The Israeli Journal of Aquaculture - Bamidgeh, IJA_65.2013.932, 8 pages
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Antibacterial Activity of Spirulina (Arthospira platensis Geitler) against Bacterial Pathogens in Aquaculture G. Rathi Bhuvaneswari1*, S.P. Shukla1, M. Makesh1, S. Thirumalaiselvan1, S. Arun Sudhagar1, D.C. Kothari2, Arvind Singh2 1
Aquatic Environment and Health Management Division, Central Institute of Fisheries Education, Versova, Mumbai 400061, India
2
National Centre for Nanoscience and Nanotechnology, University of Mumbai 400098, India (Received 14.11.12, Accepted 18.1.13)
Key words: cyanobacteria, spirulina, antibacterial properties, fish pathogens, acetone soluble fraction Abstract The antibacterial activity of seven crude extracts of a cyanobacterium, Arthospira platensis (previously called Spirulina platensis), was evaluated against seven bacterial fish pathogens. The antibacterial activity of the acetone soluble fraction of the A. platensis was considerable against all seven pathogens and equaled the antibacterial activity of the positive control (chloramphenicol) against Aeromonas hydrophila. Edwardsiella tarda was the most susceptible pathogen to this fraction. The minimum inhibitory and minimum bactericidal concentrations were lowest for the acetone soluble fraction. Atomic force microscopy of A. hydrophila cells showed that cell walls were considerably damaged after one hour of exposure to the acetone soluble fraction, i.e., pores, holes, and grooves had formed on the cell envelope. After two hours of exposure, the cells had became permeabilized and collapsed due to disintegration of the cell wall.
* Corresponding author. Tel.: +91-989-2527098, e-mail:
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Introduction Antibacterial research often focuses on novel drugs to cure diseases affecting human beings, plants, animals, fish, and live-stocks. However, due to alarming increases in infections by antibiotic-resistant microorganisms, plants containing antimicrobial compounds are gaining attention (Trias and Gordon, 1997). Cyanobacteria (blue green algae) have unique biochemical properties and are a potential source of biologically active secondary metabolites. Cyanobacteria produce intracellular and extracellular metabolites with anti-algal, anti-bacterial, anti-fungal, and anti-viral activity (Borowitzka, 1992). Spirulina is an economically important filamentous cyanobacterium that grows in aquatic habitats. It is approved in Russia as a medicinal food for treating radiation sickness (Rabadiya and Patel, 2010). Spirulina is an ideal bio-resource due to its richness in protein, phycocyanin, essential amino acids, polysaccharides, carotenoids, minerals, vitamins, and essential fatty acids (Morist et al., 2001). It is also rich in vitamins, minerals, carbohydrates, and gamma-linolenic acid. Spirulina is gaining attention not only for its food value, but also for development of pharmaceuticals. Spirulina has therapeutic effects as a growth promoter, probiotic, and booster of the immune system in animals including fish (James et al., 2006). Phycocyanin is the principal bioactive substance in spirulina and its content ranges 1015% of the dry weight (Becker, 1994). Spirulina species exhibit antiviral and antioxidant properties against human pathogens (Khan et al., 2006). This study evaluates the antimicrobial activity of Spirulina platensis (reclassified as Arthospira platensis but still commonly known as spirulina) against gram-positive and gram-negative bacteria pathogenic to fish. Findings from this investigation can serve as baseline information for further studies of fish health management and control of fish spoilage by natural compounds. Materials and Methods Culture of spirulina. A culture of the cyanobacterium, Arthospira platensis, was obtained from the algae culture laboratory of the Central Institute of Fisheries Education (CIFE), Mumbai. The pure culture was sub-cultured in a modified medium prescribed by the Nallayam Research Center (NRC) consisting of 5 g NaCl, 2.5 g NaNO3, 0.01 g FeSO4.7H2O, 0.5 g K2SO4, 0.16 g MgSO4.7H2O, 8 g NaHCO3, and 0.5 g K2HPO4 per liter in indoor airlift cultures. The culture units consisted of a magnetic stirrer, an air filter, fluorescent light panels, and a 20-l aspirator bottle. The photoperiod was 12L:12D, maintained by compact fluorescent lamps (23 W; Philips) that provided illumination of 3500±100 lux, measured with a lux meter (LX-103; Taiwan). Temperature was maintained at 24±2°C. The cultures were aerated by air injection devices connected to glass pipes that released air bubbles at the bottoms of the aspirator bottles. The air-flow rate was adjusted to a level that ensured proper mixing of the culture medium by the upward movement of the air bubbles. Estimation of biomass. 50 ml of the algal suspension was filtered through a preweighed quantitative ashless filter paper (Advantech 5A, 0.02 mg; Merck). The paper was washed repeatedly and weighed again. The difference in weight before and after filtration equaled the fresh weight of the algae cells. The filter paper with the settled algae cells was dried in a hot air oven for 6 h at 105°C and the paper was weighed a third time. The difference in weight of the filter paper before and after drying equaled the dry weight of the algae cells. Preparation of extracts. Seven extracts (acetone soluble fraction, acetone insoluble fraction, dichloromethane+methanol extract, aqueous extract, water insoluble fraction, phycocyanin, culture filtrate) were prepared from the A. platensis and used to evaluate antimicrobial properties against fish pathogens as follows. Preparation of acetone soluble and insoluble fractions. 5 g of fresh filtered A. platensis (wet wt) was homogenized with 50 ml dichloromethane/methanol (1:1 v/v). The homogenate was filtered and the filtrate evaporated under vacuum to dryness at 40°C in a rotary evaporator (Steroglass Strike 300, Germany). The dried extracts were stored at
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-20°C until further use. The cells remaining after filtration were successively extracted with 25 ml acetone. The homogenate was filtered and the filtrate was evaporated under vacuum to dryness at 40°C in a rotary evaporator. The dried compound was stored at -20°C. When used, it was dissolved in 10% dimethyl sulfoxide (DMSO). The cells remaining after extraction of the acetone soluble fraction were also stored at -20°C and 0.2 g of the acetone insoluble fraction was dissolved in 2 ml 10% DMSO to evaluate antimicrobial properties against fish pathogens. Preparation of aqueous extract and water insoluble fractions. Fresh A. platensis samples (5 g wet wt) were added to 10 ml distilled water in a 200-ml conical flask and put on a shaker (ORBITEK, Scigenics Biotech) at 120 rpm and 50°C for 3 h. The mixture was centrifuged and the aqueous extract was collected and stored at 4°C until further use. The pellet was stored at -20°C. 0.2 g of this water insoluble fraction was mixed with 2 ml 10% DMSO and used for further experiments. Preparation of crude extract and purification of phycocyanin. Freshly harvested spirulina was washed with distilled water 2-3 times to remove the culture medium. The phycobiliprotein, C-phycocyanin was isolated following the modified method of Boussiba and Richmond (1979) and estimated as described by Bennett and Bogorad (1973). In brief, 1 g biomass of A. platensis was washed twice with distilled water and centrifuged at 2500 × g for 5 min. The pellet was washed and suspended in 2.0 ml of 0.05 M phosphate buffer (pH 6.8). The aqueous phase was subjected to freezing and thawing. Then the content was centrifuged at 2500 × g for 5 min and the supernatant was collected and stored in a refrigerator. The pellet was subjected to repeated cycles of freezing and thawing until a colorless supernatant was obtained. The supernatantcontaining pigment was pooled and the final volume recorded. Pigment absorption was measured at 615 and 652 nm against a blank (0.05 M phosphate buffer). Concentrations of C-phycocyanin in the cyanobacterial cultures (mg/ml) were determined as E615 0.474(E652)/5.34, where E615 and E652 are the optical densities at 615 and 652 nm, respectively (Bennett and Bogorad, 1973). The purity of the phycocyanin was calculated as E620/E280, where E620 and E280 are the optical densities at 620 and 280 nm, respectively. The crude phycocyanin was precipitated in 50% (NH4)2S04, then recovered by centrifugation at 10,000 x g for 10 min. The colorless clear supernatant was discarded and the blue precipitate was dissolved in a small volume of 0.0025 M Na-phosphate buffer (pH 7.0) and dialyzed with a dialysis membrane (HiMedia) against the same buffer of 0.0025 M. (Boussiba and Richmond, 1979). Screening of antimicrobial properties of A. platensis. The antimicrobial properties of the seven extracts were tested against seven bacterial fish pathogens obtained from the Laboratory of Fish Pathology and Microbiology of CIFE, Mumbai (Table 1). Each bacterial strain was inoculated in 5 ml nutrient broth from a stock culture and incubated overnight at room temperature. 5 ml of each cultured broth was centrifuged at 10,000 × g for 5 min at 4°C. The pellets were washed twice with phosphate buffer and re-suspended in 0.85% saline water. Turbidity was adjusted to 0.5 McFarland Standards (10 7 CFU/ml) and confirmed by spectrophotometric reading at 600 nm. McFarland Standards were used to standardize numbers of bacteria when required by a procedure or for the susceptibility test. The basic 0.5 McFarland Standard contains approximately 1 × 10 7-108 CFU/ml.
Table 1. Bacterial fish pathogens against which seven extracts of spirulina (Arthospira platensis) were tested. Bacteria Aeromonas hydrophila Bacillus subtilis Bacillus cereus Edwardsiella tarda Micrococcus luteus Vibrio alginolyticus Vibrio parahemolyticus
Isolate Gram stain ATCC 7965 Laboratory isolate + Laboratory isolate + ATCC 15947 ATCC 10240 + Laboratory isolate Laboratory isolate -
Disease caused Hemorrhagic septicemia (Roberts, 2001) Branchio-necrosis (Austin and Austin, 1999) Branchio-necrosis (Austin and Austin, 1999) Edwardsielliosis (Roberts, 2001) Micrococcosis (Austin and Austin, 1999) Vibriosis (Roberts, 2001) Vibriosis (Roberts, 2001)
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Agar well diffusion assay. The agar well diffusion method was used for the antibacterial assay (Bagamboula et al., 2004). 20-30 ml Mueller Hinton Agar (MHA) was poured onto Petri plates and allowed to solidify. A suspension of the bacteria containing approximately 107 CFU/ml was uniformly spread with a cotton swab in three directions on a 4-mm thick MHA surface. Sterile stainless steel cylinders (6 mm diameter) were placed on the MHA plates to make wells. 100 μl extract was added to the wells. Chloramphenicol (30 μg/well) was used as a positive control and DMSO was used as a negative control. The plates with bacterial inoculums and extracts were incubated for 24 h at 37°C in a biochemical oxygen demand incubator fitted with a temperature control device. Zones of inhibition formed after treatment with extracts were measured using a Hi Antibiotic Zone Scale (HiMedia Laboratories). Tests were carried out in triplicate. Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. The MIC of the extracts was determined by the micro broth dilution method (NCCLS, 2000). 100 μl sterilized Mueller Hinton Broth (MHB) was added to each well on the tissue culture plate. Then, 100 μl extract was added and subsequent serial dilutions produced concentrations ranging 1.9-1000 μg/ml. 10 μl inoculum of each bacterium was added to the wells and plates were incubated at ambient temperature for 24 h. A positive control (containing inoculum but no extract) and a negative control (containing extract but no inoculum) were included on each plate. 10 μl of 1% tetrazolium chloride (TTC) was added to each well to determine MIC. Plates were incubated at 30°C for 1 h and results were recorded. A change to pinkish-red was considered a positive result. Wells that showed no growth during the MIC determination were selected for MBC determination. A loopful from each selected well was sub-cultured on extract-free MHA plates and incubated for 24 h at 37°C. The lowest concentration at which there was no growth was considered the MBC. Atomic force microscopy (AFM) of Aeromonas hydrophila cells. The effect of the acetone soluble fraction on the cell surfaces of the bacteria was assessed by AFM. Samples were prepared by treating 100 µl A. hydrophila suspension (0.5 McFarlands Standard) with 100 µl acetone soluble fraction and incubating them for 2 h. The control and treated samples were put onto a clean glass surface, air-dried, gently rinsed with deionized water to remove salt crystals, and air dried a second time. The samples were measured by AFM (Nanowizard II, JPK Instruments, Germany) and quantitatively analyzed using JPK image processing software. The samples were scanned using a tip moving scanner with a range of 100 x 100 µ. Cantilever resonance frequency was 253 KHz. Bacterial morphology studies were performed in tapping mode (for both trace and retrace information) using a silicon nitrite tip at ambient temperature. Statistical analysis. Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Illinois). Means and standard deviations of triplicates were calculated. Differences in percentage of zone of inhibition were compared by one-way analysis of variance. Mean separations were carried out by Duncan’s Multiple Range Test at a significance level of 0.05. Results The specific growth rate during the exponential growth phase of the A. platensis was 0.46±0.02 per day and doubling time was 1.5 days. The fresh biomass yield from 50 ml of the exponential phase culture was 0.26 g, i.e., 5.192 g/l. The dry weight of the algae was 0.86 g/l, corresponding to 16.56% of the fresh weight. Phycocyanin was extracted from the A. platensis using 0.05 M phosphate buffer, the biomass of the phycocyanin in the buffer was repeatedly frozen and thawed, and the crude extract contained 0.08 mg phycocyanin/ml with a purity of 2.42. When the crude extract was purified by precipitating the low molecular weight proteins with 50% (NH4)2SO4, the purity increased and the concentration of phycocyanin decreased. Further purification by dialysis reduced the concentration by 35% while the purity increased to 4.85±0.031. Thus, each step of purification led to a decrease in the concentration and an increase in the purity. The acetone soluble fraction had the strongest antimicrobial activity against grampositive and gram-negative bacteria and was as effective as chloramphenicol against A.
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hydrophila (Table 2). The acetone insoluble fraction also exhibited inhibitory effect against A. hydrophila, but there was a significant difference in activity between the soluble and the insoluble fractions. Before treatment with the acetone soluble fraction, atomic force microscopy (AFM) showed that the A. hydrophila cell wall had no visible pores, holes, grooves, or breakages in the envelope (Fig. 1). After treatment with the acetone soluble fraction for 1 h, the cell wall became porous and damaged. Two hours after treatment, the cell wall was disrupted. Eventually, the stability of the membrane was destroyed, the cells disintegrated, and the cell contents oozed out. Table 2. Diameter of inhibition zone, minimum inhibitory concentration, and minimum bacteriocidal concentration of extracts of spirulina (Arthospira platensis) against bacteria using the well diffusion method (mean±SEM, n = 3.) Aeromonas hydrophila
Bacillus cereus
Bacillus subtilis
Edwardsiella Micrococcus Vibrio Vibrio paratarda luteus alginolyticus hemolyticus
Diameter of inhibition zone (mm) Positive control (chloramphenicol) 34.33±0.3d 27.66±0.3c Negative control (10% dimethylsulfoxide) NAD NAD Acetone soluble fraction 33.33±0.6d 40.33±0.3d Acetone insoluble fraction 19.33±0.8c 18.66±0.3b Dichloromethane+methanol extract NAD NAD Aqueous extract NAD NAD Water insoluble fraction NAD NAD Phycocyanin 16.00±0.57b NAD Culture filtrate NAD NAD Minimum inhibitory concentration (mg/ml) Acetone soluble fraction 0.0039 Acetone insoluble fraction 6.25 Water insoluble fraction Phycocyanin 0.250 Minimum bacteriocidal concentration (mg/ml)
0.0039 6.25 -
35.33±0.3d NAD 34.33±0.3c 16.66±0.3b NAD NAD NAD NAD NAD 0.0039 12.5 -
38.33±0.6c 20.66±0.3c 24.33±06b NAD NAD NAD 41.33±0.8d 39.00±1.0d 30.66±1.6c 20.66±0.3b 15.33±0.3b NAD NAD NAD NAD NAD NAD NAD NAD 15.00±0.00b NAD NAD NAD NAD NAD NAD NAD 0.0019 25 -
0.0019 12.5 12.5 -
Acetone soluble fraction 0.00781 0.125 0.0078 0.03125 0.00781 Acetone insoluble fraction >100 >100 25 50 >100 Water insoluble fraction >100 Phycocyanin 0.500 Values for the diameter of the inhibition zone with different superscripts significantly differ (p
