Excessive use of antibiotics may enhance the resistance of bacterial species that cause human diseases1. The development of bacterial resistance to available antibiotics has stimulated researchers to find alternative antimicrobial agents, despite the role of antibiotics to prevent the growth of pathogens2.
Despite the fact that bacteria are unicellular organisms, they often show group behavior; the attachment of the bacterial biofilm to the food product is a serious public health risk3. Microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces4. Biofilm-associated cells have the ability to adhere irreversibly on a wide variety of surfaces, including living tissues and indwelling medical devices as catheters, valves, prosthesis and so forth5. Bacteria protected within biofilm exopolysaccharides up to 1,000 times more resistant to antibiotics than planktonic cells (free-floating)6 which generates serious consequences for therapy and severely complicated treatment options7. Approximately, 75% of bacterial infections involved in biofilms formation are protected by an extracellular matrix8.
Medicinal plants are important natural resources that can be constantly renewed and have an effective role in protecting people from disease; it is also an important source for containing huge amounts of antimicrobial agents9,4. Plant extracts and other biologically active compounds isolated from plants have gained wide spread interest for the treatment of disease since ancient times.
Considering the above and based on previous results obtained in our laboratory10-12, the present study was proposed to evaluate the antibacterial and antibiofilm effect of 5 extract plants against 4 clinical isolated pathogens.
MATERIALS AND METHODS
Plant material: Fresh and healthy plants of G. glabra (roots), L. nobilis (leaves), M. officinalis (leaves), M. domestica (peel) and L. siceraria (peel), growing wild around the Gaza Strip community, were obtained from the local markets.
Bacterial strains and culture conditions: Pathogenic isolates of E. coli, S. aureus, P. aeruginosa and K. pneumoniae were obtained from microbiology department of Al-Shifa hospital and maintained on brain heart infusion agar (BHA) medium slant at 4°C and microbial diagnosis were confirmed using biochemical tests.
Preparation of plant extracts: Extracts were prepared following the methodology proposed by Jameela et al.13, with minor modifications. Briefly, 10 g of air-dried plant parts powder was soaked with 150 mL of 80% ethanol and150 mL distilled water and placed in the microwave for one minute and left for a minute to cool. Extraction was repeated12 times and then the extract was filtered through Whatman filter paper number 1 and evaporated in oven at 45°C. Stock solutions (200 mg mL1) were prepared in Dimethyl sulfoxide (DMSO) and stored at -4°C in the dark for further experiments13.
Evaluation of antimicrobial activity: Antimicrobial activity of plant extracts was performed using the agar-well diffusion bioassay. Briefly, 100 μL of fresh culture (approximately 10° CFU mL1) was uniformly spread onto Mueller-Hinton agar (MHA) plates by sterile Driglasky loop. Then, inoculated plates were allowed to dry at room temperature for 20 min. After that, wells of 6 mm in diameter were made in the agar using a sterilized cup-borer and 100 μL of each extract was poured in the wells. Methanol was used as control. Plates were incubated at 37°C for 18 h. Antibacterial activity was evidenced by the presence of clear inhibition zone around each well. The diameter of this zone was measured and recorded.
Disc diffusion method: Agar disc-diffusion method was followed to determine the antibacterial activity. A suspension of bacterial inoculum was adjusted to McFarland standard 0.5 and introduced to MHA (cooled to 45-50°C) swirl gently to mix well. After solidification, sterile filter paper discs approximately 6 mm in diameter were impregnated with stock extracts of 200 mg mL1 concentration and placed on the surface of agar plate. After Incubation for 24 h at 37°C, the antibacterial activity was evaluated by measuring the diameter of zones of inhibition for microbial growth surrounding the plant extracts14.
Well diffusion method assay: MHA plates were inoculated with bacterial isolates under aseptic conditions and wells (diameter = 6 mm) were filled with 50 μL from plant crude extract (200 mg mL1). The plates were allowed to stand at room temperature for 1h for proper diffusion and thereafter incubated at 37°C for 24 h. The resulting diameter of inhibition zones were measured in millimeters (mm)15.
Determination of MIC and MBC of plant extracts: A serial dilution of plant extracts were performed using a sterile diluent of Mueller-Hinton broth (MHB) to reach a concentration range from 100-0.1953 mg mL1 and 50 μL of the inoculum was added to each well. The inoculated plates with bacterial suspension (adjusted to McFarland standard 0.5) were incubated at 37°C for 18 h. Then 2,3,5-Triphenyl Tetrazolium Chloride (TTC) was added to the wells and the plate was incubated for another one hour. Tetrazolium salt is known to be colorless and turns red when biologically active bacteria are grown, the inhibition of growth can be detected when the solution in the well remains clear after incubation with TTC15.
For MBC assay, from the lowest extract concentration that visibly inhibited the bacterial growth; 5 μL were taken and subcultures onto agar plates. The plates were incubated at 37°C for 24 h. MBC was determined according to the lowest concentration that did not exhibit any bacterial growth on the freshly inoculated agar plates16.
Biofilm formation assay
Tube method: Biofilms can be formed in test tubes; for this reason, 0.1 mL of bacterial isolates (obtained by adjusting turbidity to 0.5 McFarland standards) were transferred to glass test tubes containing 10 mL nutrient broth (NB) and incubated at 37°C for 72 h. Then, the medium was removed and the tubes were washed with distilled water (DW), air-dried and biofilm formation was assayed by crystal violet17.
Tissue culture plate method: Three wells of sterile 96-microtiter U-bottomed plate were filled with 200 μL of bacterial suspension. After incubation for 24 h at 37°C, wells were washed three times with 250 μL of DW. After 15 min, plates were stained for 10 min with 200 μL of 0.1% crystal violet per well; excess stain was removed and rinsed off by placing the plates under running tap water and the plates were air-dried. The adherent cells were re-solubilized with 160 μL of 95% (V/V) methanol per well and the optical density (OD) of each well was measured at 570 nm using plate Elisa reader18.
Biofilm inhibition assay: S. aureus and P. aeruginosa from fresh agar were inoculated in BHI broth with 1% glucose and incubated overnight at 37°C in stationary condition. The concentration at which the extract depleted the plankton growth of bacterial by at least 50% was labelled as the sub-PMIC50 and used for anti-biofilm assay. 96 well U bottom tissue culture plates were filled with sub-PMIC50 concentration of plant extract and 100 μL suspension bacteria plates were incubated overnight at 37°C; then the well contents were removed by tapping the plate, washed with sterile DW to remove planktonic bacteria19.
Adherent organisms in plates were stained with crystal violet (0.1% W/V) for 10 min, excess stain was rinsed off by DW and plates were dried; re-suspended in 200 μL 95% (v/v) methanol and transferred to 96 well-flat bottom plates. Optical density of stained adherent bacteria was determined with micro-ELISA reader at wavelength of 570 nm. The percentage of inhibition was then compared with the control20.
Antibacterial activity and MIC of plant extracts: Plant crude extracts showed varying degrees of antibacterial activities against the tested pathogenic bacteria, with a range of inhibition zone (7-25 mm in diameters) as shown in Table 1.
Antimicrobial activity of plant extracts on bacteria that used in this study by well and disc diffusion method
*E: Ethanol, W: Water, (N = 3) Values are mean±SD of three separate experiments
Our results showed that most used plant extracts have antibacterial activity against E. coli, except M. officinalis extracts which showed low inhibition zone by disc diffusion method with no effect with well diffusion method (Table 1).
The ethanolic and aquatic extracts of G. glabra roots showed strong antibacterial activities against E. coli with inhibition zone 25.3 and 15.3 mm, respectively using well diffusion method. The aqueous extract of M. domestica peels showed strong antibacterial activity against S. aureus with zone of inhibition (25.3 mm) as shown in Table 1.
The ethanolic and aquatic extracts of G. glabra roots showed strong antibacterial activities against S. aureus by well diffusion method with inhibition zones of 24 and 23.3 mm, respectively. Also, the aquatic extract of G. glabra roots showed antibacterial activity against P. aeruginosa using well diffusion method with inhibition zone of 13.3 mm (Table 1). All plant extracts showed good results of antibacterial activity against K. pneumonia with inhibition zones of 9-23.3 mm using well diffusion assay except M. officinalis; a range of inhibition zone (7.3-10.3 mm) were found by all plant extracts against all pathogens used by disc diffusion method as shown in Table 1.
All plant extracts were evaluated for their MIC against E. coli, S. aureus, P. aeruginosa and K. pneumonia. The MIC value of aquatic extract of M. domestica peels showed the best value against E. coli followed by ethanolic extract of L. nobilis which gave MIC values of 1.56 and 3.125 mg mL1, respectively (Table 2). The MIC value of ethanolic extract of G. glabra roots against P. aeruginosa was 12.5 mg mL1.
The ethanolic and aquatic extracts of G. glabra roots, L. siceraria peels and M. officinalis against S. aureus gave intermediate activity with MIC value of 25 mg mL1. The aquatic extract of G. glabra roots and L. nobilis against K. pneumonia gave intermediate activity with MIC value of 25 mg mL1. Ethanolic extract of M. officinalis and M. domestica peels against K. pneumonia gave intermediate MIC values. Also, the ethanolic and aquatic extracts of L. siceraria peels gave intermediate MIC values against K. pneumonia (Table 2).
Minimum bactericidal concentration (MBC): The ethanolic and aquatic extracts of G. glabra roots showed antibacterial activities against E. coli and S. aureus with MBC value of 200 mg mL1. The MBC of aquatic extract of M. officinalis against P. aeruginosa and K. pneumonia was 200 mg mL1. The MBC of ethanolic extract of L. siceraria peels against K. pneumonia was 200 mg mL1 but the aquatic extract of L. siceraria peels showed MBC at 100 mg mL1 against K. pneumonia. Other plant extracts gave MBC more than 200 mg mL1 against tested bacteria (Table 3).
Plant extracts activity on biofilm formation of P. aeruginosa: The five plant extracts used in our study reduced P. aeruginosa biofilm formation by ≥ 83%. Our results showed that ethanolic extract of M. domestica peels at concentration of 25 mg mL1 showed 90% inhibition on P. aeruginosa biofilm formation (Table 4).
Minimal inhibitory concentrations (MIC) of the plant extracts on bacteria that used in this study
(N = 2) Values are mean±SD of two separate experiments
Bactericidal concentrations (MBC) of the plant extracts against tested bacteria
Effects of ethanolic and aquatic extracts on biofilm formation and biofilm inhibition of P. aeruginosa
aConcentration below the MIC that not affecting on the microbial growth
Effects of ethanolic and aquatic extracts on biofilm formation and biofilm inhibition of S. aureus
aConcentration below the MIC that not affecting on the microbial growth
Ethanolic extract of G. glabra roots at concentration of 1.56 mg mL1 showed 89.6% of inhibition on biofilm formation followed by aquatic extract of L. siceraria peels and L. nobili with 89 and 88.5% inhibition on P. aeruginosa biofilm formation, respectively (Table 4).
Aquatic extract of G. glabra roots at concentration of 3.12 mg mL1 showed 87.5% inhibition of P. aeruginosa biofilm formation while ethanolic extracts of L. nobili at concentration of 6.25 mg mL1 showed 86.8% of inhibition followed by M. domestica peels aquatic extract that showed 86.7% inhibition at 25 mg mL1 (Table 4).
Ethanolic extract of M. officinalis at concentration of 1.56 mg mL1 showed 84.3% inhibition of P. aeruginosa biofilm formation while aquatic extracts of M. officinalis showed 83.2% inhibition of P. aeruginosa biofilm formation at concentration of 12.5 mg mL1. Also, ethanolic extract of L. siceraria peels at concentration of 3.12 mg mL1 showed 81.5% inhibition on biofilm formation of P. aeruginosa.
Plant extracts activity on biofilm formation of S. aureus: The five plant extracts used in our study reduced S. aureus biofilm formation by ≥86%. Our results showed that the ethanolic extract of M. domestica peels at concentration of 25 mg mL1 showed 90% inhibition of S. aureus biofilm formation while the aquatic extracts of L. nobili at concentration of 12.5 mg mL showed 86.7% inhibition followed by aquatic extracts of L. siceraria peels that showed 83.1% inhibition of S. aureus biofilm formation (Table 5).
Aquatic extracts of G. glabra roots at concentration of 12.5 mg mL1 showed 82.9% inhibition of S. aureus biofilm formation while ethanolic extracts of L. siceraria peels at concentration of 12.5 mg mL1 showed 82% inhibition (Table 5).
L. nobili ethanolic extracts concentration of 25 mg mL1 showed inhibition 81.4% followed by aquatic extract of M. domestica peels that showed inhibition of 80.6% of S. aureus biofilm formation at 25 mg mL1 (Table 5).
Aquatic extract of M. officinalis at concentration of 25 mg mL1 showed 78.3% inhibition of S. aureus biofilm formation followed by ethanolic extract of M. domestica peels that showed 77.5% inhibition at 25 mg mL1 (Table 5).
Ethanolic extract of G. glabra and M. officinalis showed 77.1% inhibition of S. aureus biofilm formation at different concentrations (Table 5).
Bacterial infectious diseases still represent an important cause of morbidity and mortality among humans worldwide21. For a long period of time, it has been found that many compounds found in herbs have antimicrobial activities and are an important source of treatment for pathogenic microbes8,22,23. Therefore, particular attention is oriented nowadays to use these compounds in control of some human pathogenic microorganisms especially multidrug resistance strains i.e. P. aeruginosa and S. aureus.
Although, antibiotics are one of the most successful forms in combating bacterial infections, frequent use and overprescribing them has resulted in the development of resistant bacteria and made them less effective or useless at all24. Therefore, increased attention has been paid to the development of antimicrobial agents to treat bacterial infections.
The results of the current study showed that well diffusion method has higher activities than disc diffusion method which could be because the paper disk which was saturated with plant extracts may retain the active component and was not allowed to diffuse into the MHA. MIC was selected to test the antimicrobial activities of plant extracts by micro broth dilution method because it provides quantitative results and is considered as the most appropriate and reliable method25.
All extracts showed low MIC value which is in agreement with Adwan and Mhanna26 and the five plant extracts that used in the presentstudy reduced P. aeruginosa biofilm formation by ≤90% and reduced S. aureus biofilm formation by ≥86%. Biofilm formed by P. aeruginosa was more sensitive against plant extracts than biofilm formed by S. aureus, which could be due to the difference in the structure of the bacterial cell wall14,27. The results of this study showed the effectiveness of plant extracts in inhibiting the formation of P. aeruginosa and S. aureus biofilm.
Our results agree with the findings of Chakotiya et al.28, who reported that the G. glabra roots extract has a significant inhibitory effect on biofilm formation against P. aeruginosa and correlated with the effectiveness of the extract to its active compounds such as glycyrrhizic acid.
The crystal violet method was used to test the activity of the plant extracts and biofilm formation17. In biofilm assay, it was noticed that the presence of glucose in broth media stimulated the formation of biofilms29. Also, the flat-bottomed 96-well plate for the bacterial adhesion was an essential step for biofilm formation30. Extract concentrations of Sub-PMIC50 were used for anti-biofilm assay to ensure that the used extract was not affecting the microbial growth. These results revealed the importance of the tested extracts in the control of common human pathogenic micro-organisms.
This study suggested that five plant extracts (Glycyrrhiza glabra roots, Laurus nobili, Malus domestica peels, Melissa officinalis and Lagenaria siceraria peels) may contain potential source of: natural antimicrobial components that may be of great use for the development of new therapies against E. coli, S. aureus, P. aeruginosa and K. pneumonia and the antibiofilm activities of these plant extracts against the biofilm formed by S. aureus and P. aeruginosa. All plant extracts used in this study contain potential antimicrobial and antibiofilm components that may be of great use for the development of therapies against common infectious bacterial isolates.
This study discovered the importance of the tested extracts in the control of common human pathogenic micro-organisms. Plant extracts used in this study may contain potential antimicrobial and antibiofilm components that may be of great use for the development of new therapies against the most common infectious bacterial isolates.
The authors are grateful to Department of biology and Biotechnology, Islamic University and Medical Technology Department of Israa University Gazafor providing excellent research facilities and El-Shifa Hospital for providing clinical bacteria strains.