Propidium Monoazide Quantitative Real-Time Polymerase Chain Reaction for Enumeration of Some Viable but Nonculturable Foodborne Bacteria in Meat and Meat Products.

Abstract

Foodborne infections due to bacterial pathogens are increasing worldwide. Given the surreptitious nature of viable but nonculturable (VBNC) bacteria, they largely remain a threat to public health and food safety due to their non-detectability through conventional plate count techniques. Hence, species-specific quantitative real-time polymerase chain reaction (PCR) (qPCR) alone and combined with the use of propidium monoazide (PMA) was used along with the plate count method to quantify VBNC Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and Enterobacteriaceae in fresh and processed meat samples. The major bacterial pathogen isolated was S. aureus (93%) followed by Enterobacteriaceae (80.33%), C. perfringens (26.33%), and B. cereus (21.33%); their total viable counts were mostly recorded in raw meat than examined meat products. PMA quantitative real-time PCR (PMA qRT-PCR) could detect and quantify VBNC bacteria in 90.48% of culture-negative samples. It affirmed the presence of VBNC S. aureus (n = 10), B. cereus (n = 8), C. perfringens (n = 6), and Enterobacteriaceae (n = 12) in either single or mixed bacterial contamination. The log10 mean values of VBNC bacterial counts were highly reported for C. perfringens and S. aureus (9.60 ± 0.449 and 8.27 ± 0.453 CFU/g, respectively) followed by Enterobacteriaceae (6.95 ± 0.564 CFU/g) and B. cereus (6.69 ± 0.749 CFU/g). Sequencing of rpoB gene of Enterobacteriaceae enabled the identification of Klebsiella pneumoniae complex, Enterobacter cloacae complex, and Salmonella Typhi, which have been reported to be capable of entry into the VBNC state. To our knowledge, this is the first report at least in Egypt that records the presence of VBNC cells in meat samples representing a strong threat to public health and food safety. Moreover, PMA qRT-PCR allowed a quick and unequivocal way of enumeration of VBNC foodborne bacteria.