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Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus



Nurul Shazwani, Mohamad Pipudin, M.Y. Jasmin, M.Y. Ina-Salwany, S.A. Harmin and Murni Karim
 
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ABSTRACT

Vibriosis caused by Vibrio alginolyticus has become one of the most threatening diseases that could limit the production of marine fish in aquaculture industry. In this study, microbe strains Micrococcus spp. (JAQ07) and Bacillus spp. (JAQ04) were used as potential probiotics. Both potential probionts were identified as gram-positive bacteria with different morphology. The antagonistic ability of each candidate probiotics towards V. alginolyticus (ATCC33839) were conducted in liquids modes via co-culture assay in three different concentrations of probiont (102, 104 and 106 CFU mL–1) and each concentration was inoculated with 105 CFU mL–1 of V. alginolyticus. The effectiveness of antagonistic activity was measured by the reduction of V. alginolyticus colonies via plate count at 24 h interval for 120 h. The co-culture assays revealed the reduction of V. alginolyticus colonies by both probiont strains compared to the control (V. alginolyticus at 105 CFU mL–1). In in-vivo assay, JAQ04 was able to enhance the survival of Artemia compared to JAQ07 after challenged with V. alginolyticus. Results revealed that at seven days after inoculation, Artemia treated with at 106 CFU mL–1 cell density followed by challenged with V. alginolyticus showed 70% of survival, while Artemia challenged with only V. alginolyticus demonstrated a 20% survival rate. Since, both strains displayed excellent probiotic activities, they are indeed suitable probiont candidates for managing Vibriosis infecting marine fish.

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Nurul Shazwani, Mohamad Pipudin, M.Y. Jasmin, M.Y. Ina-Salwany, S.A. Harmin and Murni Karim, 2015. Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus. Journal of Fisheries and Aquatic Science, 10: 300-306.

DOI: 10.3923/jfas.2015.300.306

URL: https://scialert.net/abstract/?doi=jfas.2015.300.306
 
Received: May 18, 2015; Accepted: June 29, 2015; Published: July 10, 2015



INTRODUCTION

Aquaculture is one of the fast and rapid growing food industry in the world. According to FAO (2012), the commercialization of products derived from aquaculture sector has been increased from 78 091 908 t in 2010, to 83 729 313 t in 2011. However, disease outbreaks have been the most important constraint towards its commercialization. Several contributing factors such as weather, high stocking density or bad water management will subsequently lead to infection of viruses, fungi, bacteria and parasites on the cultured fish (Moriarty, 1997).

Vibriosis is a common bacterial fish disease caused by Vibrio species, which generally affect marine fish species to a greater loss. In most cases, marine fish infected with this disease become hemorrhage with superficial skin lesion and septicemia (Egidius et al., 1986; Colwell and Grimes, 1984).

Early solution of treatment for controlling fish diseases is by means of antibiotics. The use of antibiotics to manage diseases was widely accepted in the beginning, only to be discovered that they later initiated to the emergence of numerous antibiotic-resistant bacteria (Balcazar et al., 2008). As a remedy, an alternative solution by applying bacterial probiotics such as lactic acid bacteria was introduced to promote the development of antibiotic-resistant bacteria in fish and the environment (Villamil et al., 2003; Verschuere et al., 2000).

Probiotics are bacteria that enhance the health of other organisms (Balcazar et al., 2006). The mechanisms of the probiotics include pathogen inhibition through production of antagonistic compounds, competition for essential nutrients and attachment sites, alteration of enzymatic activity for a greater immunity response, feed digestibility and modulation of interactions within the environments (Gomez and Balcazar, 2008; Bomba et al., 2002; Verschuere et al., 2000). Literally, the selection of probiotics candidate was based on their in vitro antagonism ability, the adhesion results as well as colonization and growth in intestinal mucus (Vine et al., 2006; Verschuere et al., 2000).

Thus, the present study was carried out to elucidate the antagonistic ability of two probiont candidates, strains, Bacillus JAQ04 and Micrococcus JAQ07 against V. alginolyticus by performing the in vitro and in vivo tests.

MATERIALS AND METHODS

Bacterial cultures and growth conditions: Two probiotics strains, previously identified by Nurhidayu et al. (2012) as Bacillus spp. (JAQ04) and Micrococcus spp. (JAQ07) and a pathogen strain, Vibrio alginolyticus (VA) were obtained from Aquatic Biotechnology Laboratory, Department of Aquaculture, UPM. All the bacteria were cultured using Tryptic Soy Agar (TSA; Oxoid, UK) with addition of 1.5% sodium chloride (NaCl). Both probionts and pathogen were incubated at 30 and 37°C, respectively. The strains were later cultured in Tryptic Soy Broth (TSB; Oxoid, UK) with addition of 1.5% NaCl and incubated overnight in an incubator shaker at 30°C of 120 rpm.

Morphology observation: Gram-staining procedure was performed for morphological identification of the probionts based on protocol by Gram (1884).

Co-culture assay: Each of the broth culture of probiotics treatments (102, 104 and 106 CFU mL-1) was separately inoculated into 10 mL of TSB supplied with 1.5% NaCl. All combination was done in triplicates and the remaining broth culture (control) was incubated at optimum condition for 120 h. The total viable count of V. alginolyticus was estimated by withdrawing 100 μL of each 8 fold serial dilution of each treatment into triplicates. All treatment was spreaded onto Thiosulfate-Citrate-Bile Salts-sucrose (TCBS) agar plates using a glass hockey stick and incubated for 24 h. Colonies produced were counted under a colony counter (ROCKER galaxy 230, Taiwan) and all data was recorded for further assessment. Each of the procedure was performed at 0, 24, 48, 72, 96 and 120 h.

Artemia bacterial challenge: Post-hatched Artemia nauplii were pre-incubated with probiont strains, JAQ04 and JAQ07 at three different concentrations (102, 104 and 106 CFU mL-1) in triplicates. On the following day, V. alginolyticus was added to the respective treatments at 105 CFU mL-1 cell concentrations. All tubes containing treatments and control (VA only) was kept in shaker in order to provide aeration and was maintained at the room temperature. The mortality rate of each treatment and control tubes was observed and recorded daily. The observation was dismissed once the control tube achieved a 50% mortality rate.

The Artemia were separated from culture water of each treatment by passing over the Artemia using a sterile 100 μm mesh. Artemia culture trapped in the sieve were rinsed with Sterile Sea Water (SSW) and re-suspended in 1 mL SSW. In order to determine the count of Vibrios loaded into the Artemia and culture water, a 100 μL suspension from each sample was plated on TCBS agar in triplicates. The plates were incubated for 24 h at room temperature (26°C). During the next day, colonies produced were counted using a colony counter (ROCKER galaxy 230) and calculated as CFU mL-1 using the formula:

Image for - Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus

Statistical analysis: All data was analyzed using a One-way Analysis of Variance (ANOVA). Multiple comparison tests (Duncan’s and Tukey’s-tests) were performed using SPSS Statistic 2.0 software. Results were denoted as Mean±Standard Error and the differences were considered significant at p<0.05.

RESULTS

Morphology observation: The morphology results of probiont strains, JAQ04 and JAQ07 are presented in Table 1. Based on gram-staining, strain JAQ07 was classified as gram-positive with cocci shaped (Stackebrandt et al., 1980). Instead, another probiont strain, JAQ04 was also classified as gram positive but with rod shaped (Claus and Berkeley, 1986).

Co-culture assay: Both strains JAQ04 and JAQ07 were able to inhibit the growth of V. alginolyticus at every given concentration in broth culture assays (Fig. 1 and 2).

Table 1:Morphological identification of probiont strains JAQ04 and JAQ07 based on gram staining
Image for - Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus
A1-A3- JAQ07: Micrococcus spp., B1-B3-JAQ04: Bacillus spp.

Image for - Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus
Fig. 1:
Growth pattern of Vibrio alginolyticus 105 CFU mL-1 with Bacillus spp. JAQ04 at different cell densities (102, 104 and 106 CFU mL-1) along with inoculation periods interval

Image for - Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus
Fig. 2:
Growth pattern of Vibrio alginolyticus 105 CFU mL-1 with Micrococcus spp. JAQ07 at different cell densities (102, 104 and 106 CFU mL-1) along with inoculation periods interval.

However, when there was V. alginolyticus alone with no probiont strain added, the cell count increased from 105-108 CFU mL-1 within 24 h of incubation. The cell count was consistent at 109 CFU mL-1 after 120 h incubation (48-120 h).

Multiple means comparison between different concentrations of treatments disclosed significant differences (p<0.05) for both probiotic strains (JAQ04 and JAQ07). At initial concentration of 105 CFU mL-1, the growth of V. alginolyticus was inhibited by strains JAQ04 and JAQ07 at all concentrations (102, 104 and 106 CFU mL-1) within 120 h. Moreover, all bacterial concentrations of strains JAQ04 and JAQ07 allowed an initial increment of V. alginolyticus cell density at 0-24 h, followed by decrement of the pathogen cell density at 48, 72, 96 and 120 h, respectively. At lower concentrations (102 and 104 CFU mL-1), both probiotics allowed initial growth of V. alginolyticus. At higher concentrations (106 and 106 CFU mL-1), the probiotics offered the best result by rapidly decreasing V. alginolyticus cell densities.

These experiments demonstrated that within in vitro conditions, co-culture assay of JAQ04 and JAQ07 strains at various increasing concentrations and prolonged incubation periods successfully inhibited the growth of the V. alginolyticus.

Bacterial challenge and pathogens count: After 7 days of observation, no significant difference was found on the survival of Artemia treated with both probionts JAQ04 and JAQ07 and the control (Fig. 3a-b). Thus, our results demonstrated that both probionts were not harmful to the Artemia. Meanwhile, the survival rate of the Artemia was more than 60% after challenged, indicates the potential of these probiont strains in protecting the Artemia against V. alginolyticus. Indeed, as the concentrations of the probiotics were greater, the survival rate was also increased. In contrast, Artemia challenged with only V. alginolyticus showed significant differences (p<0.05) with other treatments, with the survival rate of 20%. The results indicated that V. alginolyticus was pathogenic to Artemia.

In this experiment, we also revealed that the highest concentration of the probiont strain JAQ07 was able to reduce vibrios load both in the Artemia and the culture water as presented in Fig. 4a-b. Concentration of JAQ07 strain at 106 CFU mL-1 (T11) significantly reduced the number of vibrios in Artemia and culture water, compared to V. alginolyticus only (T2) (p<0.05). Meanwhile, JAQ04 strain was not able to reduce the number of vibrios in both Artemia and culture water at any tested concentrations.

Image for - Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus
Fig. 3(a-b):
Survival of Artemia with JAQ07 treatments after 7 days observation. (a) T1: Control, T2: VA 105 CFU mL-1, T3: JAQ07 102 CFU mL-1, T4: JAQ07 104 CFU mL-1, T5: JAQ07 106 CFU mL-1, T9: JAQ07 102+VA 105 CFU mL-1, T10: JAQ07 104 +VA 105 CFU mL-1 and T11: JAQ07 106+VA 105 CFU mL-1 and (b) T1: Control, T2: VA 105 CFU mL‾1), T6: JAQ04 102 CFU mL-1, T7: JAQ04 104 CFU mL-1, T8: JAQ04 106 CFU mL-1, T12: JAQ04 102+VA 105 CFU mL-1, T13: JAQ04 104+VA 105 CFU mL-1 and T14: JAQ04 106+VA 105 CFU mL-1. Error bars indicate the Standard Error

DISCUSSION

Probiotics have been widely used in fish culture to increase the survival and reduce the antibiotics usage. In these present studies, we have reported results that disclosed the potential of two non-pathogenic probiont strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in controlling the growth of a destructive fish pathogen, V. alginolyticus within in vivo and in vitro conditions. Initially, both of the probiont strains, which were isolated from the intestines of 51 juvenile tiger grouper were preliminary evaluated as candidate bacterial probiotics by Nurhidayu et al. (2012).

Co-culture experiments revealed that the inhibitory activity of JAQ04 and JAQ07 increased along with the increasing density of the antagonist at 120 h after incubation. In our study, we found out that both of these antagonist strains must be presented at significantly higher levels than the fish pathogen V. alginolyticus, as the degree of inhibition elevated proportionally with the level of antagonistic activity. Our results were also in accordance with Nurhidayu et al. (2012), indicated that JAQ04 and JAQ07 were able to produce inhibitory substances only after 72 h of incubation in liquid mode.

Pre-incubation of probiotics in Artemia culture showed the ability of these probionts to increase the survival of the Artemia compared to the control culture without the probionts. On the other hand, both probionts were able to confer protection to the Artemia after challenged with V. alginolyticus.

Image for - Evaluation of Antagonism Activity of Potential Malaysian Probiont Strains, Bacillus spp. JAQ04 and Micrococcus spp. JAQ07 in in vitro Condition and on Artemia fransisca against Vibrio alginolyticus
Fig. 4(a-b):
Means of Vibrio spp. loaded in Artemia together with JAQ07 treatments. (a) T2: VA 105 CFU mL-1, T9: JAQ07 102+VA 105 CFU mL-1, T10: JAQ07 104+VA 105 CFU mL-1 and T11: JAQ07 106+VA 105 CFU mL and (b) T2: VA 105 CFU mL-1, T9: JAQ07 102+VA 105 CFU mL-1, T10: JAQ07 104 +VA 105 CFU mL-1 and T11: JAQ07 106+VA 105 CFU mL. Error bars indicated the Standard Error (SE)

The level of protection was different depending on the relative concentrations of candidate probionts added. The results suggested that the probionts provided beneficial effects to the Artemia. Moreover, Bacillus and Micrococcus strains were proved not harmful and some of them could be beneficial probiotics for animals (Ryan et al., 2004).

Interestingly, we discovered that a cell density at 106 CFU mL-1 of probiont strain JAQ07 was required to inhibit the growth of V. alginolyticus. In contrast, probiont strain JAQ04 was not able to reduce V. alginolyticus loaded in both culture water and Artemia at any concentrations. These results suggested JAQ07 strain might potentially work by killing the pathogenic V. alginolyticus, perhaps with the combination of other probiotic mechanisms. In addition, JAQ04 strain acts by competing for essential nutrients and attachment sites, while the alteration of its enzymatic activity may lead to development of immunity response and increase feed digestibility and utilization (Verschuere et al., 2000).

Probiotics treatment offers a very promising alternative solution to combat diseases in fish and shrimp aquaculture. Our studies add into evidence that both JAQ04 and JAQ07 strains are safe as probiotics to be applied in aquaculture. However, further researches are needed to elucidate the exact mode of action to observe the beneficial effects and to understand the possibilities and limitations of microbial control in aquaculture.

CONCLUSION

These two probiont strains have demonstrated efficient probiotic properties, thus they can be used as potential probionts in aquaculture system against Vibriosis.

ACKNOWLEDGMENTS

The authors thank Dr. Dzarifah Zulperi from Universiti Putra Malaysia for suggestions and technical assistance on this paper. This study was supported by Universiti Putra Malaysia Grant GP-IPM/2013 and Agro-Biotechnology Institute (ABI), Ministry of Science, Technology and Innovation (MOSTI) Malaysia.

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