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Keywords:

  • detection;
  • E. coli O157: H7;
  • PMA ;
  • qPCR ;
  • sterilization

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References

Propidium monoazide is a DNA-intercalating dye. PMA-qPCR has been reported as a novel method to detect live bacteria in complex samples. In this study, this method was used to monitor the sterilization effects of UHP, ultrasound and high PEF on Escherichia coli O157:H7. Our results showed that all three sterilization techniques are successful to kill viable E. coli O157:H7 cells under their appropriate conditions. PMA-qPCR can effectively monitor the amount of DNA released from viable E. coli O157:H7 cells, and the results from PMA-qPCR were highly consistent with those from plate counting after treatment with UHP, ultrasound and high PEF. The maximal ΔCt between PMA-qPCR and qPCR obtained in this study was 10·39 for UHP, 5·76 for ultrasound and 2·30 for high PEF. The maximal sterilization rates monitored by PMA-qPCR were 99·92% for UHP, 99·99% for ultrasound and 100% for high PEF. Thus, PMA-qPCR can be used to detect the sterilization effect on food and water supplies after treatment with UHP, ultrasound and high PEF.

Significance and Impact of the Study

The reliable detection of viable foodborne pathogenic bacteria in water and food is of great importance in our daily life. However, the traditional bacteria cultivation-based methods are time-consuming and difficult to monitor all viable bacteria because of the limitation of cultivation conditions. This study demonstrated that PMA-qPCR technique is very effective to monitor viable E. coli O157:H7 after sterilization and will help to monitor the viable bacteria in food and water.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References

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 Ecoli 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.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References

UHP treatment

As shown in Fig. 1a, plate counting results showed that at a constant pressure, the sterilization efficiency increased with prolonged treatment time; similarly, at a constant time, the sterilization efficiency correlated with increased pressure. It has been reported that pressure and time are two important factors affecting the killing efficiency of UPH sterilization method (Hayashi 1996). The results from UHP treatments at 100 MPa and 400 MPa for 1 min were not significantly different from those without UHP treatment. The sterilization rate was less than 90%; thus, it cannot meet the minimal food storage requirement. However, UHP treatments at 200, 300 and 400 MPa for 10 min were effective, which showed high sterilization rates of up to 99%. These results suggest that bacterial membrane is more sensitive to treatment time, but high pressure of 300 MPa or higher and at least 10-min treatment are recommended.

image

Figure 1. Sterilization rate of UPH (a), ultrasound (b) and pulsed electric field (PEF) (c) treatments determined by plate counts. The sterilization rate = 1 – CFU (after treatment)/initial CFU. (image) 1 min; (image) 10 min.

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The similar results were found in samples processed for 1 min or 10 min using PMA-qPCR (Fig. 2a). The Ct values increased as both time and pressure increased, and the result further confirmed that 10-min treatment at 400 MPa was adequate to kill all cells, which is similar to that from traditional plate count method. These evidences validate the effectiveness of PMA-qPCR as a tool in monitoring the sterilization effect of UHP treatment.

image

Figure 2. PMA-qPCR and qPCR results of samples treated with Ultrahigh pressure (UHP) (a), ultrasound (b) and pulsed electric field (PEF) (c). (image_n/lam12052-gra-0001.png) 1 min/no PMA, (image_n/lam12052-gra-0002.png) 1 min/PMA, (image_n/lam12052-gra-0003.png), 10 min/no PMA (image_n/lam12052-gra-0004.png), 10 min/PMA; (image_n/lam12052-gra-0005.png) qPCR, (image_n/lam12052-gra-0006.png) PMA-qPCR.

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In addition, Mackey et al. (1994) have reported that the structure of DNA is quite stable at high pressures. In our study, the Ct values of samples between with and without PMA treatment were significant different after treatment at 400 MPa for 10 min (Fig. 2a, P < 0·05), but the reasons remain unknown.

Ultrasound treatment

According to the results from traditional plate count method, a prolonged treatment resulted in a rapid increase in sterilization efficiency (Fig. 1b). The number of live bacteria decreased quickly during the initial 4 min before it reached a platform. After 6 min, the sterilization rate reached 100%.

We also performed PMA-qPCR and qPCR on the cell pellet (Fig. 2b). Both PMA-treated and untreated sediment had higher Ct values with longer sterilization time. It is likely that ultrasound treatment destroys cell membranes, and the DNA is released to the extracellular environment. The result of PMA-qPCR indicated that after 8-min treatment, no positive signals for DNA amplification were detected in the pellet. However, the Ct value of qPCR was almost 35, suggesting cell survival within the sediment. Because PMA was able to bind to DNA and disrupt the subsequent PCR, the different results between the PMA-qPCR and the qPCR suggest that neither 8-min nor 10-min ultrasound treatment is able to destroy cell membrane completely, because DNA is not all released to the supernatant and detected in the cell pellet by qPCR.

High PEF treatment

After exposure to high PEF, 1·5 ml of each sample in replicate was centrifuged at 8000 g for 5 min. The UV absorption of top 1 ml supernatant was measured at the wavelength of 280 nm or 260 nm. The pellet was washed twice with 1 ml ddH2O, and then subjected to PMA treatment.

Membrane destruction, which results in increased membrane permeability and/or membrane breakdown, occurs when a critical value of electrical field strength exceeds the membrane potential (Tsong 1990). The Ct values of samples treated with and without PMA increased with increasing field intensity, and the difference between PMA-qPCR and qPCR was significant (Fig. 2c). One reason for this might be the existence of cells with a compromised membrane. The DNA of these cells could be amplified by qPCR, but PMA treatment could block the amplification because of DNA–PMA binding.

When the field intensity was lower than 20 kv cm−1, the Ct value of qPCR remained consistent. We concluded that low field intensity could not affect cell viability. The result was also confirmed by plate counting experiment (Fig. 1c).

Measurement of UV absorbance at 280 or 260 nm revealed the relative amount of intracellular substances was leaked from cells after PEF treatment, and harsher treatment caused high value of UV absorbance (Table 1).

Table 1. Absorbance of released DNA or protein after cells was treated with high pulsed electric field (PEF)
E (kv cm−1)010152025304050
OD2800·080·130·190·260·270·270·270·27
OD2600·060·070·100·150·150·160·160·16

The leakage of DNA and/or protein from cells increased with higher field intensity. Compared with untreated samples, the OD280 and OD260 values of samples treated with 10 kv cm−1 PEF were slightly elevated. The difference was more significant when the PEF was elevated from 10 to 20 kv cm−1, but the OD280 and OD260 values remained steady after 20 kv cm−1. Aronsson et al. (2001) suggested a PEF threshold that can completely destroy the membrane to release all intracellular components. In our experiment, the threshold was 20 kv cm−1.

Both the result of PMA-qPCR and the measurement of UV absorbance at 280 nm and/or 260 nm support the idea that the cell membrane is incomplete at relatively low fields of intensity, and 20 kv can completely destroy the cell membrane.

The correlation between Ct value of PMA-qPCR and plate counting

The Ct values of PMA-qPCR and the log CFU values of plate counting showed a good linear correlation with an R2 of 0·995, 0·954 and 0·928 for UHP, ultrasound and high PEF, respectively (Fig. 3). The maximal ΔCt between PMA-qPCR and qPCR obtained in this study was 10·39 for UHP, 5·76 for ultrasound and 2·30 for high PEF. The maximal sterilization rates monitored by PMA-qPCR were 99·92% for UHP, 99·99% for ultrasound and 100% for high PEF. All three sterilization methods reduced the number of live bacteria, and the PMA-qPCR method could monitor the sterilization rate accurately.

image

Figure 3. Standard curve of propidium monoazide (PMA)-qPCR for samples treated with Ultrahigh pressure (UHP) (a), ultrasound (b) and pulsed electric field (PEF) (c).

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References

Our work demonstrated that PMA-qPCR can effectively monitor the amount of DNA released from viable E. coli O157:H7 cells. All three sterilization techniques are successful to kill bacterial cells under their appropriate conditions. Thus, PMA-qPCR can be used to detect the sterilization effect on food and water supplies after treatment with UHP, ultrasound and high PEF.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References

Bacterial strain and culture conditions

A clinical E. coli O157: H7 was used for this study. Single colonies from LB (Luria-Bertani) plates were transferred to 50-ml culture tubes containing 10 ml LB medium. The E. coli O157:H7 was cultured in a shaker at 37°C for about 12 h. The cultures were then grown to log phase. Densities of E. coli O157: H7 cultures were adjusted to an OD600 of 1 by diluting with LB broth.

Sterilization conditions

Cells were treated using UPH, ultrasound and PEF. These three methods are widely used in the food processing industry. After treatment, 100 μl of cell suspension was serially diluted and plated on LB agar plates and then cultured at 37°C for 24 h to determine the loss of viability.

For UPH in our study, bacterial cultures (109 CFU ml−1) were packed in sterile bags, vacuumed and then sealed. The bags were pressurized at 100, 200, 300 and 400 MPa for 1 or 10 min at room temperature. Then, the bacteria were cultured and homogenized to guarantee a uniform population. The UHP-sterilized samples and the unsterilized control were stored at 4°C for plate counting, qPCR and PMA-qPCR experiments.

For ultrasonic treatment, a total of 30 ml bacterial cultures (109 CFU ml−1) were transferred to a 50-ml sterile tube and treated with a lab scale horn sonotrode (SCIENTZ-IID). The diameter of the horn tip was 1·3 cm, and the frequency was fixed at 20 kHz. The tip of the horn was placed in the centre of the tube and immersed for 1 cm in depth. E. coli O157:H7 cultures were treated with a discontinuous ultrasonic irradiation for 0, 2, 4, 6, 8 and 10 min. During the treatment, the tube was placed on ice to avoid extra heating. The discontinuous treatment had an intermittent ratio of 3 : 1 (3 s of sonication and 1 s of no sonication). After treatment, samples were centrifuged at 8000 g and cell pellets were resuspended in ddH2O and used for plate counting, qPCR and PMA-qPCR experiments.

For PEF treatment, the initial microbial load was approximately 109 CFU ml−1. Physiological saline solution was used as the treatment medium with an electric conductivity of 340 μS cm−1. Cultures were centrifuged at 8000 g for 5 min, and the cells were resuspended in 500 ml isovolumetric physiological saline solution. Samples were treated in a bench-scale continuous PEF system. At the beginning of each experiment, uninoculated physiological saline solution was pumped through the system as the start-up medium with a flow velocity (v) of 20 ml min−1. High voltage pulsing was then turned on, and the pulse frequency and charge voltage were adjusted. A square-waved pulse with a pulse duration of 20 μs was generated, and the applied pulse frequency (f) was 1000 Hz. The electrical field strengths were set at 0, 10, 15, 20, 25, 30, 40 and 50 KV. The first 100 ml of each 400 ml suspension was discarded. Then, 10 ml samples treated with each condition were collected into sterile bottles and were stored at 4°C for plate counting, qPCR and PMA-qPCR experiments.

All experiments were repeated for at least three times.

PMA treatment

Propidium monoazide (Biotium Inc., Hayward, CA, USA) was dissolved in 20% DMSO to generate a stock solution of 0·5 mg ml−1. The final concentration used in this study was 10 μg ml−1. A total of 10 μl PMA was added to 500-μl culture aliquots in a transparent microcentrifuge tube. The tubes were then incubated in the dark for 5 min to allow the PMA to penetrate the damaged cell membranes and bind to DNA. Subsequently, the uncapped tubes were exposed to a 650-W halogen light (Osram, Augsburg, Germany) for 5 min at a distance of 15 cm to allow PMA cross-linking with DNA. During the light exposure, samples were kept on ice to reduce the generation of extra heat. To guarantee a uniform light exposure, occasional shaking was performed. The samples were immediately centrifuged at 12,000 g for 5 min, and the supernatants were discarded. The pellets were washed with 500 μl ddH2O and then subjected to DNA isolation and qPCR assay.

DNA isolation procedures

The aliquots were boiled in water for 10 min and then cooled to room temperature. The supernatant was removed to a new tube after centrifugation at 12 000 g for 5 min and subsequently subjected to qPCR.

Real-time PCR amplification

Real-time PCR was carried out in a 25-μl reaction mixture. The sequences of primers/probe targeting RfbE gene were designed according to Enrica et al. (2009). The forward primer sequence was 5′-CTACAGGTGAAGGTGGAATGGT-3′ (22 bp). The reverse primer sequence was 5′-GTAGCCTATAACGTCATGCCAAT-3′ (23 bp). The probe sequence was 5′-FAM-TCACGAATGACAAAACACTTTATGAC-BHQ1. For all experiments, the PCR amplification was carried out in a 25-μl reaction mixture containing 2·5 μl 10 × Taq buffer, 3·5 mmol l−1 MgCl2, 400 nmol l−1 forward primer, 400 nmol l−1 reverse primer, 200 nmol l−1 probe, 2·5 U Taq DNA polymerase and 2 μl DNA template.

Relative quantitative PCR and data analysis were both performed on the Applied Biosystems Prism 7500 Sequence Detection System using the following thermal cycling parameters: 95°C for 2 min, 40 PCR cycles of 5 s at 95°C and 40 s at 60°C. Cycle thresholds were generated automatically by the ABI 7500 software. Each experiment was performed in triplicate.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References

This work was supported by the National Natural Science Foundation of China (No. 31101279 and No. 31271867), the Fundamental Research Funds for the Central Universities (No. 2011ZM0101), the Doctoral Program Foundation of Institutions of Higher Education of China (No. 20110172120034) and the Open Project Program of Provincial Key Laboratory of Green Processing Technology and Product Safety of Natural Products.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Materials and methods
  7. Acknowledgements
  8. References
  • Aronsson, K., Lindgren, M., Johansson, B.R. and Ronner, U. (2001) Inactivation of microorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae. Innov Food Sci Emerg Technol 2, 4154.
  • Bae, S. and Wuertz, S. (2009) Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide. Appl Environ Microbiol 75, 29402944.
  • Belasco, J.G. and Brawerman, G. (1993) mRNA degradation in prokaryotic cells: an overview. Control of messenger RNA stability 6, 312.
  • Besnard, V., Federighi, M. and Cappelier, J.M. (2000) Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes. J Appl Microbiol 31, 7781.
  • Brondum, J., Egebo, M., Agerskov, C. and Busk, H. (1998) On-line pork carcass grading with the autoform ultrasound system. J Anim Sci 76, 18591868.
  • Elizaquível, P., Sánchez, G. and Aznar, R. (2012) Application of propidium monoazide quantitative PCR for selective detection of live Escherichia coli O157:H7 in vegetables after inactivation by essential oils. Int J Food Microbiol 159, 115121.
  • Enrica, O., Giulia, A., Giorgio, B. and Mauro, M. (2009) A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk. Food Microbiol 26, 615622.
  • Frankenhuyzen, J., Trevors, J., Lee, H., Flemming, C. and Habash, M. (2011) Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration. J Microbiol Methods 87, 263272.
  • Gadri, N.M., Augier, G. and Carpentier, B. (2009) Characterization of bacterial strains isolated from a beef-processing plant following cleaning and disinfection—Influence of isolated strains on biofilm formation by Sakaï and EDL 933 E. coli O157:H7. Int J Food Microbiol 133, 6267.
  • Hayashi, R. (1996) An overview of the use of high pressure in bioscience and biotechnology. High Pressure Biosci Biotechnol 13, 16.
  • Liang, Z. and Keeley, A. (2012) Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples. Water Res 4, 59415953.
  • Mackey, B.M., Forestiere, K., Isaacs, N.S., Stenning, R. and Brooker, B. (1994) The Effect of High Hydrostatic Pressure on Salmonella thompson and Listeria monocytogenes examined by Electron Microscopy. Lett Appl Bacteriol 19, 429432.
  • Marouani-Gadri, N., Firmesse, O., Chassaing, D., Sandris-Nielsen, D., Arneborg, N. and Carpentier, B. (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, 96103.
  • Nataro, J.P. and Kaper, J.B. (1998) Diarrheagenic Escherichia coli. Clin Microbiol Rev 11, 142201.
  • Nocker, A., Cheung, C.Y. and Camper, A.K. (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67, 310320.
  • Nocker, A., Sossa, K.E. and Camper, A.K. (2007) Molecular monitoring of sterilization rate using propidium monoazide in combination with quantitative PCR. J Microbiol Methods 70, 252260.
  • Nocker, A., Mazza, A., Masson, L., Camper, A.K. and Brousseau, R. (2009) Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology. J Microbiol Methods 76, 253261.
  • Nogva, H., Dromtorp, S., Nissen, H. and Rudi, K. (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR. Biotechniques 34, 804812.
  • O'Brien, M.C. and Bolton, W.E. (1995) Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19, 243255.
  • Pan, Y. and 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: 80288031.
  • Pribylova, R., Kubickova, L., Babak, V., Pavlik, I. and Kralik, P. (2012) Effect of short- and long-term antibiotic exposure on the viability of Mycobacterium avium subsp. paratuberculosis as measured by propidium monoazide F57 real time quantitative PCR and culture. Vet J 194, 354360.
  • Ravel, J., Knight, I.T., Monohan, C.E., Hill, R.T. and Colwell, R.R. (1995) Temperature-induced recovery of Vibrion cholera from the viable but nonculturable state: growth or resuscitation. Microbiology 141, 377383.
  • Rudi, K., Moen, B., Dromtorp, S.M. and Holck, A.L. (2005) Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples. Appl Environ Microbiol 71, 10181024.
  • Stern, N.J., Jones, D.M., Wesley, I.V. and Rollins, D.M. (1994) Colonization of chicks by non-culturable Campylobacter spp. Lett Appl Microbiol 18, 333336.
  • Thomas, S.W., Elizabeth, A.M. and Sean, D.R. (1998) Pathogenic Escherichia coli O157:H7: a model for emerging infectious diseases. Biomed Res Reports 51, 163183.
  • Tsong, T.Y. (1990) On electroporation of cell membranes and some related phenomena. Bioelectrochem Bioenerg 24, 271295.
  • Varma, M., Field, R., Stinson, M., Rukovets, B., Wymer, L. and Haugland, R. (2009) Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in waste water. Water Res 43, 47904801.
  • Vesper, S., Mckinstry, C., Hartmann, C., Neace, M., Yoder, S. and Vesper, A. (2008) Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide(PMA). J Microbiol Methods 72, 180184.
  • Wang, S. and Levin, R.E. (2006) Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. J Microbiol Methods 64, 18.
  • Yáñez, M., Nocker, A., Soria-Soria, E., Martínez, R. and Catalán, V. (2011) Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR. J Microbiol Methods 85:124130.
  • Zhu, R., Li, T., Jia, Y. and Song, L. (2012) Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR. J Microbiol Methods 90, 262266.