Project Summary

Rapid Quantification of Culturable and Viable-but-nonculturable Escherichia coli O157:H7 in Beef Products using EMA-Real Time PCR

Principle Investigator(s):
Azlin Mustapha
University of Missouri
Completion Date:
May 2007



Escherichia coli O157:H7 causes an estimated 73,500 cases of infections in the United States ev  ery year (Buchanan et al., 1997), with a main source of infections being undercooked ground beef and other bovine products.  In order to extend beef shelf life and reduce the potential risks associated with pathogenic growth, many meat preservation methods, including heat processing, irradiation, freezing, refrigeration and addition of food grade preservation have been applied (Jay, 2000).  Although these methods have been shown to be efficient at reducing pathogenic growth, outbreaks continue to occur.   

Bacterial cells that have been exposed to unfavorable environments, such as those following typical meat processing steps like heating, cleaning and sanitizing, can undergo a unique physiological or metabolic change in order to adapt to these harsh conditions.  These cells may enter a status known as the viable but nonculturable (VBNC) state (Rowan, 2004).  This new phenomenon poses a challenge to the traditional and widely used AOAC- or FDA-approved microbial testing methods, because VBNC bacteria, which cannot grow on non-selective or selective culture media, can maintain their virulence in this state.  A previous study by our group conclusively demonstrated that VBNC E. coli O157:H7 still retained their Shiga-toxin genes in this state (unpublished data).  Therefore, the potential risk of VBNC foodborne pathogens to consumers should be a concern to the meat industry.  

For the past ten years, our group has conducted extensive work on developing rapid, sensitive and selective PCR-based detection methods for E. coli O157:H7, Salmonella and Shigella in beef and other foods.  Our most recent publication (Wang et al., 2007) showed that real-time PCR could be used to sensitively detect and quantitate pathogens in ground beef in real time.  In this current work, we added an ethidium monoazide (EMA) staining step to our established real-time PCR protocol for E. coli O157:H7 detection and quantitation to effectively decrease false-positive results from dead cells, brought about by conventional PCR methods, and false-negative results from VBNC cells, caused by traditional cultural methods.  EMA is a stain (dye) that can bind to DNA in dead cells by penetrating the disintegrated cell wall of the dead organism.  With this selective binding, DNA from dead cells cannot be amplified.  Thus, when a mixture of dead and viable cells are present in a sample, only DNA from viable cells will be amplified in a subsequent PCR reaction following EMA treatment.  This combination of EMA-real-time PCR reaction allows for a more accurate determination of the number of viable, including VBNC, cells that may be present in beef products.   

With regards to viability and pathogenicity of cells in the VBNC status, rapid identification methods, such as this developed EMA-real-time PCR, will be an effective way to detect VBNC pathogenic cells in beef before they reach retail or consumers, thus preventing the outbreaks and foodborne illnesses that might result from false negative results generated by traditional cultural techniques.  At the same time, the successful development of this method will contribute to a better understanding of the VBNC phenomenon in E. coli O157:H7 and provide valuable information for further reduction of important pathogens in the beef processing line. 

The stated objectives for this work were: 

  • To establish an EMA-real time PCR assay for quantifying viable E. coli O157:H7.  
  • To determine detection sensitivity and linear range of the EMA-real time PCR system for quantitating VBNC E. coli O157:H7. 
  • To evaluate the performance of the EMA-real time PCR system for quantitating E. coli O157:H7 in beef.

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