بررسی اثر ضد‏میکروبی عصاره ‏های الکلی گیاه اسپند (Peganum harmala) بر فرم منفرد و بیوفیلمی 6 باکتری پاتوژن

Introduction

Biofilms are complex structures consisting of surface- attached bacteria surrounded by a self- produced extracellular polymer matrix and are currently recognized as the predominant bacterial life- style (1 & 2). Typically, anywhere there is a flow of water, organisms and a solid surface, a biofilm could be formed. It is well established that bacteria contained within biofilms exhibit increased resistance to antimicrobial treatments compared to individual cells grown in suspension (3). Several mechanisms can account for the increased antibiotic resistance in biofilms, including the physical barrier formed by exopolymeric substances (4), a proportion of dormant bacteria that are inert toward antibiotics (5), and resistance genes that are uniquely expressed in biofilms (6). The ability of biofilm- embedded bacteria to resist clearance by antimicrobial agents points to the importance of a continuous search for novel agents. These agents should be effective against bacteria orwork in synergy with the currently available antimicrobial agents. Novel strategies are therefore required to deal with these biofilm- mediated infections. Currently; a renewed interest in natural substances has focused attention on plants rich in bioactive compounds well known for their antimicrobial properties. Plants produce an enormous array of secondary metabolites and it is commonly accepted that a significant part of this chemical diversity serves to protect plants against microbial pathogens (7).

Peganum is a small genus belonging to the family Zygophylaceae and mainly distributed in the Mediterranean region, also found in central Asia, North Africa and has also been distributed in America and Australia (8 and 9). From ancient times, Peganum harmala was claimed to be an important medicinal plant and it was used in global folk medicine (10). This plant has been considered for the treatment of a variety of human ailments, such as lumbago, asthma, colic, jaundice and as a stimulant emmenagogue (11). The smoke of its seeds is traditionally used as a disinfectant. Its seed extracts are known to contain β- carbolinalkaloids, anthroquinons and a small quantity of flavonoid glycosides (10 & 12). There are reports that alkaloids in P. harmala seed are mainly responsible for different pharmacological activities including antibacterial, antifungal, antiprotozoaland antiviral effects (13 & 14), in addition to vasorelaxant (15), anti- spasmodicandanti- histaminic effect (16), spontaneous effect (17), anti- hemosporidian effect (18), anticancer and anti- nociceptive effect (19), antitumor effect (20) or antineoplastic effects (21).

In some study, antibacterial activity of P. harmala was confirmed. According to these researches, P. harmala extracts can inhibit planktonic form of many bacteria such as Streptococcus pyogenes, Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli (22), Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae (23). The object of this study was to examine the antibacterial activities of alcoholic extracts of P. harmala against the biofilm of six pathogenic bacteria and their free- living forms. These bacteria included S. aureus, Bacillus cereus, Streptococcus pneumoniae, P. aeruginosa, E. coli and K. pneumoniae. However, a study with focusing on anti- biofilm activity of P. harmala has not yet been documented.

Materials and methods

Plant Collections, Identifications and extraction: Fresh plant P. harmala was collected in spring season locally in April, 2012 from Kerman, Iran. The taxonomical identification of the plant was confirmed by Dr. Mirtajadini at the department of biological sciences, Shahid Bahonar university of Kerman, Kerman, Iran.

Plants were extracted as described by El- Mahmood (24) with some modifications. Freshly collected seed and stem of P. harmala were rinsed with running water, shade- dried and then powdered using an electrical blender (Bosch Limited, Germany). For successive extraction, ten gram (10 g) of plant powder was macerated in 100 ml of ethanol 80% and methanol 96% (Pars chemic co., Kerman, Iran). Different extracts were prepared using the maceration process for 30 hat 38°C under constant shaking and filtered with What man No. 1 filter paper. The extracts were evaporated to dryness under reduced pressure using a rotary evaporator and then were incubated at 37°C to complete evaporation solvent. One hundred milligram (100 mg) of each methanolic and ethanolic extracts was carefully taken in a standard measuring flask and the appropriate volume of DMSO 1% were added to dissolve the extracts. Then each extract was made up to 1 ml by adding sterile NB medium and the solution was then filter sterilized with 0.22 µm mixed cellulose ester membranes (Millipore^TM; MA, USA). The extracts obtained were kept in sterile dark bottle at 4°C for furtheruse.

Test microorganisms and media: Microorganisms used in this study included six bacteria species, three Gram- positive (S.aureus, B.cereus and S. pneumoniae) and three Gram- negative (P. aeruginosa, E. coli and K.pneumoniae). The tested microbial species were clinical isolates provided by the Faculty of Medicine, Department of Microbiology, Kerman Medical University, Kerman, Iran.

The test microorganisms were maintained in NB/glycerol (20%) at - 80°C. Nutrient agar (NA, Merck, Germany) containing Luria- Bertani (LB, Merck, Germany) was used to activate S.pneumoniae while nutrient agar was used for other bacteria. The Mueller Hinton Agar (MHA, Merck, Germany) medium was used for disc diffusion assay and Nutrient broth (NB, Merck, Germany) was used for the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) determination. The Mueller Hinton agar was also used for the determination of the MBC on the species. The tryptic soy broth (TSB, Merck, Germany) medium was used for anti- biofilm assay. For S. pneumoniaein all assessments, medium was enriched by increasing LB.

Disc Diffusion Assay: The antimicrobial susceptibility of the tested bacteria to P. harmala alcoholic extracts was performed as recommended by the Bauer- Kirby disc diffusion method (25). Five hundred microliters (500µl) of 18 h culture of tested bacteria adjusted to 10⁸CFU/ml was poured and uniformly spread on MHA.

In the following, the sterile 6 mm blank paper disks (Padtan Teb Inc., Teh‏ran, Iran), saturated with filter sterilized plant extract at the prepared concentration (100 mg/ ml) for about 2 h and allowed to dry at 37°C for 5 h. Ciprofloxacin (2 mg/ ml) was used as a positive control; 96% of methanol and 80% of ethanol were used as a reagent control. Each of the discs was placed on the inoculated plates and they were incubated at 37°C for 18h. The diameters of the inhibition zones were measured in millimeters (26).

MIC and MBC assays: MICs and MBCs were determined by a Macrobroth dilution method as recommended by the Clinical and Laboratory Standards Institute (27) using NB as the test medium. Overnight cultures of bacteria were diluted to yield a final concentration of 5×10⁵ CFU/ml. Different concentrations of extract were prepared from serial two- fold dilutions method by dissolving extract stock concentration (100 mg/ ml) in sterile culture medium (NB). 1 ml of each standard bacterial suspension and different concentrations of extract (0.05 - 100 mg/ ml) were added to tubes containing 1 ml NB. These tubes were incubated at 37°C for 18 h. The first tube in the above series without sign of visible growth was considered as the MIC. Controls included NB plus extracts, NB plus Ciprofloxacin (2 mg/ ml) plus cells, cells plus NB plus sterile distilled water and NB plus solvents.

MBC was determined by culturing one standard loop of the tubes with no apparent growth on MHA plates and subsequent incubation at 37°C for 24 h. The least concentration that inhibited colony formation on agar was considered as MBC for the extract.

Inhibitory assay for biofilm formation: Biofilm formation was measured as described by O'Toole and Kolter (28) with some modifications. Three concentrations of extract were prepared from serial two- fold dilutions method by dissolving extract stock concentration (100 mg/ ml) in sterile culture medium (TSB). 100 µl of each concentration (12.5- 50 mg/ ml) were added into the wells of sterile 96- well polystyrene microtiter plate. The standard bacterial culture for biofilm assessments was prepared from the cells, were cultured at 37°C overnight and re- suspended at an optical density at 600 nm (OD600) of 0.2 in 1ml of TSB. Then 100µl of these suspensions were added to each well and incubated at 37°C for 24 h. Controls included TSB plus extracts (extract control), TSB plus Ciprofloxacin (2 mg/ ml) plus cells (positive control), cells plus TSB plus sterile distilled water (Negative control) and TSB (media control).

The formation of biofilm was quantified by measuring the absorbance of CV- stained biofilm at 630 nm and by total viable counts (29). After 24 h of exposure, media were discarded and each well was washed three times with phosphate buffer saline (PBS) to remove non- adherent cells. Adherent microorganisms were fixed by adding 150 µl of methanol 96% for 15 min. The contents of the wells were then aspirated and 200 μl of crystal violet 1% (Gram color- staining set for microscopy – Merck, Germany) were added to wells to stain for 20 min. Excess stain was rinsed off with running tap water and the plates were air- dried at room temperature. To measure the absorbance of adherent cells, the CV was re- solubilized with 160 µl of acetic acid glacial 33% (Merck, Germany) and the absorbance was measured with a microtiter plate reader (ELX- 800, Biotec, India) at 630 nm. The percentages of biofilm inhibition in the presence of different concentrations of extracts were calculated employing the ratio between the values of OD_630nm with and without the extracts.

Eradication of established biofilms: Initially, 100μl of the standard bacterial culture (OD 600=0.2) was transferred into sterile 96- well polystyrene microtitre plates and incubated at 37°C for 24 h. After removing the supernatant, the plates were then washed with sterile distilled PBS. Thereafter, three different concentrations (12.5- 50 mg/ ml) of herbal extracts were added to the established biofilm followed by incubation for 24 h at 37 ºC (30).The control wells were the same as those described in inhibition of biofilm formation assays. Further Eradication of a preformed biofilm was quantified by crystal violet staining and reading the absorbance at 630 nm. The percentages of reduction biofilm structures in the presence of different concentrations of extracts were calculated employing the formula as described earlier.

Effect of extracts on biofilm metabolic activity: The effect of the P. harmala on the metabolic activity of pre- formed biofilm was measured as reported by Ramage and Lopez- Ribot (31). At first established biofilms were washed twice with PBS, then extracts (12.5- 50 mg/ ml) were added to each well and plates were incubated for 24 h at 37◦C. Afterwards, 50 µl of a Triphenyl Tetrazolium Chloride (TTC, Merck, Germany) solution was added and it was further incubated in the dark at 37◦C for 3 h. The colorimetric change was measured at 490 nm using a microtiter plate reader (ELX- 800, Biotec, India). The control wells were the same as those described in inhibition of biofilm formation assays. The percentages of reduction of biofilm metabolic activity in the presence of different concentrations of extracts were calculated employing the formula as described earlier.

Statistical analysis: Statistical analysis was carried out using SPSS Version 18.0 for Windows. Analysis of variance (ANOVA) was used to evaluate any significant differences between the control and treated groups observed in the presence of different concentrations of the extracts. P value< 0.01 denoted a statistically significant difference (SPSS, version 18, USA). All tests were performed in triplicate.

Results

Inhibitory efficiency of P. harmala extract against planktonic form of pathogenic bacteria: The zone of inhibition (ZOI) for methanolic and ethanolic extracts of P. harmalawere shown in table (1), also the MIC and MBC values of these extracts were illustrated in table (2). In disc diffusion analysis, the extracts of P. harmala have the best inhibitory effect on S. aureus, B. cereus and E. coli. In this test ethanolic extracts were better growth inhibitor. Although these extracts didn’t have any inhibitory effect on other tested clinical bacterial pathogens.

The results of P. harmala extracts in broth medium, show that these extracts have sufficient inhibitory effect (below the concentration used in disc 0.19- 3.12 mg/ ml) on a tested bacteria. Then it was concluded that the inhibitory effect of these plant extracts in broth medium is more than solid medium.

Based on the disc diffusion results, the inhibitory effect of ethanolic extracts on planktonic forms is more than methanolic extracts, however for B. cereus and P. aeruginosa there wasn’t any significant difference between inhibitory effects of methanolic or ethanolic extract (Table 2). The best inhibitory effect (ZOI) of P. harmala was shown against B. cereus.

The inhibitory effects of P. harmala extracts against biofilm structures: The inhibitory efficiency of each concentration of P. harmala extracts on preventing biofilm formation, demolished of biofilm structures and inhibition of metabolic activity of biofilm were shown in fig.s (1), (2) and (3). According to the F value of the ANOVA analysis on tested data, it was confirmed that inhibitory efficiency of P. harmala extracts were significant at 0.01 level (P value<0.01).

Table 1- Antibacterial activity of P.harmala alcoholic extracts against test microorganisms by using disc diffusion method (Zone of inhibition in mm).

Table 2- P. harmala minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of test bacteria.

Fig. 1- Percent reduction of biofilm formation for test bacteria treated with different concentrations of P.harmalafor 24 h. * Differences between control (no inhibition) and treatment with extracts

 (* p < 0.05, ** p <0.01).

Fig. 2- Percent disruption of biofilm formation for test bacteria treated with different concentrations of P.harmala for 24 h. * Differences between control (no inhibition) and treatment with extracts (* p < 0.05, ** p <0.01).

Fig 3- Percent reduction of biofilm metabolic activity for test bacteria treated with different concentrations of P.harmalafor 24 h. * Differences between control (no inhibition) and treatment with extracts (* P value< 0.05, ** P value <0.01).