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MRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state ArchiMer
Coutard, Francois; Pommepuy, Monique; Loaec, Solen; Hervio Heath, Dominique.
Aims: This work investigates the maintenance of viability and potential virulence of Vibrio parahaemolyticus in a viable but nonculturable population (VBNC) state by reverse transcription-polymerase chain reaction (RT-PCR). Methods and Results: Housekeeping genes, 16S-23S rDNA and rpoS, as well as virulence genes, tdh1 and tdh2, were selected and detected by PCR in a pathogenic strain of V. parahaemolyticus (Vp4). Their expression was then studied by RT-PCR in V. parahaemolyticus Vp4 cultivated in rich medium at 37degreesC. The 16S-23S rDNA and rpoS, tdh1, tdh2 genes were transcripted at the mid-logarithmic, stationary and late stationary phases, corresponding to various physiological states. The expression of these genes was also studied by RT-PCR in a...
Tipo: Text Palavras-chave: Vibrio parahaemolyticus; Viable but nonculturable state; Tdh2; Tdh1; RT PCR; RpoS; Environment; 16S 23S rDNA.
Ano: 2005 URL: http://archimer.ifremer.fr/doc/2005/publication-1098.pdf
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Real-time reverse transcription-PCR for transcriptional expression analysis of virulence and housekeeping genes in viable but nonculturable Vibrio parahaemolyticus after recovery of culturability ArchiMer
Coutard, Francois; Lozach, Solen; Pommepuy, Monique; Hervio Heath, Dominique.
A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37°C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The...
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Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-2787.pdf
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Recovery in culture of viable but nonculturable Vibrio parahaemolyticus: regrowth or resuscitation? ArchiMer
Coutard, Francois; Crassous, Philippe; Droguet, Michael; Gobin, Eric; Colwell, Rita; Pommepuy, Monique; Hervio Heath, Dominique.
The objective of this study was to explore the recovery of culturability of viable but nonculturable ( VBNC) Vibrio parahaemolyticus after temperature upshift and to determine whether regrowth or resuscitation occurred. A clinical strain of V. parahaemolyticus Vp5 was rendered VBNC by exposure to artificial seawater ( ASW) at 4 degrees C. Aliquots of the ASW suspension of cells ( 0.1, 1 and 10 ml) were subjected to increased temperatures of 20 degrees C and 37 degrees C. Culturability of the cells in the aliquots was monitored for colony formation on a rich medium and changes in morphology were measured by scanning ( SEM) and transmission ( TEM) electron microscopy. Samples of VBNC cells were fixed and examined by SEM, revealing a heterogeneous population...
Tipo: Text Palavras-chave: Transmission electron microscopy; Scanning electron microscopy; Resuscitation; Regrowth; Vibrio parahaemolyticus; VBNC state.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-3117.pdf
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