Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR

The Bacillus cereus group includes important spore forming bacteria that present spoilage capability and may cause foodborne diseases. These microorganisms are traditionally evaluated in food using culturing methods, which can be laborious and time-consuming, and may also fail to detect bacteria in a viable but nonculturable state. The purpose of this study was to develop a quantitative real-time PCR (qPCR) combined with a propidium monoazide (PMA) treatment to analyze the contamination of UHT milk by B. cereus group species viable cells. Thirty micrograms per milliliter of PMA was shown to be the most effective concentration for reducing the PCR amplification of extracellular DNA and DNA from dead cells. The quantification limit of the PMA-qPCR assay was 7.5 x 10(2) cfu/mL of milk. One hundred thirty-five UHT milk samples were analyzed to evaluate the association of PMA to qPCR to selectively detect viable cells. The PMA-qPCR was able to detect B. cereus group species in 44 samples (32.6%), whereas qPCR without PMA detected 78 positive samples (57.8%). Therefore, the PMA probably inhibited the amplification of DNA from cells that were killed during UHT processing, which avoided an overestimation of bacterial cells when using qPCR and, thus, did not overvalue potential health risks. A culture-based method was also used to detect and quantify B. cereus sensu stricto in the same samples and showed positive results in 15 (11.1%) samples. The culture method and PMA-qPCR allowed the detection of B. cereus sensu stricto in quantities compatible with the infective dose required to cause foodborne disease in 3 samples, indicating that, depending on the storage conditions, even after UHT treatment, infective doses may be reached in ready-to-consume products.