bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.11.26.690772

A Validated Method for Detection of Bifidobacterium infantis Bi-26™ DNA in Stool by Quantitative Real-Time PCR

Godderz, M.·Sunshine, J.·Polke, M.·Rhyne, P.·Taylor, N.·Kiefer, A.·Yeung, N.·Wisplinghoff, H.·Zechner, U.·Frahm, N.
View original source ↗Export metadata ↓

Abstract

BackgroundBifidobacterium longum subspecies infantis (B. infantis) is a commensal bacterium enriched in the microbiota of breastfed infants and associated with beneficial effects on infant gut health. Clinical studies evaluating B. infantis supplementation in infants have reported increased colonization, reduced inflammation, enhanced vaccine responsiveness, and improved growth outcomes. To support rigorous evaluation of these effects in further clinical trials, reliable and validated methods for quantifying B. infantis in stool are needed. MethodsWe validated a quantitative real-time PCR (qPCR) assay against a conserved B. infantis genomic target. Assay performance was evaluated using DNA from the B. infantis strain Bi-26TM, spiked infant stool pools, and matrix controls. Validation parameters included linearity, sensitivity, accuracy, within- and between-run reproducibility, recovery, and matrix interference, with acceptance criteria informed by bioanalytical best practices. ResultsThe assay demonstrated robust performance across validation parameters. Linearity was confirmed over at least six orders of magnitude (R2 > 0.99; mean efficiency 96.4%), with a lower limit of quantification of 37.36 genome equivalents / reaction. Analytical sensitivity was high, with a 95% limit of detection of 4.67 genome equivalents / reaction. Accuracy across multiple stool pools met acceptance criteria, with mean deviation from nominal values of 0.39 log10. Reproducibility was strong, with within-run variability of 0.37% and between-run variability of 1.78%. Recovery averaged 41.3%, exceeding the predefined minimum threshold, and matrix interference was minimal. ConclusionsThis study reports a validated qPCR assay suitable for quantifying B. infantis Bi-26 DNA in stool samples collected in stabilized formats. Its robust analytical performance supports its use in clinical and translational research evaluating B. infantis colonization and its health impacts in early infant life.

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Godderz, M., Sunshine, J., Polke, M., Rhyne, P., Taylor, N., Kiefer, A., Yeung, N., Wisplinghoff, H., Zechner, U., Frahm, N.. 2025-11-26. A Validated Method for Detection of Bifidobacterium infantis Bi-26™ DNA in Stool by Quantitative Real-Time PCR. https://doi.org/10.1101/2025.11.26.690772

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

KEEP EXPLORING

Related preprints

bioRxiv2026

Extreme temperature exposure has negative demographic consequences for Sulfolobus acidocaldarius

Microorganisms inhabiting geothermal springs and volcanic systems experience fluctuating temperatures that can periodically exceed their upper thermal limits, but the demographic consequences of such exposure remain poorly understood. Here, we investigated demographic responses of the thermophilic archaeon Sulfolobus acidocaldarius to an extreme temperature (94.1{degrees}C) under two regimes: sustained exposure varying in duration, and episodic exposure interspersed with recovery at a permissive temperature (75{degrees}C). Under sustained exposure, populations showed no detectable loss of viability after 15 min but declined thereafter, decreasing by approximately five orders of magnitude after 120 min. Under episodic exposure, populations remained viable across nine exposure-recovery cycles but declined in density with successive cycles. Similar responses were observed for three strains, including a DNA mismatch repair knockout ({Delta}nucS), indicating that mismatch repair deficiency did not affect viability or recovery. Together, these results demonstrate that S. acidocaldarius can withstand brief and repeated exposure to near-boiling temperatures, with mortality determined primarily by cumulative exposure duration rather than a fixed thermal threshold.

microbiology↗
bioRxiv2026

Bacteriophage and Antibiotic Resistance Are Positively Associated across a Phylogenetically Diverse Set of Clinical Pseudomonas aeruginosa Isolates

Co-administration of phages and antibiotics has been proposed as a therapeutic approach against antibiotic-resistant bacteria. The relationship, however, between antibiotic resistance and phage resistance in clinical isolates is unclear. Here, we examine associations between phage and antibiotic resistance profiles across a panel of Pseudomonas aeruginosa clinical isolates from the Centers for Disease Control (CDC) and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate (ARI) Bank comprising 55 clinical strains with full genome sequences and antibiotic susceptibility testing (AST) data for 11 clinically relevant antibiotics. As phages in this study, we use three well-characterized, morphologically distinct phages, OMKO1, Luz19, and PAML31-1. We screen for phage resistance using a growth suppression assay, then conduct statistical analysis against antibiotic MIC (Minimum Inhibitory Concentration) data provided by the CDC to define association patterns across this dataset. We find multiple significant susceptibility correlations between pairs of antibiotics and phages, and a positive overall association between average phage resistance and antibiotic resistance across the 55 strains, even controlling for phylogenetic associations (=0.358, p<0.005). We conclude that phage and antibiotic resistance are positively associated across this clinical isolate collection, suggesting that the two resistance phenotypes are not independent in P. aeruginosa. These findings have implications for the development of phage-antibiotic cocktails.

microbiology↗
bioRxiv2026

The Estuary Effect: Variations in Temperature and Salinity Alter msh Promoter Activity in Vibrio cholerae

Vibrio cholerae, the facultative pathogen underlying cholera, naturally inhabits warm aquatic estuaries. Environmental persistence is enhanced by the ability of V. cholerae to colonize host reservoirs and form multicellular biofilms, causing seasonally endemic outbreaks in many tropical regions. Most toxigenic strains utilize the type IVa mannose-sensitive hemagglutinin (MSHA) pilus for host reservoir colonization and biofilm formation. Temperature and salinity can alter V. cholerae biofilm formation, yet their impact on MSHA production specifically remains largely unknown. Here, we utilized transcriptional reporters of predicted msh promoters (msh-P1/msh-P2/msh-P3) and functional assays, to determine temperature and salinity impacts on msh expression and pilus biogenesis. Under standard laboratory conditions (30{degrees}C, 1% NaCl) only msh-P1/P2 are active and inversely-coordinated with one another. Both msh-P1/P2 activity were elevated by high temperature (37{degrees}C) and low salinity (0.25%/0.5% NaCl), and reduced by low temperature (20{degrees}C/25{degrees}C) and high salinity (2%/3% NaCl). Temperature-mediated alterations in promoter activity were not immediately reflected in changes to cell-surface MSHA levels, whereas high salinity led to decreased MSHA production. Combining high temperature (37{degrees}C) and high salinity (2%/3% NaCl), attenuated the salinity-mediated reduction of msh-P1/P2 activity. Biofilm biomass levels were only substantially heightened at 25{degrees}C and 20{degrees}C, likely a result of no temperature-dependent changes in cell-surface MSHA, and additional temperature-controlled biofilm regulation previously described. We also found msh-P1/P2 promoter activity and MSHA production varies widely across toxigenic O1 and O139 serogroups despite complete sequence homology. These results shed new light on how key signals regulate MSHA pilus production to support V. cholerae persistence in aquatic environments.

microbiology↗