the human population, highlighting how differences in genetic makeup could be related to specific ecological properties. Furthermore, comparative genomic analyses provided details about the G. vaginalis pan- and core genome contents, including putative genetic elements involved in the adaptation to the ecological niche as well as many putative virulence factors. Among these putative virulence factors, particularly noteworthy genes identified were the gene encoding cholesterol-dependent cytolysin (CDC) toxin vaginolysin and genes related to microbial biofilm formation, iron uptake, adhesion to the vaginal epithelium, as well as macrolide antibiotic resistance.IMPORTANCE The identification of nine different genotypes among members of G. vaginalis allowed us to distinguish an uneven distribution of virulence-associated genetic traits within this taxon and thus suggest the potential occurrence of putative pathogen and commensal G. vaginalis strains. These findings, coupled with metagenomics microbial profiling of human vaginal microbiota, permitted us to get insights into the distribution of the genotypes among the human population, highlighting the presence of different structural communities in terms of G. vaginalis genotypes.

Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach.

16S small-subunit (SSU) rRNA gene-based bacterial profiling is the gold standard for cost-effective taxonomic reconstruction of complex bacterial populations down to the genus level. However, it has been proven ineffective in clinical and research settings requiring higher taxonomic resolution. We therefore developed a bacterial profiling method based on the internal transcribed spacer (ITS) region employing optimized primers and a comprehensive ITS database for accurate cataloguing of bacterial communities at (sub)species resolution. Performance of the microbial ITS profiling pipeline was tested through analysis of host-associated, food, and environmental matrices, while its efficacy in clinical settings was assessed through analysis of mucosal biopsy specimens of colorectal cancer, leading to the identification of putative novel biomarkers. The data collected indicate that the proposed pipeline represents a major step forward in cost-effective identification and screening of microbial biomarkers at (sub)species level, with relevant impact in research, industrial, and clinical settings.IMPORTANCE We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings.

The human gallbladder microbiome is related to the physiological state and the biliary metabolic profile.

BACKGROUND: The microbial populations of the human intestinal tract and their relationship to specific diseases have been extensively studied during the last decade. However, the characterization of the human bile microbiota as a whole has been hampered by difficulties in accessing biological samples and the lack of adequate methodologies to assess molecular studies. Although a few reports have described the biliary microbiota in some hepatobiliary diseases, the bile microbiota of healthy individuals has not been described. With this in mind, the goal of the present study was to generate fundamental knowledge on the composition and activity of the human bile microbiota, as well as establishing its potential relationship with human bile-related disorders. RESULTS: Human bile samples from the gallbladder of individuals from a control group, without any record of hepatobiliary disorder, were obtained from liver donors during liver transplantation surgery. A bile DNA extraction method was optimized together with a quantitative PCR (qPCR) assay for determining the bacterial load. This allows the selection of samples to perform functional metagenomic analysis. Bile samples from the gallbladder of individuals suffering from lithiasis were collected during gallbladder resection and the microbial profiles assessed, using a 16S rRNA gene-based sequencing analysis, and compared with those of the control group. Additionally, the metabolic profile of the samples was analyzed by nuclear magnetic resonance (NMR). We detected, for the first time, bacterial communities in gallbladder samples of individuals without any hepatobiliary pathology. In the biliary microecosystem, the main bacterial phyla were represented by Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Significant differences in the relative abundance of different taxa of both groups were found. Sequences belonging to the family Propionibacteriaceae were more abundant in bile samples from control subjects; meanwhile, in patients with cholelithiasis members of the families Bacteroidaceae, Prevotellaceae, Porphyromonadaceae, and Veillonellaceae were more frequently detected. Furthermore, the metabolomics analysis showed that the two study groups have different metabolic profiles. CONCLUSIONS: Our results indicate that the gallbladder of human individuals, without diagnosed hepatobiliary pathology, harbors a microbial ecosystem that is described for the first time in this study. Its bacterial representatives and metabolites are different from those detected in people suffering from cholelithiasis. In this regard, since liver donors have been subjected to the specific conditions of the hospital's intensive care unit, including an antibiotic treatment, we must be cautious in stating that their bile samples contain a physiologically normal biliary microbiome. In any case, our results open up new possibilities to discover bacterial functions in a microbial ecosystem that has not previously been explored.

Fecal microbiota profile in a group of myasthenia gravis patients.

The intestinal microbiota plays a key role in the maintenance of human health. Alterations in this microbiota have been described in several autoimmune diseases, including nervous system diseases. Nevertheless, the information regarding neuromuscular conditions is still limited. In this study, we aimed at characterizing the intestinal microbiota composition in myasthenia gravis patients (MG). To this end fecal samples were taken from ten patients, with antibodies against the acetylcholine receptor, and ten age and sex matched controls from the same population (Asturias region, Spain). Fecal samples were submitted to microbiota analyses by 16S rRNA gene profiling, bifidobacterial ITS-region profiling and qPCR. The fecal levels of short chain fatty acids were determined by gas chromatography. MG patients were found to harbor lower relative proportions of Verrucomicrobiaceae and Bifidobacteriaceae, among others, and increased of the phylum Bacteroidetes and the family Desulfovibrionaceae. The increase of these latter microbial groups was also confirmed at quantitative level by qPCR. In contrast, no statistically significant differences were found between MG patients and the control group in the bifidobacterial population at the species level or in short chain fatty acids profiles. Our data indicates an altered fecal microbiota pattern in MG patients and point out at specific microbiota targets for intervention in this population.

Mucosal microbiota of intestinal polyps reveals putative biomarkers of colorectal cancer.

The human intestine retains a complex microbial ecosystem, which performs crucial functions that impact on host health. Several studies have indicated that intestinal dysbiosis may impact on the establishment of life-threatening intestinal diseases such as colorectal cancer. An adenomatous polyp is the result of abnormal tissue growth, which is benign but is considered to be associated with a high risk of developing colorectal cancer, based on its grade of dysplasia. Development of diagnostic tools that are based on surveying the gut microbiota and are aimed at early detection of colorectal cancer represent highly desirable target. For this purpose, we performed a pilot study in which we applied a metataxonomic analysis based on 16S rRNA gene sequencing approach to unveil the composition of microbial communities of intestinal polyps. Moreover, we performed a meta-analysis involving the reconstructed microbiota composition of adenomatous polyps and publicly available metagenomics datasets of colorectal cancer. These analyses allowed the identification of microbial taxa such as Faecalibacterium, Bacteroides and Romboutsia, which appear to be depleted in cancerogenic mucosa as well as in adenomatous polyps, thus representing novel microbial biomarkers associated with early tumor formation. Furthermore, an absolute quantification of Fusubacterium nucleatum in polyps further compounded the important role of this microorganism as a valuable putative microbial biomarker for early diagnosis of colorectal cancer.

Bifidobacterium bifidum PRL2010 alleviates intestinal ischemia/reperfusion injury.

Mesenteric ischemia/reperfusion is a clinical emergency with high morbidity and mortality due to the transient reduction of blood supply to the bowel. In recent years, the critical contribution of gut microbiome to human health and proper gastrointestinal functions has gradually emerged. In the current study, we investigated the protective effects of five days supplementation with Bifidobacterium bifidum PRL2010 in a murine model of gut ischemia/reperfusion. Our findings indicate that animals pretreated with B. bifidum PRL2010 showed lower neutrophil recruitment in the lungs, remarkably reduced bacterial translocation and decreased transcription levels of TNFalpha and IL-10 both in liver and kidneys, at the same time increasing those of IL-12 in kidneys. Inhibiting the adhesion of pathogenic bacteria and boosting host innate immunity responses are among the possible protective mechanisms enacted by the probiotic. These results demonstrate that short-period treatment with B. bifidum PRL2010 is a potential strategy to dampen remote organ injury due to mesenteric ischemia/reperfusion.

A Gene Homologous to rRNA Methylase Genes Confers Erythromycin and Clindamycin Resistance in Bifidobacterium breve.

Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.

Identification of universal gut microbial biomarkers of common human intestinal diseases by meta-analysis.

Intestinal diseases, such as Crohn's disease (CD), ulcerative colitis (UC) and pseudomembranous colitis (CDI), are among the most common diseases in humans and may lead to more serious pathologies, e.g. colorectal cancer (CRC). Next generation sequencing has in recent years allowed the identification of correlations between intestinal bacteria and diseases, although the formulation of universal gut microbial biomarkers for such diseases is only in its infancy. In the current study, we selected and reanalyzed a total of 3048 public datasets obtained from 16S rRNA profiling of individuals affected by CD, UC, CDI and CRC. This meta-analysis revealed possible biases in the reconstruction of the gut microbiota composition due to the use of different primer pairs employed for PCR of 16S rRNA gene fragments. Notably, this approach also identified common features of individuals affected by gut diseases (DS), including lower biodiversity compared to control subjects. Moreover, potential universal intestinal disease microbial biomarkers were identified through cross-disease comparisons. In detail, CTRL showed high abundance of the genera Barnesiella, Ruminococcaceae UCG-005, Alistipes, Christensenellaceae R-7 group and unclassified member of Lachnospiraceae family, while DS exhibited high abundance of Lactobacillus, unclassified member of Erysipelotrichaceae family and Streptococcus genera.

Gut microbiota composition is associated with polypharmacy in elderly hospitalized patients.

Reduced biodiversity and increased representation of opportunistic pathogens are typical features of gut microbiota composition in aging. Few studies have investigated their correlation with polypharmacy, multimorbidity and frailty. To assess it, we analyzed the fecal microbiota from 76 inpatients, aged 83 ± 8. Microbiome biodiversity (Chao1 index) and relative abundance of individual bacterial taxa were determined by next-generation 16S rRNA microbial profiling. Their correlation with number of drugs, and indexes of multimorbidity and frailty were verified using multivariate linear regression models. The impact of gut microbiota biodiversity on mortality, rehospitalizations and incident sepsis was also assessed after a 2-year follow-up, using Cox regression analysis. We found a significant negative correlation between the number of drugs and Chao1 Index at multivariate analysis. The number of drugs was associated with the average relative abundance of 15 taxa. The drug classes exhibiting the strongest association with single taxa abundance were proton pump inhibitors, antidepressants and antipsychotics. Conversely, frailty and multimorbidity were not significantly associated with gut microbiota biodiversity. Very low Chao1 index was also a significant predictor of mortality, but not of rehospitalizations and sepsis, at follow-up. In aging, polypharmacy may thus represent a determinant of gut microbiota composition, with detrimental clinical consequences.

The Sortase-Dependent Fimbriome of the Genus Bifidobacterium: Extracellular Structures with Potential To Modulate Microbe-Host Dialogue.

Bifidobacteria are important gut commensals of mammals, including humans, of any age. However, the molecular mechanisms by which these microorganisms establish themselves in the mammalian gut and persist in this environment are largely unknown. Here, we analyzed the genetic diversity of the predicted arsenal of sortase-dependent pili of known and sequenced members of the Bifidobacterium genus and constructed a bifidobacterial sortase-dependent fimbriome database. Our analyses revealed considerable genetic variability of the sortase-dependent fimbriome among bifidobacterial (sub)species, which appears to have been due to horizontal gene transfer events and for which we were able to perform evolutionary mapping. Functional assessment by transcriptome analysis and binding assays involving different substrates demonstrates how bifidobacterial pili are pivotal in promoting various abilities for adhesion to glycans and extracellular matrix proteins, thereby supporting the ecological success of bifidobacteria in the mammalian gut.IMPORTANCE Adhesion of bifidobacterial cells to the mucosa of the large intestine is considered a hallmark for the persistence and colonization of these bacteria in the human gut. In this context, we analyzed the genetic diversity of the predicted arsenal of sortase-dependent pili of known and sequenced members of the Bifidobacterium genus, and constructed a bifidobacterial sortase-dependent fimbriome database. Our analyses revealed considerable genetic variability of the sortase-dependent fimbriome among bifidobacterial (sub)species, which appears to have been due to horizontal gene transfer events. In addition, functional assessment by transcriptome analysis and binding assays involving different substrates demonstrates how bifidobacterial pili are crucial in promoting various abilities for adhesion to glycans and extracellular matrix proteins, thereby supporting the ecological success of bifidobacteria in the mammalian gut. This study represents a complete genomic study regarding the presence of fimbriae in the genus Bifidobacterium.

Exploring Amino Acid Auxotrophy in Bifidobacterium bifidum PRL2010.

The acquisition and assimilation strategies followed by members of the infant gut microbiota to retrieve nitrogen from the gut lumen are still largely unknown. In particular, no information on these metabolic processes is available regarding bifidobacteria, which are among the first microbial colonizers of the human intestine. Here, evaluation of amino acid auxotrophy and prototrophy of Bifidobacterium bifidum, with particular emphasis on B. bifidum strain PRL2010 (LMG S-28692), revealed a putative auxotrophy for cysteine. In addition, we hypothesized that cysteine plays a role in the oxidative stress response in B. bifidum. The use of glutathione as an alternative reduced sulfur compound did not alleviate cysteine auxotrophy of this strain, though it was shown to stimulate expression of the genes involved in cysteine biosynthesis, reminiscent of oxidative stress response. When PRL2010 was grown on a medium containing complex substrates, such as whey proteins or casein hydrolysate, we noticed a distinct growth-promoting effect of these compounds. Transcriptional analysis involving B. bifidum PRL2010 cultivated on whey proteins or casein hydrolysate revealed that the biosynthetic pathways for cysteine and methionine are modulated by the presence of casein hydrolysate. Such findings support the notion that certain complex substrates may act as potential prebiotics for bifidobacteria in their ecological niche.

Human colon-derived soluble factors modulate gut microbiota composition.

The commensal microbiota modulates immunological and metabolic aspects of the intestinal mucosa contributing to development of human gut diseases including inflammatory bowel disease. The host/microbiota interaction often referred to as a crosstalk, mainly focuses on the effect of the microbiota on the host neglecting effects that the host could elicit on the commensals. Colonic microenvironments from three human healthy controls (obtained from the proximal and distal colon, both in resting conditions and after immune - IL-15- and microbiota - LPS-in vitro challenges) were used to condition a stable fecal population. Subsequent 16S rRNA gene-based analyses were performed to study the effect induced by the host on the microbiota composition and function. Non-supervised principal component analysis (PCA) showed that all microbiotas, which had been conditioned with colonic microenvironments clustered together in terms of relative microbial composition, suggesting that soluble factors were modulating a stable fecal population independently from the treatment or the origin. Our findings confirmed that the host intestinal microenvironment has the capacity to modulate the gut microbiota composition via yet unidentified soluble factors. These findings indicate that an appropriate understanding of the factors of the host mucosal microenvironment affecting microbiota composition and function could improve therapeutic manipulation of the microbiota composition.

TgaA, a VirB1-like component belonging to a putative type IV secretion system of Bifidobacterium bifidum MIMBb75.

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium-host interactions.

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.

Exploration of the genomic diversity and core genome of the Bifidobacterium adolescentis phylogenetic group by means of a polyphasic approach.

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum, by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays.