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

نوع مقاله: پژوهشی- انگلیسی

نویسندگان

1 دانشیار مرکز تحقیقات تولیدات گیاهی، دانشگاه شهید باهنر کرمان، کرمان، ایران

2 استادیار مرکز تحقیقات تولیدات گیاهی، دانشگاه شهید باهنر کرمان، کرمان، ایران

3 کارشناس ارشد گروه زیست شناسی، دانشکده علوم، دانشگاه شهید باهنر کرمان، کرمان، ایران

چکیده

مقدمه: استفاده از عصاره های گیاهی تهیه شده از گیاهان دارویی در کشورهای در حال توسعه بصورت گسترده‌ای متداول است. گیاه زوفا (Hyssopus officinalisis) به خانواده Lamiaceae متعلق است. هدف از پژوهش حاضر ارزیابی فعالیت مهاری و ضد باکتریایی گیاه زوفا بر روی شش باکتری مقاوم به انتی بیوتیک است.
مواد و روش‏‏ها: در این پژوهش عصاره های متانولی و اتانولی گیاه زوفا تهیه شد. اثر ضدمیکروبی عصاره‌های اتانولی و متانولی بوسیله روش انتشار دیسک بررسی شد. غلظت حداقل مهار کننده (MIC) و غلظت حداقل کشنده (MBC) عصاره ها بر روی شش باکتری پاتوژن مشخص گردید. فعالیت ضد بیوفیلمی (تشکیل بیوفیلم و تخریب بیوفیلم) این عصاره های گیاهی بوسیله روش میکروتیتر پلیت بررسی شد. ترکیب شیمیایی عصاره زوفا با روش گاز کروماتوگرافی تعیین مشخص گردید.
نتایج: حداکثر اثر مهاری این عصاره ها بر علیه باکتری.S. aureus و P.aeruginosa در فرم منفرد مشاهده شد. در بین همه باکتری های بیماریزا باکتری A. baumannii بالاترین حساسیت را به عصاره گیاه زوفا در محیط مایع نشان داد (12/3 MIC=  میلی گرم بر میلی لیتر). بالاترین اثر مهاری عصاره گیاه زوفا روی تشکیل بیوفیلم علیه باکتری E. coli بود (99 درصد). نتایج تخریب بیوفیلم نشان داد که باکتری K. pneumonia دارای مقاوم‌ترین ساختار بیوفیلمی در بین کلیه باکتری های مورد مطالعه بود (41/16). نتایج گاز کروماتوگرافی نشان داد که پنج ترکیب فعال و موثره در عصاره این گیاه وجود دارد.
بحث و نتیجهگیری: عصاره‌های گیاه زوفا می‌توانند در درمان برخی عفونت های باکتریایی استفاده شوند.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Antimicrobial properties of Hyssopus officinalis extract against antibiotic-resistant bacteria in planktonic and biofilm form

نویسندگان [English]

  • Mehdi Hassanshahiyan 1
  • Amir Saadatfar 2
  • Fatmeh Masoumi 3
1 Associate Professor, Research and Technology Institute of Plant Production (RTIPP), Shahid Bahonar University of Kerman, Iran
2 Assistant Professor, Research and Technology Institute of Plant Production (RTIPP), Shahid Bahonar University of Kerman, Iran
3 M.Sc., Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran
چکیده [English]

Introduction: Extensive use of antibiotics led to the development of bacterial resistant to antibiotics. Medicinal plants can be alternative choice for antibiotics. The plant (Hyssopus officinalis) belongs to Lamiaceae family recently were attracted as a source for antimicrobial agents. The aim of this study was to evaluate the antibacterial and inhibitory activity of H. officinalis extract on the growth of six antibiotic-resistant bacteria.
Materials and methods: In this study, ethanolic and methanolic extracts of H. officinalis were prepared. Antibacterial activity of the ethanolic and methanolic extracts was evaluated by paper disc diffusion method. Also, MIC and MBC of these extracts were determined for six pathogenic bacteria. The effect of these extracts on biofilm of bacteria (biofilm formation and destruction) was evaluated by microtiter plate method. The chemical composition of the extract was identified by GC-MS.
Results: The results of study showed the maximum inhibitory effect of these extracts against planktonic forms belong to Staphylococcus aureus and Pseudomonas aeruginosa. Between all studied bacteria, Acinetobacter baumannii showed the greatest sensitivity to H. officinalis extracts in Muller Hinton broth (MIC= 3.125 mg ml-1). The highest inhibitory effects of H. officinalis ethanolic extract on biofilm formation were observed against Escherichia coli (95 %). The results of biofilm destruction showed that Klebsiella pneumoniae biofilm had a resistant biofilm structure between all tested bacteria (16.41 %). The GC-MS analysis revealed that five active compounds were present in the extract of this plant.
Discussion and conclusion: The data obtained in this study confirmed that H.officinalis extract inhibit growth and biofilm formation of some pathogenic bacteria. It can be proposed for future studies that the compounds of this plant used for design a antimicrobial agent.

کلیدواژه‌ها [English]

  • Antimicrobial
  • Biofilm
  • Extract
  • Hyssopus officinalis
  • Pathogenic bacteria

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 37C 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

 

Bacteria

 

Disk Diffusion

MIC

MBC

Methanolic extract (mm)

Ethanolic extract (mm)

Methanolic extract (mg/ml)

Ethanolic extract (mg/ml)

Methanolic extract (mg/ml)

Ethanolic extract (mg/ml)

S. aureus

20±0.8

18±0.3

0

6.25

50

25

B. cereus

12±0.4

10±0.6

0

6.25

12.5

25

p. aeruginosa

8±0.7

7±0.2

12.5

12.5

25

50

A. baumannii

13±0.5

8±0.1

3.125

12.5

25

25

E. coli

12±0.2

15±0.3

0

25

6.25

50

K pneumoniae

12±0.9

13±0.2

25

25

0

50


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

Sources Change

 

DF

MS

Sig

Biofilm formation

Destruction biofilm

Inhibition enzyme

Biofilm formation

Destruction biofilm

Inhibition enzyme

Biofilm formation

Destruction biofilm

Inhibition enzyme

Bacteria

5

5

5

0.028

0.135

0.150

*

***

**

Extract

1

1

1

0.862

0.006

0.083

*

-

-

Concentration

2

2

3

0.322

0.288

3.671

-

**

***

Bacteria*Extract

5

5

5

0.008

0.032

0.018

-

**

-

Extract* Concentration

2

2

3

0.050

0.009

0.040

*

-

*

Bacteria* Concentration

10

10

15

0.007

0.013

0.025

-

-

-

Total

25

25

32

 

DF: Degree of Freedom

MS: Min Square

Sig: Signification

 

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

No.

Compounds

Formula

RT

Area

%Total

1

4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl

C6H8O4

9.443

5.858e+4

0.266

2

D-Verbenone

 

C10H14O

10.495

2.001e+4

0.091

3

2-Furancarboxaldehyde, 5-(hydroxylmethyl)

C6H6O3

10.657

2.181e+5

0.990

4

Phenol, 2,6-dimethoxy

C8H10O3

12.413

6.317e+4

0.287

5

1H-Cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-4-methylene

C15H24O

15.503

8.681e+4

0.103

 

 

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

 


Discussion and conclusion

Widespread use of antibiotics in the recent years causes the emergence and spread of drug-resistant bacteria resistant to treatment of infections. One of the most important reasons for antibiotic resistance is the remaining of microbes in biofilm structures. This structure prevents the penetration of antimicrobial agents and also inhibits the effective function of these agents. So, acquiring new pathway to deal with bacteria, especially in the biofilm forms is necessary (21, 22). Research in this field especially focused on biological compounds, because the biological nature of these materials reduced side effects in comparison to conventional chemical agents. Between biological derivatives, the medicinal plant as options draw more attention as a good way for dealing with pathogenic bacteria (23, 24).

In this study, antimicrobial properties of H. officinalis were investigated against six pathogenic bacteria. Disk diffusion test confirmed that H. officinalis extracts had high ability to prevent the growth of selected bacteria. As shown in Tables (1) and (2) the methanolic extract better inhibited planktonic forms of bacteria than ethanolic extract, except that the effects of ethanolic extract against E. coli and K. pneumonia were better than methanolic extract. In disk diffusion test, S. aureus was the most sensitive bacterium, while the inhibitory effect of this plant extracton P.aeruginosa was weak.

Since the inhibitory effects of H. officinalis on all tested bacteria in broth medium (MHB) are higher than solid medium (MHA), it can be concluded that the active compounds of the extracts, like many other herbal extracts, has lower diffusion in Muller Hinton agar medium and for display optimum inhibitory effects in solid medium, it is necessary to use higher concentration of extract than broth medium.

Khaled et al (2014) studied antioxidant and antibacterial activities of Lamiaceae family. Their results confirmed that these extracts have antibacterial effect against E. coli and S. aureus. However, E. coli was more sensitive to the extracts than S. aureus and the most effective extract was H. officinalis (25).

Our results are in accordance with those results reported by Khaled et al (2014). This contradiction between these two studies can be related to geographic areas where these plants have been collected (26).

The ability of H. officinalis ethanolic extract in the inhibition of biofilm formation was more than the demolish of biofilm. These results revealed that the components of H. officinalis extract had better efficacy to inhibit biofilm formation compared to biofilm destruction.

There was very low publication that studied the antibiofilm activity of H. officinalis. Elmasri et al (2014) assessed the chemical composition and antibacterial activity of H. officinalis. They isolated sixteen compounds from methanolic extract of the aerial parts of the plant. Antibacterial activity of the crude extract as well as four of the isolated metabolites were measured. Also, they studied the antibiofilm activity of these metabolit against S. aureus. Their results confirmed that these metabolits from H. officinalis dramatically inhibited S. aureus biofilm formation. Our results in the current research also confirmed methanolic extract of H. officinalis had sufficient inhibitory effect against biofilm formation of S. aureus (%70). Thus, our results are in accordance with the results reported by Elmasri et al (2014) (27).

The chemical composition of H. officinalis extract were determined in this study. Similar to other medicinal plants, the chemical constitute of this plant revealed phenolic and aromatic metabolites. The antibacterial activity of these metabolites were described in some publication in other herbs. For example, Lin-fang et al (2013) by GC-MS analysis of Picrasma vassioides identified 46 compounds. The major compounds in volatile oil are terpenoids, hydroxyl compounds and other acyclic alkane compounds (28).

Kamel and Sanidra (1994) analyzed the volatile oil of two H. officinalis varieties. They detected 93 compounds in the two oils. Their results confirmed that the major part of the extract was sesqui  terpenoids. In our chemical analysis, we reach these metabolits and our results are in accordance with the mentioned study (29-31).

In this current research, the antimicrobial and antibiofilm activity of H. officinalis extracts were confirmed. The extracts of this plant can be used for treatment of some bacterial infections especially against bacteria that acquired resitance to conventional antibiotics.

The results of current study confirmed antibacterial and anti-biofilm activity of H. officinalis. It can be concluded that the extract of this plant has potential to design new antimicrobial agent against pathogenic bacteria that are resistance to conventional antibiotics.

 

Acknowldgments

This research was supported financially by Research and Technology Institute of Plant Production (RTIPP), Shahid Bahonar University of Kerman, Kerman, Iran.

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