Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/348664

Design and assessment of species-level qPCR primers targeting comammox

Keene-Beach, N.·Noguera, D. R.
View original source ↗Export metadata ↓

Abstract

Published PCR primers targeting the ammonia monooxygenase gene (amoA) were applied to samples from activated sludge systems operated with low dissolved oxygen (DO) to quantify total and clade-level Nitrospira that perform complete ammonium oxidation (comammox); however, we found these existing primers resulted in significant artifact-associated non-target amplification. This not only overestimated comammox amoA copies but also resulted in numerous false positive detections in the environmental samples tested, as confirmed by gel electrophoresis. Therefore, to more accurately quantify known comammox, we designed specific and sensitive primers targeting three candidate species: Candidatus (Ca.) Nitrospira nitrosa, Ca. N. inopinata, and Ca. N. nitrificans. The primers were tested with amoA templates of these candidate species, and used to quantify comammox at the species level in low DO activated sludge systems. We found that comammox related to Ca. N. nitrosa were present and abundant in the majority of samples from low DO bioreactors and were not detected in samples from a high DO system. In addition, the greatest abundance of Ca. N. nitrosa was found in bioreactors operated with a long solids retention time. Ca. N. inopinata and Ca. N. nitrificans were only detected sporadically in these samples, indicating a minor role of these comammox in nitrification under low DO conditions.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC=\"FIGDIR/small/348664_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@2e42fcorg.highwire.dtl.DTLVardef@12fc927org.highwire.dtl.DTLVardef@3f44faorg.highwire.dtl.DTLVardef@1c9aa73_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract artC_FLOATNO C_FIG

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Keene-Beach, N., Noguera, D. R.. 2018-06-15. Design and assessment of species-level qPCR primers targeting comammox. https://doi.org/10.1101/348664

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Lipid nanoparticle-encapsulated mRNA encoding CagW, a conserved component of the Helicobacter pylori type IV secretion system, protects mice against H. pylori challenge

Background: Roughly one person in two carries Helicobacter pylori, a pathogen classified by the World Health Organization as a group I carcinogen, and the steady loss of antibiotic efficacy has made prophylaxis an urgent rather than an optional goal. No mRNA vaccine against this organism has yet been tested in the clinic, and CagW, a conserved inner-membrane subunit of the H. pylori type IV secretion system (T4SS), has never been evaluated as an mRNA-encoded antigen. Methods: A codon-optimised construct carrying a signal peptide, the CagW coding sequence and a human IgG Fc tag was synthesised, transcribed into mRNA, encapsulated in lipid nanoparticles (LNPs) and shown to direct antigen expression in HEK293T cells. C57BL/6 mice received three intramuscular doses of CagW-Fc mRNA-LNPs (5 ug mRNA each) at 14-day intervals; animals given alum alone, luciferase mRNA-LNPs or CagW protein adsorbed to alum served as comparators. Humoral readouts (IgG, IgG1, IgG2a, IgM, IgA and secretory IgA), lymphocyte proliferation, CD4+/CD8+ subsets, intracellular IFN-{gamma}, IL-4 and IL-17A and circulating cytokines were quantified before challenge, and protection was assessed after live challenge. Results: The formulation (93.2 nm; PDI 0.13; approximately 90% encapsulation) produced CagW-specific protein in vitro. Vaccination raised high-titre CagW-specific IgG (maximal on day 42), IgM and mucosal sIgA, and drove Th1/Th2-type cellular activation (IL-2, IL-4, IL-12, IFN-{gamma} and IL-17A). After challenge, immunised animals carried markedly fewer gastric bacteria and showed lower urease activity than controls. Conclusions: CagW-Fc mRNA-LNPs are therefore a credible mRNA vaccine platform for H. pylori and warrant further preclinical development.

microbiology↗
bioRxiv2026

Experimental evolution reveals temporally opposing roles for Csu pili during Acinetobacter baumannii respiratory infection

Multidrug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen responsible for a wide range of infections, including ventilator-associated pneumonia, bloodstream infections, and urinary tract infections. Most studies investigating A. baumannii pulmonary pathogenesis rely on acute pneumonia models that use high bacterial inocula and do not recapitulate the prolonged infections observed in some patients. Recently, our laboratory developed a murine model of chronic pulmonary infection that enabled the identification of virulence factors specifically required for long-term lung persistence. To investigate how A. baumannii adapts during chronic pulmonary infection, we performed an experimental evolution study using this model. This approach revealed an unexpected role for the archaic chaperone-usher (Csu) pili in A. baumannii pathogenesis. At early time points, Csu expression reduced bacterial burdens in both acute and chronic pneumonia models, an effect associated with enhanced host inflammatory responses. In contrast, at later stages of chronic infection, Csu promoted bacterial persistence, potentially by facilitating adhesion to pulmonary epithelial cells. These opposing effects were conserved across two distinct A. baumannii clinical isolates. We also found that Csu contributes to bacterial colonization in a catheter-associated urinary tract infection (CAUTI) model in a strain-dependent manner. Together, these findings reveal an infection-stage-dependent role for Csu in A. baumannii pathogenesis, demonstrating that a bacterial surface structure can impair early colonization while promoting long-term persistence.

microbiology↗
bioRxiv2026

A Vma13-targeted thiadiazole-succinimide disrupts V-ATPase dependent arginine-polyamine homeostasis to block Candida albicans escape from macrophages

Invasive candidiasis caused by Candida albicans is a life-threatening nosocomial infection with limited therapies and high morbidity and mortality among immunocompromised patients. Within host phagocytes, C. albicans catabolizes arginine to produce ammonia and fuels polyamine biosynthesis, which correlates with phagosomal alkalinization and yeast-to-hypha morphogenesis, facilitating macrophage escape and systemic dissemination. However, new antifungal strategies blocking this fungal escape remain scarce. Here, we characterize the thiadiazole-succinimide derivative ETSI, an arginine-dependent antifungal small molecule with minimal direct growth inhibition under arginine-free conditions yet robust anti-C. albicans activity in arginine-rich microenvironments mimicking the macrophage intracellular niche. Mechanistically, ETSI targets the vacuolar Vma13 to inhibit vacuolar H-ATPase (V-ATPase), dissipating vacuolar proton (H+) gradients and leading to cytoplasmic H+ retention. The accumulated cytoplasmic H+ traps intracellular ammonia and blocks its external release, while impaired vacuolar metabolite sequestration further triggers cytotoxic accumulation of arginine and polyamine metabolites. This dual metabolic perturbation suppresses phagosomal alkalinization, retains C. albicans in yeast form and blocks macrophage escape. ETSI is the first small molecule blocking C. albicans escape via disrupted Vma13/V-ATPase dependent arginine-polyamine homeostasis. This work identifies an intracellular metabolic vulnerability and establishes a niche-targeted antifungal strategy distinct from classic growth-suppressive drugs for treating invasive candidiasis.

microbiology↗