Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains.

Sarah J. Z. Hansen,Wesley Morovic,Connie Wong,Anthony Kiefer,Kevin Galles,Peipei Tang

doi:10.3389/fmicb.2019.03025

Abstract

Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability – up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

Highlights

Bacteria have been used in fermented food applications and for human health benefits as probiotics (Ouwehand et al, 2000; Heller, 2001)
Probiotic products are quantified throughout the production process (Fenster et al, 2019), which is factored into the formulation of multistrain products or as individual offerings (Ouwehand et al, 2018)
Cell quantifications were performed on each batch with four methods: plate count enumeration (Plate), droplet digital PCR (ddPCR) (PCR.total), viable ddPCR (PCR.live), and flow cytometry gated for viable cells (Flow.live), dead cells (Flow.dead), and total cells (Flow.total)

Summary

Introduction

Bacteria have been used in fermented food applications and for human health benefits as probiotics (Ouwehand et al, 2000; Heller, 2001). Defined as “live microorganisms, which when administered in adequate amounts, confer a health benefit” (Hill et al, 2014), probiotics in dietary supplements typically include species of Lactobacillus and Bifidobacterium (Morovic et al, 2016; Lugli et al, 2019). Per the definition, it is crucial for the correct quantities of microbes to be ingested to accurately recapitulate clinical study doses and their associated health benefits (Fenster et al, 2019). Robust methods for quantifying probiotics are paramount to ensure efficacy, safety, and quality

Methods

Results

Conclusion