Sahar Wefky Mostafa Hassan

Marine Microbiology Department, National Institute of Oceanography and Fisheries,
Alexandria, Egypt.

Abstract

A total of 11 bacteria were isolated from sea water and screened for their antibacterial activity against five bacterial pathogens. The most potent isolate (S1) was identified as Bacillus cereus S1 using 16S rRNA sequence analysis. B. cereus S1 exhibited antibacterial potentiality against, Pseudomonas florescence, P. aeruginosaStaphylococcus aureus, Vibrio damsela and Aeromonas hydrophila. The inhibition zones diameter were 11, 12, 12, 12 and 16 mm respectively. Maximization of the productivity with 1.3 fold increase was achieved using Plackett Burman experimental design. The optimized medium was formulated as (g/l): peptone, 7; beef extract, 1.5; sea water concentration (50%) with pH 5 and inoculum size (0.5ml for each 25 ml medium) and incubated for 12 h. Immobilization using adsorption on pumice improved the productivity by 1.6 fold compared to the basal medium while loss of antibacterial activity was up on using entrapment technique. The bioactive compounds were characterized by thermal stability even at 100 oC while they were inactivated up on exposing to ultraviolet radiation for 20  min. Moreover, the anticoagulant activity of B. cereus S1 was tested using activated Partial Thromboplastin Time (APTT) and  Prothrombin Time (PT) tests. It succeed to prolong the clotting time to 40 sec and 253 sec respectively which represents about 3.3 fold and 7.2 fold compared to the control. Hexane extract was compared with other standard antibiotics and it was superior in its antibacterial effect. The crude extract of B.cereus S1 was analyzed using gas chromatography–mass spectrometry (GC–MS). The main constituents were Cycloheptasiloxane, tetradecamethyl-, cyclooctasiloxane hexadecamethyl-, cyclononasiloxane  octadecamethyl- , Heptasiloxane, hexadecamethyl- and 1,2-Benzenedicarboxylic acid, diisooctyl ester (phthalate).

Keywords: Antibacterial, Anticoagulant, Antiinflammatory, Bacillus cerus S1.

Introduction

The resistance of pathogenic bacteria became the upcoming challenge in therapeutic agents, which is increasing with time due to misuse of antibiotics1,2. Gram-negative bacteria such as Aeromonas, Vibrio, Flavobacterium, Pseudomonas, and Francisella and Gram-positive bacteria from the genera Streptococcus and Lactococcus3,4,5,6,7,8,9,10, are some of the pathogens responsible for different diseases and economic losses11, 12.

Recently, side effects of new therapeutic agents which have entered the clinical area, have been reported13. The challenge for scientists is  to explore new antimicrobial agents from natural sources including bacterial strains with broad antimicrobial activity against different pathogens14, 15.

Most of the biological activities have borne from oceans. Every year, marine organisms produce hundreds of new compounds 16 such as photo protective, anti-microtubule, anti-proliferative, anti-tumor, cytotoxic, and antifouling properties. Isolation of new microbes from marine environment has been reported17, 18

Marine bacteria associated to invertebrate surface often produce secondary metabolites with amazing antagonistic activities including antivirulence, anticancer and antibacterial19. Search of secondary metabolites produced Bacillus species isolated from diverse groups of invertebrates has under laid objectives by the scientific community In the past two decades and obtained therapeutic enzymes, novel metabolites and bactericidal agents20,21,22. All these natural compounds with broad biological activities enable the bacterium to survive in its natural environment.

The environmental and nutritional conditions have a great influence on production of the antimicrobial substances. In order to develop an efficient product of antimicrobial  substances, knowledge regarding the environmental factors affecting this process needs to be well identified23,24. Optimal production is achieved by using experimental designs as an excellent tool for optimization of culture conditions.

Thus the purpose of the present study is to isolate a marine invertebrate associated bacteria, evaluation of the antibacterial activity against different bacterial pathogens. Moreover the study would extend to optimization of the fermentation conditions using statistical design. Improvement of the production was also carried out. Furthermore, extraction and characterization of the bioactive compounds will be carried out and also identified by gas chromatography–mass spectral (GC–MS) and infrared spectroscopic analysis. Other different applications such as anticoagulation and anti-inflammation will be studied.

Materials and methods

Microorganism and culture conditions
The marine bacterial isolate was isolated from the surface of shrimp. It was isolated upon seawater agar medium and was maintained on nutrient agar slants25

Pathogenic indicators
Different Gram positive and Gram negative pathogens and including  Staphylococcus aureus ATCC 6538, Streptococcus faecalis, Pseudomonas aeruginosa ATCC 8739, Vibrio anguillarum and Vibrio  fluvialis were used as target strains for detecting the antagonistic properties. These indicator bacteria were kindly provided from the Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University

Antibacterial activity
B.cereus S1was precultured in marine nutrient broth at 30 oC on a rotary shaker incubator until the absorbance of the culture at A 550=1. The culture broth was centrifuged at 10000 rpm for 15 minutes to remove bacterial cells26. The ability of the cell free supernatant to inhibit the growth of the indicator bacteria was performed using the well-cut diffusion technique. Briefly, five-millimeter-diameter wells were punched in agar plates (using a sterile gel puncher) inoculated with bacterial pathogenic strains. 50 µl of each tested compound was added in each well. After incubation at 30oC for 24h, the radius of the clear zone around each well was linearly measured in mm27.

Bacterial identification
DNA was isolated, purified using standard procedures28 and the region of 16S rDNA was amplified using universal primers (27F (5`-AGAGTTTGATCCTGGCTCAG-3`), 1492R (5`-GGTTACCTTGTTACGACTT-3`).16S sequence analysis was used to perform the genotypic characterization. Multiple alignments with the most closely members sequences similarity levels were carried out using Blast program (http://www.ncbi.nlm.nih.gov/blast). Sequences of rRNA genes, for comparison, were obtained from the NCBI database. A phylogenetic tree was reconstructed by Bioedit software .

Optimization of fermentation conditions using Plackett Burman experimental design
The  Plackett -Burman experimental design29,30 was  applied  to  reflect  the  relative importance of various factors on the production of the bioactive compounds by B.cereus S1. High (+)   and   low (-) levels for  each   variable were tested.  The  examined  variables  in  this experiment  and  their   levels  are  shown  in  Table  1.  Duplicate of the eight different trials were performed.  The   different trials (Row  no.9  represents  the  basal  control) are presented in Table 1. The  main  effect  of  each variable  was  determined  with  the  following  equation:

Exi  =  (Mi+  –  Mi-) /  N

Where  Exi  is  the  variable  main  effect,  and  Mi+, Mi-  are   the   inhibition zone  (mm)   in  the  trials,  each   independent  variable  was  used  in  high  and  low concentrations,  respectively,  and  N  is  the  number   of  trials  divided  by  2.  Microsoft Excel was used to calculate t-values   for   equal unpaired   samples to determine the variable significance.

Table 1. The applied Plackett–Burman experimental design for seven cultural variables

Trial Factors
Peptone
(P)(g/l)
Beef
(B)
(g/l)
pH Inoculum size
(IS)
(ml)
Incubation period
(IP)
(ml)
Seawater concentration
(SW)
 (%)
Culture Volume
(CV)
(ml)
Diameter of inhibition zone (mm)
1 – [3]   -[1.5] -[5]   +[1.5] +[48] +[150] -[25] 13
2 +[7] – [1.5] −[5] -[0.5] -[12] +[150] +[75] 13
3 −[3] +[4.5] -[5] −[0.5] +[48] -[50] +[75] 15
4 +[7] +[4.5] −[5] +[1.5] −[12] -[50] -[25] 17
5 −[3] _[1.5] +[9] +[1.5] -[12] −[50] +[75] 13
6 +[7] −[1.5] +[9] −[0.5] + [48] -[50] −[25] 13
7 −[3] +[4.5] +[9] _[0.5] −[12] + [150] _[25] 14
8 +[7] +[4.5] +[9] +[1.5] +[48] + [150] +[75] 0
9 0[5] 0[3] 0[7] 0[1] 0[24] 0[100] 0[50] 16
Main effect 3.75 -1.5 -4.5 -3 -4 -4 -4
t-value -0.8 -0.39 -1.1 0.1 -1.12 -1.3 -1.12

Standard t-values are obtained from statistical methods (Cochran &Snedecor, 1989)31


Preliminary characterization of the bioactive compounds
Effect of immobilization technique on production of antimicrobial agents
Immobilization was carried out by using adsorption of B.cereus S1 cells on different solid porous  supports including luffa pulp, pumice, sponge and clay,  1.5  ml of bacterial  suspension  were added  to  250 ml  sterile  flasks  containing five  grams  of  porous support materials  in 50   ml   of optimized  culture  medium. Luffa  pulp   and   sponge  pieces were  around  0.5  cm  in  diameter. The   flasks   were then shacked slowly at 120 rpm under the optimized conditions. The antibacterial activity was estimated using well cut diffusion technique and compared with the free cells32.

Effect of temperature
Ten millilitres each of the cell free culture supernatant of B.cereus S1 were dispensed in various 50 ml screw capped conical flasks. The flasks were subjected to different temperature treatment (30, 60, 100 oC) for 20 min33. The antibacterial activity was estimated using well cut diffusion technique.

Effect of UV
The effect of UV light on antimicrobial activity was determined as follows: 10 mL of filter-sterilized cell-free supernatant was exposed to UV irradiation at a distance of 25 cm for 15, 30, 45 and 60 minutes34. After each time interval, antimicrobial activity was analyzed by well-cut diffusion technique.

Scanning electron microscopy
The bacterial isolate was grown on optimized medium. A plug of cells was removed and  prepared  for  fixation  and dehydration procedures according to  Bozzola and Russell35. The samples were dried completely in a critical point dryer and finally  coated  with  gold  in  SPI-MODULE  sputter  coater.  Then the specimens were viewed with a JEOL–JSM 5500LV.

Anticoagulant activity
Evaluation of Activated Partial Thromboplastin Time (APTT)
All clotting assays were carried out using normal citrated human plasma  according to the manufacturers’ specifications. For this, 50 µL of citrated normal human plasma was mixed with 50 µL of a supernatant solution before adding 50 µL of APTT reagent. The mixture was then incubated at 37°C for 3 min. Then, 50 µL of 0.025 M calcium chloride reagent was added to the mixture to trigger the coagulation cascade. The clotting time was recorded in seconds. Heparin was used as the standard36.

Evaluation of Prothrombin Time (PT)
All clotting assays were carried out using normal citrated human plasma according to the manufacturers’ specifications. For this, 50 µL of human plasma was mixed with 50 µL of a supernatant and incubated at 37°C for 3 min. Then, 50 µL of 0.025 M PT reagent was added to the mixture to trigger the coagulation cascade. The clotting time was again recorded in seconds36.

Anti-inflammatory activity
Inhibition of albumin denaturation
Anti-inflammatory activity of the supernatant was estimated using methods  of  Sakat et  al (2010)37. The reaction mixture was consisting  of  test  extracts  and  1%  aqueous  solution of  bovine albumin  fraction,  pH  of  the  reaction  mixture  was  adjusted  using small amount at 37°C HCl. The sample extracts were  incubated at 37°C for  20 min  and then  heated to  51°C for  20 min  after cooling the  samples  the  turbidity  was  measured  spectrophotometrically  at 660  nm.  The experiment was  performed  in  triplicate. Percent inhibition of protein denaturation was calculated a s follows:

% inhibition= [{Abs control- Abs sample}/Abs control] ×100

Extraction of bioactive compounds
Different organic solvents including ethanol, chloroform, methanol and hexane were used for extraction of the antimicrobial agents. 50 ml of each solvent was added to 50 ml fermented broth in a 250 ml separating funnel. The mixture was shaken vigorously for 20 min and kept to separate the solvent from aqueous phase. The organic phase was collected and evaporated by using a rotary evaporator and then dissolved in appropriate solvent38. Antibacterial activity was determined each time using well cut diffusion technique.

Antibiogram
Sensitivity of A.hydrophila to different standard antibiotics including Streptomycin (10µg), Chloramphenicol (30 µg) and Ampicillin sulbactam (20 µg), Tetracyclin (30 µg) and Amoxixicillin (10 µg) was tested . The antagonistic effect of these antibiotics were compared to that of the active fraction using disk diffusion method39 .

Spectral analysis
The active extract was analyzed using GC–MS. The peaks were identified by comparing with WILEY MASS SPECTRAL DATA BASE Library40.

Infrared
Pellets for infrared analysis were prepared by grinding a mixture of 1mg sample with 200 mg dry KBr, followed by pressing the mixture into a 16 mm diameter mold. The Fourier transform-infrared (FT-IR) spectra were recorded on a Bruker Vector 22 instrument with a resolution of  4 cm-1 in the 4000-400 cm-1 region.

Results

Screening for antibacterial activity
The marine isolate S1 isolated from the surface of shrimp was tested for its potentiality in production of antimicrobial agents against some fish pathogens including V. damsel, P.aeruginosa, S. faecalis, S.aureus and A. hydrophila with maximum zone of inhibition comparatively to others. Results in Figure 1 showed that the highest antimicrobial activity was against A. hydrophila (16mm) followed by P. aeruginosa and S. aureus (12 mm), while the lowest antimicrobial activity was against P. florescence (11 mm). On the other hand there was lack of activity against S. faecalis and thus A. hydrophila was chosen to complete the study as it is the most susceptible bacterial pathogens. Results concluded that Gram negative bacteria was the most susceptible for the antagonistic compounds which is coincide with Telesmanich et al. (2000)40. The difference in the susceptibility may be due to physiological characteristic and metabolism of each strain, cell wall structure and degree of contact 41.

Fig. 1. Antibacterial activity of B.cereus S1 against some bacterial pathogens

Antimicrobial activity by Bacillus spp. was proven in previous studies 42, 43, 44. The results obtained in the present study indicated the potential production of bioactive compounds by marine Bacillus sp.  Anand et a l(2006)45 reported the production of a highly active metabolites by marine Bacillus sp. Mohan et al (2016)46  stated that Bacillus sp. isolated as sponge endosymbiotic bacteria showed antimicrobial activity with broad range against virulent marine fish pathogens such as Vibrio alginolyticus,Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas salmonicida, Flavobacterium sp., Edwardsiella sp., Proteus mirabilis and Citrobacter brackii with zones of inhibition (16-23 mm). Many scientists isolated Bacillus sp. from diverse group of invertebrates and confirmed the production of novel active metabolites against aquatic fish pathogens 20, 21, 47.

Molecular identification of the marine isolate S1
DNA of the promising Bacillus sp. was extracted and the extracted 16SrRNA gene was amplified using the universal primers. The produced amplicons was analyzed using agarose gel electrophoresis as shown in Figure 2a. The amplified DNA was partially sequenced. This sequence was compared with those which gave the highest homology using Blast search computer based program. The resulting data indicated that the isolate under study was identified as B.cereus S1.The obtained similarity was 99% with session number KX683220. The phylogenetic relationships of this experimental isolate and the closely related relatives were analyzed as shown in Figure 2b.

Fig. 2. 1 6S   agarose gel electrophoreses of the extracted and amplified DNA. Lanes 1, 2, 3 are the purified PCR products. Lane 4 is molecular weight marker (a); Phylogenetic relationships among the representative experimental strain and the most closely related Bacillus species (b). The dendogram was generated using tree view program

Optimization of fermentation conditions using Plackett- Burman experimental design
Two phases of the application of Plackett-Burman statistical design was carried out. The first step was to screen for important factors and their levels that affect production process in shake flasks (Table 1). The second was the verification experiments to validate the results under specific optimized medium. All experiments were carried out in duplicates. Table 1 shows results of the experimental design. It was observed that the production of the antimicrobial agent was negatively affected by beef extract, inoculum size, incubation period, sea water (%), culture volume and positively affected by peptone concentration, which means that increasing levels of peptone cause increase in the antagonistic activity which in accordance with other previous studies48 , while decreasing levels of the other variables cause increase in the antagonistic activity. The effect of some of these factors on the production was similar to that reported by  Ali (2012)49 who stated that beef extract, culture volume, inoculum size and incubation period had negative effect on antimicrobial agents production by marine Pseudomonas piscicida B12. The pH optimum for antimicrobial agent production was 6 which is in accordance with Wen Zhou et al (2010)24 who stated that optimum pH for production of bioactive compounds by B. cereus S1 was in the acidic range as was also reported by Sana et al (2008)50. Regarding effect of NaCl, Balakrishnan et al (2014)44 observed that effect of NaCl is dependent on the mechanism of expression of the bacterium to the particular salt concentration.

The main effect of each variable on the production of the bioactive compounds as  well  as t-values were estimated for each independent variable as shown in Table 1 and graphically  presented in Figure 3. Results in this Figure indicated that the main effect of all variables were negative on the production of the antagonistic agents except for peptone which exhibited positive effect on the production. Results of t-test indicated that variations in sea water (%), culture volume and incubation period in the tested ranges had the most considerable effects on production of the antimicrobial agents by B.cereus S1. The interacting effect of sea water (%), culture volume and incubation period in three -dimensional representation is illustrated in Figure 4.

Fig. 3. Elucidation of fermentation conditions affecting the production of the antagonistic agents by B.cereus S1

Fig. 4. Interaction effect between  culture volume (CV) and  sea water (%) (SW) (a); culture volume (CV) and incubation period (IP) (b) and seawater (%) (SW) and incubation period (IP) (c) Levels, with respect to inhibition zone (IZ)(mm) based on Plackett-Burman results

Abd-Elnaby et al (2016)51 reported that increasing levels of pH, inoculum size caused an increase in the antagonistic activity by about 1.3 fold for Streptomyces parvus. Conversely to the present study, some of this finding was reported by Wefky et al. (2009)48

According to the obtained results, the predicted medium for cultivation of   B.cereus S1 to enhance maximum production of the bioactive compounds was formulated as follows:  (g /l) :peptone,7; beef extract, 1.5; concentrated seawater (50%), adjusted pH 5 and inoculum size (0.5ml for each 25 ml  medium) all of which are incubated for 12 h.

Verification experiment
A verification experiment was carried out in order to evaluate the accuracy of the applied Plackett-Burman statistical design, predict the near optimum levels of independent variables. The obtained data were examined and compared to the basal and anti-optimized medium. Data revealed that antagonistic activity produced by B.cereus  raised to 21 mm and realized 1.3 fold increase when growing in optimized medium (Figure 5).

Fig. 5. Verification experiment of the applied Plackett-Burman statistical design by comparing the antagonistic activity of B.cereus S1 growing on the resulting optimized medium, the basal medium and the anti-optimized medium

Effect of immobilization on production of the bioactive compounds
Immobilization on different support materials was investigated to enhance the production of the antibacterial agents. As shown in Table 2, the antagonistic activity was increased by 1.2 fold compared to the free cells, while adsorption on the other support materials caused complete disappearance of the antagonistic activity. On the other hand, antagonistic activity was also disappeared up on using entrapped cells. These results may be due to poor mechanical stability of the support.  Diffusion limitation of the bioactive compounds is an important factor affecting this process too52. Abd-Elnaby et al. (2016) 51 also reported the potentiality of the immobilized cells in raising the antimicrobial activity of Streptomyces parvus compared to free cells.

Table 2. Antibacterial activity using free and immobilized cells

Response Antagonistic activity using different support materials
Free cells Luffa pulp Sponge Pumice Clay Ca alginate beads
IZ.(mm)  21 0 11 25 0 0

Preliminary characterization of the antibacterial metabolites in cell free supernatant
Stability of antimicrobial compounds produced by B.cereus toward temperature and UV was examined. Screening the effect of different temperature on stability of the bioactive components (Figure 6) showed that the antibacterial compounds produced by B.cereus were relatively stable even after boiling at 100 oC. Extreme temperatures, boiling for 10 min, had no affect on the antimicrobial agents produced by B. subtilis as was reported by Sabaté and Audisio (2013)53. The same finding was observed by Risøen et al. (2004)54 who stated that the antimicrobial compound retained activity over a wide range of temperatures even up to 100oC. in other study by Chalasani et al (2015)55, the antimicrobial compound was also stable at different temperatures 80% activity was retained a 80◦C for 1 h, 75% at 100◦C for 30 min and 60% at 121◦C for 20 min. On the other hand, exposing the cell free supernatant to UV radiation for different time intervals caused complete absence of antagonistic activity. It can be stated that bioactivity of the compounds is dependent on the mutation effect on the active gene which responsible for the production of antibiotics56.

Fig. 6. Effect of temperature on stability of the inhibitory activity produced by B.cereus S1

Fig. 7. SEM photomicrographs showing lysis of  A.hydrophila cells by a 24 hrs culture filtrate (0.45 µm) of  B.cereus S1a) control (no addition)  (b) and (c) treatment  with supernatant after 24 hrs incubation

Assay of bacteriolytic activity
The bacteriolytic activity of a 0.45 µm culture filtrate from 24 hrs of B. cereus S1culture against A. hydrophila was detected using SEM. Figure 8 represents the morphological changes of the nontreated and treated A. hydrophila. Figure 8a shows the SEM micrographs of bacterial cells without the hexane extract treatment. The figure revealed the normal rod shape cell structure without any shrinkage or cavity formation as the surface was smooth and regular. Figure 8b shows the morphology of the cell after 24 h of treatment with the supernatant. The bacterial cells started to show multiple defects with many of cells exhibited crumpled or shrunken cell surface. Figure 8c revealed that more formation of crumpled cells and cells lysis were formed up on treatment with hexane extract for 24 h. It was reported that the cell wall of Gram negative bacteria (A.hydrophila) is surrounded by an outer membrane consisting of lipopolysaccharides, phospholipids and lipoproteins and thus they are less sensitive to bacteriolytic enzymes than Gram positive  bacteria57. Only a few active compounds have been reported to lyse cells of Gram negative bacteria58; therefore, the present study is interesting that  the isolate B.cereus  S1 was active against A.hydrophila which can be applied as probiotic in aquaculture.

Fig. 8. Antibacterial activity of hexane extract compared with other different standard antibiotics

Fig. 9. Gas chromatogram spectral analysis showing the major peak of the active compound (a), mass spectral analysis of phthalic acid in the crude extract (b) and IR spectrum of the phthalic acid derivatives (c)

Table 3. Anticoagulant and anti-inflammatory activities of the bioactive metabolites

 

 

Anticoagulant activity Anti-inflammatory
activity
Prothrombin time
(PT) (sec.)
Partial Thromboplastin Time
(APTT) (sec.)
Supernatant 35 253 85%
 
Heparin  87

Anticoagulant activity
Pharmaceutical importance of the bioactive compounds depends up on the positive chemical interactions with microorganisms59. Bacterial supernatant was tested for blood coagulation effects in normal human plasma using heparin as standard. Results in Table 4 revealed that the supernatant exhibited grater activity with prolonged the clotting time 40 sec which represents about 3.3 fold compared to the control which suggests that the metabolites produced by B. cereus S1 is an effective antithrombotic agent. On the other hand the tested supernatant was capable of increasing the normal coagulation time up to 253 sec with 7.2 fold in relation to normal APTT time. Hassanein et al (2011)60 reported that the clotting time of human blood serum in the presence of active metabolites produced by B. subtilis K42 reached a relative PTT of 241.7% with a 3.4-fold increase, Similarly Wei et al (2011)61 stated that the fermented chickpeas from B. amyloliquefaciens showed anticoagulant activity, and the purified anticoagulant component showed higher anticoagulant activity than heparin sodium.

In vitro anti-inflammatory activity
Inhibition of albumin denaturation
Denaturation  of  proteins  is  a  well  documented  cause  of inflammation.  As  part  of  the  investigation  on  the mechanism  of  the  invitro anti inflammation  ability  of the  metabolites produced by B.cereus S1, protein denaturation was studied. It showed relatively good antiinflamatory activity (86%). Our finding is supported by study of Kurian et al (2015)62 who prove the anti-inflammatory activity of Bacillus spp. BTCZ31

Extraction of the antimicrobial agents
Different solvents were screened for their efficiency in extracting the bioactive compounds. Hexane was the most efficient solvent exhibiting the highest value of inhibition zone (21 mm). The other solvents showed varied efficiencies against the indicator bacteria (Table 4). The antibacterial effect was not observed up on using methanol, ethanol and chloroform once the extraction procedures had attempted to isolate and concentrate the biologically active compound. This may have been a consequence of the organic solvents denaturing the compound during the procedure or that the compound is labile or that the compound is a type of molecule not extracted into these solvents63 .

Table 4. Efficiency of different solvents for extraction of the active compounds

Solvent
Inhibition zone diameter (mm)
Ethnol
0
Methanol
0
Chloroform
0
Hexane
21

 

Antibiotic susceptibility test
Sensitivity of A.hydrophila to  different  standard antibiotics including   was tested . The antagonistic effect of these antibiotics were compared to that of the active fraction. It was superior in its effect than Streptomycin (10 µg), Chloramphenicol (30 µg) and Ampicillin sulbactam (20 µg) with 2.7, 2.1 and 1.5 fold respectively and also than Tetracyclin (30 µg) and Amoxixicillin (10 µg) which showed no antibacterial activity against A. hydrophila (Figure 7) this was confirmed by study of  Barakat and Beltagy, 201564 who reported the sensitivity of A. hydrophila to the bioactive compounds produced by  Streptomyces ruber EKH2 rather than that of some tested commercial antibiotics.

Spectral analysis
GC-MS analysis of hexane extract (Figure 9a) was carried out to identify the components in the extract  The major compound was at retention time 45sec. corresponds to a molecular formula of C24H38O4, is identified as 1,2-Benzenedicarboxylic acid, diisooctyl ester (phthalate). The mass spectra of this compound is shown in Figure 9b. Antibacterial activity of phthalate against Staphylococcus aureus (A TCC6538), Streptococcus faecalis (ATCC8043), Pseudomonas aeruginosa (A TCC 8739),  Escherichia coli (A TCC 8739),  Micrococcus luteus (A TCC 10240) and  Candida albicans was evidenced in previous studies65, 66. Barakat and Beltagy (2015)64 stated that phthalate showed antibacterial activity with broad spectrum against Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas aeruginosa and Vibrio ordalii. IR spectra showed peaks at 2994 (CH2), 1763 (C‚O), 1637 (C‚C), 1242 (C–O) and 1056 (C– H) cm-1 (Figure 9c).

Conclusions

The present research indicates that the marine bacterium B.cereus S1 has the potentiality to target the growth of both Gram negative and Gram positive bacteria which supports the successful use of the strain as a biological control agent. Moreover, it was proved as anticoagulant and anti-inflammatory agent. One of the challenges in future will be the large scale production of these compounds to meet the demand for different applications which can be applied as probiotic in aquaculture system. Heat stability of  the  inhibitory  substances  produced  by  B. cereus S1 can effectively be  used as bio-preservative in food. Further studies are needed for complete identification of the active compound.

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