Antibacterial activity of Ulva fasciata against Multidrug Resistant Bacterial Strains

The present study was conducted to evaluate the antibacterial activity of different organic solvent increasing polarity viz. , hexane, chloroform, ethyl acetate, acetone and methanol extracts of Ulva fasciata (Chlorophyceae) were collected from Kanniyakummari, Gulf of Mannar biosphere Reserve, Tamilnadu, India. Marine green algae extracts of U. fasciata against multi-drug resistant standard and clinical bacterial strains viz., Bacillus subtilis, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio cholerae, Shigella flexneri, Proteus mirabilis and P. vulgaris. The ethyl acetate extracts of U. fasciata showed highest antibacterial activity against all the bacterial strains tested. The mean zone of inhibition produced by the extracts in disc diffusion assays were ranged from 7.1 mm to 15.0 mm. The Minimum Inhibitory Concentrations (MIC) were between 125 µg/ml and 500 µg/ml, while the Minimum Bactericidal Concentrations (MBC) were between 250 µg/ml and 1000 µg/ml. The highest mean of zone inhibition (15.0 mm) and lowest MIC (125 µg/ml) and MBC (250 µg/ml) values were observed in ethyl acetate extract of U. fasciata against B. subtilis . The ethyl acetate extract of the U. fasciata showed the presence of phytochemicals, terpenoids, tannins and phenolic compounds in U. fasciata than the other solvents extracts. The present results of the ethyl acetate extract of U. fasciata can be used as an antibacterial substance for the treatment of multi drug resistant bacterial infections.


INTRODUCTION
Chlorophyceae seaweeds, popularly known as green algae, are widely distributed in both inter-tidal and deep-water regions of the seas. These seaweeds are of immense pharmaceutical and agricultural value. U. fasciata Delile, a green alga (Division: Chlorophycota; Class: Ulvophyceae; Order: Ulvales), belonging to the family Ulvaceae, commonly known as ''sea lettuce". Plants to several decimeters tall; characteristically deeply lobed or divided with clefts often extending to near holdfast; divisions somewhat digitately arranged from broadened basal region; blades plane, margins simple, crisped, or slightly undulate, bright gold when reproductive, margins then eroding; blades mostly (25-) 45-110 µm thick in central part, thinner toward margins. Cells tending taller than broad (10-25 µm) in many specimens, or more quadrate and of equal dimensions; near base of blades, extracellular material between two cell layers thickens blade; margins relatively smooth, but irregular from erosion of spent reproductive cells; with occasional coarse spines on basal portions. U. fasciata occupies a major share amongst different green algae in the coastal region of southern India (Selvin and Lipton, 2004).
The Ulva are a group of edible algae that are widely distributed along the coasts of the world's oceans (Wolf et al., 2012), and they have an interesting chemical composition that makes their commercial exploitation attractive to produce functional or health promoting food (Messyasz and Rybak, 2010). Seaweed U. fasciata have showed antimicrobial activities against S. aureus and P. aeruginosa that are commonly found in human infections (Selvin and Lipton, 2004). In recent years, wide concern on the antioxidant effects of U. fasciata has been aroused from scholars all over the word .
In recent years, the escalation of multidrug resistance in bacteria has gained worldwide attention due to the high impact on public health. Increased usage of antimicrobial agents to treat bacterial infections has lead to the emergence of multi drug resistant (MDR) strains the increasing of MDR incidence in the genetic and mechanisms of resistance evolved by bacteria, as such information could lead to strategies for counter acting the effect of antimicrobial resistance (Bonnet, 2004).
There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases, appearance of undesirable side effects of certain antibiotics, as well as the increasing development of resistance to the must be taken to control the use of antibiotic, to better understand the genetic mechanisms of resistance and to continue studies to develop new drugs. There are different approaches to cure and control the infection caused by the MDR strains of bacteria (Cowan, 1999).
Hence the present investigation was made to evaluate the antibacterial activity of different extracts of U. fasciata against clinical and standard MDR bacterial strains.

1. Sample collection and Preparation of extracts
Ulva fasciata (Chlorophyceae) were collected from the rocky shores of Kanniyakumari at (Lat. 9°11′N; Long. 79°24′E) Kanniyakumari district, Gulf of Mannar Marine biosphere Reserve, Tamil Nadu, India. The collections were made from the month of October 2011. The fresh seaweed species were handpicked during low tide and washed thoroughly with sea water to remove any associated with impurities, epiphytes, animal casting, and adhering sand particles and other suspend materials. Morphologically distinct thallus of algae were placed separately in new polythene bags and were kept in an ice box containing slush ice and transported to the laboratory. Then, the samples were blot dried using sterile tissue paper. The seaweed sample were shade dried followed by hot air oven drying (50 °C) and milled in an electric blender. vacuum in rotary evaporator (Heidolph, Germany) at 40 °C and the dried extracts were stored at 4 °C for antibacterial assay.

Collection of bacterial strains
The antibacterial activity was tested using seaweed extracts from each individual against two strains of gram positive bacteria viz. In vitro antibacterial activity was determined by using Muller Hinton Agar (MHA) and Muller Hinton Broth (MHB) was obtained from Himedia, Mumbai.

3. Phytochemical screening
The qualitative phytochemical analyses studies hexane, chloroform, ethyl acetate, acetone and methanol extracts of Ulva fasciata Phytochemicals like terpenoids, tannin, cardic glycosides, steroids, alkaloids, phenolic compounds and coumarins were carried out according to the method described by (Harborne 1973).

5. 1. Disc Diffusion Method
The antibacterial activity of extracts of U. fasciata was determined by disc diffusion method according to Bauer et al. (1966) with modifications. Petri plates were prepared by pouring 20 ml of sterilized molten MHA. Then the plates were allowed to solidify and used in susceptibility test. MHA plates were inoculated by streaking the swab over the entire agar surface using bacterial suspensions containing 10 8 colony forming units (CFU) per ml and allowed to dry for 10 minutes. The crude extracts were dissolved in 10 % DMSO and under aseptic conditions sterile discs were loaded with different extracts impregnated with 20 µl of three different concentrations (500, 250, 125 µg/disc) of crude extracts. The discs with extract

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were placed on the surface on the medium with sterile forceps and gently pressed to ensure contact with inoculated agar surface. Ampicillin (10 µg/disc) was used as positive control and 10 per cent DMSO was used as blind control in all the assays. Finally, the inoculated plates were incubated at 37 C for 24 h. The zone of inhibition was recorded in millimeters. The assay in this experiment was repeated for three times.

5. Determination of the Minimum inhibitory concentration (MIC)
The MIC of the U. fasciata crude extracts, a modified reaszurin microtitre plate assay was carried out according to methods of Sarker et al. (2007). 50 l of Sterile MHB were transferred in to each well of a sterile 96-well micro titer plate (Hi-Media TPG 96). The U. fasciata extracts was dissolved in 10 per cent DMSO to obtain 1000 µg/ml stock solution. 50 l of crude extract stock solution was added into the first well. After fine mixing of the crude extracts and broth 50μl of the solution was transferred to the second well and in this way, the serial dilution procedure was continued to a twofold dilution to obtain concentrations like 1000 to 15.625 µg/ml of the extract in each wells. To each well 10 µl of resazurin indicator solution was added. (The resazurin solution was prepared by dissolving a 270 mg tablet in 40 mL of sterile distilled water. A vortex mixer was used to ensure that it was a well-dissolved and homogenous solution). Then 30 µl of MHB was added to each well. Finally, 10 μl of the standardized bacterial suspensions to each well to achieve a concentration of approximately 5 ×10 5 CFU/ml were transferred in to all wells. Each plate had a set of controls: a column with all solutions with the exception of the crude extracts; a column with all solutions with the exception of the bacterial solution adding 10 µl of MHB instead and a column with 10 % DMSO solution as a negative control.The plates were incubated at 37 C for 24 h for all bacterial strains. The color change was then assessed visually. The growth was indicated by color changes from purple to pink (or colorless). In this study, the MIC was the lowest concentration of U. fasciata extracts that exhibited the growth of the organisms in the values by visual reading.

5. 3. Determination of the Minimum Bactericidal Concentration (MBC)
MBC of the U. fasciata extracts were determined by plating loop full of bacterial solution from each MIC assay well with growth inhibition into freshly prepared MHA. The plates were incubated at 37 C for 24 h for all bacterial strains. The MBC was recorded as the lowest concentration of the extract that did not permit any visible bacterial growth after the period of incubation.

5. 4. Statistical Analysis
The results were expressed as the mean  SD. All statistical analyses were performed using SPSS version 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Student's t-test was performed to determine any significant difference between different extracts for in vitro antibacterial assays. Comparison of means for in vitro antibacterial assessment was carried out using one-way analysis of variance (ANOVA) and Duncan test. P value < 0.05 was considered statistically significant.

RESULTS
The hexane, chloroform, ethyl acetate, acetone and methanol extracts of U. fasciata were used analysed for phytochemicals, terpenoid, tannin, cardiac glycosides, steroid, alkaloids, phenolic compounds and coumarins. The ethyl acetate extract of U. fasciata contained presence of phytochemicals terpenoids, tannins, and phenolic compounds than the other solvent extracts. Among the phytochemicals, cardiac glycosides were present in all the extracts except acetone and methanolic extracts. Steroids, alkaloids and coumarins were absent in all the extracts of U. fasciata.
The multi drug resistance resistance profile, of bacterial strains of both clinical and  Tables 1 and 2. When the different extracts were assayed against the test bacteria by disc diffusion assays, the mean zone of inhibition obtained were between 7.1 and 14.5 mm. Ampicillin (30 µg/disc) antibacterial positive control produced mean zone of inhibition ranged from 7.1 to 12.8 mm. The blind control (10% DMSO) did not produce any zone of inhibition for all the bacterial strains tested. The highest mean of zone inhibitions (15.0 mm), and the lowest MIC (125µg/ml) and MBC values (250 µg/ml) were observed in ethyl acetate extracts of U. fasciata against B. subtilis. a -diameter of zone of inhibition (mm) including the disc diameter of 6 mm; b -mean of three assays; ± -standard deviation; * significant at p < 0.05 a -diameter of zone of inhibition (mm) including the disc diameter of 6 mm; b -mean of three assays; ± -standard deviation; * significant at p < 0.05  negative bacteria towards antibacterial substances is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharides molecules, presenting a barrier to the penetration of numerous antibiotic molecules. The membrane is also associated with the enzymes in the periplasmic space which are capable of breaking down the molecules introduced from outside (Shan et al., 2007).

CONCLUSION
Based on the present study, the different crude extracts of U. fasciata were possessed antibacterial activity. The use of this plant in folk medicine for the treatment of human gastrointestinal tract and various diseases whose symptoms might involve bacterial infections. The ethyl acetate extract of U. fasciata highest antibacterial activity against MDR bacterial pathogens. This study recommended seaweed extracts as antibacterial substance for treating multi drug resistant microbes causing acquired infection.