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 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.

Point-of-Care Lateral Flow Detection of Viable Escherichia coli O157:H7 Using an Improved Propidium Monoazide-Recombinase Polymerase Amplification Method

“…No significant difference was observed in VBNC detection for all three spiked samples, supporting the real-time application of the proposed assay for multiple matrices with reliable reproducibility. Our results support and extend the results from previous studies in determining dead/live or VBNC E. coli O157:H7 in various complex matrices combining PMA dye with LAMP, qPCR, and PCR [ 1 , 4 , 10 , 14 , 46 , 54 ]. Some reports suggest that VBNC cells can reacquire their active state and become viable in the presence of nutrients and favourable conditions, However, no resuscitation of E. coli O157:H7 cells was observed in the presented study when VBNC cells were transferred to water, apple juice, and milk [ 11 , 15 , 46 ].…”

“…Our results support and extend the results from previous studies in determining dead/live or VBNC E. coli O157:H7 in various complex matrices combining PMA dye with LAMP, qPCR, and PCR [ 1 , 4 , 10 , 14 , 46 , 54 ]. Some reports suggest that VBNC cells can reacquire their active state and become viable in the presence of nutrients and favourable conditions, However, no resuscitation of E. coli O157:H7 cells was observed in the presented study when VBNC cells were transferred to water, apple juice, and milk [ 11 , 15 , 46 ]. The revival capacity of different cells may vary with different food matrices or environmental conditions.…”

“…To overcome these issues, a DNA intercalating dye, PMAxx has been proposed to be combined with nucleic acid amplification methods. PMAxx can enter membrane-compromised cells and covalently bind to cellular DNA by photocatalytic cross-linking, resulting in no DNA amplification [ 28 , 29 , 44 , 45 , 46 ]. The application of PMAxx with qPCR, LAMP, and PCR has been reported to quantify live E. coli O157:H7 in various food samples [ 1 , 4 , 14 , 47 , 48 , 49 ].…”

Antibacterial activity of bioactive compounds extracted from red kidney bean (Phaseolus vulgaris L.) seeds against multidrug-resistant Enterobacterales

“…This aligns with previous research ( Enayat et al, 2012 ; Elhawary et al, 2016 ) in which Enterobacterales isolates were detected in meat samples with percentages of 67 and 54%, respectively. However, Abd El-Aziz et al (2018) reported Enterobacterales in 80.33% of examined meat samples. The variations in the results obtained by different investigators may be due to differences in handling, manufacturing practices, and the time of exposure.…”

Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk

“…When milk samples were inoculated with 3 × 10 4 cfu/ml of viable S. agalactiae cells and 3 × 10 3 cfu/ml of dead cells or 3 × 10 3 cfu/ml of viable cells and 3 × 10 4 cfu/ml of dead cells, the difference in results for PMA–qPCR compared with SDS–PMA–qPCR indicated that the presence of dead cell debris with an intact outer membrane was more efficiently affected by the SDS–PMA combination than by PMA alone. Previous studies showed that DNA of dead cells of various pathogens in milk, meat homogenates or water samples can be inactivated for PCR by treatment with PMA or SDS–PMA (Tomás et al, 2009; Gensberger et al, 2014; El-Aziz et al, 2018). Compared with these reports, the PMA–qPCR assay with SDS developed in the present study was faster and had higher sensitivity than previously reported methods.…”