Articles

Inhibition of Quorum Sensing Regulated Virulence Factors and Biofilm Formation by Eucalyptus globulus against Multidrug-Resistant Pseudomonas aeruginosa
1Department of Biotechnology, Bundelkhand University, Jhansi (U.P.), India
2Department of Zoology, Indira Gandhi National Tribal University (A Central University), Amarkantak, Madhya Pradesh, India
3Department of Horticulture, Aromatic and Medicinal Plants, Mizoram University (A Central University), Aizawl, Mizoram, India
Correspondence to:Rambir Singh
Department of Horticulture, Aromatic and Medicinal Plants, Mizoram University (A Central University), Aizawl, Mizoram 796004, India
Tel: +91-947-358-3251
E-mail: rambirsehrawat@gmail.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Pharmacopuncture 2022; 25(1): 37-45
Published March 31, 2022 https://doi.org/10.3831/KPI.2022.25.1.37
Copyright © The Korean Pharmacopuncture Institute.
Abstract
Methods: The preliminary anti-quorum–sensing (AQS) activity of eucalyptus was investigated against a biosensor strain Chromobacterium violaceum ATCC 12472 (CV12472) by using the agar well diffusion method. The effect of sub-minimum inhibitory concentrations (sub-MICs) of the methanol extract of eucalyptus on different quorum-sensing–regulated virulence factors, such as swarming motility, pyocyanin pigment, exopolysaccharide (EPS), and biofilm formation, against clinical isolates (CIs 2, 3, and 4) and reference PA01 of Pseudomonas aeruginosa were determined using the swarm diameter (mm)-measurement method, chloroform extraction method, phenol (5%)-sulphuric acid (concentrated) method, and the microtiter plate assay respectively, and the inhibition (%) in formation were calculated.
Results: The preliminary AQS activity (violacein pigment inhibition) of eucalyptus was confirmed against Chromobacterium violaceum ATCC 12472 (CV12472). The eucalyptus extract also showed concentration-dependent inhibition (%) of swarming motility, pyocyanin pigment, EPS, and biofilm formation in different CIs and PA01 of P. aeruginosa.
Conclusion: Our results revealed the effectiveness of the E. globulus extract for the regulation of quorum-sensing–dependent virulence factors and biofilm formation at a reduced dose (sub-MICs) and suggest that E. globulus may be a therapeutic agent for curing and controlling bacterial infection and thereby reducing the possibility of resistance development in pathogenic strains.
Keywords
INTRODUCTION
The increasing emergence of microbial multi-drug resistance has proven to be an obstacle for the effective treatment of microbial infection worldwide. This severe problem has necessitated the development of new therapeutic agents as well as drug targets.
The use of higher doses of antibiotics for growth inhibition and biofilm removal may lead to serious adverse effects in patients and facilitate the emergence of drug resistance in the microorganism. The common mechanisms of drug resistance in
MATERIALS AND METHODS
1. Collection of plant material and extract preparation
Fresh leaves of
2. Bacterial isolates
All the clinical isolates of
3. Determination of antibacterial activity and MIC
The antibacterial activity of the eucalyptus extract against clinical isolates (CIs) and the reference strain of
4. Determination of AQS activity
The AQS activity of the eucalyptus extract was confirmed by showing pigment (violacein) inhibitory activity in CV12472 using the standard agar well diffusion method [29] with slight modification. Briefly, a Mueller–Hinton agar plate with 100 µL of freshly grown and aseptically diluted bacterial suspension with an inoculum size of 2.5 × 106 CFU/mL was prepared. Wells of 6-8 mm diameter were made on the plates by using a sterile cub borer, and the bottom was sealed using soft agar. Next, 50 μL of eucalyptus extract (500 mg/mL) was poured aseptically in the wells. The plates were incubated for 18-20 h at 28℃. DMSO (dimethyl sulfoxide) was used as a negative control. The inhibition zone diameter of pigment inhibition (mm) around the well was measured to determine AQS activity.
5. Inhibition of QS-regulated virulence factors and biofilm formation
1) Assay for swarming motility
The effect on swarming motility was determined using a swarm plate prepared using sterile nutrient broth (8 gm/L) supplemented with 5 gm/L glucose and solidified by bacto agar (0.5%) with sub-MICs of eucalyptus extract (50 μL) [30]. Swarm plates were point-inoculated with freshly grown culture of
The effect of eucalyptus extract on pyocyanin formation in CIs and the reference strain (PA01) of
The bacterial isolates were grown in LB broth containing 150 µL of sub-MIC eucalyptus extract at 37℃ for 18-24 h. After incubation, the bacterial culture was centrifuged, and the supernatant was filtered and transferred to fresh sterile tubes. Three volumes of chilled ethanol (100%) were added, and the tube was incubated at 4℃ for 24 h to precipitate extraction and EPS quantification [32]. Quantification of EPS was performed by measuring the sugar concentration with the phenol (5%) - sulfuric acid (concentrated) method [33]. Percentage inhibition of EPS formation was calculated by determining the absorbance of treated and untreated cultures (as control) at 490 nm.
4) Assay for biofilmThe effects of sub-MICs of the eucalyptus extract on biofilm formation were determined by the microtiter plate assay [34], with slight modifications. Briefly, biofilms were developed in the presence of eucalyptus extract (at different sub-MICs) at 37℃ for 18-24 h. The wells were drained properly the next day and washed with sterile PBS (phosphate-buffered saline) three times to remove planktonic cells. For biofilm staining, 150 µL of crystal violet solution (0.1%) was poured into the wells and plate was incubated at room temperature for 5-10 min. After incubation, excess dye was removed by washing with sterile PBS, and the plate was kept at room temperature for air drying. Next, 150 µL of glacial acetic acid (33% v/v) was added to wells to solubilize the dye taken in by the cells in the biofilm. Percentage inhibition in biofilm formation was calculated by determining the absorbance of treated and untreated culture (as control) at 570 nm.
6. Statistical analysis
Experiments were performed in triplicate, and mean ± standard deviation was calculated. The data were statistically analyzed with one-way ANOVA, and the level of significance was expressed as the p value (p < 0.001, p < 0.005, p < 0.01 and p < 0.05 were denoted with ****, ***, **, and *, respectively) when inhibition in PA01 was compared to those in clinical isolates. Comparison of inhibition in the reference strain/clinical isolates by sub-MICs was made using Tukey’s HSD post-hoc test, and the mean difference was expressed as significant at the 0.05 level. Values sharing a common letter (a, b, c) were not significant (at p < 0.05) when comparisons were made between percentage inhibition (%) of strain/clinical isolates by sub-MICs. Statistical analyses were performed in IBM SPSS statistics 20 (Version 20.0. Armonk, NY: IBM Corporation).
RESULTS
In preliminary evaluation of the QS-inhibitory activity of eucalyptus, the eucalyptus extract showed significant inhibition of violacein pigment formation in CV12472 (Fig. 1). The effects of the eucalyptus extract on the growth of PA01 and CIs 2, 3, and 4 at its sub-MICs (1/2 MIC, 1/4 MIC, 1/8 MIC) were determined using spectroscopic growth curve analysis, and the results showed that the growth of the reference strain/isolates was not affected at sub-MICs (data not shown). A significant (p < 0.05) concentration-dependent reduction in swarming motility was observed in PA01 and CIs 2 and 4 at all tested sub-MIC levels (1/2, 1/4, 1/8 MIC), while the reduction in CI 3 was slightly similar at both 1/4 and 1/8 MIC. The reduction in swarming motility in PA01 (approximately 44%) was significantly higher than those in CIs 2, 3, and 4 (approximately 38%, 28%, and 35%, respectively, at 1/2 MIC; Fig. 2). At 1/2 MIC, the maximum and significantly higher inhibition of pyocyanin formation (%) was observed in CI 3 (p < 0.05) and CI 4 (p < 0.01) than in PA01, while inhibition (%) at 1/4 MIC was significantly higher in PA01 than in CIs 2 and 3 but lower than that in CI 4. At 1/4 MIC and 1/8 MIC, a significant concentration-dependent pyocyanin inhibition (%) was found in PA01 and CI 4 while inhibition was slightly similar in both the CI 2 and CI 3 (Fig. 2). A significant concentration-dependent inhibition (%) in EPS formation was found in all of the tested CIs and the reference strain (PA01) of
DISCUSSION
Over the last several decades, plant-based components have been traditionally used as medicines for curing and controlling microbial infections and diseases. Previous research studies have reported the AQS activity of plant materials [35-40]. The methanol extract of eucalyptus leaves was prepared because this solvent allows easy degradation of the cell wall and releases a range of phytoconstituents from the plant cells because of its polar nature [41, 42]. In the preliminary step of research, the growth inhibitory activity and MIC of methanol extract of eucalyptus leaves against the CIs and reference strain (PA01) of
Thus, reduction in EPS formation may be beneficial to control biofilm formation and the emergence of antibiotic resistance. In the present study, a significant reduction in biofilm formation as well as EPS was observed in CIs and the reference strain PA01 and as a result, the chances of resistance development in
CONCLUSION
Because of its AQS potential at a low dose,
ACKNOWLEDGMENTS
The authors would like to thank the Central Council for Research in Ayurvedic Sciences-Regional Ayurveda Research Institute, Jhansi (Uttar Pradesh), India for providing and authenticating the
CONFLICT OF INTEREST
There are no conflicts of interest.
FUNDING
Nil.
Figures




References
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Related articles in JoP

Article
Original Article
J Pharmacopuncture 2022; 25(1): 37-45
Published online March 31, 2022 https://doi.org/10.3831/KPI.2022.25.1.37
Copyright © The Korean Pharmacopuncture Institute.
Inhibition of Quorum Sensing Regulated Virulence Factors and Biofilm Formation by Eucalyptus globulus against Multidrug-Resistant Pseudomonas aeruginosa
Pankaj Kumar Sagar1 , Poonam Sharma2
, Rambir Singh3*
1Department of Biotechnology, Bundelkhand University, Jhansi (U.P.), India
2Department of Zoology, Indira Gandhi National Tribal University (A Central University), Amarkantak, Madhya Pradesh, India
3Department of Horticulture, Aromatic and Medicinal Plants, Mizoram University (A Central University), Aizawl, Mizoram, India
Correspondence to:Rambir Singh
Department of Horticulture, Aromatic and Medicinal Plants, Mizoram University (A Central University), Aizawl, Mizoram 796004, India
Tel: +91-947-358-3251
E-mail: rambirsehrawat@gmail.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Objectives: The quorum-sensing–inhibitory and anti-biofilm activities of the methanol extract of E. globulus leaves were determined against clinically isolated multidrug-resistant Pseudomonas aeruginosa.
Methods: The preliminary anti-quorum–sensing (AQS) activity of eucalyptus was investigated against a biosensor strain Chromobacterium violaceum ATCC 12472 (CV12472) by using the agar well diffusion method. The effect of sub-minimum inhibitory concentrations (sub-MICs) of the methanol extract of eucalyptus on different quorum-sensing–regulated virulence factors, such as swarming motility, pyocyanin pigment, exopolysaccharide (EPS), and biofilm formation, against clinical isolates (CIs 2, 3, and 4) and reference PA01 of Pseudomonas aeruginosa were determined using the swarm diameter (mm)-measurement method, chloroform extraction method, phenol (5%)-sulphuric acid (concentrated) method, and the microtiter plate assay respectively, and the inhibition (%) in formation were calculated.
Results: The preliminary AQS activity (violacein pigment inhibition) of eucalyptus was confirmed against Chromobacterium violaceum ATCC 12472 (CV12472). The eucalyptus extract also showed concentration-dependent inhibition (%) of swarming motility, pyocyanin pigment, EPS, and biofilm formation in different CIs and PA01 of P. aeruginosa.
Conclusion: Our results revealed the effectiveness of the E. globulus extract for the regulation of quorum-sensing–dependent virulence factors and biofilm formation at a reduced dose (sub-MICs) and suggest that E. globulus may be a therapeutic agent for curing and controlling bacterial infection and thereby reducing the possibility of resistance development in pathogenic strains.
Keywords: anti-quorum sensing (AQS) activity, eucalyptus (Eucalyptus globulus), exopolysaccharide (EPS), minimum inhibitory concentration (MIC), multidrug-resistant, Pseudomonas aeruginosa
INTRODUCTION
The increasing emergence of microbial multi-drug resistance has proven to be an obstacle for the effective treatment of microbial infection worldwide. This severe problem has necessitated the development of new therapeutic agents as well as drug targets.
The use of higher doses of antibiotics for growth inhibition and biofilm removal may lead to serious adverse effects in patients and facilitate the emergence of drug resistance in the microorganism. The common mechanisms of drug resistance in
MATERIALS AND METHODS
1. Collection of plant material and extract preparation
Fresh leaves of
2. Bacterial isolates
All the clinical isolates of
3. Determination of antibacterial activity and MIC
The antibacterial activity of the eucalyptus extract against clinical isolates (CIs) and the reference strain of
4. Determination of AQS activity
The AQS activity of the eucalyptus extract was confirmed by showing pigment (violacein) inhibitory activity in CV12472 using the standard agar well diffusion method [29] with slight modification. Briefly, a Mueller–Hinton agar plate with 100 µL of freshly grown and aseptically diluted bacterial suspension with an inoculum size of 2.5 × 106 CFU/mL was prepared. Wells of 6-8 mm diameter were made on the plates by using a sterile cub borer, and the bottom was sealed using soft agar. Next, 50 μL of eucalyptus extract (500 mg/mL) was poured aseptically in the wells. The plates were incubated for 18-20 h at 28℃. DMSO (dimethyl sulfoxide) was used as a negative control. The inhibition zone diameter of pigment inhibition (mm) around the well was measured to determine AQS activity.
5. Inhibition of QS-regulated virulence factors and biofilm formation
1) Assay for swarming motility
The effect on swarming motility was determined using a swarm plate prepared using sterile nutrient broth (8 gm/L) supplemented with 5 gm/L glucose and solidified by bacto agar (0.5%) with sub-MICs of eucalyptus extract (50 μL) [30]. Swarm plates were point-inoculated with freshly grown culture of
The effect of eucalyptus extract on pyocyanin formation in CIs and the reference strain (PA01) of
The bacterial isolates were grown in LB broth containing 150 µL of sub-MIC eucalyptus extract at 37℃ for 18-24 h. After incubation, the bacterial culture was centrifuged, and the supernatant was filtered and transferred to fresh sterile tubes. Three volumes of chilled ethanol (100%) were added, and the tube was incubated at 4℃ for 24 h to precipitate extraction and EPS quantification [32]. Quantification of EPS was performed by measuring the sugar concentration with the phenol (5%) - sulfuric acid (concentrated) method [33]. Percentage inhibition of EPS formation was calculated by determining the absorbance of treated and untreated cultures (as control) at 490 nm.
4) Assay for biofilmThe effects of sub-MICs of the eucalyptus extract on biofilm formation were determined by the microtiter plate assay [34], with slight modifications. Briefly, biofilms were developed in the presence of eucalyptus extract (at different sub-MICs) at 37℃ for 18-24 h. The wells were drained properly the next day and washed with sterile PBS (phosphate-buffered saline) three times to remove planktonic cells. For biofilm staining, 150 µL of crystal violet solution (0.1%) was poured into the wells and plate was incubated at room temperature for 5-10 min. After incubation, excess dye was removed by washing with sterile PBS, and the plate was kept at room temperature for air drying. Next, 150 µL of glacial acetic acid (33% v/v) was added to wells to solubilize the dye taken in by the cells in the biofilm. Percentage inhibition in biofilm formation was calculated by determining the absorbance of treated and untreated culture (as control) at 570 nm.
6. Statistical analysis
Experiments were performed in triplicate, and mean ± standard deviation was calculated. The data were statistically analyzed with one-way ANOVA, and the level of significance was expressed as the p value (p < 0.001, p < 0.005, p < 0.01 and p < 0.05 were denoted with ****, ***, **, and *, respectively) when inhibition in PA01 was compared to those in clinical isolates. Comparison of inhibition in the reference strain/clinical isolates by sub-MICs was made using Tukey’s HSD post-hoc test, and the mean difference was expressed as significant at the 0.05 level. Values sharing a common letter (a, b, c) were not significant (at p < 0.05) when comparisons were made between percentage inhibition (%) of strain/clinical isolates by sub-MICs. Statistical analyses were performed in IBM SPSS statistics 20 (Version 20.0. Armonk, NY: IBM Corporation).
RESULTS
In preliminary evaluation of the QS-inhibitory activity of eucalyptus, the eucalyptus extract showed significant inhibition of violacein pigment formation in CV12472 (Fig. 1). The effects of the eucalyptus extract on the growth of PA01 and CIs 2, 3, and 4 at its sub-MICs (1/2 MIC, 1/4 MIC, 1/8 MIC) were determined using spectroscopic growth curve analysis, and the results showed that the growth of the reference strain/isolates was not affected at sub-MICs (data not shown). A significant (p < 0.05) concentration-dependent reduction in swarming motility was observed in PA01 and CIs 2 and 4 at all tested sub-MIC levels (1/2, 1/4, 1/8 MIC), while the reduction in CI 3 was slightly similar at both 1/4 and 1/8 MIC. The reduction in swarming motility in PA01 (approximately 44%) was significantly higher than those in CIs 2, 3, and 4 (approximately 38%, 28%, and 35%, respectively, at 1/2 MIC; Fig. 2). At 1/2 MIC, the maximum and significantly higher inhibition of pyocyanin formation (%) was observed in CI 3 (p < 0.05) and CI 4 (p < 0.01) than in PA01, while inhibition (%) at 1/4 MIC was significantly higher in PA01 than in CIs 2 and 3 but lower than that in CI 4. At 1/4 MIC and 1/8 MIC, a significant concentration-dependent pyocyanin inhibition (%) was found in PA01 and CI 4 while inhibition was slightly similar in both the CI 2 and CI 3 (Fig. 2). A significant concentration-dependent inhibition (%) in EPS formation was found in all of the tested CIs and the reference strain (PA01) of
-
Figure 1. Anti-quorum sensing (violacein pigment inhibition) activity of the methanol extract of eucalyptus against
C. violaceum CV12472. (A: methanol extract ofEucalyptus globulus ; B: negative control).
-
Figure 2. Inhibition of the formation of various virulence factors (A), swarming motility (B), pyocyanin formation (C), and exopolysaccharide (EPS) formation by sub-MICs of eucalyptus extract in the reference PA01 strain and the clinical isolates (CI 2, CI 3, and CI 4) of
P. aeruginosa , and comparison of the reference strain PA01 to the clinical isolates. Each bar represents the mean value of three independent replicates, and the error bar shows the standard deviation. ****p < 0.001, ***p < 0.005, **p < 0.01, *p < 0.05, NS: not significant when the reference PA01 strain was compared to CI 2, 3, and 4. Values sharing a common letter (a, b, c) were not significant (at p < 0.05) when comparison was made between inhibitions by sub-MICs with in strain/clinical isolates.
-
Figure 3. Inhibition of biofilm formation. (A) OD at 570 nm. (B) Percent inhibition (%) by sub-MICs of eucalyptus extract in the reference strain PA01 and clinical isolates (CI 2, CI 3 and CI 4) of
P. aeruginosa . Each bar represents the mean value of three independent replicates, and the error bar shows the standard deviation. ****p < 0.001, ***p < 0.005, **p < 0.01, *p < 0.05, NS: not significant. Values sharing a common letter (a, b, c) were not significant (at p < 0.05) when comparison was made between percent inhibitions (%) by sub-MICs within strain/clinical isolates.
DISCUSSION
Over the last several decades, plant-based components have been traditionally used as medicines for curing and controlling microbial infections and diseases. Previous research studies have reported the AQS activity of plant materials [35-40]. The methanol extract of eucalyptus leaves was prepared because this solvent allows easy degradation of the cell wall and releases a range of phytoconstituents from the plant cells because of its polar nature [41, 42]. In the preliminary step of research, the growth inhibitory activity and MIC of methanol extract of eucalyptus leaves against the CIs and reference strain (PA01) of
-
Table 1
MIC and sub-MICs of methanol extract of eucalyptus against clinical isolate (CI) 2, 3, 4 and PA01 of
P. aeruginosa .Strain/Clinical isolates (CIs) MIC and sub-MICs (mg/mL) of methanol extract of Eucalyptus globulus leavesMIC 1/2 MIC 1/4 MIC 1/8 MIC PA01 7.81 3.90 1.95 0.97 CI 2 15.62 7.81 3.90 1.95 CI 3 62.5 31.25 15.62 7.81 CI 4 31.25 15.62 7.81 3.90
-
Figure 4. Inhibition (%) of quorum-sensing–regulated virulence factors and biofilm formation in the (A) reference strain PA01, (B) CI 2, (C) CI 3, and (D) CI 4 of
P. aeruginosa by sub-MICs of the eucalyptus extract. Each bar represents the mean value of three independent replicates, and the error bar shows the standard deviation.
Thus, reduction in EPS formation may be beneficial to control biofilm formation and the emergence of antibiotic resistance. In the present study, a significant reduction in biofilm formation as well as EPS was observed in CIs and the reference strain PA01 and as a result, the chances of resistance development in
CONCLUSION
Because of its AQS potential at a low dose,
ACKNOWLEDGMENTS
The authors would like to thank the Central Council for Research in Ayurvedic Sciences-Regional Ayurveda Research Institute, Jhansi (Uttar Pradesh), India for providing and authenticating the
CONFLICT OF INTEREST
There are no conflicts of interest.
FUNDING
Nil.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

-
Table 1 . MIC and sub-MICs of methanol extract of eucalyptus against clinical isolate (CI) 2, 3, 4 and PA01 of
P. aeruginosa .Strain/Clinical isolates (CIs) MIC and sub-MICs (mg/mL) of methanol extract of Eucalyptus globulus leavesMIC 1/2 MIC 1/4 MIC 1/8 MIC PA01 7.81 3.90 1.95 0.97 CI 2 15.62 7.81 3.90 1.95 CI 3 62.5 31.25 15.62 7.81 CI 4 31.25 15.62 7.81 3.90
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