1887

Abstract

Foodborne illness due to bacterial pathogens is increasing worldwide as a consequence of the higher consumption of fresh and minimally processed food products, which are more easily cross-contaminated. The efficiency of food pasteurization methods is usually measured by c.f.u. plate counts, a method discriminating viable from dead cells on the basis of the ability of cells to replicate and form colonies on standard growth media, thus ignoring viable but not cultivable cells. Supercritical CO (SC-CO) has recently emerged as one of the most promising fresh food pasteurization techniques, as an alternative to traditional, heat-based methods. In the present work, using three SC-CO-treated foodborne bacteria (, and ) we tested and compared the performance of alternative viability test methods based on membrane permeability: propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM). Results were compared based on plate counts and fluorescent microscopy measurements, which showed that the former dramatically reduced the number of cultivable cells by more than 5 log units. Conversely, FCM provided a much more detailed picture of the process, as it directly quantifies the number of total cells and distinguishes among three categories, including intact, partially permeabilized and permeabilized cells. A comparison of both PMA-qPCR and FCM with plate count data indicated that only a fraction of intact cells maintained the ability to replicate . Following SC-CO treatment, FCM analysis revealed a markedly higher level of bacterial membrane permeabilization of with respect to and . Furthermore, an intermediate permeabilization state in which the cellular surface was altered and biovolume increased up to 1.5-fold was observed in , but not in or . FCM thus compared favourably with other methods and should be considered as an accurate analytical tool for applications in which monitoring bacterial viability status is of importance, such as microbiological risk assessment in the food chain or in the environment.

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2013-06-01
2024-12-06
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References

  1. Anon. ( 2002). Risk profile on the microbiological contamination of fruits and vegetables eaten raw: Report of the Scientific Committee on Food Brussels: SCF/CS/FMH/SURF;
    [Google Scholar]
  2. Bae Y. Y., Choi Y. M., Kim M. J., Kim K. H., Kim B. C., Rhee M. S.( 2011). Application of supercritical carbon dioxide for microorganism reduction in fresh pork. J Food Saf 31:511–517 [View Article]
    [Google Scholar]
  3. Ballestra P., Abreu da Silva A., Cuq J. L.( 1996). Inactivation of Escherichia coli by carbon dioxide under pressure. J Food Sci 61:829–831 [View Article]
    [Google Scholar]
  4. Barbesti S., Citterio S., Labra M., Baroni M. D., Neri M. G., Sgorbati S.( 2000). Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria. Cytometry 40:214–218 [View Article][PubMed]
    [Google Scholar]
  5. Berney M., Hammes F., Bosshard F., Weilenmann H. U., Egli T.( 2007). Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290 [View Article][PubMed]
    [Google Scholar]
  6. Devlieghere F., Vermeiren L., Debevere J.( 2004). New preservation technologies: possibilities and limitations. Int Dairy J 14:273–285 [View Article]
    [Google Scholar]
  7. Erkmen O.( 2000). Effect of carbon dioxide pressure on Listeria monocytogenes in physiological saline and foods. Food Microbiol 17:589–596 [View Article]
    [Google Scholar]
  8. Erkmen O., Karaman H.( 2001). Kinetic studies on the high pressure carbon dioxide inactivation of Salmonella typhimurium. J Food Eng 50:25–28 [View Article]
    [Google Scholar]
  9. Ferrentino G., Spilimbergo S.( 2011). High pressure carbon dioxide pasteurization of solid foods: current knowledge and future outlooks. Trends Food Sci Technol 22:427–441 [View Article]
    [Google Scholar]
  10. Foladori P., Quaranta A., Ziglio G.( 2008). Use of silica microspheres having refractive index similar to bacteria for conversion of flow cytometric forward light scatter into biovolume. Water Res 42:3757–3766 [View Article][PubMed]
    [Google Scholar]
  11. Foladori P., Bruni L., Tamburini S., Ziglio G.( 2010). Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry. Water Res 44:3807–3818 [View Article][PubMed]
    [Google Scholar]
  12. Gandhi M., Chikindas M. L.( 2007). Listeria: a foodborne pathogen that knows how to survive. Int J Food Microbiol 113:1–15 [View Article][PubMed]
    [Google Scholar]
  13. Garcia-Gonzalez L., Geeraerd A. H., Spilimbergo S., Elst K., Van Ginneken L., Debevere J., Van Impe J. F., Devlieghere F.( 2007). High pressure carbon dioxide inactivation of microorganisms in foods: the past, the present and the future. Int J Food Microbiol 117:1–28 [View Article][PubMed]
    [Google Scholar]
  14. Garcia-Gonzalez L., Geeraerd A. H., Mast J., Briers Y., Elst K., Van Ginneken L., Van Impe J. F., Devlieghere F.( 2010). Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment. Food Microbiol 27:541–549 [View Article][PubMed]
    [Google Scholar]
  15. Jung W. Y., Choi Y. M., Rhee M. S.( 2009). Potential use of supercritical carbon dioxide to decontaminate Escherichia coli O157 : H7, Listeria monocytogenes, and Salmonella typhimurium in alfalfa sprouted seeds. Int J Food Microbiol 136:66–70 [View Article][PubMed]
    [Google Scholar]
  16. Keer J. T., Birch L.( 2003). Molecular methods for the assessment of bacterial viability. J Microbiol Methods 53:175–183 [View Article][PubMed]
    [Google Scholar]
  17. Kim H. T., Choi H. J., Kim K. H.( 2009). Flow cytometric analysis of Salmonella enterica serotype Typhimurium inactivated with supercritical carbon dioxide. J Microbiol Methods 78:155–160 [View Article][PubMed]
    [Google Scholar]
  18. Klein D.( 2002). Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8:257–260 [View Article][PubMed]
    [Google Scholar]
  19. Liao H., Zhang F., Liao X., Hu X., Chen Y., Deng L.( 2010). Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining. Int J Food Microbiol 144:169–176 [View Article][PubMed]
    [Google Scholar]
  20. Mantoan D., Spilimbergo S.( 2011). Mathematical modeling of yeast inactivation of freshly squeezed apple juice under high-pressure carbon dioxide. Crit Rev Food Sci Nutr 51:91–97 [View Article][PubMed]
    [Google Scholar]
  21. Müller S., Nebe-von-Caron G.( 2010). Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiol Rev 34:554–587[PubMed]
    [Google Scholar]
  22. Nocker A., Camper A. K.( 2006). Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol 72:1997–2004 [View Article][PubMed]
    [Google Scholar]
  23. Nocker A., Sossa-Fernandez P., Burr M. D., Camper A. K.( 2007a). Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 73:5111–5117 [View Article][PubMed]
    [Google Scholar]
  24. Nocker A., Sossa K. E., Camper A. K.( 2007b). Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. J Microbiol Methods 70:252–260 [View Article][PubMed]
    [Google Scholar]
  25. Oliver J. D.( 2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev 34:415–425[PubMed]
    [Google Scholar]
  26. Spilimbergo S., Bertucco A.( 2003). Non-thermal bacterial inactivation with dense CO2. Biotechnol Bioeng 84:627–638 [View Article][PubMed]
    [Google Scholar]
  27. Suo B., He Y., Tu S. I., Shi X.( 2010). A multiplex real-time polymerase chain reaction for simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products. Foodborne Pathog Dis 7:619–628 [View Article][PubMed]
    [Google Scholar]
  28. Velusamy V., Arshak K., Korostynska O., Oliwa K., Adley C.( 2010). An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnol Adv 28:232–254 [View Article][PubMed]
    [Google Scholar]
  29. WHO( 2007). Global Public Health Security in the 21st Century Geneva: World Health Organization;
    [Google Scholar]
  30. Wouters P. C., Bos A. P., Ueckert J.( 2001). Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Appl Environ Microbiol 67:3092–3101 [View Article][PubMed]
    [Google Scholar]
  31. Ziglio G., Andreottola G., Barbesti S., Boschetti G., Bruni L., Foladori P., Villa R.( 2002). Assessment of activated sludge viability with flow cytometry. Water Res 36:460–468 [View Article][PubMed]
    [Google Scholar]
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