Gobert Guillaume, Cotillard Aurélie, Fourmestraux Candice, Pruvost Laurence, Miguet Jean, Boyer Mickaël
Danone Research Centre Daniel Carasso, RD 128 Avenue de la Vauve, 91700 Palaiseau Cedex, France.
Soladis, 6 et 8 Rue Bellecombe, 69006 Lyon, France.
J Microbiol Methods. 2018 May;148:64-73. doi: 10.1016/j.mimet.2018.03.004. Epub 2018 Mar 14.
Analysing correlations between the observed health effects of ingested probiotics and their survival in digestive tract allows adapting their preparations for food. Tracking ingested probiotic in faecal samples requires accurate and specific tools to quantify live vs dead cells at strain level. Traditional culture-based methods are simpler to use but they do not allow quantifying viable but non-cultivable (VBNC) cells and they are poorly discriminant below the species level. We have set up a viable PCR (vPCR) assay combining propidium monoazide (PMA) treatment and either real time quantitative PCR (qPCR) or droplet digital PCR (ddPCR) to quantify a Lactobacillus rhamnosus and two Lactobacillus paracasei subsp. paracasei strains in piglet faeces. Adjustments of the PMA treatment conditions and reduction of the faecal sample size were necessary to obtain accurate discrimination between dead and live cells. The study also revealed differences of PMA efficiency among the two L. paracasei strains. Both PCR methods were able to specifically quantify each strain and provided comparable total bacterial counts. However, quantification of lower numbers of viable cells was best achieved with ddPCR, which was characterized by a reduced lower limit of quantification (improvement of up to 1.76 log compared to qPCR). All three strains were able to survive in the piglets' gut with viability losses between 0.78 and 1.59 log/g faeces. This study shows the applicability of PMA-ddPCR to specific quantification of small numbers of viable bacterial cells in the presence of an important background of unwanted microorganisms, and without the need to set up standard curves. It also illustrates the need to adapt PMA protocols according to the final matrix and target strain, even for closely related strains. The PMA-ddPCR approach provides a new tool to quantify bacterial survival in faecal samples from a preclinical and clinical trial.
分析摄入的益生菌对健康产生的影响与其在消化道中的存活率之间的相关性，有助于调整其食品制剂。在粪便样本中追踪摄入的益生菌，需要准确且特异的工具来在菌株水平上定量活细胞与死细胞。传统的基于培养的方法使用起来更简单，但无法定量活的但不可培养（VBNC）的细胞，且在种以下水平的鉴别能力较差。我们建立了一种活菌PCR（vPCR）检测方法，该方法结合了单叠氮碘化丙啶（PMA）处理以及实时定量PCR（qPCR）或微滴数字PCR（ddPCR），以定量仔猪粪便中的鼠李糖乳杆菌和两株副干酪乳杆菌亚种副干酪乳杆菌。为了准确区分死细胞和活细胞，需要调整PMA处理条件并减少粪便样本量。该研究还揭示了两株副干酪乳杆菌之间PMA效率的差异。两种PCR方法都能够特异性地定量每种菌株，并提供相当的总细菌计数。然而，对于较低数量的活细胞，使用ddPCR能最好地实现定量，其特征在于定量下限降低（与qPCR相比提高了1.76个对数）。所有三株菌株都能够在仔猪肠道中存活，每克粪便中的存活率损失在0.78至1.59个对数之间。本研究表明，PMA-ddPCR适用于在存在大量不需要的微生物背景的情况下，对少量活细菌细胞进行特异性定量，且无需建立标准曲线。它还说明了即使对于密切相关的菌株，也需要根据最终基质和目标菌株调整PMA方案。PMA-ddPCR方法为在临床前和临床试验中定量粪便样本中的细菌存活率提供了一种新工具。
