Development and Use of a Real-Time Quantitative PCR Method for Detecting and Quantifying Equol-Producing Bacteria in Human Faecal Samples and Slurry Cultures

Abstract

This work introduces a novel real-time quantitative PCR (qPCR) protocol for detecting and quantifying equol-producing bacteria. To this end, two sets of primers targeting the dihydrodaidzein reductase (<i>ddr</i>) and tetrahydrodaidzein reductase (<i>tdr</i>) genes, which are involved in the synthesis of equol, were designed. The primers showed high specificity and sensitivity when used to examine DNA from control bacteria, such as <i>Slackia isoflavoniconvertens, Slackia equolifaciens, Asaccharobacter celatus, Adlercreutzia equolifaciens</i>, and <i>Enterorhabdus mucosicola</i>. To demonstrate the validity and reliability of the protocol, it was used to detect and quantify equol-producing bacteria in human faecal samples and their derived slurry cultures. These samples were provided by 18 menopausal women under treatment of menopause symptoms with a soy isoflavone concentrate, among whom three were known to be equol-producers given the prior detection of the molecule in their urine. The <i>tdr</i> gene was detected in the faeces of all these equol-producing women at about 4-5 log₁₀ copies per gram of faeces. In contrast, the <i>ddr</i> gene was only amplified in the faecal samples of two of these three women, suggesting the presence in the non-amplified sample of reductase genes unrelated to those known to be involved in equol formation and used for primer design in this study. When <i>tdr</i> and <i>ddr</i> were present in the same sample, similar copy numbers of the two genes were recorded. However, no significant increase in the copy number of equol-related genes along isoflavone treatment was observed. Surprisingly, positive amplification for both <i>tdr</i> and <i>ddr</i> genes was obtained in faecal samples and derived slurry cultures from two non-equol producing women, suggesting the genes could be non-functional or the daidzein metabolized to other compounds in samples from these two women. This novel qPCR tool provides a technique for monitoring gut microbes that produce equol in humans. Monitoring equol-producing bacteria in the human gut could provide a means of evaluating strategies aimed at increasing the endogenous formation of this bioactive compound.