# Refining microbial biomarker identification in rumen microbiome studies: a viability PCR-based approach

## TL;DR

This study improves the identification of viable microbes in the rumen by using PMA-based v-PCR, offering a more accurate and cost-effective method than traditional DNA or RNA approaches.

## Contribution

The study identifies optimal PMA conditions for in vitro rumen experiments and demonstrates v-PCR as a reliable alternative to RNA-based methods for viable microbial analysis.

## Key findings

- PMA treatment reduced bacterial evenness and altered key bacterial and archaeal taxa in rumen cultures.
- PMA increased the relative abundance of Ruminobacter and Succinivibrio while decreasing Xylanibacter after 24 h incubation.
- PMA-based v-PCR aligns with RNA-based results, showing higher Succinivibrionaceae than Prevotellaceae abundance.

## Abstract

The rumen microbiome significantly affects host performance, influencing feed efficiency, nitrogen utilization, and methane emission. However, conventional DNA-based marker gene sequencing cannot distinguish between viable and non-viable microbes, leading to inaccurate microbiota analyses. Viability PCR (v-PCR) with propidium monoazide (PMA) can inhibit DNA amplification from membrane-compromised cells, allowing the detection of viable microbes in rumen cultures. Therefore, this study aims to identify conditions for applying PMA in rumen culture experiments using qPCR and to examine its effect on the rumen microbial community using 16S rRNA gene sequencing in standard in vitro experiments. PMA treatment conditions were applied using a fivefold inoculum dilution, 100  µM PMA concentration, 30 min dark incubation, and 20 min light exposure, validated by a decrease in absolute abundance in heat-treated samples. When applied to in vitro rumen experiments, PMA treatment reduced bacterial evenness and induced shifts in key bacterial and archaeal taxa. Additionally, it affected major functional profiles of the microbiota. PMA treatment increased the relative abundance of Ruminobacter [log fold change (LFC) = 0.52] and Succinivibrio (LFC = 0.68) at 0 h (no incubation), along with Ruminobacter (LFC = 0.83) after 24 h of incubation, while decreasing that of Xylanibacter (LFC = −0.39) at 24 h. These shifts align with those of RNA-based studies showing higher Succinivibrionaceae abundance than Prevotellaceae, supporting the effectiveness of PMA in capturing active microbial dynamics. PMA-based v-PCR offers a reliable alternative to RNA-based methods, improving microbial community assessments and facilitating the identification of viability-associated microbial biomarkers in rumen studies.

This study identifies the optimal conditions for applying propidium monoazide (PMA) in in vitro rumen experiments to selectively amplify DNA from viable microorganisms while suppressing amplification from nonviable ones. PMA-based viability PCR (v-PCR) improves the accuracy of microbial community analysis by selectively detecting viable microorganisms, addressing the limitations of conventional DNA-based methods. Additionally, this approach provides a potential cost-effective alternative to RNA-based analyses, offering a practical tool for studying rumen microbial ecology.