Effect of post-treatment holding conditions on detection of tufA mRNA in ethanol-treated Escherichia coli: implications for RT-PCR-based indirect viability tests

Sheridan, G. E. C.; Szabo, Elizabeth; Mackey, B.M.

doi:10.1046/j.1472-765x.1999.00644.x

Cited by 43 publications

(25 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…mRNA might persist for longer or shorter periods in vivo. Based on experience with other microorganisms, persistence of borrelial mRNA is likely to be dependent on the cause of cell death, on the size, region, and type of the mRNA being targeted, on the level and activity of the ribonucleases in B. burgdorferi cells, on the physiological state of the bacterial cell population, and on the extracellular environmental conditions (34)(35)(36). mRNA might, in theory, remain intact for relatively long periods of time if cells are destroyed by treatments that inactivate RNase but not the mRNA (37).…”

Section: Discussionmentioning

confidence: 99%

Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability

et al. 2013

View full text Add to dashboard Cite

The persistence of dormant, noncultivable Borrelia burgdorferi after ceftriaxone treatment was examined. B. burgdorferi isolates were cultivated in Barbour-Stoenner-Kelly medium in the presence or absence of ceftriaxone, and cultures were monitored for up to 56 days. Viability of B. burgdorferi was assessed by subculture, growth, morphology, and pH (as a surrogate for metabolic activity). In addition, the presence of B. burgdorferi DNA and mRNA was assayed by PCR and by real-time reverse transcription (RT)-PCR, respectively. Spirochetes could not be successfully subcultured by day 3 after exposure to ceftriaxone. In cultures treated with ceftriaxone, the pH of the culture medium did not change through day 56, whereas it declined by at least 1 pH unit by 14 days in untreated cultures. These results suggest that B. burgdorferi viability is rapidly eliminated after antibiotic treatment. Nevertheless, DNA was detected by B. burgdorferi-specific PCR for up to 56 days in aliquots from both ceftriaxone-treated and untreated cultures. In addition, although ceftriaxone treatment resulted in a reduction in the quantities of transcript for ospC, ospA, flaB, and pfk, certain mRNAs could be detected through day 14. Transcript for all 4 genes was essentially undetectable after 28 days of treatment. Taken together, the results suggest that B. burgdorferi DNA and mRNA can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters. PCR positivity in the absence of culture positivity following antibiotic treatment in animal and human studies should be interpreted with caution.

show abstract

Section: Discussionmentioning

confidence: 99%

Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Prelimi- In principle, the presence of RNA in bacterial cells may serve as an indicator for viable cells (32), and mRNA species are supposed to have an average half-life of only minutes in metabolizing cells (2). However, in several studies it has been shown that mRNA species may persist in a detectable form for many hours after cell death (6,46,47). The persistence of mRNA species may vary depending on the method of cell death (46,47) and the physiological state of the cell population before killing.…”

Section: Discussionmentioning

confidence: 99%

Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

Fykse

Skogan

Davies

et al. 2007

Appl Environ Microbiol

View full text Add to dashboard Cite

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.Vibrio cholerae is the etiological agent of epidemic cholera, which causes watery diarrhea that can result in the rapid dehydration and death of infected persons. Coastal waters are an important reservoir of V. cholerae, and cholera is generally transmitted to humans via contaminated water or seafood (12, 13). Of more than 200 known serogroups of V. cholerae, the two well-known serogroups O1 and O139 have been associated with epidemic cholera (11). The serogroup O1 can be divided into three serotypes, Inaba, Ogawa, and Hikojima, and each serotype can be divided into two biotypes, classical and El Tor (30). The other serogroups of V. cholerae, collectively referred to as non-O1 and non-O139 serogroups, have not been associated with epidemics but have been associated with occasional outbreaks of cholera-like disease. These other serogroups are usually isolated from patients with mild diarrhea or from the environment (27,39). Vibrio species such as V. parahaemolyticus, V. cholerae, and V. vulnificus are found in blue mussels harvested along the coastline of Norway (5).The pathogenesis of cholera is a complex process, and the major virulence factors of V. cholerae are the cholera toxin (CT) encoded by the ctxAB genes and the toxin-coregulated pilus (TCP) encoded by the tcpA gene (30). CT leads to increased intestinal secretion of electrolytes and water into the lumen. TCP is a ...

show abstract

“…Therefore, detection of mRNA by reverse transcriptase PCR (RT-PCR), as opposed to DNA-based methods, is considered a better indicator of cell viability (25). The relationship between detection of microbial mRNA and cell viability has been investigated in a number of studies concerning bacterial pathogens and standard indicators of fecal contamination (2,4,5,16,22,24,26). Recently, Vaitilingom et al (29) described a method for the detection of viable bacteria, molds, and yeasts by RT-PCR, with primers specific for an elongation factor gene, in heat-treated milk samples.…”

mentioning

confidence: 99%

Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products

Bleve

Rizzotti

Dellaglio

et al. 2003

Appl Environ Microbiol

156

View full text Add to dashboard Cite

Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells. The optimized RT-PCR assay was able to detect 10 CFU of fungi ml ؊1 in pure culture and 10 3 and 10 2 CFU ml ؊1 in artificially contaminated yogurts and pasteurized fruit-derived products, respectively. Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi. The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities.

show abstract

Effect of post-treatment holding conditions on detection of tufA mRNA in ethanol-treated Escherichia coli: implications for RT-PCR-based indirect viability tests

Cited by 43 publications

References 19 publications

Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability

Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability

Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products

Contact Info

Product

Resources

About