Persistence of Vibrio parahaemolyticus in the Pacific Oyster,

Crassostrea gigas, Is a Multifactorial Process Involving Pili and Flagella

but Not Type III Secretion Systems or Phase Variation
Alisha M. Aagesen,a Sureerat Phuvasate,b Yi-Cheng Su,b Claudia C. Hsea
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon, USAa; Oregon State University Seafood Research and
Education Center, Astoria, Oregon, USAb

he genus Vibrio consists of a group of bacteria that naturally

inhabit aquatic environments worldwide. The human pathogen Vibrio parahaemolyticus is commonly found associated with
shellfish, particularly oysters (1, 2). Depuration is a controlled
process in which shellfish are placed into clean seawater to reduce
the bacterial contaminants in their tissues. However, depuration is
not very effective at reducing the numbers of V. parahaemolyticus
cells in oysters (3), and little is known about the association of V.
parahaemolyticus with oysters.
Both type I and type IV pili are important for bacterial attachment to a variety of surfaces (46). V. parahaemolyticus encodes a
homologue of type I pili that is most similar to the CsuA/B operon
from Acinetobacter baumannii (7) and homologues of two wellstudied type IV pili from vibrios, the mannose-sensitive hemagglutinin (MSHA) and the chitin-regulated pilus (PilA) (http://img
.jgi.doe.gov/) (8, 9). Vibrio vulnificus uses PilA for persistence in
Crassostrea virginica (10, 11). Both type IV pili are involved in
biofilm formation in V. parahaemolyticus (12). Flagella are often
critical during early stages of bacterial colonization of a surface
(13, 14). V. parahaemolyticus possesses a single, polar flagellum
used for movement in liquid (reviewed in reference 14) as well as
peritrichous (lateral) flagella for surface movement (1517 and
reviewed in reference 18). Many bacterial pathogens use type III
secretion systems (T3SSs) for survival in the host by injecting virulence factors directly into the host. V. parahaemolyticus encodes
two T3SSs that exhibit different phenotypes during disease (19
21). V. parahaemolyticus also exhibits phase variation and can
switch from an opaque (OP) to a translucent (TR) phenotype
based on polysaccharide production (22). In this study, we exam-

ined the role of V. parahaemolyticus pili, flagella, phase variation,

and T3SSs in persistence in the Pacific oyster, Crassostrea gigas.
Oysters were collected from Oregon Oyster Farm (Newport,
OR) and exposed to 105 CFU/ml of V. parahaemolyticus for 16
to 18 h at room temperature (20C) with a recirculating pump.
Algae were added to the tank to facilitate uptake according to the
manufacturers recommendation (Phytoplex; Kent Marine). Depuration was conducted at 19 to 20C for 48 to 72 h. At each time
point, animals from each exposure tank were weighed, homogenized, and diluted with phosphate-buffered saline (PBS) for enumeration on tryptic soy agar (TSA) supplemented with 1.5% NaCl
and either 100 g/ml streptomycin, 60 g/ml chloramphenicol,
10 g/ml phosphomycin, or 50 g/ml kanamycin, depending on
the strain (see Table S1 in the supplemental material), to eliminate
naturally occurring background flora. The data were log transformed prior to data analysis. All strains are compared with their
respective parental strain using a one-way analysis of variance
(ANOVA) and Tukeys honestly significant difference (HSD) post

Received 28 January 2013 Accepted 1 March 2013

Published ahead of print 8 March 2013
Address correspondence to Claudia C. Hse, hasec@science.oregonstate.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.00314-13.
Copyright 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.00314-13

TABLE 1 Comparison of V. parahaemolyticus wild type and type IV pilus mutants in Pacific oysters during depuration
Indicated value at each time pointa
12 h

30 h

48 h

Strain

0 h (log mean
CFU/g)

Log mean
CFU/g

Log reduction

Log mean
CFU/g

Log reduction

Log mean
CFU/g

Log reduction

ATCC 17802
VPAA9 mshA
VPAA27 pilA
VPAA28 mshA/pilA
VPAA26 pilD

5.08 0.10 A
4.66 0.14 AB
4.43 0.14 B
4.43 0.12 B
4.56 0.13 B

3.82 0.18 A
2.72 0.21 B
2.98 0.14 AB
2.14 0.28 BC
1.73 0.27 C

1.26 0.15 A
1.94 0.20 A
1.44 0.17 AB
2.3 0.26 BC
2.80 0.21 C

2.50 0.22 A
1.41 0.29 AB
1.55 0.22 AB
1.29 0.28 B
0.70 0.18 B

2.58 0.19 A
3.24 0.19 A
2.88 0.20 AB
3.15 0.35 AB
3.88 0.20 B

1.57 0.29 A
0.48 0.23 B
0.40 0.19 B
0.56 0.20 AB
0.30 0.15 B

3.62 0.27 A
4.50 0.29 A
4.16 0.24 A
3.87 0.28 A
4.50 0.28 A

a
The log mean or log reduction and standard error are shown. Different letters indicate statistical significance for each time point according to the one-way ANOVA (treatment)
and post hoc comparisons using Tukeys HSD test (P 0.05). These data represent four independent experiments with 12 animals analyzed at each time point.

May 2013 Volume 79 Number 10

Applied and Environmental Microbiology

p. 33033305

aem.asm.org

3303

Downloaded from http://aem.asm.org/ on February 17, 2016 by OCEAN UNIV

Vibrio parahaemolyticus can resist oyster depuration, suggesting that it possesses specific factors for persistence. We show that
type I pili, type IV pili, and both flagellar systems contribute to V. parahaemolyticus persistence in Pacific oysters whereas type
III secretion systems and phase variation do not.

Aagesen et al.

TABLE 2 Comparison of the V. parahaemolyticus wild type and type I pilus mutant in Pacific oysters during depuration
Indicated value at each time pointa
4h

24 h

48 h

Strain

0 h (log mean
MPN/g)

Log mean
MPN/g

Log reduction

Log mean
MPN/g

Log reduction

Log mean
MPN/g

Log reduction

ATCC 17802
VPAA8 csuB

4.31 0.21 A
4.27 0.22 A

3.86 0.25 A
3.51 0.10 A

0.44 0.17 A
0.76 0.13 A

3.04 0.18 A
2.41 0.23 A

1.27 0.14 A
1.86 0.22 B

1.88 0.15 A
1.08 0.19 B

2.43 0.06 A
3.19 0.11 B

a
The log mean or log reduction and standard error are shown. Different letters indicate statistical significance for each time point according to the one-way ANOVA (treatment)
and post hoc comparisons using Tukeys HSD test (P 0.05). These data represent two independent experiments with 10 animals analyzed at each time point.

tion (23). Although loss of type II secretion was not examined in

this study, it cannot be ruled out as playing a possible role in
persistence in the oyster during depuration.
To examine the role of flagellar systems, polar (flaM
LM5392), lateral (lafK LM7789), and completely nonmotile
(flaK/lafK LM7901) flagellar system mutants (see Table S1 in
the supplemental material) were tested for persistence in the Pacific oyster during depuration. Only flaM was defective in uptake
compared to its isogenic parent LM5431 TR (Table 3). Examining
these mutants over 48 h of depuration, all flagellar system mutants, flaM, lafK, and flaK/lafK, were recovered significantly
less (log CFU/g) than was LM5431 at 24 h and 48 h. These data
suggest that both polar and lateral flagellar systems are important
for V. parahaemolyticus persistence in the Pacific oyster.
Oysters possess an innate immune system and have macrophage-like hemocytes that are responsible for bacterial killing
(24). Therefore, to persist in the oyster, bacteria likely have to
overcome their immune defenses (25). When the T3SS double
mutant was tested (LM7341), there was no significant different
between it and the wild type (TR) (data not shown), suggesting
that V. parahaemolyticus does not utilize either T3SS for persistence in the Pacific oyster. V. parahaemolyticus may use other unidentified factors or tactics to circumvent the oyster immune system for survival in the oyster host.
One possibility for oyster immune system evasion is phase
variation in colony morphology from the opaque to the translucent phenotype, a result of altering surface polysaccharide production (26). In our analyses, we compared strains LM5431 (TR)
and LM5432 (OP) to determine if phase variation affected persistence in the oyster. Interestingly, the OP and TR strains were not
statistically different during the course of depuration (Table 3),
suggesting that phase variation is not involved in persistence in the
Pacific oyster. These findings are similar to what was observed in
V. vulnificus, where a translucent variant, capable of reverting back
to the opaque phenotype, showed no difference in recoverability

TABLE 3 Comparison of V. parahaemolyticus wild type and flagellar mutants in Pacific oysters during depuration
Indicated value at each time pointa
24 h

48 h

72 h

Strain

0 h (log mean
CFU/g)

Log mean
CFU/g

Log reduction

Log mean
CFU/g

Log reduction

Log mean
CFU/g

Log reduction

LM5432 OP
LM5431 TR
LM5392 flaM
LM7789 lafK
LM7901 flaK/lafK

4.62 0.21 AB
4.83 0.20 A
3.54 0.41 B
4.33 0.34 AB
4.73 0.30 AB

4.03 0.21 A
4.21 0.19 A
2.12 0.27 B
2.64 0.37 B
2.57 0.43 B

0.66 0.14 A
0.62 0.11 A
1.60 0.27 B
1.69 0.21 B
2.16 0.28 B

3.64 0.16 A
3.53 0.11 A
1.14 0.34 B
1.88 0.47 B
1.41 0.33 B

1.05 0.13 A
1.30 0.14 A
2.58 0.31 B
2.45 0.32 B
3.32 0.27 B

2.99 0.49 A
3.07 0.46 AB
1.26 0.38 B
1.77 0.46 AB
1.32 0.30 B

1.71 0.40 A
1.96 0.36 A
2.46 0.31 A
2.55 0.31 AB
3.42 0.29 B

a
The log mean or log reduction and standard error are shown. Different letters indicate statistical significance for each time point according to the one-way ANOVA (treatment)
and post hoc comparisons using Tukeys HSD test (P 0.05). These data represent four replicate experiments with 15 animals analyzed for each time point.

3304

aem.asm.org

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on February 17, 2016 by OCEAN UNIV

hoc comparison (P 0.05). Different letters indicate statistically

significant differences between strains for each time point.
To test the role of pili in colonizing the Pacific oyster, V. parahaemolyticus ATCC 17802-defined gene deletions were created in
the type I pilin gene csuB, the type IV pilin genes mshA and pilA, a
double gene deletion in mshA and pilA, and a single gene deletion
in the type IV pilin peptidase pilD (see Table S1 in the supplemental material). The mshA mutant was not defective in uptake, but
during depuration, there was significantly less recovered (log
CFU/g) after 12 h and 48 h of depuration (Table 1). Unlike the
mshA mutant, the pilA, mshA/pilA, and pilD mutants had
defects in uptake after the bacterial exposure (t 0), although no
difference was observed for the amount of bacteria added to each
tank (data not shown). During depuration, no difference was observed for the pilA mutant (log CFU/g) until 48 h of depuration.
The mshA/pilA and pilD mutants were recovered significantly less (log CFU/g) after 12 h and 30 h of depuration. By 48 h
of depuration, there was no difference in the mshA/pilA mutant (log CFU/g) recovery but significantly less pilD mutant (log
CFU/g) was recovered. As for the type I pilus mutant csuB, there
were no defects in uptake or after 4 h and 24 h of depuration but
there were significantly fewer bacteria recovered over the 48 h of
depuration (log most probable number [MPN]/g) (Table 2).
From these data, it appears that V. parahaemolyticus uses both
type I and type IV pili to persist in the Pacific oyster during the
depuration process. To verify that these gene deletions did not
have a polar effect on neighboring genes, biofilm formation was
performed. Type IV pilus mutants had biofilm defects similar to
those of previous reports (12), and the type I pilus mutant also had
similar defects in biofilm formation; complementation restored
these mutants to wild-type levels (data not shown). These depuration results are consistent with what was observed in V. vulnificus, where loss of pilA and pilD resulted in a decrease in bacteria
recovered from the tissues of C. virginica compared with that recovered from the wild type (10). It has been previously published
that loss of pilD in V. vulnificus also results in loss of type II secre-

V. parahaemolyticus Factors for Persisting in Oysters

8.
9.
10.
11.

12.
13.

ACKNOWLEDGMENTS
We thank Linda McCarter for her kindness in providing the opaque and
translucent strains and the flagella and T3SS mutants to test in our oyster
infection assay. We also thank Chris Langdon at the Oregon State University Hatfield Marine Science Center (Newport, OR) for the use of his
oyster quarantine facilities, where some of the oyster infection assays were
completed, and the Oregon Oyster Farm (Newport, OR) for providing all
of the oysters used in this study.
This study was funded in part by National Research Initiative grant no.
2008-35201-04580 from the USDA National Institute of Food and Agriculture, Food Safety and Epidemiology program 32.0A, and the OSU
General Research Fund.

REFERENCES
1. Johnson CN, Bowers JC, Griffitt KJ, Molina V, Clostio RW, Pei S, Laws
E, Paranjpye RN, Strom MS, Chen A, Hasan NA, Huq A, Noriea NF,
III, Grimes DJ, Colwell RR. 2012. Ecology of Vibrio parahaemolyticus and
Vibrio vulnificus in the coastal and estuarine waters of Louisiana, Maryland, Mississippi, and Washington (United States). Appl. Environ. Microbiol. 78:7249 7257.
2. Vieira RH, Costa RA, Menezes FG, Silva GC, Theophilo GN, Rodrigues
DP, Maggioni R. 2011. Kanagawa-negative, tdh- and trh-positive Vibrio
parahaemolyticus isolated from fresh oysters marketed in Fortaleza, Brazil.
Curr. Microbiol. 63:126 130.
3. Croci L, Suffredini E, Cozzi L, Toti L. 2002. Effects of depuration of
molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae 01 and Vibrio parahaemolyticus. J. Appl. Microbiol. 92:460 465.
4. Althouse C, Patterson S, Fedorka-Cray P, Isaacson RE. 2003. Type 1
fimbriae of Salmonella enterica serovar Typhimurium bind to enterocytes
and contribute to colonization of swine in vivo. Infect. Immun. 71:6446
6452.
5. Paranjpye RN, Strom MS. 2005. A Vibrio vulnificus type IV pilin contributes to biofilm formation, adherence to epithelial cells, and virulence.
Infect. Immun. 73:14111422.
6. Crepin S, Houle S, Charbonneau ME, Mourez M, Harel J, Dozois CM.
2012. Decreased expression of type 1 fimbriae by a pst mutant of uropathogenic Escherichia coli reduces urinary tract infection. Infect. Immun.
80:28022815.
7. Tomaras AP, Dorsey CW, Edelmann RE, Actis LA. 2003. Attachment to
and biofilm formation on abiotic surfaces by Acinetobacter baumannii:

May 2013 Volume 79 Number 10

14.
15.
16.
17.
18.
19.
20.

21.
22.
23.

24.

25.

26.

involvement of a novel chaperone-usher pili assembly system. Microbiology 149:34733484.

Meibom KL, Li XB, Nielsen AT, Wu CY, Roseman S, Schoolnik GK.
2004. The Vibrio cholerae chitin utilization program. Proc. Natl. Acad. Sci.
U. S. A. 101:2524 2529.
Tarsi R, Pruzzo C. 1999. Role of surface proteins in Vibrio cholerae
attachment to chitin. Appl. Environ. Microbiol. 65:1348 1351.
Paranjpye RN, Johnson AB, Baxter AE, Strom MS. 2007. Role of type IV
pilins in persistence of Vibrio vulnificus in Crassostrea virginica oysters.
Appl. Environ. Microbiol. 73:50415044.
Srivastava M, Tucker MS, Gulig PA, Wright AC. 2009. Phase variation,
capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio
vulnificus by the Eastern oyster (Crassostrea virginica). Environ. Microbiol.
11:1934 1944.
Shime-Hattori A, Iida T, Arita M, Park KS, Kodama T, Honda T. 2006.
Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm
formation. FEMS Microbiol. Lett. 264:89 97.
Yildiz FH, Visick KL. 2009. Vibrio biofilms: so much the same yet so
different. Trends Microbiol. 17:109 118.
McCarter LL. 2001. Polar flagellar motility of the Vibrionaceae. Microbiol.
Mol. Biol. Rev. 65:445 462.
Kirov SM. 2003. Bacteria that express lateral flagella enable dissection of
the multifunctional roles of flagella in pathogenesis. FEMS Microbiol.
Lett. 224:151159.
McCarter L, Silverman M. 1989. Iron regulation of swarmer cell differentiation of Vibrio parahaemolyticus. J. Bacteriol. 171:731736.
McCarter L, Silverman M. 1990. Surface-induced swarmer cell differentiation of Vibrio parahaemolyticus. Mol. Microbiol. 4:10571062.
Stewart BJ, McCarter LL. 2003. Lateral flagellar gene system of Vibrio
parahaemolyticus. J. Bacteriol. 185:4508 4518.
Hiyoshi H, Kodama T, Iida T, Honda T. 2010. Contribution of Vibrio
parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice. Infect. Immun. 78:17721780.
Matlawska-Wasowska K, Finn R, Mustel A, OByrne CP, Baird AW,
Coffey ET, Boyd A. 2010. The Vibrio parahaemolyticus type III secretion
systems manipulate host cell MAPK for critical steps in pathogenesis.
BMC Microbiol. 10:329.
Noriea NF, III, Johnson CN, Griffitt KJ, Grimes DJ. 2010. Distribution
of type III secretion systems in Vibrio parahaemolyticus from the northern
Gulf of Mexico. J. Appl. Microbiol. 109:953962.
Hsieh YC, Liang SM, Tsai WL, Chen YH, Liu TY, Liang CM. 2003.
Study of capsular polysaccharide from Vibrio parahaemolyticus. Infect.
Immun. 71:3329 3336.
Paranjpye RN, Lara JC, Pepe JC, Pepe CM, Strom MS. 1998. The type
IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in ironoverloaded mice. Infect. Immun. 66:5659 5668.
Philipp EER, Lipinski S, Rast J, Rosenstiel P. 2012. Immune defense of
marine invertebrates: the role of reactive oxygen and nitrogen species, p
236 246. In Abele D, Vazquez-Medina JP, Zenteno-Savin T (ed), Oxidative stress in aquatic ecosystems. Wiley-Blackwell, Chichester, United
Kingdom.
Duperthuy M, Schmitt P, Garzon E, Caro A, Rosa RD, Le Roux F,
Lautredou-Audouy N, Got P, Romestand B, de Lorgeril J, KiefferJaquinod S, Bachere E, Destoumieux-Garzon D. 2011. Use of OmpU
porins for attachment and invasion of Crassostrea gigas immune cells by
the oyster pathogen Vibrio splendidus. Proc. Natl. Acad. Sci. U. S. A. 108:
29932998.
Enos-Berlage JL, McCarter LL. 2000. Relation of capsular polysaccharide
production and colonial cell organization to colony morphology in Vibrio
parahaemolyticus. J. Bacteriol. 182:55135520.

aem.asm.org 3305

Downloaded from http://aem.asm.org/ on February 17, 2016 by OCEAN UNIV

compared with the opaque wild type in whole oyster homogenates

of C. virginica (11).
In conclusion, V. parahaemolyticus persistence in the Pacific
oyster is a complex process involving two types of pili, type I and
type IV, as well as polar and lateral flagellar systems but not T3SSs
or phase variation. This study has provided a glimpse into the
intricate Vibrio-oyster interplay. Future studies will expand on
this knowledge, and it will be interesting to explore the effects of
chitinous particles and/or mannose on improving depuration efficiency of V. parahaemolyticus. Also, exploring other parameters
of depuration that may affect flagellar production, i.e., salinity and
pH combinations, may also improve depuration conditions, ultimately leading to a highly efficient and cost-effective postharvest
processing technique.