Effects of Piper betle Leaf Extract on Biofilm and Rhamnolipid Formation of Pseudomonas aeruginosa
Irene Ratridewi*1,2, Shod A. Dzulkarnain3, Andreas B. Wijaya4, John T. R. Huwae4, Daniel S. M. Putra4, Wisnu Barlianto2, Sanarto Santoso3, Dewi Santosaningsih3
1Doctoral Program, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia.
2Department of Pediatrics of Saiful Anwar General Hospital, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia.
3Department of Clinical Microbiology of Saiful Anwar General Hospital, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia.
4Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia.
*Corresponding Author E-mail: irene24.fk@ub.ac.id
High mortality rate and antimicrobial resistance are still becoming world-wide problems, due to Pseudomonas aeruginosa’s (P. aeruginosa) virulence and its ability to form biofilm. Biofilm’s formation is affected by the presence of rhamnolipid, whose production is regulated by quorum sensing systems. Piper betle (P. betle) possesses antimicrobial, antioxidant, anti-inflammatory and immunomodulatory properties. The aim of our study is to investigate the effects of P. betle leaf’s extract against biofilm formation and rhamnolipid production of P. aeruginosa. Active compounds of P. betle were identified using plate chromatography. Agar dilution method was used to determine the minimum biofilm inhibitory concentration (MBIC) of methanolic leaf extract of P. betle. A biofilm-producing P. aeruginosa isolate in the polystyrene plate adherence test was selected for confirmation of biofilm production by Scanning Electron Microscopy (SEM), after P. betle administration. Rhamnolipid detection and evaluation were performed by interpreting halo formed around the well. After administration of various concentrations of P. betle leaf extract on the microplate well, it was concluded that the MBIC of P. betle leaf extract on P. aeruginosa was 0.4%. Methanolic extract of P. betle leaf extract at concentration of 0.4% showed that P. aeruginosa could not form biofilm at all, although the bacteria could still aggregate and form a matrix. After linear regression analysis, beta-coefficient was obtained at -0.931 for P. betle leaf extract. It can be concluded that P. betle leaf extract was effective in inhibiting the growth of biofilm and formation of rhamnolipid by P. aeruginosa. The increase in concentration of P. betle leaf extract was inversely proportional to the diameter of the halo rhamnolipid formed. The higher the level of P. betel leaf extract, the smaller the diameter of the halo rhamnolipid formed.
KEYWORDS: Piper betle leaf extract, Pseudomonas aeruginosa, biofilm, rhamnolipid.
Pseudomonas aeruginosa (P. aeruginosa)
is considered to be the most common
Gram-negative pathogen which responsible for health-care associated infections (HAIs)1,2.
High mortality rate and
antimicrobial resistance are still becoming
world-wide problems3, due to P. aeruginosa’s virulence
and its ability to form biofilm4,5. Biofilm’s
formation is affected by the presence of rhamnolipid, whose production is regulated by quorum sensing
systems (Rhl, Las
and Pqs)6. Aside from being the primary surfactant of the biofilm, rhamnolipid
is proven to have antimicrobial
factors assisting in its competitive growth against
other microbes and increasing its virulence factor7. The inhibition of rhamnolipid production and also quorum sensing system will be helpful to break the antibiotic resistance’s problem8.
Piper betle (P. betle, Indonesian name Sirih hijau), is widely used as traditional medicine in
Indonesia. Studies have shown that P. betle leaf’s extract possesses antimicrobial, antioxidant, anti-inflammatory and immunomodulatory properties9. One of the most abundant compound inside P. betle is
eugenol, which exhibits powerful
antioxidant activity10. Eugenol could supress the expression of P. aeruginosa’s virulence factor such rhamnolipid and also inhibit
biofilm formation10,11.
However, the influence of eugenol against rhamnolipid
has not been elucidated yet. The aim of our study
is to investigate the effects of P. betle leaf’s extract against
biofilm formation and rhamnolipid production
of
P. aeruginosa.
Pseudomonas aeruginosa clinical isolate was obtained from the bacterial culture collection in the Department of Clinical Microbiology, Faculty of Medicine, Brawijaya University and identification was confirmed by using MicrobactTM Gram-negative system12.
The P. betle leafs was collected from Malang, East Java, Indonesia. The species of Piper was identified and confirmed by plant taxonomist of UPTD Materia Medica, Batu, as herbarium unit in East Java, Indonesia. The extraction process was executed in Technical Chemistry Laboratory of State Polytechnic of Malang, Indonesia. The leafs were shade dried and ground into fine powder (30 grams), then macerated in 100 ml absolute methanol for 72 hours by the extraction apparatus. The extract was evaporated to dry at 40 oC by using rotary evaporator and then stored at 4 oC for further use.
Active compounds of P. betle were identified by using plate chromatography. Two microliters of P. betle leaf methanol extracts was separately applied on 5 cm × 10 cm chromatographic precoated silica gel plates, as the stationary phase. As mobile phase, TLC plates were developed in a twin trough glass chamber which containing mixture of ethyl acetate and n-hexane (7 : 3 v/v). When the solvent front has been moved to 15 cm from original position and also allowed to dry, the plates were removed. After drying, the spots on developed plates will be visualized under visible (white), short UV (254nm), and also long UV (366nm) light. Then, the plates were sprayed with vanillin-sulfuric acid for color reaction and allowed to dry. Visualizer and scanner were used for documentation at UV 254 nm and UV 366 nm, also under the visible light before and after application of the vanillin-sulfuric acid spray. The
movement of each separating spot of P. betle extract was expressed by its retention factor (Rf). Values were calculated for each spot using the following formula:
Distance traveled by the solute from the point of application to the center of the spot
Rf =----------------------------------------
Distance traveled by the solvent front
The P. aeruginosa isolates were tested for their ability to form biofilm according to Stepanovic et al17, with some modifications. Selected isolates, grown on tryptone soya agar plates for 24 h, were inoculated into 7 mL tryptone soya broth with 1% glucose (TSBglu) in Falcon tubes. To achieve 0.5 McFarland standard, the turbidity would be adjusted. Five mL of inoculated broth were transferred to new sterile Falcon tubes, for quantitative assessment. As negative controls, uninoculated TSBglu tubes were used. Tubes were incubated for overnight at 37ºC. The content of each tube was carefully aspirated with a pipette, and immediately stained with 2mL of 0.25% safranin for 1 minute. Then, the tubes were decanted and also inverted without washing. After overnight standing at room temperature, the tubes were examined for biofilm formation. It was considered positive when there was stained film or adherent layer on the inner surface of the tube18. The biofilm formation was estimated as negative (0), weak (+), moderate (++), or strong (+++).
Agar dilution method was used to determine the minimum biofilm inhibitory concentration (MBIC) of methanolic leaf extract of P. betle. Bacteria isolates were incubated for 24 hours inside brain heart infusion (BHI) liquid medium, then diluted to achieve 0.5 Mc Farland. Then, the suspension (100µL of suspension with a density of 108) was challenged with P. betle extract and incubated, so that the biofilm formation and MBIC could be determined. Two-fold serial dilutions of the extract (0%, 0.05%, 0.1, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.00%) were prepared in Mueller-Hinton agar. Ten microliters of bacterial inoculum was delivered onto agar with final inoculum of 104 CFU/spot. Bacterial plates were incubated at 37oC and evaluated after 24 h. After incubation, the well is washed with phosphate buffer saline (pH 7.3) and left to dry. Well is stained using crystal violet (1%) and placed upside down to observe the formation of biofilm. The lowest concentration where biofilm formation is not visible is considered as the MBIC of P. betle leaf extract towards
P. aeruginosa19.
A biofilm-producing Pseudomonas aeruginosa isolate in the polystyrene plate adherence test was selected for confirmation of biofilm production by SEM, after P. betle administration. The biofilm-producing strain was first isolated in BHI broth and 108 CFU of bacteria were transferred to a conical tube (Falcon-CORNING) containing 2mL TSB culture medium prepared with 2% glucose and a 0.5-cm segment of VYGON umbilical catheter (reference 1270.04, 0.8mm-1.5 mm diameter). The tube was incubated under constant stirring for 48 h at 100rpm/37oC for bacterial growth and biofilm formation. Then, the catheter segment was removed, washed with PBS, immersed in 2.5% glutaraldehyde solution, fixed in an increasing alcohol series (15, 30, 50, 70, 90, and 100%) for 15 min each, dried in a vacuum centrifuge for 5 min, and also visualized under SEM to prove biofilm production after P. betle administration in various concentration (0%, 0.2%, 0.4%, 0.6%).
Rhamnolipid Detection and Evaluation21,22 Pseudomonas aeruginosa was incubated in liquid BHI for 24 hours. Fifty µL of the incubated suspension with density 106 was inoculated in agar medium containing KH2PO4 0.7g/L, Na2HPO4 0.9g/L, NaNO3 2.0g/l,
MgSO4 0.4g/L, CaCl2 0.1g/L, glycerol 20mL, CTAB 0.2g/L, methylene blue 0.005g/L and bacteriology agar 20g/L at 37°C for 24 hours. After being incubated for 24 hours, the plate will be incubated at room temperature for 7 to 10 days. Halo formed around the well was measured in mm or µm using digital Vernier calliper. To evaluate the effects of P. betle leaf extract against rhamnolipid, determined concentration of extract was placed inside the well of the medium before being inoculated.
Linear regression method was used to assess the correlation between rhamnolipid halo diameter and the concentration of P. betle’s methanolic leaf extract. Negative beta coefficient value indicates negative correlation between the concentration of P. betle ‘s methanolic leaf extract and rhamnolipid halo diameter, while a positive correlation is indicated by positive beta coefficient value. The correlation relationship is considered good if the value is close to 1.
Piper betle Analysis – Thin Layer Chromatography
Table 1. Phytochemical Compounds from Methanolic Leaf Extract of Piper betle
|
Rf |
Active Compound |
Effects |
|
0.057 |
Unidentified |
Unknown |
|
0.144 |
Linalyl acetate |
Decongestant |
|
0.227 |
Borneol |
Anti-microbial and anti- inflammation |
|
0.556 |
Eugenol |
Antiseptic and anesthesia |
|
0.709 |
Methyl acetate |
Aromatic compound |
|
0.788 |
Anethole |
Insecticide and anti-microbial |
|
0.877 |
Safrole |
Beverage and candy material |
|
0.921 |
Azulene |
Marker for ROS, biomarker in several laboratory test |
To separate multiple compounds of methanolic extract of
P. betle leaf, thin layer chromatography (TLC) was performed. Mobile phase used by Zarzycki et al was applied, and to increase its relative polarity, its mobile phase was also modified. Relative polarity of mobile phase was qualitatively estimated by considering the solubility parameters of correspondent pure solvents23. When relative amount of the solvents was kept constant by using a solvent with
higher solubility parameter, it will produce binary solvent with higher polarity. Polarity of binary system will increase as the relative content of the solvent with higher value increases. Representative photographs of the TLC strips developed with indicated mobile phases are shown in Figure 1 and also their respective retention factors in Table 1.
Figure 1. Representative photographs of thin layer chromatography for a methanolic extract of Piper betle, developed with the indicated mobile phases and visualized under visible light (VL) or under ultraviolet light (UVL 254 and 366)
After administration of different concentrations of Piper betle leaf extract on the microplate well, it was concluded that the MBIC of Piper betle leaf extract on
P. aeruginosa is 0.4%.
Figure 2. Effects of Various Concentration of Piper betle Leaf Extract Against Pseudomonas aeruginosa’s Biofilm Formation or Mean Biofilm Inhibitory Concentration. No 1: 0%; 2: 0.01%; 3:
0.05%; 4: 0.1%; 5: 0.2%; 6: 0.3%; 7: 0.4%; 8: 0.5%; 9: 0.6%; 10:
0.7%; 11:0.8%; 12: 0.9%; 13: 1% (A Black and white format; B original format)
Biofilm visualization by using Scanning Electron Microscope (SEM) showed that without administration of P. betle leaf extract, P. aeruginosa bacteria can form a solid and complex biofilm. If the concentration of 0.2%
P. betle leaf extract was added to bacterial growth medium, it was able to interfere the formation of P. aeruginosa biofilms. As result, the biofilm matrix became not solid and had damage in several parts. Methanolic extract of P. betle leaf extract at concentration of 0.4% showed that P. aeruginosa could not form biofilms at all, although the bacteria could still aggregate and form matrix. A concentration of 0.6% indicated that P. aeruginosa has lost the ability to form biofilm constituent matrix and most bacteria were unable to form aggregates. This finding showed that P. betle leaf extract will inhibit the formation of biofilms not through a bactericidal pathway to planktonic cells24 (Figure 3).
Figure 3. The Effect of Various Concentration P. betle Leaf Methanolic Extract on the Morphology of P. aeruginosa Biofilm
Table 2. Rhamnolipid Halo Diameter After Methanolic Leaf Extract of Piper betle Administration
|
Concentration of Piper betle Leaf Extract [%] |
Rhamnolipid Halo Diameter (mm) |
Standard Deviation |
|
[0%] |
13.4 |
0.3 |
|
[0.05%] |
13.4 |
0.5 |
|
[0.1%} |
13.0 |
0.5 |
|
[0.2%] |
12.9 |
0.4 |
|
[0.3%] |
12.2 |
0.3 |
|
[0.4%] |
12.0 |
0.4 |
|
[0.5%] |
11.8 |
0.3 |
|
[0.6%] |
11.7 |
0.1 |
|
[0.7%] |
11.3 |
0.1 |
|
[0.8%] |
10.2 |
0.6 |
After administration of the highest concentration used in this study, it was observed that rhamnolipid was still formed by the bacteria colony. A change in the frequency or concentration may give a different result (Table 2). After data analysis with linear regression method, a beta-coefficient was obtained at -0.931 for P. betle leaf extract (p< 0.05). It can be concluded that P. betle leaf extract was effective in inhibiting the growth of biofilm and formation of rhamnolipid by P. aeruginosa. Further studies should be done to investigate the effects of P. betle leaf extract on live animals up to clinical trials as the extract has the potential to be used as medication in treating P. aeruginosa infection8,,25. The increase in concentration of P. betle leaf extract was inversely proportional to the diameter of the halo rhamnolipid formed. The higher the level of P. betel leaf extract, the smaller the diameter of the halo rhamnolipid (Figure 4).
(A) (B)
Figure 4. The Effect of Various Concentration of Methanolic Extract (%) of P. betle Leaf on the Formation Of Rhamnolipid in Pseudomonas aeruginosa (14150) 1: negative control; 2: positive
control or pure strain; 3: 0%; 4: 0.05%; 5: 0.1%; 6: 0.2%; 7:
0.3%; 8: 0.4%; 9: 0.5%; 10: 0.6%; 11: 0.7%; 12: 0.8%. (A)
Original format (B) Black and white format for easy measurement of halo rhamnolipid diameters
The mean biofilm inhibitory concentration of Piper betle leaf extract on Pseudomonas aeruginosa is 0.4%. It was showed that Pseudomonas aeruginosa could not form biofilms at all after the administration of Piper betle leaf extract (0.4%), although the bacteria could still aggregate and form a matrix. Piper betle leaf extract is effective in inhibiting the growth of biofilm and formation of rhamnolipid by Pseudomonas aeruginosa. The increase in concentration of P. betle leaf extract was inversely proportional to the diameter of the halo rhamnolipid formed. The higher the level of Piper betel leaf extract, the smaller the diameter of the halo rhamnolipid.
The authors declare no conflict of interest.
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Received on 22.10.2020 Modified on 29.11.2020
Accepted on 05.01.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5182-5186.
DOI: 10.52711/0974-360X.2021.00901