Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical
            <i>Vibrio vulnificus</i>
            Strains in Artificial Seawater

Saux, Marion Fischer-Le; Hervio-Heath, Dominique; Loaec, Solen; Colwell, Rita R.; Pommepuy, Monique

doi:10.1128/aem.68.11.5641-5646.2002

Cited by 61 publications

(41 citation statements)

References 36 publications

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“…It seems difficult to know if such pathogenic effects were due to VBNC cells or to cells that reverted to a culturable state. But, recently, Fischer-Le Saux et al [19] detected a cytotoxin-hemolysin mRNA produced by a non culturable population of Vibrio vulnificus. They concluded that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk.…”

Section: Discussionmentioning

confidence: 99%

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

et al. 2007

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-The aim of this study was to assess the efficiency of the embryonated egg model to recover Viable But Non Culturable (VBNC) cells of Listeria monocytogenes. L. monocytogenes cells were incubated in filtered sterilised distilled water. The VBNC state was obtained after a 25 to 47 days incubation period (concentration of culturable cells less than 1 cfu/mL). Fifteen days after the VBNC state was reached, non culturability was checked in various media. One milliliter of each VBNC suspension that contained 10 4 metabolically active cells (i.e. Direct Viable Count + cells) was inoculated into the vitellus fluid of embryonated and non-embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs (18/32), but not in the non-embryonated eggs (1/32). The recovery rate was higher after culture of the vitellus fluid plus embryo (18/32) than after culture of the vitellus fluid alone (6/32). The results indicate that the embryo likely plays a prominent part in the recovery process. The virulence of recovered cells was assessed by the ability to form plaques in HT-29 cell monolayers and by the ability to colonise mouse spleens. Although the cells were classified as avirulent when in the VBNC state, the virulence was recovered after resuscitation.VBNC / virulence / embryonated eggs / Listeria monocytogenes

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Section: Discussionmentioning

confidence: 99%

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

et al. 2007

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“…It has been demonstrated that vibrio species are pathogenic for humans, causing intestinal and extra-intestinal infections (21)(22)(23). Therefore, their detection as environmental contaminants is of a paramount importance for the control of both water quality and disease transmission (24,25). It has been shown that vibrio uses survival strategies such as that of entering into a VBNC state in response to unfavorable environmental conditions.…”

Section: Discussionmentioning

confidence: 99%

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination

Albertini¹,

Accorsi²,

Teodori³

et al. 2006

Cytometry Pt A

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Background:Vibrio alginolyticus is known to enter into a viable but nonculturable (VBNC) state in response to environmental conditions unfavorable to the growth. Cells in VBNC condition pose a public health threat because they are potentially pathogenic.Methods:We constructed a pathway for the identification of the most significant variables and the characterization of those variables able to discriminate the groups under investigation. Different parameters measured by the image processing software were chosen as the most representative of V. alginolyticus cell morphology (length index for dimension) and metabolic activity (density profile indexes). To detect relationships between the groups of treatment performed, we carried out a principal components analysis (PCA).Results:The PCA analysis indicated that increasing coccoid shape transformation was related to both metabolic and dimension variations, delineating a well defined graph profile. Indeed, we discovered that specific morphological variations occur when cells in the culturable state pass into VBNC condition, namely comma‐shaped culturable bacteria are converted into coccoid‐shaped VBNC cells. The results were also supported by scanning electron microscopy analysis.Conclusions:This technique allows the analysis of a large number of vibrio samples in a short period of time. The obtained multiparameter information may complement genetic/molecular analyses facilitating, in an automatic fashion, further studies to evaluate the potential risk of this pathogen in the environment. It may also be a useful tool for large‐scale cell biology studies and high content screening. © 2006 International Society for Analytical Cytology

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“…The PCR product obtained with each set of primers for the DNA from strain Vp5 of V. parahaemolyticus was purified using a column system (QIAquick PCR purification kit) according to the recommendations of the manufacturer (QIAGEN). The nucleotide sequence of the purified PCR product was determined as previously described (9).…”

Section: Methodsmentioning

confidence: 99%

Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but NonculturableVibrio parahaemolyticusafter Recovery of Culturability

Coutard

Lozach

Pommepuy

et al. 2007

Appl Environ Microbiol

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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 levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state.Vibrio parahaemolyticus is a marine bacterium of which some strains generate food-borne outbreaks of disease characterized by acute gastroenteritis. The thermostable direct hemolysin (TDH) and TDH-related hemolysin were previously considered to be the main factors at the origin of these enterotoxic phenomena. Recently, the genome sequencing of a clinical strain of V. parahaemolyticus, RIMD 2210633, has revealed several other factors of virulence, including genes for two type III secretion systems (TTSS), TTSS1 and TTSS2, present on chromosomes 1 and 2, respectively (18). TTSS1 has been described as a cytotoxic system, and TTSS2 has been described as an enterotoxic system (25). Under environmental stresses, such as a temperature downshift, V. parahaemolyticus appears to be fairly inactive metabolically and enters into a dormant state, namely, the viable but nonculturable (VBNC) state. The VBNC cells are able to respond to some environmental stimuli, such as a temperature upshift, and to become metabolically active and culturable. The recovery of culturability by the VBNC cells of V. cholerae, V. vulnificus, and V. parahaemolyticus has been demonstrated to cause in vivo pathogenicity (1,6,23). Two scenarios may explain the virulence: a high number of cells (a high infectious dose) without significant regulation of the virulence genes and/or genetic up-regulation of virulence genes under such conditions.To determine if cells induce the expression of virulence genes after the recovery o...

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Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical Vibrio vulnificus Strains in Artificial Seawater

Cited by 61 publications

References 36 publications

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination

Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but NonculturableVibrio parahaemolyticusafter Recovery of Culturability

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