scholarly journals Simultaneous Isolation and Enumeration of Virulent Vibrio Cholerae and Vibrio Vulnificus using an Advanced MPN-PCR Method

2021 ◽  
Author(s):  
Jae-Hwa Lee ◽  
Seul-Ki Park ◽  
Fazlurrahman Khan ◽  
Du-Min Jo ◽  
Do-ha Lee ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Vibrio Vulnificus ◽  
Detection Method ◽  
Low Cost ◽  
Luria Bertani ◽  
Most Probable Number ◽  
Probable Number ◽  
Pcr Method ◽  
Cell Enumeration ◽  
Differential Medium

Abstract Vibrio cholerae and Vibrio vulnificus are one of the critical foodborne pathogens that need to be intensively controlled their infection as a result of the intake and distribution of seafood, especially raw oysters. For this reason, various methods have already been developed for the detection and enumeration of these bacteria. The most probable number (MPN)-PCR (polymerase chain reaction) method is commonly used with the selective-differential medium for the efficiency and convenience of cell enumeration. One of the most frequently used for the detection of Vibrio spp. is Thiosulfate-Citrate-Bile salts-Sucrose (TCBS) agar. But this selective-differential medium can fail to distinguish between V. cholerae, V. vulnificus, and Vibrio alginolyticus. For this reason, the conventional MPN-PCR method with TCBS medium for the detection of Vibrio spp. has a problem that processing PCR to the two-times. This study suggests a simple and minimized detection method using one-time PCR and non-NaCl Luria-Bertani (LB-0) medium culture. This detection method is based on the difference in salt requirement between V. cholerae and V. vulnificus. Employing the developed methodology, the simultaneous cell enumeration of V. cholerae and V. vulnificus can be possible at a low cost. Furthermore, this study proposes a new specific primer to detect virulence-related genes from V. cholerae and V. vulnificus. This advanced MPN-PCR method was verified using bioaccumulated pacific oysters (Crassostrea gigas) by V. cholerae and V. vulnificus.

scholarly journals Rapid Proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

2015 ◽  
Vol 81 (21) ◽  
pp. 7600-7609 ◽  
Author(s):  
Kevin Esteves ◽  
Dominique Hervio-Heath ◽  
Thomas Mosser ◽  
Claire Rodier ◽  
Marie-George Tournoud ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Coastal Lagoons ◽  
Flash Floods ◽  
Low Flow ◽  
Most Probable Number ◽  
Abrupt Decrease ◽  
Probable Number ◽  
Content Type

ABSTRACTVibrio parahaemolyticus,Vibrio vulnificus, andVibrio choleraeof the non-O1/non-O139 serotype are present in coastal lagoons of southern France. In these Mediterranean regions, the rivers have long low-flow periods followed by short-duration or flash floods during and after heavy intense rainstorms, particularly at the end of the summer and in autumn. These floods bring large volumes of freshwater into the lagoons, reducing their salinity. Water temperatures recorded during sampling (15 to 24°C) were favorable for the presence and multiplication of vibrios. In autumn 2011, before heavy rainfalls and flash floods, salinities ranged from 31.4 to 36.1‰ and concentrations ofV. parahaemolyticus,V. vulnificus, andV. choleraevaried from 0 to 1.5 × 103most probable number (MPN)/liter, 0.7 to 2.1 × 103MPN/liter, and 0 to 93 MPN/liter, respectively. Following heavy rainstorms that generated severe flash flooding and heavy discharge of freshwater, salinity decreased, reaching 2.2 to 16.4‰ within 15 days, depending on the site, with a concomitant increase inVibrioconcentration to ca. 104MPN/liter. The highest concentrations were reached with salinities between 10 and 20‰ forV. parahaemolyticus, 10 and 15‰ forV. vulnificus, and 5 and 12‰ forV. cholerae. Thus, an abrupt decrease in salinity caused by heavy rainfall and major flooding favored growth of human-pathogenicVibriospp. and their proliferation in the Languedocian lagoons. Based on these results, it is recommended that temperature and salinity monitoring be done to predict the presence of theseVibriospp. in shellfish-harvesting areas of the lagoons.

scholarly journals Abundance of Vibrio cholerae, V. vulnificus, and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

2014 ◽  
Vol 80 (24) ◽  
pp. 7667-7672 ◽  
Author(s):  
Jessica L. Jones ◽  
Catharina H. M. Lüdeke ◽  
John C. Bowers ◽  
Kristin DeRosia-Banick ◽  
David H. Carey ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Vibrio Vulnificus ◽  
Long Island ◽  
Long Island Sound ◽  
The United States ◽  
Mitigation Strategies ◽  
Most Probable Number ◽  
Probable Number ◽  
Content Type ◽  
Island Sound

ABSTRACTVibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence ofVibriospp. in clams. The objective of this study was to compare the levels ofVibrio cholerae,Vibrio vulnificus, andVibrio parahaemolyticusin oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of totalV. cholerae,V. vulnificus,V. parahaemolyticus, and pathogenic (tdh+and/ortrh+)V. parahaemolyticus.V. choleraewas detected in 8.8% and 3.3% of oyster (n= 68) and clam (n= 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively.V. vulnificuswas detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively.V. parahaemolyticuswas detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences betweenV. vulnificusand total and pathogenicV. parahaemolyticuslevels in the two shellfish species were statistically significant (P< 0.001). These data indicate thatV. vulnificusand total and pathogenicV. parahaemolyticusare more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.

Prevalence and Molecular Characterization of Vibrio cholerae from Ice and Beverages Sold in Jakarta, Indonesia, Using Most Probable Number and Multiplex PCR

2012 ◽  
Vol 75 (4) ◽  
pp. 651-659 ◽  
Author(s):  
DIANA E. WATURANGI ◽  
NATANIA PRADITA ◽  
JESSICA LINARTA ◽  
SWAPAN BANERJEE
Keyword(s):  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
Virulence Gene ◽  
Watery Diarrhea ◽  
Most Probable Number ◽  
Intestinal Infection ◽  
Safety Measures ◽  
Probable Number ◽  
Tropical Regions

Vibrio cholerae is well recognized as the causative agent of cholera, an acute intestinal infection characterized by watery diarrhea that may lead to dehydration and death in some cases. V. cholerae is a natural inhabitant of the aquatic environment in the tropical regions. Jakarta has the highest percentage of individuals affected by sporadic diarrheal illness compared with other areas in Indonesia. Inadequate safety measures for drinking water supplies, improper sanitation, and poor hygiene can increase the risk of cholera outbreaks. Few studies have been conducted on the prevalence of these bacteria in ice and beverages that are popularly sold and consumed in Jakarta. In this study, we detected and quantified V. cholerae from ice and beverages collected from several areas in five regions of Jakarta. Levels of V. cholerae in both ice and beverages were determined with the three-tube most-probable-number (MPN) method and ranged from &lt;0.3 to &gt;110 MPN/ml. The presence of regulatory and virulence gene sequences was determined by using uniplex and multiplex PCR assays. Of 110 samples tested, 33 (30%) were positive for V. cholerae; 21 (64%) were ice samples and the remaining 12 (36%) were beverages. A total of 88 V. cholerae strains were isolated, based on the presence of the toxR gene sequence identified by PCR. Other genetic markers, such as hlyA (59%), ompU (16%), and ctxA (19%), also were found during the search for potential pathogenic strains. The detection and isolation of potentially harmful V. cholerae from ice and beverages in Jakarta indicate that these products pose a health risk from choleragenic vibrios, particularly because of the emergence of classical biotypes of V. cholerae O1 and potentially harmful non-O1 serovars of this species.

scholarly journals Effects of Temperature and Salinity on Vibrio vulnificus Population Dynamics as Assessed by Quantitative PCR

2004 ◽  
Vol 70 (9) ◽  
pp. 5469-5476 ◽  
Author(s):  
Mark A. Randa ◽  
Martin F. Polz ◽  
Eelin Lim
Keyword(s):  
Population Dynamics ◽  
Water Temperature ◽  
Water Column ◽  
Quantitative Pcr ◽  
Vibrio Vulnificus ◽  
Most Probable Number ◽  
Salinity Regime ◽  
Optimal Salinity ◽  
Probable Number ◽  
Barnegat Bay

ABSTRACT The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.

Selectivity and Specificity of a Chromogenic Medium for Detecting Vibrio parahaemolyticus†

2005 ◽  
Vol 68 (7) ◽  
pp. 1454-1456 ◽  
Author(s):  
YI-CHENG SU ◽  
JINGYUN DUAN ◽  
WEN-HSIN WU
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Bile Salts ◽  
Vibrio Vulnificus ◽  
Enterobacter Cloacae ◽  
Most Probable Number ◽  
Chromogenic Medium ◽  
Probable Number ◽  
Vibrio Fluvialis ◽  
Vibrio Mimicus ◽  
Vibrio Spp

The thiosulfate–citrate–bile salts–sucrose agar (TCBS) used in the most-probable-number method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus. This study examined the selectivity and specificity of Bio-Chrome Vibrio medium (BCVM), a chromogenic medium that detects V. parahaemolyticus on the basis of the formation of distinct purple colonies on the medium. A panel consisting of 221 strains of bacteria, including 179 Vibrio spp. and 42 non-Vibrio spp., were examined for their ability to grow and produce colored colonies on BCVM. Growth of Salmonella, Shigella, Escherichia coli, Enterobacter cloacae, Yersinia enterocolitica, and Aeromonas was inhibited by both BCVM and TCBS. All 148 strains of V. parahaemolyticus grew on BCVM, and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of Vibrio fluvialis, were either unable to grow or produced blue-green or white colonies on BCVM. Bio-Chrome Vibrio medium was capable of differentiating V. parahaemolyticus from other species, including V. vulnificus and V. mimicus. Further studies are needed to evaluate the sensitivity and specificity of BCVM for detecting V. parahaemolyticus in foods.

Effects of Flash Freezing, Followed by Frozen Storage, on Reducing Vibrio parahaemolyticus in Pacific Raw Oysters (Crassostrea gigas)

2009 ◽  
Vol 72 (1) ◽  
pp. 174-177 ◽  
Author(s):  
CHENGCHU LIU ◽  
JIANZHANG LU ◽  
YI-CHENG SU
Keyword(s):  
Crassostrea Gigas ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Frozen Storage ◽  
Most Probable Number ◽  
Freezing Process ◽  
Probable Number ◽  
Pacific Oysters ◽  
Subsequent Storage ◽  
Shellfish Sanitation

This study investigated the effects of flash freezing, followed by frozen storage, on reducing Vibrio parahaemolyticus in Pacific raw oysters. Raw Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus at a total level of approximately 3.5 × 105 most probable number (MPN) per gram. Inoculated oysters were subjected to an ultralow flash-freezing process (−95.5°C for 12 min) and stored at −10, −20, and −30°C for 6 months. Populations of V. parahaemolyticus in the oysters declined slightly by 0.22 log MPN/g after the freezing process. Subsequent storage of frozen oysters at −10, −20, and −30°C resulted in considerable reductions of V. parahaemolyticus in the oysters. Storing oysters at −10°C was more effective in inactivating V. parahaemolyticus than was storage at −20 or −30°C. Populations of V. parahaemolyticus in the oysters declined by 2.45, 1.71, and 1.45 log MPN/g after 1 month of storage at −10, −20, and −30°C, respectively, and continued to decline during the storage. The levels of V. parahaemolyticus in oysters were reduced by 4.55, 4.13, and 2.53 log MPN/g after 6 months of storage at −10, −20, and −30°C, respectively. Three process validations, each separated by 1 week and conducted according to the National Shellfish Sanitation Program's postharvest processing validation–verification interim guidance for Vibrio vulnificus and Vibrio parahaemolyticus, confirmed that a process of flash freezing, followed by storage at −21 ± 2°C for 5 months, was capable of achieving greater than 3.52-log (MPN/g) reductions of V. parahaemolyticus in half-shell Pacific oysters.

Survey of Postharvest-Processed Oysters in the United States for Levels of Vibrio vulnificus and Vibrio parahaemolyticus

2009 ◽  
Vol 72 (10) ◽  
pp. 2110-2113 ◽  
Author(s):  
ANGELO DePAOLA ◽  
JESSICA L. JONES ◽  
KATHY E. NOE ◽  
ROBIN H. BYARS ◽  
JOHN C. BOWERS
Keyword(s):  
Vibrio Vulnificus ◽  
Gulf Coast ◽  
Virulence Gene ◽  
Selective Advantage ◽  
The United States ◽  
Most Probable Number ◽  
Probable Number ◽  
Mild Heat ◽  
Thermostable Direct Hemolysin ◽  
The Mean

From June through October 2004, the U.S. Food and Drug Administration collected oysters (61 samples) that had been subjected to postharvest processing (PHP) methods, including mild heat treatment, freezing, and high hydrostatic pressure, from processors and retail markets in various states to determine Vibrio vulnificus and V. parahaemolyticus levels. Presence in a 25-g sample and most probable number (MPN) using standard enrichment and selective isolation procedures were utilized. Suspect colonies were isolated and identified using DNA probe colony hybridization. Neither species of vibrio was detected in 25-g portions of most samples regardless of the PHP. The lowest frequency of isolation of either pathogen (&lt;10%) was observed with the mild heat process. Few (12 to 13%) frozen samples collected at the processor but not at retail contained &gt;30 MPN/g of either pathogen. The mean levels of either organism in PHP oysters observed in the present study were 5 to 6 log less than in unprocessed raw Gulf Coast oysters. Of the 70 V. vulnificus isolates examined, only 5 possessed the putative virulence marker, type B 16S rRNA. Neither the thermostable direct hemolysin (tdh) nor the tdh-related hemolysin (trh) virulence gene was detected in any of the 40 V. parahaemolyticus isolates examined in the present study. These data suggest that if there is any selective advantage to pathogenic strains of V. vulnificus and V. parahaemolyticus, these differences are minimal. These results indicate that all PHP treatments greatly reduce exposure of V. vulnificus and V. parahaemolyticus to raw-oyster consumers. Consequently, these PHP oysters pose a much lower risk of illness to consumers due to these pathogens.

scholarly journals Microbial contamination including Vibrio cholerae in fishery auction markets in West Sea, South Korea

2019 ◽  
Vol 22 (1) ◽  
Author(s):  
Yukyung Choi ◽  
Yewon Lee ◽  
Soomin Lee ◽  
Sejeong Kim ◽  
Jeeyeon Lee ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Environmental Samples ◽  
Sea Bass ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Pcr Analysis ◽  
Probable Number ◽  
Auction Markets ◽  
E Coli ◽  
Fishery Products

Abstract Background The monitoring of pathogens of fishery auction markets is important to obtain safe fishery products regarding hygiene and sanitation. In this study, aerobic, coliform, Escherichia coli, and Vibrio cholerae were monitored in the fishery products and environmental samples obtained from fishery auction markets. Methods The fishery products (flounder, octopus, skate, rock cod, sea bass, snail, monkfish, flatfish, comb pen shell, corb shell, conger eel, hairtail, croaker, and pilchard) were placed in filter bags, and the environmental samples (samples from the water tanks at the fishery auction markets, seawater from the fishery distribution vehicles, ice from wooden or plastic boxes, and surface samples from wooden and plastic boxes used for fish storage) were collected. Aerobic bacteria, E. coli, and coliform in the samples were enumerated on aerobic count plates and E. coli/coliform count plates, respectively. For V. cholerae O1 and V. cholerae non-O1 quantification, most probable number (MPN)-PCR analysis was performed. Results Aerobic and coliform bacteria were detected in most samples, but E. coli was not detected. Wooden boxes were contaminated with high levels of aerobic and coliform bacteria in all seasons (spring, summer, and fall). During fall, V. cholerae non-O1 were detected in snails, hairtails, croakers, flatfishes, pilchards, plastic boxes, and water samples. Conclusions These results indicate an increased prevalence of V. cholerae contamination in fishery products in fall, including food contact samples, which can be vehicles for cross-contamination.

Depuration of Oysters (Crassostrea gigas) Contaminated with Vibrio parahaemolyticus and Vibrio vulnificus with UV Light and Chlorinated Seawater

2012 ◽  
Vol 75 (8) ◽  
pp. 1501-1506 ◽  
Author(s):  
ROBERTA JULIANO RAMOS ◽  
MARÍLIA MIOTTO ◽  
FRANCISCO JOSÉ LAGREZE SQUELLA ◽  
ANDRÉIA CIROLINI ◽  
JAIME FERNANDO FERREIRA ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Uv Light ◽  
Control Treatment ◽  
Most Probable Number ◽  
Brazilian Coast ◽  
Uv Treatment ◽  
Probable Number ◽  
Postharvest Treatment ◽  
Small Producers

The efficacy of depuration using UV light and chlorinated seawater for decontaminating Vibrio parahaemolyticus and Vibrio vulnificus from oysters was investigated. Oysters were contaminated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 104 to 105 CFU ml−1 for bioaccumulation. The depuration was conducted in a closed system in which 350 liters of seawater was recirculated at a rate of 7 liters/min for 48 h at room temperature. Counts of V. parahaemolyticus or V. vulnificus were determined at 0, 6, 18, 24, and 48 h. Three treatments were conducted: T1, control treatment; T2, UV treatment; and T3, UV plus chlorine treatment. After 48 h of depuration of V. parahaemolyticus, T3 reduced the count by 3.1 log most probable number (MPN) g−1 and T2 reduced the count by 2.4 log MPN g−1, while T1 reduced the count by only 2.0 log MPN g−1. After 48 h of depuration of V. vulnificus, T2 and T3 were efficient, reducing the counts by 2.5 and 2.4 log MPN g−1, respectively, while T1 reduced the count by only 1.4 log MPN g−1. The UV light plus chlorine treatment was more efficient for controlling V. parahaemolyticus in oysters. Both UV light and UV light plus chlorine were efficient for V. vulnificus. The present study is the first report showing the efficacy of depuration systems for decontaminating V. parahaemolyticus and V. vulnificus in oysters cultivated on the Brazilian coast. This study provides information on processes that can contribute to controlling and preventing such microorganisms in oysters and could be used for effective postharvest treatment by restaurants and small producers of oysters on the coast of Brazil.

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