Factors that influence the pancreatic and duodenal microbiome in patients undergoing pancreatic surgery.

BACKGROUND/OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The gastric microbiota in patients with Crohn's disease; a preliminary study.

The gastric microbiota in Crohn's disease (CD) has not been studied. The purpose of the study was to evaluate differences of stomach microbiota between CD patients and controls. DNA was extracted from gastric mucosal and fluid samples, from 24 CD patients and 19 controls. 16S rRNA gene sequencing identified 1511 operational taxonomic units (OTUs), of which 239 passed the low abundance and low variance filters. All but one CD patients were HP negative. Fifteen bacterial phyla were identified in at least one mucosal or fluid site. Of these, Bacteroidota and Firmicutes accounted for 70% of all phyla. Proteobacteria, Actinobacteriota, and Fusobacteriota combined accounted for 27%. There was significant difference in the relative abundance of Bacteroidota, Proteobacteria, Fusobacteriota, and Campilobacterota between CD patients and controls only in gastric corpus samples. In gastric liquid, there was a significant difference only in Actinobacteriota. Pairwise comparison identified 67 differentially abundant OTUs in at least one site. Of these, 13 were present in more than one comparison, and four differentiating OTUs (Neisseriaceae, Neisseria, Absconditabacteriales, and Microbacteriaceae) were identified at all tested sites. The results reveal significant changes in gastric microbial profiles (beta diversity, phylum, and individual taxa levels) between H. pylori-negative CD patients and controls.

mbImpute: an accurate and robust imputation method for microbiome data.

A critical challenge in microbiome data analysis is the existence of many non-biological zeros, which distort taxon abundance distributions, complicate data analysis, and jeopardize the reliability of scientific discoveries. To address this issue, we propose the first imputation method for microbiome data-mbImpute-to identify and recover likely non-biological zeros by borrowing information jointly from similar samples, similar taxa, and optional metadata including sample covariates and taxon phylogeny. We demonstrate that mbImpute improves the power of identifying disease-related taxa from microbiome data of type 2 diabetes and colorectal cancer, and mbImpute preserves non-zero distributions of taxa abundances.

Sneathia: an emerging pathogen in female reproductive disease and adverse perinatal outcomes.

Sneathia is an emerging pathogen implicated in adverse reproductive and perinatal outcomes. Although scarce, recent data suggest that vaginally residing Sneathia becomes pathogenic following its ascension into the upper urogenital tract, amniotic fluid, placenta, and foetal membranes. The role of Sneathia in women's health and disease is generally underappreciated because the cultivation of these bacteria is limited by their complex nutritional requirements, slow growth patterns, and anaerobic nature. For this reason, molecular methods are typically required for the detection and differential diagnosis of Sneathia infections. Here, we review the laboratory methods used for the diagnosis of Sneathia infections, the molecular mechanisms underlying its virulence, and its sensitivity to antibiotics. We further review the evidence of Sneathia's contributions to the pathogenesis of bacterial vaginosis, chorioamnionitis, preterm prelabour rupture of membranes, spontaneous preterm labour, stillbirth, maternal and neonatal sepsis, HIV infection, and cervical cancer. Collectively, growing evidence indicates that Sneathia represents an important yet underappreciated pathogen affecting the development and progression of several adverse clinical conditions diagnosed in pregnant women and their neonates, as well as in non-pregnant women.

Gastric Adenocarcinomas and Signet-Ring Cell Carcinoma: Unraveling Gastric Cancer Complexity through Microbiome Analysis-Deepening Heterogeneity for a Personalized Therapy.

Gastric cancer (GC) is the fifth most prevalent cancer worldwide and the third leading cause of global cancer mortality. With the advances of the omic studies, a heterogeneous GC landscape has been revealed, with significant molecular diversity. Given the multifaceted nature of GC, identification of different patient subsets with prognostic and/or predictive outcomes is a key aspect to allow tailoring of specific treatments. Recently, the involvement of the microbiota in gastric carcinogenesis has been described. To deepen this aspect, we compared microbiota composition in signet-ring cell carcinoma (SRCC) and adenocarcinoma (ADC), two distinct GC subtypes. To this purpose, 10 ADC and 10 SRCC and their paired non-tumor (PNT) counterparts were evaluated for microbiota composition through 16S rRNA analysis. Weighted and unweighted UniFrac and Bray-Curtis dissimilarity showed significant community-level separation between ADC and SRCC. Through the LEfSe (linear discriminant analysis coupled with effect size) tool, we identified potential microbial biomarkers associated with GC subtypes. In particular, SRCCs were significantly enriched in the phyla Fusobacteria, Bacteroidetes, Patescibacteria, whereas in the ADC type, Proteobacteria and Acidobacteria phyla were found. Overall, our data add new insights into GC heterogeneity and may contribute to deepening the GC classification.

Chronic fatigue syndrome patients have alterations in their oral microbiome composition and function.

Host-microbe interactions have been implicated in the pathogenesis of chronic fatigue syndrome (CFS), but whether the oral microbiome is altered in CFS patients is unknown. We explored alterations of the oral microbiome in Chinese Han CFS patients using 16S rRNA gene sequencing and alterations in the functional potential of the oral microbiome using PICRUSt. We found that Shannon and Simpson diversity indices were not different in CFS patients compared to healthy controls, but the overall oral microbiome composition was different (MANOVA, p < 0.01). CFS patients had a higher relative abundance of Fusobacteria compared with healthy controls. Further, the genera Leptotrichia, Prevotella, and Fusobacterium were enriched and Haemophilus, Veillonella, and Porphyromonas were depleted in CFS patients compared to healthy controls. Functional analysis from inferred metagenomes showed that bacterial genera altered in CFS patients were primarily associated with amino acid and energy metabolism. Our findings demonstrate that the oral microbiome in CFS patients is different from healthy controls, and these differences lead to shifts in functional pathways with implications for CFS pathogenesis. These findings increase our understanding of the relationship between the oral microbiota and CFS, which will advance our understanding of CFS pathogenesis and may contribute to future improvements in treatment and diagnosis.

Influences of pH and Iron Concentration on the Salivary Microbiome in Individual Humans with and without Caries.

This study aimed to identify the differences in the oral microbial communities in saliva from patients with and without caries by performing sequencing with the Illumina MiSeq platform, as well as to further assess their relationships with environmental factors (salivary pH and iron concentration). Forty-three volunteers were selected, including 21 subjects with and 22 without caries, from one village in Gansu, China. Based on 966,255 trimmed sequences and clustering at the 97% similarity level, 1,303 species-level operational taxonomic units were generated. The sequencing data for the two groups revealed that (i) particular distribution patterns (synergistic effects or competition) existed in the subjects with and without caries at both the genus and species levels and (ii) both the salivary pH and iron concentration had significant influences on the microbial community structure. IMPORTANCE: The significant influences of the oral environment observed in this study increase the current understanding of the salivary microbiome in caries. These results will be useful for expanding research directions and for improving disease diagnosis, prognosis, and therapy.

Isolation and identification of Caviibacter abscessus from cervical abscesses in a series of pet guinea pigs (Cavia porcellus).

An organism reported in the early literature to be a rare cause of cervical lymphadenitis in guinea pigs, Streptobacillus moniliformis, has been reclassified as Caviibacter abscessus We describe a series of sequential cases of abscesses in guinea pigs that were presented to our clinic from which the only agent isolated was a unique, serum-requiring bacterium. Discrete colonies were not detected in 6.5% CO2 or anaerobically on routine primary isolation media containing up to 5% whole sheep blood, with and without cysteine, vitamin K, and hemin supplementation after 7 days of incubation at 37°C. Based on subsequently determined growth requirements, the organisms were best described as serum-requiring, aerotolerant anaerobes. Colonies were detectable within 24 h at 37°C in an anaerobic atmosphere on a mycoplasma agar-based medium containing 10% pig serum and reached 3 mm in diameter within 3-5 days. Microscopic appearance consisted of small gram-negative rods and coccobacilli with occasional filaments. However, in direct smears from clinical specimens and from weak or dysgonic growth on plates incubated under suboptimal growth conditions (e.g., in 6.5% CO2), irregular rods with occasional small bulbous forms or numerous long wavy filaments were observed. All of the isolates generated unique spectral profiles similar to that of C. abscessus when examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phylogenetic analysis of partial 16S rRNA gene sequences showed that the isolates were identical to each other and shared 99.9% sequence identity with C. abscessus.

Influence of Endogenous and Exogenous Estrogenic Endocrine on Intestinal Microbiota in Zebrafish.

Gender is one of the factors influencing the intestinal microbial composition in mammals, but whether fish also have gender-specific intestinal microbial patterns remains unknown. In this decade, endocrine disrupting chemicals in surface and ground water of many areas and increasing observation of freshwater male fish displaying female sexual characteristics have been reported. Here we identified the difference in intestinal microbiota between male and female zebrafish, and revealed the influence of endocrine disrupting chemicals on zebrafish intestinal microbiota by using high-throughput sequencing. The results indicated that Fusobacteria, Bacteroidetes and Proteobacteria were dominant in the gut of zebrafish and there were no obvious gender-specific intestinal microbial patterns. Two endocrine disrupting chemicals, Estradiol (E2) and Bisphenol A (BPA), were selected to treat male zebrafish for 5 weeks. E2 and BPA increased vitellogenin expression in the liver of male zebrafish and altered the intestinal microbial composition with the abundance of the phylum CKC4 increased significantly. Our results suggested that because of the developmental character and living environment, gender did not influence the assembly of intestinal microbiota in zebrafish as it does in mammals, but exposure extra to endocrine disrupting chemicals disturbed the intestinal microbial composition, which may be related to changes in host physiological metabolism.

Acute Appendicitis in Children Is Associated With a Local Expansion of Fusobacteria.

BACKGROUND: Lumenal obstruction has typically been regarded as the cause of acute appendicitis (AA). Recent evidence including data from "antibiotics first" trials suggests that this disease may result from invasion of the appendix by specific pathogens. Small studies have identified an abundance of bacteria from the genus Fusobacterium in appendixes from patients with AA. We aimed to validate these findings in a larger cohort of children with appendicitis in addition to profiling the appendiceal microbiota in a population of children without appendicitis. METHODS: Appendix swabs were collected from children undergoing appendectomy for AA (n = 60), incidental appendectomy for reasons other than appendicitis (n = 18), or ileocecectomy for inflammatory bowel disease (n = 7), in addition to samples from other sites. Bacterial 16S ribosomal RNA gene sequences from each sample were amplified, sequenced, and analyzed with the UPARSE and QIIME programs. RESULTS: We found that the normal human appendix harbors populations of Fusobacteria that are generally absent in fecal samples from healthy adults and children. In patients with AA, Fusobacteria populations proliferate and often persist despite several weeks of broad-spectrum antibiotics prior to surgery. Relative to non-AA samples, AA samples were depleted of sequences from the genus Bacteroides Phylogenetic analysis of sequence data indicates that F. nucleatum, F. necrophorum, and F. varium are the species of Fusobacterium observed in AA samples. CONCLUSIONS: These results indicate that the appendiceal niche harbors distinct microbial populations that likely contribute to the pathogenesis of appendicitis, which may one day be leveraged to improve the diagnosis and/or treatment of patients with AA.

Does the intestinal microbial community of Korean Crohn's disease patients differ from that of western patients?

BACKGROUND: Intestinal microbiota play an important role in maintaining the homeostasis of the host immune system. To analyze the alteration of the intestinal microbial community structure in Korean Crohn's disease (CD) patients, we performed a comparative metagenomic analysis between healthy people and CD patients using fecal samples and mucosal tissues of ileocecal valve. METHODS: 16S rRNA genes from fecal samples or mucosal tissues of 35 CD patients and 15 healthy controls (HC) were amplified using a universal primer set and sequenced with GS FLX Titanium. The microbial composition and diversity of each sample were analyzed with the mothur pipeline, and the association between microbial community and clinical characteristics of the patients were investigated. RESULTS: The contribution of bacterial groups to the intestinal microbial composition differed between CD and HC, especially in fecal samples. Global structure and individual bacterial abundance of intestinal microbial community were different between feces and ileocecal tissues in HC. In CD patients with active stage, relative abundances of Gammaproteobacteria and Fusobacteria were higher in both fecal and mucosal tissue samples. Moreover, the intestinal microbial community structure was altered by anti-tumor necrosis factor (anti-TNF) treatment. CONCLUSIONS: Our 16S rRNA sequence data demonstrate intestinal dysbiosis at the community level in Korean CD patients, which is similar to alterations of the intestinal microbial community seen in the western counterparts. Clinical disease activity and anti-TNF treatment might affect the intestinal microbial community structure in CD patients.

Distinct microbiome in pouchitis compared to healthy pouches in ulcerative colitis and familial adenomatous polyposis.

BACKGROUND: Pouchitis occurs in up to 50% of patients with ulcerative colitis (UC) undergoing ileal pouch anal anastomosis (IPAA). Pouchitis rarely occurs in patients with familial adenomatous polyposis (FAP) who undergo IPAA. Our aim was to compare mucosal and luminal flora in patients with UC-associated pouchitis (UCP), healthy UC pouches (HUC), and healthy FAP pouches (FAP). METHODS: Nineteen patients were enrolled in this cross-sectional study (nine UCP, three HUC, seven FAP). Patients with active pouchitis were identified using the Pouchitis Disease Activity Index (PDAI). Ileal pouch mucosal biopsies and fecal samples were analyzed with a 16S rDNA-based terminal restriction fragment length polymorphism (TRFLP) approach. Pooled fecal DNA from four UCP and four FAP pouches were sequenced for further speciation. RESULTS: TRFLP data revealed statistically significant differences in the mucosal and fecal microbiota between each group of patients. UCP samples exhibited significantly more TRFLP peaks matching Clostridium and Eubacterium genera compared to HUC and FAP pouches and fewer peaks matching Lactobacillus and Streptococcus genera compared to FAP. DNA Sanger sequencing of a subset of luminal samples revealed UCP having more identifiable sequences of Firmicutes (51.2% versus 21.2%) and Verrucomicrobia (20.2% versus 3.2%), and fewer Bacteroidetes (17.9% versus 60.5%) and Proteobacteria (9.8% versus 14.7%) compared to FAP. CONCLUSIONS: The pouch microbial environment appears to be distinctly different in the settings of UC pouchitis, healthy UC, and FAP. These findings suggest that a dysbiosis may exist in pouchitis which may be central to understanding the disease.

Two distinct proton binding sites in the ATP synthase family.

The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.

Validation of 16S rDNA sequencing in microdissected bowel biopsies from Crohn's disease patients to assess bacterial flora diversity.

The bowel flora is implicated in Crohn's disease (CD) pathogenesis but its precise role is still unclear. Several non-mutually exclusive hypotheses have been proposed: an unidentified persistent pathogen; excessive bacterial translocation; an immune system abnormality in response to normal bacteria; or a breakdown in the balance between protective and harmful bacteria. These hypotheses can be tested by identifying bacteria in specific microscopic bowel structures or lesions. The present paper describes a novel technique to assess bacterial flora diversity in bowel biopsies, by combining laser capture microdissection with broad-range 16S rDNA sequencing. Fifty-four samples comprising histologically normal and pathological mucosa, MALT, ulcers, submucosal lymphangiectasias, epithelioid granulomas, and lymph nodes were microdissected out of 30 bowel biopsies from five CD patients. Bacterial 16S rDNA was successfully amplified by PCR in all samples, and PCR products from 15 samples were selected for cloning and sequence analysis. A total of 729 bacterial DNA sequences were analysed, which could be attributed to six different phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, and Planctomycetes). DNA from typical bowel bacteria (Enterobacteriaceae, Clostridiales, Bacteroidetes, Fusobacteria) was detected in all microdissected areas. It was thus convincingly demonstrated that 16S rDNA sequencing can be combined with microdissection to study the bowel flora. However, no specific persistent pathogen causal for CD was identified. The results suggest that Enterobacteriaceae may initiate or colonize ulcers in CD. Translocation of bacteria through established mucosal lesions or as a result of increased permeability may be involved in the evolution towards chronic inflammation and in the establishment of persistent lesions. Further study is needed to confirm these preliminary findings.

Structural evidence for a constant c11 ring stoichiometry in the sodium F-ATP synthase.

The Na+-dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria.