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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.
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.
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 ....
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.
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.......
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 .......
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.
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.
....Name | Company | Catalog Number | Comments |
Reagent/Equipment | |||
Agar | Fisher Scientific | DF0140-15-4 | may use other brands |
Autoclave | Any | ||
BHI powder | Fisher Scientific | DF0418-17-7 | may use other brands |
Blender | Any | to blend oyster meat | |
CampyGen gas generator | Hardy Diagnostics | CN035A | to provide a microaerophilic atmosphere; may use other brands |
Chocolate agar plates | Hardy Diagnostics | E14 | may use other brands |
Common PCR reagents (dNTPs, MgCl2, Taq Polymerase) | Any | or use PCR beads (Fisher Sci 46-001-014) | |
Culture tubes | Fisher Scientific | S50712 | may use other brands |
Eppendorf tubes | Fisher Scientific | S348903 | may use other brands |
Gel doc | Any | ||
HardyChrom Vibrio agar plates | Hardy Diagnostics | G319 | This study evaluates this medium |
Incubator | Any | ||
Inoculating loops | Fisher Scientific | 22-363-606 | 10 microliter-size was used in this study |
NaCl | Fisher Scientific | BP358-212 | may use other brands |
Oysters | Any | ||
PBS | Fisher Scientific | R23701 | may use other brands |
Petri dish | Fisher Scientific | FB0875713 | may use other brands |
Pipette and tips | Any | Sterilized tips | |
Primers for tlh | IDT DNA | ||
Scale | Any | ||
Spreader | Fisher Scientific | 08-100-11 | Beads may be used instead |
Stomacher blender | Stomacher | 400 | Samples were homogenized at 200 rpm for 30 sec. Other homogenizer can be used. |
Sterile filter bags for blenders | Fisher Scientific | 01-812-5 | |
TCBS powder | Hardy Diagnostics | 265020 | This study evaluates this medium |
Thermocycler | Any | ||
TSB powder | Fisher Scientific | DF0370-07-5 | may use other brands |
UV viewing cabinet | Any | Emit long-wave UV light | |
Water bath | Any | ||
Name | Sources | Catalog Number | Comments |
Bacterial species and strains | |||
Aeromonas hydrophila | ATCC | ||
Candida albicans | ATCC | ||
Campylobacter jejuni | ATCC | ||
Escherichia coli | ATCC | ||
Proteus mirabilis | ATCC | ||
Pseudomonas aeruginosa | ATCC | ||
Staphylococcus aureus | ATCC | ||
Salmonella Choleraesuis | ATCC | ||
Shigella boydii | ATCC | ||
Shigella flexneri | ATCC | ||
Shigella sonnei | ATCC | ||
Vibrio alginolyticus | ATCC | ||
V. cholerae (serotypes include O139, O1, non O1, El Tor biovars) | FDA, ATCC | ||
V. damsela | FDA | ||
V. fisherii | Environment | ||
V. fluvialis | CDC | ||
V. furnissii | CDC | ||
V. hollisae | FDA | ||
V. metschnikovii | ATCC | ||
V. mimicus | FDA | ||
V. parahaemolyticus(serotypes include O3:K6, O1:K56, O4:K8, O5:K15, O8, etc) | ATCC, FDA, CDC, Environment | ||
V. proteolyticus | FDA | ||
V. vulnificus | FDA |
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