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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS Title: Quantificative Analysis of Viable, Stressed and Dead Cells of Campylobacter Jejuni Strain 81-176

item He, Yiping
item Chen, Chin-Yi

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 26, 2009
Publication Date: April 22, 2010

Interpretive Summary: Campylobacter jejuni is the leading cause of bacterial food-borne illness in USA, and can be found in a high proportion of poultry products. It requires atmospheres containing lower levels of oxygen and high levels of carbon dioxide for growth which renders detection and isolation of the bacteria difficult. The ability to detect C. jejuni in foods is critical for preventing food-borne illnesses, achieving public health goals, and minimizing the spread of the pathogen. Molecular methods to detect and quantify live, stressed, and dead C. jejuni cells, including a technique involving selective amplification of DNA from live cells, were developed and tested. The results showed that the conventional plate counting method was less sensitive than the DNA amplification method or a bacterial live/dead staining method for detection of viable cells. Therefore, conventional plate counting methods should be used in conjunction with more sensitive molecular methods for detection and enumeration of viable C. jejuni.

Technical Abstract: Campylobacter jejuni is an important foodborne gastrointestinal pathogen. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses such as low nutrient availability, entry into stationary phase, or low CO2/high O2 conditions. In this study, molecular and microscopic methods, including real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. With the real-time PCR assay, highly sensitive and specific quantification of total cell numbers of C. jejuni in different growth phases was achieved. In addition, our results revealed that real-time PCR can be used for direct quantification of Campylobacter genome release into the PBS as an indicator of cell death and is suitable for quantitative determination of dead cells in suspensions. By EMA-PCR, we obtained a dynamic range of greater than 3 log for differentiating viable versus dead cells. The viability and morphological characteristics of the stressed cells after one week incubation at 25 degrees C in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99 percent of the stressed cells were converted from the spiral to the coccoid form and became non-culturable within 1 week. However, more than 96 percent of the coccoid cells retained their viability based on membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus the cell culturability obtained by plate counting is the least sensitive method in detecting viable cells, and should be used in conjunction with other molecular methods such as EMA-PCR for detection of food-borne pathogens that are particularly sensitive to stresses.

Last Modified: 4/22/2015
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