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Antimicrobial activity of herbal extracts against Vibrio spp. bacteria isolated from white feces syndrome on white leg shrimp (Litopenaeus vannamei) in some provinces in the mekong delta

Received 05 Mar 2021 Revised 30 Jun 2021 Accepted 19 Jul 2021 The study was conducted to determine the antibacterial activity of three herbal extracts: guava (Psidium guajava), leafflower (Phyllanthus urinaria L), beach daisy (Wedelia biflora (L.) DC ) against Vibrio spp. isolated from white feces syndrome infected shrimp in some provinces in the Mekong Delta. The antibacterial activity of the three herbal extracts was evaluated by the well diffusion method, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The results indicated that there were 102 isolates in total of 378 shrimp samples which were clarified into 7 groups. Among these group, Vibrio alginolyticus group were the most abundant species with the percentage of 50 %, followed by Vibrio cholerae group with the percentage of 17.7%. The herbal extracts of P. guajava and P. urinaria L showed a broad spectrum antibacterial activity against ten isolates which were selected for MIC and MBC test. The extract of P. urinaria L had the strongest antibacterial activity against V. harveyi CM3HPA2 and V. alginolyticus CM3IB2 (MIC of 0.2 mg/ml, MBC of 0.39 mg/ml). The obtained results indicated that the herbal extract of P. urinaria L will be a good candidate for reducing opportunistic pathogens Vibrio spp. abundant in gastrointestinal tract of shrimp. Keywords


INTRODUCTION
The Mekong Delta is the most productive area for both brackish and freshwater aquacultures in Viet Nam with high potential development. White leg shrimp (Liptopennaeus vannamei) is a type of shrimp that is widely cultivated in Viet Nam and also in some countries of Southern Asia as a source of rich nutrient food. The more intensive farming shrimps are grown, the more disease outbreaks occur. To improve production, farmers try to increase stocking density; therefore, it leads to disease outbreak easily. Aquatic animal diseases can be caused by bacteria, parasites, viruses, and fungi (FAO, 2018).
The white feces syndrome (WFS) which refers to the presence of a floating white fecal string in ponds rearing shrimp (Penaeus monodon & L. vannamei) has caused significant economic damage in the cultured shrimp industry in China, Indonesia, Malaysia, Thailand, Viet Nam, and other countries in Southeast Asia (Mires & Board, 2010). The syndrome is found to be associated with the opportunistic pathogens Vibrio spp in combination with other possible unknown pathogens (Aranguren et al., 2019).
The growth of several bacterial diseases was initially controlled by the use of antibiotics. It increases the risks of antibiotic residues in shrimp tissues and shrimp products. It is pointed out that the antimicrobial resistant agents of the Vibrio species isolated from the shrimps farming is high (Jeyasanta et al., 2017). In that case, herbal extracts gradually become the priority as good alternative prevention for aquatic diseases due to its natural product availability.
There is a possibility for developing new antiviral drugs against white spot syndrome virus (WSSV) infection from P. guajava (Velmurugan et al., 2012). Other studies have proved that Phyllanthus urinaria L has pharmacological effect such as hepatoprotective (Hau et al., 2009), anticancer (Huang et al., 2006) based on that it includes flavonoids, tannins, and other benzenoid constituents (Liu et al., 1999). Several medical plants used as shrimp feed supplement include bael fruit (Aegle marmelos), garlic (Allium sativum), Indian birthwort (Aristolochia indica), Indian lilac (Azadirachta indica), golden shower tree (Cassia fistula), madagasca periwinkle (Catharanthus roseus), turmeric (Curcuma longa) (Balasubramanian et al., 2007). Therefore, this study was conducted to evaluate the antimicrobial activity of Psidium guajava L., Phyllanthus urinaria L. and Wedelia biflora (L.) DC. for reducing opportunistic pathogens Vibrio spp. in the gastrointestinal tract and improving disease resistance of shrimp with highly promising to the aquaculture industry.

Sample collection
Shrimp samples were collected from 63 ponds of farms in Dam Doi, Cai Nuoc, Phu Tan districts -Ca Mau province, Thanh Phu district -Ben Tre province, My Xuyen, Vinh Chau districts -Soc Trang province. From each pond, 3 diseased shrimps (the presence of gross signs such as reduced feeding, retarded growth, floating white fecal string in ponds and in the feeding tray, etc.) and 3 healthy shrimps were collected. The shrimp samples were collected at shrimp farm. Shrimp samples were put into plastic bags with oxygen supplied and transported to the laboratory.

Bacteria isolation and storage
First of all, shrimp samples were put on clean trays for observing and recorded the external and internal clinical signs. Secondly, the inoculating loop was used to get samples of bacterial from the intes-tine, hepatopancreatic of shrimp specimens and then streaking onto the Thiosulfate Citrate Bile Salts Sucrose agar (Merck) plate. After that, the plates were incubated for 24 hours at 28 o C. The dominant bacterial colonies were subcultured onto Trypto-casein Soy Agar supplemented with 1.5% NaCl (TSA+1.5% NaCl) media and incubated for 24 hours at 28 0 C. A pure well-isolated colony was selected and incubated into Brain Heart Infusion Broth supplemented with 1.5% NaCl (BHIB+1.5% NaCl) for 24 hours at 28 0 C. These served as stock cultures by mixing with glycerol (20%). The stock cultures were stored in the lowest compartment of the freezer (-80 0 C) (Lightner, 1996).

Bacterial identification
Bacterial identification was done by primary tests including (Gram staining, motility, oxidase, and catalase). The commercially available kit API 20E (BioMerieux, France) was used for determining the biochemical profiles of isolates. The 16S rRNA gene of these isolates was sequenced and compared to GenBank database -NCBI BLAST for species identification. According to information on clinical signs recorded from farmers in different locations, 22 isolates with typical clinical signs were randomly selected for identification.

Herbal extraction
P. guajava, P. urinaria L and W. biflora (L.) DC extracts were supplied by Vibo Company. Firstly, the collected of whole plant samples were cleaned to get rid of dust particles, then were cut into small pieces and ground into fine powder by an electrical blender. The powder samples were macerated in methanol 96% (the ratio 1:10) for 3 days at room temperature. After that, the extracted solutions were filtered through Whatman no. 1 filter paper and released the solvent by a rotary vacuum evaporator at 40 o C.

Antibacterial activity
Ten isolates selected randomly from 22 identified isolates were used to evaluate the antibacterial activities of herbal extracts by the standard well diffusion method (Magaldi et al., 2004). A cotton swab was dipped into the bacterial suspension of approximately 1 x 10 8 CFU/mL tube and streaked on TSA+1.5% NaCl discs. The wells were made by a 6 mm diameter standard sterile cork. The herbal extracts were diluted with dimethyl sulfoxide (DMSO) solution to reach the final concentration at 100, 50 and 25 mg/mL. These wells were filled up with 50 μL of herbal extracts. The plates were in-cubated at 28 o C for 24 hours. DMSO was used as a negative control. Antibacterial activity was determined by measuring the diameter of the inhibition zone (Oonmetta-aree et al., 2006).

Minimal inhibitory concentration (MIC)
Two herbal extracts (P. guajava, P. urinaria L) were selected for the Minimum Inhibitory Concentration (MIC) test. In the broth dilution method, 3 mL of herbal extract in the BHIB+1.5% NaCl solutions were prepared by two fold dilutions from 25 to 25/1024 (0.02) mg/mL (10 times) in the test tubes. The standardized bacterial suspension of 10 isolates (1x10 6 CFU/mL) was inoculated in the series of broth dilution at 28 o C for 24 hours. MIC was determined as the herbal extracts that completely inhibit the growth of bacteria on the lowest concentration (Oonmetta-aree et al., 2006).

Minimum bactericidal concentration (MBC)
Thirty µl of ten isolates from MIC asays were streaked onto TCBS plate, and counted colonies after 24-hour incubation for 28 o C. MBC was determined as the lowest concentration of the herbal extracts in which no bacteria growth (Oonmettaaree et al., 2006).

Data analysis
The average value, percentage and standard deviation were analyzed by using Microsoft Office Excel 2010 software and the 16S-RNA gene sequenced results of isolates were compared to Gen-Bank database -NCBI Blast.

Bacterial isolation and identification
A total of 63 shrimp ponds including 39 farms in Ca Mau province (located in Dam Doi, Cai Nuoc, Phu Tan districts), 10 farms in Soc Trang province (located in My Xuyen, Vinh Chau districts), and 14 farms in Ben Tre province (located in Thanh Phu district) from October 2019 to August 2020. The clinical signs included an empty and discontinuous gut, a paler and white hepatopancreas, retarded growth, reduced feeding, some ponds showed white feces strings in the feeding trays according to information obtained from the farmers. There were 102 isolates in total 378 shrimp samples collected from shrimp ponds. According to phenotypic characteristics, colony morphology on TCBS and TSA+1.5% NaCl media, these isolated strains were clarified into 7 groups (Table 1).
Presently, the causative agents of white feces syndrome in shrimp remains unknown. This syndrome has been associated with a microsporidian -EHP (Enterocytozoon hepatopenaei) (Rajendran et al., 2016). In addition, EHP also was proposed as the causative agent in P. monodon (Ha et al., 2013) and L. vannamei (Tang et al., 2016). However, it was failed to illustrate white feces syndrome in the experiment infected EHP (Tangprasittipap et al., 2013). Woraprayote et al. (2020) reported that Vibrio abundances increase in shrimp haemolymph and intestine has been also related to white feces syndrome.
Among these groups in this study, V. alginolyticus (B2 group) was the most abundant species with the percentage of 50 % and V. cholerae (B1 group) was the second high abundant species with the percentage of 17.7%. Similarly, V. alginolyticus and V. fluvialis have been recorded as the major pathogen contribute the occurrence of white feces syndrome in grow-out ponds of P. monodon in Sri Lanka (Sandaruwan Kumara & Hettiarachchi, 2017). Moreover, Cao et al. (2015) found that V.
cholerae isolate was a pathogen for white feces syndrome on L. vannamei in China.

Screening of antimicrobial activity of different herbal extracts on ten isolated Vibrio spp. bacteria
The antibacterial activity of herbal extracts using standard well diffusion method (Magaldi et al., 2004) with the results shown in Figure 3 and Table  4. The results indicated that three tested herbal extracts exhibited different antibacterial activities against ten isolates. Noticeably, among three tested herbal extracts, P. urinaria L showed the highest antibacterial activity against all ten isolates with the growth inhibition zone fluctuated from 12.7 to 24.85 mm. In detail, the results pointed that the highest antibacterial activity against V. alginolyticus CM3IB2 with the inhibition zone was 24.85 mm, 23.4 mm, 20.45mm at three concentrations.
Besides, DMSO played a role as the negative control of all tests showed the zone at 0 mm (Table 4).
In this study, P. guajava showed the intermediate antibacterial activity with the highest inhibition zone were 22.95, 20.7, 17.7 mm on V. alginolyticus CM3IB2 (Table 4). The extract of W. biflora (L.) DC showed the lowest antibacterial activity than the other two herbal extracts with the highest inhi-bition zone were 16.45, 14.35, 11.45 mm on V. parahaemolyticus BTIA1 and the lowest inhibition zone on V. cholerae CM1HPB1 (Table 4).
In addition, mangrove leaf extract (Rhizophora apiculata) had the best effect on V. parahaemolyticus with the inhibition zone of 5.61 mm at 700 mg/L (Supono et al., 2019). According to Widowati et al. (2018), microalgae Dunaliella salina and Tetraselmis chui were applied as biocontrol agents for 21 days showing a decrease of bacteria amount and its capable against Vibrio. Moreover, supplementation of hen egg white lysozome (HEWL) at 0.125 g/kg into feed was demonstrated as an effective method not only to avoid antibiotic treatment in aquaculture but also stimulated resistance to Vibrio infection as well as white feces syndrome (Woraprayote et al., 2020). Notes: Growth inhibition zone: Resistant ≤ 9 mm; Intermediate: ≥ 10 -13 mm; Susceptible: ≥ 15 mm (Chaweepack et al., 2015)

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
MIC and MBC were determined based on the results of the antibacterial activity test. Amongst tested herbal extracts, P. guajava and P. urinaria L with higher antibacterial activity were continuously tested to determine MIC and MBC. The result is shown in As reported by Canillac and Mourey. (2001), the ratio of MBC/MIC is equal to or less than 4 means that the extraction considered bactericidal ability. On the other hand, this ratio is larger than 4 that shows the bacteriostatic ability. In this study, P. urinaria L extract showed higher values of the ratio of MBC/MIC compared to P. guajava on all ten isolates with the MBC/MIC ratio values was 4 on V. parahaemolyticus BTIA1, V. harveyi CM3HPA2, V. alginolyticus CM3IB2 while it was 16 only on V. vulnificus CM2HPA4, and the ratio values were 8 on 6 isolates other.

Conclusions
There were 102 isolates in total shrimp samples which had clinical signs of white feces syndrome. Among these groups, V. alginolyticus group was the most abundant species with the percentage of 50 %, followed by V. cholerae group with a percentage of 17.7%. The herbal extracts of P. guajava, P. urinaria L showed a broad spectrum antibacterial activity against 10 isolates in diseased shrimp.
The extract of P. urinaria L had the strongest antibacterial activity against V. harveyi CM3HPA2 and V. alginolyticus CM3IB2 (MIC of 0.2 mg/mL, MBC of 0.39 mg/mL).

Recommendations
Among isolated bacterial strains, V. alginolyticus group was the most abundant species with a percentage of 50%. It should be carried out in the challenge test for the confirmation of the possible causative agents and the recurrence of white feces syndrome. The herbal extracts of P. guajava, P. urinaria L should be further studied in in vivo studes.