Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia, Egypt .
Foodborne Pathog Dis. 2018 Apr;15(4):226-234. doi: 10.1089/fpd.2017.2356. Epub 2018 Jan 3.
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 log 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.
食源性细菌病原体感染在全球范围内呈上升趋势。鉴于存活但非可培养（VBNC）细菌的隐匿性质，由于它们不能通过常规平板计数技术检测到，因此它们在很大程度上仍然是公共卫生和食品安全的威胁。因此，单独使用物种特异性实时定量聚合酶链反应（PCR）（qPCR）和结合使用吖啶单甲醚（PMA）以及平板计数法来定量新鲜和加工肉样品中的 VBNC 金黄色葡萄球菌、蜡样芽孢杆菌、产气荚膜梭菌和肠杆菌科。分离出的主要细菌病原体是金黄色葡萄球菌（93%），其次是肠杆菌科（80.33%）、产气荚膜梭菌（26.33%）和蜡样芽孢杆菌（21.33%）；它们的总活菌计数大多记录在生肉中，而不是检查的肉类产品中。PMA 定量实时 PCR（PMA qRT-PCR）可以检测和定量 90.48%的培养阴性样品中的 VBNC 细菌。它证实了 VBNC 金黄色葡萄球菌（n=10）、蜡样芽孢杆菌（n=8）、产气荚膜梭菌（n=6）和肠杆菌科（n=12）的存在，无论是单一细菌污染还是混合细菌污染。VBNC 细菌计数的对数平均值报告较高的是产气荚膜梭菌和金黄色葡萄球菌（分别为 9.60±0.449 和 8.27±0.453 CFU/g），其次是肠杆菌科（6.95±0.564 CFU/g）和蜡样芽孢杆菌（6.69±0.749 CFU/g）。肠杆菌科 rpoB 基因测序能够鉴定出肺炎克雷伯菌复合体、阴沟肠杆菌复合体和伤寒沙门氏菌，这些菌已被报道能够进入 VBNC 状态。据我们所知，这是至少在埃及首次报告在肉样中存在 VBNC 细胞，这对公共卫生和食品安全构成了严重威胁。此外，PMA qRT-PCR 允许快速、明确地对食源性 VBNC 细菌进行计数。
