Detection of Viable but Nonculturable Escherichia coli O157:H7 in Ground Beef by Propidium Monoazide real-time PCR

Escherichia coli O157:H7 can enter into a viable but nonculturable (VBNC) state under stress conditions. Pathogens in this dormant state may escape detection if conventional methods are employed, and potentially pose serious threats to human health. Studies have shown that many intervention and preservation processes that are commonly used in the food industry may instead induce a VBNC state rather than kill the intended pathogens. This study aimed to detect whether E. coli O157:H7, an important and dangerous foodborne pathogen, could adapt to the stress caused by lactic acid exposure by entering the VBNC state. A propidium monoazide (PMA) quantitative PCR (qPCR) method was used for detection and quantifi cation of VBNC E. coli O157:H7 cells. The performance of this PMA-qPCR method was assessed using pure culture and ground beef samples inoculated with VBNC E. coli O157:H7 cells. The applied assay could detect as low as 103 CFU/mL VBNC E. coli O157:H7 in pure culture and 4 × 104 CFU/g VBNC cells in ground beef. Results indicate that PMA qPCR could accurately quantify E. coli O157:H7 in a VBNC state. Research Article Detection of Viable but Nonculturable Escherichia coli O157:H7 in Ground Beef by Propidium Monoazide real-time PCR Jehan Mahmoud Mahmoud Ouf1, Yuan Yuan2, Prashant Singh2 and Azlin Mustapha2* 1Food Hygiene Department, Animal Health Research Institute, Dokki, Giza, Egypt 2Food Science Program, University of Missouri, Columbia, Missouri, USA Dates: Received: 06 April, 2017; Accepted: 19 May, 2017; Published: 20 May, 2017 *Corresponding author: Azlin Mustapha, Food Science Program, Division of Food Systems and Bioengineering, 246 William Stringer Wing, Eckles Hall, University of Missouri, Columbia, MO 65211, USA, Tel: +1-573-882-2649; Fax: +1-573-884-7964; E-mail:


Introduction
Escherichia coli O157:H7 is an important foodborne pathogen that causes gastrointestinal illness as well as life-threatening diseases [1]. This pathogen can colonize the intestinal tract of cattle and make its way into beef products during slaughtering and subsequent processing. The infectious dose of E. coli O157:H7 ranges from 10-100 cells and as low as fewer than 50 viable E. coli O157:H7 cells can lead to serious outbreaks [2].
Furthermore, this pathogen has the potential to enter into the viable-but-nonculturable state [3]. In such a state, cells fail to grow and form colonies on commonly used selective media for their detection, but remain alive and retain their metabolic activities [4]. In fact, pathogenic bacteria can be avirulent in the VBNC state but regain virulence after resuscitation into culturable cells under suitable conditions [5]. Reissbrodt et al. (2002) [6], reported that VBNC cells may resuscitate in the presence of certain growth promoters or enrichments. Some VBNC cells are still virulent and even cause fatal infections, which may be due to their rapid resuscitation in suitable hosts [5,7]. In E. coli O157:H7, the expression of multiple virulence genes, including the Shiga toxin genes, stx1 and stx2 genes, can still occur in VBNC cells [8] and strains of this bacterium in the VBNC state can become culturable again in the presence of the antioxidant, oxyrase, the enterobacterial autoinducer or sodium pyruvate [9].
The distinction between viability and culturability is especially critical for pathogens, because loss of culturability may not guarantee loss of pathogenicity. If pathogenicity persists, pathogens in the dormant (VBNC) state may, in fact, pose a heretofore unrecognized public health threat [10]. The occurrence and persistence of VBNC cells that retain pathogenicity or are able to recover from this state is a public health concern since they may constitute an unrecognized source of infection [11]. In fact, because cells of E. coli O157:H7 in the VBNC state retain virulence, they should be considered as risks to public health [12].

PMA treatment
Serial dilutions (10 -2 to 10 -8 ) of a freshly grown culture of E. coli O157:H7 cells were prepared in 0.1% peptone water. Two milliliters of each diluted suspension were withdrawn and divided into two 1-mL suspensions in separate tubes. One set was used for DNA extraction without PMA treatment, whereas the other set was stained in the dark for 5 min with 25 μM PMA (Biotium Inc., Hayward, CA, USA), placed in ice and exposed to a 650-W halogen light at a distance of 20 cm for 10 min, as previously optimized and described by our group [17]. PMA treated cells were centrifuged at 12,000 ×g for 5 min, then washed under the same conditions in 1 mL 0.1% peptone water.

DNA isolation
DNA from the obtained cell pellets were isolated by resuspending in 100 μL of PrepMan ® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions with minor modifi cations.
In order to achieve a higher DNA yield, cell suspensions were heated at 100 °C in a dry bath incubator for 20 min. Boiled cell suspensions were centrifuged at 12000 ×g for 5 min and 10 μL of the supernatant was used as the template DNA to construct standard curves for quantitative purposes by real-time PCR assay.

Real-time PCR
Primers and probes targeting E. coli O157:H7 were designed by Wang et al. [23]. pUC19 plasmid DNA was used as an internal amplifi cation control (IAC). Primers and probes targeting pUC19 were as designed by Fricker et al., 2007 [24] (Table 1).
Real time PCR was performed in a LightCycler ® 96 real-time PCR platform (Roche Diagnostics Corporation, Indianapolis, USA). A 25 μL PCR reaction mix consisted of 12.5 μL of 2× TaqMan™ Universal PCR Master Mix (Applied Biosystems), 0.5 μM of each E. coli O157:H7 primer, 0.4 μM of each IAC primer, 0.2 μM of E. coli and IAC probe, 0.25 pg of pUC19 (8.62 × 10 4 copies) (Promega, Madison, WI, USA), and 5 μL template DNA. Nuclease-free water (Promega) was used to adjust the reaction volume to 25 μL [17]. The real-time PCR program consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The concentration of E. coli O157:H7 (log CFU/mL) was determined based on a standard curve ( Figure 1). Data from mean values of three independent experiments with duplicates were used to calculate the coeffi cient of regression (R 2 ) values.

Culturability of E. coli O157:H7
Bacterial samples were serially diluted in 0.1% peptone water. One hundred microliters of appropriate dilutions were spread-plated on TSA and incubated at 37 C for 48 h to enumerate acid-stressed E. coli O157:H7 cells. When colonies ceased to occur on the agar, 1 mL of the samples was transferred to 9 mL of TSBY and incubated at 37 °C for 48 h to further test the culturability of the cells on TSA for another 48 h at 37 o C.

Preparation of artifi cially spiked samples
Ground beef (10% fat, 90% lean) was purchased from a local supermarket (Columbia, MO, USA) and tested for the presence of E. coli O157:H7 using standard culture methods [25]. Twenty-fi ve grams of the ground beef samples were weighed in fi ltered stomacher bags (Fisherbrand, Houston, TX, USA) and artifi cially inoculated with different concentrations (10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 CFU/g) of VBNC E. coli O157:H7. Cells were allowed to attach at room temperature for 15 min. Then, each sample was diluted with 225 mL of lactose broth (Difco Labs.), and stomached in a Stomacher 400 (Seward Lab. Systems, Inc., Bohemia, NY and USA) for 2 min at high intensity. The homogenized ground beef suspension samples were briefl y centrifuged (2000 ×g for 2 min) to separate out beef and fat particles. Two milliliters of the supernatant were divided into two 1-mL portions, each of which was transferred to two fresh centrifuge tubes. Bacterial cells were collected by centrifugation at 12,500 ×g for 5 min, and the obtained cell pellets were washed in sterile distilled water under the same centrifugation conditions. Real time PCR with or without PMA pre-treatment were conducted as described in the next section.

Detection of low concentrations of VBNC E. coli O157:H7 in ground beef by PMA real-time PCR
PMA treatment of one set of samples from the previous section was applied prior to DNA extraction, using PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems) as described above. One negative (un-inoculated) control for each sample was also included in the study. The extracted DNA was diluted 1:2. PCR conditions used for the two sets of DNA samples were the same as described above. Real-time PCR with IAC was performed to recognize the limit of detection of E. coli O157:H7 counts in spiked ground beef samples.

Statistical analysis
All experiments were performed in triplicate. Mean and pairwise mean comparisons were performed using Microsoft Offi ce Excel 2007.

Results and Discussion
Quantitative differentiation of the live fraction of pathogens in raw food samples is highly critical from a public health risk perspective, as many studies have shown that under stressed conditions, major foodborne pathogens can enter a VBNC state.
In this state, bacteria can remain alive for long periods of time and maintain their potential for virulence [26]. E. coli O157:H7 lost the ability to propagate after starvation in sea salt medium at 5 ºC for 70 days [3]. Wang and Doyle (1998) [27], reported that E. coli O157:H7 became nonculturable after 77 days in reservoir water or 70 days in lake water held at 25 ºC. The VBNC state of E. coli O157:H7 was also induced in 13% NaCl or after exposure to chlorine [28,29]. Previous research indicate that pH changes in media are related to a loss of culturability of bacterial cells. At 4 ºC, E. coli O157:H7 suspended in phosphatebuffered saline at pH 4 entered the VBNC state more rapidly than at pH 7 [29].
Lactic acid is widely used in food processing for reducing and eliminating pathogenic and spoilage organisms and helps  Table 2). A high degree of acid tolerance is an important feature of E. coli O157:H7 pathogen [30]. The minimum pH for E. coli O157:H7 growth is 4.0 to 4.5 [31].
To construct the standard curves, a fresh overnight grown E. coli O157:H7 culture was serially diluted using 0.1% peptone water and culturability was detected on TSA for determination of the initial bacterial count (2 × 10 9 CFU/mL). Standard curves were generated (typical coeffi cient of determination above 98%) using PMA and non PMA real time PCR to estimate the counts of viable E. coli O157:H7. Figure 1a [21], who mentioned that PMA is less likely to permeate viable cells and probably VBNC with intact membranes, as well as with our previous study [17].   [17], who stated that the PMA real-time PCR could detect a range from 10 5 to 10 8 CFU/g of viable E. coli O157:H7, in ground beef samples regardless of whether dead cells were present or not. They added that substances naturally found in environmental and food samples may inhibit the amplifi cation of target DNA in real-time PCR. This may be the reason for the relatively high detection limit of VBNC E. coli O157:H7 cells in ground beef (4 ×10 4 CFU/g) in our study. Španová et al. (2000) [33], reported that food samples contain many organic and inorganic substances, such as phenolic compounds, fat, enzymes, polysaccharides, proteins and salts, all of which can either inhibit PCR amplifi cation or lead to a reduction in amplifi cation effi ciency of PCR reactions. This detection limit (4 × 10 4 CFU/g) in ground beef was not satisfactory with respect to the low infectious dose of E. coli O157:H7. Therefore, to use this procedure, a resuscitation or enrichment step would have to be incorporated especially when the initial concentration is low. In pure culture or spiked ground beef, the bacterial cells treated with PMA staining showed lower counts (the Ct values of PMA qPCR were 1-2 cycles higher than those of qPCR at the same numbers of cells), further highlighting the importance of an enrichment step.
The differing results between the viable count detected by PMA real-time PCR and that from the non-PMA real-time PCR ( Figure 2) indicated that E. coli O157:H7 cells after lactic acid treatment entered a VBNC state. The addition of an internal amplifi cation control (IAC) in a real-time PCR reaction system allows one to monitor the effi ciency of each reaction and prevent false-negative results [34][35][36]). pUC19 is a small, high copy number E. coli plasmid with a molecular weight of 2686 bp. It has been previously used with success as an IAC in realtime PCR [24]. Further, a negative control (water) was added to ensure that no DNA cross-contamination occurred in the PCR reaction. A real-time PCR assay is more sensitive than traditional PCR and can be quantitative for pathogenic bacterial detection in food.

Conclusion
The results confi rm that lactic acid, widely used in food processing, can induce E. coli O157:H7 to enter the VBNC state.
The nonculturable cells of E. coli O157:H7 failed to propagate even in TSBY and maintained intact membranes for 10 hat