Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

Blackstone, George M.; Nordstrom, Jessica L.; Vickery, Michael C. L.; Bowen, Michael D.; Meyer, Richard F.; DePaola, Angelo

doi:10.1016/s0167-7012(03)00020-4

Cited by 155 publications

(101 citation statements)

References 20 publications

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“…Number of V. alginolyticus (log CFU ml -1 of samples) more sensitive than detection of V. vulnificus (Takahashi et al 2005), V. cholerae (Gubala 2006) and as sensitive as detection of V. parahaemolyticus (Blackstone et al 2003;Cai et al 2006) and V. vulnificus (Panicker et al 2004). Vibrio alginolyticus may enter into a viable but nonculturable (VBNC) state, and enrichment can help the recovery and multiplication of the cells, avoiding false-negative detection.…”

Section: Discussionmentioning

confidence: 99%

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

Zhou

Hou

et al. 2007

Journal of Applied Microbiology

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Aim: Vibrio alginolyticus is an economically important micro‐organism. The main aim of the present study was to develop a real‐time polymerase chain reaction (PCR) assay for rapid, sensitive and effective quantification of V. alginolyticus in seawater and seafood. Methods and Results: Purified DNA of V. alginolyticus, artificially inoculated seawater and seafood tissue homogenates were subjected to the gyrB‐targeted real‐time PCR assay. Natural seawater and seafood samples were analysed by this real‐time PCR protocol. Specificity tests showed that positive result was obtained only with V. alginolyticus strains. The detection sensitivity was determined to be 0·4 pg of genomic DNA equivalent to 72 cells per PCR in pure culture and 100 cells in 1 ml of seawater or seafood tissue homogenates. Single cell detection is achieved after 3 h of sample enrichment. Conclusions: A sensitive and specific SYBR Green I‐based real‐time PCR assay targeting gyrB gene was successfully developed to quantify V. alginolyticus within 6 h in seawater and seafood samples. Significance and Impact of the Study: No report on the molecular‐based method was available for quantitative detection of V. alginolyticus. This work will provide a novel method for evaluation of the risk of V. alginolyticus to marine environmental health and seafood safety.

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Section: Discussionmentioning

confidence: 99%

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

Zhou

Hou

et al. 2007

Journal of Applied Microbiology

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“…Among these species, Vibrio parahaemolyticus inhabits natural environments with moderate salinity and warm temperature conditions, and its life cycle has consequently been associated with estuarine systems (Kaneko and Colwell, 1973, 1975, 1978Joseph et al, 1982). Moreover, V. parahaemolyticus has been regularly isolated from the microbiome of different marine organisms, such as corals (Chimetto et al, 2008), fish (Cabrera-Garcia et al, 2004;Herrera et al, 2006;Terzi et al, 2009), molluscs (Blackstone et al, 2003;Martinez-Urtaza et al, 2008b), sponges (Hoffmann et al, 2010), shrimp (Cabanillas-Beltrán et al, 2006) and zooplankton (Kaneko and Colwell, 1973;Baffone et al, 2006). The interaction with planktonic organisms has a central role in the pelagic ecology of Vibrio populations.…”

Section: Introductionmentioning

confidence: 99%

Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas

et al. 2011

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The life cycle of Vibrio parahaemolyticus has been conventionally associated with estuarine areas characterized by moderate salinity and warm seawater temperatures. Recent evidence suggests that the distribution and population dynamics of V. parahaemolyticus may be shaped by the existence of an oceanic transport of communities of this organism mediated by zooplankton. To evaluate this possibility, the presence of V. parahaemolyticus in the water column of offshore areas of Galicia was investigated by PCR monthly over an 18-month period. Analysis of zooplankton and seawater showed that the occurrence of V. parahaemolyticus in offshore areas was almost exclusively associated with zooplankton and was present in 80% of the samples. The influence of environmental factors assessed by generalized additive models revealed that the abundance and seasonality of V. parahaemolyticus in zooplankton was favoured by the concurrence of downwelling periods that promoted the zooplankton patchiness. These results confirm that offshore waters may be common habitats for V. parahaemolyticus, including strains with virulent traits. Additionally, genetically related populations were found in offshore zooplankton and in estuaries dispersed along 1500 km. This finding suggests that zooplankton may operate as a vehicle for oceanic dispersal of V. parahaemolyticus populations, connecting distant regions and habitats, and thereby producing impacts on the local community demography and the spread of Vibrio-related diseases.

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“…Vibrio parahaemolyticus is also the leading cause of seafoodassociated bacterial gastroenteritis in the United States and Asia (5, 6), which usually stems from the consumption of raw or undercooked shellfish (7,8). Typically, infection by this organism leads to nausea, vomiting, fever, and a diarrhea distinct from that of the related Vibrio cholerae.…”

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Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

2014

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bVibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation in rpoN (VP2670) in V. parahaemolyticus RIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effects in vivo using a streptomycintreated mouse model of colonization. We confirmed that deletion of rpoN rendered V. parahaemolyticus nonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. In in vivo competition assays between the rpoN mutant and a wild-type RIMD2210633 strain marked with the ␤-galactosidase gene lacZ (WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that the rpoN mutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as the rpoN mutant, which suggested that lack of motility is not the sole cause of the fitness effect. In an in vitro growth competition assay in mouse intestinal mucus, the rpoN mutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ⌬rpoN mutant than in the wild type. These data suggest that in V. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization. Vibrio parahaemolyticus is a Gram-negative bacterium ubiquitous in the marine and estuarine environments worldwide (1-4). Vibrio parahaemolyticus is also the leading cause of seafoodassociated bacterial gastroenteritis in the United States and Asia (5, 6), which usually stems from the consumption of raw or undercooked shellfish (7,8). Typically, infection by this organism leads to nausea, vomiting, fever, and a diarrhea distinct from that of the related Vibrio cholerae. Less commonly, infection by V. parahaemolyticus can cause wound infection and septicemia, leading to mortality in immunocompromised individuals (9-12).Much effort has gone into understanding the mechanisms that contribute to V. parahaemolyticus pathogenesis, with a particular focus on virulence factors produced by this bacterium. Strains that caused disease often possessed either the thermostable direct hemolysin (TDH) or the TDH-related hemolysin (TRH), while nonpathogenic strains typically lacked these two markers (13-15). Additionally, sequence analysis of RIMD2210633, an O3:K6 isolate (TDH ϩ TRH Ϫ ), revealed the presence of two type 3 secretion systems (T3SS), one on each chromosome (T3SS-1 and T3SS-2) (16, 17). T3SS-1 is common to both clinical and nonclinical strains of V. parahaemolyticu...

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Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

Cited by 155 publications

References 20 publications

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas

Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

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