mRNA Detection by Reverse Transcription-PCR for Monitoring Viability over Time in an
            <i>Enterococcus faecalis</i>
            Viable but Nonculturable Population Maintained in a Laboratory Microcosm

Lleò, Maria del Mar; Pierobon, Sabrina; Tafi, Maria Carla; Signoretto, Caterina; Canepari, Pietro

doi:10.1128/aem.66.10.4564-4567.2000

Cited by 169 publications

(67 citation statements)

References 30 publications

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“…It is possible that carbenicillin, gentamicin and levofloxacin could not completely inactivate P. aeruginosa PAO1 cells, as in the PMA experiment above. Also, the step of having a 7 day starvation before the stimulation could induce VBNC cells because they have intact cell membranes, low metabolic activity and continued mRNA synthesis (Lleo et al ., 2000). It has been known that many species of bacteria enter VBNC state when they are subjected to various environmental stressors, such as UV disinfection, osmotic pressures, high/low temperature and starvation (Colwell et al ., 1985; Byrd et al ., 1991; Oliver et al ., 2005; Pawlowski et al ., 2011).…”

Section: Discussionmentioning

confidence: 99%

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Lee

Bae

2017

Microbial Biotechnology

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SummaryNucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNC P. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells.

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Section: Discussionmentioning

confidence: 99%

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Lee

Bae

2017

Microbial Biotechnology

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“…This phenomenon represents a state of dormancy and allows survival and persistence of bacterial cells in the natural or host environment. To date, at least 18 species undergoing a VBNC-type shift have been documented (77), and more-sophisticated methods are being employed that confirm continued viability (39). The ability to adapt to unfavorable conditions may be a key factor in the success of V. cholerae as an opportunistic pathogen and is most likely explained by its 760 LIPP ET AL.…”

Section: Vibrios and V Choleraementioning

confidence: 99%

Effects of Global Climate on Infectious Disease: the Cholera Model

2002

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Recently, the role of the environment and climate in disease dynamics has become a subject of increasing interest to microbiologists, clinicians, epidemiologists, and ecologists. Much of the interest has been stimulated by the growing problems of antibiotic resistance among pathogens, emergence and/or reemergence of infectious diseases worldwide, the potential of bioterrorism, and the debate concerning climate change. Cholera, caused by Vibrio cholerae, lends itself to analyses of the role of climate in infectious disease, coupled to population dynamics of pathogenic microorganisms, for several reasons. First, the disease has a historical context linking it to specific seasons and biogeographical zones. In addition, the population dynamics of V. cholerae in the environment are strongly controlled by environmental factors, such as water temperature, salinity, and the presence of copepods, which are, in turn, controlled by larger-scale climate variability. In this review, the association between plankton and V. cholerae that has been documented over the last 20 years is discussed in support of the hypothesis that cholera shares properties of a vector-borne disease. In addition, a model for environmental transmission of cholera to humans in the context of climate variability is presented. The cholera model provides a template for future research on climate-sensitive diseases, allowing definition of critical parameters and offering a means of developing more sophisticated methods for prediction of disease outbreaks

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“…However, conventional PCR methods do not distinguish among viable, viable but nonculturable (VBNC), and dead cells. VBNC cells are defined as those cells that have lost the ability to express genes but may return to a culturable state (5). The presence of these cells limits the use of PCR for microbiological monitoring of food samples, where metabolically injured or nonviable cells are generally present after the stresses imposed during food processing.…”

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

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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.

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mRNA Detection by Reverse Transcription-PCR for Monitoring Viability over Time in an Enterococcus faecalis Viable but Nonculturable Population Maintained in a Laboratory Microcosm

Cited by 169 publications

References 30 publications

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Effects of Global Climate on Infectious Disease: the Cholera Model

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

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