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

OD

60

0 (

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

OD

60

0 (

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

OD

60

0 (

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

OD

60

0 (

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.