Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR

• Main Authors: Mohammad Khezri, Masoud Rezaei, Ashraf Mohabbati Mobarez, mehdi zolfaghari
• Format: Article
• Language: English
• Published: Farname 2019-02-01
• Series: Iranian Journal of Medical Microbiology
• Subjects: Escherichia coli O157:H7; Bacteria viability; Reverse Transcriptase PCR
• Online Access: http://ijmm.ir/article-1-889-en.html

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.