Isolation and selection of bacteria against shrimp pathogenic vibrio parahaemolyticus from shrimp pond water on Duyen Hai district, Tra Vinh province
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52 43
Isolation and selection of bacteria against shrimp pathogenic
vibrio parahaemolyticus from shrimp pond water on Duyen Hai
district, Tra Vinh province
Tran Vu Phuong1*, Dang Thi Ngoc Thanh2, Cao Ngoc Diep1
1Biotechnology R&D Institute, Can Tho Universty, Vietnam
2Sai Gon University, Vietnam
*Corresponding author: [email protected]
ARTICLE INFO
ABSTRACT
DOI:10.46223/HCMCOUJS.
tech.en.10.1.359.2020
Antibiotic has frequently been used in the shrimp-farming
process in Vietnam. This leads to the status that antibiotic-
resistant bacteria and products do not receive in the market.
Bacteria had the resistant ability to pathogenic bacteria in water,
and they have an important role in sustainable aquaculture. This
study aimed to isolate and select good bacterial strains against
Vibrio parahaemolyticus, pathogenic bacteria, on shrimp from 8
samples of shrimp pond water at 3 villages Ngu Lac, Phuoc An
and Long Toan of Duyen Hai district, Tra Vinh province on NB
agar medium. As a result, fifty-nine bacterial isolates were
isolated and 10/59 isolates (16.95%) were identified as resistant
to Vibrio parahaemolyticus by the good diffusion method. In 10
isolates, there were 7 isolates that had good resistance to selection
for PCR technique and sequencing. The result indicated that these
seven strains, including DH1m, DH2f, DH4d, DH8i, DH8m,
DH8n, belonged to Bacilli and the DH1n strain belonged to
Streptomyces sp.
Received: October 1st, 2019
Revised: November 13th, 2019
Accepted: November 21st, 2019
Keywords:
antimicrobial, bacteria,
shrimp-pond water, Vibrio
parahaemolyticus
1. Introduction
Shrimp cultivation has been faced with pathogenic bacterial infections, such as
luminous vibriosis and acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio
harveyi and Vibrio parahaemolyticus, respectively (Chumpol et al., 2019). Shrimp production
in Southeast Asia steadily averaged 6.0% annual growth from 2008 to 2011; however, the
production declined from 3.45 million metric tons (MMT) to 3.25 MMT in 2012 (down 5.8%)
and 3.21 MMT in 2013 (down 1.1%) due to the impact of early mortality syndrome (EMS) in
China, Thailand, Vietnam, and Malaysia (Anderson, 2016). EMS is also called the acute
hepatopancreatic necrosis disease (AHPND) and is infectious with the symptoms including
sloughing of hepatopancreas (HP) cells, enlarged HP nuclei, and lack of B, F, R cells and E cell
mitosis (Food and Agriculture Organization of the United Nations (FAO), 2013). The outbreak
of AHPND/ EMS in Thailand was first reported in late 2012 in pacific white shrimp farming
44
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52
on the eastern coast, and then spreading to the west coast facing the Gulf of Thailand (Flegel,
2012; Joshi et al., 2014). It caused 100% mortality early on in shrimp cultures, at around 20-30
days of cultivation; and V. parahaemolyticus is recognized as the bacterial strain that caused
AHPND/EMS (Tran et al., 2013).
To solve these problems, shrimp farmers normally use antibiotics to eliminate
pathogenic bacteria; however, antibiotics can be harmful to consumer health. Also, long term
use of antibiotics leads to residual compounds in sediment and water, and the bacteria can adapt
themselves by selecting for antibiotic-resistant genes (Zhang, Li, & Sun, 2011). For sustainable
shrimp cultivation, probiotics and/or their anti-vibrio compounds as biocontrol agents have
been explored to control vibriosis in shrimp farming. Gram-negative and gram-positive bacteria
have been identified as potential probiotics for aquaculture, with effects against various
pathogens (Brunt, Newaj-Fyzyl, & Austin, 2007). The aim of this work was to isolate, select,
and identify bacterial isolates having good resistance to V. parahaemolyticus in the water of
shrimp pond on Duyen Hai district, Tra Vinh province, Vietnam to produce a probiotic for
shrimp cultivation.
2. Materials and methods
2.1. Materials
Water samples were collected at the depth of 0.2m and distance shore 4m, from 8
shrimp-ponds of 3 villages (Long Toan, Ngu Lac, Dinh An) (9o64’37” to 9o67’88” East and
106o45’29” to 106o51’73” North) of Duyen Hai district, Tra Vinh province, Vietnam, they
stored in an icebox and transferred to Can Tho University laboratory, stored -4oC in the
refrigerator until analysis.
Vibrio parahaemolyticus provided by Dr. Đang Thi Hoang Oanh, Department of
Aquaculture Pathology, College of Aquaculture, Can Tho University.
Nutrient Agar (Difco) medium supplemented with Aginalxic (10µg/l) and Nystatin
(25µg/l) into the medium after autoclaving; Luria Bertani medium (Sambrook, Fritsch, &
Manlatis, 1989).
2.2. Methods
2.2.1. Isolation and culture
Water samples were serially diluted with sterile saline water (0.01%). A hundred
microliters of the suspensions were spread onto the NB agar medium. All plates were inoculated
at room temperature for 24h; the disjointed colonies were recorded and re-streaked to obtain a
pure culture. The colonies bearing distinct morphological characteristics were picked up and
transferred to freshly prepared media until pure cultures were obtained.
2.2.2. Screening assays for antibacterial activity
The liquid cultures were grown with shaking at 150rpm for 1 day depending on their
growth rate at 30°C. The broth was centrifuged in 50mL falcon tubes (5000rpm, 15min at room
temperature; Megafuge 1.0R, Heraeus) and the supernatant was stored at 4°C. The bacterial test
organisms were plated in the LB medium. The antimicrobial extract was added to the wells; the
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52 45
plates were incubated at 4°C for 2h for the diffusion of antimicrobial extract and observed for
the zones of inhibition at 28°C for 48h.
2.2.3. The Agar well diffusion method
The active isolates were cultured by the method given in the previous step. The
supernatants were used for testing extracellular antimicrobial activity by agar well diffusion
method. By using a sterile cork borer, wells were punctured in an appropriate agar medium
previously seeded with one of the test organisms. One hundred microliters of the culture
supernatants were added to each well. The plates were then incubated at 4oC for at least 2h to
allow the diffusion of crude extracts followed by incubation for 24h at 37oC for bacteria and
48h at 28oC for yeast. The diameters of inhibition zones were monitored and measured
(Galindo, 2004) and positive control was penicillin.
2.2.4. Genomic DNA extraction
Bacterial cells from these cultures were collected by centrifugation and genomic DNA
was extracted (Sambrook et al., 1989).
2.2.5. PCR amplification and sequencing of 16S rDNA
PCR was used with primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) (Weisburg,
Barns, Pelletier, & Lane, 1991) and 1492R (5’-TACGGTTACCTTGTTACGACTT-3’)
(Reysenbach, Giver, Wickham, & Pace, 1992) Cycling condition was as follows: initial
denaturation at 95°C for 5min, 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 90
sec, and a final extension of 5min for 72°C.
2.2.6. Sequence analysis
The 16S rRNA gene sequences were compared with those from the type strains
Tool (BLAST) (Altschul, Gish, Miller, Myers, & Lipman, 1990).
For phylogenetic analysis, multiple sequence alignment was performed using
CLUSTALX, version 1.81. The phylogenetic tree was constructed using Mega 7.0 (Kumar,
Stecher, Li, Knyaz, & Tamura, 2018). The consistency of the trees was verified by
bootstrapping (1000 replicates) for maximum likelihood.
2.2.7. Data analyses
The experimental results were analyzed as a two-way ANOVA with the isolates and
with levels of diameters of inhibition zones. All analyses were conducted using the program
MSTATC, Minitab 16. The data were considered significantly different at P<0.01. Duncan test
at P=0.01 was used to differentiate between statistically.
3. Results and discussions
3.1. Isolation of bacteria
A total of 59 isolates of bacteria was purified from 8 water samples collected at 3 sites
(Long Toan, Phuoc An and, Ngu Lac).
46
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52
Figure 1. Shape and size of several colonies
Almost their colonies have round-shaped; milky or less yellow, diameter size of colony
>2mm, and yellow, entire or lobate margin, pulvinate elevation; diameter size of these colonies
varied from 0.2 to 3.0mm (Figure 1) and all of them have Gram-positive. Seven of 59 tested
isolates could produce antimicrobial active metabolites inhibiting V. parahaemolyticus (Table
1).
Table 1
Antimicrobial activity of 10 bacterial isolates to Vibrio parahaemolyticus
Inhibition zone
diameter
[D = d1 - d2] (mm)
Inhibition zone
diameter
[D = d1 - d2] (mm)
Bacterial
isolates
Bacterial
isolates
No
No
1
2
3
DH 1m
DH 1n
DH 2f
20.33 a
21.33 a
17.33 bc
5
6
7
DH 8i
DH 8m
DH 8n
13.67 ef
14.33 de
16.33 de
Positive control
(penicillin)
4
DH 4d
19.33 ab
8
11.67g
Source: The researcher’s data analysis
Means within a column followed by the same letter/s are not significantly different at
p<0.05.
2).
Antimicrobial activity of two bacterial isolates showed through the sterile halo (Figure
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52 47
Figure 2. Antimicrobial activity of two bacterial isolates (DH8n and DH1n) against V.
parahaemolyticus
3.2. The identification of bacterial isolates
Seven good bacterial isolates as follows: DH1m, DH1n, DH2f, DH4d, DH8i, DH8m,
and DH8n were selected for PCR and sequencing. The result was presented in Table 3.
Table 3
Phylogenetic affiliation of isolates based on 16S rRNA gene sequences by using BLAST
program in the GenBank database based on sequences similarity
Taxonomic group and
strain
Similarity
(%)
Closest species relative
Bacilli
DH1m
MK834692 Bacillus subtilis strain TBMAX53
KX668274 Bacillus tequilensis strain MS01
MF077125 Bacillus tequilensis strain 52-LR1-2
GU980947 Bacillus subtilis strain CICC 10023
EU153188 Bacillus subtilis strain JPC-2
MK014304 Bacillus sp. strain DM2
98.90
98.90
99.36
99.26
99.84
99.76
99.60
99.60
99.84
99.84
99.11
DH2f
DH4d
DH8i
MK719884 Bacillus siamensis strain Q13
KY652945 Bacillus amyloliquefaciens strain
isolate G12
DH8m
DH8n
KF922381 Bacillus sp. B1(2014b)
CP033576 Bacillus velezensis strain NY12-2
48
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52
Taxonomic group and
Similarity
(%)
Closest species relative
strain
KJ496372 Bacillus flexus strain MS14-1
99.11
KR999908 Geobacillus stearothermophilus
strain NB3-8
Proteobacteria
DH1n
99.77
JF751041 Streptomyces sp. 2011
Source: The researcher’s data analysis
A sequence of DN1n isolate searched on NCBI data bank, its similarity with
Pseudomonas… with 99.77%, in this table, however, there is only a strain Streptomyces sp. (a
positive-gram bacteria) which has also the similarity with DN1n isolate with 99.77% we can
select this strain to replace Pseudomonas sp. in the phylogenetic tree. Furthermore, the result
of gram strain showed that all of the isolated bacteria were gram-positive therefore DH1n isolate
was selected as gram-positive instead of gram-negative.
A UPGMA phylogenetic tree (Figure 3) in these strains showing the two clusters:
cluster A with 6 strains including two smaller clusters as cluster A1 with 4 strains (Bacillus
subtilis DH1m, Bacillus velezensis DH8m, Bacillus amyloliquefasciens DH8i, B. tequilensis
GH2f) while cluster A2 with 2 strains were Streptomyces sp. DH1n and Bacillus subtilis DH4d.
Cluster B only had one strain Bacillus flexus DH8n. Even though 7 strains originated from 3
sites far from many kilometers, but they had their genetics closely.
Figure 3. UPGMA phylogenetic tree of partial 16S rRNA gene sequences from the bacterial
isolates from water pond-shrimp and closely related type strains. Numbers are the percentage
bootstrap values that were calculated for 1000 replicates. Bar, 0.02 was per nucleotide
position
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52 49
Vibrio species are among the most important bacterial pathogens of cultured shrimp.
They are responsible for several diseases and mortalities of up to 100% due to vibriosis have
been reported (Karunasagar, Pai, Malathi, & Karunasagar, 1994). In the search for more
effective and environmentally friendly treatments, probiotics have emerged as a viable
alternative (Balca´zar et al., 2006; Verschuere, Rombaut, Sorgeloos, & Verstraete, 2000).
Probiotics are defined as live microorganisms that confer a health benefit on the host when
consumed in adequate amounts (Reid et al., 2003). The genus Bacillus has been isolated from
crustacean intestine (Rengpipat, Rukpratanporn, Piyatiratitivorakul, & Menasaveta, 2000),
bivalves (Sugita, Tanaami, Kobashi, & Deguchi, 1981), and marine fish (Sugita, Hirose,
Matsuo, & Deguchi, 1998). Some species of this genus have shown inhibitory activity against
various pathogens (Rengpipat, Phianphak, Piyatiratitivorakul, & Menasaveta, 1998; Sugita et
al., 1998).
In addition, several studies have reported that Bacillus produces polypeptide antibiotics,
such as bacitracin, gramicidin S, polymyxin, and tyrotricidin, which are active against a wide
range of Gram-positive and Gram-negative bacteria (Drablos, Nicholson, & Ronning, 1999;
Morikawa, Ito, & Imanaka, 1992; Perez, Suarez, & Castro, 1993).
Balca´zar and Rojas-Luna (2007) discovered the bacterial strain Bacillus subtilis UTM
126 produced antimicrobial activity against pathogenic Vibrio species, including V.
alginolyticus, V. parahaemolyticus, and V. harveyi. The treatment with B. subtilis UTM 126
decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus
subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp
aquaculture.
Recently, Vidal, Pessoa, Dos Santos, Mendes, and Mendes (2018) recognized that B.
cereus isolate from the intestine of shrimps Litopenaeus vannamei can colonize the intestine of
post-larvae shrimps of the same species and promote a significant reduction of pathogens, with
great effectiveness in reducing the pathogenic bacteria V. parahaemolyticus and V.
alginolyticus in shrimps grown in the laboratory. Chumpol et al. (2019) also found the probiotic
purple non-sulfur bacteria (PNSB) are safe for use in producing antivibration compounds
against shrimp pathogenic vibrios. The probiotic PNSB, Rhodobacter sphaeroides (SS15,
TKW17) and Afifella marina STW181, released antivibration compounds inhibiting various
shrimp pathogenic Vibrio spp.
4. Conclusion
Fifty-nine bacterial isolates were isolated from 8 water samples of pond-shrimp at 3
villages of Duyen Hai district, Tra Vinh province, Vietnam. Seven/59 isolates that had good
resistance to Vibrio parahaemolyticus were selected to identify. The result showed that 6 strains
belonged to Bacilli and one was Pseudomonas sp.
Two strains Streptomyces sp. DH1n and Bacillus subtilis DH1m had the highest
antibacterial activity against Vibrio parahaemolyticus, they have a great potential to local
probiotic production in shrimp farming.
50
Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52
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