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
Received: 06 April, 2017; Accepted: 19 May, 2017; Published: 20 May, 2017
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:
Mahmoud Ouf JM, Yuan Y, Singh P, Mustapha A (2017) Detection of Viable but Nonculturable Escherichia coli O157:H7 in Ground Beef by Propidium Monoazide real-time PCR. Int J Agric Sc Food Technol 3(2): 026-031. DOI: 10.17352/2455-815X.000018
© 2017 Mahmoud Ouf JM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
E. coli O157; qPCR; PMA; Lactic acid; Stress
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 quantification 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.
Escherichia coli O157:H7 is an important foodborne pathogen that causes gastrointestinal illness as well as life-threatening diseases . 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 . Furthermore, this pathogen has the potential to enter into the viable-but-nonculturable state . 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 . In fact, pathogenic bacteria can be avirulent in the VBNC state but regain virulence after resuscitation into culturable cells under suitable conditions . Reissbrodt et al. (2002) , 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  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 .
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 . 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 . In fact, because cells of E. coli O157:H7 in the VBNC state retain virulence, they should be considered as risks to public health .
Conventional culture-based methods involving enrichment, isolation and confirmation steps, are commonly used for detection of foodborne pathogens . However, culture-based methods may considerably underestimate true bacterial cell counts when a fraction of E. coli O157:H7 cells in a sample is nonculturable . Molecular methods, such as the polymerase chain reaction (PCR), are increasingly used for detection of VBNC cells , because they do not rely on colony growth. However, conventional PCR cannot differentiate viable cells from dead cells because DNA from dead cells can serve as a template during PCR amplification . Our previous publications [17-19], have shown that DNA-intercalating agents, such as propidium monoazide (PMA) and ethidium monoazide (EMA) can be used in conjunction with a PCR assay to selectively detect viable cells. These dyes can penetrate membrane-damaged cells and covalently bind to cellular DNA, thus preventing DNA amplification from dead bacteria and enabling exclusive detection of viable cells [20-22].
This study aimed to determine if E. coli O157:H7 could adapt to the stress caused by lactic acid by entering the VBNC state, and to evaluate the applicability of a PMA-qPCR to detect and quantify VBNC cells of this pathogen. The performance of this PMA-qPCR method was assessed using pure culture and ground beef samples spiked with lactic acid induced VBNC E. coli O157:H7 cells.
Materials and Methods
Preparation of viable E. coli O157:H7 cells
E. coli O157:H7 strain 505B (a beef isolate) was obtained from the culture collection of the Food Microbiology Laboratory, University of Missouri Columbia (Columbia, MO, USA). The strain was grown overnight in Tryptic Soy broth supplemented with 0.5% yeast extract (TSBY, Difco Labs., Sparks, MD, USA) at 37 °C until the numbers reached ~109 CFU/mL as determined by plate counting on Tryptic Soy Agar (TSA) (Difco Labs.). Fresh overnight grown E. coli O157:H7 culture was serially diluted using 9 mL 0.1% peptone water, plated in TSA, and colonies enumerated after an overnight incubation at 37 oC.
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 . 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 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 modifications. 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.
Primers and probes targeting E. coli O157:H7 were designed by Wang et al. . pUC19 plasmid DNA was used as an internal amplification control (IAC). Primers and probes targeting pUC19 were as designed by Fricker et al., 2007  (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 × 104 copies) (Promega, Madison, WI, USA), and 5 μL template DNA. Nuclease-free water (Promega) was used to adjust the reaction volume to 25 μL . 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 coefficient of regression (R2) values.
- Carol AP (1999) The epidemiology, detection and control of Escherichia coli O157. J Sci Food Agric 79: 1367–1381. Link: https://goo.gl/CN9nF5
- Marouani-Gadri N, Firmesse O, Chassaing D, Sandris-Nielsen D, Arneborg N, et al. (2010) Potential of Escherichia coli O157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment. Int J Food Microbiol 144: 96–103. Link: https://goo.gl/2Q47mY
- Rigsbee W, Simpson LM, Oliver JD (1997) Detection of the viable but nonculturable state in Escherichia coli O157:H7. J Food Safety 16: 255–262. Link: https://goo.gl/S2A9pj
- Oliver JD (2000) The public health significance of viable but nonculturable bacteria. In Nonculturable Microorganisms in the Environment 277-300. Link: https://goo.gl/GtMQrs
- Du M, Chen J, Zhang X, Li A, Li Y, et al. (2007) Retention of virulence in à viable but nonculturable Edwardsiella tarda isolate. Appl Environ Microbiol 73: 1349–1354. Link: https://goo.gl/1cyxYX
- Reissbrodt R, Rienaecker I, Romanova JM, Freestone PPE, Haigh RD, et al. (2002) Resuscitation of Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat stable enterobacterial autoinducer. Appl Environ Microbiol 68: 4788–4794. Link: https://goo.gl/kRJfr6
- Baffone W, Citterio B, Vittoria E, Casaroli A, Campana R, et al. (2003) Retention of virulence in viable but nonculturable halophilic Vibrio spp. Int J Food Microbiol 89: 31-39. Link: https://goo.gl/LbZ3tD
- Yaron S, Matthews KR (2002) A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes. J Appl Microbiol 92: 633–640. Link: https://goo.gl/H1lfia
- Asakura H, Igimi S, Kawamoto K, Yamamoto S, Makino S (2005) Role of in vivo passage on the environmental adaption of enterohemorrhagic Escherichia coli O157:H7: cross-induction of the viable but nonculturable state by osmotic and oxidative stresses. FEMS Microbiol Lett 253: 243–249. Link: https://goo.gl/lj2EYQ
- Rahman I, Shahamat M, Chowdhury R (1996) Potential virulence of viable but nonculturable Shigella dysenteriae Type 1. Appl Environ Microbiol 62: 115-120. Link: https://goo.gl/zUbXY8
- Dinu LD, Bach S (2011) Induction of viable but non-culturable Escherichia coli O157:H7 in the hyllosphere of lettuce: a food safety risk factor. Appl Environ Microbiol 77: 8295-8302. Link: https://goo.gl/EsFA7f
- Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43: 93–100. Link: https://goo.gl/JyzK9G
- Murakami T (2012) Filter-based pathogen enrichment technology for detection of multiple viable foodborne pathogens in one day. J Food Prot 75: 1603–1610. Link: https://goo.gl/ZkU64V
- Rowan NJ (2004) Viable but nonculturable forms of food and waterborne bacteria: QuoVadis? Trends Food Sci Technol 15: 462–467. Link: https://goo.gl/PmJBXk
- Oliver JD (2010) Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev 34: 415–425. Link: https://goo.gl/Oogkva
- Wang S, Levin RE (2006) Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. J Microbiol Methods 64: 1–8. Link: https://goo.gl/swlWKM
- Liu Y, Mustapha A (2014) Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR. Int J Food Microbiol 170: 48–54. Link: https://goo.gl/VTUdFa
- Wang L, Li Y, Mustapha A (2009) Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR. J Appl Microbiol 107: 1719-1728. Link: https://goo.gl/ssc1el
- Wang L, Mustapha A (2010) EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs. J Food Sci 75: M134-M139. Link: https://goo.gl/UCiyDm
- Lee JL, Levin RE (2009) A comparative study of the ability of EMA and PMA to distinguish viable from heat killed mixed bacterial flora from fish fillets. J Microbiol Methods 76: 93–96. Link: https://goo.gl/dCMs9m
- Nocker A, Cheung CY, Camper AK (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cell. J Microbiol Methods 67: 310–320. Link: https://goo.gl/SDPJrH
- Pan Y, Breidt F (2007) Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73: 8028–8031. Link: https://goo.gl/5H6TkI
- Wang L, Li Y, Mustapha A (2007) Rapid and simultaneous quantitation of Escherichia coli O157:H7, Salmonella and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. J Food Prot 70: 1366-1372. Link: https://goo.gl/AhfF5F
- Fricker M, Messelha¨ußer U, Busch U, Scherer S, Ehling-Schulz M (2007) Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Appl Environ Microbiol 73: 1892–1898. Link: https://goo.gl/1K1Wqo
- FDA (1995) Bacteriological Analytical Manual, 8th edition. Gaitherburg, MD, USA:AOAC International
- Dinu LD, Bach S (2013) Detection of viable but non-culturable Escherichia coli O157:H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays. Food Control 31: 268-273.Link: https://goo.gl/gMA4hq
- Wang G, Doyle MP (1998) Survival of enterohemorrhagic Escherichia coli O157:H7 in water. J Food Prot 61: 662–667. Link: https://goo.gl/2sVmdA
- Makino SI, Kii T, Asakura H, Shirahata T, Ikeda T, et al. (2000) Does enterohemorrhagic Escherichia coli enter the viable but nonculturable state in salted salmon roe? Appl Environ Microbiol 66: 5536–5539. Link: https://goo.gl/U8Y053
- Zhao L, Matthews KR (2000) Influence of starvation, temperature, and pH on culturability of enterohemorrhagic Escherichia coli O157:H7. J Food Safety 20: 193–208. Link: https://goo.gl/Hd0aj9
- Benjamin MM, Datta AR (1995) Acid tolerance of enterohemorrhagic Escherichia coli. Appl Environ Microbiol 61: 1669-1672. Link: https://goo.gl/w6kz4I
- Buchanan RL, Bagi LK (1994) Expansion of response surface models for the growth of Escherichia coli O157:H7 to include sodium nitrite as a variable. Int J Food Microbiol 23: 317-322. Link: https://goo.gl/R2wiZJ
- Xiao Xl, Tian C, Yu YG, Wu H (2013) Detection of viable but nonculturable Escherichia coli O157:H7 using propidium monoazide treatments and qPCR. Can J Microbiol 59: 157-163. Link: https://goo.gl/8OH8xn
- Španová A, Rittich B, Karpíšková R, Čechová L, Škapová D (2000) PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation. Bioseparation 9: 379–384. Link: https://goo.gl/rkW6W2
- Lambertz ST, Ballagi-Pordany A, Lindqvist R (1998) A mimic as internal standard to monitor PCR analysis of food-borne pathogens. Lett Appl Microbiol 26: 9–11. Link: https://goo.gl/7fB3ct
- Hoorfar J, Cook N, Malorny B, Wagner M, De Medici D, et al. (2004) Diagnostic PCR: making internal amplification control mandatory. J Clin Microbiol 96: 221–222. Link: https://goo.gl/tGX3AC
- Murphy NM, McKauchlin J, Ohai O, Grant KA (2007) Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica. Int J Food Microbiol 120: 110–119. Link: https://goo.gl/2EVpfJ