Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selectively suppress the DNA amplification of dead cells. The results showed the optimum concentration of PMA is 5.0 µg/mL. The detection limit of this assay by mPCR was 103 CFU/mL in the culture broth, and by PMA-mPCR was 104 CFU/mL both in pure culture and a food matrix (milk and ground beef). In addition, the detection of mixed viable and dead cells was also explored in this study. The detection sensitivity ratio of viable and dead counts was less than 1:10. Therefore, the PMA-mPCR assay proposed here might provide an efficient detection tool for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella and also have great potential for the detection and concentration assessment of VBNC cells.

Induction of Viable but Nonculturable Escherichia coli O157:H7 by Low Temperature and Its Resuscitation.

Viable but non-culturable (VBNC) cells are alive bacteria cells, but lose their culturability in conventional culture media, usually escape detection by the plate count method and pose a serious threat to food safety and public health. Therefore, it is urgent to study the VBNC status, and to provide theoretical basis and scientific basis for food processing and safety control caused by pathogenic microorganisms. In this study, Escherichia coli O157:H7 was induced to the VBNC state at two different temperatures (-20°C and 4°C) and its resuscitation and morphological changes under different nutritional conditions were studied. The initial inoculum of 2.1 × 107 CFU/mL E. coli O157:H7 cells were induced into the VBNC state in normal saline, distilled water, LB broth at -20 °C after 176, 160, 80 days, respectively. The results showed that E. coli O157:H7 reserved at -20°C, and LB culture medium were easier to enter VBNC state than others conditions, the cells still had metabolic activity and the cell morphology changed from the typical rod shape to short rod and the cell size decreased. The resuscitate ways including the direct warming resuscitation, gradual warming resuscitation, adding chemical substance resuscitation, and adding nutrients resuscitation were studied. The results showed that the optimal conditions of 5% Tween 80 and 3% Tween 80 acculated the resuscitation of E. coli O157:H7 VBNC state cells induced by low temperature LB medium and low temperature saline. E. coli O157:H7 VBNC state failed from resuscitation when incubating in LB broth, respectively using direct warming and adding nutrients substance. This study provides new insights into induction and resuscitation of VBNC E. coli O157:H7 and offers an approach for investigating the formation mechanism of VBNC foodborne pathogens in food safety.

Simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus and Salmonella by multiplex PCR in milk.

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are food-borne pathogens that cause serious gastrointestinal illness and frequent food safety accidents. This study aimed to develop a practical multiplex polymerase chain reaction (mPCR) technique for the simultaneous detection of these food-borne pathogens in culture broth and artificial food matrix. Pathogen-specific DNA sequences in the rfbE, nuc, and invA genes were used as targets to design primers for the identification of E. coli O157:H7, S. aureus, and Salmonella, respectively. As expected, the method produced species-specific bands of amplified products without any contaminating non-specific bands. The highest species specificity was established with primer concentrations of 0.1, 0.2, and 0.4 µM for E. coli O157:H7, S. aureus, and Salmonella, correspondingly. The detection sensitivity of this assay was 103 CFU/mL in culture broth, and the limit of detection was consistent with singleplex PCR in the food sample. The mPCR assay proposed here is an easy and convenient detection method, which will be valuable for microbial epidemiology and food safety investigations.

Surface-Enhanced Raman Scattering (SERS) With Silver Nano Substrates Synthesized by Microwave for Rapid Detection of Foodborne Pathogens.

Rapid and sensitive methods have been developed to detect foodborne pathogens, a development that is important for food safety. The aim of this study is to explore Surface-enhanced Raman scattering (SERS) with silver nano substrates to detect and identify the following three foodborne pathogens: Escherichia coli O157: H7, Staphylococcus aureus and Salmonella. All the cells were resuspended with 10 mL silver colloidal nanoparticles, making a concentration of 107 CFU/mL, and were then exposed to 785 nm laser excitation. In this study, the results showed that all the bacteria can be sensitively and reproducibly detected directly by SERS. The distinctive differences can be observed in the SERS spectral data of the three food-borne pathogens, and the silver colloidal nanoparticles can be used as highly sensitive SERS-active substrates. In addition, the assay time required only a few minutes, which indicated that SERS coupled with the silver colloidal nanoparticles is a promising method for the detection and characterization of food-borne pathogens. At the same time, principle component analysis (PCA) and hierarchical cluster analysis (HCA) made the different bacterial strains clearly differentiated based on the barcode spectral data reduction. Therefore, the SERS methods hold great promise for the detection and identification of food-borne pathogens and even for applications in food safety.

Current Perspectives on Viable but Non-culturable State in Foodborne Pathogens.

The viable but non-culturable (VBNC) state, a unique state in which a number of bacteria respond to adverse circumstances, was first discovered in 1982. Unfortunately, it has been reported that many foodborne pathogens can be induced to enter the VBNC state by the limiting environmental conditions during food processing and preservation, such as extreme temperatures, drying, irradiation, pulsed electric field, and high pressure stress, as well as the addition of preservatives and disinfectants. After entering the VBNC state, foodborne pathogens will introduce a serious crisis to food safety and public health because they cannot be detected using conventional plate counting techniques. This review provides an overview of the various features of the VBNC state, including the biological characteristics, induction and resuscitation factors, formation and resuscitation mechanisms, detection methods, and relationship to food safety.