Prediction of high fecal propionate-to-butyrate ratios using 16S rRNA-based detection of bacterial groups with liquid array diagnostics.

Butyrate and propionate represent two of three main short-chain fatty acids produced by the intestinal microbiota. In healthy populations, their levels are reportedly equimolar, whereas a deviation in their ratio has been observed in various diseased cohorts. Monitoring such a ratio represents a valuable metric; however, it remains a challenge to adopt short-chain fatty acid detection techniques in clinical settings because of the volatile nature of these acids. Here we aimed to estimate short-chain fatty acid information indirectly through a novel, simple quantitative PCR-compatible assay (liquid array diagnostics) targeting a limited number of microbiome 16S markers. Utilizing 15 liquid array diagnostics probes to target microbiome markers selected by a model that combines partial least squares and linear discriminant analysis, the classes (normal vs high propionate-to-butyrate ratio) separated at a threshold of 2.6 with a prediction accuracy of 96%.

HumGut: a comprehensive human gut prokaryotic genomes collection filtered by metagenome data.

BACKGROUND: A major bottleneck in the use of metagenome sequencing for human gut microbiome studies has been the lack of a comprehensive genome collection to be used as a reference database. Several recent efforts have been made to re-construct genomes from human gut metagenome data, resulting in a huge increase in the number of relevant genomes. In this work, we aimed to create a collection of the most prevalent healthy human gut prokaryotic genomes, to be used as a reference database, including both MAGs from the human gut and ordinary RefSeq genomes. RESULTS: We screened > 5,700 healthy human gut metagenomes for the containment of > 490,000 publicly available prokaryotic genomes sourced from RefSeq and the recently announced UHGG collection. This resulted in a pool of > 381,000 genomes that were subsequently scored and ranked based on their prevalence in the healthy human metagenomes. The genomes were then clustered at a 97.5% sequence identity resolution, and cluster representatives (30,691 in total) were retained to comprise the HumGut collection. Using the Kraken2 software for classification, we find superior performance in the assignment of metagenomic reads, classifying on average 94.5% of the reads in a metagenome, as opposed to 86% with UHGG and 44% when using standard Kraken2 database. A coarser HumGut collection, consisting of genomes dereplicated at 95% sequence identity-similar to UHGG, classified 88.25% of the reads. HumGut, half the size of standard Kraken2 database and directly comparable to the UHGG size, outperforms them both. CONCLUSIONS: The HumGut collection contains > 30,000 genomes clustered at a 97.5% sequence identity resolution and ranked by human gut prevalence. We demonstrate how metagenomes from IBD-patients map equally well to this collection, indicating this reference is relevant also for studies well outside the metagenome reference set used to obtain HumGut. All data and metadata, as well as helpful code, are available at http://arken.nmbu.no/~larssn/humgut/ . Video Abstract.

Effects of Dietary Fibres on Acute Indomethacin-Induced Intestinal Hyperpermeability in the Elderly: A Randomised Placebo Controlled Parallel Clinical Trial.

The effect of dietary fibres on intestinal barrier function has not been well studied, especially in the elderly. We aimed to investigate the potential of the dietary fibres oat ß-glucan and wheat arabinoxylan to strengthen the intestinal barrier function and counteract acute non-steroid anti-inflammatory drug (indomethacin)-induced hyperpermeability in the elderly. A general population of elderly subjects (≥65 years, n = 49) was randomised to a daily supplementation (12g/day) of oat ß-glucan, arabinoxylan or placebo (maltodextrin) for six weeks. The primary outcome was change in acute indomethacin-induced intestinal permeability from baseline, assessed by an in vivo multi-sugar permeability test. Secondary outcomes were changes from baseline in: gut microbiota composition, systemic inflammatory status and self-reported health. Despite a majority of the study population (85%) showing a habitual fibre intake below the recommendation, no significant effects on acute indomethacin-induced intestinal hyperpermeability in vivo or gut microbiota composition were observed after six weeks intervention with either dietary fibre, compared to placebo.

Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae.

The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae.

Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili.

Several major bacterial pathogens and related commensal species colonizing the human mucosa express phosphocholine (PC) at their cell surfaces. PC appears to impact host-microbe biology by serving as a ligand for both C-reactive protein and the receptor for platelet-activating factor. Type IV pili of Neisseria gonorrhoeae (Ng) and Neisseria meningitidis, filamentous protein structures critical to the colonization of their human hosts, are known to react variably with monoclonal antibodies recognizing a PC epitope. However, the structural basis for this reactivity has remained elusive. To address this matter, we exploited the finding that the PilE pilin subunit in Ng mutants lacking the PilV protein acquired the PC epitope independent of changes in pilin primary structure. Specifically, we show by using mass spectrometry that PilE derived from the pilV background is composed of a mixture of subunits bearing O-linked forms of either phosphoethanolamine (PE) or PC at the same residue, whereas the wild-type background carries only PE at that same site. Therefore, PilV can influence pilin structure and antigenicity by modulating the incorporation of these alternative modifications. The disaccharide covalently linked to Ng pilin was also characterized because it is present on the same peptides bearing the PE and PC modifications and, contrary to previous reports, was found to be linked by means of 2,4-diacetamido-2,4,6-trideoxyhexose. Taken together, these findings provide new insights into Ng type IV pilus structure and antigenicity and resolve long-standing issues regarding the nature of both the PC epitope and the pilin glycan.