Detection and Quantification of Probiotic Strains in Clinical Fecal Samples of Healthy Adults by Real‐time PCR

INTRODUCTION A randomized, double‐blind, placebo‐controlled, crossover clinical study (NCT01879098) on healthy, middle‐aged adults investigated the effects of three probiotic strains, Bifidobacterium animalis subsp. lactis B94, Lactobacillus plantarum HA119, and Bacillus subtilis R0179 on bile acid metabolism, satiety, and inflammation. In order to substantiate positive outcomes, probiotic strains must persist during transit through the gastrointestinal tract of participants. The objective of this sub‐study was to detect and quantify HA119 and R0179 at the strain level in fecal samples using previously designed primers for real‐time PCR (qPCR). METHODOLOGY Participants of the clinical study were randomized to receive one capsule per day of a probiotic strain or placebo for six weeks. Following a four week washout period, participants were crossed to the other treatment (probiotic to placebo or placebo to probiotic). Fecal samples were collected at the baseline and final time points of each intervention for a total of four stool samples per participant. DNA isolated from fecal samples of participants in the L. plantarum HA119 (n = 136) and B. subtilis R0179 (n = 124) groups were analyzed by a SYBR‐green based qPCR method for HA119 and R0179, respectively. Strain‐specific primer targets included a gene encoding a phage protein for HA119, and a hypothetical protein for R0179. All fecal samples from participants in the B. lactis B94 group were not analyzed for B94 due to the lack of strain‐specific primers. RESULTS The HA119 strain was detected in 97% of the participants in the HA119 arm. The mean HA119 level in the feces from the final time point of probiotic intervention was 7.34 ± 0.86 log bacteria/g of feces. R0179 was detected in 97% of the participants in the R0179 group at mean 6.52 ± 0.38 log bacteria/g of feces in the final time point of probiotic intervention. The absence of the probiotic strains in the feces following the four week washout period suggests that the duration of the washout period was adequate between the intervention periods. CONCLUSIONS Overall, these results demonstrate the use of strain‐specific primers for the detection and quantification of probiotic strains HA119 and R0179 in complex fecal matrices containing numerous gut microbes. The ability to show persistence of specific probiotic strains in feces could help monitor participant compliance in clinical trials. This qPCR study also suggests that a four week washout period between intervention periods is sufficient for crossover clinical studies with probiotic intervention. Support or Funding Information Lallemand Health Solutions

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