- Top of page
- Results and discussion
- Materials and methods
Escherichia coli O157:H7 can cause foodborne diseases with the symptoms of bloody diarrhoea, haemorrhagic colitis and haemolytic-uremic syndrome (Nataro and Kaper 1998). In North America, Europe and Japan, it has caused large-scale epidemic outbreak and thousands of sporadic cases of gastrointestinal illnesses (Thomas et al. 1998). These infections may be caused by food and environmental contaminations (Gadri et al. 2009).
The reliable detection of viable bacteria is a great challenge to the monitoring of water and food safety. Moreover, the monitoring of viable foodborne pathogenic bacteria is a critical and necessary procedure in the food industry and is of great importance in our daily life. Plate counts or other cultivation-based approaches are traditional methods to detect viable cells. However, some viable bacteria may not be culturable because of environmental conditions (Stern et al. 1994). Furthermore, cultivation is time-consuming and may cause biased evaluation due to different media selection in different culture methods. As viable but not culturable (VBNC) cells remain potentially pathogenic under favourable conditions (Besnard et al. 2000), they are difficult to be detected by traditional culturing methods (Ravel et al. 1995). The presence of VBNC is a potential, but significant risk factor in the food industry.
Reverse transcriptase PCR (RT-PCR) has been used to detect mRNA expression from viable cells (Brondum et al. 1998; Nocker et al. 2006), but RNAs are degraded rapidly after cell death (Belasco and Brawerman 1993). Another method to discriminate live and dead cells is flow cytometry. The major difficulty in flow cytometry is that the observation of viable cells spans a narrower detection range than PCR or plate counts (Rudi et al. 2005). Moreover, in many environmental samples, bacteria form aggregates that are difficult to separate into single cell for flow cytometric analysis, causing an underestimation of the cell count.
Based on RT-PCR and flow cytometry techniques, DNA binding dye like Ethidium bromide monoazide (EMA) has been used to differentiate viable from dead cells (O'Brien and Bolton 1995; Nogva et al. 2003; Wang and Levin 2006). Despite the effectiveness of EMA in reducing PCR signals from dead cells, a disadvantage remains in this application because this compound can cross intact membranes of some bacterial species and lead to false negative cell counts (Nocker et al. 2006). Therefore, a new method, propidium monoazide (PMA) combined with qPCR (PMA-qPCR), has been developed. Live cells with intact membranes can completely exclude PMA (perhaps due to the 2+ charge on the PMA molecule compared with the 1+ charge on EMA). This difference gives PMA an advantage in differentiating live/dead cells in a bacterial population (O'Brien and Bolton 1995; Pan and Breidt 2007; Varma et al. 2009; Frankenhuyzen et al. 2011). Indeed, PMA has successfully been applied as an useful reagent to detect viable cells in many microbiological species including Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Serratia marcescens, E. coli O157:H7 (Nocker et al. 2009), Vibrio vulnificus (Wang and Levin 2006), fungi in air and water samples (Vesper et al. 2008), faecal Bacteroidales bacteria (Bae and Wuertz 2009), Legionella pneumophila (Yáñez et al. 2011), as well as, Vibrio parahaemolyticus (Zhu et al. 2012) and fresh Cryptosporidium oocysts (Liang and Keeley 2012).
PMA-qPCR has been used to monitor the effect of different disinfection methods such as hypochlorite, benzalkonium, heat (Nocker et al. 2007), antibiotic exposure (Pribylova et al. 2012) and ammonia or hydrogen peroxide treatment (Liang and Keeley 2012). For detection of live cells or evaluation of sterilization rate to E. coli O157:H7, PMA-qPCR was proved to be a good method. A study shows that conditions leading to E. coli O157:H7 persistence are not likely to arise when good refrigeration and hygiene practices are applied and highlights the usefulness of EMA or PMA-qPCR as a complement to CFU determination in studying bacterial survival after cleaning and disinfection (Marouani-Gadri et al. 2010). Elizaquível et al. (2012) applied the PMA-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water PMA-qPCR, and the results indicate that the PMA-qPCR method is a suitable technique for evaluating ultrasonic disinfection of vegetable wash water, being able to distinguish between live and dead bacteria. Thus, PMA-qPCR has shown promise as a rapid and reproducible method for assessing cell viability and therefore is a valuable alternative method to plate counts.
Ultrahigh pressure (UHP), ultrasound and high-pulsed electric field (PEF) are three sterilization methods that have little influence on nutritional components during the sterilization process. Due to this advantage, they are expected to be widely used in the food industry in the future. The aim of this study was to evaluate the applicability of PMA-qPCR method in monitoring the sterilization effect of three different methods. Our results will help to understand the mechanical process of these methods in killing potential pathogens.