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Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2-/- Il10-/- gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice.
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Colo/microbiologia , Proteínas de Ligação a DNA/genética , Infecções por Helicobacter/genética , Helicobacter hepaticus/patogenicidade , Interleucina-10/genética , Mucosa Intestinal/microbiologia , Mutagênese/genética , Animais , Células Epiteliais/microbiologia , Feminino , Infecções por Helicobacter/microbiologia , Inflamação/genética , Inflamação/microbiologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Interleucina-17/genética , Masculino , Camundongos , Mutação/genética , RNA Mensageiro/genética , Fatores Sexuais , Fator de Necrose Tumoral alfa/genéticaRESUMO
A novel Helicobacter species Helicobacter japonicum was isolated from the stomach and intestines of clinically normal mice received from three institutes from Japan. The novel Helicobacter sp. was microaerobic, grew at 37°C and 42°C, was catalase and oxidase positive, but urease negative. It is most closely related to the 16S rRNA gene of H.muridarum (98.6%); to the 23S rRNA gene of H.hepaticus (97.9%); to the hsp60 gene of H.typhlonius (87%). The novel Helicobacter sp. has in vitro cytolethal distending toxin (CDT) activity; its cdtB gene sequence has 83.8% identity with that of H.hepaticus The whole genome sequence of H.japonicum MIT 01-6451 has a 2.06-Mb genome length with a 37.5% G + C content. When the organism was inoculated into C57BL/129 IL10-/- mice, it was cultured from the stomach, colon and cecum of infected mice at 6 and 10 weeks post-infection. The cecum had the highest H.japonicum colonization levels by quantitative PCR. The histopathology of the lower bowel was characterized by moderate to severe inflammation, mild edema, epithelial defects, mild to severe hyperplasia, dysplasia and carcinoma. Inflammatory cytokines IFNγ, TNFα and IL17a, as well as iNOS were significantly upregulated in the cecal tissue of infected mice. These results demonstrate that the novel H.japonicum can induce inflammatory bowel disease and carcinoma in IL10-/- mice and highlights the importance of identifying novel Helicobacter spp. especially when they are introduced from outside mouse colonies from different geographic locations.
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Carcinoma/microbiologia , Helicobacter/patogenicidade , Doenças Inflamatórias Intestinais/microbiologia , Intestinos/microbiologia , Animais , Carcinoma/patologia , Helicobacter/genética , Helicobacter/isolamento & purificação , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Doenças Inflamatórias Intestinais/patologia , Interferon gama/biossíntese , Interleucina-10/genética , Interleucina-17/biossíntese , Intestinos/patologia , Japão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Tiflite/microbiologia , Tiflite/patologiaRESUMO
Background: Fecal microbiota, live-jslm (RBL; REBYOTA®), is the first Food and Drug Administration (FDA)-approved, single-dose, rectally administered, microbiota-based live biotherapeutic product for preventing Clostridioides difficile infection (CDI) recurrence. Alternative routes of administration are of clinical interest. Objectives: Evaluate the safety and efficacy of RBL administration via colonoscopy. Design: Retrospective analysis of electronic medical records of participants administered RBL via colonoscopy under FDA enforcement discretion. Methods: The number of participants with treatment and/or procedure-emergent adverse events (TEAEs) was evaluated. Treatment success and sustained clinical response, defined as the absence of CDI recurrence within 8 weeks and 6 months, respectively, were evaluated. Results: TEAEs were experienced by 75% (6/8) of participants; most were mild to moderate in severity, and none due to RBL or its administration. Most participants had treatment success (80%; 8/10); 75% (6/8) had sustained clinical response. Conclusion: Real-world safety and efficacy of RBL administered via colonoscopy were consistent with clinical trials of rectally administered RBL.
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While Helicobacter pylori infects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors in H. pylori pathogenesis, global gene expression of six H. pylori isolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factors cagA, vacA, and babB and were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin of H. pylori strains may promote increased gastric disease.
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Células Epiteliais/microbiologia , Mucosa Gástrica/citologia , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Adaptação Fisiológica , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Apoptose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Técnicas de Cocultura , Células Epiteliais/patologia , Genes Bacterianos , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Pessoa de Meia-Idade , Movimento , Filogeografia , TranscriptomaRESUMO
Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes (P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFNgamma, IL-17, TNFalpha, and iNOS at 6 and 12 mo, as well as H. pylori-specific IgG1 levels at 12 MPI (P < 0.05) and IgG2c levels at 6 and 12 MPI (P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI (P < 0.05) and also than those in infected males at 12 MPI (P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.
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Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/genética , Helicobacter pylori/patogenicidade , Mutação , Animais , Anticorpos Antibacterianos/sangue , Citocinas/genética , Proteínas de Escherichia coli/genética , Feminino , Mucosa Gástrica/imunologia , Gastrite/genética , Gastrite/metabolismo , Gastrite/microbiologia , Gastrite/patologia , Expressão Gênica , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/imunologia , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese , Óxido Nítrico Sintase Tipo II/genética , Estresse Oxidativo , Pentosiltransferases/genética , Caracteres Sexuais , Fatores de TempoRESUMO
Along with Helicobacter pylori infection, the gastric microbiota is hypothesized to modulate stomach cancer risk in susceptible individuals. Whole metagenomic shotgun sequencing (WMS) is a sequencingâ¯approach to characterize the microbiome with advantages over traditional culture and 16S rRNA sequencing including identification of bacterial and non-bacterial taxa, species/strain resolution, and functional characterization of the microbiota. In this study, we used WMS to survey the microbiome in extracted DNA from antral gastric biopsy samples from Colombian patients residing in the high-risk gastric cancer town Túquerres (n = 10, H. pylori-positive = 7) and low-risk town of Tumaco (n = 10, H. pylori-positive = 6). Kraken2/Bracken was used for taxonomic classification and abundance. Functional gene profiles were inferred by InterProScan and KEGG analysis of assembled contigs and gene annotation. The most abundant taxa represented bacteria, non-human eukaryota, and viral genera found in skin, oral, food, and plant/soil environments including Staphylococus, Streptococcus, Bacillus, Aspergillus, and Siphoviridae. H. pylori was the predominant taxa present in H. pylori-positive samples. Beta diversity was significantly different based on H. pylori-status, risk group, and sex. WMS detected more bacterial taxa than 16S rRNA sequencing and aerobic, anaerobic, and microaerobic culture performed on the same gastric biopsy samples. WMS identified significant differences in functional profiles found between H. pylori-status, but not risk or sex groups. H. pylori-positive samples were significantly enriched for H. pylori-specific genes including virulence factors such as vacA, cagA, and urease, while carbohydrate and amino acid metabolism genes were enriched in H. pylori-negative samples. This study shows WMS has the potential to characterize the taxonomy and function of the gastric microbiome as risk factors for H. pylori-associated gastric disease. Future studies will be needed to compare and validate WMS versus traditional culture and 16S rRNA sequencing approaches for characterization of the gastric microbiome.
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Gastrite , Microbioma Gastrointestinal , Infecções por Helicobacter , Helicobacter pylori , Microbiota , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/microbiologia , Colômbia , RNA Ribossômico 16S/genética , Infecções por Helicobacter/microbiologia , Gastrite/patologia , Helicobacter pylori/genética , Biópsia , Fatores de Risco , América do SulRESUMO
Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.
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Callithrix , Hematologia , Envelhecimento , Animais , Callithrix/fisiologia , Feminino , Masculino , Gravidez , Valores de Referência , Reprodução/fisiologiaRESUMO
Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets (Callithrix jacchus). Despite standardized housing, diet and husbandry, a recently described gastrointestinal syndrome characterized by duodenal ulcers and strictures was observed in a subset of marmosets sourced from the New England Primate Research Center. As changes in the gut microbiome have been associated with GI diseases, the gut microbiome of 52 healthy, non-stricture marmosets (153 samples) were compared to the gut microbiome of 21 captive marmosets diagnosed with a duodenal ulcer/stricture (57 samples). No significant changes were observed using alpha diversity metrics, and while the community structure was significantly different when comparing beta diversity between healthy and stricture cases, the results were inconclusive due to differences observed in the dispersion of both datasets. Differences in the abundance of individual taxa using ANCOM, as stricture-associated dysbiosis was characterized by Anaerobiospirillum loss and Clostridium perfringens increases. To identify microbial and serum biomarkers that could help classify stricture cases, we developed models using machine learning algorithms (random forest, classification and regression trees, support vector machines and k-nearest neighbors) to classify microbiome, serum chemistry or complete blood count (CBC) data. Random forest (RF) models were the most accurate models and correctly classified strictures using either 9 ASVs (amplicon sequence variants), 4 serum chemistry tests or 6 CBC tests. Based on the RF model and ANCOM results, C. perfringens was identified as a potential causative agent associated with the development of strictures. Clostridium perfringens was also isolated by microbiological culture in 4 of 9 duodenum samples from marmosets with histologically confirmed strictures. Due to the enrichment of C. perfringens in situ, we analyzed frozen duodenal tissues using both 16S microbiome profiling and RNAseq. Microbiome analysis of the duodenal tissues of 29 marmosets from the MIT colony confirmed an increased abundance of Clostridium in stricture cases. Comparison of the duodenal gene expression from stricture and non-stricture marmosets found enrichment of genes associated with intestinal absorption, and lipid metabolism, localization, and transport in stricture cases. Using machine learning, we identified increased abundance of C. perfringens, as a potential causative agent of GI disease and intestinal strictures in marmosets.
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Microbioma Gastrointestinal , Animais , Callithrix , Constrição Patológica , Disbiose/microbiologia , Trato GastrointestinalRESUMO
Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets. To understand the role of the microbiome in GI diseases, we characterized the gut microbiome of 91 healthy marmosets (303 samples) and 59 marmosets diagnosed with inflammatory bowel disease (IBD) (200 samples). Healthy marmosets exhibited "humanized," Bacteroidetes-dominant microbiomes. After up to 2 years of standardized diet, housing and husbandry, marmoset microbiomes could be classified into four distinct marmoset sources based on Prevotella and Bacteroides levels. Using a random forest (RF) model, marmosets were classified by source with an accuracy of 93% with 100% sensitivity and 95% specificity using abundance data from 4 Prevotellaceae amplicon sequence variants (ASVs), as well as single ASVs from Coprobacter, Parabacteroides, Paraprevotella, Phascolarctobacterium, Oribacterium and Fusobacterium. A single dysbiotic IBD state was not found across all marmoset sources, but IBD was associated with lower alpha diversity and a lower Bacteroides:Prevotella copri ratio within each source. IBD was highest in a Prevotella-dominant cohort, and consistent with Prevotella-linked diseases, pro-inflammatory genes in the jejunum were upregulated. RF analysis of serum biomarkers identified serum calcium, hemoglobin and red blood cell (RBC) counts as potential biomarkers for marmoset IBD. This study characterizes the microbiome of healthy captive common marmosets and demonstrates that source-specific microbiomes can be retained despite standardized diets and husbandry practices. Marmosets with IBD had decreased alpha diversity and a shift in the ratio of Bacteroides:Prevotella copri compared to healthy marmosets.
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Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Animais , Callithrix/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Humanos , Doenças Inflamatórias Intestinais/veterinária , PrevotellaRESUMO
In populations with similar prevalence of Helicobacter pylori infection, cancer risk can vary dramatically. Changes in composition or structure of bacterial communities in the stomach, either at the time of exposure or over the course of H. pylori infection, may contribute to gastric pathology. In this study, a population of 37 patients from the low-gastric-cancer-risk (LGCR) region of Tumaco, Colombia, and the high-gastric-cancer-risk (HGCR) region of Túquerres, Colombia, were recruited for gastric endoscopy. Antral biopsy specimens were processed for histology and bacterial isolation. Fifty-nine distinct species among 26 genera were isolated by aerobic, anaerobic, and microaerobic culture and confirmed by 16S rRNA analysis. Urease-positive Staphylococcus epidermidis and Streptococcus salivarius were frequently isolated from gastric biopsy specimens. We asked whether coinfection of H. pylori with urease-positive S. salivarius and/or S. epidermidis had a demonstrable effect on H. pylori-induced gastritis in the germfree (GF) INS-GAS mouse model. Coinfections with S. salivarius and/or S. epidermidis did not affect gastric H. pylori colonization. At 5 months postinfection, GF INS-GAS mice coinfected with H. pylori and S. salivarius had statistically higher pathological scores in the stomachs than mice infected with H. pylori only or H. pylori with S. epidermidis (P < 0.05). S. epidermidis coinfection with H. pylori did not significantly change stomach pathology, but levels of the proinflammatory cytokine genes Il-1ß, Il-17A , and Il-22 were significantly lower than in H. pylori-monoinfected mice. This study demonstrates that non-H. pylori urease-positive bacteria may play a role in the severity of H. pylori-induced gastric cancer in humans. IMPORTANCE Chronic infection with H. pylori is the main cause of gastric cancer, which is a global health problem. In two Colombian populations with high levels of H. pylori prevalence, the regional gastric cancer rates are considerably different. Host genetic background, H. pylori biotype, environmental toxins, and dietary choices are among the known risk factors for stomach cancer. The potential role of non-H. pylori gastric microbiota in gastric carcinogenesis is being increasingly recognized. In this study, we isolated 59 bacterial species from 37 stomach biopsy samples of Colombian patients from both low-gastric-cancer-risk and high-gastric-cancer-risk regions. Urease-positive S. epidermidis and S. salivarius commonly cultured from the stomachs, along with H. pylori, were inoculated into germfree INS-GAS mice. S. salivarius coinfection with H. pylori induced significantly higher gastric pathology than in H. pylori-monoinfected mice, whereas S. epidermidis coinfection caused significantly lower H. pylori-induced proinflammatory cytokine responses than in H. pylori-monoinfected mice. This study reinforces the argument that the non-H. pylori stomach microflora play a role in the severity of H. pylori-induced gastric cancer.
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Coinfecção , Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Streptococcus salivarius , Animais , Coinfecção/complicações , Citocinas , Modelos Animais de Doenças , Infecções por Helicobacter/complicações , Humanos , Imunidade , Camundongos , RNA Ribossômico 16S/genética , Staphylococcus epidermidis/genética , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/patologia , Streptococcus salivarius/genética , UreaseRESUMO
Cyclomodulins are virulence factors that modulate cellular differentiation, apoptosis, and proliferation. These include colibactin (pks), cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt). Pathogenic pks+, cnf+, and cdt+ E. coli strains are associated with inflammatory bowel disease (IBD) and colorectal cancer in humans and animals. Captive marmosets are frequently afflicted with IBD-like disease, and its association with cyclomodulins is unknown. Cyclomodulin-encoding E. coli rectal isolates were characterized using PCR-based assays in healthy and clinically affected marmosets originating from three different captive sources. 139 E. coli isolates were cultured from 122 of 143 marmosets. The pks gene was detected in 56 isolates (40%), cnf in 47 isolates (34%), and cdt in 1 isolate (0.7%). The prevalences of pks+ and cnf+ E. coli isolates were significantly different between the three marmoset colonies. 98% of cyclomodulin-positive E. coli belonged to phylogenetic group B2. Representative isolates demonstrated cyclomodulin cytotoxicity, and serotyping and whole genome sequencing were consistent with pathogenic E. coli strains. However, the presence of pks+, cnf+, or cdt+ E. coli did not correlate with clinical gastrointestinal disease in marmosets. Cyclomodulin-encoding E. coli colonize laboratory common marmosets in a manner dependent on the source, potentially impacting reproducibility in marmoset models.
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Toxinas Bacterianas/metabolismo , Callithrix/microbiologia , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Peptídeos/metabolismo , Policetídeos/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Escherichia coliRESUMO
The microbiota is heavily involved in both health and disease pathogenesis, but defining a normal, healthy microbiota in the common marmoset has been challenging. The aim of this review was to systematically review recent literature involving the gastrointestinal microbiome of common marmosets in health and disease. Twelve sources were included in this review. The gut microbiome composition was reviewed across institutions worldwide, and taxonomic shifts between healthy individuals were described. Unlike the human gut microbiome, which is dominated by Firmicutes and Bacteroidetes, the marmoset gut microbiome shows great plasticity across institutions, with 5 different phyla described as dominant in different healthy cohorts. Genera shared across institutions include Anaerobiospirillum, Bacteroides, Bifidobacterium, Collinsella, Fusobacterium, Megamonas, Megasphaera, Phascolarctobacterium, and Prevotella. Shifts in the abundance of Prevotella or Bifidobacterium or invasion by pathogens like Clostridium perfringens may be associated with disease. Changes in microbial composition have been described in healthy and diseased marmosets, but factors influencing the severe changes in microbial composition have not been established. Multi-institutional, prospective, and longitudinal studies that utilize multiple testing methodologies are required to determine sources of variability in the reporting of marmoset microbiomes. Furthermore, methods of microbial manipulation, whether by diet, enrichment, fecal microbiome transplantation, etc, need to be established to modulate and maintain robust and resilient microbiome communities in marmoset colonies and reduce the incidence of idiopathic gastrointestinal disease.
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Microbioma Gastrointestinal , Animais , Bactérias , Callithrix/microbiologia , Fezes/microbiologia , Estudos ProspectivosRESUMO
A fast-growing Mycobacterium species was cultured from draining, purulent lesions on the caudal abdomen of a 12-year-old male domestic long-haired cat. Whole-genome sequencing identified the organism as Mycobacterium porcinum.
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Mice deficient in the IL-10 pathway are the most widely used models of intestinal immunopathology. IL-17A is strongly implicated in gut disease in mice and humans, but conflicting evidence has drawn IL-17's role in the gut into question. IL-22 regulates antimicrobial and repair activities of intestinal epithelial cells (IECs) and is closely associated with IL-17A responses but it's role in chronic disease is uncertain. We report that IL-22, like IL-17A, is aberrantly expressed in colitic Il10-/- mice. While IL-22+ Th17 cells were elevated in the colon, IL-22-producing ILC3s were highly enriched in the small intestines of Il10-/- mice. Remarkably, Il10-/-Il22-/- mice did not develop colitis despite retaining high levels of Th17 cells and remaining colonized with colitogenic Helicobacter spp. Accordant with IL-22-induced IEC proliferation, the epithelia hyperplasia observed in Il10-/- animals was reversed in Il10-/-Il22-/- mice. Also, the high levels of antimicrobial IL-22-target genes, including Reg3g, were normalized in Il10-/-Il22-/- mice. Consistent with a heightened antimicrobial environment, Il10-/- mice had reduced diversity of the fecal microbiome that was reestablished in Il10-/-Il22-/- animals. These data suggest that spontaneous colitis in Il10-/- mice is driven by IL-22 and implicates an underappreciated IL-10/IL-22 axis in regulating intestinal homeostasis.
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Colite/etiologia , Colite/metabolismo , Suscetibilidade a Doenças , Interleucina-10/deficiência , Interleucinas/genética , Interleucinas/metabolismo , Animais , Biópsia , Colite/patologia , Modelos Animais de Doenças , Expressão Gênica , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunofenotipagem , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Modelos Biológicos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Interleucina 22RESUMO
Infection with Helicobacter pylori causes chronic inflammation and is a risk factor for gastric cancer. Antibiotic treatment or increased dietary folate prevents gastric carcinogenesis in male INS-GAS mice. To determine potential synergistic effects, H. pylori-infected male INS-GAS mice were fed an amino acid defined (AAD) diet with increased folate and were treated with antibiotics after 18 weeks of H. pylori infection. Antibiotic therapy decreased gastric pathology, but dietary folate had no effect. However, the combination of antibiotics and the AAD diet induced anemia, gastric hemorrhage, and mortality. Clinical presentation suggested hypovitaminosis K potentially caused by dietary deficiency and dysbiosis. Based on current dietary guidelines, the AAD diet was deficient in vitamin K. Phylloquinone administered subcutaneously and via a reformulated diet led to clinical improvement with no subsequent mortalities and increased hepatic vitamin K levels. We characterized the microbiome and menaquinone profiles of antibiotic-treated and antibiotic-free mice. Antibiotic treatment decreased the abundance of menaquinone producers within orders Bacteroidales and Verrucomicrobiales. PICRUSt predicted decreases in canonical menaquinone biosynthesis genes, menA and menD. Reduction of menA from Akkermansia muciniphila, Bacteroides uniformis, and Muribaculum intestinale were confirmed in antibiotic-treated mice. The fecal menaquinone profile of antibiotic-treated mice had reduced MK5 and MK6 and increased MK7 and MK11 compared to antibiotic-free mice. Loss of menaquinone-producing microbes due to antibiotics altered the enteric production of vitamin K. This study highlights the role of diet and the microbiome in maintaining vitamin K homeostasis.
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Antibacterianos/uso terapêutico , Disbiose/etiologia , Alimentos Formulados/efeitos adversos , Hemorragia Gastrointestinal/etiologia , Microbioma Gastrointestinal , Infecções por Helicobacter/tratamento farmacológico , Deficiência de Vitamina K/etiologia , Aminoácidos/administração & dosagem , Anemia/dietoterapia , Anemia/etiologia , Animais , Antibacterianos/efeitos adversos , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Dieta , Suplementos Nutricionais , Ácido Fólico/administração & dosagem , Ácido Fólico/biossíntese , Ácido Fólico/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Vitamina K 1/administração & dosagem , Vitamina K 1/metabolismo , Vitamina K 2/metabolismoRESUMO
The common marmoset (Callithrix jacchus) is increasingly used as an animal model for biomedical research; however, gastrointestinal diseases causing significant morbidity are endemic in many captive marmoset colonies. Establishing gut microbiome patterns in a marmoset colony may aid in clinical decision-making and model reproducibility. A standardized method of sample collection and storage is essential for proper interpretation of microbiome data. While microbiome studies commonly utilize fecal samples, the goal of this study was to determine whether the microbiome profile from a rectal swab performed on a sedated animal was comparable to the microbiome profile from a fecal sample. During routine physical exams, paired fecal and rectal swab samples were collected from each of 23 marmosets. DNA was extracted from all fecal and rectal swab samples and 16S ribosomal RNA gene sequences were amplified and analyzed. Initial comparison of the relative abundance of bacterial phyla between paired samples had a r2 value of 0.70 with S of 0.08 with no significant differences in α and ß diversity metrics between fecal and rectal samples. Initial analysis however, revealed 5 discordant fecal-rectal pairs which corresponded only with the 5 rectal swabs that were classified as free of visible fecal matter during collection. Exclusion of these 5 pairs resulted in an optimized fit of the data as evidenced by a r2 value of 0.91 with S of 0.05. These results demonstrate that rectal swabs are a reliable method for profiling the fecal microbiome in the marmoset since the bacterial composition from a rectal swab with visible fecal contents correlated well with the bacterial composition from a fecal sample from the same marmoset. This study highlights the importance of standardized sample collection methods and exclusion of inappropriate samples.
Assuntos
Microbioma Gastrointestinal , Reto/microbiologia , Manejo de Espécimes/métodos , Animais , Bactérias/isolamento & purificação , Callithrix , Fezes/microbiologia , Feminino , MasculinoRESUMO
Current methods for detecting mites in mouse colonies have limitations in terms of cost, accuracy, and throughput. To address these limitations, we developed PCR assays to detect Myocoptes musculinus in fecal samples. Using a newly generated ribosomal RNA sequence of M. musculinus (MC28S), we developed PCR and qPCR assays capable of detecting M. musculinus mites or eggs ingested during grooming. To determine our ability to detect mites, we tested fur swabs and feces from mouse colonies experimentally infested with M. musculinus and Demodex musculi, 2) Myobia musculi and Radfordia affinis, 3) M. musculinus and M. musculi, and 4) no mites (negative control). The MC28S PCR and qPCR assays positively identified M. musculinus in groups 1 and 3. The MC28S PCR assay detected M. musculinus in 9 of 10 fecal samples from known-positive animals, whereas the qPCR assay correctly identified M. musculinus in all 10 fecal samples. To our knowledge, this report is the first description of PCR-based detection of murine mites in feces. By eliminating the need for pelt examinations, mite detection from fecal samples can facilitate mite detection in sentinel or quarantine programs.
Assuntos
Pelo Animal/parasitologia , Fezes/parasitologia , Infestações por Ácaros/veterinária , Ácaros/classificação , Doenças dos Roedores/diagnóstico , Animais , Camundongos , Infestações por Ácaros/diagnóstico , Ácaros/genética , RNA Ribossômico/química , Reação em Cadeia da Polimerase em Tempo Real , Doenças dos Roedores/parasitologiaRESUMO
C57BL/6 (B6) mice from Taconic Sciences (Tac) and the Jackson Laboratory (Jax) were infected with H. pylori PMSS1 (Hp) for 16 week; there was no significant difference in the gastric histologic activity index between Hp infected Tac and Jax B6. However, the degree of gastric mucous metaplasia and Th1-associated IgG2c levels in response to Hp infection were increased in Tac mice over Jax mice, whereas the colonization levels of gastric Hp were higher by 8-fold in Jax B6 compared with Tac B6. Additionally, mRNA expression of gastric Il-1ß, Il-17A and RegIIIγ were significantly lower in the infected Tac compared to the infected Jax mice. There were significant differences in the microbial community structures in stomach, colon, and feces between Jax and Tac B6 females. Differences in gastric microbial communities between Jax and Tac B6 females are predicted to affect the metagenome. Moreover, Hp infection perturbed the microbial community structures in the stomach, colon and feces of Jax mice, but only altered the colonic microbial composition of Tac mice. Our data indicate that the GI microbiome of Tac B6 mice is compositionally distinct from Jax B6 mice, which likely resulted in different pathological, immunological, and microbial responses to Hp infection.
Assuntos
Microbioma Gastrointestinal , Infecções por Helicobacter , Helicobacter pylori/imunologia , Imunidade Inata/fisiologia , Estômago/microbiologia , Estômago/patologia , Animais , Feminino , Gastrite/imunologia , Gastrite/microbiologia , Gastrite/patologia , Microbioma Gastrointestinal/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Interações Hospedeiro-Patógeno/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade da Espécie , Estômago/imunologiaRESUMO
Purpose-bred common marmosets from domestic sources housed in a US research facility, and used in multiple drug discovery programmes, were noted to have a high incidence of spontaneous inflammatory bowel disease and sporadic cholecystitis and cholangiohepatitis. Inflammatory infiltrates increased in incidence and severity with age. Because Helicobacter spp. have been linked to gastrointestinal diseases, samples from the gastrointestinal tracts of 39 marmosets were screened for Helicobacter spp. by culture and PCR. Helicobacter spp. were frequently detected in marmosets; 28.2% of the marmosets were positive for a proposed novel species, Helicobacter jaachi sp. nov., by culture, and 48.7% were positive by Helicobacter genus-specific PCR. Seventeen strains of Helicobacter sp. from 11 marmosets were cultured from various gastrointestinal sites. Older animals (age 6-11 years) had a higher helicobacter prevalence rate (57.1%) compared with younger animals (age 3-5 years), which had a 27.2% prevalence rate. Cells of H. jaachi sp. nov. were catalase, urease and oxidase positive and had fusiform morphology, with periplasmic fibres and multiple bipolar, sheathed flagella. All isolates had similar 16S and 23S rRNA sequences, which clustered as representatives of a novel Helicobacter species closely related to 'Helicobacter sanguini' (97%), a species isolated from cotton-top tamarins and 'Helicobacter callitrichis' (96%) isolated previously from the faeces of common marmosets. The whole genome sequence of one of the liver isolates, H. jaachi sp. nov. MIT 09-6949(T), had a 1.9 Mb genome length with a 41 mol% DNA G+C content. The type strain of Helicobacter jaachi sp. nov., MIT 09-6949(T), has been deposited in the BCCM/LMG Bacteria Collection as LMG 28613(T). These findings add to the increasing number of animal species with gastrointestinal disease in which novel enterohepatic Helicobacter spp. have been isolated.
Assuntos
Callithrix , Gastroenteropatias/veterinária , Infecções por Helicobacter/veterinária , Helicobacter/genética , Doenças dos Macacos/microbiologia , Animais , DNA Bacteriano/genética , Feminino , Gastroenteropatias/microbiologia , Genoma Bacteriano , Helicobacter/classificação , Helicobacter/isolamento & purificação , Infecções por Helicobacter/microbiologia , Masculino , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genéticaRESUMO
The draft genome sequences of eight enterohepatic Helicobacter species, H. muridarum, H. trogontum, H. typhlonius, and five unnamed helicobacters, are presented here. Using laboratory mice pervasively infected with helicobacters, we characterized the presence of known virulence factors.