Detection of Viable but Nоnculturable Microbial Cells in Chicken Mince

There is a potential for existence of hazardous viable but nonculturable (VBNC) cells of pathogenic microorganisms in foodstuffs that can be formed under the influence of various factors. Their detection and determination of conditions for formation of VBNC cells of various bacteria are relevant for preventing contamination of meats. This was the aim of the present study. The search was conducted for VBNC cells in chicken mince in real time and during experimental infection of it by Staphylococcus aureus 209P. In order to detect VBNC cells in chicken mince, total number of microbes, number of bacterial colonies (CFU), and the portion of living (dead) cells were determined in 1g of the product using a commercial set of fluorescent dyes. A second study was carried out after 5 h of incubation of tested samples at room temperature. In samples of minced meat on the 4th day after production, more than 99 % of all detected living cells were VBNC. After 5-hour incubation of the sample, the number of CFU/g increased by 22.5 times, but the portion of VBNC cells remained higher than 99 % of viable bacteria. During artificial infection of the same batch of mince with S. aureus in broth culture at the stage of logarithmic growth, the amount of VBNC cells for 0 hours was 97.33 %. After 5 hours their number increased to 99.99 %. Probably, in the introduced culture of Staphylococcus at the stage of active reproduction, formation of VBNC bacteria did not occur, which initially reduced their number in the sample. After 5-h incubation, transition of bacteria to VBNC state was accelerated, possibly due to unfavorable conditions for the cell population (changes in trophic substrate, temperature, pH, etc.). Experimental data confirm presence of VBNC bacteria in chicken products that don’t grow on traditional nutrient media and showing a false negative result in traditional microbiological expertise. Because of the biohazard of such dormant cells, it is advisable to provide regulated testing of foodstuffs for presence of VBNC cells.

1. Abdullaeva, A. M., Blinkova, L. P., & Pakhomov, Yu. D. (2019). Significance of viable but nonculturable bacteria for safety of food products. Problemy Veterinarnoy, Sanitarii, Gigieny i Ekologii [Problems of Veterinary, Sanitation, Hygiene and Ecology] 2(30), 183-189. http://dx.doi.org/10.25725/vet.san.hyg.ecol.201902012

2. Al-Qadiri, H. M., Lu, X., Al-Alami, N. I., & Rasco, B. A. (2011). Survival of Escherichia coli O157: H7 and Campylobacter jejuni in bottled purified drinking water under different storage conditions. Journal of Food Protection, 74(2), 254–260. https://doi.org/10.4315/0362-028X.JFP-10-368

3. Asakura, H., Makino, S.-I., Takagi, T., Kuri, A., Kurazono, T., Watarai, M., & Shirahata, T. (2002). Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation from the viable but non-culturable state. FEMS Microbiology Letters, 212(1), 87-93. https://doi.org/10.1111/j.1574-6968.2002.tb11249.x

4. Aurass, P., Prager, R., & Flieger, A. (2011). EHEC/ EAEC O104: H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief. Environmental Microbiology, 13(12), 3139–3148. https://doi.org/10.1111/j.1462-2920.2011.02604.x

5. Ayrapetyan, M., & Oliver, J. D. (2016). The viable but non-culturable state and its relevance in food safety. Current Opinion in Food Science, 8, 127–133. https://doi.org/10.1016/j.cofs.2016.04.010

6. Barron, J. C., & Forsythe, S. J. (2007). Dry stress and survival time of Enterobacter sakazakii and other Enterobacteriaceae in dehydrated powdered infant formula. Journal of Food Protection, 70(9), 2111–2117.https://doi.org/10.4315/0362-028X-70.9.2111

7. Bates, T. C., & Oliver, J. D. (2004). The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus. Journal of Microbiology, 42(2), 74–79.

8. Blinkova, L., Martirosyan, D., Pakhomov, Yu., Dmitrieva, O., Vaughan, R., & Altshuler M. (2014). Nоnculturable forms of bacteria in lyophilized probiotic preparations. Functional Foods in Health and Disease, 4(2), 66-76.

9. Capozzi, V., Di Toro, M. R., Grieco, F., Michelotti, V., Salma, M., Lamontanara, A., Russo, P., Orru, L., Alexandre, H., & Spano, G. (2016). Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: new insights on molecular basis of VBNC behaviour using a transcriptomic approach. Food Microbiology, 59, 196–204. https://doi.org/10.1016/j.fm.2016.06.007

10. Chaisowwong, W., Kusumoto, A., Hashimoto, M., Harada, T., Maklon, K., & Kawamoto, K. (2012). Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat. Journal of Veterinary Medical Science, 74(1), 43–50. https://doi.org/10.1292/jvms.11-0305

11. Cunningham, E., O’Byrne, C., & Oliver, J. D. (2009). Effect of weak acids on Listeria monocytogenes survival: evidence for a viable but nonculturable state in response to low pH. Food Control, 20(12), 1141–1144. https://doi.org/10.1016/j.foodcont.2009.03.005

12. Ding, T., Suo, Y., Xiang, Q., Zhao, X., Chen, S., Ye, X., & Liu, D. (2017). Significance of viable but nonculturable Escherichia coli: induction, detection, and control. Journal of Microbiology and Biotechnology, 27(3), 417–428 https://doi.org/10.4014/jmb.1609.09063.

13. Dinu, L. D., & Bach, S. (2013). Detection of viable but non-culturable Escherichia coli O157: H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays. Food Control, 31(2), 268-273.

14. Divol B. & Lonvaud-Funel A. (2004). Evidence for viable but nonculturable yeasts in botrytis – affected wine. Journal of Applied Microbiology, 99(1), 85-93.

15. Fakruddin, M., Mannan, K. S., & Andrews, S. (2013). Viable but nonculturable bacteria: food safety and public health perspective. ISRN Microbiology, 2013:703813. https://doi.org/10.1155/2013/703813

16. Gunasekera, T. S., Sørensen, A., Attfield, P. V., Sørensen, S. J., & Veal, D. A. (2002). Inducible gene expression by nonculturable bacteria in milk after pasteurization. Applied and Environmental Microbiology, 68(4), 1988–1993. https://doi.org/10.1128/AEM.68.4.1988-1993.2002

17. Hu, Y., & Coates, A. (2012). “Nonmultiplying bacteria are profoundly tolerant to antibiotics,” in Antibiotic Resistance, eds R. M. Anthony and A. Coates (Berlin: Springer Press), 99–119. https://doi.org/10.1007/978-3-642-28951-4_7

18. Kramer, B., & Muranyi, P. (2014). Effect of pulsed light on structural and physiological properties of Listeria innocua and Escherichia coli. Journal of Applied Microbiology, 116(3), 596–611. https://doi.org/10.1111/jam.12394

19. Li, L., Mendis, N., Trigui, H., Oliver, J. D., & Faucher, S. P. (2014). The importance of the viable but nonculturable state in human bacterial pathogens. Frontiers in Microbiology, 5, 258. https://doi.org/10.3389/fmicb.2014.00258

20. Liu, J., Li, L., Li, B., Peters, B. M., Deng, Y., Xu, Z. & Shirtliff, M. E. (2017). First study on the formation and resuscitation of viable but nonculturable state and beer spoilage capability of Lactobacillus lindneri. Microbial Pathogenesis, 107, 219-224. https://doi.org/ 10.1016/j.micpath.2017.03.043

21. Makino, S.-I., Kii, T., Asakura, H., Shirahata, T., Ikeda, T., Takeshi, K., & Itoh, K. (2000). Does Enterohemorrhagic Escherichia coli O157: H7 enter the viable but nonculturable state in salted salmon roe? Applied and Environmental Microbiology, 66(12), 5536–5539. https://doi.org/10.1128/AEM.66.12.5536-5539.2000

22. Masmoudi, S., Denis, M., & Maalej, S. (2010). Inactivation of the gene katA or sodA affects the transient entry into the viable but nonculturable response of Staphylococcus aureus in natural seawater at low temperature. Marine Pollution Bulletin, 60(12), 2209–2214. https://doi.org/10.1016/j.marpolbul.2010.08.017

23. Nicolò, M. S., Gioffrè, A., Carnazza, S., Platania, G., Silvestro, I. D., & Guglielmino, S. P. (2010). Viable but nonculturable state of foodborne pathogens in grapefruit juice: a study of laboratory. Foodborne Pathogens and Disease, 8(1), 11–17. https://doi.org/10.1089/fpd.2009.0491

24. Nicolò, M. S., & Guglielmino, S. P. P. (2012). “Viable but nonculturable bacteria in food,” in Public Health–Methodology, Environmental and Systems Issues, ed. J. Maddock (Rjeka: InTech), 189–216. https://doi.org/10.5772/38118

25. Nyström, T. (2003). Nonculturable bacteria: programmed survival forms or cells at death’s door? Bioessays, 25(3), 204–211. https://doi.org/10.1002/bies.10233

26. Oh, E., McMullen, L., & Jeon, B. (2015). Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions. Frontiers in Microbiology, 6, 295. https://doi.org/10.3389/fmicb.2015.00295

27. Oliver, J. D. (2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiology Reviews, 34(4), 415-425. https://doi.org/10.1111/j.1574-6976.2009.00200.x

28. Peneau, S., Chassaing, D., & Carpentier, B. (2007). First evidence of division and accumulation of viable but nonculturable Pseudomonas fluorescens cells on surfaces subjected to conditions encountered at meat processing premises. Applied and Environmental Microbiology, 73(9), 2839–2846. https://doi.org/10.1128/AEM.02267-06

29. Pinto D., Santos M. A., & Chambel L. (2015). Thirty years of viable but nonculturable state research unsolved molecular mechanisms. Critical Reviews in Microbiology, 41(1), 61-76.

30. Ramamurthy, T., Ghosh, A., Pazhani, G. P., & Shinoda, S. (2014). Current Perspectives on Viable but Non- Culturable (VBNC) Pathogenic Bacteria. Frontiers in Public Health, 2, 103. https://doi.org/10.3389/fpubh.2014.00103

31. Rao, N. V., Shashidhar, R., & Bandekar, J. R. (2014). Induction, resuscitation and quantitative realtime polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water. World Journal of Microbiology and Biotechnology, 30, 2205–2212. https://doi.org/10.1007/s11274-014-1640-1

32. Rowan, N. J. (2004). Viable but non-culturable forms of food and waterborne bacteria: quo vadis? Trends in Food Science & Technology, 15(9) 462–467. https://doi.org/10.1016/j.tifs.2004.02.009

33. Rowan, N. J., Valdramidis, V. P., & Gomez-Lopez, V. M. (2015). A review of quantitative methods to describe efficacy of pulsed light generated inactivation data that embraces the occurrence of viable but non culturable state microorganisms. Trends in Food Science & Technology, 44(1), 79–92. https://doi.org/10.1016/j.tifs.2015.03.006

34. Schottroff, F., Fröhling, A., Zunabovic-Pichler, M., Krottenthaler, A., Shlüter, O., & Jäger, H. (2018). Sublethal injury and viable but nonculturable (VBNC) state in microorganisms during preservation of food and biological materials by non-thermal processes. Frontiers in Microbiology, 9, article 2773, p. 1-19. https://doi.org/10.3389/fmicb.2018.02773.

35. Serpaggi, V., Remize, F., & Recorbet, G. (2012). Characterization of the “viable but nonculturable” (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiology, 30(2), 440-447.

36. Zhang, S., Ye, C., Lin, H., Lv, L., & Yu, X. (2015). UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa. Environmental Science & Technology, 49(3), 1721–1728. https://doi.org/ 10.1021/es505211e

37. Zhao, X., Zhong, J., Wei, C. Lin C.-W., & Ding T. (2017). Current perspectives on viable but nonculturable state in foodborne pathogens. Frontiers in Microbiology, 8, 1-32. https://doi.org/10.3389/fmicb.2017.00580

38. Zhao, X., Wei, C., Zhong, J., & Jin, S. (2016). Research advance in rapid detection of foodborne Staphylococcus aureus. Biotechnology & Biotechnological Equipment, 30(5), 1–7. https://doi.org/ 10.1080/13102818.2016.1209433

39. Ziprin, R. L., Droleskey, R. E., Hume, M. E., & Harvey, R. B. (2003). Failure of viable nonculturable Campylobacter jejuni to colonize the cecum of newly hatched leghorn chicks. Avian Diseases, 47(3), 753–758. https://doi.org/10.1637/7015