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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Abstract

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 °C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Introduction

As a member of the Vibrio genus, V. parahaemolyticus is a Gram-negative, non-spore-forming, curved, rod-shaped bacterium. It exhibits high motility in both liquid and semi-solid environments. Most V. parahaemolyticus strains are non-pathogenic to humans, yet the pathogenic subtypes have caused epidemics and pandemics, hence this species is considered to be an important foodborne pathogen in many countries1,2. The incidence of Vibrio infection in the US has shown an upward trend since 20003. Among Vibrio spp., V. parahaemolyticus is the most frequently reported species causing illnesses in the ....

Protocol

1. Media and Culturing of Microbial Strains

NOTE: Use aseptic techniques in all experiments. Use sterile materials. Sterilize all containers, tools and reagent prior to use. Autoclave all waste materials prior to disposal because they are considered biohazardous. Autoclave temperature and time combination is ≥121 °C x ≥15 min for all of the following procedures.

  1. To make ~1-L tryptic soy agar (TSA), first add 1 L deionized water in a 2-L Erlenmeyer flask containing .......

Representative Results

In this study, 54 microbial strains were assembled, which included 22 strains within the V. parahaemolyticus species, 19 other Vibrio species, and 13 non-Vibrio species (Table 1). Most V. parahaemolyticus strains were either received from FDA, CDC or other state health departments. They represent diverse serotypes and isolation sources. These strains were previously identified by the regulatory agencies. We further confirmed the identit.......

Discussion

This study focuses on culture media development and evaluation. Conventionally, TCBS is the selective and differential medium used for isolating and detecting V. parahaemolyticus, V. cholerae and V. vulnificus12. However, limitations have been reported for this medium, such as the inability to differentiate V. cholerae from other Vibrio species. Sucrose and pH indicator are the differentiation agents of TCBS. Thus, acid production by sucrose fermenter causes color change of .......

Disclosures

Some media were generously provided by Hardy Diagnostics, Santa Maria, CA. Thorsen conducted this study while a student at California Polytechnic State University. He is currently an employee of Hardy Diagnostics.

Acknowledgements

We thank M. Channey, E. Chau, and K. Tomas for their assistance on the project. Project supplies were partially funded by California Polytechnic State University.

....

Materials

NameCompanyCatalog NumberComments
Reagent/Equipment
AgarFisher ScientificDF0140-15-4may use other brands
AutoclaveAny
BHI powderFisher ScientificDF0418-17-7may use other brands
BlenderAnyto blend oyster meat
CampyGen gas generatorHardy DiagnosticsCN035Ato provide a microaerophilic atmosphere; may use other brands
Chocolate agar platesHardy DiagnosticsE14may use other brands
Common PCR reagents (dNTPs, MgCl2, Taq Polymerase)Anyor use PCR beads (Fisher Sci 46-001-014)
Culture tubesFisher ScientificS50712may use other brands
Eppendorf tubesFisher ScientificS348903may use other brands
Gel docAny
HardyChrom Vibrio agar platesHardy DiagnosticsG319This study evaluates this medium
IncubatorAny
Inoculating loopsFisher Scientific22-363-60610 microliter-size was used in this study
NaClFisher ScientificBP358-212may use other brands
OystersAny
PBSFisher ScientificR23701may use other brands
Petri dishFisher ScientificFB0875713may use other brands
Pipette and tipsAnySterilized tips
Primers for tlhIDT DNA
ScaleAny
SpreaderFisher Scientific08-100-11Beads may be used instead
Stomacher blenderStomacher400Samples were homogenized at 200 rpm for 30 sec.  Other homogenizer can be used.
Sterile filter bags for blendersFisher Scientific01-812-5
TCBS powderHardy Diagnostics265020This study evaluates this medium
ThermocyclerAny
TSB powderFisher ScientificDF0370-07-5may use other brands
UV viewing cabinetAnyEmit long-wave UV light
Water bathAny
NameSourcesCatalog NumberComments
Bacterial species and strains
Aeromonas hydrophilaATCC
Candida albicansATCC
Campylobacter jejuniATCC
Escherichia coliATCC
Proteus mirabilisATCC
Pseudomonas aeruginosaATCC
Staphylococcus aureusATCC
Salmonella CholeraesuisATCC
Shigella boydiiATCC
Shigella flexneriATCC
Shigella sonneiATCC
Vibrio alginolyticusATCC
V. cholerae (serotypes include O139, O1, non O1, El Tor biovars)FDA, ATCC
V. damselaFDA
V. fisheriiEnvironment
V. fluvialisCDC
V. furnissiiCDC
V. hollisaeFDA
V. metschnikoviiATCC
V. mimicusFDA
V. parahaemolyticus(serotypes include O3:K6, O1:K56, O4:K8, O5:K15, O8, etc)ATCC, FDA, CDC, Environment
V. proteolyticusFDA
V. vulnificusFDA

References

  1. Yeung, P. S., Boor, K. J. Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections. Foodborne Pathog. Dis. 1 (2), 74-88 (2004).
  2. Yeung, P. S. M., Boor, K. J., Faruque, S. M.

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