year 12, Issue 6 (January - February 2019)                   Iran J Med Microbiol 2019, 12(6): 390-398 | Back to browse issues page

XML Persian Abstract Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Khezri M, Rezaei M, Mohabbati Mobarez A, zolfaghari M. Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR. Iran J Med Microbiol 2019; 12 (6) :390-398
1- Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran
2- Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran ,
3- Department of Medical Bacteriology, Faculty of Medical, Tarbiat Modares University, Tehran, Iran
4- Department of Seafood Processing, Faculty of Fisheries and Environmental Sciences, Agricultur and Natural Resource University of Gorgan, Gorgan, Iran
Abstract:   (8758 Views)
Background and Aims: Many bacteria including Escherichia coli may enter into a viable but non-culturable (VBNC) state under unfavorable stresses, which are unable to be detected by culture-based methods. In this study, the use of Reverse Transcription PCR (RT-PCR) for detection of VBNC state of E. coli O157:H7 was investigated.
Materials and Methods:  Escherichia. coli O157:H7 was inoculated in distilled water and 30 percent salt water at room temperature. The cultivability of bacteria was determined using routine culture and colony counting on MacConkey agar. The RT-PCR of 16S rRNA gene involved direct extraction and purification of RNA, DNase I treatment for removing DNA contamination, cDNA synthesis and electrophoresis of PCR products of cDNA was used to detect viable E. coli O157:H7 under studied treatments and was compared with the results of RT-PCR of 16S rRNA gene of heat- killed bacteria.
Results: The cultivability of bacteria was maintained during the study period in distilled water; however, the use of 30percent NaCl caused the bacteria to be non-cultivable on day 4. The RT-PCR of 16S rRNA showed the positive expression of this gene in cultivable and non-cultivable bacteria during the study period, whereas heat-killed bacteria were negative for this gene, which indicated the efficacy of RT-PCR of 16S rRNA in differentiation of alive from dead bacteria.
Conclusions: Escherichia coli O157:H7 entered into the VBNC under 30 percent NaCl which can be associated with serious human health problems. RT-PCR can be used to detect bacteria in the VBNC state.

Full-Text [PDF 748 kb]   (2230 Downloads)    
Type of Study: Original Research Article | Subject: Food Microbiology
Received: 2018/11/15 | Accepted: 2019/01/22 | ePublished: 2019/03/29

1. Liu Y, Gilchrist A, Zhang J, Li XF. Detection of viable but nonculturable Escherichia coli O157: H7 bacteria in drinking water and river water. Appl Environ Microbiol. 2008; 74(5): 1502-7. [DOI:10.1128/AEM.02125-07] [PMID] [PMCID]
2. McIngvale SC, Chen XQ, McKillip JL, Drake MA. Survival of Escherichia coli O157: H7 in buttermilk as affected by contamination point and storage temperature. J Food Prot. 2000; 63(4): 441-4. [DOI:10.4315/0362-028X-63.4.441] [PMID]
3. Yaron S, Matthews KR. A reverse transcriptase‐polymerase chain reaction assay for detection of viable Escherichia coli O157: H7: investigation of specific target genes. J Appl Microbiol. 2002; 92(4): 633-40. [DOI:10.1046/j.1365-2672.2002.01563.x] [PMID]
4. Kargar M, Homayoon M. Prevalence of shiga toxins (stx1, stx2), eaeA and hly genes of Escherichia coli O157: H7 strains among children with acute gastroenteritis in southern of Iran. Asian Pac J Trop Med. 2015; 8(1): 24-8. [DOI:10.1016/S1995-7645(14)60182-6]
5. Oliver JD. The viable but nonculturable state in bacteria. The journal of microbiology. 2005; 43(1): 93-100.
6. Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev. 2010; 34(4): 415-25. [DOI:10.1111/j.1574-6976.2009.00200.x] [PMID]
7. Li L, Mendis N, Trigui H, Oliver JD, Faucher SP. The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol. 2014; 5: 258. [DOI:10.3389/fmicb.2014.00258] [PMID] [PMCID]
8. Makino SI, Kii T, Asakura H, Shirahata T, Ikeda T, Takeshi K, et al. Does Enterohemorrhagic Escherichia coli O157: H7 Enter the Viable but Nonculturable State in Salted Salmon Roe?. Appl Environ Microbiol. 2000; 66(12): 5536-9. [DOI:10.1128/AEM.66.12.5536-5539.2000] [PMID] [PMCID]
9. Zhao X, Zhong J, Wei C, Lin CW, Ding T. Current perspectives on viable but non-culturable state in foodborne pathogens. Front Microbiol. 2017; 8: 580. [DOI:10.3389/fmicb.2017.00580] [PMID] [PMCID]
10. Ding T, Suo Y, Xiang Q, Zhao X, Chen S, Ye X, et al. Significance of viable but nonculturable Escherichia coli: induction, detection, and control. J Microbiol Biotechnol. 2017; 27(3): 417-28. [DOI:10.4014/jmb.1609.09063] [PMID]
11. Ramamurthy T, Ghosh A, Pazhani GP, Shinoda S. Current perspectives on viable but non-culturable (VBNC) pathogenic bacteria. Frontiers in public health. 2014; 2: 103. [DOI:10.3389/fpubh.2014.00103] [PMID] [PMCID]
12. Molaee N, Abtahi H, Ghannadzadeh MJ, Karimi M, Ghaznavi-Rad E. Application of Reverse Transcriptase-PCR (RT-PCR) for rapid detection of viable Escherichia coli in drinking water samples. J Environ Health Sci Eng. 2015; 13(1): 24. [DOI:10.1186/s40201-015-0177-z] [PMID] [PMCID]
13. Koochakzadeh A, Askari Badouei M, Zahraei Salehi T, Aghasharif S, Soltani M, Ehsan M. Prevalence of Shiga toxin-producing and enteropathogenic Escherichia coli in wild and pet birds in Iran. Rev Bras Cienc Avic. 2015; 17(4): 445-50. [DOI:10.1590/1516-635X1704445-450]
14. Sue D, Boor KJ, Wiedmann M. σB-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes. Microbiology. 2003; 149(11): 3247-56. [DOI:10.1099/mic.0.26526-0] [PMID]
15. Tan Q, Xu H, Chen T, Li P, Aguilar ZP, Xu D, et al. Differential expression of virulence and stress fitness genes during interaction between Listeria monocytogenes and Bifidobacterium longum. Biosci Biotechnol Biochem. 2012; 76(4): 699-704. [DOI:10.1271/bbb.110832] [PMID]
16. Spano G, Beneduce L, Terzi V, Stanca AM, Massa S. Real‐time PCR for the detection of Escherichia coli O157: H7 in dairy and cattle wastewater. Lett Appl Microbiol. 2005;40(3):164-71 [DOI:10.1111/j.1472-765X.2004.01634.x] [PMID]
17. Lothigius Å, Sjöling Å, Svennerholm AM, Bölin I. Survival and gene expression of enterotoxigenic Escherichia coli during long‐term incubation in sea water and freshwater. J Appl Microbiol. 2010; 108(4): 1441-9. [DOI:10.1111/j.1365-2672.2009.04548.x] [PMID]
18. Dinu LD, Bach S. Induction of viable but non-culturable Escherichia coli O157: H7 on the phyllosphere of lettuce: a food safety risk factor. Appl Environ Microbiol. 2011; 77(23): 8295-302. [DOI:10.1128/AEM.05020-11] [PMID] [PMCID]
19. Trevors JT. Viable but non-culturable (VBNC) bacteria: gene expression in planktonic and biofilm cells. J Microbiol Methods. 2011; 86(2): 266-73. [DOI:10.1016/j.mimet.2011.04.018] [PMID]
20. Kim M, Young Park S, Jung Park T, Ha SD. Effect of Sodium Chloride on the Reduction of Bacillus Cereus in Shrimp Jeotgal During Refrigerated Storage. Journal of Food Safety. 2017; 37(1): e12281. [DOI:10.1111/jfs.12281]
21. Casasola-Rodríguez B, Ruiz-Palacios GM, Pilar RC, Losano L, Ignacio MR, Orta de Velásquez MT. Detection of VBNC Vibrio cholerae by RT-Real Time PCR based on differential gene expression analysis. FEMS Microbiol Lett. 2018; 365(15): fny156. [DOI:10.1093/femsle/fny156] [PMID]
22. del Mar Lleò M, Pierobon S, Tafi MC, Signoretto C, Canepari P. mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl Environ Microbiol. 2000; 66(10): 4564-7. [DOI:10.1128/AEM.66.10.4564-4567.2000] [PMID] [PMCID]
23. Coutard F, Pommepuy M, Loaec S, Hervio‐Heath D. mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state. J Appl Microbiol. 2005; 98(4): 951-61. [DOI:10.1111/j.1365-2672.2005.02534.x] [PMID]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2024 CC BY-NC 4.0 | Iranian Journal of Medical Microbiology

Designed & Developed by : Yektaweb | Publisher: Farname Inc