Karner, T. | bioRxiv Science

bioRxiv2025

Revised 16S rRNA V4 hypervariable region targeting primers enhance detection of Patescibacteria and other lineages across diverse environments

Primer bias in 16S rRNA gene amplicon sequencing can distort microbial diversity estimates by underrepresenting key taxa. We introduce a modified primer pair (V4-EXT) targeting the hypervariable V4 region of bacterial and archaeal 16S rRNA genes, with improved in silico taxonomic inclusivity. To benchmark performance, we analyzed 938 samples from terrestrial, aquatic, and host-associated habitats, comparing microbial community profiles derived with V4-EXT and the currently most widely used V4-targeted primers. V4-EXT substantially improved the detection of Patescibacteria and other underrepresented lineages, such as Chloroflexota and Iainarchaeota, while enhancing recovery of novel amplicon sequence variants across sample types. Overall, V4-EXT provides broader taxonomic coverage and more inclusive microbial community profiles, particularly in high-diversity ecosystems such as groundwater and soils. We propose V4-EXT as a robust successor for comprehensive microbial community analysis across diverse habitats.

microbiology↗

bioRxiv2025

Gut microbial diversity and inferred capacity to produce butyrate modulate cortisol reactivity following acute stress in healthy adults

Acute stress triggers the release of stress hormones such as cortisol, increasing stress reactivity and aiding post-stress recovery. Rodent studies revealed that stress reactivity is modulated by the gut microbiota, and few interventional studies have provided evidence for an effect on human cortisol dynamics. However, it remains unclear whether stress reactivity is related to interindividual variations in gut microbial composition and to ones capacity to produce microbial metabolites such as short-chain fatty acids (SCFAs). To close this gap, we analyzed data from 74 healthy human adults who completed the study in the laboratory and were either exposed to a well-established, standardized intervention that induced acute stress or to a non-stressful control condition (n = 35/39 per stress/control group). Stool samples were obtained at baseline, and the gut microbiota were characterized through 16S rRNA gene amplicon sequencing. Cortisol changes were assessed from repeated saliva sampling, paralleled by measurements of subjectively experienced stress. We found that higher gut microbial alpha diversity was associated with higher cortisol and subjective stress reactivity across individuals of the stress group, but not in controls. Cortisol stress reactivity was also associated with the relative abundance of bacterial taxa inferred to encode metabolic pathways for the production of butyrate and propionate, two key SCFAs. The results are the first to highlight the link between gut microbial diversity, inferred SCFA production capacity, and the acute stress response in healthy adults, underscoring the microbiotas potential to flexibly modulate human psychophysiology in the aftermath of stress.

neuroscience↗