Alterations in common marmoset gut microbiome associated with duodenal strictures

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.National Institutes of Health/[T32 OD010978]/NIH/Estados UnidosNational Institutes of Health/[P30-ES002109]/NIH/Estados UnidosUniversidad de Costa Rica/[803-C1-163]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Direct Bacterial Killing In Vitro by Recombinant Nod2 Is Compromised by Crohn's Disease-Associated Mutations

Background: A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn’s disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations. Methodology/Principal Findings: In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn’s-associated mutations. Wildtype, but not Crohn’s-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity. Conclusions/Significance: The lack of anti-bacterial activity demonstrated with Crohn’s-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphism

The effects of acetate and selected inhibitors on the phenotypic and metabolic traits of {\it Cytophaga johnsonae\/}

Cytophaga johnsonae belongs to a group of microorganisms known as the gliding bacteria. Characteristically, cells of these bacteria are able to translocate across solid surfaces but are unable to swim through liquids. Their motility is unusual in that it does not arise from detectable organelles, such as flagella, and the mechanism(s) for this motility is unknown. Since these species must make contact with solid surfaces to move, their cell envelope must play an indispensable role in translocation. The membrane of C. johnsonae contains high levels of branched-chain fatty acids, which is unusual for Gram-negative organisms. To ascertain the role that these lipids play in gliding motility, biochemical features of the cell envelope were studied by growing cells with acetate in an attempt to alter their lipid composition. Although cells grown with acetate did not demonstrate any significant differences in the cellular fatty acids, sulfonolipid synthesis did show a proportionate decrease in the presence of acetate.^ Polystyrene latex bead movement on the cell surface is considered by some to be a manifestation of the gliding machinery; however, this relationship has been questioned by others due to contradictory observations. Growth in acetate stopped the movement of beads on the cell surface, while gliding motility was unaffected. The results of this study confirm that bead movement on the cell surface is not a prerequisite for or equivalent to the phenomenon of gliding motility.^ Since acetate is a weak acid, it may act as an uncoupler of oxidative phosphorylation, and thereby decrease the energy supply of the cell. The protonmotive force has been considered to be the source of energy for gliding motility (and presumably, bead movement), and if the electrochemical gradient of the membrane were diminished, the ability of cells to glide could be affected. Intracellular ATP, respiratory activity, and protonmotive force were assessed in cells grown with acetate, as well as select inhibitors of energy-related functions. It was found that ATP and/or O\sb2 consumption of the cell could be dramatically decreased without appreciably affecting the protonmotive force. In addition, gliding motility does not depend upon the energy derived from ATP, whereas, bead movement on the cell surface may be influenced by ATP levels.

The effects of acetate and selected inhibitors on the phenotypic and metabolic traits of {\it Cytophaga johnsonae\/}

Cytophaga johnsonae belongs to a group of microorganisms known as the gliding bacteria. Characteristically, cells of these bacteria are able to translocate across solid surfaces but are unable to swim through liquids. Their motility is unusual in that it does not arise from detectable organelles, such as flagella, and the mechanism(s) for this motility is unknown. Since these species must make contact with solid surfaces to move, their cell envelope must play an indispensable role in translocation. The membrane of C. johnsonae contains high levels of branched-chain fatty acids, which is unusual for Gram-negative organisms. To ascertain the role that these lipids play in gliding motility, biochemical features of the cell envelope were studied by growing cells with acetate in an attempt to alter their lipid composition. Although cells grown with acetate did not demonstrate any significant differences in the cellular fatty acids, sulfonolipid synthesis did show a proportionate decrease in the presence of acetate.^ Polystyrene latex bead movement on the cell surface is considered by some to be a manifestation of the gliding machinery; however, this relationship has been questioned by others due to contradictory observations. Growth in acetate stopped the movement of beads on the cell surface, while gliding motility was unaffected. The results of this study confirm that bead movement on the cell surface is not a prerequisite for or equivalent to the phenomenon of gliding motility.^ Since acetate is a weak acid, it may act as an uncoupler of oxidative phosphorylation, and thereby decrease the energy supply of the cell. The protonmotive force has been considered to be the source of energy for gliding motility (and presumably, bead movement), and if the electrochemical gradient of the membrane were diminished, the ability of cells to glide could be affected. Intracellular ATP, respiratory activity, and protonmotive force were assessed in cells grown with acetate, as well as select inhibitors of energy-related functions. It was found that ATP and/or O\sb2 consumption of the cell could be dramatically decreased without appreciably affecting the protonmotive force. In addition, gliding motility does not depend upon the energy derived from ATP, whereas, bead movement on the cell surface may be influenced by ATP levels.

Mechanism of action of, and mechanism of reduced susceptibility to the novel anti-Clostridium difficile compound LFF571

LFF571 is a novel semi-synthetic analog of the thiopeptide natural product GE2270 A and a potent inhibitor of Gram-positive aerobic and anaerobic bacteria. The parent compound is a translational inhibitor that binds elongation factor Tu (EF-Tu) and blocks its function. Here we report an analogous mechanism of action for LFF571 against Clostridium difficile. In exponentially growing cultures, LFF571 inhibited protein synthesis with an IC50 of 0.06 µg/ml. We also determined the frequency and mechanism of reduced susceptibility to the compound in vitro. Single-step, spontaneous mutants of C. difficile were selected on super-inhibitory concentrations of LFF571 at a frequency of <4.5 x 10-11 to 1.2 x 10-9. No C. difficile mutants with loss of LFF571 susceptibility emerged over ten serial passages in vitro. Sequence analysis of strains with reduced susceptibility to LFF571 revealed a single nucleotide substitution (g782a) in tufB or in tufA and tufB, resulting in a G260E change in the thiopeptide binding pocket of EF-Tu. This mutation did not confer cross-resistance to the clinically-used antimicrobials vancomycin or metronidazole, nor to the newly approved drug fidaxomicin. These results support the development of LFF571 as a treatment for C. difficile infection

Long-Term Colonization Dynamics ofEnterococcus faecalisin Implanted Devices in Research Macaques

Enterococcus faecalis is a common opportunistic pathogen that colonizes cephalic recording chambers (CRCs) of macaques used in cognitive neuroscience research. We previously characterized 15 E. faecalis strains isolated from macaques at the Massachusetts Institute of Technology (MIT) in 2011. The goal of this study was to examine how a 2014 protocol change prohibiting the use of antimicrobials within CRCs affected colonizing E. faecalis strains. We collected 20 E. faecalis isolates from 10 macaques between 2013 and 2017 for comparison to 4 isolates previously characterized in 2011 with respect to the sequence type (ST) distribution, antimicrobial resistance, biofilm formation, and changes in genes that might confer a survival advantage. ST4 and ST55 were predominant among the isolates characterized in 2011, whereas the less antimicrobial-resistant lineage ST48 emerged to dominance after 2013. Two macaques remained colonized by ST4 and ST55 strains for 5 and 4 years, respectively. While the antimicrobial resistance and virulence factors identified in these ST4 and ST55 strains remained relatively stable, we detected an increase in biofilm formation ability over time in both isolates. We also found that ST48 strains were typically robust biofilm formers, which could explain why this ST increased in prevalence. Finally, we identified mutations in the DNA mismatch repair genes mutS and mutL in separate ST55 and ST4 strains and confirmed that strains bearing these mutations displayed a hypermutator phenotype. The presence of a hypermutator phenotype may complicate future antimicrobial treatment for clinically relevant E. faecalis infections in macaques.National Institutes of Health (U.S.) (Grant T32-OD010978)National Institutes of Health (U.S.) (Grant P30-ES002109

4-AMINOTHIAZOLYL ANALOGS OF GE2270 A: DESIGN, SYNTHESIS AND EVALUATION OF IMIDAZOLE ANALOGS

Imidazole analogs of the antibiotic natural product GE2270 A (1) were designed, synthesized, and evaluated for Gram positive bacteria growth inhibition. A recently reported, copper-mediated synthesis was exploited to prepare the 4-thiazolyl imidazole analogs of GE2270 A. The synthesis described represents a structurally complex, natural product-based application of this recently reported synthetic methodology. In addition, the biological evaluation of the imidazole-based analogs further define the SAR of the 4-aminothiazolyl-based template

Shotgun Metagenomics of Gastric Biopsies Reveals Compositional and Functional Microbiome Shifts in High- and Low-Gastric-Cancer-Risk Populations from Colombia, South America

ABSTRACTAlong with Helicobacter pylori infection, the gastric microbiota is hypothesized to modulate stomach cancer risk in susceptible individuals. Whole metagenomic shotgun sequencing (WMS) is a sequencing 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

Analysis of gut microbiome profiles in common marmosets (Callithrix jacchus) in health and intestinal disease

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.Universidad de Costa Rica/[803-C1-163]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET