A poster presentation which discussed whether the presence of hemolysin genes correlate with the competitive fitness of Vibrio parahaemolyticus in Northeast.

1. Does the presence of hemolysin genes correlate with the competitive fitness
of Vibrio parahaemolyticus in Northeast?
Saba Ilyas, Feng Xu, and Cheryl A. Whistler
Department of Molecular, Cellular, and Biomedical Sciences, UNH, Durham, NH
Background
•Vibrio parahaemolyticus is a saltwater bacterium found naturally
in coastal waters. (1)
•Rare pathogenic variants of Vibrio parahaemolyticus can cause
human gastric infections most often from the consumption of raw
or improperly handled seafood (1).
•Infections typically occur seasonally during warmer months when
total populations of these bacteria rise, and with them, the risk of
exposure to an infectious dose of pathogens increases. (1)
•Although it is difficult to distinguish pathogenic strains from vast
majority of harmless V. parahaemolyticus strains, the presence of
hemolysin genes (tdh & trh) has been used as a marker to identify
pathogens(3).
•In recent years, the abundance of hemolysin producers has risen.
Concurrently, a strain harboring both hemolysin genes invaded the
region causing many infections (1). This suggest hemolysins
encoding islands could provide a competitive advantage.
The purpose of this project was to examine: A) the
competitiveness of hemolysin producers over non-producers, and
B) the correlation of the trh gene with urease activity.
•The urease test is a simple, quick, and cost-effective method to
detect the presence of trh gene, and it was used to evaluate its
effectiveness to replace a more complex and costly gene detection
method, PCR, for identifying pathogenic V. parahaemolyticus
during surveillance.
Methods
Competitions:
•Isolated colonies were picked from blue and white tagged strain A and
strain B, mixed in single tube containing HI Kan50 broth, and spread plated
right away (10^-5) on LBS Kan50 plates in triplicates, along with its
reciprocals and control groups.
•The tube culture and spread plates were incubated at 37°C overnight.
•50 random colonies, from input plates, were picked and patch-plated on
x-gal plate LBS Kan50 to check for starting cell count for each strain.
•After ~20 hours, the cultures were spread plated (10^-7 & 10^-8) on
LBS Kan50, incubated at 37°C overnight, and were later observed for
ending cell counts of each strain by patch-plating 50 random output
colonies on x-gal LBS Kan50 plate.
Urease test:
•Clinical and environmental strains harboring either one, both, or no
hemolysin genes were grown in HI broth at 28°C or 27°C for ~ 6 hours, and then inoculated in
triplicate as 10 μl samples onto 200 μl modified Christensen's urea agar in 96-well plates.
•The plate was covered with Breathe-Easy membrane.
• The plate was incubated at 37°C overnight. A positive reaction was
observed as a change in color from yellow to pink.
Figure 2: Streaked out
competition strain
Figure 3:Patch-plating
on x-gal LBSKan50 plate
Figure 4: Urease plate showing
urease activity in pink wells.
References:
1) N.P., “Increase in Vibrio parahaemolyticus illnesses associated with consumption of shellfish from several Atlantic coast harvest areas, United States, 2013,
Center for Disease Control, October 21, 2013. Web. February 10, 2014. <http://www.cdc.gov/vibrio/investigations/index.html>
2) Xu F., Ilyas S., Hall J.A., Jones S. H., Cooper V. S., Whistler C.A. Genetic characterization of clinical and environmental Vibrio parahaemolyticus from the
Northeast USA reveals emerging resident and non-indigenous pathogen lineages. Front in Microbiol. 2015; (6): 1-15.
3) DePaola, A., J. Ulaszek, C.A.Kaysner, B.J.Tenge, J.L. Nordstorm, J. Wells, N.Puhr and S.M. Gendel. Molecular, Serological, and Virulence Characteristics of Vibrio
parahaemolyticusIsolated from Environmental, Food, and Clinical Sources in North America and Asia. Appl. Environ. Microbiol. 2003. 69(7): 3999-4005
Figure 1: Phylogeny tree showing various ST groups.
ST631 strains caused seven infections in the past. All clinical
strains collected from this group contained both hemolysin
genes, however the environmental strain of this ST was not
hemolysin gene carrier (2).
ST674 strains were linked to a single infection. Four
environmental strains collected in this group harbored both
hemolysin genes, while one clinical strain didn’t harbor any
hemolysin gene (2).
ST1127 strains caused four infections in the past. Each of the
collected strains in this group harbor different hemolysin gene
combinations. Unlike above two ST groups, this group
contained the most variation in hemolysin genes (2).
Results for competitions:
Results for urease test:
0.1
1
10
0 5 10 15 20
OD600(log)
Time (hours)
MA-25 vs MA-15
MA-25B
MA-25W
MA-15B
MA-15W
0.1
1
10
0 5 10 15 20
OD600(log)
Time (hours)
MA-M vs MA-15
MA-MB
MA-MW
MA-15B
MA-15W
0.1
1
10
0 5 10 15 20
OD600(log)
Time (hours)
MA-Q vs G-149
MA-QB
MA-QW
G-149B
G-149W
0.1
1
10
0 5 10 15 20
OD600(log)
Time (hours)
G-3599 vs MA-21
G-3599B
G-3599W
MA-21B
MA-21W
Sequence Type (ST) Competition
pairs
Competition
RF
Control
RF
p-value Hemolysin
genes
631 MA-Q 1.02 0.94 0.272 tdh+/trh+
G149 0.99 1.00 0.8414 None
674 G-3599 0.95 0.98 0.5091 tdh+/trh+
MA-21 1.06 0.95 0.181 None
1127 MA-M 0.69 0.92 0.2028 tdh+/trh+
MA-15 1.54 0.94 0.1108 trh+
1127 MA-25 0.96 0.93 0.8138 tdh+
MA-15 1.05 0.95 0.3254 trh+
Table 1: Correlation of Urease activity with the presence of trh.
Figure 5: Growth curve on MA-Q and G -
149 tagged blue and white.
Figure 6: Growth curve on G-3599 and MA-21
tagged blue and white
Figure 7: Growth curve on MA-M and MA-15
tagged blue and white.
Figure 8: Growth curve on MA-25 and MA-15
tagged blue and white.
Table 2: Results of competitions showing relative fitness of competitive strains and control
groups along with their p-values and hemolysin genes distribution.
Hemolysin genotype % of urease positive (# of strains tested)
tdh trh
For clinical isolates
+ + 100% (67)
+ - 25% (8)
- + 100%(8)
- - 33%(11)
For environmental isolates
+ + 100%(7)
+ - 0%(1)
- + 100%(2)
- - 0%(10)
Conclusion:
•In simple binary competitions, there was no significant
growth advantage for hemolysin producers over non-
producers regardless of whether they were
environmental or clinical isolates. The competition
strain’s relative fitness did not significantly differ from
their control strain, meaning that the hemolysin
harboring strain in the experimental group did not
show any significant growth advantage over non-
hemolysin strain when compared with their control
group.
•Analysis of urease experiment showed a significant
correlation between the presence of trh gene and
urease positive test, indicating urease is a suitable
surrogate for surveillance.
Acknowledgements: Tiffany DeGroot, Bethany Parker, Ashley Marcinkiewicz. This Research was
funded by SURF at UNH, Durham.