Isolation and determination of Vibrio spp. pathogen from hemorrhagic disease in Sciaenops ocellatus in cage culture

In this study, 18 strains of Vibrio bacteria were identied from 27 samples of Red drum sh (Sciaenops ocellatus) suffering from the haemorrhagic disease from cage culture in Vietnam. The bacterial strains were identied with the 16S rRNA sequencing method and checked for morphological, physiological, and biochemical characteristics by using the API 20E KIT. Twelve strains of V. alginolyticus, three strains of V. uvialis, and three strains of V. orientalis were recorded. All Vibrio strains have gene similarities with those on the gene bank ranging from 98 to 100%. The biochemical characteristics of these 18 isolates were similar. These bacteria are susceptible to tetracycline and doxycycline and entirely resistant to ampicillin, amoxicillin, and erythromycin.


Introduction
Red drum (Sciaenops ocellatus) is a marine sh species with high economic value, commonly farmed in Texas and Florida, USA, to provide alternatives to wild-caught sh and stock enhancement efforts (FAO, 2009). Red drums have also become the primary sh breed in China since 1991 (Hong & Zhang, 2003;Shen, Qian, Xu, Gu, & Shao, 2005) and that was imported in Vietnam in 1999, this sh is cultured mainly in the central coastal provinces from Ba Ria Vung Tau to Quang Ninh (Ministry of Fisheries, 2005). Vibriosis is an intestinal disease. Vibrio is considered one of the most common pathogens in farmed marine sh and water environments (Novriadi, 2016). Vibrio bacterium is also identi ed as a pathogen in marine sh cultures, such as Lates calcarifer (Gibson Kueh, Terence, Chew, Uichanco, & Shen, 2021), (Hoa, Oanh, & Phuoc, 2018) and Mugil cephalus (Raju & Sreeramulu, 2017). Some studies have shown that the causative agent of disease on Red drum sh is Streptococcus iniae infection in cage-cultured Red drums in Eastern China (Zhou et al., 2008), (Mmanda et al., 2014). Vibrio harveyi (V. carchariae) causes infectious gastroenteritis in cultured Red drums in Taiwan (P. C. Liu, Chuang, & Lee, 2003). V. vulni cus, V. brasiliensis, V. cholera, and V. parahaemolyticus cause Red drums' haemorrhagic disease (Hoang Tan Quang et al., 2020). According to our research in 2020, the causative agent of haemorrhagic disease on S. ocellatus in Thuan An, Thua Thien Hue, Vietnam is mainly V. alginolyticus (50%), V. azureus (27.67%), V. uvialis (16.67%), and V. orientalis (6.67%) (Yen, Linh, & Tram, 2021). The rst evidence of Red drums infected by Nocardia seriolae is from a farm in Mexico (Rio-Rodriguez et al., 2021). In addition, V. alginolyticus bacterium is an opportunistic pathogen related to infections in humans and marine animals. V. alginolyticus and V. parahaemolyticus strains are also pathogens in coastal aquaculture systems in Guangdong, China (Xie Z.Y., Hu C.Q., Chen C., Zhang L. P., & C.H, 2005). The rst study to develop vaccine VaBGs can cause a stronger humoral and cellular immune response to protect mice and sh from V. alginolyticus compared with the conventional FKC vaccine (Cao et al., 2018). V. alginolyticus and S. iniae have also been identi ed as the causative agent of Cobia with the haemorrhagic disease, ulcers, n erosion, and blindness (Nguyen Bao Trung & Dung, 2018).

| Physical and chemical water quality
Page 3/17 The pH (HANNA HI98107 pH handheld pH meter, Romania), temperature (mercury thermometer), salinity (refractometer), and dissolved oxygen (sera test KIT, Virtue) of the sites were also recorded.

| Bacteria isolation and identi cation
The sh were washed thoroughly under tap water and dried. The outer surface of the sh was disinfected with 70% ethanol, and the sh was opened with a sterilized scalpel. The internal pathological signs were recorded. The damaged tissues from the brain, liver, kidney, and spleen were separated with sterilized culture rods. They were then inoculated in the thiosulfate citrate bile salts sucrose medium (TCBS, Himedia, India) and cultured at 28 °C for 24 hours. The prevalent, loose colonies were further cultured in the TCBS medium under the same conditions for total DNA extraction. The presence of toxin genes was identi ed, and their morphological, physiological, and biochemical characteristics and antibiotic susceptibility were determined.

| Vibrio molecular identi cation
Total DNA extraction The Vibrio bacterial cell lines isolated from Red drum sh with haemorrhagic signs were grown proliferatively in the trypto-casein soy broth (TSB) supplemented with 2% NaCl and shaken at 180 rpm at 30 °C for 24 hours. Cell biomass was obtained by centrifugation at 8,000 rpm/min for 2 min at 4 °C. The total DNA of Vibrio cell lines was extracted with the modi ed phenol/chloroform method according to Neumann et al. (1992). The step using SDS/lysozyme or proteinase K was eliminated, and the bacterial cells were directly extracted with phenol (Neumann, Pospiech, & Schairer, 1992 ) (Table  1).

16S rRNA gene ampli cation with PCR and nucleotide sequence analysis
The 16S rRNA gene regions of bacterial strains isolated by using PCR with speci c primer pairs according to Frank et al. (2008) are 27F: AGAGTTTGATCMTGGCTCAG and 1492R: TACGGYTACCTTGTTACGACTT (Jeremy A Frank et al., 2008).
The PCR was performed on an Applied Biosystems-Life Technologies-Thermo Fisher Scienti c-USA system with the reaction components presented in Table 1 and the thermal cycle shown in Table 2.
The PCR product was subjected to electrophoresis on 1% agarose gels stained with ethidium bromide, and the electrophoretic images were analyzed with a DyNA Light, Dual Intensity UV Transillumninator system. Then, the PCR product of the 16S rRNA gene region was puri ed and directly sequenced with the Sanger method on an ABI PRISM® 3100 Avant Genetic Analyzer (Applied Biosystems) system at Maccrogen Company, Korea (dna.macrogen.com). The nucleotide sequence of the genomic region was determined and aligned by using the program Clustal-X (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) and edited by using the BioEdit 7.0.5 software (Hall, 1999). The nucleotide sequences of the genomic region were compared with the 16S rRNA sequences of the microorganisms published on the World GenBank (GenBank) with the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). The phylogenetic tree was generated according to the maximum likelihood algorithm of the MEGA X software.

| Physiological and biochemical characterization
The physiological and biochemical characteristics include Gram staining, oxidase, catalase, oxidase fermentation, bacterial motility, and hemolysis. The bacterial motility was determined with the hanging drop method and hemolysis by culturing on agar supplemented with 5% horse blood, with the Buller's method (2014) (Buller, 2004). The bacterial strains were tested by using the API 20E KIT (Bio-Mérieux, France) following the manufacturer's instructions. The reactions were carried out at 28 °C, and the results were read after 24 hours.
In addition, several biochemical reactions were also carried out to fully understand the characteristics of the isolated strains of Vibrio, namely, the Voges-Prosakauer test and the fermentation of carbohydartes with the purple Broth Base (Difco, UK) supplemented with 5% glucose, fructose, galactose, glucose, glycerol, maltose, mannose, or ribose. The viability of Vibrio strains at different salt concentrations was conducted on the TSB medium supplemented with 0, 1, 6, 8, and 10% NaCl. The Vibrio strains isolated in this study were compared with those isolated by Buller (Buller, 2004).

| Antibiotic susceptibility test
The antibiotic susceptibility of Vibrio spp. was tested with the agar plate diffusion method following Bauer et al. (1966) (Bauer, 1966). The bacterial strains Vibrio spp. were grown proliferatively in the TSB medium supplemented with 2% NaCl and inoculated at 28 °C for 24 h in a culture cabinet with a shaking speed of 150 rpm. Then, the bacterial cell density was determined by determining the optical density (OD) at 600 nm on a UV-VIS spectrophotometer (U2900, Hitachi, Japan). The preparation was diluted to a concentration corresponding to OD 1 and further to 10 6 CFU/mL for the following experiments.
One hundred micro-littre of the 10 6 CFU/mL bacterial solution was evenly spread on the Muhler Hilton agar-agar medium (MHA, Himedia, India), supplemented with 1.5% NaCl and dried at ambient temperature for 30 minutes. Next, the paper plates impregnated with antibiotics were transferred to the media with a pair of sterilized forceps. The antibiotics in the study are tetracycline 30 ug (Te), amoxicillin 10 ug (Ax), neomycin 30 ug (Ne), ampicillin 10 ug (Am), kanamycin 30 ug (Kn), doxycycline 30 ug (Dx), enro oxacin 5 ug (Ef), erythromycin 15 ug (Er), and cefotaxime 30 ug (Ct). The plates were placed in an incubator at 28°C. The diameter of the sterile ring was measured after 24 hours.
The bacteria's antibiotic susceptibility or resistance to antibiotics was evaluated from the diameter of the sterile ring following the standards of the Clinical and Laboratory Standards Institute (2018) (Sahu, Jain, Mishra, & Prasad, 2018).

Results And Discussion
3.1 | Physical, chemical water parameters and sample characteristics Table 3 displays water pH, temperature, salinity, and dissolved oxygen. The values are within the acceptable range for nurturing Red drums. Out of 35 samples collected from the sites, 27 are infected, accounting for 77.14%. The sh show the following symptoms: tail amputation, haemorrhaging in gills, body, and skin and uid accumulation in the abdominal cavity. The sh weight ranges from 23 to 25 g with a length between 11 and 16 cm (Table 4).

| Isolation and identi cation
In haemorrhaged Red drums (Fig. 2), blue and yellow colonies of Vibrio spp. appear on the TCBS medium (Fig. 3) after 24 h of culture. Different parts of the sh body exhibit different degrees of infection, from 11.11% in the brain to 38.89% in the liver (Table 5).

| Molecular identi cation and phylogenetic tree generation PCR product electrophoresis
The 16S rRNA region's PCR ampli cation of all 18 bacterial strains isolated from haemorrhaged Red drums collected at the study sites gives a single band with an ampli cation rate of 100%. All PCR products of bacterial strains are highly concentrated and sharp. The size of the PCR product is approximately 1,500 bp, consistent with the original expected size (Fig. 4).
Nucleotide sequence of the 16S rRNA gene region The nucleotide sequence analysis in the 16S rRNA gene region of the bacterial strains shows that the PCR ampli cation rate and the successful nucleotide sequence analysis rate equal 100%. The sequence edited with the Bioedit software results in the gene regions sized from 1381 to 1448 bp with a mean at 1441 bp. The occurrence rate of each type of nucleotide in the region indicates that guanidin (G) occupies the rst place (31.43-32.04%, mean 31.86%), followed by adenine (A) (24.93-25.35%, mean 25.19%) and timin (T) (uracin, (U)) being the last place (20.51-20.86%, mean 20.64%). The prevalence (G + C) accounts for 53.89-54.37%, mean 54.17% (Table 6).
The 16S rRNA gene region analysis with the MEGA X software shows that the conserved area for bacterial populations isolated from Red drums has 83/1448 modi ed nucleotide positions, accounting for 5,732% of the total length of the gene (Table 7).

Molecular identi cation
The BLAST results from the world gene bank show that the 18 bacterial strains belong to the genus Vibrio, with V. alginolyticus being the most signi cant occurrence (67%), followed by V. uvialis and V. orientalis with the same rate (17%). Their similarities range from 98.05 to 100%. All nucleotide sequences of the bacterial strains are registered on the world gene bank (Genbank Database) with corresponding reference codes (Table 8).

Phylogenetic tree
The phylogenetic tree generated with the UPGMA method shows the genetic relationships of the 18 strains of Vibrio bacteria isolated from Red drums (Fig 5).

| Biochemical characteristics of isolated bacterial strains
The 18 bacterial strains identi ed in Table 10 were subjected to their morphological, physiological, and biochemical characterization (Fig 6, Table 9).
The physiological and biochemical test with the API 20E KIT (Bio Mérieux, France) indicates that among the 18 strains causing haemorrhage in Red drums, 12 strains belong to V. alginolyticus, 3 to V. uvialis, and 3 to V. orientalis. According to the Buller's taxonomy key, these strains correspond to codes 414725, 3246126, and 4066106 (Table 8).
Speci cally, 67.67% of the strains have yellow colonies on the TCBS medium, belonging to the group of Gram-negative bacteria. They are comma-shaped ( Fig. 6. A, D), indole unproductive, capable of degrading nitrate, producing catalase, oxidase, and positive Voges-Proskauer reaction. They can also ferment glucose, sucrose, mannitol, sorbitol, and arabinose. One of these strains is capable of producing H 2 S (8.33%).
Three over eighteen (16.67%) bacterial strains have yellow colonies on the TCBS medium, belonging to the group of Gram-negative. They are short rod-shaped ( Fig. 6. B, E), oxidase-positive, capable of producing catalase and indole, fermenting glucose, mannitol, sucrose, and arabinose but not sorbitol. The rest 3/18 (16.67%) bacterial strains have blue colonies. They appear as short rods and belong to the group of Gram-negative bacteria ( Fig. 6. C, F), and they can grow at the NaCl concentration of 1, 6, and 8% but fail in 0 and 10% saline. The reaction is oxidase, catalase, mannitol, and arabinose positive but glucose, sorbitol, sucrose, and H 2 S-negative.

| Antibiotic susceptibility of Vibrio strains
All 18 strains of Vibrio are entirely resistant to ampicillin, amoxicillin, and erythromycin (Table 10). Generally, the Am resistance of Vibrio strains isolated from the marine environment is high (Stabili, Gravili, Boero, Tredici, & Alifano, 2010) Nguyen and Tu (2018) also reported that V. alginolyticus causing disease in the caged Cobia cultured in Kien Giang, Vietnam, is resistant to ampicillin, streptomycin, and erythromycin (Nguyen Bao Trung & Dung, 2018).
The isolates are also sensitive to tetracycline and doxycycline. Tetracycline is an antibiotic allowed in aquaculture, so farmers often use it to prevent and treat the diseases caused by bacteria in sh and shrimp farms. Our ndings agree well with those of Nguyen and Tu (Nguyen Bao Trung & Dung, 2018). Tetracycline has long been the most commonly used antibiotic in aquaculture in Korea, especially for the species severely infected by Vibrio (J.-W. Liu et al., 2006;Morris & Tenney, 1985). The less tetracycline-resistant Vibrio strains have also been reported in other studies. According to Liu et al. (2004), the V. alginolyticus strains causing disease in Cobia in Taiwan are sensitive to doxycycline, erythromycin, nalidixic acid, oxolinic acid, oxytetracycline, streptomycin, sulphonamide, and tetracycline but resistant to ampicillin and neomycin (P.-C. Liu, Lin, Hsiao, & Lee, 2004).

Conclusions
Our ndings indicate twelve V. alginolyticus strains, three V. uvialis strains, and three V. orientalis strains isolated from the haemorrhaged Red drums cultured in cages in Thua Thien Hue, Vietnam. These Vibrio strains have 16S rRNA gene homology on the GenBank, ranging from 98.05 to 100% and correspond to codes 4147125, 3246126, and 4066106. They are all susceptible to tetracycline and doxycycline. The V. uvialis and V. orientalis strains are entirely resistant to ampicillin, amoxicillin, and erythromycin.

CONFLICT OF INTEREST
Thre are no any con icts of interest regarding to this article and all of authors agreed to submit this manuscrip for publishing.

DATA AVAIL ABILIT Y STATEMENT
The authors declare that we do not have any shared data available ANIMAL ETHICS STATEMENT The use of experimental animals follows the regulation of ethics for animal use in scienti c work and was approved by international, and rules in Vietnam. The Hue University's scienti c Committee agreed for setting up the experiments and monitoring the outcomes by No. DHH2021-02-153.