Identification, Growth Profile and Probiotic Properties Of

Biocontrol Science, 2019, Vol. 24, No. 1, 1－11

Original Identification, Growth Profile and Probiotic Properties of Autochthonous Intestinal Bacteria of Sagor catfish （Hexanematichthys sagor）

PANG SING TUNG＊, JULIAN RANSANGAN, AND KISHIO HATAI

Microbiology and Fish Disease Laboratory, Borneo Marine Research Institute, University Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia

Received 2 April, 2017/Accepted 22 February, 2018

The prevalence of antibiotic resistant bacteria in aquaculture has reached alarming proportions and intensified the search for microbe derived antimicrobial compounds. This study isolated bacteria from the intestine of Sagor catfish（ Hexanematichthys sagor） and screened it for antagonistic properties. Five out of 334 bacterial isolates inhibited growth of fish pathogens. The 5 bacterial strains included relatives of Shewanella haliotis, Myroides odoratimimus, Vibrio harveyi, Vibrio alginolyticus and Alcaligenes faecalis. The growth profiles and probiotic properties of these bacteria were examined. The results showed that the isolate 9（ 3） 7.5.2.1, whose closest relative was S. haliotis exhibited growth and probiotic advantage compared to the other bacterial strains, such as highest doubling time and the ability to survive at all experimental temperatures（ 18 to 60℃）, and bile concentrations（ 0.01 to 1.00%） and pH（ pH2 to 9）. While the bacteria with probiotic properties were successfully isolated. Further study is necessary to examine the efficiency of the probiotic candidate bacteria in boosting fish immunity against pathogens.

Key words ： Intestinal bacteria / Growth proﬁle / Antagonistic activity / Probiotic properties / Shewanella haliotis.

INTRODUCTION larly viable to investigate the probiotic properties of the intestinal flora of the marine catfish due to its carnivo- Fish disease outbreaks resulted in huge losses in rous and predacious feeding behaviour（ Parida et al., aquaculture production. Diseases are controlled almost 2015）. This study describes the probiotic properties of exclusively using chemicals or antibiotic drugs world- bacterial flora isolated from the intestine of the tropical wide（ Hai, 2015; Anand et al.,2011; Primavera, 2006）, Sagocatfish, Hexanematichthys sagor. and the effect of these on human populations constitute a major public health concern（ Lupin 1999）. MATERIALS AND METHODS Alternative to chemicals or antibiotics are necessary. Probiotics are recognized as one of the alternative ther- Screening of Bacteria apies for fish health management in aquaculture（ Ige, A total of 20 Sagor catfish（ Fig. 1） were captured using 2013; Panigrahi et al., 2007）, as these are beneficial to hook and line from Mangkabong Bay（ 6°08'34.4"N fish health, minimize disease risk in fish and improve 116°12'23.2"E）, Malaysia. The fish were dissected right aquaculture production（ Hai, 2015; Micheal et al., 2014; after the fish were delivered to the fish disease labora- Hong et al., 2005; Nikoskelainen et al. 2003）. tory at University Malaysia Sabah. The high potential of microflora in marine fish intes- The abdomen of each fish was cut open and the tines as a cure for disease has amazed microbiologists intestines were cut into pieces（ approximately 0.5 cm）. （Jayanth et al., 2002; Sugita et al., 1991）. It is particu- About 10 pieces of intestine were randomly selected and homogenized using a sterile mortar and pestle in ＊Corresponding author. Tel: +601116880804, E-mail : kisia_ the presence of 1 ml Phosphate Buffered Saline（ PBS） pang（a）yahoo.com solution（ pH7.2）. One mililiter of homogenate was 2 P. S. TUNG ET AL.

FIG. 1．The Sagor ﬁsh which was captured using hook and line from Mangkabong Bay, Malaysia.

FIG. 3．Gram staining of the bacterial isolates obtained from Sagor catfish intestine. The isolates with laboratory reference numbers 9（3）7.5.2.1（ above） and 6（2）1.3.2（ below） are shown as representative of Gram-negative bacilli and Gram- FIG. 2．Purification of the Sagor catfish intestinal bacteria on positive cocci, respectively. the Tryptic Soy Agar plate. Colonies of the isolate whose laboratory reference number 8（3）1.2.1, cultivated for 2 days at 27℃, is shown as a representative to differentiate the bacteria isolated from marine and freshwater fish. mixed with 9 ml of sterile PBS solution. The dilution was Gram staining（ Johnson and Case, 2001） was repeated 10 times and 100 µl of mixture was spread performed to confirm the purity of the bacterial isolates. evenly on Tryptic Soy Agar（ TSA）（ Merck, Germany） Bacteria which appeared purple under the light micro- using a sterile bend rod stick. scope were recorded as Gram-positive; while those All the TSA plates were incubated at room tempera- which looked pink were noted as Gram-negative. The ture（ 28℃） and the bacterial growth was monitored at bacteria were compared based on their cell morphology 24 hours intervals. Bacterial colonies exhibiting different and the results of the Gram staining（ Fig. 3）. Strains shapes or colours were picked and serially isolated until which did not present the same morphology or colours a single colony was obtained（ Fig. 2）. The isolation of were subjected to the isolation process again until the the bacterial colonies was conducted every day consec- pure bacteria were obtained. Subsequently, the bacteria utively for 15 days. However, newly grown bacteria which were briefly subjected to oxidase, motility and catalase exhibited the same shapes and colours were excluded. tests devised by Chauhan（ 2012）. Replicates of each The laboratory reference number was labelled based bacterial isolate were subsequently stored at 4℃ and on the number of streaking. For example bacteria 9（3） -80℃ following Lauber et al.（ 2010）. For short term 7.5.2.1, the 9 means this bacterial was the ninth picked storage, bacteria were cultured at 4℃ in semi-solid TSA bacteria colony from the first isolation. The second whose concentration was 3% of the original formula. number, 3 meaning that this bacterial was the third For long term preservation, overnight bacterial culture colony from second isolation, and so on. There are all was suspended in 70% Tryptic Soy Broth（ TSB） and together 6 numbers, so this bacterial was screened for 30% glycerol in cryovial tubes before being stored at 6 times before getting to the pure colony. The bracket -80℃. at the second number showing that this bacteria is Subsequently the bacterial isolates were grown on isolated from marine Sagor catfish. This is for the author selective media, including Thiosulphate-citrate-bile INTESTINAL BACTERIA 3

TABLE 1．Details of pathogens used in this study Pathogen Host / Case Origin Strain Reference Streptococcosis Streptococcus agalactiae developed tilapia Malaysia KT 869025 Nur-Nazifah et al.（ 2017）（Oreochromis niloticus） Aeromonas caviae NA NA ATCC 15468 Ruimy et al.（ 1994） Japanese Ee（l Anguilla Nagasaki University, Aeromonas hydropila A 10 Kanai and Takag（i 1986） japonica） Japan Aeromonas salmonicida NA NA ATCC 33658 Ruimy et al.（ 1994） Universiti Malaysia Seabass Ransangan and Mustafa Vibrio harveyi Sabah, Sabah, JR 7 （Lates calcarifer）（2009） Malaysia Poisoned food related Vibrio parahaemolyticus Japan ATCC 17802 Ruimy et al.（ 1994） to Shirasu case Spoiled horse mackerel Vibrio alginolyticus Japan ATCC 17749 Ruimy et al.（ 1994） ﬁsh Photobacterium damselae Marine ﬁsh Japan ATCC 51805 Kimura et al.（ 2000） Note: The full spelling of NA is Not Available. The related information is not found on the internet.

salts-sucrose agar（ TCBS）, Mac Conkey（ Merck, catfish were cultured on TSA overnight at room temper- Germany） and Pseudomonas isolating agar（ Merck, ature（ 36℃）. The overnight bacteria colonies were Germany）. This selective media test was conducted in collected and dissolved in 0.5 ml of TSB. In order to accordance with the method formulated by Pfeffer and concentrate the bacteria in the broth, the cultures were Oliver（ 2003）. centrifuged at 13,000 rpm for 2 minutes and the supernatant discharged. Antagonism Assay The extractions of bacterial DNA was conducted using The antagonistic properties of the bacteria were the standard CTAB DNA extraction method as employed examined using the disc diffusion method utilised by by Nishiguchi and Doukakis（ 2002）. A total of 3 ml of Zaidan et al.（ 2005）. Overnight pathogen cultures bacterial culture was spun down and the supernatant （adjusted to OD600 of 0.2） were spread on the TSA removed. Then, 600 µl of CTAB solution was added using sterile cotton swabs. Then the plates were dried and vortexed for a few seconds. After that, the mixture for 5 minutes prior to the antagonistic assay. A total of was incubated at 75℃ for 5 minutes in the case of the 100 µl of each filter-sterilized culture fluid was absorbed for Gram-negative bacteria, and for while 30 minutes in by a 9 mm diameter sterile disc（ Whatman filter paper the case of the Gram-positive bacteria. After incubation, No.1）. These discs were aseptically placed on inocu- the mixture was cooled to room temperature before lated TSA. The TSA with blank disc（ without bacteria） vortexing and spinning down. Then, 700 µl of chloro- served as control. Each bacterial sample was examined form was added to the mixture before vortexing for 25 in triplicate. seconds and centrifuging at 12,000 rpm for 5 minutes. Eight bacterial pathogens including Vibrio harveyi, V. After the centrifugation, the upper aqueous phase of parahaemolyticus, V. alginolyticus and Photobacterium the mixture was transferred into a new 1500 µl tube and damselae, Streptococcus agalactiae, Aeromonas then 100 µl CTAB and 900 µl of distilled water were caviae, A. hydrophila and A. salmonicida were tested in added into the tube. The mixture was mixed well by this assay. The details of the pathogens used in this vortexing before being incubated at 75℃ for 5 min. It study are listed in TABLE 1. The TSA were incubated at was then cooled down to room temperature before being 37℃ and monitored for 10 days. The inhibition zone centrifuged again at 12,000 rpm for 5 minutes. Then was measured using a Vernier caliper at 24 hour inter- the supernatant in the tube was transferred into a new vals and recorded according to the methods of Zaidan tube with 300 µl of 95% ethanol. The mixture was mixed et al.（ 2005）. by vortexing for 10 seconds before being centrifuged at 12,000 rpm for 5 minutes. Lastly, the supernatant was Polymerase Chain Reaction（ PCR） removed and the remaining white pellet which was Extraction and purification of DNA sticking on the tube was dried at room temperature Bacteria isolated from the intestines of the Sagor （27℃）. A total of 50 µl TE buffer was added into the 4 P. S. TUNG ET AL.

tube and mixed well with the crude DNA extracts（ white well before another ml of diluted bacteria was trans- pellet） before being stored at 4℃. ferred to the second test tube contained 9ml of distilled water. The same procedure was repeated until the 10th Primer and Master Mix test tube. After that, 100 µl of diluted bacteria was One µl of each 10 µM forward and reverse oligonu- transferred from each test tube into the TSA. The diluted cleotide primers（ IDT, Singapore） were added into 48µl bacteria was spread evenly using sterile bended rod of Master Mix（ Promega, Madison, Wisconsin）（ TABLE stick. The TSA was sealed and incubated over night at 5）. The PCR was carried out in a thermocycler with a room temperature. The colony forming unit in one millil- heater lid for 1 cycle at 95℃ for 1 minute, then 30 itre（ CFU/ml was calculated on the next day following cycles were run at 95℃ for 1 minute, 58℃ for 1 minute the formula below: and 72℃ for 1 minute. Upon conclusion of the thermal cycling, reaction mixtures were held at 72℃ for 5 Number of bacteria colonies X Dilution factor CFU/ml = minutes and cooled to room temperature. PCR samples Volume of culture plate were electrophoresed in a 1.5% agarose gel containing 4.0 µg ml-1 ethidium bromide and visualized on an ultra- violet transilluminator. The PCR products were purified Heat Tolerance using a AccuPrepTM PCR purification Kit（ Bioneer The heat tolerance assay was conducted according Corporation, Korea） in accordance with the manufac- to the method devised by Kim et a（l 2001）. Prior to this turer’s instruction. assay, bacteria were grown to log phase on the TSA and kept at room temperature（ 27℃）. Cloning and Sequencing To begin the assay, a colony of bacteria was picked After purification, two microlitres（ 2 µl） of the PCR from the TSA and cultured in the TSB until the log phase product were ligated into a cloning vector pGEM-T easy of the bacteria. The Colony Forming Unit（ CFU/ml-1） of Vector（ Promega, USA） and transformed into the the bacterial culture was determined before it was chemically competent Escherischia coli JM109 exposed to various temperatures（ 18, 25, 37, 45, 50, （Promega, USA） the next day. The recombinant plas- 55 and 60℃） for an hour. Triplicates of the control and mids were purified using a DNA spinTM Plasmid DNA every examined bacterial were exposed at each stated Purification Kit（ iNtRON Biotechnology, Korea）. Finally, temperature and a TSB without bacteria culture was set all plasmids were sent to AIT, Singapore Pt Ltd for bidi- as control in this assay. After an hour of heat exposure, rectional sequencing to get all the nucleotide sequences the CFU in 1 ml of five selected intestinal bacteria strains of the 16S rRNA genes. was again determined. Increase or reductions in examined bacterial density were determined through obser- BLAST analysis vation of changes in the CFU after heat treatment. The homology search for the DNA sequences of the bacterial isolates in this study（ against DNA sequences Bile Tolerance in the public databases） was carried out using the The bile tolerance test was carried out following the Basic Local Alignment Search Tool（ BLAST）（ http:// method described by Buntin et al.（ 2008）. A batch of blast.ncbi.nlm.nih.gov）. 10 ml TSB tubes containing 0 – 1.0% bile（ Sigma, USA）（ w/v） was prepared. For each bacteria selected Growth Profile for experimentation, 5 ml of log phase bacteria were Five intestinal bacteria strains which exhibited strong inoculated into the TSB. After incubation in different antagonism were selected for growth profiling for 36 concentrations of bile for 3 hours at room temperature hours. For every selected bacterium, 5 µl of an over- （27℃）, the bacteria from the TSB tubes were inocu- night culture was inoculated in a tube containing 8 ml of lated on the TSA. Bacterial growth on the TSA the next Typtic Soy Broth（ TSB）. The tubes were incubated at day indicated a positive result, whereas no bacterial room temperature（ 27℃） and the optical density（ OD） growth indicated a negative result. was recorded at 600 nm at 1 hour intervals over a 36 hour period. The experiment was carried out in triplicate Acid Tolerance and the OD readings averaged. In order to examine how long the bacterial isolates On the next day, the concentration of bacteria were were able to survive at different pH levels, an acid toler- adjusted to the recorded OD before subjected to 10 ance test was conducted by inoculating 5 ml of log fold serial dilution. The 10 fold serial dilution was starting phase bacteria in 10 ml of TSB containing 0.01 – 1% with 1 ml of bacteria was transferred into 9 ml of sterile hydrochloric acid（ w/v）（ Sigma, USA）. To increase the distilled water in the first test tube. The mixture was mix pH value, sodium hydrochloric（ NaOH） was added into INTESTINAL BACTERIA 5

the broth. The bacteria were incubated for 1 and 24 hours at room temperature. After incubation, bacteria from the TSB tubes were inoculated on TSA. From observation the next day, the growth of bacteria on the TSA indicated a positive result; while no bacterial growth indicated a negative result.

RESULTS

Bacterial composition A total of 334 strains of bacteria were isolated from the intestines of Sagor catﬁsh in this study. The isolated FIG. 4．Antagonistic activity of the bacterial isolates obtained bacteria, were found to consist of Gram-positive cocci from Sagor catfish intestine. The result of a disc diffusion （39.52%）, Gram-positive bacilli（ 24.25%）, Gram- method, in which antagonism of the isolate 8（3）7.3.1 and 7 negative bacilli（ 19.76%） and Gram-negative cocci （3）6.1.2 against Vibrio alginolyticus are examined, was （16.47%）. shown as a representative.

Antagonistic Assay To reduce the number of bacteria, all samples were bacteria decreased at the end of the experiment（ 35th examined for their antagonistic ability towards fish hour） indicating a decline in the growth rate of the pathogens. The results show 2.69% of Sagor catfish bacteria（ Fig.6）. intestinal bacteria consist antagonistic abilities towards In order to minimize the bias in stress examination, the pathogens in this study. However, only 5 bacteria the selected bacteria were examined for their growth strains displaying the highest antagonism activity parameters. The experiment to analyse the growth towards pathogens were selected for further analysis. proﬁle was conducted at room temperature（ 27℃） over Only the bacteria displaying antagonistic activity a 36 hours. Each strain of bacteria was prepared in a towards pathogens（ Fig. 4） were listed in TABLE 2. The bijou bottle and the OD was adjusted at 0.2 prior to data shown in TABLE 2 represents the minimum values starting the examination. Based on the result shown in （mm） derived from the triplicate data. TABLE 6, bacteria 9（3）7.5.2.1 displayed the highest growth rate（ 0.4） within the first 13 hours with the Characterization of Bacteria highest biomass yield（ 4.9）. It also had the shortest Selected bacteria were examined using Gram staining, doubling time（ 1.7） compared to the other selected biochemical tests and selective media tests. All of these bacteria. strains were Gram negative. The phenotypes of the selected bacteria are listed in TABLE 3. Heat Tolerance The selected bacteria were examined for their toler- Assessment of closest relatives of bacteria sequences ance to different temperatures ranging from 18 to 60℃. The selected bacteria strains showed high gene simi- Based on the results shown in TABLE 7, four bacterial larity to the Shewanella haliotis, Myroides odoratimimus, isolates（ 9（3）7.5.2.1, 8（3）1.2.1, 6（9）8.4 and 7（3） V. harveyi, V. parahaemolyticus and Alcaligenes faecalis 6.1.2） showed tolerance to temperatures up to 60℃. strains. The accession number of the bacteria in However, the bacterium 9（2）7.3.2 was only able to GenBank, their nucleotide similarity and the closest survive up to 45℃. relatives of the selected bacteria are listed in TABLE 4. The phylogenetic tree for the selected bacteria is shown Bile Tolerance in Fig. 5. The selected bacteria were examined for their tolerance to bile, whose concentrations ranged from 0.01% Growth Proﬁles to 1.00%（ w/v）. Bile tolerance results for the selected It was noticed that the isolate 9（3）7.5.2.1 multiplied bacteria are shown in TABLE 8. rapidly compared to the other bacteria. Most bacteria reached log phase at the second hour of incubation and pH Tolerance reached the stationary phase at sixth hour. Interestingly, The selected bacteria were examined for their toler- the isolate 9（3）7.5.2.1 reached log phase at the first ance to pH ranging from pH2 to pH9. The acid hydro- hour and entered stationary phase at the third hour. chloric（ HCl） was added into the TSB to decrease the Nevertheless, the absorbance value（ OD） of all the pH value; the sodium hydroxide（ NaOH） was added to 6 P. S. TUNG ET AL.

TABLE 2．Antagonistic activity of the ﬁve selected bacteria against ﬁsh bacterial pathogens Lab V. parahae- reference S. agalactiae A. caviae A. hydrophilla A. salmonicida V. harveyi P. damselae V. alginolyticus molyticus number 8（3）1.2.1 10 11 Day 0 6（9）8.4 13 7（3）6.1.2 11 8（3）1.2.1 15 11 7（3）6.1.2 10 Day 1 6（9）8.4 12 9（3）7.5.2.1 11 8（3）1.2.1 16 10 13 10 Day 2 7（3）6.1.2 15 15 6（9）8.4 12 9（3）7.5.2.1 11 12 12 8（3）1.2.1 15 14 14 12 Day 3 9（2）7.3.2 13 6（9）8.4 13 7（3）6.1.2 13 9 19 13 11 13 9（3）7.5.2.1 18 11 15 8（3）1.2.1 13 11 12 13 Day 4 9（2）7.3.2 13 6（9）8.4 14 7（3）6.1.2 13 12 Day 5 9（3）7.5.2.1 15 14 17 Day 6 9（2）7.3.2 8（3）1.2.1 12 15 9（2）7.3.2 14 12 8 Day 7 6（9）8.4 16 10 7（3）6.1.2 17 8 9（3）7.5.2.1 18 15 17 8（3）1.2.1 18 Day 8 9（2）7.3.2 15 6（9）8.4 14 12 7（3）6.1.2 20 13 9（3）7.5.2.1 15 20 21 8（3）1.2.1 18 Day 9 9（2）7.3.2 13 18 6（9）8.4 18 7（3）6.1.2 20 14 9（3）7.5.2.1 17 16 15 18 8（3）1.2.1 18 12 16 18 Day 10 9（2）7.3.2 18 13 12 6（9）8.4 20 17 20 7（3）6.1.2 16 14 13 The empty cells represent no antagonism activity of the bacteria towards pathogen. INTESTINAL BACTERIA 7

TABLE 3．The phenotypes of 5 selected intestinal bacteria from Sagor catfish intestine Biochemical tests Selective media tests Lab reference Gram Morphology Mac Pseudomonas number staining Motility Oxidase Catalaze TCBS TSA Conkey isolation agar 9（3）7.5.2.1 - bacillus + + + + - + + 8（3）1.2.1 - bacillus + + + + - + + 9（2）7.3.2 - bacillus + + + + + - + 6（9）8.4 - bacillus + + + - + + + 7（3）6.1.2 - bacillus + + + - - - + Note: The + at selective media tests shows growth of bacteria on specific media; - means no growth of bacteria on the specific media

TABLE 4．Identity of the selected intestinal bacterial strains Laboratory reference Nucleotide Accession number Closest relative species number similarity 9（3）7.5.2.1 MF 175883 99% Shewanella haliotis strain CE08 8（3）1.2.1 MF 175885 99% Myroides odoratimimus strain PR63039 9（2）7.3.2 MF 175882 99% Vibrio harveyi isolate EHP7 6（9）8.4 MF 175884 99% Vibrio parahaemolyticus strain BF-1 7（3）6.1.2 MF 175881 99% Alcaligenes faecalis strain LZU-52

FIG. 5．Phylogenetic tree inferred from the 16S rRNA gene nucleotide sequences by using the neighbor-joining method. Five antagonistic isolates obtained from Segor catﬁsh intestine as well as their closest relatives are included. Scale bar, nucleotide substitutions 8 P. S. TUNG ET AL.

TABLE 5．Chemicals and quantity needed for 6 Shewanella haliotis the master mixture in the DNA extraction Alcaligenes faecalis 5 Chemicals Quantity Vibrio harveyi Myroides odoratimimus 4 Bacteria DNA 2.0 µl Vibrio alginolyticus m n Forward primer 1.0 µl 3 60 0 at Reverse primer 1.0 µl c e

a n 2 b r

dNTP 1.0 mM o s A b Taq polymerase 0.5 U 1 Magnesium chloride 3.4 mM 0 Distilled water 31.1 µl 0 1 2 3 6 8 9 10 11 12 13 35 36 5 x PCR buffer 10.0 µl -1 Time (h) Total volume 50 µl FIG. 6．Growth proﬁles of the antagonistic bacterial isolates obtained from Sagor catﬁsh intestines cultured in tryptic soy broth. The standard deviation shows the different of data from TABLE 6．Growth parameters of selected bacteria the mean value. -1 Laboratory Growth parameters（ h ） reference number µ λ A t d increase the pH value. The pH value was measure using 9（3）7.5.2.1 0.4 4.7 4.9 1.7 pH meter. Results for the selected bacteria are indicated 8（3）1.2.1 0.2 2.7 2.9 2.9 in TABLE 9. 9（2）7.3.2 0.1 1.6 1.7 4.9 6（9）8.4 0.3 3.5 3.8 2.2 DISCUSSION 7（3）6.1.2 0.1 1.6 1.8 4.2 The digestive tracts of vertebrates are colonized by Note: µ specific growth rate; λ maximum specific growth complex assemblages of micro-organisms, collectively rate; A maximum biomass; td doubling time of bacteria

TABLE 7．Heat tolerance of the selected bacteria. Temperature（ ℃） Laboratory reference number 18 25 37 45 50 55 60 9（3）7.5.2.1 +728 +635 +4151 +10584 +216 +364 +1441 8（3）1.2.1 -116 +6660 +218513 +74636 +46843 +10757 -209 9（2）7.3.2 -218 +181 +4405 +3114 X X X 6（9）8.4 +33 +13 +362 +1212 +147 +411 -154 7（3）6.1.2 +141 +896 +2946 +5716 +8069 +145 +265 Note: + indicates increase number of bacteria; - indicates decrease number of bacteria; X indicates no bacteria growth The number shown is difference between the mean number of bacterial before and after the experiment.

TABLE 8．Bile tolerance of the probiotic bacteria Laboratory reference Bile concentration（ w/v） number 0.01% 0.10% 0.20% 0.30% 0.40% 0.50% 0.60% 0.70% 0.80% 0.90% 1.00% 9（3）7.5.2.1 +10 +109 +51 +8 +1 -6 -2 +8 +12 +4 +35 8（3）1.2.1 +4345 +107 +408 +5 +215 +1450 -47 -59 +4 +40 -6 9（2）7.3.2 +8 +2105 +491 +2491 +1267 +148 -2 +894 +23 +8 +91 6（9）8.4 +30 +272 +741 -27 +3 +145 -43 -26 0 +60 -31 7（3）6.1.2 +17 +712 +16 +49 +36 +8 +96 +47 +11 -4 +181 + indicates increase number of bacteria; - indicates decrease number of bacteria INTESTINAL BACTERIA 9

TABLE 9．pH tolerance of the probiotic bacterial for 24 hours Laboratory reference pH2 pH3 pH4 pH6 pH8 pH9 number 1 hr 24 hr 1 hr 24 hr 1 hr 24 hr 1 hr 24 hr 1 hr 24 hr 1 hr 24 hr 9（3）7.5.2.1 - + - + + + + + + + + + 8（3）1.2.1 - - - + - - - + + + + + 9（2）7.3.2 ------+ + + + + + 6（9）8.4 - + - + - - + + + + + + 7（3）6.1.2 - - - - + - + + + + + + Note: + indicates growth of bacteria; - indicates no growth of bacteria

called gut microbiota（ Wong and Rawls, 2012）. perhaps the crystal violet and bile salts in MacConkey Differences in the intestinal microbiota of fish are presum- inhibited the growth of bacteria 6（9）8.4 and 7（3）6.1.2. ably caused by differences in the types of food sources, The pseudomonas isolation agar was apply in the selec- gut morphology and digestion capability, and the physi- tive media test is for target the pseudomonas bacteria ological behaviour among fishes（ Ye et al., 2014）. which are known to growth on soil and water. However, This study has revealed the intestinal microbiota of bacteria 9（3）7.5.2.1, 8（3）1.2.1 and 6（9）8.4 which the Sagor catfish through screening, growth profiling, growth on pseudomonas isolation agar did not have antagonism and tolerant abilities of bacteria. A total of closest relative to the bacteria Pseudomonas. 334 bacteria strains were successfully isolated from After the phenotype screening of intestinal bacteria of intestine of Sagor Catfish. The isolation was done on Sagor catfish, the bacteria were carried on to antago- Tryptic Soy Agar（ TSA）（ Merck, USA）. The bacteria nism examination. Pathogens used in this study are was sub-culture everyday for 60 days. Only bacteria responsible for some common disease affecting appearing in single colour and shape were proceed to Malaysian aquaculture, such as streptococcosis（ S. the next stage of examination. agalactiae）, vibriosis（ V. harveyi, V. alginolyticus and V. To confirm the bacteria are in single and pure colonies, parahaemolyticus）, pasteurellosis（ Photobacterium Gram staining was conducted on them. The bacteria daselae） and motile Aeromonas septicemia（ MAS） appeared in one shape and one colour under light （Aeromanas hydrophilla, A. caviae and A. salmonicida） microscope is consider as purified bacteria. Based on （Amal and Zamri-Saad, 2011; Spanggaard et al. 2000）. the results of Gram staining, most of the bacteria isolated The details of pathogens in this study was listed at from the Sagor catfish intestine were Gram positive bacilli TABLE 1. Results in this study revealed that an average （132 out of 334 isolated strains）. These findings agreed of 2.69% of probiotic bacteria exhibited growth of with those of Banerjee et al.（ 2015） who studied bacte- pathogens. The bacteria with strong antagonistic prop- rial composition in the gastrointestinal tract of catfish. erties in this study were listed as common gut bacteria They found that Gram positive bacilli bacteria are domi- by Jayanth et al.（ 2002）. He reported that gut bacteria nant in gastrointestinal tract of catfish. mostly belong to the genus Bacillus, Micrococcus, Then the bacteria were subjected to selective media Pseudomonas, Vibrio, Flavobactrium, Alcaligenes, test, Thiosulfate-citrate-bile salts-sucrose（ TCBS）, Xanthomonas and Achoromobacter. However, only 5 MacConkey and Pseudomonas isolation agar were bacteria which display antagonism activities on both involving in this test. TCBS agar was originally devel- freshwater and marine pathogens were chosen for oped for the isolation of Vibrio spp. pathogenic for further characterization. humans（ West et al., 1982）. The results shows that The characterization of probiotic candidates in this bacteria strains 9（2）7.3.2 and 6（9）8.4 on this study study was carried out using Polymerase Chain Reaction growth on TCBS, this indicates that these bacteria are （PCR） which is commonly application for bacteria identi- related to the bacteria Vibrio spp. This results were fication in researches. The DNA sequences of selected once again assured by the blasting of sequences which probiotic candidates were blasted in NCBI blast machine shows that bacteria strain 9（2）7.3.2 and 6（9）8.4 are and the closest relatives were listed at TABLE 4. The closest relatives to Vibrio harveyi and Vibrio parahaemo- closest relative of the probiotic candidates were found lyticus. Meanwhile, MacConkey is a selective media for to be either originally from the fish gut or from the Gram-negative and bacillus bacteria（ Lee et al., 2010）. surrounding of fish（ sediment and water）. According to The results shows that only bacteria 9（3）7.5.2.1, 8（3） Chaklader et al.（ 2014）, fish takes in the bacteria into 1.2.1 and 9（2）7.3.2 growth on MacConkey. This is their guts through inhale of the water surrounding them. 10 P. S. TUNG ET AL.

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