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.

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