In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus

The main objective of this research is to investigate the anti-biofilm and anti-bacterial activity of Sesbania grandiflora (S. grandiflora) against Staphylococcus aureus. S. grandiflora extract were prepared and analyzed with UV –Vis spectroscopy, Fourier transform infrared spectroscopy, Dynamic light scattering. Biofilm forming pathogens were identified by congo-red assay. Quantification of Extracellular polymeric substance (EPS) particularly protein and carbohydrate were calculated. The efficacy of the herbal extract S. grandiflora and its inhibition against the pathogenic strain of S. aureus was also evaluated. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora. The antibacterial activity of S. grandiflora extract against the bacterial strain S. aureus showed that the extract were more active against the strain. To conclude, anti-biofilm and antibacterial efficacy of S. grandiflora plays a vital role over biofilm producing pathogens and act as a good source for controlling the microbial population.


Introduction
Bio films are defined as microbial communities of cells attached either to a biotic or abiotic surface enclosed in a complex EPS (Extra cellular Polymeric Substance). Biofilms are formed with interaction among microbial aggregates, filamentous bacterial strains, organic and inorganic particles, which are held together by EPS [1,2]. EPS majorly comprised of four substance called carbohydrate, protein, lipids and extracellular DNA (e DNA). The biofilm acts as a protective barrier and provides resistance against antibiotics, degrading enzymes, protozoan grazers and host immune response [3,4]. Some bacterial biofilms have been reported to have useful effects on sewage treatment plants, food chains etc [5]. Moreover, biofilms also posses diverse problems in food industry, medicine, biofouling of boats, cooling towers, water pipes etc. The regular use of antibiotics makes the biofilm producing bacteria resistant to antimicrobial agents [6]. Hence, the development of antibiofilm strategies is therefore a major interest and currently constitutes an important field of investigation in which environmentally friendly anti-biofilm molecules or organisms are highly valuable.
Traditionally, humans have utilized crude extracts of medicinal plants as curative agents for various ailments. Plant extracts and other biologically active compounds isolated from leaves, stems, and roots have gained interest in anti-biofilm activity [7][8][9]. Taking this as an initiative, S. grandiflora were selected to investigate the anti-biofilm and anti-bacterial activity.
The aim of this study was to investigate the anti-biofilm and antibacterial activity of S. grandiflora against S. aureus.

Plant material
The leaves of S. grandiflora were collected from Vellore district. The collected leaves were washed with double distilled water and were airdried at room temperature for two weeks and coarsely powdered [13] ( Fig. 1).

Preparation of S. grandiflora seed extract
The powdered sample was boiled with 100 ml of sterile distilled water at 60°C for 1 h. Then, the extract was filtered through Whatman No. 1 filter paper and used for further experiments (Fig. 2).

Antimicrobial activity
The antimicrobial activities of the leaves extract was determined by disc diffusion method. Nutrient agar (NA) plates were prepared, sterilized and solidified. The culture Staphylococcus aureus was swabbed uniformly onto the NA plates using sterile cotton swabs. Plates were incubated at 37°C for 24 h [11,14,15].

Biofilm formation in Congo red agar
NA plates were prepared, inoculated and incubated at 37°C for 24 h. The cultured plate was flooded with 0.2% congo red dye. After 15 min, the plates were destained with 1 M NaCl [16].

EPS production
Staphylococcus aureus culture of 20 µl (222 CFU) was added in freshly prepared 50 ml of sterile nutrient broth and were treated with Sesbania grandiflora plant extract powder of 3 different concentration (250, 500,750 mg) and one is maintained as control without the addition of plant extract then the samples were incubated at 37°C for 24 h at 140 rpm. Control and the treated samples were centrifuged at 12,000 rpm for 15 min at 4°C and to the supernatants were collected, to that three volumes of ice cold isopropanol was added and incubated overnight at −25°C. The incubated samples were centrifuged at 5000 rpm for 30 min and pellets were collected (EPS) and air dried at room temperature. The collected EPS of control and treated were purified, and stored for further analysis [17].

EPS characterization
The control and treated EPS samples were characterized by UV -Visible Spectroscopy analysis, Fourier Transform Infrared Spectroscopy (FTIR), Light Scattering analysis [18,19].

Emulsifying activity
The control and treated EPS solution (2 mg/ml) was heated at 100°C for 15 min followed by cooling (25°C) and the volume was made upto 2 ml using phosphate-buffered saline. 1 ml of olive oil was added, vortexed for 1 min and the absorbance was measured after 30 min at 540 nm. The emulsifying activity was calculated in terms of percentage decline in the absorbance at different time intervals [19].

Quantification and inhibition of EPS components
EPS was quantified by measuring proteins (Lowry's method) and carbohydrates (Anthrone method) [17].
The percentage of EPS inhibition was calculated using the formula = × % inhibition control OD -Treated OD/Control OD 100

Antimicrobial activity
The anti microbial potential of S. grandiflora was evaluated according to their zone of inhibition against S. aureus pathogens and the results were compared with the activity of the standards viz, Ampicillin. 40 µl of S. grandiflora were found to be more effective against S. aureus when compared with the standards

Biofilm formation in Congo red agar
Biofilm formation of S. aureus on NA plates supplemented with congo red appeared as dark red around the bacteria colonies. The biofilm produced by bacteria is made up of exopolysaccharide matrix that protects microbes from host immune system and anti-microbial therapy. Based on our results, S. aureus displayed highest biofilm forming ability (Fig. 3).

EPS production
Maximum EPS was produced after 48 h in Nutrient broth by S. aureus.

UV visible spectroscopy
The UV-Vis analysis was used for the identification of optical property of S. grandiflora.
The high exciton binding energy was responded at 270 nm as clear absorption band (Fig. 4).

FTIR
Fourier Transform Infrared spectroscopy was used to find out the biomolecules. FTIR spectrum shows peak at 707 cm −1 , 1075 cm −1 , 1659 cm −1 , 2942 cm −1 , 3462 cm −1 which confirms the presence of carboxylic group, O-acetyl ester linkage bond, C-H stretching of sugar molecules, C-H functional groups and hydroxyl group respectively. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora (Fig. 5).

Dynamic light scattering
The stability of EPS was analyzed by Zeta potential which is high in the control when compare to treated EPS. The value number of the zeta potential showed the stability of the EPS. This analysis is mainly used to check the cell wall penetration of EPS and stability of the bacteria ( Fig. 6; Table 1).

Emulsifying activity
A stable emulsion of the EPS was observed with Olive oil. This yielded 48.51%, 53.62%, 72.11% (at different concentration 250, 500, 750 mg/ml) emulsifying activity with olive oil after 30 min of incubation. The emulsifying activity of EPS was measured before 30 min because emulsification will break within 30 min, due to the stability of the sample. The presence of both hydrophilic (hydroxyl) and hydrophobic (aliphatic CH 2 ) functional groups may be responsible for its emulsifying property (Fig. 7).

Quantification and inhibition of EPS components
The optical density of the control and treated EPS revels that the amount and inhibition percentage of the protein in the given sample by measuring at 660 nm and inhibition percentage of carbohydrate in the given sample was measured at 620 nm ( Fig. 8; Table 2).
The gradual decrease in the amount of protein and carbohydrate in control and treated EPS were calculated in terms of percentage. The amount of protein in EPS decreases with the increase of plant extract. This variation in control and treated EPS is due to increase in plant extract concentration ( Fig. 9; Table 3).

Discussion
S. grandiflora was extensively studied by different scientists and academicians for its phytopharmacological potentials especially on leaves, flowers and seeds. Recent investigation demonstrated that use of leaf extract of S. grandiflora act as a reducing agent and synthesized spherical shaped silver nanoparticles [20]. Moreover, the S. grandiflora flower acts as a promising material to develop the active ingredient of anti-plaque toothpaste as well as mouthwash solution [21]. It has been reported that a biofilm is strongly associated with the drug resistance property [22]. Hence, eradication of biofilm is often considered to be a difficult task and therefore use of plant products to inhibit biofilm may be a viable alternative [23].
In this study, the biofilm inhibition potential of S. grandiflora against S. aureus was evaluated by using quantitative spectroscopic techniques. 40 µg of S. grandiflora leaf extract showed potential antibacterial activity against S. aureus. S. grandiflora extract have been known to contain alkaloids, flavonoids, saponins, tannins and steroids [24]. These active compounds have the potential to inhibit adhesions and prevent matrix formation. There were reports which state that biofilms acquire resistances to inhibitors under nutrient limited or depleted conditions in contrast to their susceptibility conditions. S. grandiflora leaf extract shown to have anti-biofilm efficacy under both nutrient repleted and nutrient depleted conditions, which indicate the presence of bioactive agent in the leaf extract [25].
Carbohydrate and proteins were considered as a main constituent of EPS in pure culture. In this study, the ratio of carbohydrate and protein of the pure culture was comparatively less after treatment with S. grandiflora leaf extract [26].
UV-Vis spectroscopy analysis of control and treated EPS revealed a major absorption peak at 270 nm, which revealed that the intensity of the peak is directly proportional to the concentration of the EPS. This was similar to surface Plasmon vibration with the previous work. FTIR results represented that the active biomolecules present in the leaves of S. grandiflora was responsible to inhibit the biofilm formation [27]. The broad stretching of 3462 cm −1 indicates the presence of hydroxyl group. The band at 2942 cm −1 was due to the presence of C-H functional groups, which revealed the presence of sugar. Another peak at 1659 cm −1 could attribute to the C-H stretching of mannose or  Biochemistry and Biophysics Reports 12 (2017) 193-197 galactose [28]. The absorption peak at 1075 cm −1 may be attributed to O-acetyl ester linkage bond. DLS showed that the decrease in volume number indicate the degradation of the protein molecule in EPS, and inturn revealed the decreased stability than the Control [29].

Conclusion
In this study, a suitable and efficient method for the EPS extraction from S. grandiflora biofilms was achieved. This study also suggests that S. grandiflora extract possesses compounds with potential antimicrobial properties. To conclude, the aqueous extract of S. grandiflora leaf extract showed evidence of high anti-biofilm and anti-bacterial activity property against S. aureus. Further research is needed to elucidate the antimicrobial agents from S. grandiflora that are pharmacologically important using advance techniques in an eco-friendly approach   Reports 12 (2017) 193-197