A high-throughput LC-MS/MS method for the measurement of the bile acid/salt content in microbiome-derived sample sets

Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode – ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period

Investigating Colonization of the Healthy Adult Gastrointestinal Tract by Fungi

A wide diversity of fungi have been detected in the human gastrointestinal (GI) tract with the potential to provide or influence important functions. However, many of the fungi most commonly detected in stool samples are also present in food or the oral cavity. Therefore, to recognize which gut fungi are likely to have a sustained influence on human health, there is a need to separate transient members of the GI tract from true colonizers. To identify colonizing fungi, the eukaryotic rRNA operon’s second internal transcribed spacer (ITS2) was sequenced from the stool, saliva, and food of healthy adults following consumption of different controlled diets. Unlike most bacterial 16S rRNA genes, the only fungal ITS2 operational taxonomic units (OTUs) detected in stool DNA across multiple diets were also present in saliva and/or food. Additional analyses, including culture-based approaches and sequencing of the 18S rRNA gene, ITS2 cDNA, and DNA extracted using alternative methods, failed to detect additional fungi. Two abundant fungi, Saccharomyces cerevisiae and Candida albicans, were examined further in healthy volunteers. Saccharomyces became undetectable in stool when a S. cerevisiae-free diet was consumed, and the levels of C. albicans in stool were dramatically reduced by more frequent cleaning of teeth. Extremely low fungal abundance, the inability of fungi to grow under conditions mimicking the distal gut, and evidence from analysis of other public datasets further support the hypothesis that fungi do not routinely colonize the GI tracts of healthy adults

The gut mycobiome of the Human Microbiome Project healthy cohort

Background: Most studies describing the human gut microbiome in healthy and diseased states have emphasized the bacterial component, but the fungal microbiome (i.e., the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. To date, human gut mycobiome studies have primarily been disease centric or in small cohorts of healthy individuals. To contribute to existing knowledge of the human mycobiome, we investigated the gut mycobiome of the Human Microbiome Project (HMP) cohort by sequencing the Internal Transcribed Spacer 2 (ITS2) region as well as the 18S rRNA gene. Results: Three hundred seventeen HMP stool samples were analyzed by ITS2 sequencing. Fecal fungal diversity was significantly lower in comparison to bacterial diversity. Yeast dominated the samples, comprising eight of the top 15 most abundant genera. Specifically, fungal communities were characterized by a high prevalence of Saccharomyces, Malassezia, and Candida, with S. cerevisiae, M. restricta, and C. albicans operational taxonomic units (OTUs) present in 96. 8, 88.3, and 80.8% of samples, respectively. There was a high degree of inter- and intra-volunteer variability in fungal communities. However, S. cerevisiae, M. restricta, and C. albicans OTUs were found in 92.2, 78.3, and 63.6% of volunteers, respectively, in all samples donated over an approximately 1-year period. Metagenomic and 18S rRNA gene sequencing data agreed with ITS2 results; however, ITS2 sequencing provided greater resolution of the relatively low abundance mycobiome constituents. Conclusions: Compared to bacterial communities, the human gut mycobiome is low in diversity and dominated by yeast including Saccharomyces, Malassezia, and Candida. Both inter- and intra-volunteer variability in the HMP cohort were high, revealing that unlike bacterial communities, an individual’s mycobiome is no more similar to itself over time than to another person’s. Nonetheless, several fungal species persisted across a majority of samples, evidence that a core gut mycobiome may exist. ITS2 sequencing data provided greater resolution of the mycobiome membership compared to metagenomic and 18S rRNA gene sequencing data, suggesting that it is a more sensitive method for studying the mycobiome of stool samples

A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ?CD08011, ?CD418, ?CD1801, and ?CD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ?CD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ?CD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI

Complete genome sequence of Candidatus Ruthia magnifica

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Mollusca) is a member of the Vesicomyidae. Species within this family form symbioses with chemosynthetic Gammaproteobacteria. They exist in environments such as hydrothermal vents and cold seeps and have a rudimentary gut and feeding groove, indicating a large dependence on their endosymbionts for nutrition. The C. magnifica symbiont, Candidatus Ruthia magnifica, was the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced (Newton et al. 2007). Here we expand upon the original report and provide additional details complying with the emerging MIGS/MIMS standards. The complete genome exposed the genetic blueprint of the metabolic capabilities of the symbiont. Genes which were predicted to encode the proteins required for all the metabolic pathways typical of free-living chemoautotrophs were detected in the symbiont genome. These include major pathways including carbon fixation, sulfur oxidation, nitrogen assimilation, as well as amino acid and cofactor/vitamin biosynthesis. This genome sequence is invaluable in the study of these enigmatic associations and provides insights into the origin and evolution of autotrophic endosymbiosis

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.United States. Public Health Service (Grant GM050895

Temporal changes in gastrointestinal fungi and the risk of autoimmunity during early childhood: the TEDDY study

Fungal infections are a major health problem that often begin in the gastrointestinal tract. Gut microbe interactions in early childhood are critical for proper immune responses, yet there is little known about the development of the fungal population from infancy into childhood. Here, as part of the TEDDY (The Environmental Determinants of Diabetes in the Young) study, we examine stool samples of 888 children from 3 to 48 months and find considerable differences between fungi and bacteria. The metagenomic relative abundance of fungi was extremely low but increased while weaning from milk and formula. Overall fungal diversity remained constant over time, in contrast with the increase in bacterial diversity. Fungal profiles had high temporal variation, but there was less variation from month-to-month in an individual than among different children of the same age. Fungal composition varied with geography, diet, and the use of probiotics. Multiple Candida spp. were at higher relative abundance in children than adults, while Malassezia and certain food-associated fungi were lower in children. There were only subtle fungal differences associated with the subset of children that developed islet autoimmunity or type 1 diabetes. Having proper fungal exposures may be crucial for children to establish appropriate responses to fungi and limit the risk of infection: the data here suggests those gastrointestinal exposures are limited and variable.</p

The Calyptogena magnifica chemoautotrophic symbiont genome

Chemoautotrophic endosymbionts are the metabolic cornerstone of hydrothermal vent communities, providing invertebrate hosts with nearly all of their nutrition. The Calyptogena magnifica (Bivalvia: Vesicomyidae) symbiont, Candidatus Ruthia magnifica, is the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced, revealing a suite of metabolic capabilities. The genome encodes major chemoautotrophic pathways as well as pathways for biosynthesis of vitamins, cofactors, and all 20 amino acids required by the clam

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated