Received: 28 August 2018 Revised and accepted: 22 April 2019

DOI: 10.1111/jwas.12617

FUNDAMENTAL STUDIES

Characterization and growth conditions of Vibrio

parahaemolyticus strains with different virulence
degrees that cause acute hepatopancreatic
necrosis disease in Litopenaeus vannamei

Sonia A. Soto-Rodriguez | Rodolfo Lozano-Olvera |

Daniel A. Palacios-Gonzalez | Carmen Bolan-Mejia |
Karla G. Rendon-Aguilar

Centro de Investigación en Alimentación y

Desarrollo, AC Unidad Mazatlán en
Acuicultura y Manejo Ambiental, Mazatlán,
Mexico
Correspondence
Sonia A. Soto-Rodriguez, Centro de
Investigación en Alimentación y Desarrollo,
AC Unidad Mazatlán en Acuicultura y Manejo
Ambiental, Avenida Sábalo-Cerritos s/n,
Mazatlán, Sinaloa, Mexico.
Email: ssoto@ciad.mx
Funding information
National Fishery and Aquaculture Institute
(INAPESCA)
Abstract
The phenotypic characteristics and growth kinetics at sev-
eral temperatures, salinities, and pH values of three Vibrio
parahaemolyticus (Vp) strains with different virulence and
one nonpathogenic strain were evaluated. Independent of
the virulence of the strain, a high metabolic diversity was
found, which yielded different colored phenotypes on the
CHROMagar™ Vibrio. All strains were resistant to ampicillin
and carbenicillin, and Vp AHPND+ organisms were the most
sensitive to enrofloxacin. The exponential growth of Vp
strains started at 1–2 hr of incubation, although no relation-
ship was observed between the bacterial density and
degree of virulence. Moreover, the growth of the most viru-
lent strain was independent of the nutrients in the incuba-
tion media during the initial hour postinoculation. No strain
grew at 4 C in 0% NaCl and pH 4, but only Vp AHPND+
grew at 44 C. For all strains, the lag phase was proportional
to the NaCl concentration, and the growth was better at
pH 8–9. However, the Vp AHPND− strain displayed a
greater variability, was more sensitive to extreme condi-
tions, and showed a lag phase of 9 hr independent of
the pH.

© Copyright by the World Aquaculture Society 2019

1002 wileyonlinelibrary.com/journal/jwas J World Aquacult Soc. 2019;50:1002–1015.

SOTO-RODRIGUEZ ET AL. 1003

KEYWORDS
AHPND, environmental parameters, growth conditions, Vibrio
parahaemolyticus, virulence degree

1 | I N T RO D UC T I O N

Vibrio parahaemolyticus (Vp) inhabit marine and estuarine environments of various salinities throughout the world;
have been implicated in diarrhea; and are also associated with some of the most serious diseases in fish, shellfish,
and penaeid shrimp (Food and Agriculture Organization of the United Nations/World Health Organization, 2011),
but there have only been a few studies of opportunistic Vp isolates causing shrimp vibriosis. Specific strains of Vp
cause an emerging disease known as acute hepatopancreatic necrosis disease (AHPND), which was first reported in
China in 2009 and spread to other Asian (Network of Aquaculture Centers in Asia-Pacific/Food and Agriculture
Organization of the United Nations, 2011) and Latin-American (Restrepo, Bayot, Betancourt, & Pinzón, 2016) coun-
tries and the United States (OIE World Health Organization, 2017). Vp strains that cause AHPND carry a pVA1 plas-
mid that encodes a toxin homologous to the PirAB binary toxin produced by Photorhabdus luminescens (Lee et al.,
2015). There are major differences among Vp strains with respect to pathogenicity—human pathogenic Vp strains
are different from Vp AHPND+ strains because they involve specific virulence factors. Virulent strains cause mass
mortalities of cultivated shrimp, Penaeus (Litopenaeus) vannamei (Boone) and Penaeus monodon (Hong, Xu, Zhuo,
Liu, & Lu, 2016). The disease typically affects juvenile shrimp within 20–35 days of stocking. Since early 2013, the
pathogen has spread in Mexico (Nunan, Lightner, Pantoja, & Gomez-Jimenez, 2014) and resulted in a significant
(70%) decrease in shrimp production compared to 2012 (Anuario Estadistico de Acuacultura, 2013). AHPND cur-
rently continues to be a problem for the shrimp industry. The pathognomonic lesions of AHPND indicate a severe
desquamation of the tubular epithelial cells of the hepatopancreas produced by PirAB (Sirikharin et al., 2015; Soto-
Rodriguez, Gomez-Gil, Lozano-Olvera, Betancourt-Lozano, & Morales-Covarrubias, 2015). Differences in Vp
virulence have been observed in Asian and Mexican strains using immersion challenges with juvenile shrimp. Some
Mexican AHPND+ strains cause 100% mortality within 17 hr postinoculation (p.i.), whereas some of the less virulent
strains do not induce 100% mortality (Soto-Rodriguez et al., 2015). Moreover, Mexican strains are more virulent than
the strains previously reported by Tran et al. (2013), Nunan et al. (2014) and Joshi et al. (2014). In addition, pirA and
pirB genes have recently been reported for Vibrio harveyi, Vibrio owensii, and Vibrio campbellii strains (Dong et al.,
2017; Xiao et al., 2017), indicating the complexity of the problem. Despite this, to date, there are few studies that
compare virulent and nonpathogenic strains in terms of the use of substrates, antimicrobial susceptibility, and growth
conditions. The enhanced metabolism of a virulent Vp strain has likely contributed to its ability to rapidly populate
the environment and kill shrimp (Williams, Jensen, Kuhn, & Stevens, 2017). Virulent Vp strains around the world
might have intraspecific phenotypic variability that may enable them to live in nearly any aquatic environment. In
addition, Vp strains in the environment exhibit a halophilic and seasonal distribution, which is directly related to the
salinity and temperature (Johnson et al., 2010; Zimmerman et al., 2007). Vp environmental isolates have been associ-
ated with high temperatures and salinities, which explains their ubiquitous nature in tropical areas. However, there
have been no studies of Vp growth patterns at different pH levels, and how the seawater parameters affect the
growth or virulence of Vp that cause AHPND is still uncertain. Considering that there are broad differences in the
systems of shrimp culture around the world and knowing the importance of characterizing the Vp strains, the aims of
the present study were to evaluate the phenotypic characteristics (biochemical and physiological profiles), antibiotic
sensitivity, growth kinetics, and survival under several conditions of temperature, salinity, and pH of Vp AHPND+
with different degrees of virulence and AHPND− strains.
1004 SOTO-RODRIGUEZ ET AL.

2 | MATERIALS AND METHODS

2.1 | Inoculum preparation

The Vp AHPND− (M0702−) strain and Vp AHPND+ strains (M0904+++, M0603++, and M0905+) (Soto-Rodriguez
et al., 2015) were selected according to their degree of virulence. All bacteriological media and saline solutions were
supplemented with NaCl at a final concentration of 2.5% (+). The strains were recovered from cryovials, inoculated
in 5 or 10 mL of tryptic soybroth (TSB) + 2.5% NaCl (TSB+ Bioxon, Mexico City, Mexico), and incubated in a rotary
shaker (nb-205L N-BIOTEK Co., Ltd. Mexico City, Mexico) at 30 C for 24 hr.

2.2 | Biochemical profile

Methods described by Alsina and Blanch (1994) and Holt, Krieg, Sneath, Staley, and Williams (1994) were followed
for all strains. Tests included Gram staining, cell morphology, motility, swarming on TSA+ (Bioxon), growth on thiosul-
fate citrate bile salt sucrose agar (TCBS, Bioxon), growth on CHROMagar™ Vibrio (Monterey, Nuevo Leon, Mexico),
luminescence, sensitivity to the vibriostatic agent O/129, oxidase, catalase, O–F test, arginine dihydrolase, ornithine
decarboxylase, and lysine decarboxylase. Indole and sulfhydric acid production, gas from D-glucose, nitrate and
nitrite reduction, Voges–Proskauer, urease and gelatinase, β-galactosidase, and acetoin were also evaluated. Further
characterization was conducted using API® 20E and API™ ZYM (bioMerieux™, Mexico City, Mexico). All strains were
incubated at 30 C for 24–48 hr.

2.3 | Antibiotic susceptibility

The antimicrobial susceptibility test of the Vp strains was performed using a disk diffusion assay according to Bauer,
Kirby, Sherris, and Turck (1966). Antimicrobial agents were tested using a Bio-Rad (Hercules, CA) Sensi–Disk antimi-
crobial susceptibility test multidisk with the following antimicrobial agents: amikacin, ampicillin, cephalothin,
cefotaxime, ceftriaxone, chloramphenicol, gentamicin, netilmicin, nitrofurantoin, carbenicillin, kanamycin,
trimethoprim-sulfamethoxazole, and ciprofloxacin. The tests for nalidixic acid (Sanofi Diagnostics Pasteur, Mexico
City, Mexico), oxytetracycline (Sigma–Aldrich Co., Mexico City, Mexico), norfloxacin (Schering-Plough, Mexico City,
Mexico), nitrofurantoin (Sigma–Aldrich Co.), and florfenicol (Sigma–Aldrich Co.) were conducted separately using a
6.0-mm sterile Sensi-Disc (Oxoid, Basingstoke, England), according to Hindler (1992). The inoculum was adjusted to
an optical density at 600 nm (OD600) = 1.0 (approximately 108 bacterial cells/mL). Duplicates of the strains were
suspended in a sterile saline solution, spread on Mueller-Hinton agar (Difco, Mexico City, Mexico), and incubated for
24 hr at 30 C. Following the method of Hindler (1992), the minimum inhibitory concentrations (MICs) for the Vp
strains for enrofloxacin, norfloxacin, oxytetracycline (Sinbiotik International S.A de C.V, Mexico City, Mexico), and
florfenicol (Cheminova, Mexico City, Mexico) were estimated, using 7–10 concentrations in triplicate, ranging from
0.015 to 300 μg/mL.

2.4 | Growth kinetics: in vitro and in vivo assays

The OD600 of the bacterial suspension in duplicate was measured at 0, 1, 2, 3, 4, 5, 6, 7, and 24 hr p.i.. To determine
Vp growth in a nutrient-rich media (TSB+), their natural media (seawater), and seawater with shrimp (host), the Vp
AHPND+++ M0904 strain was used. Glass tubes with 10 mL of TSB+ (M0904/TSB) or 10 mL of filtered (0.45 μm)
and sterilized seawater (M0904/seawater) were inoculated in triplicate, including control tubes without bacteria. The
tubes were incubated overnight, and at 0, 0.5, 1, 2, 4, 8, 12, and 24 hr p.i., the total CFU/mL were estimated on
TCBS agar (Bioxon). An additional assay was performed using 2-L Erlenmeyer glass flasks filled with 300 mL of
0.45-μm filtered and sterilized seawater. The flasks were then aerated through hydrophobic 0.20-μm filters (Millex-
SOTO-RODRIGUEZ ET AL. 1005

FG Merck Mexico Estado de Mexico, Mexico). Three shrimp (weighing 1.1 g) were placed in each flask in triplicate
and fed a commercial 35% protein diet. Then, the flasks were inoculated with the bacterial suspension (M0904/
shrimp). The control group was not inoculated with bacteria. At 0 min, 20 min, and 40 min and at 1, 2, 4, 8, 12, and
24 hr p.i., 100 μL was plated on TCBS agar (Bioxon) to estimate the CFU/mL, and the green and yellow colonies were
counted.

2.5 | Growth kinetics for distinct environment parameters

To compare the tolerance of Vp strains with different virulence to environmental parameters of seawater, the strains
were grown at several temperatures, salinities, and pH values. Strains were incubated at 4, 21, 30, 37, and 44 C;
from 0.0 to 10.0% NaCl; and at pH values of 4, 5, 6, 7, 8, 9, and 10, for which the OD600 was measured from 0 to
24 hr. All experiments were conducted in triplicate and included control tubes without bacteria. Graphs of the incu-
bation time versus OD600 from the tested conditions were obtained, and the exponential growth of strains incubated
under all environmental parameters was analyzed from 0 to 20 hr corresponding to OD600 > 0.200. To compare the
growth kinetics between strains for the given conditions, the maximum specific growth rate (μmax/hr) was calculated
from the exponential growth phase consistent with Dalgaard and Koutsoumanis (2001) as follows:

Ln OD600 2 −Ln OD600 1 −1

μ max = h
t2 −t1

where Ln OD6002 and Ln OD6001 are the natural logarithm of the optical density corresponding to Time 2 and Time
1, respectively, and h is hours.

2.6 | Statistical analysis

All data were first analyzed to determine whether they were normally distributed, and then, an a posteriori analysis
was conducted. For the bacterial growth data, a nonparametric statistical analysis (Kruskal–Wallis) was performed,
and if significant differences were found, Dunn's post hoc test was conducted. A two-way analysis of variance was
used to analyze the effect of the factors (temperature, salinity, and pH) and strain (M0904+++, M0603++, M0905+,
and M0702−) on μmax/hr. If significant differences were found, Fisher's least significant difference post hoc test was
performed. All analyses were performed using a significance of .05.

3 | RESULTS

3.1 | Biochemical profile

Vp cells were motile, Gram-negative rods and did not swarm on TSA+. The colonies on TSA+ were smooth, circular,
nonluminescent, and did not have a pigment (Table S1). All colonies on the TCBS agar were green, but they had a dif-
ferent color phenotype on CHROMagar™ Vibrio. Regardless of the degree of virulence of the strain, the colonies
were mauve and navy blue, with the color also dependent on the incubation time. The strains had high metabolic
diversity in physiological and biochemical characteristics. All of the strains were oxidase, catalase, and lysine decar-
boxylase positive. They were also Vogues-Proskauer and urease negative, used fermentation/oxidation metabolism,
did not produce sulfhydric acid or gas from glucose, produced acetoin, and were D-glucose positive. Furthermore,
the Vp strains were inositol, melibiose, rhamnose, sorbitol, sucrose, and tryptophan deaminase negative. Only
AHPND+ strains were ornithine decarboxylase and L-arabinose positive. In addition, the AHPND− M0702 strain
was arginine dihydrolase positive and L-arabinose negative. The susceptibility to vibriostatic 0/129 (10 and 150 μg)
was variable.
1006 SOTO-RODRIGUEZ ET AL.

3.2 | Antibiotic susceptibility

Independent of the degree of virulence, all strains exhibited the largest zone of inhibition to nalidixic acid
(0.05 mg/mL, Table 1); however, the strains were resistant to ampicillin (0.50 mg/mL) and carbenicillin (5.0 mg/mL),
and Vp AHPND− was also resistant to florfenicol. AHPND+ strains showed low sensitivity to oxytetracycline
(8.00 mg/mL), whereas Vp AHPND− did not. For the MIC test, the most used antibiotics in Mexican shrimp farms
were selected. Again, independent of the degree of virulence, the lowest MIC was found for enrofloxacin (from 0.25
to 2.0 μg/mL, Table S2). The M0904+++ and M0905+ virulent strains exhibited the highest MICs for oxytetracycline,
reaching 300 μg/mL.

3.3 | Growth kinetics: in vitro and in vivo assays

Independent of the degree of virulence, strains began the exponential growth phase from 1 to 2 hr of incubation,
and after 7 hr, all Vp strains continued to grow (Figure 1a). However, the bacterial density did not correspond with
their virulence degree. For example, at 7 hr p.i., AHPND+ (M0904+++, M0603++, and M0905+) showed a similar
OD600 nm = 0.603, 0.647, 0.562, respectively; however, they caused different cumulative mortality (80, 57, and 36%,
respectively). Moreover, the OD600 nm = 0.513 of the AHPND− M0702 strain was slightly lower than the virulent
strains and caused 20% cumulative mortality (Figure 1b).
When the AHPND+++ M0904 strain was incubated on different media, the vibrio colonies on TCBS from water
samples of M0904/TSB+, M0904/seawater, M0904/shrimp, and seawater/shrimp (control group) were counted.
Green colonies showed exponential growth starting at 0.5 hr and exhibited similar growth in all treatments from 0 to
8 hr p.i. (Figure 2). However, there were significant differences between the treatments at 0.5 hr (p = .003, n = 3),
1 hr (p < .001, n = 3), 12 hr (p = .004, n = 3), and 24 hr p.i. (p = .001, n = 3). All treatments reached their highest

TABLE 1 Mean of inhibition halo (cm) of Vibrio parahaemolyticus strains

AHPND+ AHPND−

Antibiotic Concentration (mg/mL) M0904+++ M0603++ M0905+ M0702−

Nalidixic acid 0.05 2.6 2.3 2.5 2.8
Chloramphenicol 1.50 2.4 2.1 2.2 2.3
Cefotaxime 1.50 2.5 1.8 2.1 2.1
Nitrofurantoin 20.00 2.2 1.7 1.9 2.1
Cephalothin 1.50 1.9 1.8 2.0 2.1
Ciprofloxacin 0.25 1.9 1.4 1.8 1.9
Norfloxacin 0.50 1.8 1.3 1.8 1.8
Gentamicin 0.50 1.5 1.5 1.5 1.7
Amikacin 1.50 1.3 1.3 1.2 1.4
TSX 1.25 1.6 1.6 1.6 2.0
Florfenicol 0.30 0.9 1.0 1.1 0.0
Kanamicine 1.50 1.1 1.2 1.3 0.8
Netilmicin 1.50 1.1 1.2 1.1 1.3
Oxytetracycline 8.00 0.0 1.6 0.0 2.7
Ampicillin 0.50 0.0 0.0 0.0 0.0
Carbencillin 5.00 0.0 0.0 0.0 0.0

Note: AHPND+: Vibrio parahaemolyticus causing AHPND. AHPND−: V. parahaemolyticus non-causing AHPND.
Abbreviations: AHPND, acute hepatopancreatic necrosis disease; TSX, trimethoprim-sulphamethoxazole.
SOTO-RODRIGUEZ ET AL. 1007

(A) M0904 AHPND+++ M0603 AHPND++ M0905 AHPND+ M0702 AHPND-

0.8

0.7

0.6

0.5
OD (600 nm)

0.4

0.3

0.2

0.1

0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

(B) M0904 AHPND+++ M0603 AHPND++ M0905 AHPND+ M0702 AHPND-

100
90
Cumulative mortality (%)

80
70
60
50
40
30
20
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

F I G U R E 1 Growth kinetic of Vibrio parahaemolyticus with different virulence (AHPND+) and nonpathogenic
V. parahaemolyticus (AHPND−) (a) and (b) percentage of cumulative mortality of Litopenaeus vannamei inoculated
with the V. parahaemolyticus strains at 106 CFU/mL. OD, Optical density at 600 nm

density at 8 hr p.i., although the growth of M0904+++ in TSB was faster (9.95 Log), followed by M0904/seawater
(9.52 Log) and M0904/shrimp (9.53 Log). By the end of the experiment (24 hr), the inoculated shrimp reached 78%
cumulative mortality, and no mortality was observed in the control group. Moribund shrimp showed typical gross
signs of AHPND (atrophied and pale hepatopancreas, empty gut, lethargy, and expanded chromatophores).

3.4 | Growth kinetics at several temperatures, salinities, and pH levels

Independent of virulence, Vp strains incubated at several temperatures showed exponential growth starting at 2 hr,
although AHPND+ strains, in general, displayed slightly higher growth than AHPND− M0702 (Figure 3). However,
all strains grew at 4 C and at 30 and 37 C, with the AHPND+ strains reaching an OD600 between 0.7 and 0.8. In
contrast, the M0702− strain had an OD600 less than 0.7. In addition, only the most virulent strains (M0904+++ and
M0603++) grew at 44 C.
Independent of virulence, no strains grew at 0.0% NaCl, and all strains displayed exponential growth at 0.5%
NaCl; however, starting from 6.0% NaCl, the strains exhibited a delay in their growth (Figure 4). The AHPND−
M0702 strain showed a larger growth variability, and the strain did not grow at extreme conditions of 9.0 and
10.0% NaCl.
1008 SOTO-RODRIGUEZ ET AL.

M0904+++/TSB M0904+++/Seawater M0904+++/Shrimp

11.0
*
10.0 *
9.0
8.0
7.0
Log CFU/mL

6.0
5.0 **
4.0
3.0
2.0
1.0
0.0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time post infection (h)

F I G U R E 2 Green colonies' growth (CFU/mL) on TCBS of water samples from each treatment. Points are mean
values, and bars indicate SD. There are significant differences (p < .005, n = 3) between the treatments at 0.5, 1, 12,
and 24 hr postinoculation

(a) 4 21 30 37 44 (b) 4 21 30 37 44
0.9 0.9
0.8 0.8
0.7 0.7
0.6 0.6
OD (600 nm)

0.5
OD (600nm)

0.5
0.4 0.4
0.3 0.3
0.2 0.2

0.1 0.1

0.0 0.0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)

(c) 4 21 30 37 44 (d) 4 21 30 37 44
0.9 0.9

0.8 0.8

0.7 0.7

0.6 0.6
OD (600nm)

OD (600nm)

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)

F I G U R E 3 Growth kinetic of Vibrio parahaemolyticus with different degrees of virulence, at distinct temperatures.
(a) M0904+++; (b) M0603++; (c) M0905+; and (d) M0702− strains. Bars indicate SD (n = 3)
SOTO-RODRIGUEZ ET AL. 1009

0.0 0.5 3.0 4.0 0.0 0.5 3.0 4.0

(a) (b) 6.0 7.0 9.0 10.0
6.0 7.0 9.0 10.0
0.9
1
0.8
0.9
0.7 0.8
0.6 0.7

OD (600 nm)
OD (600nm)

0.5 0.6
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)

(c) 0.0 0.5 3.0 4.0 (d) 0 0.5 3.0 4.0

6.0 7.0 9.0 10.0 6.0 7.0 9.0 10.0
0.9
0.9
0.8 0.8
0.7 0.7
0.6 0.6
OD (600nm)
OD (600 nm)

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)

F I G U R E 4 Growth kinetic of Vibrio parahaemolyticus with different degrees of virulence, at distinct salinities.
(a) M0904+++; (b) M0603++; (c) M0905+, and (d) M0702− strains. Bars indicate SD (n = 3)

All strains grew better at pH 8–9, but no strains grew at pH 4. The AHPND+ strains displayed higher growth
(OD600 was over 0.8) and less variability than the M0702 strain (Figure 5). It is remarkable that this strain showed a
delayed time to growth until 9 hr and also started to grow at pH 5 after 20 hr.
To compare the growth rate between Vp strains and to relate this rate to their degree of virulence, the μmax was
calculated for each strain incubated at several temperatures, salinities, and pH, and then, the significant differences
were estimated. Regarding temperature, the analysis of μmax/hr data showed a significant effect of the interaction
temperature*strain (p < .05). No significant differences were found at 4 and 21 C (p > .05, Figure S1), but at 30 and
37 C, the μmax/hr values of the AHPND+ strains (M0904+++, M0603++, and M0905+) were significantly lower
(p < .05) than the AHPND− M0702 strain. At 44 C, the M0603 strain presented the highest values of μmax/hr,
followed by M0904.
The analysis of μmax/hr data at different salinities showed a significant effect for the interaction salinity*strain
(p < .05). The AHPND+ and AHPND− strains displayed large fluctuations in salinities (Figure S2). The AHPND− strain
had the highest μmax/hr compared with AHPND+ strains (p < .05), followed by AHPND+ M0603++, M0904+++, and
M0905+ strains. Among salinities, the highest μmax/hr values were recorded at 4.0 and 0.5% NaCl (p < .05).
Results of the analysis of μmax/hr data at different pH values were also significant for the interaction pH*strain
(p < .05). The AHPND− strain (M0702) had the lowest μmax/hr values at most of pH levels, except pH 9 and
1010 SOTO-RODRIGUEZ ET AL.

(a) 4 5 6 7 (b) 4 5 6 7
8 9 10 8 9 10
0.9 0.9
0.8 0.8

0.7 0.7

0.6 0.6
OD (600 nm)

OD (6oo nm)
0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h) Time (h)

(c) 4 5 6 7 (d) 4 5 6 7
8 9 10 8 9 10
0.9 0.9

0.8 0.8

0.7 0.7

0.6 0.6
OD (600nm)
OD (600 nm)

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0.0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)

F I G U R E 5 Growth kinetic of Vibrio parahaemolyticus with different degree of virulence, at distinct pH values.
(a) M0904+++; (b) M0603++; (c) M0905+; and (d) M0702− strains. Bars indicate SD (n = 3)
10 (p < .05, Figure S3). The strains with the overall highest values were AHPND+ M0603++, M0904+++, and
M0905+, with no significant differences among them (p > .05).

4 | DISCUSSION

Most scientific papers on Vp that cause AHPND have focused on the isolation, identification, molecular detection
methods, and whole genome of strains. To date, little information has been reported on phenotypic characteristics
and antibiotic sensitivity assays. There were no distinguishable phenotypic traits between Vp AHPND+ and AHPND
− strains regarding the degree of virulence. The physiological and morphological traits showed variability between
strains in the use of substrates or enzyme production. Williams et al. (2017) found that a pathogenic Vp AHPND
strain had significantly better growth and utilized nutrient sources more efficiently than the other two clinical Vp
strains. This high metabolic diversity allowed the bacteria to live in a wide range of aquatic environmental conditions.
Moreover, this is the first report of the different phenotype colors of Vp isolated from shrimp affected with AHPND.
In this study, depending on the incubation time, the colony growth of Vp strains on CHROMagar™ Vibrio demon-
strated the typical mauve and navy blue color independent of the virulence. Therefore, it is impractical to use
CHROMagar™ Vibrio to isolate and identify AHPND+ isolates from shrimp systems because it may miss pathogenic
isolates. Therefore, we recommend the use of polymerase chain reaction techniques to properly identify isolates.
SOTO-RODRIGUEZ ET AL. 1011

Traditionally, vibrios have been considered highly susceptible to virtually all antimicrobials. All Vp strains from
this study were susceptible to most of the antibiotics tested, particularly to nalidixic acid, but Vp was resistant to
ampicillin and carbenicillin, previously reported for Vp from farmed shrimp affected with AHPND (Kongrueng et al.,
2015; Lai et al., 2015). In contrast, the results of the disk diffusion test with Vp AHPND+ showed that the 2S01
strain was only susceptible to florfenicol, and this susceptibility level was intermediate to that of ceftriaxone,
nitrofurantoin, and norfloxacin (Dong et al., 2017). In this study, independent of the degree of virulence, the lowest
MIC was for enrofloxacin, and AHPND+ strains were the least sensitive to oxytetracycline. Vibrios that are patho-
genic to shrimp showed sensitivity to the antibiotics traditionally used in Mexican shrimp culture to control vibrio
infections (Soto-Rodriguez, Gomez-Gil, Lozano, & Roque, 2010). In addition, the genomes of Vp AHPND+ strains iso-
lated from Mexico and China harbor several tet genes, and the gene encoding for β-lactam resistant is on chromo-
somes and plasmids (Dong et al., 2017; Fu, Wang, Tian, Wei, & Liu, 2018; Han, Mohney, Tang, Pantoja, & Lightner,
2015). The origin of these antibiotic resistance genes may stem from the massive use of antibiotics in aquaculture
facilities since the 1980s (Cabello, 2006). Vp AHPND+ does not cause a typical vibriosis infection, but rather, the
delivery of PirA and PirB toxins in the water produces an acute intoxication in shrimp, such that most antibiotics fail
to control AHPND in shrimp farms. The increased resistance to these antibiotics is a serious problem for shrimp
aquaculture.
In this study, no clear relation was observed between the bacterial density, measured as optical density, and the
strain virulence. The virulence of Vp causing AHPND possibly depends on the concentration of the PirAB toxin
(Tinwongger et al., 2016) or intrinsic factors of the strain (Feng, Liu, Wang, Sun, & Pan, 2017).
To understand Vp AHPND+ survival, microcosm assays were conducted under sterile conditions and in contact
with shrimp. The yellow colonies on TCBS were mainly from the bacterial community (native and transient) of the
organism's digestive tract. Green colony growth at the end of the experiment (attributable to the M0904+++ strain)
was better in TSB+ compared with seawater and seawater+shrimp. Vp M0904+++ reached the highest bacterial den-
sity (Log 9) because TSB+ is a medium rich in nutrients. In parallel experiments (data not shown), we found that
tryptone and glucose were the main components of the TSB used by the bacteria. However, from 2 to 8 hr, the bac-
terial growth was similar among treatments, possibly because the bacteria take advantage of their metabolic versatil-
ity to survive.
It is important to study the relationship between environmental conditions and the survival of Vp AHPND+
strains. Wendling, Batista, and Wegner (2014) reported that the pathogenicity of vibrios is tightly related to seawater
temperature. Furthermore, environmental factors, such as temperature, salinity, and pH of the culture water, might
be involved in the disease outbreaks (Prayitno & Latchford, 1995; Randa, Polz, & Lim, 2004).
To compare the kinetic growth between strains, the most important parameter is μmax/hr, calculated from the
inflection of the slope of the growth curve in the exponential phase of the bacteria (Zwietering, Jongenburger,
Rombouts, & Riet, 1990). Few studies calculate the μmax/hr of Vp incubated under diverse environmental parameters.
In this study, the μmax/hr values obtained were consistent with the optical density for each incubation condition.
Independent of the degree of virulence, no Vp strains grew at 4 C, 0% NaCl, and pH 4, so there were no relationship
between pathogenicity and these environmental parameters. The minimum growth rate of clinical Vp was 8.3 C
(Miles, Ross, Olley, & McMeekin, 1997). Vp growth, as for most vibrios, is dependent on temperature, and the organ-
ism can grow well between 15 and 30 C (Thompson et al., 2004); in temperatures of under 15 C, Vp displayed
decline the growth profile (Wang et al., 2018). In this study, temperature was strongly associated with vibrio densi-
ties; all strains grew better at 30 and 37 C, but only AHPND+ strains reached the highest density, over the infective
threshold, and only these strains grow at 44 C. Johnson et al. (2010) suggest that human pathogenic Vp responds
differently to temperature than non-pathogenic Vp. Liu, Liu, Pan, Xie, and Zhao (2016) reported that the maximum
specific growth rate of 50 Vp isolates from different sources was between 0.02 and 0.44/hr, incubated at 2.5% NaCl
and 37 C; meanwhile, in this study, the μmax/hr of the Vp strains at similar culture conditions were between 0.688
and 1.099/hr.
1012 SOTO-RODRIGUEZ ET AL.

In this study, at 0.5% NaCl, strains started their exponential phase of growth and displayed a lag phase propor-
tional to the NaCl concentration. Vp is a moderate halophile that requires a minimum of 0.086 M (0.5%) NaCl for
growth and inhabits marine, brackish, and estuarine waters worldwide. The higher μmax/hr for AHPND+ ranged from
0.5 to 4% NaCl and for AHPND− was at 5% NaCl, but only virulent Vp grew at 10% NaCl. Culture media containing
lower or higher concentrations of NaCl resulted in slower Vp generation times (Beuchat, 1975). The mean μmax/hr of
Vp strains incubated at 30 C and 3.0% NaCl were from 0.48 to 0.609/hr, 10 times greater than the μmax
(0.05–0.035/hr) reported by Liu et al. (2016). Johnson et al. (2010) found a nonlinear relationship when salinities vary
over a sufficiently wide range such as in this study. Fluctuations in salinity pose a constant challenge to the osmotic
stress response of the organism, which, as observed in this study, can grow in the range of 1–9% NaCl (Whitaker
et al., 2010). Vp isolates from shrimp culture also showed a positive correlation between salinity and bacterial density
(Lekshmy, Mohandas, & Radhakrishnan, 2014), which suggests that salinity is an important growth factor for the bac-
teria. Prayitno and Latchford (1995) reported that exposure of the pathogen V. harveyi BP04 strain to low salinities
(10, 15 g/L) for 12 hr before use in challenge experiments with P. monodon larvae enhanced shrimp mortalities.
Because of its utilization of a wide variety of energy sources, the maximum bacterial growth rate indicates that the
environment, such as temperature and salinity of the seawater, can affect the growth of Vp (Liu et al., 2016).
Research regarding the relationship between pH and Vp growth is scarce. In this study, no Vp strains grew at
4, and all strains grew better at pH 8–9, independent of the degree of virulence, and only AHPND+ strains grew over
pH 5; however, these strains reached higher bacterial densities. The AHPND− strain was more sensitive to pH than
the AHPND+ strains. Whitaker et al. (2010) determined whether growth in differing NaCl concentrations alters the
susceptibility of clinical Vp O3:K6 to other environmental stresses. The authors found that growth of Vp at 3% NaCl
protects the organism from low organic and inorganic pH values. Vanderzant and Nickelson (1972) also found that
Vp inoculated on the supernatant of shrimp homogenate was very sensitive to pH values below 6.0. Beuchat (1973)
found that Vp strains did not grow well at 5 C and pH 7.0, but when Vp was cultivated over pH 7.0, strains grew at
5 and 9 C. Exposure of the V. harveyi BP04 strain to pH 5.5 reduced their virulence to shrimp larvae (Prayitno &
Latchford, 1995).
In this study, no significant differences were found in growth conditions among Vp strains with different degrees
of virulence, although wide growth fluctuations in the AHPND− strain were observed. Liu et al. (2016) reported that
considerable variability among Vp strains has been demonstrated, with more stressful conditions (e.g., suboptimal
temperatures and salinities) resulting in more variable growth responses. The wide tolerance of the Vp strains tested
here could be part of ecological fitness, a set of metabolic characteristics that enhance the survival, spread, and/or
transmission of microorganisms within an ecological niche (Hacker & Carniel, 2001), which may enable them to adapt
and survive in almost all marine–estuarine environments where shrimp farms are located. The growth environments
and the bacterial genotypes affect the strain growth variability of Vp (Liu et al., 2016). Vp survival in different envi-
ronments follows responses to specific conditions, such as different carbon and energy sources, water pH, and tem-
perature, including starvation. Chonsin et al. (2016) reported that Vp AHPND+ strains acquired AHPND virulence
genes, such as the VpA1 virulent plasmid, via horizontal gene transfer, which converts nonpathogenic Vp into viru-
lent strains. Thus, different Vp strains are expected to have variations in both chromosomes and, therefore, a differ-
ent phenotypic profile; however, the AHPND− strain was more sensitive to the extreme conditions tested.
Environmental factors can stimulate horizontal gene transfer mechanisms in bacteria. The AHPND+ strains grew
better under stress conditions, which may indicate a risk of AHPND outbreaks and disease dispersion to tropical
zones. Future research must evaluate the ability of the bacterial cell to adapt and relate this adaptation to PirAB
toxin production. As the occurrence of AHPND in shrimp is dependent on the bacterial density (Soto-Rodriguez
et al., 2015), and the optimal growth conditions of Vp AHPND+ are prevalent in the grow out ponds (30 C, 0.5 to
4% NaCl, pH 8–9), it would be desirable to prevent the stocking of shrimp postlarvae under these environmental
conditions. Studies under laboratory conditions control some variables present in nature; however, it allows us to
identify potential variables that could affect the infection process of AHPND during the shrimp culture. So, caution
SOTO-RODRIGUEZ ET AL. 1013

must be taken because of the limits of in vitro assays. In future work, chitin substrate must be added to bacterial cul-
ture during experiments to simulate a shrimp farm condition.

ACKNOWLEDGMENTS

This work was supported by the National Fishery and Aquaculture Institute (INAPESCA). The local shrimp postlarval
producer Fitmar Co. provided the organisms used in the bioassays. We thank H. López, K. Enciso, and C. Aguayo for
technical assistance.

CONF LICT OF IN TE RE ST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

S.A.S. and R.L. were responsible for the experimental design, data analysis, and in vivo assay; D.A.P. carried out the
statistical analysis; and C.B. and K.G.R. were responsible for the biochemical analysis, antibiotic susceptibility test,
and growth kinetics.

ORCID

Sonia A. Soto-Rodriguez https://orcid.org/0000-0003-0760-4029

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SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of this
article.

How to cite this article: Soto-Rodriguez SA, Lozano-Olvera R, Palacios-Gonzalez DA, Bolan-Mejia C,
Rendon-Aguilar KG. Characterization and growth conditions of Vibrio parahaemolyticus strains with different
virulence degrees that cause acute hepatopancreatic necrosis disease in Litopenaeus vannamei. J World
Aquacult Soc. 2019;50:1002–1015. https://doi.org/10.1111/jwas.12617