A comprehensive dataset on antimicrobial activity of indigenous Oscillatoria spp. against common bacteria causing diseases in fish and shellfish

The antibacterial activity of phenolic extracts was assessed using the disk diffusion technique on two marine and two freshwater species of Oscillatoria. Three Gram positive bacteria (Staphylococcus sp. and Streptococcus sp.) and nine Gram negative bacteria (Escherichia coli, Vibrio spp., Salmonella sp., Shigella sp., Pseudomonas spp., Aeromonas sp.) were isolated from diseased marine fish, shrimp and crab. Filamentous Oscillatoria sp. indicated higher antibiotic activity than planktonic one. Gram positive bacteria showed higher susceptibility to phenol compared to the gram-negative bacteria. Filamentous Oscillatoria sp. showed highest inhibition zone of 34.06 ± 0.08mm against Staphylococcus sp., while planktonic Oscillatoria sp. showed lower inhibition zone against Pseudomonas sp. about 17.11 ± 0.18mm. Minimum inhibitory concentration value was found to be 100 µg/ml for filamentous Oscillatoria sp., 150 µg/ml for planktonic Oscillatoria sp. These findings suggest that, Oscillatoria spp. contain potential antibacterial substances. It also paves the way for detailed analysis of Oscillatoria spp. bioactive compound for the creation of novel antibiotics and serve both the aquaculture and pharmaceutical industries.


a b s t r a c t
The antibacterial activity of phenolic extracts was assessed using the disk diffusion technique on two marine and two freshwater species of Oscillatoria .Three Gram positive bacteria ( Staphylococcus sp. and Streptococcus sp.) and nine Gram negative bacteria ( Escherichia coli, Vibrio spp., Salmonella sp., Shigella sp., Pseudomonas spp., Aeromonas sp.) were isolated from diseased marine fish, shrimp and crab.Filamentous Oscillatoria sp.indicated higher antibiotic activity than planktonic one.Gram positive bacteria showed higher susceptibility to phenol compared to the gram-negative bacteria.Filamentous Oscillatoria sp.showed highest inhibition zone of 34.06 ± 0.08mm against Staphylococcus sp., while planktonic Oscillatoria sp.showed lower inhibition zone against Pseudomonas sp. about 17.11 ± 0.18mm.Minimum inhibitory concentration value was found to be 100 μg/ml for filamentous Oscillatoria sp., 150 μg/ml for planktonic Oscillatoria sp.These findings suggest that, Oscillatoria spp.contain potential antibacterial substances.It also paves the way for detailed analysis of Oscillatoria spp.bioactive compound for the creation of novel antibiotics and serve both the aquaculture and pharmaceutical industries.

Value of the Data
• Findings of this study supported the possibility of treating pathogenic bacterial diseases in fish and shellfish by means of Oscillatoria sp.extracts.• Cyanobacterial phenolic compounds exhibit the potentialities to exploit them as valuable resource for the development of novel antimicrobial agents which will help to boost up the production of aquaculture industry.• These data might create the future prospectives for the further research on antifungal and anti-cancerous properties based on detailed bioactive compound analysis of Oscillatoria sp.

Data Description
Pathogenic bacteria were isolated from the diseased marine fish and shellfish (Shrimp, Crab) ( Table 1 ) [1] .Each isolated bacterium was cultured in their respective selective agar medium.Table 1 represents the isolated bacteria with its source, growth media and type.

Minimum Inhibitory Concentration (MIC)
The Minimum Inhibitory Concentration (MIC) value of each filamentous Oscillatoria species ( Oscillatoria sp. 2, Oscillatoria sp. 3, Oscillatoria sp. 4) was found to be 100 μg/ml against all the test bacteria.On the contrary, planktonic strain ( Oscillatoria sp. 1) required MIC value of 100 μg/ml to be effective against all the test bacteria.

Collection and preparation of pure inoculums
Pure isolates of two freshwater and two marine water Oscillatoria sp.obtained from Live Feed Culture laboratory of Department of Aquaculture, Faculty of Fisheries, Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh.Isolates were inoculated in the Bold's Basal Medium (BBM) and Conway medium using the formula described by Bischoff and Bold.(1963) [2] and Tompkins et al. (1995) [3] respectively.Hundred ml pure seed of Oscillatoria sp. was inoculated into 900 ml of culture medium for the stock culture maintenance.Then the cultures of Oscillatoria sp. 1 (planktonic; marine), Oscillatoria sp. 2 (highly filamentous; marine), Oscillatoria sp. 3 (moderate filamentous; freshwater), Oscillatoria sp. 4 (highly filamentous; freshwater) were incubated in controlled indoor condition at 24 °C temperature for 13 days, 10 days, 11 days, 9 days respectively.The cultures were maintained under continuous light conditions of 12 h light: 12 h dark regime, with 20 0 0 lux light intensity [4] .

Mass Culture in plastic jars
Filtered and autoclaved (for 15 mins in 121 °C and 15 lbs pressure) distilled water and sea water were used to prepare BBM and Conway media for mass culture sequentially.Growth media were prepared using required nutrient solutions; in case of marine stocks, salinity was maintained according to the salinity found in its sampling site.Pure stocks were then transferred to 20L plastic tanks for mass culture at 25 °C with a photoperiod of 12 h:12 h (light: dark), commencing with a 5L volume and eventually scaling up to around 16 L.Every two days, media was added to the culture tank until it reached the desired volume.Using a centralized air pump, individual culture tanks were continuously aerated.PVC pipe substrates were used for Oscillatoria sp.mass culture and samples from each tank were examined under a light microscope weekly to check the purity of the stock.

Harvesting and dried biomass of Oscillatoria sp
After respective mass cultures, Oscillatoria were harvested by centrifugation at 50 0 0 rpm for 5 minutes by using centrifugation machine (TL5R Free Standing low speed refrigerated centrifuge, Herexi).The wet microalgae biomass obtained from post-centrifugation was subsequently oven-dried overnight at 40 °C through a high-quality hot air oven (JSR Korea's Natural Convention Oven LNO-150) and the dried biomass was crushed into fine powders by using a mortar and pestle.The powdered microalgae were then stored in a standard freezer at 4 °C until further use.

Collection and incubation of sample
Sterile cotton swab sticks were used to obtain samples from the diseased fish skin, gills, intestine and other affected areas.Sterile swabs were also used to swab the abdominal flap, mouthparts, claws, and carapace region of the crab, and the joints of the appendages, uropod, endoskeleton, and cephalothorax region of the shrimp which were subsequently inoculated into nutrient broth.The samples were then incubated overnight at 37 °C to allow the optimal growth and proliferation of the bacteria present in the samples.

Isolation and identification of bacteria by culture and staining
Bacterial culture from nutrient broth was streaked onto MacConkey agar, Mannitol Salt Agar (MSA), Thiosulfate citrate bile salts sucrose (TCBS), blood agar, Pseudomonas agar, Salmonella Shigella Agar (SS) agar and then incubated at 37 °C for 24 hours.The growth of E. coli, Vibrio sp., Staphylococcus saprophyticus , and Aeromonas hydrophila was indicated by bright-pink large colonies in MacConkey agar, green and yellow color colonies in TCBS agar, medium-sized yellow colonies in MSA, and round, smooth, and glistening colonies, looking like dewdrops with or without hemolysis in Blood agar, respectively.The bacteria were identified by further confirmation through biochemical tests.

Preparation of Smear
Gram's staining was carried out to identify the type of bacterium [5] .A sterile inoculating loop was used to transfer a drop of distilled water onto a glass slide, and it was combined with a loop full of bacterial culture that had been taken from agar plates.A room-temperature air drying period was subsequently given to the resultant smear.It was momentarily placed over a flame for the fixation of bacteria.

Staining protocol
The principal stain crystal violet was added to the prepared slide and left on for one minute.To eliminate of any unbound crystal violet, the slide was washed with a gentle stream of water for up to two seconds.The mordant Gram's iodine was then used for 1 minute to attach the crystal violet to the bacterial cell wall.The slide was then rinsed for 2 seconds.Then acetone was applied for 15 seconds and washed with a gentle stream of water.Afterwards, the secondary stain safranin was applied to the slide for 1 minute and washed again with a gentle stream of water.Once the slide was air dried, Light microscopy was used to investigate it.Gram-negative bacteria displayed pink, but Gram-positive bacteria showed up as blue/purple.

Biochemical tests
Bacterial samples underwent biochemical assays using the VITEK 2 system at the Marine Biotechnology Laboratory, Universiti Malaysia Terengganu, Malaysia.Aeromonas hydrophila and E. coli were among the bacteria that the VITEK 2 system identified.Additional biochemical tests such as Catalase, Production of H 2 S, Triple Sugar Iron, and motility tests were conducted at the Disease and Microbiology laboratory, CVASU to confirm the presence of other bacteria.

Preparation of crude Oscillatoria spp. extract
2g dried cyanobacterial biomass was pulverized and sieved to obtain a fine powder, which was subsequently mixed with 20 ml 95 % pure ethanol (1g sample/10 ml ethanol) to yield the crude ethanolic extract [6] .It was soaked in the solvent in the sterile screw-capped bottles of 100 ml volume for a day (24 h) at room temperature.Then the obtained solution was fine filtered using Whatman No.1 filter paper under vacuum to remove cellular materials [7] .

Extraction of Oscillatoria spp. phenolic compounds
Isolation of the potent extracts of phenolic compounds from Oscillatoria sp. was carried out in this study [8] .Filtered crude ethanolic extract solution was concentrated under reduced pressure through a rotary evaporator at their respective boiling points.The concentrated extract was then securely stored at 4 °C until subjected to the next step of extraction.For this, an acidic degradation method was employed [9] which involved addition of 2 ml of the concentrated extract to hydrochloric acid (2M, 200 ml) and heating the mixture in a water bath (90-100 °C) with a stirrer for 30 minutes.After cooling the extract to room temperature, the solution was transferred to a separating funnel where ethyl acetate (100 ml) was added (this process was repeated twice).The two layers were then carefully separated, with the top layer comprising the ethyl portion rich in free phenolic acids.The extract was concentrated under pressure using a rotary evaporator, resulting in a precipitate which was stored at 4 °C until it was ready for use in diagnosing phenols.Meanwhile, the aqueous layer was discarded.

Antibacterial activity of crude and phenolic extracts
The disk diffusion method was used to evaluate the antibacterial activity of the ethanolic raw extract and phenolic extract of Oscillatoria sp.[10] , which involved Mueller-Hinton agar (MHA) plates.A turbidity of 0.5 McFarland standards was met by adjusting the bacterial suspension with 0.85 % physiological saline, which corresponded to approximately 1.5 × 10 8 CFU/ml [11] .To assess the antimicrobial activity, six replicates of each bacterial species were inoculated onto MHA plates using sterile cotton swabs.To screen for inhibitory effects, sterile filter paper discs (6mm) were impregnated with 30 μl of the cyanobacterial extracts and then air-dried.Using sterilized forceps, the dried discs were placed on the Muller-Hinton agar plate.Colistin (10 μg/disc) was used as the standard antimicrobial disk for gram-negative bacteria such as Aeromonas hydrophila, E. coli , and Vibrio spp., Salmonella, Shigella sp., Pseudomonas spp., while Ceftriaxone (30 μg/disc) was used as the positive control for gram-positive bacteria including, Streptococcus saprophyticus, Staphylococcus spp.Sterile and clean Whatman filter paper was utilized as the negative control.The plates were then kept at 37 °C overnight while being inverted.The extracts created the distinct, circular inhibition zones surrounding the discs.Digital slide calipers (Robotics BD shipment) were used to measure the zone of inhibition from edge to edge.

Antimicrobial Activity Index (AI)
Comparison of the specific Oscillatoria sp.extract's zone of inhibition to that of a reference antibiotic, activity index (AI) was determined using the following equation [ 12 , 13 ]).

Act i v it y index =
Diamter of the inhibition zone of extract Diamter of the inhibition zone of the r e f er ence antimicrobial

Minimal inhibitory concentration (MIC)
Minimum inhibitory concentration of the potent Oscillatoria sp. was evaluated by tube microdilution method [14] .250 mg Oscillatoria sp.power was dissolved in 1 ml 100 % dimethyl sulfoxide (DMSO) and diluted with sterilized deionized water to 10 % DMSO.To achieve 1 % DMSO final concentrations, the stock solution was diluted with 10 % DMSO.After that, the samples were placed in a Sonicator machine for 10 minutes using pulsed, high frequency sound waves to agitate, and lyse the cells.The extract concentrations were then serially diluted to 300, 250, 200, 150, and 100 μg extracts/mL of 1 % DMSO.In a 96-well microtiter plate, each dilution was transferred using micropipette.Bacterial suspension was prepared and matched the standard of 0.5 McFarland.150 μl bacterial suspensions were added in each well of a microtiter plate (96 well) from the column 1 to 12. Positive growth controls included both bacterial suspension and DMSO, whereas negative growth controls included DMSO with Oscillatoria extracts.Then the plates were incubated at 37 °C.Using a POLARstar Omega Plate from BMG LABTECH, Offenbur g, Germany, the optical density of each well was measured spectrophotometrically at 630 nm.The lowest concentration of each test solution that prevented the development of any of the microorganisms in the wells was recorded as the MIC value following an overnight incubation.For each sample, the tests were carried out in triplicate, and the average concentration for each triplicate was determined.
The Author(s).Published by Elsevier Inc.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Data on antimicrobial activity test were collected through disk diffusion method.Bacteria were isolated by selective agar media streaking, Gram's staining, biochemical and VITEK-2 test.MIC value was determined by ELISA test using spectrophotometer (630nm).The collected data were analyzed by using MS Excel and IBM SPSS (v.26.0) software.

Table 1
Isolated pathogenic bacteria from marine diseased fish and Shellfish.

Table 2
Diameter of the inhibition zone (mm) exhibited by Oscillatoria species against isolated pathogenic bacteria.Values are expressed as mean of the six replicates with standard error (mean ± SE) and values with the different letters within each series indicate significant differences (p < 0.05) among the species.Concentration of phenolic extracts of Oscillatoria spp.were 30μl/disc.Colistin (10μg) and Ceftriaxone (30 μg) were used for reference antibiotic for Gram's negative and Gram's positive bacteria respectively.BacteriaOscillatoria sp. 1 Oscillatoria sp. 2 Oscillatoria sp. 3 Oscillatoria sp. 4 Antibiotic

Table 3
Antimicrobial index (AI) (mean ± SE) of Oscillatoria strains against isolated pathogenic bacteria.Values with the distinct letters in each series indicate significant distinctions (p < 0.05) among the Oscillatoria species.