Introduction  

Due to rise in fish and shellfish farming, the rate of mortalities as a result of pathogenic microorganisms has increased (Shariff et al., 2001). The antibiotics that are employed in disease management encourage multiple antibiotic resistance genes development in microorganisms (Balcázar et al., 2006a). Therefore, alternatives are needed and probiotic research is receiving substantial consideration (Kesarcodi-Watson et al., 2008).

Quite a number of probiotics have been employed in aquaculture. They are lactic acid bacteria (Balcázar et al., 2006b ; Vaazquez et al., 2005), Pseudomonas sp. (Alavandi et al., 2004; Chythanya et al., 2002; Vijayan et al., 2006), Bacillus spp. (Far et al., 2009; Lallo et al., 2007; Zokaeifar et al., 2012) and commercial probiotics (Balasundaram et al., 2012). The employment of Shewanella algae and Halomonas aquamarina as indigenous probiotics enhanced the survival rate of white shrimp (Litopenaeus vannamei Boone) (Suantika et al., 2013). Ariole and Eddo (2015) reported the use of Shewanella algae as a biocontrol agent against Aeromonas infection in Clarias gariepinus. Probiotics have the capability to produce antimicrobial compounds and have been employed to inactivate numerous pathogens like Aeromonas hydrophila and Vibrio alginolyticus. They have the potential to inhabit the intestinal mucus and can be used in prophylaxis or be employed as therapeutic agents (Vine et al., 2004).

Verschuere et al. (2000) stressed the importance of isolating indigenous microorganisms for use as biocontrol agents in shrimp aquaculture in view of the fact that they are established   within the system in a short time. In Nigeria, indigenous probiotics are required to go well with the explicit requirement in aquaculture. Hence, the aim of the present study was to screen an indigenous antagonistic Shewanella algae previously isolated from healthy shrimp (Penaeus monodon) intestine and found to protect Clarias gariepinus from Aeromonas hydrophila infection (Ariole and Eddo, 2015) for antibiotic susceptibility, enzymatic activities and abiotic stress tolerance. Optimization of the cultivation conditions and identification of its bioactive compounds were also carried out. 

1 Materials and Methods

1.1 Source of Aeromonas hydrophila and Shewanella algae

Aeromonas hydrophila and Shewanella algae were previously isolated from moribund shrimp (Penaeus monodon) intestine (Ariole and Aungwa, 2013) and healthy shrimp (Penaeus monodon) intestine (Ariole and Eddo, 2015) respectively and maintained as part of culture collection in Microbiology Laboratory, University of Port Harcourt, Nigeria.  

1.2 Extracellular enzyme production

Shewanella algae isolate was tested on starch agar (1% soluble starch in nutrient agar), skim milk agar (1% skim milk in nutrient agar), Egg yolk agar (15% egg yolk in nutrient agar), cellulose agar (2% cellulose in nutrient agar) and gelatin agar (0.4% gelatin in nutrient agar) for amylase, protease, lipase, cellulase and gelatinase activities respectively. The isolate was streaked as a line on the various media. The plates were incubated for 24 h at 37℃. A zone of clearance around the line of growth on skim milk agar and gelatin agar indicated production of protease and gelatinase respectively. The starch agar plates were flooded with iodine solution whereas cellulose agar plates were flooded with congo red solution. A zone of clearance around the line of growth indicated production of amylase and cellulase respectively. A thin iridescent layer overlying the growth on Egg yolk agar indicated production of lipase. The diameters of the clearance zones were recorded in millimetres. The catalase activity of the isolate was detected by adding three drops of 10% hydrogen peroxide to a small portion of bacterial growth on a clean slide. The presence of effervescence caused by the liberation of oxygen indicated catalase production.

1.3 Antibiotic susceptibility of Shewanella algae

The antibiotic susceptibility was performed in Oxoid Muller- Hinton agar plates with multodisc using agar diffusion method. A total of 15 antibiotic discs namely Ciproflox (10 µg), Erythromycin (30 µg), Levofloxacin (20 µg), Gentamycin (10 µg), Ampiclox (20 µg), Rifampicin (20 µg), Amoxil (20 µg), Streptomycin (30 µg), Norfloxacin (10 µg), Chloramphenicol (30 µg), Tarivid (10 µg), Nalidixic acid (30 µg), Reflacine (10 µg), Augumentin (30 µg) and Septrin (30 µg) were employed. The bacterial suspension with the same density as the McFarland 0.5 was streaked with a sterile swab over the entire surface of Muller- Hinton agar plates and the antibiotic discs were placed on the plates. The plates were incubated at 37℃ for 24 h. Inhibitory zone was measured in millimetres.

1.4 Optimal pH, temperature and salinity for Shewanella algae growth

The most favorable conditions for growth of Shewanella algae were assessed by growing the strain at various pH (4, 5, 6, 7, 8, 9, 10 and 11), salinities (5, 10, 15, 20, 25 and 30 ppt) and temperatures (28, 37, 40 and 45℃) in 100 mL nutrient broth for a period of 24 h. Samples for cell count were drawn at zero hour and every 6 h. The cell counts were measured by serial dilution and spread plate method.

1.5 Environmental stress tolerance assay

The tolerance of Shewanella algae to varying concentrations of pH, salinity and temperature was studied. The pH tolerance was examined by growing the organism in nutrient agar with different pH values from pH 4 to pH 11. The tolerance to salinity was tested in nutrient agar supplemented with different concentrations of sodium chloride (0, 2, 5, 10, 15, 20, 25 and 30%). The temperature resistance of the organism was studied on nutrient agar plates by growing them in a wide range of temperatures (28-55℃).

1.6 Extraction of bioactive compounds (metabolites)

The Shewanella algae was grown on nutrient broth for 48 hr. Cell free supernatant (crude extract) was obtained by centrifuging the broth culture at 8000 rpm for 10 min. Equal amount of methanol was added to extract the bioactive compounds, the solvent was vaporized using a rotary evaporator. The residue was then reconstituted with Dimethyl sulphoxide (DMSO) for GC-MS analysis.

1.7 Antibacterial assay using crude and methanolic extracts

The antimicrobial activity was determined using agar diffusion method. Exactly 0.1 ml of 24 hr nutrient broth culture of the pathogen (Aeromonas hydrophila) was evenly spread over Mueller Hinton agar plates using sterile glass spreader. Wells were punched on the agar plates with sterile cork-borer (6 mm diameter) and 0.1 ml of the crude and methanolic extracts were introduced into the wells. The plates were incubated at 37℃ for 48 hours. After incubation, the plates were observed for clear zones of inhibition around the wells. The zone diameters were measured and recorded.

1.8 Gas chromatography-mass spectrometry (GC-MS) analysis of bioactive compounds

GC-MS analysis on the methanolic extract was carried out using Agilent 7890A-5975C GC-MS system employing the following conditions: HP5-column  (30 m x 0.25 mm x 0.25 um), operating in electron impact mode at 70 eV; Carrier gas flow (a constant) =1ml/min, Injection volume =0.5 ul, Split ratio =10:1Injector temperature = 250℃, Ion source temperature =280℃, Oven temperature, Initial =110℃ (hold 2 mins), 110℃ to 200℃ at 10℃/ min, 200℃ to 280℃ at 5℃/ min (hold 9 mins). Mass spectra were taken at 70 Ev.  Interpretation of mass spectrum was conducted using the National Institute of Standards and Technology (NIST) Database. The spectra of the unknown components were compared with spectra of known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.

1.9 Statistical analysis

Data generated from this study were analyzed using one-way analysis of variance (ANOVA) and values for P  ≤ 0.05 was considered to be statistically significant.

2 Results

2.1 Enzymatic activity

The Shewanella algae was positive for protease, lipase, gelatinase, amylase and catalase but negative for cellulase (Table 1). 

Table 1 Qualitative enzyme assay for Shewanella algae

2.2 Antibiotic susceptibility test

Shewanella algae was susceptible to all tested antibiotics (Table 2).

Table 2 Antibiotic susceptibility of Shewanella algae

2.3 Optimal pH, salinity and temperature

It was observed that the highest growth of Shewanella algae occurred at pH 6-8 (Figure 1). The isolate showed optimum growth at salinities from 5 to 15 ppt since these salinities did not result in significant variation in the growth of Shewanella algae (Figure 2). The optimum temperature for growth was 37℃ (Figure 3). 

Figure 1 Effect of pH on growth of antagonistic Shewanella algae

Figure 2 Effect of salinity on growth of antagonistic Shewanella algae

Figure 3 Effect of temperature on growth of antagonistic Shewanella algae

2.4 Environmental stress tolerance assay

It was observed that a salinity gradient of 0-100 ppt in the growth medium supported confluent growth of the isolate. The maximum temperature tolerance for the Shewanella algae was 40oC although it grew between 28 and 40oC. Any pH between 4 and 11 supported growth of the isolate (Table 3).

Table 3 Abiotic tolerance assay of antagonistic Shewanella algae

2.5 Gas Chromatogram and Quantitative composition of metabolite

The gas chromatogram and quantitative composition of methanolic extract of antibacterial substance produced by Shewanella algae are presented in Figure 4 and Table 4 respectively. Two peaks were observed and two bioactive compounds (Tromethamine and Pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro-) were identified from the mass spectra of the compounds.

Table 4 Quantitative composition of metabolite

Figure 4 Gas-chromatogram of methanolic extracted metabolite of Shewanella algae

3 Discussion

In the present research, studies on an indigenous probiotic (Shewanella algae) previously isolated from healthy shrimp (Penaeus monodon) intestine and found to protect Clarias gariepinus from Aeromonas hydrophila infection (Ariole and Eddo, 2015) were carried out. Bacteria with antibacterial properties are widely used in aquaculture to control bacterial infection by adopting different methods to introduce them into the host and the system. As probionts, they can control pathogenic bacteria proliferation by colonizing surfaces, competitive exclusion and by producing inhibitory compounds (Kennedy et al., 2009).

The antagonistic isolate was capable of producing catalase and hydrolytic enzymes such as amylase, lipase, protease and gelatinase (Table 1) which could support digestion and enhance nutrient uptake by the host (Pai et al., 2010). In addition, this could help to degrade the unconsumed feed and feaces in the culture pond. The capacity of intestinal shrimp microbiota to produce different extracellular enzymes with amylolytic, lipolytic and proteolytic activities has been reported elsewhere (Tuyub et al., 2014). Catalase producers can reduce the harmful effects of active oxygen molecules or free radicals which are generated during metabolic process (Patel et al., 2010). This catalase positive strain if inhabited in the gut can act as a good antioxidant.

Shewanella algae was susceptible to all tested antibiotics (Table 2). Bacteria selected as prospective probionts should not carry the genes for antibiotic resistance (Saarela et al., 2000). Antibiotics used in aquaculture have a serious negative impact to the environment. Residual antibiotics in the environment increase the resistance in aquatic bacteria and change the bacterial flora in sediment (Lalumera et al., 2004). The Shewanella algae was sensitive to all the antibiotics tested. This ensures its inability to transfer antibiotic resistance.

It was observed that the highest growth of Shewanella algae occurred at pH 6-8 (Figure 1). The isolate showed optimum growth at salinities from 5 to 15 ppt since these salinities did not result in significant variation in the growth of Shewanella algae (Figure 2). The optimum temperature for growth was 37℃ (Figure 3). These optimal parameters will be useful for industrial production of this strain. The growth of microorganisms is strongly influenced by medium components as well as cultural parameters like temperature and pH (Mukesh Kumar et al., 2012).

The result of environmental stress tolerance assay (Table 3) showed that a salinity gradient of 0-100 ppt in the growth medium supported confluent growth of the isolate. The maximum temperature tolerance for the Shewanella algae was 40oC although it grew between 28 and 40oC. Any pH between 4 and 11 supported growth of the isolate (Table 3). The growth response of Shewanella algae over a wide range of temperature, pH and sodium chloride concentrations has also been reported by Shahram et al. (2010). The ability of the antagonistic Shewanella algae to grow over a wide range of environmental conditions such as pH, temperature and salinity would help its survival in the host and the aquatic environment if employed as a biocontrol agent (Vine et al., 2006).

The crude and methanolic extracts were active against the pathogen with inhibition zone of 13.0 ±0.02 mm and 18.0 ± 0.03 mm respectively. Two bioactive compounds (Tromethamine and Pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro-) were identified (Figure 4 and Table 4).

The antagonistic activity showed by Shewanella algae could be as a result of the two compounds produced since marine secondary metabolites can easily impede other micro organisms (Jeffrey et al., 2011). However, Tromethamine is a drug used intravenously to correct acidosis and also as an alkalizing agent and a buffer in enzymic reactions while Pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro-, the major compound produced, is an antioxidative agent. Therefore, further research to examine its antioxidant activity is necessary. These results suggest that the Shewanella algae could probably be used as a potential probiotic in aquafeed formulation and a good source of new drug invention.

Author’s contributions

CNA conceived, designed and supervised the study and wrote the manuscript. JIE performed the laboratory and statistical analyses of the study. All authors read and approved the final manuscript.

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