Droplet digital PCR improves absolute quantification of viable lactic acid bacteria in faecal samples

Area of Science:

• Microbiology
• Gastroenterology
• Food Science

Background:

• Assessing probiotic efficacy requires accurate methods to track live bacteria in the gut.
• Traditional culture methods struggle to quantify viable but non-culturable (VBNC) cells and lack strain-level specificity.
• Developing precise tools for quantifying ingested probiotics in fecal samples is essential for food applications.

Purpose of the Study:

• To establish and validate a viable quantitative PCR (vPCR) assay for precise strain-level quantification of probiotics in complex matrices.
• To compare the performance of real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) when combined with propidium monoazide (PMA) treatment.
• To assess the survival and viability of specific Lactobacillus strains in piglet gastrointestinal tracts.

Main Methods:

• A viable PCR (vPCR) assay was developed using propidium monoazide (PMA) treatment followed by either qPCR or ddPCR.
• The assay was optimized for piglet fecal samples to accurately discriminate between live and dead bacterial cells at the strain level.
• Three specific strains – Lactobacillus rhamnosus and two Lactobacillus paracasei subsp. paracasei – were quantified.

Main Results:

• Optimized PMA treatment and reduced sample size enabled accurate discrimination of live vs. dead cells, with varying PMA efficiency observed between L. paracasei strains.
• Both qPCR and ddPCR accurately quantified each target strain, with ddPCR demonstrating a superior lower limit of quantification for detecting lower viable cell numbers.
• All tested probiotic strains survived in the piglet gut, exhibiting viability losses between 0.78 and 1.59 log10/g feces.

Conclusions:

• The PMA-ddPCR method offers a robust and sensitive tool for quantifying low numbers of viable bacterial cells in complex samples like feces, without requiring standard curves.
• This approach is valuable for preclinical and clinical trials investigating bacterial survival and probiotic efficacy.
• Protocol optimization for PMA treatment is crucial and must be adapted to the specific matrix and bacterial strain, even for closely related ones.