بررسی خواص ضد میکروبی گیاه زوفا (Hyssopus officinalisis) علیه باکتری‌های بیماریزای مقاوم به انتی بیوتیک در فرم منفرد و بیوفیلم

Introduction

Despite extensive progress in scientific knowledge and medicine, infectious diseases remain a leading cause of worldwide morbidity and mortality. The main chemical agents for eradication of pathogenic fungal and bacteria are antibiotics. Antibiotics were gold key for the treatment of pathogens for long times. However, in recent years, antibiotic resistant bacteria were increased and the use of antibiotics for treatment of pathogenic microbes had less efficiency (1). Bacterial biofilms are complex and vast-structured communities of adhesive microorganisms enclosed in a self-produced extracellular matrix of exopolysaccharide and irreversibly connecting to various surfaces (2, 3). The capability of microorganisms to attach to both biotic and abiotic surfaces and to form biofilm are accountable for a number of diseases of chronic nature, indicate high resistance to antibiotics (4, 5). Accordingly, biofilms can cause important problems in very areas, both in medical settings and in non-medical  settings (6, 7).

In recent years, attentions drawn on herbs, because they increase in antibiotic-resistant bacteria and side effect of synthetic antibiotics. Medicinal plants are known for having synthetica diversity of compounds to protect themselves against many of their own pathogens and therefore can serve as antimicrobial materials (8).

Hyssopus officinalis is a flowering plant, which belongs to Lamiaceae family (9) and a herb widely growing in Iran (10). This plant has been used in folk medicine for various aims such as anti-inflammatory, anti-bacterial, anti-pyretic, anti-spasmodic, antihypertensive and anti-hyperlipidemic purposes (11). The aim of this research was to evaluate the antibacterial and antibiofilm activity of H. officinalis. Also the effective compounds of this plants were determined.

Materials and Methods

Plant samples collection and extraction: The fresh plant of H. officinalis was prepared in May, 2015, from Kerman, Iran. The taxonomical identification of the plant was confirmed by a plant taxonomist and the DH9541 code were registered as herbarium code for this plant. The plants dried in air and powdered. Fifty grams of H. officinalis powder were soaked in 500 ml of ethanol and methanol (96 %) solvents. Different extracts were prepared using the cold maceration process for 72h at room temperature under constant shaking and filtered with Whatman No.1 filter paper. The residue was further macerated and filtered twice with the same solvent overnight. The filtrates obtained from each extraction were mixed and concentrated under vacuum. The extracts obtained were kept at 4°C for further use (12, 13)

Bacterial strains: Six pathogenic bacteria were used in this study. The Gram-positive bacteria were Bacillus cereus (ATCC1298) and Staphylococcus aureus (ATCC1189), and Gram-negative bacteria were Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC35218), Acinetobacter baumannii (ATCC1611) and Klebsiella pneumonia (ATCC700603).

Disk Diffusion Method: Antimicrobial activity of the ethanolic and methanolic extracts was evaluated by paper disc diffusion method. Saved culture of test bacteria was grown in Notrient Broth (NB ) medium at 37 °C for 24 h. Final cell concentrations were adjusted to 10⁸ cfu ml^-1 with reference to the McFarland standard (OD= 0.5). This inoculum 1ml was added on the surface of Mueller-Hinton Agar (MHA, Oxoid) plates by sterile cotton swab. Then, 6 mm paper discs (0.15 mg ml^-1 of each extract) were placed into each of these concentrations for 1 h. The disc was put for 30 minutes at room temperature and transferred to the medium. Disc solvent-free extract used as negative control. The inhibition zone around each disc was measured in millimeter and the assay was carried out three times for each extract (14, 15).

MIC and MBC determination: Determination of the Minimal Inhibitory Concentration (MIC) was carried out using the macrobroth dilution method as recommended by the Clinical and Laboratory Standards institution (CLSI). The concentration of bacterial culture was serialy diluted to reach 5×10⁵ cfu ml^-1. Different concentrations of extract were prepared by dissolving extract stock concentration (100 mg ml^-1) in sterile culture medium (NB). Then, 1 ml of standardized inoculum (5×10⁵ cfu ml^-1) was added to 1 ml of each extract concentration. Then, all tubes were incubated at 37°C for 18 h. MIC was calculated as the lowest concentration able to inhibit bacterial growth. Two controls were used for this experiment: 1) tube with extract and growth medium, and 2) the tube with growth medium and inoculum. Minimal Bactericidal Concentration (MBC) was determined by inoculate negative tubes (tubes that had not any visible growth) into MHA plates and subsequent incubation at 37°C for 24 h. The minimum concentration inhibited colony formation on agar was considered as MBC for the extract (16).

Inhibition of biofilm formation: Biofilm formation in polystyrene microtiter plates was assayed according to O’Toole and Kolter (1998) method with some modifications. Three concentrations of each extract (12.5, 25 and 50 mg ml^-1) were pipetted (100 µl) into the wells of the microtiter plates. For biofilm assessment, bacteria were cultured at 37°C overnight and re-suspended at an optical density at 600 nm (OD₆₀₀) of 0.2 in 1 ml of TSB. Afterwards microtiter plates were incubated for 24 h at 37°C. In this experiment, three controls were used in microplate including: 1) Extract control: this well inoculated with NB medium and extract 2) Negative control: this well inoculated with bacteria without any extract 3) Media control: only NB pour into this well (17).

The formation of biofilm was quantitated by measuring the absorbance of crystal violet stained biofilm at 630 nm and by total viable counts (18). After incubation, the media were aspirated and non-adherent cells were removed by washing the wells three times with sterile phosphate buffer saline (PBS). Adherent microorganisms were fixed by adding 150 µl of methanol 96% for 15 min. The microtiter plates were stained with 200 µl of crystal violet (1%) (Merck, Germany) for 20 min, excess stain rinsed off with running tap water. To measure the absorbance of adherent cells, the crystal violet 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, Biotek, USA) at 630 nm. Percent prevention of biofilm formation was calculated using the ratio between the values of OD630 _nm wells with and without the extracts.

% inhibition = [(OD negative control-OD media control)-(OD test-OD extract control)] /(OD negative control-OD media control) ×100

Disruption of established biofilm: Disruption of established biofilm structures was measured as described by Sandasi (2008) with some modifications. 100 μl 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 incubation, the medium was aspirated and the planktonic cells were removed by washing the biofilms three times with sterile PBS. Thereafter, three different concentrations (12.5-50 mg ml^-1) of H. officinalis extracts were added to each well and plates were then placed back into the 37°C for 24 h. The control wells were the same as described in section 2.5. The percentages of reduction biofilm structures in the presence of different concentrations of extracts were calculated employing the formula as described in section 2.5.

Assessment of biofilm metabolic activity: The effect of the H. officinalis upon the metabolic activity of pre-formed biofilm was measured according to Ramage and Lopez-Ribot (2005). Briefly, pre-formed biofilms were washed twice with PBS, extracts (12.5-50 mg ml^-1) 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 to each well and allowed the reaction to occur in the dark at 37^◦C for 3 h. Final absorbance was read at 490 nm using microtiter plate reader. The percentages of reduction of biofilm metabolic activity in the presence of different concentrations of extracts were calculated by comparing the absorbance of control bacteria and treated inoculation with each extract (19).

Gas Chromatography–Mass Spectrometry (GC/MS): The chemical compound of H. officinalis were determined by GC-MS. The chromatograph (Agilent 6890 UK) was equipped with an HP-5MS capillary column (30 × 0.25 mm ID × 0.25 mm film thickness) and the data were taken under the following conditions: initial temperature 50°C, temperature ramp 5°C/min, 240°C/min to 300°C (holding for 3 min), and injector temperature at 290°C. The carrier gas was helium and the split ratio was 0.8 ml/min. For confirmation of analysis results, essential oil was also analyzed by GC/MS (Agilent 6890 gas chromatograph equipped with an Agilent 5973 mass-selective detector; Agilent UK) and the same capillary column and analytical conditions as above. The MS was run in electron ionization mode with ionization energy of 70 eV (20).

Statistical analysis: Differences for individual parameters between control and treated groups were tested by Duncan analysis of variance (ANOVA) using SPSS version 18.0. Differences were considered significant if the P value was less than 0.05, 0.01 and 0.001. All experiments were performed in triplicate and repeated three times.

Results

The inhibitory effects of H. officinalis xtracts against planktonic forms of bacteria: The zone of inhibition (ZOI) for methanolic and ethanolic extracts of H. officinalis were shown in Table (1), also the MIC and MBC of these extracts were illustrated in this table. The big and small ZOI for methanolic and ethanolic extracts related to S. aureus and P. aeruginosa, respectively.

The inhibitory effects of H. officinalis extracts in broth medium (MHB) were more than solid medium (MHA). This result can be expected because the concentration of extract used in solid medium (paper disks) were lower than broth medium (3.125-50 mg ml-1). Between all pathogenic bacteria, A. baumannii show the greatest sensitivity to H. officinalis extracts in broth medium (MIC=3.125 mg ml^-1). Among six pathogenic bacteria studied in this study the maximum MIC (25 mg ml^-1) belonged to K.pneumonia.

Table 1- The antimicrobial effect of H. officinalis against six planktonic bacteria and MIC/MBC values

The inhibitory effects of H. officinalis extracts against biofilm structures: The efficacy of each concentrations of H. officinalis extract to inhibit biofilm formation, demolished of biofilm, and metabolic activity were shown in Figures (1) and (2). The maximum inhibitory effects of H. officinalis ethanolic extract on biofilm formation were observed on E. coli (95 %), although methanolic extract had low efficiency to inhibit biofilm formation of B. cereus (28.03%) (Fig1-a).

The eradication of bacterial biofilm treated with different concentrations of H. officinalis extracts were varied. The P. aeruginosa biofilm was susceptible (95.4 %), Between six pathogenic bacteria that studied in this research K. pneumoniae had the minimum sensitivity to inhibitory effect of H. officinalis (Fig.1-b).

The effect of H. officinalis extracts on inhibition of dehydrogenase enzyme of pathogenic bacteria were illustrated in Figure (2). As shown in this figure metabolic activity of bacteria in biofilm dramatically decreased, the maximum reduction observed in P. aeruginosa (OD=0.7) and minimum reduction were recorded for K. pneumoniae (OD=2.1).

Fig. 1- The effect of H. officinalis extractagainst biofilm of pathogenic bacteria(a) inhibition of biofilm formation (b) disruption of biofilm structures with different concentrations of H. officinalis extracts.

Fig. 2- The effect of H. officinalis extract on dehydrogenase enzyme activity of six pathogenic bacteria (a) ethanolic extract (b) methanolic extract

According to ANOVA analysis, it was confirmed that inhibitory efficiency of H. officinalis extracts were significant at 0.05, 0.01 and 0.001 level (Table 2). As shown in Table 2, the inhibitory effect of extracts on formation, destruction and metabolic activity of bacterial biofilm was significant at 0.05, 0.01, and 0.01 percent levels, respectively. The inhibition and destruction of bacteria biofilm in treatment with extract of H. officinalis was independent from solvent used for extraction.

Chemical composition of H. officinalis extract: The chemical mixture of H. officinalis extract was determined by GC-MS. The results were shown in Table (3) and also the chromatographs were presented in Figure (3). As shown in Table (3), the main active compounds illustrated in the extract of this plant was: Verbenone, Pyrane, Phenol,  Furan, and Cyclopropan. All detected compounds in the extract of this plant  have aromatic ring. In other hands, the phenolic compounds were predominat in this medicinal plant.

Table 2- The results of biofilm inhibition and destruction by extract of H. officinalis were statistically analyzed by Duncan's test

DF: Degree of Freedom

MS: Min Square

Sig: Signification

Table 3- Chemical composition of H. officinalis extracts analysis by GC-MS

Fig. 3- The GC-MS Chromatogram of H. officinalis extract. Five main compounds were detected in the GC-MS analysis include (from left to right): Verbenone, Pyrane, Phenol, Furan and Cyclopropan