Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR

Wu, Bin; Liang, Weili; Kan, Biao

doi:10.1016/j.mimet.2015.05.016

Cited by 32 publications

(30 citation statements)

References 40 publications

(47 reference statements)

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“…DNA copies of live Vibrio cholerae after PMA qPCR were gradually underestimated, with increasing PMA concentrations and incubation times (Wu et al . ). Nevertheless, V. parahaemolyticus was shown to be viable based on its ability to recover from a VBNC state when it persisted in ASW at 4°C for 150 days.…”

Section: Resultsmentioning

confidence: 97%

“…The complete repression of genomic DNA signals from V. parahaemolyticus cells incubated in ASW microcosms for 150 days would be caused by the inability of PMA to bind only to nonviable cells. DNA copies of live Vibrio cholerae after PMA qPCR were gradually underestimated, with increasing PMA concentrations and incubation times (Wu et al 2015). Nevertheless, V. parahaemolyticus was shown to be viable based on its ability to recover from a VBNC state when it persisted in ASW at 4°C for 150 days.…”

Section: Resultsmentioning

confidence: 99%

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Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Yoon

Moon

Choi

et al. 2019

Lett Appl Microbiol

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Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation‐based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real‐time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15–16·69. During cold‐starvation, PMA qPCR selectively excluded DNAs from heat‐killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). Significance and Impact of the Study Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold‐starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real‐time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold‐starvation conditions.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Yoon

Moon

Choi

et al. 2019

Lett Appl Microbiol

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“…As described in our previous study ( Wu et al, 2015 ), viable cells were enumerated using propidium monoazide (PMA, Biotium, USA) combined with quantitative PCR (PMA-qPCR) by counting DNA copies of live cells. Briefly, 200 μL aliquots of cells cultured in each condition were treated with 20 μM of PMA for 20 min in the dark, then exposed to light on ice for 15 min using a 650W double-ended halogen lamp.…”

Section: Methodsmentioning

confidence: 99%

Growth Phase, Oxygen, Temperature, and Starvation Affect the Development of Viable but Non-culturable State of Vibrio cholerae

Liang

Kan

2016

Front. Microbiol.

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Vibrio cholerae can enter into a viable but non-culturable (VBNC) state in order to survive in unfavorable environments. In this study, we studied the roles of five physicochemical and microbiological factors or states, namely, different strains, growth phases, oxygen, temperature, and starvation, on the development of VBNC of V. cholerae in artificial sea water (ASW). Different strains of the organism, the growth phase, and oxygen levels affected the progress of VBNC development. It was found that the VBNC state was induced faster in V. cholerae serogroup O1 classical biotype strain O395 than in O1 El Tor biotype strains C6706 and N16961. When cells in different growth phases were used for VBNC induction, stationary-phase cells lost their culturability more quickly than exponential-phase cells, while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains, suggesting that heterogeneity of cells should be considered. Aeration strongly accelerated the loss of culturability. During the development of the VBNC state, the culturable cell count under aeration conditions was almost 106-fold lower than under oxygen-limited conditions for all three strains. The other two factors, temperature and nutrients-rich environment, may prevent the induction of VBNC cells. At 22 or 37°C in ASW, most of the cells rapidly died and the culturable cell count reduced from about 108 to 106–105 CFU/mL. The total cell counts showed that cells that lost viability were decomposed, and the viable cell counts were the same as culturable cell counts, indicating that the cells did not reach the VBNC state. VBNC state development was blocked when ASW was supplied with Luria-Bertani broth (LB), but it was not affected in ASW with M9, suggesting that specific nutrients in LB may prevent the development of VBNC state. These results revealed that the five factors evaluated in this study had different roles during the progress of VBNC induction. Changing a single factor could influence and even block the development of the VBNC state. These findings provide new insight to help design further studies to better understand the mechanisms which trigger the development and regulation of the VBNC state.

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“…Here we report the modification of a standard qPCR assay used for laboratory diagnosis of B. pertussis , through treatment of samples with propidium monoazide (PMA) that inhibits PCR-mediated amplification of DNA from dead cells and allows distinguishing of viable from dead cells (16–20). The use of PMA involves an initial incubation of samples with PMA in darkness, during which it diffuses into dead cells, followed by light activation of PMA that permanently modifies the gDNA of dead cells, preventing it from acting as a template in PCR.…”

Section: Introductionmentioning

confidence: 99%

A qPCR assay for Bordetella pertussis cells that enumerates both live and dead bacteria

Ramkissoon¹,

MacArthur²,

Ibrahim³

et al. 2019

Preprint

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AbstractBordetella pertussis is the causative agent of whooping cough, commonly referred to as pertussis. Although the incidence of pertussis was reduced through vaccination, during the last thirty years it has returned to high levels in a number of countries. This resurgence has been linked to the switch from the use of whole-cell to acellular vaccines. Protection afforded by acellular vaccines appears to be short-lived compared to that afforded by whole cell vaccines. In order to inform future vaccine improvement by identifying immune correlates of protection, a human challenge model of B. pertussis colonisation has been developed. Accurate measurement of colonisation status in this model has required development of a qPCR-based assay to enumerate B. pertussis in samples that distinguishes between viable and dead bacteria. Here we report the development of this assay and its performance in the quantification of B. pertussis from human challenge model samples. This assay has future utility in diagnostic labs and in research where a quantitative measure of both B. pertussis number and viability is required.

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Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR

Cited by 32 publications

References 40 publications

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Growth Phase, Oxygen, Temperature, and Starvation Affect the Development of Viable but Non-culturable State of Vibrio cholerae

A qPCR assay for Bordetella pertussis cells that enumerates both live and dead bacteria

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