Ferneyhough, B. | bioRxiv Science

bioRxiv2026

Long-read shotgun metagenomics identifies distinct urogenital microbial community states associated with functional potential and urinary tract infection during pregnancy

Urinary tract infections (UTIs) are among the most common bacterial infections during pregnancy and are associated with adverse maternal and neonatal outcomes. Although previous studies used enhanced culture, 16S rRNA gene sequencing, and/or short-read shotgun metagenomic sequencing to characterize the urogenital microbiome of adult females, few have assessed the urogenital microbiome during pregnancy and thus this microbiome remains poorly understood. Here, we applied Oxford Nanopore long-read shotgun metagenomic sequencing to characterize urogenital microbial composition, functional potential, metagenome-assembled genomes (MAGs), and the urogenital virome of pregnant females. Taxonomic profiling identified three distinct microbial community states predominated by Lactobacillus crispatus, Gardnerella species, and L. iners. The Gardnerella-predominated community was associated with a higher frequency of UTI during pregnancy. Functional profiles strongly reflected these community states, demonstrating that taxonomic differences were accompanied by distinct functional potential. Genome-resolved analysis recovered 415 MAGs, including 29 high-quality genomes, revealing substantial genomic diversity among urogenital microbes. Viral sequences, predominantly bacteriophages, also were detected in most samples, highlighting an additional component of the pregnancy-associated urogenital microbiome. These findings demonstrate that pregnant females possess distinct urogenital microbial community states and show that they differ not only in taxonomic composition but also in their functional potential.

microbiology↗

bioRxiv2026

A comparison of two culture and three sequencing methods for detection of the urobiome

Standard urine culture (SUC), the clinical reference method, is optimized for fast-growing aerobes and misses much of the urinary microbiota. Enhanced culture and sequencing methods have rarely been compared on the same specimens. We compared five methods (SUC, expanded quantitative urine culture [EQUC], short-read V4 16S rRNA gene sequencing, long-read near full-length 16S rRNA gene sequencing, and PCR-free long-read shotgun metagenomic sequencing) on parallel aliquots of urine from pregnant participants screened for asymptomatic bacteriuria. Of 98 cultured samples, 98 (100.0%) were EQUC-positive while 85 (86.7%) SUC-positive. EQUC detected 102 species, but SUC only detected 41. Short-read 16S rRNA gene sequencing, long-read 16S rRNA gene sequencing, and shotgun sequencing were positive for 188/188, 186/188 and 139/139 samples, detecting 195, 69 and 528 species, respectively. In the 79 samples positive by EQUC and all three sequencing methods, sequencing profiles resembled each other more than culture profiles, and SUC captured only a subset of taxa detected by every other method. Shotgun sequencing gave the broadest taxonomic coverage and was the only sequencing method to identify Escherichia coli to the species level. With EQUC, it outperformed both 16S sequencing methods in detecting Gardnerella vaginalis and distinguishing Lactobacillus species, and it resolved six Gardnerella species, three detected by no other method. Long-read 16S rRNA gene sequencing gave the most confident amplicon-based species assignments, whereas culture better recovered Corynebacterium and Staphylococcus species. No single method was best across all endpoints, but long-read shotgun metagenomics provided the broadest species-level view. Method choice should match the research or clinical question; outcome-based studies are needed to establish clinical utility.

bioinformatics↗

bioRxiv2021

Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction

BackgroundBiopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. MethodsReproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. ResultsReproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001s bioactivity : MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal. NOTEPlease see the end of the paper for links to ten files of supplementary information and for an independent peer review. KEY POINTSO_LISynthetic hexapeptides have upregulated and downregulated nematode reproduction, following similar work in other nematode species and in fish and frogs. C_LIO_LIPeptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro and influence circulating levels of ovine pituitary hormones in vivo via intracerebroventricular peptide infusion. C_LIO_LIThe amino acid sequence of the 14mer master peptide arose from a (rat) bioassay- guided fractionation using ovine materials, whose aim was to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. C_LIO_LIThe bioactive peptides mimic the non-contiguous six-residue receptor-binding active face of a polypeptide hormone, which in mammals is a derivative of the secretory vesicle prohormone secretogranin II (SgII), taking the form of a novel, structually complex 70mer dubbed SgII-70. C_LIO_LIThe ability has been gained to reinforce or block a default reproductive and tissue- building OFF signal, using peptide mimetics. C_LI

physiology↗