Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.


INTRODUCTION
In recent years, the outbreak of foodborne diseases caused by pathogens and its related virulence factors is a major threat for many countries, although much attention had been paid to food safety issues. Foodborne pathogens including enterohemorrhagic Escherichia coli (EHEC), Staphylococcus aureus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa have posed great concern in the food industry and public health (Miao et al., 2016(Miao et al., , 2019Xie et al., 2017a;Xu et al., 2019), especially their acquisition of antimicrobial resistance (Xu et al., 2007(Xu et al., , 2011aYu et al., 2016;Xie et al., 2017b;Liu et al., 2018c,d), biofilm formation (Lin et al., 2016;Bao et al., 2017a;Miao et al., 2017b), and toxin production (Liu et al., 2016a). EHEC causes more than 63,000 illnesses, 2,100 hospitalizations, and 20 deaths each year in the United States (Jiang et al., 2016). E. coli O157:H7 is the main EHEC serotype that causes the majority of EHEC human infections. As one of the most commonly found foodborne pathogens, it can be transmitted by contaminated food such as cattle, milk, eggs and vegetables, rice cakes, and others (Ackers et al., 1998;Jacob et al., 2013;Marder et al., 2014). The methods are laborintensive and time-consuming and cannot meet the requirement of rapid monitoring. Hence, developing a rapid, sensitive, and accurate method to detect E. coli O157:H7 in various foods with a complex matrix is crucial in preventing disastrous E. coli O157:H7 outbreaks and associated human infections.
In 1982, Xu et al. (1982) firstly reported "viable but nonculturable" (VBNC) state, which was considered to be a survival strategy of non-spore-forming bacteria in response to adverse conditions (Xu et al., 1982;Oliver, 2010). Bacteria can enter into VBNC state by the stimulation of adverse environmental conditions, such as low temperature, nutrient-limited conditions, high salt, low pH, and even ultraviolet-induced (Foster, 1999;Ramaiah et al., 2002;Cunningham et al., 2009;Deng et al., 2015;Liu et al., 2017aLiu et al., ,b, 2018aGuo et al., 2019). For now, 85 species of bacteria have been confirmed that can enter into VBNC state, including 18 non-pathogenic and 67 pathogenic species . In recent years, many studies have determined that VBNC bacteria still can produce harmful substances (Deng et al., 2015;Liu et al., 2017aLiu et al., ,b, 2018a. Shigella dysenteriae and E. coli O157 still retain Shiga toxin encoding gene (stx) and produce toxin in VBNC state (Rahman et al., 1996;Liu et al., 2017d). Moreover, bacteria in VBNC can resuscitate and grow again with suitable conditions (Pinto et al., 2015). Nowadays, the traditional culture method and a nucleic acid detection method are widely used. However, the discovery of VBNC in recent years has brought difficulties to the detection method. The VBNC cell cannot grow on the plate medium due to low metabolic activity, which means that normal culture methods can lead to false-negative results of detection. Hence, rapid and accurate identification of VBNC bacteria is of utmost importance. Bacteria in the VBNC state remain metabolically and physiologically viable and continuing to express virulence genes (Liu et al., 2016b(Liu et al., ,c,d, 2017a(Liu et al., ,c, 2018aBao et al., 2017b;Xu et al., 2017a). Concerning conventional detection methods that are time-consuming and only suitable for experimental use, alternative rapid and cost-effective detection methods are desperately required to detect VBNC pathogens (Lin et al., 2016;Xu et al., 2016;Miao et al., 2017a;Zhao et al., 2018). As the ethidium bromide monoazide (EMA) or propidium monoazide (PMA) penetrates only into dead bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes, EMA/PMA treatment to cultures with both viable and dead cells results in selective removal of DNA from dead cells. Therefore, scientists had utilized EMA/PMA combined with nucleic acid amplification techniques to complete the detection of VBNC cells. Recently, nucleic acid amplification methods combined with EMA/PMA have been widely used for the detection of pathogenic bacteria in the VBNC state, such as PMA-polymerase chain reaction (PCR) (Liu Y. et al., 2018;Zhong and Zhao, 2018).
Although numerous quick methods based on nucleic acids, such as PCR (Gehring and Tu, 2005;Xu et al., 2011b) and real-time PCR (O'Hanlon et al., 2004), have been developed and used for detecting E. coli O157:H7 in food, a quicker and less expensive technology is always most preferred. Isothermal amplification is a novel method for DNA amplification at a constant temperature, providing simple, fast, independent of sophisticated instruments and cost-effective techniques to detect biological targets, especially for less well-equipped laboratories, as well as for filed detection. Isothermal technologies mainly include loop-mediated isothermal amplification, rolling circle amplification, single primer isothermal amplification, polymerase spiral reaction, strand displacement amplification, and recombinase polymerase amplification (Walker et al., 1992;Notomi et al., 2000;Haible et al., 2006;Zhao et al., 2009Zhao et al., , 2010Zhao et al., , 2011Wang et al., 2011;Xu Z. et al., 2012;Xu et al., 2020;Liu et al., 2015;Yang et al., 2017). Cross priming amplification (CPA) is a novel isothermal method that relies on five primers (2a/1s, 2a, 3a, 4s, and 5a) to amply the target nucleotide sequences (Xu G. et al., 2012). It does not require any special instrumentation and presents all the features, including rapidity, specificity, and sensitivity. Recently, CPA assays have been used for the detection of E. coli O157:H7, Listeria monocytogenes, Enterobacter sakazakii, Salmonella enterica, Yersinia enterocolitica, and other pathogens (Yulong et al., 2010;Wang et al., 2014Wang et al., , 2018Zhang et al., 2015;Xu et al., 2020). Introducing the cross-priming principles, CPA is advantageous on reproducibility and stability with a similar level of sensitivity, specificity, and rapidity comparing with loopmediated isothermal amplification, the most broadly applied isothermal methodologies. Therefore, CPA is a potentially valuable tool for the rapid diagnosis of foodborne pathogens, as well as those in the VBNC state. However, there is no report of PMA-CPA assay for the detection of VBNC E. coli O157:H7.
This study aimed to develop a rapid PMA-CPA assay to detect E. coli O157:H7 in VBNC state targeting on rfbE, stx1, and stx2 genes combining visual methods with the addition of calcein and applying this assay to detect the E. coli O157:H7 strains from food samples.

Induction of Entry Into the Viable But Non-culturable State in Saline and Food Sample
Induction of VBNC cells and the establishment of PMA-CPA assays were performed on E. coli O157 ATCC43895. The bacterial strain was incubated in trypticase soy broth (Huankai Microbial, China) to reach the exponential phase [∼10 9 colony-forming units (CFUs)/ml]. To induce the entry of VBNC state, the culture was diluted to the final density of 10 8 CFU/ml with saline (pure culture system) and food homogenate (Cantonese rice cake, Guangzhou Restaurant, Guangzhou) (food system) and stored at −20 • C.

Determination of the Culturable and Viable But Non-culturable State of E. coli O157
The conventional plate counting method was used to determine the cultivability of E. coli O157. The induction culture was serial diluted with 0.9% sodium chloride and inoculated on trypticase soy agar at 37 • C for 24 h. When the number of colonies was <1 CFU/ml for 3 days, it was considered that the survived cells might have entered into the non-culturable state (Deng et al., 2015). Also, the final determination of the VBNC cell was evaluated by the LIVE/DEAD BacLight bacterial viability kit (Thermo Fisher Scientific, China) (Liu et al., 2017d). The stained induction culture was observed by fluorescence microscopy.

Design of Crossing Priming Amplification Primers
As mentioned before (Xu et al., 2020), the CPA primers were designed for specific O-antigen rfbE gene and Shiga toxin genes stx1 and stx2 of E. coli via Primer Premier 5. For each of the target

Development of Propidium Monoazide-Crossing Priming Amplification Assay in Pure Bacterial Culture and Food Sample
For the pure bacterial culture, 500 µl of 10-10 6 -CFU/ml VBNC culture was transformed into 1.5-ml centrifuge tubes. For the food sample culture, the 10-10 6 -CFU/ml cultures were washed three times by saline to avoid the effect of substances in a food sample and placed in 1.5-ml centrifuge tubes. Then, PMA reagent was added to the final concentration of 5 µg/ml. Subsequently, the detection samples mixed with PMA were incubated in the dark at room temperature for 10 min before the tubes were placed horizontally on ice exposed to a halogen lamp (650 W) at a distance of 15 cm for 15 min to complete the combination of DNA and PMA (Chen et al., 2020). The mixed samples were centrifuged at 10,000 r/min for 5 min, and the precipitation under the tubes was processed by DNA extraction kit (Dongsheng Biotech, Guangzhou) followed the instruction of the manufacturer, which were prepared as DNA samples for PMA-CPA. The CPA reaction was performed as mentioned before (Xu et al., 2020)   chromogenic agent (mixture with calcein and Mn 2+ ), and 1µl template DNA, and the volume was made up to 26 µl with nuclease-free water. The mixed chromogenic agent consists of 0.13-mM calcein and 15.6-mM MnCl 2 .4H 2 O.
The mixed reaction solution was incubated at 65 • C for 60 min and heated at 80 • C for 2 min to terminate it. PMA-CPA amplified products were visualized under visible light or the appearance of the laddering pattern on 1.5% agarose gel electrophoresis.
However, instead of the laddering pattern, apparent bright strip might present, which can also be regarded as positive results. This experiment was performed in triplicate to ensure reproducibility.

Observation of E. coli O157 in Viable But Non-culturable State With Fluorescence Microscopy
The E. coli O157 in normal viable and VBNC state was analyzed using the LIVE/DEAD R BacLight Bacterial Viability Kit. Under a fluorescence microscope, the VBNC cells showed green, whereas dead cells exhibited red (Figure 1). The results showed that the VBNC cell might change their morphological characterization from rod-shaped (normal state) to shorter rods or coccoid (VBNC state) (Liu et al., 2017c).

Propidium Monoazide-Crossing Priming Amplification Assay for Detection of Viable But Non-culturable Cells of E. coli O157 in Food Samples
Serial diluted DNA of VBNC cells evaluated the sensitivity of PMA-CPA. There was an obvious color change at the 10 3 -10 6 CFU/ml DNA, and the ladder-like pattern was clearly observed under ultraviolet light. The detection limits of E. coli O157 VBNC were 10 3 , 10 5 , and 10 5 CFU/ml for rfbE, stx1, and stx2 genes, respectively.
The PMA-CPA assays for the detection of E. coli O157 VBNC in food samples were successfully conducted. The detection limits of VBNC cells in the food system were 10 3 , 10 5 , and 10 5 CFU/ml for rfbE, stx1, and stx2 genes, respectively, which were the same as the limits in pure VBNC cells (Figure 2).

DISCUSSION
Escherichia coli O157:H7 is currently a widespread foodborne pathogen throughout the world and has promoted a heightened interest and concern for the low-level detection of these foodborne pathogens (Pennington, 2010). There is a fastincreasing and urgent demand for high-performance techniques for monitoring bacteria in complex foods to reduce the risk of the associated food poisoning. In evaluating detection methodologies for ecologic and epidemiological purposes, a series of attributes should be considered and assessed, including specificity, sensitivity, simplicity, expense, and time. In this study, PMA-CPA assay targeting rfbE, stx1, and stx2 successfully detected detection E. coli O157:H7 VBNC cell in pure culture and food samples. Notably, Shiga toxins 1 and 2 (Stx1 and Stx2) encoded by stx1 and stx2 can result in gastrointestinal symptoms, such as diarrhea and hemorrhagic colitis, and may also progress to a hemolytic uremic syndrome, a severe sequela of this infection (Saeedi et al., 2017). Considered to be an important health risk in the food testing, the detection of Shiga toxin, especially rapid and easy operating detection assay, may be of utmost significance and FIGURE 2 | Sensitivity of PMA-CPA assay for detection of VBNC cells of rfbE (A), stx1 (B), and stx2 (C) genes by mixed chromogenic agent in food samples. M-DNA marker; lanes 1-7, 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 CFU/ml, negative control. urgent necessity. Therefore, we established a PMA-CPA method for the detection of E. coli O157:H7 in VBNC cell, as well as its virulence factors. The detection limits of PMA-CPA assay showed consistency with that of CPA assay, no matter in pure bacterial culture or food samples, which had been performed previously (Xu et al., 2020). To the best of our knowledge, this is the first report of a PMA-CPA assay to detect E. coli O157:H7 in VBNC state from food samples.

CONCLUSION
In conclusion, the designed CPA primers targeted the rfbE, stx1, and stx2 genes for the effective detection of E. coli O157:H7 VBNC cell. Therefore, being simple, rapid, sensitive, and specific, PMA-CPA assay can be a useful and powerful method in the field and also an alternative diagnostic tool for the detection of E. coli O157:H7 in VBNC cell and its related virulence factors in testing as part of an outbreak investigation.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/supplementary material.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.