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Detection and Evaluation of Viable but Non-culturable Escherichia coli O157:H7 Induced by Low Temperature with a BCAC-EMA-Rti-LAMP Assay in Chicken Without Enrichment

  • Hu Chen
  • Yuan Yang Zhao
  • Mei Shu
  • Tian Tian Zhang
  • Yan Bi
  • Yan Yan Gao
  • Guo Ping Wu
Article
  • 16 Downloads

Abstract

In this study, a rapid and sensitive method that combines ethidium bromide monoazide (EMA) staining with real-time loop-mediated isothermal amplification (Rti-LAMP) assay was tested in detecting and evaluating viable but non-culturable state (VBNC) Escherichia coli (E. coli) O157:H7 induced by low temperature. When 1.7 × 104 CFU/ml of E. coli O157:H7 was 3 cycles of freeze-thaw, the cells were all dead. However, E. coli O157:H7 in 1.7 × 106 CFU/ml and 1.7 × 108 CFU/ml gradually transferred into VBNC state reaching 6.8 × 102 CFU/ml (0.04%) and 4.1 × 105 CFU/ml (0.24%) after 6 cycles of freeze-thaw, respectively. Keeping E. coli O157:H7 (1.7 × 108 CFU/ml) at 4 °C and − 20 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells increased from 0 to 1.1 × 106 CFU/ml and 5.5 × 106 CFU/ml detected by both EMA-Rti-LAMP and direct epifluorescence method (DEM) up to 258-day storage at 4 °C and − 20 °C, respectively. The EMA-Rti-LAMP had similar accuracy with DEM in detecting viable including VBNC cells, the former had specificity but not the later. Furthermore, the EMA-Rti-LAMP could detect as low as 25 CFU/g of VBNC E. coli O157:H7 derived from contaminated chicken combined with bentonite-coated activated carbon (BCAC) treatment to remove DNA amplification inhibitors, and the entire assay could be completed in 5 h. In addition, one same sample for E. coli O157:H7 positive was detected by both BCAC-EMA-Rti-LAMP and plate count method from 24 retail chicken samples, but four positive samples by BCAC-Rti-LAMP assay. The results obviously suggested that the BCAC-EMA-Rti-LAMP assay might be a rapid and sensitive method for detection of viable including VBNC E. coli O157:H7 cells in food without enrichment.

Keywords

Escherichia coli O157:H7 (E. coli O157:H7) Viable but non-culturable state (VBNC) Chicken Rti-LAMP Ethidium bromide monoazide (EMA) Bentonite-coated activated carbon (BCAC) 

Notes

Funding

This study was funded by the National Natural Science Foundation of China (No. 31560480, 31760483), Jiangxi Natural Science Foundation of China (No. 20171ACB20013), and the Special Funds for Collaborative Innovation of Modern Agricultural Science and Research in Jiangxi, China (No. JXXTCX201703).

Compliance with Ethical Standards

Conflict of Interest

Hu Chen declares that he has no conflict of interest. Yuan Yang Zhao declares that he has no conflict of interest. Mei Su declares that she has no conflict of interest. Tian Tian Zhang declares that she has no conflict of interest. Yan Bi declares that she has no conflict of interest. Yan Yan Gao declares that she has no conflict of interest. Guo Ping Wu declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Not applicable.

References

  1. Abolmaaty A, Vu C, Oliver J, Levin RE (2000) Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction. Microbios 101:181–189PubMedGoogle Scholar
  2. Almasoud A, Hettiarachchy N, Rayaprolu S, Babu D, Kwon YM, Mauromoustakos A (2016) Inhibitory effects of lactic and malic organic acids on autoinducer type 2 (AI-2) quorum sensing of Escherichia coli O157:H7 and Salmonella typhimurium. LWT Food Sci Technol 66:560–564CrossRefGoogle Scholar
  3. Arana I, Muela A, Orruno M, Seco C, Garaizabal I, Barcina I (2010) Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli. FEMS Microbiol Ecol 74:500–509CrossRefGoogle Scholar
  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. Environ Microbiol 13(12):3139–3148CrossRefGoogle Scholar
  5. Baena-Ruano S, Jiménez-Ot C, Santos-Dueñas IM, Cantero-Moreno D, Barja F, García-García I (2006) Rapid method for total, viable and non-viable acetic acid bacteria determination during acetification process. Process Biochem 41:1160–1164CrossRefGoogle Scholar
  6. Branco P, Monteiro M, Moura P, Albergaria H (2012) Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization. Int J Food Microbiol 158:49–57CrossRefGoogle Scholar
  7. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Writtwer C (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefGoogle Scholar
  8. 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 31:268–273CrossRefGoogle Scholar
  9. Dong HJ, Cho AR, Hahn TW, Cho S (2014) Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples. J Food Prot 77:1593–1598CrossRefGoogle Scholar
  10. Ferens WA, Hovde CJ (2011) Escherichis coli O157:H7 : animal reservoir and sources of human infection. Foodborne Pathog Dis 8(4):465–487CrossRefGoogle Scholar
  11. Galloway WRJD, Hodgkinson JT, Bowden SD, Welch M, Spring DR (2011) Quorum sensing in gram-negative Bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways. Chem Rev 111:28–67CrossRefGoogle Scholar
  12. Hara-Kudo Y, Nemoto J, Ohtsuka K, Segawa Y, Takatori K, Kojima T, Ikedo M (2007) Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J Med Microbiol 56:398–406CrossRefGoogle Scholar
  13. Ivanova IA, Kambarev S, Popova RA, Naumovska EG, Markoska KB, Dushkin CD (2010) Determination of Pseudomonas putida live cells with classic cultivation and staining with “live/dead Baclight bacterial viability kit”. Biotechnol Biotechnol Equip 24:567–570CrossRefGoogle Scholar
  14. Kobayashi H, Oethinger M, Tuohy MJ, Hall GS, Bauer TW (2009) Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide. Lett Appl Microbiol 48:633–638CrossRefGoogle Scholar
  15. Lee JL, Levin RE (2006) Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction. J Microbiol Methods 67:456–462CrossRefGoogle Scholar
  16. LeStrange K, Markland SM, Hoover DG, Sharma M, Kniel K (2017) An evaluation of the virulence and adherence properties of avian pathogenic Escherichia coli. One Health 4:22–26CrossRefGoogle Scholar
  17. Liu Y, Mustapha A (2014) Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR. Int J Food Microbiol 170:48–54CrossRefGoogle Scholar
  18. Liu J, Zhou R, Li L, Peters BM, Li B, Lin CW, Chuang TL, Chen D, Zhao X, Xiong Z, Xu Z, Shirtliff ME (2017) Viable but non-culturable state and toxin gene expression of enterohemorrhagic Escherichia coli O157 under cryopreservation. Res Microbiol 168:188–193CrossRefGoogle Scholar
  19. Luan C, Levin R (2008) Use of activated carbon coated with bentonite for increasing the sensitivity of PCR detection of Escherichia coli O157:H7 in Canadian oyster (Crassostrea gigas) tissue. J Microbiol Methods 72:67–72CrossRefGoogle Scholar
  20. 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? Appl Environ Microbiol 66(12):5536–5539CrossRefGoogle Scholar
  21. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63CrossRefGoogle Scholar
  22. Rigsbee W, Simpson LM, Oliver JD (1997) Detection of the viable but nonculturable state in Escherichia coli O157:H7. J Food Saf 16(4):255–262CrossRefGoogle Scholar
  23. Roszak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment. Microbiol Rev 51:365–379PubMedPubMedCentralGoogle Scholar
  24. Simpson Beauchamp C, Byelashov OA, Geornaras I, Kendall PA, Scanga JA, Belk KE, Smith GC, Sofos JN (2010) Fate of Listeria monocytogenes during freezing, thawing and home storage of frankfurters. Food Microbiol 27:144–149CrossRefGoogle Scholar
  25. Strawn LK, Danyluk MD (2010) Fate of Escherichia coli O157:H7 and Salmonella spp. on fresh and frozen cut mangoes and papayas. Int J Food Microbiol 138:78–84CrossRefGoogle Scholar
  26. Sturbelle RT, de Avila LF, Roos TB, Borchardt JL, da Conceicao Rde C, Dellagostin OA, Leite FP (2015) The role of quorum sensing in Escherichia coli (ETEC) virulence factors. Vet Microbiol 180:245–252CrossRefGoogle Scholar
  27. Wang Y, Claeys L, van der Ha D, Verstraete W, Boon N (2010) Effects of chemically and electrochemically dosed chlorine on Escherichia coli and legionella beliardensis assessed by flow cytometry. Appl Microbiol Biotechnol 87(1):331–341CrossRefGoogle Scholar
  28. Wang L, Li P, Zhang Z, Chen Q, Aguilar ZP, Xu H, Yang L, Xu F, Lai W, Xiong Y, Wei H (2014) Rapid and accurate detection of viable Escherichia coli O157:H7 in milk using a combined IMS, sodium deoxycholate, PMA and real-time quantitative PCR process. Food Control 36:119–125CrossRefGoogle Scholar
  29. Wu GP, Chen SH, Levin RE (2015) Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification. J Microbiol Methods 117:41–48CrossRefGoogle Scholar
  30. Xiao XL, Tian C, Yu YG, Wu H (2013) Detection of viable but nonculturable Escherichia coli O157:H7 using propidium monoazide treatments and qPCR. Can J Microbiol 59:157–163CrossRefGoogle Scholar
  31. Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwell RR (1982) Survival and viability of non-culturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb Ecol 8(4):313–323CrossRefGoogle Scholar
  32. Yan M, Xu L, Jiang H, Zhou Z, Zhou S, Zhang L (2017) PMA-LAMP for rapid detection of Escherichia coli and Shiga toxins from viable but non-culturable state. Microb Pathog 105:245–250CrossRefGoogle Scholar
  33. Zhang S, Zhu X, Wu Q, Zhang J, Xu X, Li H (2015) Prevalence and characterization of Escherichia coli O157 and O157:H7 in retail fresh raw meat in South China. Ann Microbiol 65:1993–1999CrossRefGoogle Scholar
  34. Zhao YY, Tang KJ, Zhang TT, Gao YY, Lin LP, Wu GP (2017) Quantification and discrimination of viable and dead Escherichia coli O157:H7 cells from chicken without enrichment by ethidium bromide Monoazide real-time loop-mediated isothermal amplification. Food Anal Methods 11:1257–1266CrossRefGoogle Scholar
  35. Zhong J, Zhao X (2018) Detection of viable but non-culturable Escherichia coli O157:H7 by PCR in combination with propidium monoazide. 3 Biotech 8:28CrossRefGoogle Scholar
  36. Zhong Q, Tian J, Wang B, Wang L (2016) PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state. Food Control 63:230–238CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Hu Chen
    • 1
  • Yuan Yang Zhao
    • 1
  • Mei Shu
    • 1
  • Tian Tian Zhang
    • 1
  • Yan Bi
    • 1
  • Yan Yan Gao
    • 1
  • Guo Ping Wu
    • 1
  1. 1.College of Food Science and EngineeringJiangxi Agricultural UniversityNanchangChina

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