International Journal of Food Microbiology 170 (2014) 48–54

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR Yarui Liu, Azlin Mustapha ⁎ Food Science Program, Division of Food Systems and Bioengineering, University of Missouri, Columbia, United States

a r t i c l e

i n f o

Article history: Received 1 July 2013 Received in revised form 6 October 2013 Accepted 27 October 2013 Available online 6 November 2013 Keywords: Propidium monoazide Real-time PCR Viable Escherichia coli O157:H7 Ground beef

a b s t r a c t Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157: H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 100 to 108 CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85 °C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 108 dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 102 CFU/mL viable E. coli O157:H7 in pure culture and 105 CFU/g in ground beef, in the presence of 106/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 106 dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Escherichia coli O157:H7 is one of the most notorious foodborne pathogens, with an infectious dose of as low as a few hundred cells (Karmali, 2004). Beef and dairy products, juices and fresh produce are foods that are often associated with E. coli O157:H7 outbreaks. E. coli O157:H7 infections can lead to nonspecific diarrhea, hemorrhagic colitis and even hemolytic uremic syndrome (HUS) (Banatvala et al., 2001). According to the Centers for Disease Control and Prevention (CDC), E. coli O157:H7 leads to an estimated 73,480 illnesses each year, resulting in more than 2000 hospitalizations and 60 deaths (Mead et al., 1999). It has been estimated that the annual cost of E. coli O157: H7 infections was $405 million from 1996 to 2004 (Frenzen et al., 2005). Thus, accurate and sensitive methods to detect E. coli O157:H7 in food products are urgently needed. Conventional culture-based methods, involving enrichment, isolation and confirmation steps, have been used for over a century due to their sensitivity, low cost, ease of use, and ability to monitor cell viability (Murakami, 2012). However, they require four to five days to achieve ⁎ Corresponding author at: Food Science Program, 256 WCS Wing, Eckles Hall, University of Missouri, Columbia, MO 65211, United States. Tel.: +1 573 882 2649; fax: +1 573 884 7964. E-mail address: [email protected] (A. Mustapha). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.10.026

confirmatory results. On the other hand, the polymerase chain reaction (PCR), a widely used nucleic acid-based technique, can identify target species within 3 h. However, conventional PCR cannot differentiate viable cells from dead cells (Wang and Levin, 2006). DNA from dead cells can lead to false-positive PCR results, leading to unnecessary product recalls and economic losses. Membrane integrity is considered the most important criterion for distinguishing between viable and irreversibly damaged cells (Nocker et al., 2006). Propidium monoazide (PMA), a DNA intercalating dye, can only penetrate dead or membrane-compromised cells and covalently bind to cellular DNA through photolysis. Consequently, the covalent link will render the DNA insoluble and inhibit PCR amplification of DNA from dead or membrane-compromised cells (Nocker et al., 2006). PMA is less likely to penetrate viable but nonculturable (VBNC) cells with intact membranes. Xiao et al. (2013) combined PMA staining with real-time PCR and successfully detected as low as 100 CFU/mL of E. coli O157:H7 in a VBNC state with this assay. PMA has been successfully used in combination with real-time PCR to detect viable lactic acid bacteria (García-Cayuela et al., 2009), T4 phage (Fittipaldi et al., 2010), Campylobacter (Josefsen et al., 2010), Salmonella (Liang et al., 2011) and E. coli (Yang et al., 2011; Taskin et al., 2011) in environmental and food samples. Ethidium monoazide (EMA) is another dye that covalently binds to cellular DNA. Both PMA and EMA have specific advantages and disadvantages. Compared with PMA, EMA penetrates dead

Y. Liu, A. Mustapha / International Journal of Food Microbiology 170 (2014) 48–54

cells more effectively but exerts a greater inhibitory effect on viable cells (Lee and Levin, 2006). EMA, at high concentrations, penetrates viable cells and leads to false-negative PCR results (Nocker et al., 2006). Wang et al. (2009) developed an EMA real-time PCR assay to detect viable E. coli O157:H7 cells in ground beef. According to their results, EMA, at a concentration of 100 μg/mL in the cell suspension (approximately 120 μM), completely prevented DNA amplification from 108 CFU viable E. coli O157:H7 cells via real-time PCR, while a 12-h enrichment step was necessary to detect 101 CFU/g of viable E. coli O157:H7 in spiked ground beef samples. Real-time PCR is a rapid and sensitive method that can be used for the quantification of microorganisms of interest. However, substances naturally found in environmental and food samples may inhibit the amplification of target DNA in real-time PCR. To solve this problem, an internal amplification control (IAC) is included in a real-time PCR assay to monitor the PCR's efficiency and to prevent false-negative results due to PCR inhibitors present in food samples (Murphy et al., 2007). The objectives of this study were to optimize a PMA staining procedure, and to combine it with real-time PCR for the detection and quantification of only viable E. coli O157:H7 cells in contaminated ground beef samples.

49

2.4. Influence of light exposure time on DNA amplification from viable cells Six viable E. coli O157:H7 samples (108 CFU/mL each) were prepared as described previously. PMA stock solution in the amount of 1.25 μL was added to each 1 mL of cell suspension to achieve a final concentration of 25 μM. Samples were incubated in the dark at room temperature for 5 min and exposed to a 650-halogen light for varying lengths of time (0, 2, 5, 10, 15, 20 min). After PMA staining, cell pellets were collected by centrifugation at 13,400 ×g for 5 min and washed with sterile distilled water. The influence of light exposure time on DNA amplification from viable cells was evaluated by real-time PCR after DNA extraction. 2.5. DNA extraction Cell pellets were resuspended in 100 μL of PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA), vortexed for 10 s and boiled for 25 min. Boiled cell suspension was centrifuged at 13,400 ×g for 3 min and 5 μL of the supernatant was used as template DNA for the real-time PCR assay.

2. Materials and methods 2.6. Real-time PCR 2.1. Preparation of viable and dead E. coli O157:H7 cells E. coli O157:H7 strains G5310, C7927, and 505B were from our own culture collection at the Food Microbiology Laboratory, University of Missouri, Columbia, MO. Each of the three strains was grown in tryptic soy broth supplemented with 0.5% yeast extract (TSBY; Difco Labs., BD Diagnostic Systems, Sparks, MD, USA) at 37 °C overnight (~109 CFU/mL). One milliliter of each strain was then mixed together, cells were harvested by centrifugation at 13,400 ×g for 5 min, and washed and serially diluted in 0.1% peptone water to yield cell suspensions ranging from 100 to 108 CFU/mL. To obtain dead cells, the cell suspensions were heated at 85 °C for 35 min. Cell viability was checked by plating in plate count agar (PCA; Difco Labs.). 2.2. Minimum PMA amount needed to bind dead cell DNA Propidium monoazide (PMA) was purchased from Biotium Inc. (Hayward, CA, USA), and a 20 mM stock solution was prepared in sterile distilled water and stored in the dark at −20 °C. Six dead E. coli O157: H7 samples (108 cells/mL each) were prepared as described above. Different amounts of PMA (0, 0.5, 1.25, 2.5, 5, 10 μL) were added to 1 mL of each sample to achieve PMA concentrations of 0, 10, 25, 50, 100, and 200 μM, respectively. Following a 5-min incubation period in the dark at room temperature on a Labquake Rotisserie (Barnstead International, Dubuque, IA, USA), samples were exposed to a 650-W halogen light for 10 min. The samples were placed on ice at a distance of 20 cm from the light source to avoid excessive heating. After the photo-induced crosslinking, cells were collected by centrifugation at 13,400 ×g for 5 min, and washed in sterile distilled water under the same centrifugation conditions prior to DNA extraction. 2.3. Influence of PMA concentration on amplification of DNA from viable cells Fresh viable E. coli O157:H7 cells (108 CFU/mL) were prepared as described previously. Varying amounts of PMA (0, 10, 25, 50, 100, and 200 μM) were added to 1 mL of each cell suspension. PMA staining was conducted as described earlier (5 min in the dark followed by a 10-min light exposure). Cell pellets were collected by centrifugation at 13,400 ×g for 5 min and washed with sterile distilled water under the same centrifugation conditions. After DNA extraction, the influence of PMA concentration on DNA amplification from viable cells was evaluated by real-time PCR.

Primers and probes targeting E. coli O157:H7 and pUC 19 were used as previously described by Wang et al. (2007, 2009) with minor modifications. The sequence of E. coli O157:H7 primer 1 is 5′-TTGACCCACACT TTGCCGTAA-3′, and that of E. coli O157:H7 primer 2 is 5′-GCGAAAA CTGTGGAATTGGG-3′. The sequence of E. coli O157:H7 probe is 5′5HEX-TGACCGCATCGAAACGCAGCT-3BHQ_1-3′. pUC 19 was used as an IAC. The sequence of the IAC primer 1 is 5′-GCAGCCACTGGTAACA GGAT-3′ and that of the IAC primer 2 is 5′-GCAGAGCGCAGATACCAA AT-3′, and the sequence of the IAC probe is 5′-56FAM-AGAGCGAG GTATGTATGTAGGCGG-3BHQ_1-3′ (Fricker et al., 2007). A 7500 real-time PCR system (Applied Biosystems) was used. A PCR reaction of 50 μL contained 25 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 × 104 copies; Promega, Madison, WI, USA), and 3 μL DNA extract. Nuclease-free water (Promega) was used to adjust the reaction volume to 50 μL. The real-time PCR program consisted of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. 2.7. Application of PMA real-time PCR to viable E. coli O157:H7 cells One milliliter of viable E. coli O157:H7 cells (101 to 108 CFU/mL) was treated or untreated with PMA. For PMA staining, 1.25 μL of PMA stock solution was added to each sample to achieve a final concentration of 25 μM. Samples were incubated in the dark for 5 min and exposed to a 650-W halogen light for 10 min. DNA extraction and real-time PCR with IAC were conducted as described earlier. Standard curves were constructed by plotting Ct values generated from real-time PCR against E. coli O157:H7 cell concentrations (log CFU/mL). 2.8. Application of PMA real-time PCR to mixed viable and dead E. coli O157:H7 cells One milliliter of dead E. coli O157:H7 cells (106/mL) was mixed with 1 mL of fresh viable cells with different concentrations (101 to 108 CFU/mL) and centrifuged at 13,400 ×g for 5 min. The mixed cell pellets were resuspended in 1 mL of 0.1% peptone water and untreated or treated with PMA, as was optimized previously. DNA extraction and real-time PCR with IAC were conducted as described earlier. Real-time PCR with IAC was conducted to check results.

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2.9. Application of PMA real-time PCR to artificially contaminated ground beef

40

Ground beef with two different fat contents (10% and 27%) was purchased from a local grocery store. It was determined to be free of E. coli O157:H7 using standard culture methods (FDA, 1995) and the real-time PCR method used in this study. Twenty-five grams of each ground beef sample was placed in sterile stomacher bags containing filter membranes (Filtra-Bag®,VWR International, Edmonton, AB, Canada). Two sets of samples were prepared. The first set of nine samples was prepared by adding only viable E. coli O157:H7 cells at different concentrations (100 to 108 CFU/g) into each sample. Considering it unlikely that as high as 108 or 107/mL dead cells of E. coli O157:H7 would be present in food products in reality, the second set of nine samples was prepared by adding 106/g dead E. coli O157:H7 cells and various concentrations of viable E. coli O157:H7 cells (100 to 108 CFU/g) into each 25 g of ground beef sample. The spiked beef samples were massaged by hands for 2 min to allow for adequate distribution and attachment of cells to the meat. Samples were individually mixed with 225 mL TSBY and homogenized for 2 min by stomaching. One milliliter of homogenized suspension from each sample was removed for DNA extraction with prior PMA staining, as was optimized earlier. Real-time PCR was conducted as described previously.

30

E. coli O157

35

IAC

Ct

25 20 15 10 5

UD

UD

UD

UD

UD

0 0

10

25

50

100

200

IAC

PMA concentration (µM) Fig. 1. Minimum PMA concentration necessary for binding DNA from 108 cells/mL of dead E. coli O157:H7. Results were from two repeated experiments. UD, undetected after 40 cycles in real-time PCR.

for binding DNA obtained from the same concentration of dead E. coli O157:H7 cells and preventing their subsequent PCR amplification.

2.10. Detection of low concentrations of viable E. coli O157:H7 in ground beef samples with enrichment by PMA real-time PCR

3.2. Influence of PMA concentration on amplification of DNA from viable cells

Twenty-five grams of each ground beef sample was inoculated with 106/g of dead E. coli O157:H7 cells, as well as viable E. coli O157:H7 cells at concentrations of 100, 101, 102, 103 and 104 CFU/g. Each sample was added to 225 mL TSBY and homogenized for 2 min by stomaching. Samples were incubated in a shaker incubator set at approximately 200 rpm at a temperature of 37 °C. Ten milliliters of each sample were collected at different enrichment times (0, 2, 4, 6, and 8 h). The collected suspensions were first centrifuged at 2000 ×g for 2 min to precipitate the meat tissues and fat. Then, cell pellets were collected by centrifuging at 8000 ×g for 15 min. Cell pellets were washed and resuspended in 1 mL of 0.1% peptone water. PMA staining, DNA extraction and realtime PCR were conducted as described previously.

Fig. 2 shows that when the PMA concentration was ≤50 μM, no significant differences were found between Ct values generated from PMAtreated and those from PMA-untreated viable cells (P N 0.05). However, when the PMA concentration was ≥100 μM, the Ct values generated from 108 CFU/mL of PMA-treated viable cells were significantly higher than those generated from PMA-untreated cells (P ≤ 0.05). The results indicated that high concentrations of PMA (≥100 μM) could also inhibit the amplification of DNA from viable cells, causing an underestimation of viable cells in real-time PCR. Data from Figs. 1 and 2 confirmed that a PMA concentration of 25 μM could completely inhibit PCR amplification of DNA from dead cells, without influencing DNA amplification from viable cells. Thus, 25 μM of PMA was decided to be used for further studies. EMA is another DNA binding dye which has also shown to have similar inhibitory effects at higher concentrations. Wang et al. (2009) reported that the higher the EMA concentration, the greater its inhibition of DNA amplification from viable E. coli O157:H7. EMA, at a concentration higher than 120 μM in combination with a 10-min

2.11. Statistical data analysis The SAS GLM procedure (SAS 9.2, Copyright 2002–2007; SAS Institute Inc., Cary, NC, USA) was used to evaluate the effect of PMA staining on dead and viable cells. Tukey's test was applied to determine differences between various PMA staining and light exposure treatments. Differences were compared at a significance level of 0.05.

40

3. Results and discussion

E. coli O157

35

IAC

30

3.1. Minimum PMA amount needed to bind dead cell DNA

Ct

25

As shown in Fig. 1, DNA from dead cells that were not stained with PMA generated false-positive results in real-time PCR with a Ct value of 18.82, confirming the fact that DNA persists after cell death and PCR cannot differentiate live from dead cells. By staining dead cells with various concentrations of PMA, it was shown that a minimum of 25 μM PMA was able to completely inhibit real-time PCR amplification of DNA from 108 cells/mL of dead E. coli O157:H7 (Fig. 1). The amplification of the IAC (pUC 19) in each reaction successfully monitored the efficiency of the PCR reaction. According to Wang et al. (2009), a treatment of 12 μM EMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 108/mL dead E. coli O157:H7 cells. In this study, a treatment of 25 μM PMA with the same light exposure conditions was found to be optimum

20 15 10 5 0 0

10

25

50

100

200

IAC

PMA concentration (µM) Fig. 2. Influence of PMA concentration on amplification of DNA from 108 CFU/mL of viable E. coli O157:H7 cells. Results were from two repeated experiments. UD, undetected after 40 cycles in real-time PCR.

Y. Liu, A. Mustapha / International Journal of Food Microbiology 170 (2014) 48–54

40 35 30 25

Ct

light exposure, completely prevented DNA amplification from 108 CFU/mL viable E. coli O157:H7 cells, generating false-negative results. However in this study, unlike EMA, higher concentrations (≥100 μM) of PMA only lead to an underestimation of viable E. coli O157:H7 cells while positive results could still be obtained in realtime PCR. By comparing these results, PMA was found to be less toxic to viable E. coli O157:H7 cells than similar concentrations of EMA.

20 15 10

3.3. Influence of light exposure time on DNA amplification from viable cells Fig. 3 shows that the length of light exposure time did not significantly influence the Ct values generated from 108 CFU/mL viable E. coli O157:H7 cells (P N 0.05). The light exposure step is necessary to activate the PMA that is bound to DNA from dead cells and to inactivate any excess PMA that have not entered cells (Fittipaldi et al., 2012). Previously published studies on PMA staining varied on the optimal light exposure time for DNA amplification. This difference might be because of the different light sources and bacterial species used in different studies. In this study, a light exposure time of 10 min was found to be effective for cells treated with 25 μM of PMA to completely prevent DNA amplification from 108/mL dead E. coli O157:H7 cells (Fig. 1). Fig. 3 demonstrates that viable cells treated with a 10-min light exposure showed a Ct value close to that of untreated controls (P N 0.05). Therefore, a 10-min light exposure was used for further development of the assay.

3.4. Application of PMA real-time PCR to viable E. coli O157:H7 cells Without PMA treatment, the detection range of the real-time PCR assay was from 102 to 108 CFU/mL viable E. coli O157:H7 cells (Fig. 4). With PMA staining, the lowest detection limit of 102 CFU/mL did not change. However, a slight increase (approximate 1.5 cycles) in the Ct values from PMA-treated samples was observed. The increase in Ct values might be because a small portion of E. coli O157:H7 cells is lost during the washing step after PMA treatment (Liang et al., 2011). Another possible reason was that there could be a small portion of dead cells present in the overnight culture used in this experiment. The compromised membranes of dead cells in the overnight culture were penetrated by PMA and the amplification of their DNA was then inhibited in the following PCR reactions, resulting in larger Ct values of PMA-treated samples. The PMA treatment, as optimized previously, had no adverse effect on detection of viable E. coli O157:H7 by real-time PCR. To compare PMA with EMA, another set of viable cells was treated by EMA following Wang et al. (2009) prior to real-time PCR. It turned out that the detection limit of viable E. coli O157:H7 in pure culture using EMA real-

5

a. without PMA

y = -3.6488x + 47.518 R² = 0.9473

b. with PMA

y = -3.5991x + 48.825 R² = 0.9635

0 0

2

4

6

8

10

Cell count (Log CFU/mL) Fig. 4. Standard curves, without PMA (a) and with PMA (b) for detection of viable E. coli O157:H7 cells, in the absence of dead cells, by real-time PCR. Results are from two repeated experiments.

time PCR assay was also 102 CFU/mL, which was the same as that of PMA real-time PCR (data not shown). 3.5. Application of PMA real-time PCR to mixed viable and dead E. coli O157:H7 cells Without PMA treatment, the real-time PCR was unable to differentiate between DNA from viable E. coli O157:H7 cells and that from dead cells (Fig. 5). Because DNA from both viable and dead cells served as templates for real-time PCR, the number of viable cells was significantly overestimated. For example, when 101 CFU/mL of viable cells were present together with 106/mL of dead cells, the real-time PCR without PMA treatment generated a Ct value of 25.8, whereas the PMA realtime PCR assay could not detect any E. coli O157:H7 (neither viable nor dead) in the mixture (Fig. 5). The Ct value obtained above corresponded to the Ct value yielded from 106 CFU/mL of viable cells by using real-time PCR without PMA treatment (Fig. 4). These results confirm that PMA staining prior to DNA extraction can effectively prevent false-positive results in real-time PCR that was caused by the presence of DNA from dead cells. With PMA treatment, this real-time PCR assay could detect a range of 102 to 108 CFU/mL of viable E. coli O157:H7 cells even in the presence of 106/mL dead cells (Fig. 5). Comparing Fig. 5 with Fig. 4, the detection range of PMA real-time PCR for viable cells, when both viable and dead cells were present, was the same as when only viable cells were present. Although similar slopes of the two standard curves were observed, the Ct values from viable and dead cell mixtures (y = −3.2767x + 46.42; R2 = 0.9368) were slightly smaller than that from only viable cells

40

40 E. coli O157

35

35

IAC

30

30 25

Ct

25

Ct

51

20

20 15

15

a. without PMA

10

10

y = -3.2767x + 46.42

5

b. with PMA R² = 0.9368

5 UD

0 0

2

5

10

15

20

IAC

0 0

2

4

6

8

10

Cell count (Log CFU/mL)

Light exposure time (min) Fig. 3. Influence of light exposure time on amplification of DNA from 108 CFU/mL of viable E. coli O157:H7 cells. Results were from two repeated experiments. UD, undetected.

Fig. 5. Detection range of E. coli O157:H7 cells without PMA (a) and with PMA (b), when viable and 106 dead cells/mL are present, by real-time PCR. Results are from two repeated experiments.

Y. Liu, A. Mustapha / International Journal of Food Microbiology 170 (2014) 48–54

(y = −3.5991x + 48.825; R2 = 0.9635), especially when the concentrations of viable cells were low (≤103 CFU/mL), indicating a slight overestimation of viable cells in the mixture. For example, the average Ct value yielded from 102 CFU/mL viable and 106/mL dead cells was 36.4, whereas that generated from 102 CFU/mL viable alone was 38.5. However, no Ct value was yielded from the control when only 106/mL dead cells were present (data not shown), which again proved that the PMA treatment was sufficient to eliminate DNA from dead cells when only dead cells were present. These indicate that the PMA treatment was not able to completely remove signals from a high number of dead E. coli O157: H7 cells when both viable and dead cells were present in the sample. Incomplete suppression of dead cell signals using PMA real-time PCR has been reported previously (Pan and Breidt, 2007; Kralik et al., 2010; Løvdal et al., 2011). Pan and Breidt (2007) stated that the number of viable Listeria monocytogenes was significantly overestimated when the ratio of dead cells to live cells exceeded 104 and the concentration of live cells was less than 103 CFU/mL. We found that the detection limit of viable E. coli H157:H7 in a mixture of viable and dead cells using EMA real-time PCR, was the same as that of PMA real-time PCR. However, the Ct values from EMA-treated viable and dead cell mixtures were larger than those from only viable cells (data not shown), which indicates that EMA also penetrates viable cells, thus influencing the Ct values generated by real-time PCR. Therefore, based on the results, it was concluded that PMA overestimates viable E. coli O157:H7 cells in viable and dead cell mixture, whereas EMA underestimates viable cells in the mixture.

40

(a)

35 30 25 20 15

y = -4.1025x + 55.727 R² = 0.9362

a. only viable

10

y = -4.5275x + 58.147

b. viable and dead mixture R² = 0.9568

5 0 4

Ct

52

40

5

6

7

8

9

8

9

(b)

35 30 25 20 15

y = -3.8588x + 54.412 R² = 0.9253

a. only viable

10

b. viable and dead mixture y = -3.7335x + 52.407

5

R² = 0.9946

0 4

5

6

7

Cell count (Log CFU/g) 3.6. Application of PMA real-time PCR to artificially contaminated ground beef Both culture-based and real time PCR-based tests confirmed that the ground beef was originally free of E. coli O157:H7. The PMA real-time PCR assay was applied to ground beef samples inoculated with only viable E. coli O157:H7 from 101 to 108 CFU/g. It was also applied to ground beef samples which were simultaneously inoculated with 106/g of dead E. coli O157:H7 cells and different concentrations of viable E. coli O157: H7 cells (100 to 108 CFU/g). As shown in Fig. 6, the PMA real-time PCR could detect a range from 105 to 108 CFU/g of viable E. coli O157:H7, in ground beef samples regardless of whether dead cells were present or not. Again, the PMA treatment was proven to be effective at penetrating dead E. coli O157: H7 cells without being negatively influenced by meat tissues and background microflora. The relatively high detection limit of viable E. coli O157:H7 cells in ground beef (105 cells/g) could be caused by: 1) the high counts of background microflora in the ground beef interfering with the detection of target E. coli O157:H7 cells (the aerobic plate count of the 10% fat content ground beef was 1.7 × 106 CFU/g, and that of the 27% fat content ground beef was 1.5 × 105 CFU/g); 2) the food samples which contain many organic and inorganic substances, such as phenolic compounds, fat, enzymes, polysaccharides, proteins and salts, all of which can either inhibit PCR amplification or lead to a reduction in amplification efficiency of PCR reactions (Španová et al., 2000); and 3) a small portion of viable cells that may not have recovered after homogenization by stomaching and the intact membranes of some viable cells that might have been injured during homogenization and penetrated by PMA, leading to fewer DNA templates in the following PCR reactions. Standard curves of ground beef with 10% and 27% fat contents exhibited very similar linear relationships between Ct values and the concentrations of viable E. coli O157:H7 in ground beef. Nonetheless, the detection limit of 105 CFU/g in ground beef was still not satisfactory, since the infectious dose of E. coli O157:H7 is low. Therefore, a preenrichment step was needed to increase the number of viable cells to a detectable level when the initial concentration is low. Yang et al. (2013) reported that 103 CFU/g of viable E. coli O157:H7 in ground beef could be detected by using magnetic nanobead-based

Fig. 6. Application of PMA real-time PCR to artificially contaminated 10% ground beef (a) and 27% fat ground beef (b), and its detection range for viable or viable and dead E. coli O157:H7 cells. Results are from two repeated experiments.

immunomagnetic separation (IMS) combined with PMA multiplex PCR (mPCR). Although their IMS–PMA–mPCR assay achieved a better detection limit of E. coli O157:H7 in ground beef (103 cells/g) than this study (105 cells/g) did, it was still not sensitive enough to detect the pathogen at its infectious dose. On the other hand, with an 8-h enrichment step, the PMA real-time PCR assay described in this study could detect as low as 1 CFU/g of viable E. coli O157:H7 in ground beef, which is even lower than its infectious dose. Moreover, a real-time PCR assay is more sensitive than traditional PCR and can be quantitative for pathogenic bacterial detection in food. By plugging the Ct value of an unknown sample into the standard curve (Fig. 6), the concentration of viable E. coli O157:H7 in the original sample could be estimated. Continuation of this study by combining IMS with PMA real-time PCR would be an interesting next step.

3.7. Detection of low concentrations of viable E. coli O157:H7 in ground beef samples by PMA real-time PCR with enrichment To detect low concentrations of viable E. coli O157:H7 cells in ground beef, enrichment in TSBY was conducted prior to the PMA real-time PCR. A 6-h enrichment was required to detect initial concentrations of 102, 103 and 104 CFU/g viable E. coli O157:H7. After an 8-h enrichment, PMA real-time PCR could detect as low as 1 CFU/g of viable E. coli O157: H7 in ground beef (Table 1). The results from 10% fat content and those from 27% fat content ground beef were the same. In addition, it was also found that the sample inoculated with only 106/g dead cells generated negative results during the entire enrichment process using PMA realtime PCR, while the same dead cell samples generated false-positive results using real-time PCR without PMA treatment (Table 1). These results demonstrated that the addition of PMA prior to DNA extraction effectively eliminated the DNA from dead E. coli O157:H7 cells in ground beef. Blank food samples without any artificial inoculation of E. coli O157:H7 were also enriched, showing that there was no E. coli O157:

Y. Liu, A. Mustapha / International Journal of Food Microbiology 170 (2014) 48–54 Table 1 Application of PMA real-time PCR to ground beef (10% and 27% fat contents) contaminated with low concentrations of viable and 106 dead cells/g E. coli O157:H7. Results are from two repeated experiments. Enrichment time (h)

2h

4h

6h

8h

Concentration of viable cells (CFU/g)

Concentration of dead cells (cells/g)

PMA treatment

No PMA treatment

E. coli O157:H7

IAC

E. coli O157:H7

IAC

104 103 102 101 100 0 104 103 102 101 100 0 104 103 102 101 100 0 104 103 102 101 100 0

106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106

− − − − − − − − − − − − + + + − − − + + + + + −

+ + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + +

H7 contamination in the original samples. The addition of an IAC in this study successfully monitored the efficiency of the real-time PCR, preventing the occurrence of false-negative results. Both viable and dead cells can be present in food products. However, only viable pathogens pose a risk to consumers' health. Therefore, effective methods for the detection of only viable bacterial pathogens are needed. A pre-enrichment step is commonly included in pathogen detection methods, allowing the ratio of live cells to dead cells to increase and decreasing the influence of DNA from dead cells in a subsequent PCR. However, as shown in this study, enrichment steps alone cannot completely prevent the false-positive result generated from 106/g dead E. coli O157:H7 cells after the 8-h enrichment period. With PMA staining, DNA amplification from dead cells in the subsequent PCR and the generation of false-positive results yielded from dead cells were successfully prevented. Compared with the literature, a shorter enrichment time (8 h) and a lower detection limit (1 CFU/g) was achieved in this study. This might be because a shaker incubator was used for the enrichment period in this study, which facilitated faster growth of E. coli O157: H7. Additionally, instead of 1 mL of the enriched cell suspension, 10 mL of sample was used for DNA isolation. This modification resulted in a higher yield of final DNA which, in turn, facilitated and improved its amplification in the subsequent real-time PCR. In September 2011, the U.S. Department of Agriculture (USDA) announced that serogroups O26, O45, O103, O111, O121, and O145 of Shiga toxin producing E. coli would also be considered adulterants when present in raw or nonintact beef products (USDA, 2011). The PMA-real time PCR assay developed in this study shows great promise and could be optimized for detection of viable cells of these other pathogenic serogroups in ground beef in future studies. 4. Conclusions Overall, a PMA treatment can effectively prevent the amplification of DNA from dead E. coli O157:H7 cells in ground beef. The PMA realtime PCR assay described in this study can selectively detect only viable

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E. coli O157:H7 with a sensitivity of 102 CFU/mL in pure culture, and 105 CFU/g in ground beef, in the presence of 106 dead cells/mL or g. With an 8-h enrichment, the assay could detect as low as 1 CFU/g viable E. coli O157:H7 in ground beef. The whole process can easily be completed in about 5 h after an 8-h enrichment step. Hence, PMA realtime PCR assay has the potential to be employed as a rapid, yet selective and sensitive, detection method for the food industry to detect only viable E. coli O157:H7 in food products. Acknowledgments We sincerely thank Dr. Luxin Wang and Prashant Singh for their invaluable assistance and suggestions in this research. 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