research article antibacterial activities of different ...

Egypt. J. Exp. Biol. (Bot.), 10(1): 75 – 85 (2014)

© The Egyptian Soc iety of Experim ent al Biology

RESEARCH ARTICLE Mos t a f a M. E l- S hee k h S hym a a M. E l- S haf a ay At ef M. Abo u- S had y E nas M. E l- B al lat

AN T IB AC T ER I AL AC T IV IT IES OF D IFFER EN T EXTR AC T S OF SOME FR E SH AND M AR IN E AL G AE

ABSTRACT: The in v itro antimicrobial activity of algal extracts belonging t o two species of microalgae; Chlorella vulgaris, Spirulina p lat ensis and two species of seaweeds; Sargassum vulgare and Sargassum w ightii were tested against m ethicillin-resistant Staphylococcus aureus (MRS A) and m et hicillin-sensitive S. aureus (MSS A) clinical isolates. In this study, microtiter plate reader assay m ethod was used to eluci date the effects. The ext racts were prepared in four different solvents, out of whi ch methanol extracts showed better and promising result s. The results also confirm ed the potential use of algal extracts as a source of antimicrobi al compounds. KEY WORDS: Antimicrobial activity, Microtiter plate assay

Algae,

Bacteri a,

CORRESPONDENCE: Mos t af a Moh am ed El - S h eek h Botant D epartment, Faculty of Sci ence, Tanta University, Egypt. E-mail: [email protected] S hym a a Mo ha m ed E l- S ha f aa y At ef Mo ha m e d Abo u- S ha dy E nas Mos t a f a E l- B a lla t

Botany Department, Faculty of Science, Tanta University, Egypt

ART ICLE CODE : 07.02.14 ISSN: 1687-7497

INTRO DU CTIO N: Bact erial resistance has been the m ai n factor responsible f or the increase of morbidity, mortality and health care costs of bacterial infections. The defense m echani sm agai nst anti biotics is widel y present i n bacteria and became a world health probl em. The increasing prevalence of multidrug resistance strains of bact eria and the recent appearance of strains with reduced susceptibility to anti biotics raises the specter of untreatabl e bacterial infections and adds urgency to t he search for new infectionfighting strategies to control microbial infections (Mala et al., 2009). It is not onl y the resistance but also the cost of synthetic chemicals l ead to search f or alternate m edicine such as antimicrobial compounds from natural sources. Al gae derived natural products and antibiotics are found to be the effective alternative recognized from natural environm ental resources. The first investigation on anti biotic activity of algae was carried out by Pratt et al. (1944). One of the potential groups of natural resource i s algae whi ch are known to possess promising novel bioactive substances (Vadas, 1979; Met zger et al., 2002). Since algae have been used i n traditional m edicine for a l ong time and also som e al gae have bacteriostatic, bactericidal, antif ungal, antiviral and antitumor activity, they have been extensively st udied by several researchers (Justo et al., 2001). Many investigators have reported antibacterial activities of microalgae as due t o fatty acids (Cooper et. al. , 1983; Findlay and Patil, 1984). Compounds with cytostatic, antiviral, anthelmintic, antifungal, and antibacterial activities have been detect ed i n green, brown and red algae (Lindequist and Schweder, 2001; Newman et al., 2003). Cyanobacteria are pot ential sources of hi gh value chemicals and pharmaceuticals. Spirulina i s one of the m ost potential cyanobacteria used in medici ne. S pirulina i s safe for human consumption as m edicine, because it is free of microcystin toxin and the long term dietary suppl em entation of up to 5%

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of the S piru lina m ay be consum ed without evident toxi c side eff ects (Yang et al., 2011). It is known to produce intracellular and extracell ular m etabolites with diverse biological activities such as antifungal (Mac Millan et al., 2002), antiviral (Hayashi et al., 1996), and antibacterial activities (Kaushi k and Chauhan, 2008). The ability of seaweeds to produce secondary metabolites of potential int erest has been extensively docum ented (Faul kner, 1993). There are num erous reports of compounds derived from macroalgae with a broad range of biological activities, such as antibiotics (antibacterial and antifungal properti es), antiviral diseases (Trono, 1999), antitumor and anti-infl ammatory (S cheuer, 1990), as well as neurotoxins (Kobashi, 1989). Chemical structure types include sterols (Ahmad et al. , 1993), i soprenoids amino acids, terpenoids, phlorotannins, steroids, phenolic compounds, hal ogenated ketones and alkanes, cyclic polysulphi des, fatty acids and acrylic acid can be counted (Mtolera and Sem esi, 1996). The aim of thi s research was to study the antibacteri al activities of different extract s of marine macroalgae and fresh wat er microalgae. M ATER IAL AND METHO DS: Organisms and culture conditions: Microalgae: The two microalgal strai ns were obtained from the culture collection of the Botany Department, Faculty of Science, Mansoura University, Egypt. Zarrouk's (1966) and Kuhl 's (1962) m edia were used for cultivation of S. platensis and C. vulgaris, respectively. The culture flasks were aerated with air mixed with 3% CO 2 to accelerate algal growth and incubated at 30ºC under continuous illuminati on provided from day light fluorescent tubes giving light intensity of 80 µEm - 2 s - 1 f or C. vulgaris and 2500 Lux for S. platensis. Before bubbling into t he culture, the mixture was allowed to pass through bacteri al filter (0.2 µm diameter). S. platensis was grown until the late expon ential phase of the growth (the 10 t h day), while C. vulgaris was grown and harvested till (the 12 th day) and the cultures were harvested by centrifugation at 3500 rpm for 15 minutes. The pellet w as rinsed three times and resuspended in sterilized distilled water to remove traces of growth m edium. The collected biomass was dried in an oven at (40-60ºC) and then powdered by m anual mort er. Macroalgae (seaweeds): In this study, two species of seaweeds were collected from the rocky areas i n few m et ers bel ow the water surface i n Red sea, Seuz beach, E gypt during November 2012. The seaw eeds were brought to the laboratory ISSN: 1687-7497

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in plastic bags containing sea water t o prevent evaporation. Algae were then cl eaned from epiphytes and rock debris and given a quick fresh water rinse to remove surface salts. The seaweeds were i dentified following Abbott and Hollenberg (1976), Aleem (1993), and Taylor (1960). The sam pl es were air dried in the shade at room temperature 25-30ºC on absorbent paper, cut into small pieces and grounded to fi ne powder. These powd ered sampl es were stored in light plastic bags. Tested bacteria: The sam ples were collected from different patients clinically diagnosed t o have bacterial infection (impetigo). This was achieved by visiting the outpatient cli nic of Dermatology Departm ent at Tanta University Hospital. The samples i ncluded different isol ates of Staphylococcus aureus. The best purifi ed isolates were used in the study. The patient samples were transferred in 2 ml phosphate–buffered saline (PBS; NaCl, 8 g/L; KCl, 0.2 g/ L; Na2HPO 4, 1.15 g/L; K H2PO4, 0.2 g/L) and were forwarded to the Bacteriology Laboratory in Botany Department, Faculty of Science, Tant a Uni versity. All culture swabs were processed in the sam e day that t hey were coll ected. Each specim en was pl ated to a m annitol salt agar m edia. Culture plates were incubated up to 24 h at 37°C, and then examined for colony morphology consistent with S. aureus. Identification of S. aureus coloni es i ncluded test for catalase, slide coagulase and DNase (Koneman et al., 1997), as well as Api-St aph strip system (API Syst em S.A., MontalïeuVercieu, France). A positive bacteri al growth (baseline) on nutrient agar, the diagnosis of skin lesions i n clinical samples, was based on the presence of > 105 col ony forming unit (CFU) of bacteria/ml in the culture (W illiams et al., 1990). Methods of determination of the physiological and biochemical characteristics of bacterial isolates: A. Growth on Mannitol Salt Agar (MSA): This type of medi um is both selective and differential. The MSA is be selected for organisms such as Staphylococcus species whi ch can live in hi gh salt concentration. The differential ingredi ent in MSA is the sugar mannitol. Organisms whi ch are capable of using mannitol as a food source will produce acidic byproducts of ferm entation that will lower the pH of the medi a. The acidity of the m edia will cause t he pH i ndicator, phenol red, to turn yellow. Staphylococcus aureus is capable of ferm enting mannitol and covert the color of the m edia from pink into yellow. B. Catal ase test: A few drops of 3% (v/v) hydrogen peroxide were added to t he bacterial growt h appeared on the surface of nutrient agar htt p://www.egyseb.org

E l - S h e ek h e t a l . , A nt ib ac t e r i a l A c t i v it i es o f D i f f e r en t E xt r ac t s o f S om e Fr e s h a n d M ar i n e A l g a e

plates. G as bubbles either i nstantly or after 5 minutes if the bacteria were cat alase positive. C. D eoxyri bonuclease (DNase) test: DNase agar (oxoi d) was i noculated by the i solated bacteria for 2 days. At the end of incubation period, the plates w ere treated with 1% HCl. The DNA molecules hydrolyzed t o a mixture of mono and polynucleotide by the action of enzym e DNase produced by the organism. HCl reacted with t he nucleotides in the m edi um forming a cloudy precipitat e and a clear area contai ned the nucl eotide fracti ons which were not precipitated by the acid. D. C oagulase test: Coagul ase is a protein enzym e produced by several microorganisms that enabl es the conversion of fibrinogen to fi brin. In laboratory, it is used to distinguish between different types of Staphylococcus isolates. Antibiotic susceptibility of the tested isolates: The susceptibility of the test ed S. aureus isolates to 9 antimicrobi al agents was perform ed by modifi ed Kirby-Bauer singl e-disk diffusi on technique on Muller Hinton agar (Robert et al. , 2003). The used antimicrobial s were amoxicillin, oxacillin, imipenem, gentamicin, chloramphenicol, vancomyci n, deoxycycline, clindamycin and nitrofurantoi n. The results of the susceptibility tests were interpreted according to the criteria established by the Clinical and Laboratory Standards Institute (CLSI, 2010). Methicillinresi stance w as tested by usi ng 1 mg oxacillin disk (O xoid). Determination of the suitable extracti on time of antimicrobial material: A sample of Chlorella vulgaris was extracted by soaking in 70% m ethanol as an exam ple for solvents in a coni cal flask at room tem perature 25°C-30°C for diff erent periods 24h, 48h, 72h and 96h (1:15 v/v) (Rosell and Srivastava, 1987). The extracts were filt ered and evaporated under reduced pressure. The residues were concentrated to constant concentrati on and the antimicrobi al activity against Staphylococcus aureus was assayed using hollow well technique by making five w ell s of 7 mm diameter cut with sterilized cork borer and 50 µL of algal extract and methanol as control. The diameter of inhibition zones w as calculated at t he end of incubation period and the results were compared with each other and the best extraction time was used in our study. Determination of the best al gal extract with the best sol vent using microtiter plates assay method: The extraction was carri ed out with different solvents (i.e., 70% ethanol, 70% m et hanol, 70% et hyl acetate and 70% chloroform) by soaki ng all algal samples in the respective solvents. Antibacterial activity assay of the algal extract s was conducted I ISSN: 1687-7497

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using microtiter pl ates reader assay m et hod according to Bechert et al. (2000) with som e modifications. Aliquot of 100 µl of bacterial isol ate (10 6 CFU/ml) in Muller Hi nton Brot h m edia w as transferred to each well of 96 well pl ates, 50 µl of extracts were added to each well in replica. The pl ates were incubated under microaerophilic conditions at 37ºC f or 24h. After incubation, the absorbance of the sampl es was determined using automated ELIZA microplate reader adj usted at 630 nm. The i nhibition percentage of algal extracts was calculated according to the following equation (Mulyono et al., 2012). The t est was carried out in triplicat e (in sam e 96-well plat e) and repeated twice f or each strain and each tested agent. Inhibition percenta ge (%) = 100 – (A bacteria + NB + extract – A NB + extract) / (A bacteria + NB + solvent - A NB + solvent) x 100 Where: *A= Absorbance; *NB= Nutrient Broth m edia; Treatm ent = A b a c t e r i a + N B + e xt r ac t ; (-Ve) control = A b ac t e r ia + N B + s ol ven t ; Blank' = ANB + e x t r ac t ; Blank'' = A NB + s ol ven t Fractionation and characterization of antimicrobial crude extract using colum n chromatography: Selected active crude extracts (4g) were fractionated by column chromatography on silica gel G (EDW C, 60-120 m esh). Column (2 cm x 40 cm) was set up in chloroform wit h silica gel (30-40 g), eluted with gradients of solvents from 7: 3% of chloroform: m ethanol to 3: 7% chloroform: methanol and the coll ected fractions were evaporated t o dryness with the rotary evaporator. The dried sampl es were di ssolved i n pure m ethanol and assayed for their antimicrobi al activit y. The maximum absorption of the active fractions was measured by spectrophotom eter (UV 2101/ pc) using quartz cuvette containing the different fractions. After detection of the material which has antagonistic activity agai nst bacteria, it is lyophilized and was subjected to the following analysis in order t o reveal its structure as f ar as possible. Agar diffusion method for determining the minimum inhibitory concentration (MIC) for bacteria: MIC was determined by agar m et hod (Chattopadhyay et al., 1998). Serial dilutions (200, 100, 50, 25, 12.5, 6.25, 3, and 1 mg/ml) of the antimicrobi al material were tested agai nst the most resistant pathogenic strain of Staphylococcus aureus isolate wit h concentration (10 8 CFU). The inoculated pl ates were then i ncubated at 37°C for 24h. The lowest concentration which did not show any visible growth was considered as the MIC. Statistical analysi s: Results are presented as m ean ± SD (standard deviation) for three replicates. The

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statistical analysis were carri ed out using SAS and SYSTAT statistical softw are packages, the degree of signifi cance of the obtained results was test ed. One and three way analyses of vari ance were carried out. RES ULTS : Determination of the suitable extracti on time of antimicrobial material: This experiment was carri ed out in order to determine the suitable extraction tim e of the antimicrobial material using Chlorella vulgar is as an exam ple for algae extracted by soaki ng in 70% m et hanol at room tem perature. The antimicrobial activity against a bacterial strain (Staphylococcus aureus) was determined as a di am et er of inhibition zone using agar diffusion method. The obtained results i ndicated that the inhibition zone of Staphylococcus aureus at different extraction times (24h, 48h, 72h, and 96h) were 1.5, 1.7, 1.9, and 1.9 mm, respectivel y indicating that the 72h ext raction tim e w ould be sufficient to extract the antimicrobi al substance (Fig. 1). Therefore, w e used it in the rest of our study.

and coverting the color of the medi a from pi nk into yellow. All the recovered Staphylococcus aureus isolates were shown positive results (Fig. 2).

Fig. 2. Photo 2. The eff ect of Staphylococcus aureus on mannit ol s alt ag ar medium.

Catalase test: A few drops of 3% (v/v) hydrogen peroxide were added to t he bacterial growt h appeared on the surface of nutrient ag ar pl ates. Gas bubbl es were appeared i nstantly. All the Staphylococcus aureus isolates were catalase positive (Fig. 3).

Fig. 1. Effect of different extraction times at room temperature f or extracting the ant imicrobial substanc e from Chlorella v ulgaris us ing 70% met hanol against Staphy loc occus aur eus; 1 = 24h; 2 = 48h; 3 = 72h; 4 = 96h; c = Control (70% met hanol).

Determination of the physiological and bi ochemical characteristics of bacterial isolates: Growth on Mannitol Salt Agar (MSA): MSA was sel ected for organisms such as Staphylococcus species which can live in areas of high salt concentration. The differential ingredient in MSA is the sugar mannitol. Organisms capabl e of using mannitol as a food source would produce acidic byproducts of fermentation that would lower t he pH of t he medi a. The acidity of the m edia would cause the pH indicator, phenol red, to turn yellow. Staphylococcus aureus was capable of f erm enting mannitol ISSN: 1687-7497

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Fig. 3. Formation of gas bubbles when dr ops of hydrog en peroxide were added to the Staphy loc occus aureus growth appeared on the surface of nutr ient agar plat es

Deoxyribonuclease (DNase) test: DNase agar w as i noculated by the isol ated bacteria for 2 days. At the end of incubation period, the plates were treated wit h 1% HCl. The DNA mol ecules hydrolyzed to a mixture of m ono and polynucl eotide by the action of enzyme DNase produced by the Staphylococcus aureus. HCl reacted with the nucleotides i n the medi um forming a cloudy precipitate and a cl ear area contained the nucleotide fractions which were not htt p://www.egyseb.org


precipitated by the acid. All the Staphylococcus aureus isolates were DNase positive (Fig. 4).

with resistant phenotypes included those that were classified as intermediate resistance. Table

1. Inc idenc e of antibact er ial among S. aureus is olates.

Antibacterial agent

Fig. 4. Formation of a cloudy pr ec ipitate and a clear area c ontain ed the nucleotide fractions whic h wer e not prec ipit at ed by the HC l.

Coagulase test: Coagul ase is a protein enzym e produced by several microorganisms t hat enabl es the conversion of fibrinogen to fibrin. In the laboratory, it is used to distinguish between different types of Staphylococcus isolates. All the recovered Staphylococcus aureus isol ates were coagulase positive (Fi g. 5).

Fig.

5. Clumps were not m ixed unif ormly int o c oagulas e plasma, repres ent ing a pos itive slid e c oagulas e t est which is indic ative of Staphy loc occus aureus.

Susceptibility of S. aureus isolates to different antibacterial agents: The susceptibility of S. aureus isolates to 9 different antimicrobial agents representing 9 classes was conducted using disk diffusion method. The used antimicrobial discs were as follow: Amoxicillin (AMX), Oxacillin (OX), Imipenem (IPM), Gentamicin (GEN), Chloramphenicol (CL), Vancomycin (VA), Deoxycycline (DO), Clindamycin (DA) and Nitrofurantoin (F). The sizes of the inhibition zones were interpreted by referring to Clinical and Laboratory Standards Institute standards and the results were reported as being susceptible, intermediate or resistant to the antibiotics that were tested. The obtained data showing the incidence of antimicrobial resistance among isolates are shown in table 1. Isolates

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AminoPenicillins Amoxicillin (AMX) Penicillins Oxacillin (OX) Carbapenems Imipenem (IPM) Aminoglycosides Gentamicin (GEN) Phenicols Chloramphenicol (CL) Glycopeptides Vancomycin (VA) Tetracyclines Deoxycycline (DO) Lincosamides Clindamycin (DA) Nitrofurantions Nitrofurantoin (F)

res istanc e

No. (%) of resistant isolates 8 (100 %) 7 (87.5 %) 1 (12.5 %) 5 (62.5 %) 6 (75 %) 5 (62.5 %) 4 (50 %) 8 (100 %) 4 (50 %)

As shown in table 1, the incidence of resistance to different tested antibiotics ranged between 12.5% (imipenem) and 100% (amoxicillin and clindamycin). For pencillins, the incidence of resistance to oxacillin was 87.5% but for aminopenicillin was 100%. In case of aminoglycosides, phenicols, glycopeptides, tetracyclines and nitrofurantions, there was moderate incidence of resistance; gentamicin (62.5%), chloramphenicol (75%), vancomycin (62.5%), deoxycycline (50%) and nitrofurantion (50%), respectively. The only class of antibiotics showed very low incidence of resistance with most isolates was carbapenems. Determination of the best solvent for extracting the antibacterial material from different micro and macroalgae using microtiter plates: This experiment was carried out in order to determine the best solvent (70% ethanol, 70% methanol, 70% ethyl acetate and 70% chloroform) for extracting the antibacterial material from different tested algae (Chlorella Vulgaris , Spirulina platensis, Sargassum vulgare and Sargassum wightii) against methicillinresistant S. aureus (MRSA) and methicillinsensitive S. aureus (MSSA) clinical isolates. Antibacterial activity assay of the al gal extracts was conducted usi ng microtiter plates reader assay m et hod. The results presented in t able 2 show that ethanol, methanol, ethyl acetate and chl oroform extracts of different al gae possessed antibacterial activity against the bacteria tested. The bli nd control (ethanol, m ethanol, ethyl acetate and chloroform) di d not inhibit t he tested bact eria. 70% m ethanol extract s showed the strongest inhibition against the tested bacteria wit h inhibition activity percentage* on 26.92%, followed by 70% chloroform extract s wit h inhibition activity percentage 25.54%,

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followed by 70% ethyl acetate extracts with inhibition activity percentage 24.86%, whereas 70% et hanol showed the weakest inhibition with inhibition activity percentage 22.68% of all algae against all tested

bacteria. Therefore, we use 70% m ethanol as solvent for extractions. *Inhibiti on activity percentage = (average diameter of inhibition zone of each sol vent/ average diameter of inhibition zone of all solvents) x 100

Table 2. One way analys is of varianc e (ANOVA) of different algal extracts against different str ains of Staphy loc occus aureus using different s olvents Solvent (70%)

Algae

Spirulina platensis

Isolate (C)

Isolate (D)

Ethanol Methanol Ethyl acetate Chloroform

99.60 ± 0.02

99.98 ± 0.01

99.95 ± 0.01

96.83 ± 0.01

99.56 ± 0.01

99.04 ± 0.01

95.90 ± 0.02 97.08 ± 0.01

96.17 ± 0.01

93.37 ± 0.02

95.02 ± 0.01

92.85 ± 0.02

89.71 ± 0.01

96.06 ± 0.02

92.03 ± 0.02 92.71 ± 0.02

89.63 ± 0.02

80.05 ± 0.02

83.54 ± 0.02

50.50 ± 0.0

62.63 ± 0.02

53.82 ± 0.02

74.84 ± 0.03

92.71 ± 0.01

88.47 ± 0.02

75.51 ± 0.02

79.97 ± 0.02

87.56 ± 0.02

90.54 ± 0.01

81.85 ± 0.02 70.30 ± 0.01

1.33**

3.00*

1.33**

1.33**

Ethanol Methanol Ethyl acetate Chloroform

72.74 ± 0.01

82.78 ± 0.02

69.38 ± 0.0

99.84 ± 0.02

58.76 ± 0.0

48.62 ± 0.02

99.85 ± 0.01

99.95 ± 0.01

99.94 ± 0.06

99.97 ± 0.02

99.85 ± 0.02

99.28 ± 0.01

99.95 ± 0.01

99.32 ± 0.02

99.81 ± 0.01

99.96 ± 0.02

99.32 ± 0.0

99.96 ± 0.01

99.32 ± 0.02

99.06 ± 0.01

2.00*

6.00*

3.00*

1.50*

74.81 ± 0.01

72.22 ± 0.02

97.63 ± 0.01

80.72 ±0.01

99.96 ± 0.03

99.32 ± 0.03

94.80 ± 0.01

75.72 ± 0.01

74.16 ± 0.01

70.76 ± 0.01

59.71 ± 0.0

83.40 ± 0.01

94.06 ± 0.01

F-Value Chlorella vulgaris

Staphylococcus aureus Isolate Isolate (H) (F)

Isolate (E)

(ns)

1.33**

F-Value Ethanol Sargassum Methanol vulgare Ethyl acetate Chloroform F-Value Ethanol Sargassum Methanol wightii Ethyl acetate Chloroform F-Value

0.10

Isolate (C')

Isolate (A)

( ns)

0.10

Isolate (R)

95.40 ± 0.0 2.67*

94.81 ± 0.01

89.40 ± 0.0

99.98 ± 0.01

99.99 ± 0.0

99.98 ± 0.01

99.82 ± 0.01

99.97 ± 0.02 99.96 ± 0.01

99.42 ± 0.02

99.67 ± 0.02

99.63 ± 0.55

3.00*

1.33**

2.67*

2.67*

67.04 ± 0.0

54.25 ± 0.02

78.27 ± 0.01

99.80 ± 0.0

99.96 ± 0.02

99.96 ± 0.0

99.92 ± 0.02

99.83 ± 0.02

96.80 ± 0.0

99.93 ± 0.02

99.95 ± 0.05

99.54 ± 0.01

84.62 ± 0.02

99.60 ± 0.0

99.01 ± 0.01

98.86 ± 0.02

99.03 ± 0.0

99.07 ± 0.02 99.13 ± 0.01

99.90±0.07

2.67*

1.50*

1.33**

2.67*

4.00*

2.68*

1.33**

1.33**

85.46 ± 0.01

42.45 ± 0.01

50.00 ± 0.0

91.36 ± 0.03

87.83 ± 0.06

97.75 ± 0.01

64.00 ± 0.0

83.20 ± 0.0

89.22 ± 0.02

97.92 ± 0.02

99.28 ± 0.01

99.82 ± 0.02

99.60 ± 0.01

99.95 ± 0.01

99.93 ± 0.01 99.96 ± 0.01

93.86 ± 0.01

98.37 ± 0.02

99.57 ± 0.02

99.71 ± 0.02

99.95 ± 0.02

99.94 ± 0.04

99.86 ± 0.02 99.93 ± 0.01

95.34 ± 0.01

99.00 ± 0.0

98.56 ± 0.01

95.59 ± 0.01

95.42 ± 0.02

95.27 ± 0.01

96.96 ± 0.01 92.28 ± 0.02

1.33**

2.67*

3.00*

1.50*

1.50*

1.50*

2.00*

3.00*

(±) standard deviation of the m eans (n=3); * Significant at P ≤ 0. 01,** Significant at P ≤ 0. 001, and (ns) Non-s ignif ic ant at P ≥ 0.01

O ne way ANOVA analysis confirm ed that the variation in antibact erial activities i n relation to algae and different bacteri al isolates were significant at p ≤ 0. 01 for almost treatm ents except Spirulina p latensis against isolates (E, D, F, and C'), Chlorella vulgar is against isol ate (C'), Sargassum vulgare against isol ates (D, A, and R) and Sargassum w ightii against isolate (E) were significant at P ≤ 0. 001. O n the other hand, Spiru lina p latensis against isolat es (C and A) were non-significant at p ≥ 0. 01 (Tabl e 2). However three-way ANOVA analysi s confirm ed that the variation in the antibacterial activity in relation to al gae, bacteri a, solvents and their interactions on antibacterial activity were significant at p ≤ 0.0001 for all treatm ents (Table 3). Table 3. Three-way analys is of variance (ANOVA) of different algal extracts against diff erent strains of St aphylococcus aureus using different solvents 2

Source

DF

F-Value

P-Value

R

Algae Solvent Bacteria Algae*Solvent Algae*Bacteria Solvent*Bacteria Algae*Solvent*Bacteria

3 3 7 9 21 21 63

99999.99 99999.99 88665.46 99999.99 99999.99 80146.77 99999.99

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

99.9%

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Fractionation and characterization of the antimicrobial crude extract using colum n chromatography: The antibacterial material i solated from Chlore lla vulgar is was transferred to a column containing Silica gel (EDWC, 60-120 m esh). The material was el uted using gradients of solvents from 7:3% of chl oroform: methanol t o 3:7% chl oroform: m ethanol. The different fractions were collected every 5 min and exami ned for antimicrobial activity usi ng agar diffusion assay m ethod. The results showed that 22 fractions were collected and only 3 fractions had antimicrobial activity. UV absorption spectrum of these fractions was determined usi ng spectrophotom eter (UV 2101/ pc) at range of 250-800 nm. The results show ed that the 3 fractions had the sam e absorption peaks (2 peaks at 410 and 664 nm) (Fig. 6). The different pi gm ents and impuriti es were rem oved by filtration using charcoal. The UV spectrum of the purified antimicrobial material was t hen carried out i n pure m ethanol. This spectrum shows one absorption peak at 410 nm, indicating the presence of an aromatic compound (Fig. 7). The obt ained com pound was examined f or antimicrobial activity. The results showed that the compound still has antimicrobial activity (Fig. 8), indicating that the com pound having



a peak at 664 nm antimicrobial activity.

does

not

have

any

purific ation by charc oal.

81

*Abs. =Abs orption

B A

C

Fig. 8. The antimicrobial activity of the act ive fraction bef ore and after purification b y charc oal; A=active fraction after purification, B=act ive fraction bef ore purification, an d C=control (Methanol).

Fig. 6. UV spectrophot om eter sc anning of dif ferent active f ract ions separ at ed by c olum n chromatography; A=fraction 1, B=f raction 2, C=f raction 3. *Abs. =Abs orption

Fig. 7. UV s pectrum of t he ant imicrobial material is olated from Chlorella vulgaris af ter I ISSN: 1687-7497

Determination of minimum inhibitory concentration (MIC) of the purified antagoni stic antimicrobial material extracted from Chlorella vulgaris: MIC was determined by agar diffusion m ethods. Serial diluti ons (200, 100, 50, 25, 12.5, 6.25, 3, and 1 m g/ml) of the antimicrobial materi al extracted from Chlorella vulgar is were tested agai nst the most resistant isol ate of bacteria (St aphylococcus aureus) as Gram -positive bacteria. The concentration of the tested St aphylococcus aureus was 10 8 CFU. The pl ates were inocul ated and incubated at 37°C for 24h. The lowest concentration of the antimicrobial material, which di d not show any visibl e growth, was considered as the MI C. The results presented i n (Fig. 9), showed the MIC value for Staphylococcus aureus is (6.25 mg/ml).

Fig. 9. Minimum inhibitory c oncentration (MIC) of the purified antagonistic material extracted from Chlorella v ulgaris extract ed by 70% met hanol: A) 200mg/ml; B) 100mg/ml; C) 50mg/m l; D) 25m g/ml; E) 12.5 mg/ml; F) 6.25 mg/ml; G ) 3 mg/ml; H) 1 mg/ml.

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DISC USSIO N: This st udy depended on the phenotypic m et hods commonl y used in the identificati on of Staphylococcus aureus. Firstly, mannit ol salt agar (MSA) w as used for i dentification of S. aureus clinical sampl es. Mannitol Salt Agar (MSA) is bot h a selecti ve and diff erenti al m edium used in the isolation of staphylococci. Early work by G ordon indi cated that the ferm entation of mannitol could be used as a m eans of differentiating pathogeni c staphylococci from nonpathogeni c staphylococci (Gordon, 1904). Although hi s work was confirmed and expanded upon by several i nvestigators (Dudgeon, 1908), mannitol f erm entation was not used routinel y as an identification tool. The results showed that all the isolates are positive reaction for manitol salt agar. This is because Staphyl ococci can withstand the osmotic pressure created by 7.5% NaCl, while thi s concentration will inhibit the growth of most other gram -positive and gramnegati ve bacteri a (K och, 1942). A dditionally, MSA contai ns mannitol and uses phenol red as a pH indicator (pH = 7.8) i n the medium. W hen mannitol is fermented by a bacterium, acid i s produced, which low ers the pH and results in the formation of a yellow area surrounding an isol ated colony on MSA. The second test for identification of S. aureus was cat alase test which showed that all clinical isolates are catalase positive. These results agree with that reported by Amini et al. (2012). Staphylococci have the ability to produce en zym es such as catal ase consi dered to be virulence determinants. Thi s enzym e allows bacteria to better resist intraand extra-cellular killing by hydrogen peroxide (Grüner, 2007). Species of the genus Staphylococcus are characterized by the production of catalase. Among them, onl y two species, Staphylococcus saccharolyticus and Staphylococcus aureus subsp. anaerobius, are not able to produce catalase (Kl oos and Bannerman, 1999; S anz et al., 2000). In order to support the previ ous biochemical t ests, DN ase tests and slide coagulase tests were carried out in our present study. The combination of all the biochemical t ests i ncreased the s ensitivity to identify t he S. aureus among the bacteri al isolates. DNase and tube coagul ase tests were carried out and the results were positive for both. These results agree with that reported by Amini et al. (2012) and Koneman et al. (1997). The posi tive results of S. aureus in case of DN ase test might be due to t he ability of these bacteria to produce DNase enzym es (deoxyribonuclease) which breaks down the DNA by adding HCl which reacts with the nucleotides in the m edium forming a cloudy ISSN: 1687-7497

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precipitate and a clear area that contai n the nucleotide fractions which were not precipitated by the aci d. The organi c extraction solvent s we used were ethanol, m ethanol, ethyl acetate and chloroform. This is because organic solvent always provi des a hi gh efficiency i n extracting compounds for antimicrobial acti vities compared to wat er-based m ethods (Parekh and Chanda, 2007; Axelsson and G entili, 2014). Lipi d-soluble ext racts from fresh wat er microalgae and m arine m acroalgae have b een investigated as a source of substances wit h pharmacological properties. Moreover, several different organic solvents have been used for screening al gae for antimicrobial activity. Sastry and Rao (1994) showed antibacterial activity against Gram -positive and Gram -negative pathogenic strains aft er successive extraction with benzene, chloroform, and methanol. Likewise, Mahasneh et al. (1995) showed anti biotic activity in organic extracts of six speci es of marine algae against multi-antibiotic resistant bacteria. Regarding the extracti on tim e, Rosell and Srivast ava (1987) used diff erent lengths of time 24h, 48h and 72h for extracting the components of algae at room tem perature. They found t hat no significant difference am ong the different extraction peri ods. I n contrast, our present study showed that 72h extraction of antimicrobial material has been found suffici ent. This might be due to that the extraction of antimicrobial mat erial increased with soaking tim e until saturati on. Our results agreed with that reported by El shobary (2010) who reported that the m ost active ext ract was at 72h. Many in vitro m ethods have been used to detect the antimicrobial activity of algal material. The most commonly used methods are the agar disk/well diffusi on assay and the agar/broth dilution assay (Patton et al., 2006; Elshobary, 2010). Studies of date have used the popul ar agar disk/well diffusion m et hod (Almajano et al., 2008; G utierrez et al., 2008) despite its MIC data being potentially subjective due to t he different measurem ent m ethods of the inhibiti on zone. There has been an i ncrease in the use of the brot h dilution assay and, i n particular, the 96 -well microplat e assay in recent years (Eloff, 1998; Casey et al., 2004; Turcotte et al., 2004; Patton et al. , 2006; Ruﬁán-Henares and Morales 2006). Thi s assay has been i denti ﬁed as bei ng more accurate than the agar/disk diffusion, less tedious and can be automated. This becom es very attractive as the throughput of samples is high and the increased accuracy of the m ethod allows results to be easily compared si nce the MICs can be determined. On contrast, Ostrosky et al. (2008) and C hiheb et al. (2009) reported that agar dilution is the most widely used htt p://www.egyseb.org


m et hod due to its simplicit y of implem entation and low cost. The obtained results in our study reveal ed that the strongest antibacteri al activities were exhibited usi ng 70% m ethanol. These results were in accordance with de Cano et al. (1990) who reported that Nostoc muscorum extract produced using methanol showed the highest antibacterial and antifungal activities agai nst two human pathogens; staphylococcus aureus and Candida albicans. Pandi an et al. (2011) also test ed petrol eum -ether, chloroform and m et hanol of Acanthaphora spicifera in vitro for thei r antibacterial and antifungal activity against Escherich ia coli, Bacillus subt ilis, Bacillus palmitus, Pseudomonas aeruginosa, and agai nst Candida albicans, Microsporum gypseum, Aspergillus n iger , respectively by disc diffusion techni ques. The methanolic extract of Acanthaphora spicifera showed

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hi gher antibacterial and antifungal activity compared to the other two extracts. Simic et al. (2012) demonstrated that the m ethanolic extract of green microalgae, Trentepohlia umbrina, has a strong antimicrobi al activity agai nst St aphylococcus aureus, E scherichia coli, K lebsie lla pneumonia, Pseudomonas aeruginosa, Ent erococcus f aecalis, Aspergillus n iger , Candida a lb icans, Fusarium oxysporum, Penicillium purpurescens and Trichoderma harsianum. Fi nally, we conclude that algae are potential source of bioacti ve compounds especially green algae (Chlorella vu lgar is). Thus, the l owest concentrati ons of the algal extracts show a great eff ect agai nst the m ost resistant isolates of Staphylococcus aureus. So, further studies can be done to evaluat e the in v ivo effect of these extracts and introduce these extracts to t he pharmaceutical uses.

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‫اﻷﻧﺸﻄﺔ اﻟﻤﻀﺎدة ﻟﻠﺒﻜﺘﺮﻳﺎ ﻟﻠﻤﺴﺘﺨﻠﺼﺎت اﻟﻤﺨﺘﻠﻔﺔ ﻣﻦ ﺑﻌﺾ اﻟﻄﺤﺎﻟﺐ‬ ‫ إﻳﻨﺎس ﻣﺼﻄﻔﻰ اﻟﺒﻼط‬،‫ ﻋﺎطﻒ ﻣﺤﻤﺪ أﺑﻮﺷﺎدي‬،‫ ﺷﯿﻤﺎء ﻣﺤﻤﺪ اﻟﺸﺎﻓﻌﻲ‬،‫ﻣﺼﻄﻔﻰ ﻣﺤﻤﺪ اﻟﺸﯿﺦ‬ ‫ ﻣﺼﺮ‬،‫ ﺟﺎﻣﻌﺔ طﻨﻄﺎ‬،‫ ﻛﻠﯿﺔ اﻟﻌﻠﻮم‬،‫ﻗﺴﻢ اﻟﻨﺒﺎت‬ ‫ھﺬه اﻟﺪراﺳﺔ أن ﻣﺴﺘﺨﻠﺼﺎت اﻟﻤﯿﺜﺎﻧﻮل أﻋﻄﺖ أﻓﻀﻞ ﻧﺘﺎﺋﺞ وﻗﺪ‬ ‫أﻛﺪ ّت ﻧﺠﺎح اﺳﺘﺨﺪام ﻣﺴﺘﺨﻠﺼﺎت اﻟﻄﺤﺎﻟﺐ ﻛﻤﺼﺪر ﻟﻠﻤﺮﻛﺒﺎت‬ .‫اﻟﻀﺪ ﻣﯿﻜﺮوﺑﯿﺔ‬

: ‫اﻟﻤﺤﻜﻤﻮن‬ ‫ ﻋﻠﻮم اﻟﻘﺎھﺮة‬،‫ﻗﺴﻢ اﻟﻨﺒﺎت‬

‫ ﻋﻔﺖ ﻓﮫﻤﻲ ﺷﺒﺎﻧﻪ‬.‫د‬.‫أ‬

‫ ﻋﻠﻮم اﻟﻘﺎھﺮة‬،‫ﻗﺴﻢ اﻟﻨﺒﺎت‬

‫ زﻳﻨﺐ ﺧﻠﯿﻞ إﺑﺮاھﯿﻢ‬.‫د‬.‫أ‬

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‫ﺗﮫﺪف ھﺬه اﻟﺪراﺳﺔ إﻟﻰ دراﺳﺔ اﻟﻨﺸﺎط ﺿﺪ ﻣﯿﻜﺮوﺑﻲ‬ ‫ﻟﻤﺴﺘﺨﻠﺼﺎت اﻟﻄﺤﺎﻟﺐ اﻟﺘﻰ ﺗﻨﺘﻤﻰ إﻟﻰ ﻧﻮﻋﯿﻦ ﻣﻦ اﻟﻄﺤﺎﻟﺐ‬ (‫اﻟﺪﻗﯿﻘﺔ ﻣﺜﻞ )ﻛﻠﻮرﻳﻠﻼ ﻓﻮﻟﺠﺎرﻳﺰ وﺳﺒﯿﺮوﻟﯿﻨﺎ ﺑﻼﺗﯿﻨﺴﯿﺲ‬ ‫وﻧﻮﻋﯿﻦ ﻣﻦ اﻷﻋﺸﺎب اﻟﺒﺤﺮﻳﺔ ﻣﺜﻞ )ﺳﺮاﺟﺴﯿﻮم ﻓﻮﻟﺠﺎر‬ ‫اﻟﺒﻜﺘﺮﻳﺎ‬ ‫ﻣﻦ‬ ‫ﺳﻼﻻت‬ ‫ﺿﺪ‬ (‫وﻳﺠﺘﺎى‬ ‫وﺳﺮاﺟﺴﯿﻮم‬ ‫ وﻗﺪ ﻗﻤﻨﺎ ﺑﺈﺳﺘﺨﺪام‬.‫) اﺳﺘﺎﻓﯿﻠﻮﻛﻮﻛﺲ أورﻳﻮس( ﺗﻢ ّ ﻋﺰﻟﮫﺎ‬ ّ ‫طﺮﻳﻘﺔ أطﺒﺎق اﻟﻤﯿﺮﻛﻮﺗﯿﺘﺮ ﻟﺘﻘﯿﯿﻢ اﻟﻨﺸﺎط اﻟﻀﺪ ﻣﯿﻜﺮوﺑﻰ وﺗﻢ‬ ‫ وأوﺿﺤﺖ‬.‫ﺗﺤﻀﯿﺮ اﻟﻤﺴﺘﺨﻠﺼﺎت ﺑﺈﺳﺘﺨﺪام أرﺑﻊ ﻣﺬﻳﺒﺎت ﻣﺨﺘﻠﻔﺔ‬

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