1. The effects of temperature and salinity conditions of Vibrio parahaemolyticus growth in vitro
Jermaine D. Dorsey1, Mamie T. Coats1, & Crystal N. Johnson2
1Alabama State University, Montgomery, Alabama 36104
2Louisiana State University, Baton Rouge, Louisiana 70803
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DISCUSSION
ACKNOWLEDGMENTS
ABSTRACT
Vibrio parahaemolyticus (Vp) is a halophilic Gram-negative bacterium found
abundantly in estuarine and marine environments and is known to cause
gastroenteritis in humans. Exposure is linked to the consumption of raw or
under-cooked oysters and other shellfish. The objective of this study was to
examine the effects of temperature and salinity on the in vitro growth rates of
Vp containing the gene thermostable direct hemolysin (tdh+) and tdh-related
hemolysin (trh+), which are pathogenicity factors. Samples were collected from
the Gulf Coast regions of Louisiana from oysters and sediment. Isolates were
cultivated from collected samples through a method of direct plating and colony
hybridization, which is a probing method to identify and quantify the various
trh+, tdh+, and tlh+ genes associated with Vp. In this study, several isolates
containing only tdh, trh, both genes, or neither gene were grown to calculate
the number of generations over a set period of time. Vp was incubated at 10°C,
21°C, 25°C, 31°C, and 37°C and in salinity concentration of 2% and 3.5% to
stimulate temperature changes in estuaries and in the open ocean, respectively.
All groups of isolates had more generations at 2% than 3.5%. The temperature
to achieve the maximal number of generations varied among the groups.
Isolates with neither virulence gene experienced highest levels of growth in
37°C while isolates containing both genes had maximal generations at 31°C.
These data suggest that estuaries are a more likely environment for all groups
of isolates due to the salinity requirements. Overall, this study provides a
deeper understanding of tdh+ and trh+ containing environmental isolates that
have the potential to become pathogenic in humans.
INTRODUCTION
V. parahaemolyticus is a halophilic bacteria that is abundantly present in marine
environments, including the coastal waters of the United States. The bacterium is
estimated to cause more than 4500 cases of infection in the US each year.
Hospitalization is required in only 7% of reported cases.
This microorganism can be the causative agent in seafood-borne gastroenteritis
following the consumption of raw oysters, or undercooked shellfish.
Gastroenteritis due to V. parahaemolyticus usually occurs within 24 hours of
exposure and includes symptoms such as abdominal cramps and watery diarrhea.
More symptoms including vomiting and nausea are also possible. V.
parahaemolyticus illness is usually self-limiting and resolves within 7 days.
Infected individuals are encouraged to drink plenty of liquids to replace fluid loss.
V. parahaemolyticus has several individual virulence factors. The thermostable
direct hemolysin (tdh+) is a major virulence factor associated with the V.
parahaemolyticus and is found in more than 90% of clinical V. parahaemolyticus
isolates and about 1% of the environmental strains. During infection tdh+ acts on
cellular membranes as a pore-forming toxin that alters ion flux in intestinal cells
leading to a secretory response and diarrhea. A second virulence factor which is
similar to tdh+ is the tdh-related hemolysin (trh+) which causes Ca2+ activated Cl-
channels to open resulting in ion flux and fluid accumulation. Both of these
virulence factors are present in infectious V. parahaemolyticus but how they
correlate with growth under varying conditions remains to be defined.
The goal for this project was to examine the effects of temperature and salinity on
growth rates in the presence of virulence determinants tdh+ and trh+.
• MARC U STAR program and Biomedical Research
and Training Program at Alabama State
University, Montgomery, Al. 36104. The MARC
program is supported by the National Institutes
of Health (NIH) MARC GRANT #5T34GMOO8167 .
• Dr. Crystal N. Johnson Laboratory at Louisiana
State University, Baton Rouge, LA. Dr. Johnson’s
research is supported by NSF grant # EF-1003943
as part of the joint NSF-NIH Ecology of Infectious
Diseases program.
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GENERATIONS
TEMPERATURE °C
2% SALINITY
TDH TRH TDH/TRH TLH
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GENERATIONS
TEMPERATURE °C
3.5% SALINITY
TDH TRH TDH/TRH TLH
RESULTS
Overall, V. parahaemolyticus grew better in 2% than 3.5%
salinity. This suggests that the pathogen would thrive more in
estuaries than in the open ocean.
Regardless of the pathogenic potential, all strains grew more
robustly as the incubation temperature increased. Established
trends for V. parahaemolyticus infection support this
assessment as infection rates increase during warmer months.
It is also noteworthy that in 3.5% salinity trh+ containing
isolates experienced enhanced growth at 37°C when compared
to tdh+ and tdh+/trh+ containing isolates. The need for this
high level of salinity and temperature correlates with decreases
in this (trh+ only) genotype among infectious isolates.
Future studies will examine the activity of each gene locus
under the described growth condition.
MATERIALS AND METHODS
V. parahaemolyticus isolates were isolated from sediment and oysters from Louisiana Gulf coast.
Isolates were probed for the presence of tdh+, trh+, and tlh+.
Initial culturing of Isolates was done in a 10x alkaline phosphate water (APW) solution with incubation
at 33°C for 4 hours. Bacteria were quantitated via serial dilutions and plating on T1N3 agar.
To determine the effect of the presence of tdh+ and trh+ growth at varying temperatures and degrees
of salinity. Isolates were inoculated into APW solution containing either 3.5% or 2% saline. Cultures
were incubated at either 10°C, 21°C, 25°C, 31°C or 37°C for 4 hours. Both the initial (before incubation)
and final (after incubation) number of colony forming units (CFU) were determined.
The number of generations was calculated using the formula (logB) - (logA) / log2. {LogB= final CFU;
LogA=initial CFU}.