Marine Pollution Bulletin 160 (2020) 111551

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Tropical shrimp aquaculture farms harbour pathogenic Vibrio T

parahaemolyticus with high genetic diversity and Carbapenam resistance
Sreejith V. Narayanana,b, , Toms C. Josepha, Shaheer Peeralila, Reshmi Koombankallila,
⁎

Murugadas Vaiyapuria, Mukteswar P. Mothadakaa, Kuttanapilly V. Lalithaa,1

a
Microbiology Fermentation and Biotechnology Division, ICAR-Central Institute of Fisheries Technology, Willingdon Island, Cochin 682029, Kerala, India
b
Cochin University of Science and Technology, Kalamassery, Cochin 682022, Kerala, India

ARTICLE INFO ABSTRACT

Keywords: In characterization of food borne pathogens from the environment, assessment of virulence, genetic diversity and
Vibrio parahaemolyticus AMR are essential preludes to formulate preventive strategies and to combat the spread. This study aimed to
Aquaculture identify and characterize pathogenic Vibrio parahaemolyticus in the coastal aquaculture farms of Kerala, India.
Tdh Twenty-seven β-haemolytic V. parahaemolyticus were isolated from 7 out of 40 farms studied. Among the 27
Trh
isolates, 15 possessed the tdh gene and 4 had trh. ERIC PCR and PFGE illustrated the presence of pathogenic
Genetic diversity
isolates that shared genetic similarity with clinical strains. One pathogenic isolate was identified to be multidrug
Antimicrobial resistance (AMR)
resistant (MDR) and 59% exhibited a MAR index of 0.2 or above. Seventy four percent of the pathogenic isolates
were ESBL producers and 3.7% of them were carbapenemase producers phenotypically. This asks for adoption of
control measures during farming to prevent the transmission of pathogenic V. parahaemolyticus to the en­
vironment and food chain.

1. Introduction detrimental to the seafood industry (Letchumanan et al., 2014; Shaheer

Seafood is considered as an alternative source of protein for the
growing human population and aquaculture is the fastest growing food
producing sector in the world contributing 46.2% of the global pro­
duction of capture fisheries (FAO, 2018). The FAO recognizes the
contribution of aquaculture to food and nutritional security and pro­
motes sustainable aquaculture development to meet the 2030 agenda of
the UN SDG (sustainable development goals). However, the farm
management techniques adopted to ensure better productivity has re­
sulted in serious environmental problems and development of anti­
microbial resistant pathogens (Cabello et al., 2013). As the coastal
farms depend on brackish water, these pathogens could easily spread
into surrounding environment and to the marine world. Maintaining
the microbial quality of aquaculture farms is vital for sustainable pro­
duction and public health.
Due to its natural presence in marine and estuarine environments
Vibrio parahaemolyticus is often found associated with the seafood
produced in aquaculture. The presence of pathogenic V. para­
haemolyticus in seafood intended for human consumption can cause
significant health issues like gastrointestinal illness and is also
et al., 2019). Apart from the implications on human health, V. para­
haemolyticus is also known to infect aquatic animals including fish and
shellfish, especially shrimp where it causes AHPND or EMS resulting in
huge economic losses (Tran et al., 2013). Besides, V. parahaemolyticus is
also a cause of Vibriosis responsible for the mortality of cultured shrimp
worldwide (Mastan and Begum, 2016).
Two major virulence factors responsible for the pathogenicity of V.
parahaemolyticus in humans are the thermostable direct haemolysin
(TDH) and the TDH-related haemolysin (TRH) (Letchumanan et al.,
2014). Both the TDH and TRH have similar virulence mechanisms and
share 70% sequence homology (Ceccarelli et al., 2013; Wang et al.,
2015). The presence of either of these genes has been reported to be
responsible for the haemolysis of erythrocytes by V. parahaemolyticus
called as the Kanagawa phenomenon (KP) (Ceccarelli et al., 2013;
Wang et al., 2015). Mostly these virulence genes are detected only in
clinical isolates and are very rarely seen in the environmental isolates
(Deepanjali et al., 2005; Letchumanan et al., 2014). However, clinical
strains negative for tdh and trh genes are also widely reported
(Raghunath, 2014; Ronholm et al., 2016).
The study of V. parahaemolyticus gained much significance after the

Corresponding author at: Microbiology Fermentation and Biotechnology Division, ICAR-Central Institute of Fisheries Technology, Willingdon Island, Cochin
⁎

682029, Kerala, India.

E-mail address: sreejithvnz@gmail.com (S.V. Narayanan).
1
Saparya, Vennala, Cochin 682028, Kerala, India.

https://doi.org/10.1016/j.marpolbul.2020.111551
Received 7 May 2020; Received in revised form 5 August 2020; Accepted 5 August 2020
Available online 15 August 2020
0025-326X/ © 2020 Elsevier Ltd. All rights reserved.
S.V. Narayanan, et al. Marine Pollution Bulletin 160 (2020) 111551

Table 1
Details of aquaculture farms screened for pathogenic V. parahaemolyticus.
Sl. No Place Geographical coordinates Period Culture No of farms No of toxR positive isolates

1 Aroor 9°52′05.9″N 76°17′48.4″E January 2015 P. monodon 6 79

2 Pattanakkad 9°49′41.3″N 76°17′13.4″E January 2015 P. monodon 2 22
3 Thuravoor 9°47′0.24″N 76°17′15″E April 2015 P. monodon 2 43
4 Kodungallur4, pullut 10°15′52.5″N 76°12′28.14″E April 2015 L. vannamei 2 40
5 Thaikatussery 9°46′36.16″N 76°20′0.0096″E May 2015 P. monodon 1 16
6 Ponnamveli 9°44′29.58″N 76°19′51.25″E June 2015 P. monodon 4 63
7 Kodungallur2 10°14′39.78″N 76°13′35.07″E June 2015 L. vannamei 1 31
8 Panagadu 9°54′40.1″N 76°18′56.0″E June 2015 P. monodon 4 41
9 Kodungallur3 10°13′44.2″N 76°12′31.6″E July 2015 P. monodon 4 40
10 Kodungallur1 9°14′34.60″N 76°12′39.75″E September 2015 L. vannamei 3 56
11 Kumbalangi 9°52′25.0″N 76°17′11.6″E December 2015 P. monodon 1 25
12 Ezhupunna 9°49′29.5″N 76°17′15.1″E January 2016 P. monodon 3 40
13 Thrissur, vellukara 10°16′41.44″N 76°11′17.96″E February 2016 L. vannamei 2 28
14 Kandakadavu 9°51′21.3″N 76°16′06.9″E March 2016 P. monodon 1 9
15 Chellanam 9°51′15.9″N 76°16′10.4″E April 2016 P. monodon 2 24
16 Kannamaly 9°52′1.87″N 76°16′1.41″E April 2016 P. monodon 2 33

emergence of O3:K6 clone in Kolkata, India in 1996, which later spread
throughout the world to cause the pandemic. Later, serovariants of this
strain, O4:K68, O1:K25 and O1:KUT, that followed a spreading pattern
similar to that of O3:K6 were identified ( Matsumoto et al., 2000). The
unique sequences in these strains were identified as markers in various
PCR methods employed for the identification of pandemic strains. This
include, the pandemic strain specific toxRS sequence targeted in GS
PCR, a portion of the gene that codes for a hypothetical protein similar
to the Mn2+ and Fe2+ transporter of V. vulnificus targeted in PGS PCR
and a modification in the C terminal amino acid sequence of histone
like DNA binding protein HU-α which is targeted in the HU-α PCR (
Matsumoto et al., 2000; Okura et al., 2004; Williams et al., 2004).
Besides these PCR based techniques, the genetic construction of the V.
parahaemolyticus isolates can be compared with pandemic strains using
the molecular subtyping methods which enables the identification of
isolates that share genetic similarity with the pandemic strains (van
Belkum et al., 2007). Molecular subtyping is an important tool in epi­
demiological investigations and is helpful in recognizing outbreaks,
delineating the source of infection, identifying virulent strains and to
study the genetic diversity (Olive and Bean, 1999). Among the mole­
cular subtyping systems commonly used for V. parahaemolyticus the
ERIC-PCR, PFGE and MLST were identified to have better dis­
criminative index (W. Chen et al., 2012; Shaaly et al., 2005).
V. parahaemolyticus was traditionally known to be susceptible to
most of the antibiotics. The haphazard use of antibiotics in the aqua­
culture farms has resulted in the development of multidrug resistant
pathogens some of which are shown to be capable of interspecies
transfer of antimicrobial resistant genes (Cabello et al., 2013; Lee et al.,
2018; Loo et al., 2020). The Vibrio infections are usually treated with
quinolones, tetracyclines and extended-spectrum cephalosporins
(Elmahdi et al., 2016; Wong et al., 2015). However, in the past two
decades multidrug resistant and multiple antibiotic resistant V. para­
haemolyticus isolates have been identified from various sources and
recently V. parahaemolyticus strains resistant to extended-spectrum ce­
phalosporins, tetracyclines and carbapenams were also reported
(Elmahdi et al., 2016; Lee et al., 2018; Letchumanan et al., 2019; Wong
et al., 2012; Zhao et al., 2018). Industrial effluents and aquaculture has
been reported to be the major contributors of antibiotic resistance
genes. The antibiotics disseminated into the environment from various
sources, persists in their active form bound to the sediments of aquatic
environments causing contamination of animals and thereby the food
chain during harvest (Loo et al., 2020).
The identification and characterization of V. parahaemolyticus in
aquaculture farms is essential to determine the prevalence of strains
with pathogenic and pandemic potential and also to formulate the
preventive strategies to check the transmission of pathogenic strains
into the food chain. In this study, we have examined the presence of
pathogenic V. parahaemolyticus in the coastal shrimp aquaculture farms
of central Kerala and performed a comprehensive characterization of
the isolates to determine their virulence properties, pandemic potential,
genetic diversity and antibiotic susceptibility pattern.
2. Materials and methods
2.1. Reference strains used
V. parahaemolyticus ATCC 17802, Escherichia coli ATCC 25922,
clinical isolates of V. parahaemolyticus from National Institute of
Cholera and Enteric Diseases (NICED), Kolkata, NICED.VP458,
NICED.VP459 and NICED.VP460 were used as controls for various
parameters tested in this study. Salmonella Braenderup H9812 was used
as a standard marker in PFGE.
2.2. Sample collection
One hundred samples comprising of water, sediment, and shrimp
were collected from 40 aquaculture farms rearing Penaeus monodon and
Litopenaeus vannamei, located in 3 districts of Kerala, along the south-
west coast of the Indian subcontinent (Table 1). The farms were se­
lected from a list of farms meeting predefined criteria, using RANDB­
ETWEEN function in Microsoft Excel. Shrimp samples were collected
only if the animals were of considerable size (at least 40–45 count) at
the time of sampling, as collection of small animals would affect the
productivity of farm. Shrimp samples were collected in sterile poly­
ethylene bags and transported to the laboratory with ice. Water samples
were collected by inverting sterile 1 litre polystyrene bottles into the
water to about 30–40 cm below the surface. Sediment samples were
scooped out into sterile polyethylene bags. Water and sediment samples
were collected from water inlet (feeder canal) area, the four corners and
the centre. Samples collected from all the collection points were pooled
before analysis and tests were initiated within 2–3 h. Replicate analysis
was performed for all the parameters tested in this study.
2.3. Isolation and identification of V. parahaemolyticus
The procedure described by Blanco-Abad et al. (2009) was followed
with minor modification for the selective enrichment of V. para­
haemolyticus in aquaculture samples. Unless specified, all media used
for the identification of V. parahaemolyticus contained 3% NaCl. All
samples were enriched in Alkaline Peptone Water (APW) (pH 8.5–9.0)
and incubated for 18 h at 37 °C. After incubation, 1 mlitre from the
surface of this enrichment broth was taken and added to Salt Polymyxin
Broth (SPB) containing 250 U of polymyxin and incubated further for
18 h. After incubation, 1 ml from SPB was serially diluted up to 10−4

2
S.V. Narayanan, et al.
dilutions and 0.1 ml from 10−2, 10−3 and 10−4 dilutions were surface
plated on to Thiosulphate Citrate Bile salts Sucrose (TCBS) Agar (BD,
USA) and incubated overnight at 37 °C. Characteristic dark centered
blue-green colonies were picked on to TSA (BD, USA) slants and pur­
ified by streaking on TSA plates. The initial isolates were subjected to
further biochemical tests such as Oxidase, Catalase, Salt tolerance
(growth in 0%, 3%, 6%, 8% and 10% NaCl) and O/129 susceptibility
for presumptive identification of V. parahaemolyticus. V. para­
haemolyticus ATCC 17802 was used as a positive control for the bio­
chemical tests.
2.4. Molecular confirmation
A total of 689 presumptive V. parahaemolyticus isolates were sub­
jected to toxR gene amplification using the primers described by Kim
et al. (1999). Each of the 25μl reactions consisted of 0.5 pmol/μl of
primers, 200 μM of dNTPs (Thermo Fisher Scientific, USA), 2 mM
MgCl2 (Thermo Fisher Scientific, USA), 1.25 units Taq DNA polymerase
(Thermo Fisher Scientific, USA) and 2 μl of crude DNA extract as
template. The PCR was performed in an Veriti™ 96-Well Thermal Cycler
(Thermo Fisher Scientific, USA) and the protocol consisted of 30 cycles
with initial denaturation at 94 °C for 5 min, denaturation at 94 °C for
1 min, annealing at 63 °C for 1 min, extension at 72 °C for 1 min and a
final extension at 72 °C for 7 min. V. parahaemolyticus ATCC 17802 was
used as a positive control.
2.5. Identification of pathogenic isolates
All the 590 isolates confirmed to be V. parahaemolyticus by toxR
gene amplification was tested for Kanagawa phenomenon (KP) in
Wagatsuma agar as described by Miyamoto et al. (1969). Briefly, 5 ml
of freshly drawn human blood was centrifuged at 5000 g for 5 min to
recover the erythrocytes which were washed twice in normal saline and
made up to a final volume of 5 ml. The washed erythrocytes were added
to the basal Wagatsuma medium and plated. Overnight culture from
TSA slants were used to inoculate the Wagatsuma agar plates and in­
cubated at 37 °C and observed for β-haemolysis after 24 and 48 h. V.
parahaemolyticus strain NICED.VP459, belonging to the O3:K6 serotype
was used as positive control.
2.6. Detection of virulence genes and pandemic potential
All the 27 KP positive V. parahaemolyticus isolates were subjected to
PCR amplifications for the identification of virulent and pandemic
strains. The tdh and trh gene amplifications were employed for the
identification of pathogenic isolates and GS-PCR, PGS-PCR, and HU-α
PCR were used to check the pandemic potential of the pathogenic iso­
lates. The primers used for various PCR amplifications are shown in
Table 2. For the tdh (Gutierrez West et al., 2013 primers), trh and PGS-
PCR amplifications, 0.5pmols/μl primer, 1.5 mM MgCl2 (Thermo Fisher
Scientific, USA) and 1.25 units Taq DNA polymerase (Thermo Fisher
Scientific, USA) were used. 1 pmol/μl primer, 2 mM MgCl2 and 1.25
units Taq DNA polymerase (Thermo Fisher Scientific, USA) was used for
tdh gene amplifications with the Bej et al. (1999) primers. The GS-PCR
amplifications were performed with 0.2 pmols/μl primer, 1.5 mM
MgCl2 (Thermo Fisher Scientific, USA) and 1.25 units Taq DNA poly­
merase (Thermo Fisher Scientific, USA). V. parahaemolyticus strain NI­
CED.VP459 was used as a positive control during detection of virulence
genes and pandemic potential of the isolates.
2.6.1. Cloning and sequencing of virulence genes
The uncertain amplification bands produced by three pathogenic
isolates during the amplification of tdh gene using the Bej et al. (1999)
primers were cloned into the vector pTZ57R/T and transformed to E.
coli JM109 using InsTAclone PCR Cloning Kit (Thermo Fisher Scientific,
USA) (Soleimani et al., 2012). The insert in the vector was sequenced
3
Marine Pollution Bulletin 160 (2020) 111551
using the Sanger sequencing technology (Scigenome Pvt. Ltd., India).
BLAST algorithm was used for similarity searches in the GenBank Se­
quence Database.
2.7. Multiplex O serotyping
The O serotypes of the pathogenic V. parahaemolyticus isolates were
identified using multiplex PCR method described by M. Chen et al.
(2012). Briefly, two multiplex PCR amplifications were set up for each
isolate where the PCR group 1 targets the serotypes O1, O2, O4, O5, O6,
and O10, and the PCR group 2 targets the serotypes O3/O13, O7, O8,
O9, O11, and O12. Each of the 25 μL reaction mixture contained
2.5 mM MgCl2, 0.2 mM dNTP, 1.5 units Taq DNA polymerase (Thermo
Fisher Scientific, USA) and the respective primer concentrations for
each set of primers as mentioned in the reference. All amplifications
were carried out in a Veriti™ 96-Well Thermal Cycler (Thermo Fisher
Scientific, USA) and the protocol consisted of 30 cycles denaturation at
95 °C for 30 s, annealing at 60 °C for 45 s and extension at 72 °C for
1 min. Laboratory strains with known serotypes were used as positive
control.
2.8. Antibiotic susceptibility
The antibiotic susceptibility of 27 pathogenic V. parahaemolyticus
isolates was determined using disc diffusion method in Mueller-Hinton
agar (BD, USA) plates supplemented with 2% NaCl. Twelve antibiotics
were tested and the results were interpreted as Sensitive (S),
Intermediate (I) and Resistant (R) based on Clinical and Laboratory
Standards Institute (CLSI) breakpoints for Vibrio sp. with the help of
WHONET software (O'Brien and Stelling, 1995). The antibiotics tested
were Ampicillin (10 μg), Amoxycillin-Clavulanate (20/10 μg), Cefepime
(30 μg), Cefotaxime (30 μg), Cefoxitin (30 μg), Ceftazidime (30 μg),
Meropenem (10 μg), Gentamicin (10 μg), Tetracycline (30 μg), Cipro­
floxacin (5 μg), Trimethoprim/Sulfamethoxazole (1.25/23.75 μg) and
Chloramphenicol (30 μg). The Multiple Antibiotic Resistance (MAR)
index for the isolates were calculated as described previously
(Krumperman, 1983). E. coli ATCC 25922 strain with known antibiotic
sensitivity pattern was used as a control.
2.9. Molecular subtyping
2.9.1. ERIC PCR
The ERIC-PCR was performed using the protocol described by Wong
and Lin (2001). All the 27 pathogenic V. parahaemolyticus isolated in
this study and the reference strains were subjected to ERIC-PCR. Each
reaction (50 μl) consists of 1 pmol/μl primers (ERIC1R, 5-ATGTAAGC
TCCTGGGGATTCAC-3, and ERIC2, 5-AAGTAAGTGACTGGGGTGAGCG-
3), 1.5 mM MgCl2 and 1.25 units DreamTaq DNA polymerase (Ther­
mofisher Scientific, USA). The amplification conditions used for ERIC-
PCR include an initial denaturation at 95 °C for 5 min, denaturation at
95 °C for 30 s, annealing at 52 °C for 1 min, extension at 72 °C for 5 min
and a final extension at 72 °C for 10 min. The PCR products were se­
parated on a 2% agarose gel and imaged using G:Box Imaging System
(Syngene).
2.9.2. PFGE
The PFGE typing of the 27 pathogenic V. parahaemolyticus isolates
and the reference strains was performed in line with the standard op­
erating procedure for PFGE of V. cholerae and V. parahaemolyticus from
the Pulsenet (https://www.cdc.gov/pulsenet). The genomic DNAs of V.
parahaemolyticus isolates were digested with the SfiI (New England
Biolabs, USA) restriction enzyme and the digestion of Salmonella
Braenderup H9812 was performed with XbaI (New England Biolabs,
USA). The digested plugs were loaded in a 1% agarose gel (Bio-Rad
Certified™ Megabase Agarose, USA) and the fragments separated in
Chef Mapper Pulsed Field Electrophoresis System (Bio-Rad, USA) for
S.V. Narayanan, et al.
Table 2
Primers and PCR conditions used for characterizing V. parahaemolyticus isolated from aquaculture farms
Gene Sequence Amplicon (bp)
toxR 5′-GTCTTCTGACGCAATCGTTG-3′ (forward) 368
5′-ATACGAGTGGTTGCTGTCATG-3′ (reverse)
tdh 5′-GTAAAGGTCTCTGACTTTTGGAC -3′ (forward) 269
5′-TGGAATAGAACCTTCATCTTCACC-3′ (reverse)
5′-CTGTCCCTTTTCCTGCCCCCG-3′ (forward) 245
5′-AGCCAGACACCGCTGCCATTG-3′ (reverse)
trh 5′-GGCTCAAAATGGTTAAGCG-3′ (forward) 250
5′-CATTTCCGCTCTCATATGC-3′ (reverse)
GS PCR 5′-TAATGAGGTAGAAACA-3′ (forward) 651
5′-ACGTAACGGGCCTACA-3′ (reverse)
PGS PCR 5′-TTCGTTTCGCGCCACAACT-3′ (forward) 235
5′-TGCGGTGATTATTCGCGTCT-3′ (reverse)
HU-α 5′-CGATAACCTATGAGAAGGGAAACC-3′ (forward) 474
5′-TAGTAAGGAAGAATTGATTGTCAAATAATG-3′ (reverse)
19 h with an initial switch time of 10 s and final switch time of 35 s.
2.9.3. Analysis of fingerprints
The ERIC PCR and PFGE patterns were analyzed using the
GelCompar II version (5.1) (Applied Maths NV, Belgium). The patterns
from TIFF images were normalized conforming to the standards and
dendrogram representing the relationship between the isolates was
created in the software using the Dice Similarity coefficient and cluster
analysis by UPGMA (unweighted pair group method with arithmetic
averages) method with an optimization and position tolerance value of
1.5% (Kai et al., 2008).
3. Results and discussion
3.1. Incidence of pathogenic V. parahaemolyticus in aquaculture samples
Presence of V. parahaemolyticus was confirmed in 90 of the 100
samples collected from 40 shrimp aquaculture farms. All the sediment
samples (40/40), 85% of the water samples (34/40) and 80% of the
shrimp samples (16/20) harboured V. parahaemolyticus. Of the initial
689 isolates, 590 were confirmed to be V. parahaemolyticus after pre­
liminary biochemical tests and the toxR gene amplification. The
number of V. parahaemolyticus isolates obtained from each geographical
location is presented in Table 1. V. parahaemolyticus is of significant
importance with regard to seafood, especially shrimp, as both of these
shares the habitat with respect to the halophillic conditions. Environ­
mental conditions like temperature and salinity have been shown to
significantly influence the V. parahaemolyticus abundance (DePaola
et al., 2003). The warm climate of the tropics makes the presence of V.
parahaemolyticus in aquaculture farms throughout the year (Lekshmy
et al., 2014). However, during the monsoon months the salinity of
aquaculture ponds will be reduced thereby affecting the detectable V.
parahaemolyticus count (Sanjeev, 1999). In this study, V. para­
haemolyticus was isolated from sediment and water samples during the
monsoon months. This is consistent with the previous finding that low
salinity is not much detrimental to the growth of V. parahaemolyticus,
provided the presence of organic matter in the environment (Deepanjali
et al., 2005; DePaola et al., 2003). The sediments in the aquaculture
ponds are a rich source of nutrients and organic matter due to the ac­
cumulation of feed and sedimentation of plankton and other organic
materials. The 1-cm layer of pond bottom is said to have 10 or more
times higher amount of nutrients than a 1 m deep water column, thus
making it a favorable site for microbial development (Avnimelech and
Ritvo, 2003).
To identify the pathogenic V. parahaemolyticus , all the 590
4
Marine Pollution Bulletin 160 (2020) 111551
Reference PCR cycling conditions
Kim et al., 1999 95 °C-30 s
63 °C-30 s
72 °C-30 s
Bej et al., 1999 94 °C-1 min 58 °C-1 min 72 °C-1 min
Gutierrez West et al., 2013 95 °C-1 min 62 °C-1 min 72 °C-1 min
Tada et al., 1992 95 °C-1 min 55 °C-1 min 72 °C-1 min
Matsumoto et al., 2000 96 °C-1 min
45 °C-2 min
72 °C-3 min
Okura et al., 2004 94 °C-30 s
60 °C-30 s
72 °C-30 s
Williams et al., 2004 95 °C-30 s
56 °C-30 s
72 °C-30 s
confirmed isolates were subjected to haemolysis test in Wagatsuma
agar. Twenty seven isolates from seven farms that showed β-haemolysis
(Kanagawa phenomenon positive) were selected for further character­
ization. Pathogenic V. parahaemolyticus was observed only in 7 out of
the 40 aquaculture farms (17.5%) subjected to study and bulk of these
isolates were from sediments (Table 3). Shrimps, being detritus feeders
and as an animal that lives near the bottom, are exposed to microbes
and the presence of pathogenic bacterium like V. parahaemolyticus can
be hazardous both for shrimp and humans. Pathogenic V. para­
haemolyticus possessing the tdh gene is reported to cause disease in P.
monodon (Shaheer et al., 2019). The incidence of pathogenic V. para­
haemolyticus from aquaculture environments has been reported from
different places in India (Alagappan et al., 2013; Gopal et al., 2005;
Sanjeev, 1999; Silvester et al., 2015).
3.2. Molecular characterization of pathogenic V. parahaemolyticus isolates
Of the 27 isolates, 13 belonged to the tdh+/trh− genotype, 2 were
tdh /trh+, another 2 tdh+/trh+ and rest of them were tdh−/trh−.
−
During the amplification of tdh gene, positive amplifications were ob­
served for 14 isolates when the primers of Bej et al. (1999) were em­
ployed and for 7 isolates using the primers of Gutierrez West et al.
(2013). A total of 15 isolates exhibited the presence of tdh gene when
either of these primers was used and four isolates were positive for trh
gene. The presence of pathogenic V. parahaemolyticus in aquaculture
farms in India has been earlier studied but the existence of tdh or trh
positive V. parahaemolyticus is rarely reported (Chakraborty and
Surendran, 2009; Gopal et al., 2005; Silvester et al., 2015). Globally,
there are considerable number of reports on isolation of V. para­
haemolyticus from seafood from various sources, but very few have re­
ported isolation of pathogenic strains from shrimp aquaculture en­
vironment (Paria et al., 2019; Sujeewa et al., 2009; Zhao et al., 2018). It
was reported earlier that pathogenic strains of V. parahaemolyticus were
less frequently encountered in environmental samples (Deepanjali
et al., 2005; Letchumanan et al., 2014). But recent studies have shown
higher level of TDH producing V. parahaemolyticus among the en­
vironmental isolates (de Jesús Hernández-Díaz et al., 2015; Gutierrez
West et al., 2013; Velazquez-Roman et al., 2014). Often, the pathogenic
strains in the environment go undetected due to poor amplification of
tdh gene (Gutierrez West et al., 2013; Narayanan et al., 2020). Unlike
the clinical isolates, non-specific amplifications and faint amplification
bands were often observed when amplifying the tdh gene from the
environmental isolates using the conventional primers (Fig. 1a). In our
previous study, we had reported faint bands during the amplification of
tdh gene using conventional primers which were identified as tdh gene
S.V. Narayanan, et al. Marine Pollution Bulletin 160 (2020) 111551

Table 3
Molecular characteristics of pathogenic V. parahaemolyticus isolated in this study.
Sl.No Isolate code Source Location O serotype tdh trh PGS PCR

1 F.K.VP22 Sediment Shrimp farm, Kannamaly O1 + − −

2 F.C.VP37 Water Shrimp farm, Chellanam O5 + − +
3 F.C.VP39s Water Shrimp farm, Chellanam O2 + − −
4 F.C.VP41 Shrimp Shrimp farm, Chellanam O3 − − −
5 F.C.VP49 Sediment Shrimp farm, Chellanam O5 − − +
6 F.K.VP56 Sediment Shrimp farm, Kodungalur1 O1 − − −
7 F.K.VP57 Sediment Shrimp farm, Kodungalur1 O1 + − −
8 F.K.VP58 Sediment Shrimp farm, Kodungalur1 O1 + − −
9 F.C.VP60 Sediment Shrimp farm, Chellanam O1 + − −
10 F.K.VP79 Shrimp Shrimp farm, Kodungalur1 O5 + − −
11 F.A.SPOT31 Sediment Shrimp farm, Aroor O10/O12 + + −
12 F.A.SPOT38 Sediment Shrimp farm, Aroor O10/O12 + + −
13 F.A.SPOT4111 Water Shrimp farm, Aroor O2 − − +
14 F.A.SPOT4112 Water Shrimp farm, Aroor O2 − − +
15 F.P.3AM2 Sediment Shrimp farm, Ponnamveli O7 − − +
16 F.P.3BM1 Sediment Shrimp farm, Ponnamveli O7 + − +
17 F.P.3BM1A Sediment Shrimp farm, Ponnamveli O7/O12 − − +
18 F.P.3BM3 Sediment Shrimp farm, Ponnamveli O7 − − −
19 F.P.3BM5-1 Sediment Shrimp farm, Ponnamveli O7 + − +
20 F.P.3BM8A-M Sediment Shrimp farm, Ponnamveli O7 + − −
21 F.P.3BM8B Sediment Shrimp farm, Ponnamveli O7 − − +
22 F.P.3BP11 Water Shrimp farm, Ponnamveli O7 − − +
23 F.P.3BP4 Water Shrimp farm, Ponnamveli O7 + − +
24 F.P.3BP5B Water Shrimp farm, Ponnamveli O7 + − +
25 F.K.4AM4A Sediment Shrimp farm, Kodungallur2 O1 − + −
26 F.K.4AM4B Sediment Shrimp farm, Kodungallur2 O5 + − −
27 F.K.4 AM5 Sediment Shrimp farm, Kodungallur2 O1 − + −
28 NICED.VP458 Clinical NICED, Kolkata O4 + − −
29 NICED.VP459 Clinical NICED, Kolkata O3 + − +
30 NICED.VP460 Clinical NICED, Kolkata O4 + − −
31 ATCC17802 Reference strain ATCC O1 − + −

Fig. 1. Gel electrophoresis images showing the separation

of PCR products a) Image representing faint amplification
bands and non-specific amplifications produced during the
PCR amplification of tdh gene from aquaculture isolates
using Bej et al. (1999) primers b) Image representing O
serotyping of V. parahaemolyticus isolates using multiplex
PCR c) Image representing the pandemic specific PGS-PCR
amplifications.

5
S.V. Narayanan, et al.
after sequencing (Narayanan et al., 2020). Gutierrez West et al. (2013)
reported inconsistent and usually faint bands during the amplification
of tdh gene after employing the conventional primers described by Bej
et al. (1999), which necessitated band purification, reamplification and
sequencing to confirm the identity of amplicons. In this study, we
employed both the conventional primers and the primers designed by
Gutierrez West et al. (2013) for the amplification of tdh gene. The faint
bands produced by three such isolates whilst the conventional primers
were employed for tdh gene amplification were subjected to band
purification, reamplification and sequencing and were confirmed to be
tdh gene (Gene bank accession number: MK075116, MN813981,
MN813982). But, in contrast to the observations of Gutierrez West et al.
(2013), higher frequency of amplification was observed when the Bej
et al. (1999) primers were used, though many of them produced only
faint amplification bands. KP positive V. parahaemolyticus isolates with
tdh−/trh− genotype were detected in this study. This is in consensus
with the previous reports where similar strains were identified
(Gutierrez West et al., 2013; Silva et al., 2018).
Multiplex PCR for determining the O serotype of the pathogenic V.
parahaemolyticus isolated in this study revealed the presence of the O1,
O2, O3, O5, O7 and O10 serotypes among the 27 isolates (Fig. 1b). The
serotypes of three isolates (F.P.3BM1A, F.A.SPOT31, F.A.SPOT38) gave
amplifications for multiple serotypes (Table 3). These strains are hy­
pothesized to be undergoing serotype conversion and hence showing
amplifications for both. Such polymorphisms have been earlier reported
in the pandemic strains (Espejo et al., 2017). Whole-genome sequence
analysis of pandemic isolates revealed that recombination events in the
regions encoding the O and K antigens to be the cause of serotype
conversion from O3:K6 to O4:K68 (Chen et al., 2011).
The PCR amplification of the pandemic specific genes revealed that
12 among the 27 KP positive isolates were positive for PGS-PCR.
However, there is uncertainty with respect to the pandemic nature of
these 12 isolates as none of them exhibited similar results for GS-PCR
and Hu-α PCR. Variance in results among the methods employed for
detection of pandemic specific genes was earlier reported (Li et al.,
2016; Narayanan et al., 2020; Parthasarathy et al., 2016). Not all
strains that showed positive results for PGS-PCR possessed the tdh or trh
genes. This is consistent with the findings of Li et al. (2016) where they
have reported that the clinical isolates negative for tdh and trh genes
were positive for the pandemic specific PCR amplifications. Never­
theless, the results of ERIC-PCR and PFGE analysis shows that some of
these isolates share genetic similarities with the pandemic and clinical
isolates used in this study.
3.3. Molecular subtyping of pathogenic V. parahaemolyticus
DNA based molecular subspecies typing techniques are useful in
food borne disease surveillance and source attribution, and facilitate
tracking the transmission of pathogens from pond to table. Also, a
comparative typing of environmental isolates with known clinical
strains facilitates the identification of potential pathogens present in the
environment (van Belkum et al., 2007). Both ERIC-PCR and PFGE has
been used successfully for studying the relationship between clinical
and environmental isolates (Suffredini et al., 2011; Xie et al., 2017). In
this study, the analysis of ERIC-PCR fingerprints distributed the isolates
into four clusters (E1, E2, E3, E4) at a Dice similarity coefficient cut off
of 70% (Fig. 2). E2 formed the largest cluster with 12 isolates. Two
isolates from aquaculture farms F.P.3BP5B and F.K.VP79, clustered
along with the clinical isolates (NICED.VP458, NICED.VP459 & NI­
CED.VP460) and the ATCC reference isolate in cluster E3 with a simi­
larity of 80.5% and 76.3% respectively.
The analysis of PFGE fingerprints distributed the isolates into 6
clusters (P1, P2, P3, P4, P5, P6) at a Dice similarity coefficient cut off of
70%, among which P1 formed the largest with 12 isolates (Fig. 3). The
two non-pandemic clinical isolates (NICED.VP458 & NICED.VP460)
and the V. parahaemolyticus ATCC 17802, clustered with three isolates
6
Marine Pollution Bulletin 160 (2020) 111551
from aquaculture farm (F.K.4AM4A, F.K.4AM4B, F.K.4AM5) in cluster
P3. The pandemic clinical isolate (NICED.VP459) was grouped in
cluster P4 along with three isolates from aquaculture farm (F.K.VP79,
F.A.SPOT31, F.A.SPOT38) all of which were tdh positive. Isolate
F.K.VP9 clustered with the pandemic strain in both ERIC-PCR and
PFGE. These results suggest that, the isolates clustered with the clinical
strains share similarities in their genetic features and might possess the
potential to cause an outbreak when transmitted through the food.
Detection of clusters with genotype identical to that of clinical strains
provides an ‘early warning’ of potential outbreak (van Belkum et al.,
2007). This will also serve the purpose of a database during an out­
break.
In a study of PFGE typing scheme of V. parahaemolyticus from 15
countries, it was reported that the pandemic isolates of V. para­
haemolyticus and related strains which dominated in outbreaks from
1996 to 1999 formed separate clusters (Wong et al., 2007). It is hard to
infer from the clustering of this study that these isolates have a pan­
demic nature, as most of these isolates were negative for the pandemic
specific PCR (Table 3). But it should be taken into consideration that
unlike the above mentioned study these isolates were purely environ­
mental isolates and the changes that occur to these isolates when in­
gested i.e. the characteristics during colonization in the intestinal mu­
cosa, would have to be studied before making similar inferences.
In this study, we have shown the high genetic diversity among the
pathogenic V. parahaemolyticus population in Cochin, India represented
by a Dice similarity coefficient of 62.7% in ERIC PCR. This is further
corroborated by the results of PFGE where the minimum Dice similarity
coefficient among the isolates was found to be 61.6%. Previous studies
using the ERIC PCR fingerprints have highlighted the genetic hetero­
geneity of V. parahaemolyticus isolated from Cochin, India (Chakraborty
and Surendran, 2009; Silvester et al., 2017). Recent reports suggest that
PFGE is being widely used to study the genetic diversity and geo­
graphical distribution of environmental isolates (Chen et al., 2017;
Suffredini et al., 2011; Zhao et al., 2018). All the clusters had isolates
from multiple geographic locations thus negating any geographic effect
on clustering of isolates. This is concurrent with the observations of
Silvester et al. (2017) who reported the absence of any bio-geographic
or seasonal effect on the genotypic distribution of V. parahaemolyticus
from Cochin estuary. Moreover, clustering based on the virulence pat­
tern with respect to the tdh/trh genotype was also not observed.
3.4. Antimicrobial resistance pattern of pathogenic isolates
The antibiotic sensitivity pattern of 27 pathogenic V. para­
haemolyticus isolates from aquaculture samples, determined using disc
diffusion method is summarized in Table 4. All the 27 isolates were
resistant to Ampicillin. V. parahaemolyticus is reported to be in­
trinsically resistant towards penicillins due to the presence of a class A
carbenicillin-hydrolyzing β-lactamase (CARB) family of β-lactamases
(Chiou et al., 2015). The resistance shown by the isolates towards other
antibiotics were as follows; cefotaxime- 74.1%, cefoxitin- 48.1%, cefe­
pine- 44.4%, ceftazidime- 29.6%, amoxicillin/clavulanic acid- 7.4%,
ciproflaxacin-3.7%, gentamicin- 3.7%, meropenem- 3.7% (Fig. 4)
(Supplementary Table S1). All the isolates were sensitive to chlor­
amphenicol, trimethoprim/sulphmethaxazole and tetracycline. Though
it has been reported that tetracycline resistance genes persists in the
aquaculture environments due to its prophylactic and therapeutic use,
V. parahaemolyticus from aquaculture are mostly sensitive to tetra­
cycline (Elmahdi et al., 2016; Prasad and Ravishankar, 2018;
Tamminen et al., 2011). 74% (20 isolates) and 3.7% (1 isolate) of the
pathogenic V. parahaemolyticus isolates were ESBL and carbapenemase
producers phenotypically. The results of antibiotic susceptibility testing
shows that 16 isolates (59%) had MAR index of 0.2 or above. The MAR
index is the ratio of the number of antibiotics to which the isolate is
resistant to the number of antibiotics to which the isolate is exposed.
The MAR index of 0.2 or above is significant and is considered ‘high-
S.V. Narayanan, et al.
Fig. 2. Dendrogram representing the analysis of ERIC PCR fingerprints in GelCompar II software. Dice Similarity coefficient and cluster analysis by UPGMA method
with an optimization and position tolerance value of 1.5% was used to generate the ERIC dendrogram.
risk’ source of contamination (Krumperman, 1983). The highest MAR
index of 0.58 was shown by an isolate (F.P.3BP5B) which was found to
be multidrug resistant (resistant to the antibiotic classes' penicillins,
cephems, aminoglycosides and carbapenems). This MDR strain was
clustered along with the clinical strains in ERIC PCR and showed more
than 80% genetic similarity with the clinical strains used in this study
(Fig. 2). The presence of high number of MDR V. parahaemolyticus in
aquaculture environments has been previously reported (Zhao et al.,
2018). But, carbapenam resistance in V. parahaemolyticus is very rare
and was once reported from freshwater and marine fish sample from
Malaysia (Lee et al., 2018). In contrast to the global trend, an increasing
resistance towards third and fourth generation cephalosporins was
observed in our data (Elmahdi et al., 2016). Though some authors have
reported resistance among V. parahaemolyticus towards extended-spec­
trum cephalosporins, ESBL activity is not very common in V. para­
haemolyticus (Elmahdi et al., 2016; Lee et al., 2018; Letchumanan et al.,
2019; Silva et al., 2018; Wong et al., 2012). In this study we have
characterized only the pathogenic isolates and the actual levels of MDR
V. parahaemolyticus with Carbapenamase and ESBL activity among the
non-pathogenic V. parhaemolytiucs in the aquaculture environment
might be much higher. As these are coastal aquaculture farms where
7
Marine Pollution Bulletin 160 (2020) 111551
there is flow of water to and from the interconnected backwaters the
MDR strains would spread into the environment causing the sur­
rounding water and sediments to become reservoirs of antibiotic re­
sistance genes.
In the coastal aquaculture farms, farmers depend on the natural
phenomenon of high and low tides for the exchange of water. The
presence of pathogenic and antibiotic resistant V. parahaemolyticus in
the aquaculture farms will result in the spread of these pathogens into
the coastal waters during the exchange of water. The presence of V.
parahaemolyticus in shell fish and coastal waters has been reported to be
highly correlated (Park et al., 2018). And hence, maintaining the mi­
crobial quality of aquaculture ponds by following good aquaculture
practices is of paramount importance. The removal of residual V.
parahaemolyticus before the start of the culture can be attained by re­
moval of top layer sediments. This will also help reduce the organic
content due to accumulation of feed, sedimentation of plankton and
dead shrimps. The organic matter in the ponds has been reported to
support the survival of V. parahaemolyticus even at low salinity levels
(Deepanjali et al., 2005). Traditional farmers identify the richness of
organic matter by the black coloration of the sediments and the sedi­
ments are removed only after three or four farming seasons. Removing
S.V. Narayanan, et al. Marine Pollution Bulletin 160 (2020) 111551

Fig. 3. Dendrogram representing the analysis of SfiI digested PFGE fingerprints of pathogenic V. parahaemolyticus isolates in GelCompar II software. Dice Similarity
coefficient and cluster analysis by UPGMA method was used with an optimization and position tolerance value of 1.5% to generate the PFGE dendrogram.

Fig. 4. Graph representing the antibiotic sensitivity profile of pathogenic V. parahaemolyticus isolates from coastal aquaculture farms (X-axis: Antibiotics tested, Y-
axis: Percentage of isolates).

8
S.V. Narayanan, et al. Marine Pollution Bulletin 160 (2020) 111551

Table 4
Antibiotic sensitivity profile of 27 pathogenic V. parahaemolyticus isolates from aquaculture samples.
Antibiotic name Antibiotic class Code Breakpoints Number %R %I %S

Amoxicillin/Clavulanic acid Beta-lactam+Inhibitors AMC 14–17 27 7.4 59.3 33.3

Ampicillin Penicillins AMP 14–16 27 100 0 0
Ceftazidime Cephems CAZ 18–20 27 29.6 37 33.3
Chloramphenicol Phenicols CHL 13–17 27 0 0 100
Ciprofloxacin Quinolones CIP 16–20 27 3.7 48.1 48.1
Cefotaxime Cephems CTX 23–25 27 74.1 18.5 7.4
Cefepime Cephems FEP 19–24 27 44.4 51.9 3.7
Cefoxitin Cephems FOX 15–17 27 48.1 33.3 18.5
Gentamicin Aminoglycosides GEN 13–14 27 3.7 11.1 85.2
Meropenem Penems MEM 20–22 27 3.7 29.6 66.7
Trimethoprim/Sulfamethoxazole Folate pathway inhibitors SXT 11–15 27 0 0 100
Tetracycline Tetracyclines TCY 12–14 27 0 0 100

the top layer of sediments after every culture will help to reduce the
residual pathogens and also the organic matter. The initial load of V.
parahaemolyticus in water can be reduced by chlorination and complete
dechlorination of water. Some farmers use storage ponds where the
water will be treated before every exchange rather than allowing direct
flow from the backwaters. The use of adjoining water storage ponds will
prevent the contamination from backwaters which could harbour a
plethora of pathogens due to the outflow from nearby farms and con­
tamination from various other sources. But maintaining water treat­
ment ponds adjacent to the culture ponds depends largely on the geo­
graphic suitability and economic feasibility. Vaiyapuri et al. (2019)
described in detail about the steps to be followed at different points in
the fish value chain to prevent the contamination and multiplication of
MRSA. Adopting good aquaculture practices with special mention to
personal hygiene, disinfection of gears before use, avoiding the use of
prohibited antibiotics and chemicals, treatment of water and usage of
antibiotic free inputs were recommended to prevent MRSA con­
tamination in aquaculture ponds (Vaiyapuri et al., 2019). These
methods in general would be useful to reduce the contamination not
only by MRSA but by any pathogens including V. parahaemolyticus.
Additionally, awareness among the farmers about antibiotic resistance
in pathogenic bacteria and their possible implications to health and
urging them to voluntarily take up these measures to ensure the quality
of their produce is vital. It is also important that microbiome of a
healthy aquaculture system is maintained by introducing probiotics. A
wide variety of probiotics with various roles including pathogen in­
hibition, nutrient digestibility, water quality improvement, growth
promotion, disease resistance, immune system enhancement and stress
tolerance are now commercially available (Martínez Cruz et al., 2012;
Sahu et al., 2008).
nutrients in the sediments due to accumulation of excessive feeds and
organic matter, and the indiscriminate use of antibiotics are conducive
to the development of antibiotic resistant V. parahaemolyticus. The
study revealed that the brackish water aquaculture farms act as a re­
servoir of pathogenic and MDR V. parahaemolyticus. As most of these
are coastal farms that depend on water bodies for the exchange of water
these pathogens could easily spread to the marine environment posing
risk to handlers. India, being an exporter of seafood worth 7 billion USD
and with domestic consumption worth 22.69 billion USD there is a
greater risk of transmission of these pathogens to the food chain. Rapid
delineation of pathogens using techniques like ERIC-PCR, PFGE or
nucleotide sequence based typing techniques will help to identify the
risk at an early stage, enabling us to adopt preventive measures to
maintain the microbial safety and quality of aquaculture farms. Future
prospects of this work include whole genome sequence profiling of
potentially pathogenic MDR isolates which would provide insight into
their genetic makeup and pathogenic potential.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.marpolbul.2020.111551.
CRediT authorship contribution statement
Sreejith V. Narayanan:Investigation, Formal analysis, Writing -
original draft.Toms C. Joseph:Methodology, Supervision.
Shaheer Peeralil:Validation.Reshmi Koombankallil:Investigation.
Murugadas Vaiyapuri:Writing - review & editing.Mukteswar P.
Mothadaka:Resources, Writing - review & editing.Kuttanapilly V.
Lalitha:Conceptualization, Writing - review & editing.
Declaration of competing interest

4. Conclusion
In this study, potentially pathogenic V. parahaemolyticus was iso­
lated from seven shrimp aquaculture farms in central Kerala, India.
Among the 590 confirmed V. parahaemolyticus isolates, 27 possessed the
β-haemolytic activity. The tdh and trh genes were amplified from 15
and 4 isolates respectively and 12 isolates showed positive for the
pandemic specific PGS-PCR. A comparative molecular subtyping re­
vealed the presence of clusters that share genetic similarities with the
clinical and pandemic isolates. The isolates that clustered with clinical
strains are estimated to have an outbreak potential and thus provide an
‘early warning’. ERIC-PCR and PFGE also revealed the genetic diversity
among the pathogenic V. parahaemolyticus isolated from Cochin, India.
The antimicrobial susceptibility testing demonstrated that most of these
pathogenic V. parahaemolyticus isolates were resistant to multiple an­
tibiotics. Among the potentially pathogenic V. parahaemolyticus isolates,
one was identified to be a MDR strain with carbapenem resistance
which showed high genetic similarity with the clinical strains. The
ambient temperature in the tropics, salinity of water, abundance of
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ­
ence the work reported in this paper.
Acknowledgment
The clinical strains of V. parahaemolyticus and the Salmonella
Braenderup H9812 used in this study were provided by Dr. Asish Kumar
Mukhopadhyay, National Institute of Cholera and Enteric Diseases,
Kolkata, India. The consumables for this work were supported by in­
stitutional funds of ICAR-Central Institute of Fisheries Technology,
Cochin, India. Special acknowledgements are due to the Director, ICAR-
Central Institute of Fisheries Technology, Cochin for all his support. We
thank Dr. Ajeeshkumar K for his assistance in preparation of this
manuscript.
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