EVALUATION OF PHYLLANTHUS EMBLICA EXTRACT AS ANTIBACTERIAL AND ANTIBIOFILM AGAINST BIOFILM FORMATION BACTERIA

The objective of this study was to evaluate the antibacterial effect of Phyllanthus emblica extract by (ethanol: methanol 1:1) against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli at different concentration started with 20, 10, 5, 2.5, 1.25 and 0.625 mg/ml. The antibacterial activity was determined by the agar well diffusion method to investigate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The alcoholic extract of Phyllanthus emblica had the highest antibacterial activity at 20 mg/mL and 5 mg/mL except in Pseudomonas aeruginosa where the value of inhibition was between 20 mg/mL and 10 mg/mL whereas The MIC concentrations were mostly very high and ranged from 5 to 1.25mg/ml while MBC range from 10 to 2.5 mg/ml against tested bacteria. In this study, we evaluated the effect of Phyllanthus emblica against Pseudomonas aeruginosa biofilm formation was evaluated and the biofilm inhibitory concentrations of the Phyllanthus emblica extract was 40-6.25mg/ml.This implies that they may contain valuable substances for application directed against pathogenic biofilms. The use of herbal extract such as Phyllanthus emblica represent a new date for antimicrobial therapy after increasing the antibiotic resistance to microbes. Key word: Phyllanthus emblica, antibacterial, antibiofilm, Pseudomonas aeruginosa biofilm ةيقا رعلا ةيعا رزلا مومعلا ةمجم – 142 : 151 ( 1 ) 49 / 8102 ةزمحو يروبجلا يويحلا ءاشغمل ةنوكملا ايرتكبلا عاونا ضعبل يويحلا ءاشغلا نيوكتل داضمو يريتكب داضمك جمملاا صمختسم مييقت *ةزمح دمحم ليسا يروبجلا لايع دمحم مغن دعاسم ذاتسا دعاسم ذاتسا ةكرتشملا ضا رملاا ةدحو * يرطيبلا بطلا ةيمك دادغب ةعماج aseelm30@yahoo.com drvet2011@yahoo.com :صمختسملا ةبدسنب دمي ل دمي ملا لودحكلا صمختدسملا ري صدت ةدسا رد تدلا ةديلاحلا ةدسا ردلا ت دده 0:0 ددض يرديتكب دادضمك جدمملاا تادبنل ب اءدددب ةدد متخم زدديكا رتب ةددينولوقلا ايددشيريشيلاا نددع ددض ةدديدوقنعلا تا روددكملاو ةدديراجنزلا اددئاوزلا 81 ، 01،1،8،1،0،81 و 1،281 مل يرديتكبلا دادضملا سرد ،لدم/مغمم طبد م زديكرت لدقا، عرزلا طدسولاب رادشتنلاا ةدقيرطب تادبن MIC لدتاق زديكرت لدقاو MBC نم تحوا رت زيكا رتب ةيداضم ةيمعا ترهظا تلاو 81 /مغمم لم تلا 1 /مغمم ةديراجنزلا ادئاوزلا اددع مي ا ردجلا اديمجل لدم يزيكرتب هتيمعا صمختسملا رهظا ثيح 81 و 01 ددق دطيب ت زديكرت لدقا ادما ،لدم/مغمم نيدب نوا ردت جئادتنلا تدمعا لجدس 1 تدلا 0،81 ندم نوا ردت لدتاق زديكرت لدقا قلذدكو لدم/مغمم 01 تدلا 8،1 لدم/مغمم يرديتكب دادضمك ، سرد لودحكلا صمختدسملا ري صدت ءددب يوديحلا ءادشغلا نيودكت تمع جمملاا تابنل زديكرت ندم ا 01 تدلا 2،81 لدم/ مدغمم صمختدسملا ندم ةمدلحتسملا جئادتنلا نا ، ةدقحلا تادسا ردب زعودي ةدسا ردلا ذده د يرديتكب دادضمك جدمملاا مادختدسا نا ، تادبنلا بديكرتلا تدلا دوعت دق مي ا رجلا دض تابنلا داضمك ةلامعتسلا مو رج ،ةيتايحلا تاداضممل ةيريتكبلا ةمواقملا ةبسن دايدزا دعب اميسلا ةيحات ملا تاممكلا لاا : يويحلا ءاشغلا داضم ،يريتكبلا داضملا ،جمم ةيراجنزلا ائاوزمل يويحلا ءاشغلا ، *Received:27/12/2016, Accepted:28/2/2017 The Iraqi Journal of Agricultural Sciences –241-252: (2) 44/ 1028 AL-Gbouri & Hamzah 143 INTRODUCTION Recently the resistance of microorganisms to any antibiotic has increased (21). Inadequate usage of antibiotics the most important factor of antibiotic resistance (51).Drug resistant bacteria, particularly , Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa, are most important in healthcare (49) because no new antimicrobial agents are currently available for treatment of infected patients (8,19,15) else bacterial virulence factor play a role in diseases mechanism which is targets in drug evolution,moreover the ability of pathogens to form biofilms award a selected advantage for bacteria to militate under harsh environmental conditions lead to resistance to antimicrobial agents (52) Pseudomonas aeruginosa, and, Escherichia coli are examples of bacteria that form biofilms (39,35).Therefore alternative therapeutic agents from plants is One strategy to avoid antibiotic resistant bacteria, safe and have low cost(2,33,36,46). Consequently this study aimed to assess the in vitro antibacterial activities of Phyllanthus emblica extracts against bacterial clinical isolates Staphylococcusaureus , Gram-negative bacilli: Escherichia coli, Klebsiella pneumonia and antibiofilm effect against Pseudomonas aeruginosa and Escherichia coli. MATERIALS AND METHODS Preparation of plant extract: The dried plant was purchased from markets in Baghdad. The powdered plant material (250 g) was extracted in a 1000 ml conical flask with 500ml ; solvents(ehanol:methanol,1:,1,v:v) for 14 days in freeze after that filterated using Whattman No 4 filter paper. The filtrate obtained was concentrated byevaporated to dryness to obtain the crude extract. and kept it at 4°C until further uses. Preparation of microorganism and inoculums Microorganisms were medical isolates collected from the culture collections of the zoonotic diseases unit /veterinary medicine college at the university of Baghdad. Organisms were as follows: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Organisms were maintained on brain heart infusion agar overnight, Inocula were prepared by diluting overnight cultures in saline and adjusted to 0.5 McFarland turbidity standards to approximately 10 8 cfu ml for each bacterium. Assay for antibacterial activity The screening of antibacterial activity was carried out by using the agar diffusion method as described by Lino and Deogracios (30) with slight modifications. Each of the bacterial cultures tested inoculated (0.2 ml each) using the sterilized swabs onto Mueller Hinton agar (MHA, Oxoid) plates (diameter: 15 cm),Then A sterilized stainless steel borer was used to formed four wells (6 mm diameter), The Phyllanthus emblica extract was separately redissolved in sterile distilled water at concentrations of (20, 10, 5 and 2.5 mg/ml),then 100 μl of each concentration of the plant extract were filled each well .The culture plates were allowed to stand then incubated at 37°C for 24 h. antibacterial activity was determined by measurement of diameter zones of inhibition (mm) (against the test organisms) around the extracts (30). Determination of Minimum Inhibitory Concentration (MIC) The MIC and the MBC of the Phyllanthus emblica were determined by using test tubes where twofold serial dilutions with muller hinton broth were made to the various concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.312 and 0.156 mg/ml) of the Phyllanthus emblica extract for each bacteria. Specifically 1ml of 0.5 McFarland turbidity standard 10 8 cfu/ml was added to each tube and incubated aerobically at 37 C o for 18-24hrs. The MIC assay was determined by visualize the bacterial growth. 0.5 ml(0.04mg/ml) of piodonitrotetrazolium violet(trazolium salt)( (INT) was added to each tube and incubate all tubes were incubated at room temperature for 6 hrs. The tubes were examined for color change and the MIC was indicated by the first clear tube that not changed to red color when compared with the control tubes or none inhibited concentrations. Minimum Bactericidal Concentration (MBC) The MBC was determined by subculturing a loop ful of the MIC tubes that showing no visible growth and no colour change onto extract free agar plates that incubated for a further 24 hours at 37 C o then the lowest The Iraqi Journal of Agricultural Sciences –241-252: (2) 44/ 1028 AL-Gbouri & Hamzah 144 concentration of MIC at which no growth on solid medium was regarded as MBC. The effect of Phyllanthus emblica extract on the bacterial biofilm formation Biofilm formation was assessed in plastic sterile test tubes, where seven appropriate concentrations (20,10,5,2.5,1.25,0.625 and 0.312 mg/ml) of extract were prepared from a serial two-fold dilutions method in muller hinton broth and eight tubes were inoculated with 1ml of the 0.5 McFarland turbidity standard and incubated for 4-5 hours to allow cell attachment and then add 1ml of each concentrations was added to each tube. All the tubes were further incubated for 24 hours at 37°C, the eight tube was containing bacteria and muller hinton broth only (negative control). The ability of adherence bacteria The adhered cell biomass was determined using 1% crystal violet staining. At first, plastic tube was emptied and washed three times with sterile Phosphate Buffered Saline (PBS). The tubes were air-dried and then oven-dried at 60 o C for 45 min. then the tubes were stained with 1ml of 1% crystal violet and incubated at room temperature for 15 min after which the tubes were washed 5 times with sterile distilled water to remove unabsorbed stain after that 1ml of ethanol was added to each tube and the absorbance was determined at 540nm using a spectrophotometer RESULTS AND DISCUSSION Antimicrobial Activities of the Extract by agar diffusion method: Table 1 shows the diameters for the zones of inhibition (mm) of Phyllanthus emblica extract at different concentrations(mg/ml). At 20mg/ml, E. coli had a higher zone of inhibition of 27mm(fig.1) while S aureus and P. aeruginosa had 25mm,15mm respectively. At 10 mg/ml of the extract concentration showed the highst zone of inhibition in s.aureus 21mm (fig.2) and E.coli 20mm while in P. aeruginosa was the least 10mm At 5 mg/ml, E. coli had the highest zone of inhibition of 14mm and the least zone of inhibition of S. aureus was 10mm while P. aeruginosa was not sensitive to the extract of Phyllanthus emblica. In contrast,all bacteria were not sensitive to the extract of Phyllanthus emblica at 2.5mg/ml (fig.3). The attention of researchers at the present time search for medicinal plants instead of antibiotics as a result of antibiotic resistant bacteria(4, 24,37, ,40) As well as the ease of access to medicinal plants and the availability and affordability(7,22,41). Actually, there is a rising interest to research the effect of natural compounds of plants extracts, on the habitation of the microorganisms. It has been reported that Zingiber officinale (3) Citrullus colocynthis (6) and Be


INTRODUCTION
Recently the resistance of microorganisms to any antibiotic has increased (21).Inadequate usage of antibiotics the most important factor of antibiotic resistance (51).Drug resistant bacteria, particularly , Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa, are most important in healthcare (49) because no new antimicrobial agents are currently available for treatment of infected patients (8,19,15) else bacterial virulence factor play a role in diseases mechanism which is targets in drug evolution,moreover the ability of pathogens to form biofilms award a selected advantage for bacteria to militate under harsh environmental conditions lead to resistance to antimicrobial agents (52) Pseudomonas aeruginosa, and, Escherichia coli are examples of bacteria that form biofilms (39,35).Therefore alternative therapeutic agents from plants is One strategy to avoid antibiotic resistant bacteria, safe and have low cost(2, 33,36,46).Consequently this study aimed to assess the in vitro antibacterial activities of Phyllanthus emblica extracts against bacterial clinical isolates Staphylococcusaureus , Gram-negative bacilli: Escherichia coli, Klebsiella pneumonia and antibiofilm effect against Pseudomonas aeruginosa and Escherichia coli.

MATERIALS AND METHODS Preparation of plant extract:
The dried plant was purchased from markets in Baghdad.The powdered plant material (250 g) was extracted in a 1000 ml conical flask with 500ml ; solvents(ehanol:methanol,1:,1,v:v) for 14 days in freeze after that filterated using Whattman No 4 filter paper.The filtrate obtained was concentrated byevaporated to dryness to obtain the crude extract.and kept it at 4°C until further uses.Preparation of microorganism and inoculums Microorganisms were medical isolates collected from the culture collections of the zoonotic diseases unit /veterinary medicine college at the university of Baghdad.Organisms were as follows: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Organisms were maintained on brain heart infusion agar overnight, Inocula were prepared by diluting overnight cultures in saline and adjusted to 0.5 McFarland turbidity standards to approximately 10 8 cfu ml for each bacterium.

Assay for antibacterial activity
The screening of antibacterial activity was carried out by using the agar diffusion method as described by Lino and Deogracios (30) with slight modifications.Each of the bacterial cultures tested inoculated (0.2 ml each) using the sterilized swabs onto Mueller Hinton agar (MHA, Oxoid) plates (diameter: 15 cm),Then A sterilized stainless steel borer was used to formed four wells (6 mm diameter), The Phyllanthus emblica extract was separately redissolved in sterile distilled water at concentrations of (20, 10, 5 and 2.5 mg/ml),then 100 μl of each concentration of the plant extract were filled each well .The culture plates were allowed to stand then incubated at 37°C for 24 h.antibacterial activity was determined by measurement of diameter zones of inhibition (mm) (against the test organisms) around the extracts (30).

Determination of Minimum Inhibitory Concentration (MIC)
The MIC and the MBC of the Phyllanthus emblica were determined by using test tubes where two-fold serial dilutions with muller hinton broth were made to the various concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.312 and 0.156 mg/ml) of the Phyllanthus emblica extract for each bacteria.Specifically 1ml of 0.5 McFarland turbidity standard 10 8 cfu/ml was added to each tube and incubated aerobically at 37 C o for 18-24hrs.The MIC assay was determined by visualize the bacterial growth.0.5 ml(0.04mg/ml) of piodonitrotetrazolium violet(trazolium salt)( (INT) was added to each tube and incubate all tubes were incubated at room temperature for 6 hrs.The tubes were examined for color change and the MIC was indicated by the first clear tube that not changed to red color when compared with the control tubes or none inhibited concentrations.

Minimum Bactericidal Concentration (MBC)
The MBC was determined by subculturing a loop ful of the MIC tubes that showing no visible growth and no colour change onto extract free agar plates that incubated for a further 24 hours at 37 C o then the lowest concentration of MIC at which no growth on solid medium was regarded as MBC.

The effect of Phyllanthus emblica extract on the bacterial biofilm formation
Biofilm formation was assessed in plastic sterile test tubes, where seven appropriate concentrations (20,10,5,2.5,1.25,0.625 and 0.312 mg/ml) of extract were prepared from a serial two-fold dilutions method in muller hinton broth and eight tubes were inoculated with 1ml of the 0.5 McFarland turbidity standard and incubated for 4-5 hours to allow cell attachment and then add 1ml of each concentrations was added to each tube.All the tubes were further incubated for 24 hours at 37°C, the eight tube was containing bacteria and muller hinton broth only (negative control).

The ability of adherence bacteria
The adhered cell biomass was determined using 1% crystal violet staining.At first, plastic tube was emptied and washed three times with sterile Phosphate Buffered Saline (PBS).The tubes were air-dried and then oven-dried at 60 o C for 45 min.then the tubes were stained with 1ml of 1% crystal violet and incubated at room temperature for 15 min after which the tubes were washed 5 times with sterile distilled water to remove unabsorbed stain after that 1ml of ethanol was added to each tube and the absorbance was determined at 540nm using a spectrophotometer RESULTS AND DISCUSSION Antimicrobial Activities of the Extract by agar diffusion method: Table 1 shows the diameters for the zones of inhibition (mm) of Phyllanthus emblica extract at different concentrations(mg/ml).At 20mg/ml, E. coli had a higher zone of inhibition of 27mm(fig.1) while S aureus and P. aeruginosa had 25mm,15mm respectively.At 10 mg/ml of the extract concentration showed the highst zone of inhibition in s.aureus 21mm (fig.2) and E.coli 20mm while in P. aeruginosa was the least 10mm At 5 mg/ml, E. coli had the highest zone of inhibition of 14mm and the least zone of inhibition of S. aureus was 10mm while P. aeruginosa was not sensitive to the extract of Phyllanthus emblica.In contrast,all bacteria were not sensitive to the extract of Phyllanthus emblica at 2.5mg/ml (fig.3).The attention of researchers at the present time search for medicinal plants instead of antibiotics as a result of antibiotic resistant bacteria(4, 24,37, ,40) As well as the ease of access to medicinal plants and the availability and affordability (7,22,41) 2. and fig4 ranging from 1.25 to 5 mg/ml fig.5,6 and 7 depending on the species of bacteria, thereby demonstrating the potential of this extract as antibacterial agents, likewise alcoholic extract of Phyllanthus emblica also showed bactericidal activity against all test strains at MBC values ranging from 2.5 to 10mg/ml fig.8,9.The statistical analysis using Anova singal factor shown in table (3).The resemblance of the MBC and MIC values of the herbal extract could be due to the sensitivity of the tube dilution method in detecting a minimum amount of turbidity which was the indicator of the growth of the test organisms than visual inspection on the other hand the MIC and MBC increased value against E. coli and P.aeruginosa rather than S.aureus that could be related with the differences in cell wall composition of testing microorganisms.The composition of cell membrane of Gram-negative bacteria involving many layer that prevent the permeation of antimicrobial agent therefore the bacteria become more resistance (16) as well P.aeruginosa have multiple technique of resistance to antibiotics and disinfectants like quartenary ammonium compounds (QACs), dyes and soaps (11,14,43,48,50) such resistance including, target structure alteration, enzymatic degradation, multidrug efflux pumps ,down regulation of outer membrane porins, βlactamases (1,5, 27,28,29,32,38) which may play a role in the low-level drug resistance else the decrease antibacterial effectiveness of this plant against P.aeruginosa shown in the present study should consequently to the resistance of bacteria strains tested that related to pump efflux system (18,42) Anti-biofilm activity on plastic tube quantification with P.aeruginosa The results of in vitro anti-biofilm activity of alcoholic extract of Phyllanthus emblica against P.aeruginosa found to inhibit the biofilm formation on the plastic surface and showed decreases in the turbidity when the optical density (OD) was taken at 590 nm (nanometer) are presented in fig.10.The bacteria used in this part of the investigation have been selected from the bacteria used for antibacterial activity depending on their biofilm formation potential.Inhibition of biofilm formation on plastic tube surfaces for P.aeruginosa by Phyllanthus emblica were additionally visualized by ccrystal violet assay which is illustrated in Fig. 11 Table 1  potential source of antibacterial agent as well as our result agree with previous study (17).This result shows that the plant might have important compounds which may active against test organisms.moreover P.aeruginosa formation of biofilms that resist the entrance of chemical agents (45,47) likewise P. aeruginosa biofilms are found on many surfaces in hospital tools like dialysis membranes and catheters (20,23,34), on the other hand the continuance of the biofilm of this bacteria on the tools makes it very difficult to eliminate and finally caused subsequent in infected patient like cystic fibrosis, otitis media (15,44,9) however the uses of conventional antibiotic for eradicate the biofilm forming is very hard because of substantial and acquired mechanisms of resistance (10,31).The plant showed a positive anti biofilm effect on the P.aeruginosa adherence formation on the plastic surface, These active herbal extract was found to inhibit the biofilm formation a dose dependent manner on the plastic surface and showed decreases in the turbidity when the OD was taken at 590 nm.The success of Phyllanthus emblica extract in inhibiting biofilm formation of P.aeruginosa in this study is a promising tool for reducing microbial colonisation on surfaces and epithelial mucosa which subsequently leads to infections .The facility of Phyllanthus emblica plant extract can inhibited the cell adherence is confirmation with prior studies that was found where it was found that inhibition of cell attachment to a substrate is easier to attain than inhibiting the growth of an already established biofilm (12).In this study, suppression of cell adherence was effective with most extracts showing suppression more than 50%.There are multiple way success for inhibiting cell adherence.Finally we found that Phyllanthus emblica can effect on the planktonic state which refers to the condition where the bacteria were allowed to grow as a suspension in the test tubes.Phyllanthus emblica can significantly damage the adhesion of the early colonizers.This subsequently will interfere with the initial stage of biofilm development.

Activities of the Phyllanthus emblica Extract by MIC and MBC The
. Actually, there is a rising interest to research the effect of natural compounds of plants extracts, on the habitation of the microorganisms.extracts of Phyllanthus emblica had strong bactericidal activity with MICvalues against Staphylococcus aureus , P.aeuroginosa and Escherichia coli are presented in Table