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: tvuphuong@ctu.edu.vn 
ARTICLE INFO ABSTRACT 
DOI:10.46223/HCMCOUJS. 
tech.en.10.1.359.2020 
Received: October 1st, 2019 
Revised: November 13th, 2019 
Accepted: November 21st, 2019 
Keywords: 
antimicrobial, bacteria, 
shrimp-pond water, Vibrio 
parahaemolyticus 
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. 
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  ... ween 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 
No 
Bacterial 
isolates 
Inhibition zone 
diameter 
[D = d1 - d2] (mm) 
No 
Bacterial 
isolates 
Inhibition zone 
diameter 
[D = d1 - d2] (mm) 
1 DH 1m 20.33 a 5 DH 8i 13.67 ef 
2 DH 1n 21.33 a 6 DH 8m 14.33 de 
3 DH 2f 17.33 bc 7 DH 8n 16.33 de 
4 DH 4d 19.33 ab 8 
Positive control 
(penicillin) 
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. 
Antimicrobial activity of two bacterial isolates showed through the sterile halo (Figure 
2). 
 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 
Closest species relative 
Similarity 
(%) 
Bacilli 
DH1m 
MK834692 Bacillus subtilis strain TBMAX53 
KX668274 Bacillus tequilensis strain MS01 
98.90 
98.90 
DH2f MF077125 Bacillus tequilensis strain 52-LR1-2 99.36 
 GU980947 Bacillus subtilis strain CICC 10023 99.26 
DH4d EU153188 Bacillus subtilis strain JPC-2 99.84 
 MK014304 Bacillus sp. strain DM2 99.76 
DH8i MK719884 Bacillus siamensis strain Q13 99.60 
DH8m 
DH8n 
KY652945 Bacillus amyloliquefaciens strain 
isolate G12 
KF922381 Bacillus sp. B1(2014b) 
CP033576 Bacillus velezensis strain NY12-2 
99.60 
99.84 
99.84 
99.11 
48 Tran Vu Phuong et al. Ho Chi Minh City Open University Journal of Science, 10(1), 43-52 
Taxonomic group and 
strain 
Closest species relative 
Similarity 
(%) 
Proteobacteria 
DH1n 
KJ496372 Bacillus flexus strain MS14-1 
KR999908 Geobacillus stearothermophilus 
strain NB3-8 
JF751041 Streptomyces sp. 2011 
99.11 
99.77 
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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