Lactic acid bacteria (LAB) have been investigated as probiotics in the feed of terrestrial and aquatic farmed animals ^{1– 7} . This is based on the fact that LAB inhabit the human and animal gastrointestinal tract. This group of bacteria also convert lactose to acetic acid, thus decreasing gastrointestinal pH and naturally preventing colonization of harmful bacteria ⁸ . This is because they have the ability to produce the inhibitory materials that inhibit pathogen growth in the digestive tract ⁹ . Besides that, the probiotics can stimulate host immunity responses ^{2, 9, 10} .

LAB are a group of gram-positive, non-sporulating bacteria, in cocci or rod form. They can live in aerobic or facultative anaerobic conditions. LAB are able to produce lactic acid through the fermentation process of carbohydrates ^{11, 12} , and the optimum growth is at pH 5.5 to 6.0, so growth is restricted to neutral and alkaline conditions ¹³ . Those naturally occurring bacteria are non-pathogenic to humans and animals, hence being generally recognized as safe (GRAS) organisms ¹⁴ .

According to Parada et al. ¹⁵ and Carr et al. ¹⁶ 17l generas of LAB have been described worldwide. They are common in food products, for instance milk, meat, fruits, and vegetables, and also in genital and alimentary tracts of humans and animals ¹⁷ , including fish intestines ^{18, 19} .

Molecular approaches based on DNA such as 16S rRNA sequencing have been used to characterize the intestinal microflora, and this method is proven to be accurate and fast ²⁰ . For instance, Balcazar et al. ²¹ have identified LAB isolates from salmonid fish using 16S rRNA genes, and they found that there were 6 species of LAB, namely Lactobacillus curvatus, L. sakei, L. plantarum, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Carnobacterium maltaromaticum, and Leuconostoc mesenteroides. Sun et al. ²² investigated microbiota from gastrointestinal tracts of the orange-spotted grouper Epinephelus coioides, using a standard isolation procedure in combination with analysis of 16S rRNA sequences, and found several bacterial species, namely Vibrio parahaemolyticus, V. harveyi, V. metschnikovi, V. alginolyticus, Delftia acidovorans, Pseudomonas putida, Acinetobacter baumannii, Burkholderia cepacia, Erwinia carotovora, S. aureus, L. lactis, L. casei, E. faecium, Bacillus pumilus, B. clausii, and Psychrobacter sp.

Nursyirwani et al. ²³ successfully isolated three LAB from the tiger grouper alimentary tract, and found that LAB have antibacterial activity against Vibrio alginolyticus. However, the taxonomic classification of these bacteria has not been examined. Therefore, the objective of the present study was to characterize the three LAB isolates from tiger grouper based on their phenotype and genotype.

The three LAB isolates were categorized as cocci, gram-positive, non-motile, and non-spore forming. The LAB did not produce catalase, oxidase, and arginine dehydrolase, and were facultative anaerobes, with growth at 45°C at concentrations of 6.5% NaCl. However, differences were observed in the ability to produce acid from carbohydrates provided in the API 50 CH kit. Acid was not produced from substrates _L-arabinose, _D-raffinose, and _D-xylose by the KSBU-12C isolate, meanwhile the KSBU-5Da isolate did not use _L-arabinose, gluconate, glycerol, _D-mannose, sorbitol, _D-tagatose, and _D-xylose; and the KSBU-9 isolate used _L-arabinose, and _D-xylose. The cell morphology, and the physiological and biochemical characteristics of the isolates are presented in detail in Table 1.

The 16S rRNA derived from the LAB isolates were amplified by PCR with two universal primers 24F (5-‘AGAG TTT GAT CCT GGC T-3’) and 1540R (5-‘AAG G AGGT GAT CC AG CC GCA-3’). The LAB isolates have a highly similar 16S rRNA sequence (99% similarity as shown in Table 2; those were 1510-bp for KSBU-12C and 1526-bp for KSBU-5Da and KSBU-9) ( Figure 1). A phylogenetic tree was constructed for the LAB isolates sequences using Clustal W, followed by the Mega 5 neighbor-joining program as shown in Figure 2.

Based on morphological, physiological, and biochemical characteristics, the LAB isolates (KSBU-12C, KSBU-5Da, and KSBU-9) show similarity to three bacterial species of the Enterococcus genus found by Ludwig et al. ²⁸ . The analysis of the 16S rDNA sequence indicated that KSBU-5Da, KSBU-12C, and KSBU-9 isolates were of the Enterococcus genus, and with high similarity to E. hirae ATCC-9790 registered in GeneBank with accession number NR_075022.1 ( Table 2). Each of the LAB isolates indicated 99% homology with E. hirae ATCC 9790. Strains of bacteria with the same or more than 97% 16S rRNA gene sequence belonged to one species ²⁹ .

E. hirae ATCC 9790 is a gram-positive LAB used as a model organism in basic research for four decades ³⁰ . E. hirae was first isolated in the intestines of pigs and chickens ³¹ . E. hirae strain NRIC 0101 was collected by the Japan Nodai Culture Collection Center, NCCC ³² . In addition, E. hirae C17456 was a species isolated from chickens ³³ . In summary , Enterococcus hirae has been found in humans, poultry, foods for instant dosa batter, and in the environment ^{34– 37} . Together with E. faecium, E. faecalis, E.casseliflavus, and E. mundtii they are enterococci which were predominant in coastal waters and sediments of South of California ³⁸ . E. hirae was one of the homofermentative strains obtained from traditional fermentation products such as pla-som and pla-chom (fermented fish) in Thailand ³⁹ . It has been shown that E. hirae K34, L. plantarum K39, and L. plantarum K50 isolated from Kung-Som (fermentative shrimp) display antimicrobial activity against pathogenic bacterial strains of Bacillus cereus, E. coli, Staphylococcus aureus, Salmonella typhimurium, Vibrio cholerae, and Listeria monocytogenes ⁴⁰ .

Information on the use of E. hirae as a probiotic in aquaculture is still limited. Mazurkiewicz et al. ⁴¹ reported that E. hirae isolated from the intestine of common carp ( Cyprinus carpio L.) and applied into feed did not have a significant effect on the growth performance of the common carp. In contrary, another report by Adnan et al. ⁴² and Carlos et al. ⁴³ showed that E. hirae from freshwater fish Catla catla and rainbow trout Oncorhynchus mykiss had a significant effect on inhibiting the growth of pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella typhi and Pseudomonas spp.

In the present study, the role of E. hirae isolated from tiger grouper fish the growth performance of fish has not been evaluated yet. Therefore, further study will be needed to examine the effect of these bacteria on the feeding of cultured fish.

This research has successfully characterized three of the LAB isolates (KSBU-12C, KSBU-5Da, and KSBU-9) based on their phenotype and genotype. All isolates were determined to be E. hirae.

Sequenced DNA of LAB isolates can be found in NCBI GenBank, with accession numbers MF977716 to MF977718.