Checking isolates LAB in MRS agar
By Nguyen Thanh Tam1, Dang Thi Thu Thao1,Sunee Nitisinprasert2,Kenji Sonomoto3 and Nguyen Van Ba1
1Faculty of Applied Biology, Tay Do University, Can Tho City, Viet Nam.
2Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University
3Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Introduction
Bacteriocins are extracellularly released peptides or protein molecules, with bacteriostatic or bactericidal mode of action against closely related species. The discovery of nisin, the first bacteriocin is used on a commercial scale as a food preservative dates back to the first half of last century but research on bacteriocins of LAB has expanded in the last two decades.
Edwardsiella ictaluri is a gram-negative, rod-shape, oxidase-negative, peritrichous, fermentative bacterium, has been reported almost exclusively as an oligate pathogen of ictalurids fish and cause enteric septicaemia of catfish (ESC), especially in channel catfish Ictaluruspunctatus (Hawke et al., 1981) and striped catfish Pangasianodon hypophthalmus (Tu Thanh Dung et al., 2008).
This research aims to find out an effective bacteriocin that inhibited the growth of Edwardsiella ictaluri.
Materials
LAB were isolated from fermented fish products using MRS agar supplemented with L-cysteine hydrochloride and 0.6% CaCO3 and were incubated anaerobically at 420C for 24h. Bacteria strains that produce lactic acid, gram-positive, non-spore forming and catalase-negative were selected for further study. Single colonies were selected and microscopically was examined for purity and then subcultured in MRS broth.
LAB strains were cultured in MRS broth test-tubes and were incubated at 420C for 48h. Cell free supernatant obtained by centrifugation at 13.000 rpm for 5 min was neutralized to pH = 6, treated by catalase and filtrated through 0.2 mm pore size filter thus obtaining cell free filtrate. These filtrates were further used to determine antimicrobial activities.
Methods
The antimicrobial effect of the isolated LAB against E. ictaluri, Bacillus coagulans JCM2257T, Pediococcus pentosaceus JCM 5885 was performed by the well diffusion assay. The pathogenic bacteria (only E.ictaluri) were incubated in Brain Heart Infusion (BHI) broth at 300C for 24h while MRS broth was used for Bacillus coagulans JCM2257T and Pediococcus pentosaceus JCM 5885. Petridishes containing 20ml of BHI agar or MRS agar were prepared and inoculated with 0.1 ml of 24h broth culture of E. ictaluri or Bacillus coagulans JCM2257T and Pediococcus pentosaceus JCM 5885, respectively. Once solidified the dishes were stored for 2h in a refrigerator. The wells made by sterile tip (six-millimetre diameter) were filled up by 50μl of previously prepared cell-free filtrate. Petri dishes were incubated at 420C (indicator strains) and 300C (E.ictaluri bacteria) for 24h. The antimicrobial activity was determined by measuring the clear zone around the wells. The diameter of the inhibition zones were measured with calipers in millimetre.
Results
A total of 80 LAB were isolated from 100 samples of fermented fish products collected in 4 cities of the Mekong Delta. Antimicrobial effect of all isolated LAB against E.ictaluri and two indicator strains were performed by agar well diffusion method (Fig 1). The result showed that 39 strains had produced inhibition zone against E.ictaluri, 30 strains with inhibition zone against B.coagulans, and 20 strains resisted P.pentosaceus. Their effect against each indicator strains and E.ictaluri varied between 37 – 72% (Fig 2). Among the 39 E.ictaluri resistant strains, there were 15 strains with largest inhibition zone (from 2.5 – 4.0 mm diameter), 15 strains with the average inhibition zone (from 1.5 to <2.5 mm diameter) and 9 strains with smallest inhibition zone (<1.5 mm diameter) (Fig 1). Besides, 30 B.coagulan resistant strains and 20 P.pentpsaceus resistant strains produced inhibition zones from 2 – 3 mm diameter.
Twenty out of the eighty isolates had broad spectrum of inhibitory activity against E.ictaluri and two indicator strains (Table 1).
Fig 1. Inhibition zones on P.pentosaceus (A; 1A, 2A, 3A-supernatant not filtered; 4A, 5A, 6A-supernatant filtered), B. coagulans (B) and E.ictaluri (C; 1C-the large; 2C-the average and 3C-the small) formed by isolated LAB from fermented fish products as detected by agar well diffusion
Fig 2. Frequency of inhibition by the eighty isolated LAB from fermented fish products against E.ictaluri and two indicator strains
Table 1. Antimicrobial effect of the twenty LABs isolated from fermented fish products against E.ictaluri two indicator strains
Tested LAB strains | Inhibition of indicator strains (mm) |
E.ictaluri | P.pentosaceus | B.coagulans |
1 | 4 | 3 | 3 |
2 | 4 | 3 | 3 |
3 | 3,8 | 3 | 3 |
4 | 3,8 | 3 | 3 |
5 | 3,7 | 3 | 3 |
6 | 3,5 | 3 | 3 |
7 | 3,5 | 3 | 3 |
8 | 3 | 3 | 3 |
9 | 3 | 3 | 3 |
10 | 3 | 3 | 3 |
11 | 3 | 2,8 | 3 |
12 | 3 | 2,5 | 3 |
13 | 3 | 2 | 2,8 |
14 | 2,8 | 2 | 2,8 |
15 | 2,8 | 2 | 2,8 |
16 | 2,7 | 2 | 2,8 |
17 | 2,6 | 2 | 2 |
18 | 2,6 | 2 | 2 |
19 | 2,5 | 2 | 2 |
20 | 2,5 | 2 | 2 |
4. Conclusion
Fermented fish products are abundant source of lactic acid bacteria, and could be considered as candidates for antimicrobial producing strains. The antimicrobial compounds produced by most of LAB studied in this work are similar to organic acids, except for 80 isolates which was found to produce antimicrobial substance other than organic acids and hydrogen peroxide.
Presence of E. ictaluri in Pangasius kidney and liver
5. References
Hawke J.P., Mc-Whorter A.C., Steigerwalt A.G. and Brenner D.J. (1981). Edwardsiella ictalurisp. no., the causative agent of enteric septicemia of catfish. Interntional Journal of Systematic Bacteriology 31, 396-400.
Tu Thanh Dung, Freddy Haesebrouck, Nguyen Anh Tuan, Patrick Sorgeloos, Maego Baele and Annemie Decostere (2008). Antimicrobial Susceptibility Pattern of Edwardsiella ictaluri Isolates from Natural Outbreaks of Bacillary Necrosis of Pangasianodon hypophthalmus in Vietnam. College of Aquaculture and Fisheries, Cantho University, Cantho, Vietnam. Department of Pathology, Bacteriology, and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Ghent, Belgium.
Mami Nishie, Makoto Sasaki, Jun-ichi Nagao, Takeshi Zendo, Jiro Nakayama & Kenji Sonomoto, Lantibiotic transporter requires cooperative functioning of the peptidase domain and the ATP binding domain, J. Biol. Chem., Vol.286,No.13,pp.11163–11169,2011.04.
Sunee Nitisinprasert, Veeraphol Nilphai, Phunjai Bunyun, Prakit Sukyyai, Katsumi Doi and Kenji Sonomoto (2000). Screening and identification of Effecrive Thermotolerant Lactic Acid Bacreia Producing Antimicrobial Activity against Eschericia coli and Salmonella sp. Resistant to Antibiotics. Department of Biotechnology, Faculty of AgroIndustry, Kasetsart University, Bangkok 10900, Thailand. Microbial Genetics Division, Institute of Genetic Resources, Kyushu University, 6-10-1 Hokozaki, Higashi-ku, Fukuoka 812-8581, Japan. Laboratory of Microbial Technology, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan.