Inhibition of quorum sensing regulated bacterial functions by plant essential oils with special reference to clove oil

Authors


Iqbal Ahmad, Associate Professor, Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, India. E-mail: iqbalahmad8@yahoo.co.in

Abstract

Aims:  To evaluate quorum sensing (QS) inhibitory activity of plant essential oils using strains of Chromobacterium violaceum (CV12472 and CVO26) and Pseudomonas aeruginosa (PAO1).

Methods and Results:  Inhibition of QS-controlled violacein production in C. violaceum was assayed using disc diffusion and agar well diffusion method. Of the 21 essential oils, four oils showed varying levels of anti-QS activity. Syzygium aromaticum (Clove) oil showed promising anti-QS activity on both wild and mutant strains with zones of pigment inhibition 19 and 17 mm, respectively, followed by activity in cinnamon, lavender and peppermint oils. The effect of clove oil on the extent of violacein production was estimated photometrically and found to be concentration dependent. At sub-MICs of clove oil, 78·4% reduction in violacein production over control and up to 78% reduction in swarming motility in PAO1 over control were recorded. Gas chromatography–mass spectrometry analysis of clove oil indicated presence of many phytocompounds. Eugenol, the major constituent of clove oil could not exhibit anti-QS activity.

Conclusions:  Presence of anti-QS activity in clove oil and other essential oils has indicated new anti-infective activity. The identification of anti-QS phytoconstituents is needed to assess the mechanism of action against both C. violaceum and Ps. aeruginosa.

Significance and Impact of the study:  Essential oils having new antipathogenic drugs principle because of its anti-QS activity might be important in reducing virulence and pathogenicity of drug-resistant bacteria in vivo.

Introduction

Quorum sensing (QS) is a mechanism by which bacterial population measures its cell density. This cell-to-cell communication is dependent on synthesis, exchange and perception of small signal molecules between bacteria. QS signals typically activate specific receptors that function as transcriptional regulators. In many Gram-negative proteobacteria, the QS signals are small diffusible compounds called N-acyl-homoserine lactones (AHLs). As the cell density of a bacterial population increases and constitutively produced AHLs reach the threshold concentration, the transcriptional regulator detects the presence of AHLs and induces expression of gene sets (Whitehead et al. 2001).

A wide spectrum of density-dependent multicellular behaviour in several bacteria have been reported such as bioluminescence, pigment production, conjugation, antibiotic production, expression of virulence factors, biofilm formation and many degradative enzymes in animal, fish and plant pathogens. Various pathogenic bacteria such as Pseudomonas aeruginosa, Vibrio ssp., Burkholderia cepacia and Yersinia enterocolitica employed QS to regulate their virulence and pathogenicity (Fuqua et al. 1996; Swift et al. 1996; Ahmad et al. 2008).

One of the major problems of antibiotic therapy is the emergence of drug-resistant bacteria both in hospital and in community-acquired infection. In view of the slow progress in developments of new antibiotics with novel mode of actions, new strategies have been suggested to combat bacterial infections including inhibition of QS (Fernandes 2006; Ahmad et al. 2009). Inhibition of QS systems regulating the expression of virulence factors as well as biofilm formation is a highly attractive target for developing novel therapeutics (Hentzer and Givskov 2003). The discovery of QS inhibition by halogenated furanones from Australian macroalgae, Delisea pulchra has generated interest among scientific community to evaluate various natural and synthetic compounds for anti-QS property (Manefield et al. 1999). However, most of the characterized anti-QS compounds have not yet qualified as chemotherapeutic agents because of its toxicity, high reactivity and instability. Therefore, there is an increasing need for discovery of new anti-QS compounds. In the recent years, some reports have been published mainly from weeds, foods and medicinal plant extracts (Cumberbatch 2002;Gao et al. 2003; Sameena 2006; Vattem et al. 2007; Allison et al. 2008). Essential oils derived from medicinal and food plants are known for their application in traditional medicine, in food industry and in a number of therapeutic uses (Bakkali et al. 2007). However, the plant essential oils, rich diverse source of bioactive compounds, have not yet been subjected to systematic scrutiny for their anti-QS activity. To the best of our knowledge, there is only one report on cinnamaldehyde exhibiting anti-QS activity (Niu et al. 2006).

Considering the multiple therapeutic properties of essential oils and wide use in infectious diseases both in modern and in traditional medicines, we have made an attempt to screen 21 commonly available essential oils for their anti-QS activity using biosensor strains, Chromobacterium violaceum CV12472 and CVO26. The anti-QS activity of four essential oils is reported here for the first time. Interestingly, most promising anti-QS activity against both C. violaceum pigment production and swarming motility of Ps. aeruginosa was demonstrated by clove oil at sub-MIC.

Materials and methods

Plant essential oils

Essential oils were obtained from different sources like Wyndmere Naturals, USA; Himalaya Drug Co., Dehradun, India; Hi-Media Laboratory, Mumbai, India; and Aroma Sales Corporation, New Delhi, India.

Bacterial strains and culture medium

The strains of C. violaceum CV12472, CVO26 and CV31532 and Ps. aeruginosa PAO1 were kindly provided by Professor Robert J.C. McLean, Texas State University, USA. Unless otherwise stated, all strains were grown in or on LB (Luria–Bertani) broth (15·0 g tryptone, 0·5% yeast extract, 0·5% NaCl) solidified with 1·5% agar (Hi-media) at 28°C when required and supplemented with appropriate antibiotic (kanamycin 20 μg ml−1 for C. violaceum CVO26).

Determination of MIC

MIC of essential oils was determined against biosensor strains (CV12472, CVO26 and PAO1) by broth macrodilution method. MIC is defined as the minimum concentration of essential oils at which there was no visible growth of test strain. Sub-MICs were selected for the assessment of anti-QS activity in the above-mentioned strains.

Extraction of natural C6-AHL from Chromobacterium violaceum CV31532

The method by Shaw et al. (1997) was adopted for isolation of AHL. Briefly, C. violaceum CV31532, a C6-AHL over-producing strain, was grown in 4 l Luria broth on shaking incubator at 28°C for 18 h. The culture was centrifuged at 12 000 g for 10 min, and the supernatant obtained was sterilized by membrane filtration (0·22 μm). The filtrate obtained was extracted with acidified ethyl acetate (0·1% v/v acetic acid) (supernatant/acidified ethyl acetate, 7 : 3, v/v) and finally concentrated and dried by rotary evaporator at 40°C and reconstituted in acetonitrile. The extract was stored at 4°C for further use in bioassay with CVO26. The amount of extracted AHL needed for CVO26-based assay was standardized by agar well diffusion plate assay.

Assay for the inhibition of violacein production in Chromobacterium violaceum

Disc diffusion assay was performed with C. violaceum CV12472 to determine the pigment inhibition at the range of sub-MICs of essential oils. Briefly, LB agar plates were spread with 0·1 ml of appropriately diluted (c. 2·5 × 106 CFU ml−1) freshly grown cultures. Sterile discs (7 mm) impregnated with different amounts of essential oils were mounted. Solvent and Luria broth were used as control. Plates were incubated for 18–24 h at 28°C to check the inhibition of pigment production around the disc. Growth inhibition, if any, was also recorded.

Anti-QS activity of essential oils using CVO26 was assayed by agar well diffusion method in the presence of appropriately standardized amount of natural C6-AHL. Briefly, LB agar plates were spread with 0·1 ml of appropriately diluted (c. 2·5 × 106 CFU ml−1) freshly grown cultures and 8-mm diameter wells were cut and varying amounts (20–100 μl) of appropriately diluted essential oils in DMSO were loaded along with natural C6-AHL. Plates were incubated for 18–24 h at 28°C to check the inhibition of pigment production around the well. Growth inhibition, if any, was also recorded.

Quantitative estimation of violacein in the presence and absence of sub-MICs of clove oil

Extent of violacein production by C. violaceum (CV12472) in the presence of clove oil was studied by extracting violacein and quantifying photometrically using the method of Blosser and Gray (2000) with little modifications. Briefly, the twofold serial dilutions of clove oil were made in Luria broth, and 50 μl of freshly grown culture (c. 1·7 × 107 CFU ml−1) was inoculated and incubated at 28°C till complete pigmentation is achieved in blank, i.e. untreated culture. First, 200 μl of treated and untreated cultures was placed in eppendorf tube and lysed by adding 200 μl of 10% SDS and mixing for 5 s with vortex and incubating at room temperature for 5 min. Further, 900 μl of water-saturated butanol (50 ml n-butanol mixed with 10 ml distilled water) was added to cell lysate, vortexing for 5 s and centrifuged at 13 000 g for 5 min. The upper (butanol) phase containing the violacein was collected and the absorbance was read at 585 nm in Spectronic 20 D+ (Hewlett Packard). Reduction in the production of pigment in the presence of oil was measured in terms of percentage inhibition as [(OD of control − OD of treated)/OD of control] ×100.

Concurrently, cell viability of bioreporter strain was determined. Cell densities were measured in treated and untreated samples by agar plate count of culture.

Effect of sub-MIC of clove oil on swarming motility of Pseudomonas aeruginosa (PAO1)

The method described by Vattem et al. 2007 was used in this assay with slight modification. Sub-MICs of clove oil and eugenol were mixed with 0·3% LB agar separately and were poured into plates and point inoculated with PAO1 (c. 1·7 × 107 CFU ml−1) and incubated at 37°C for 48 h. The extent of swarming was determined by measuring the diameter of swarm and compared with control.

Gas chromatography–mass spectrometry (GC–MS) analysis

The clove oil was subjected to GC–MS in order to identify the constituents. GC conditions used were as follows: instrument model was GCD 1800A, Hewlett Packard with HP-1 Column (30 m × 0·25 mm × 0·25 μm; Thermo Scientific); injector and detector temperatures were 250°C and 280°C, respectively; carrier gas used was helium at 1 ml min−1; initial temperature of oven was 100–250°C at the rate of 10°C min−1, hold time at 250°C for 3 min and final temperature was 250–280°C at the rate of 30°C min −1, hold time at 280°C for 2 min; solvent used for dilution was methanol.

Results

MIC of the test essential oils against bio-reporter strains (CV12472, CVO26 and PAO1) ranged from 0·2% to ≥6·4% (v/v) (Table 1). A range of sub-MICs were selected for anti-QS screening using C. violaceum CV12472 strain, while with CVO26 strain, agar well diffusion method was adopted in the presence of natural C6-AHL, produced and extracted from CV31532. Of the 21 essential oils screened, significant inhibition of pigment production was detected in clove oil with 19 and 17 mm zone of pigment inhibition against CV12472 and CVO26 strains, respectively (Figs 1a and 2; Table 1). Inhibition of pigment production was also detected in the cinnamon, peppermint and lavender oil with zone of pigment inhibition ranging from 10–12 mm against CV12472. But this activity was relatively less intense against CVO26. No effect on pigment inhibition was observed by other plant essential oils at tested concentrations. Clove oil, at relatively lower concentration (2 μl) showed no activity (Fig. 1b), but at higher concentration (20 μl) antibacterial activity was observed along with anti-QS activity (zone of inhibition 21 mm) (Fig. 1c). Further, to assess the anti-QS nature of clove oil, against Ps. aeruginosa PAO1, we investigated the effect of sub-MICs of clove oil on swarming motility. We found concentration-dependent reduction in swarming motility of PAO1 by 40%, 56%, 68% and 78% at 0·2, 0·4, 0·8 and 1·6 (% v/v) of clove oil, respectively, after 48 h of incubation (Table 3 and Fig. 3a,b). Clove oil at 3·2% v/v treatment was found to be inhibitory to the growth of PAO1.

Table 1.   Screening of plant essential oils for anti-quorum sensing (anti-QS) activity against Chromobacterium violaceum strains CV12472 and CVO26, as well as antibacterial activity in terms of minimum inhibitory concentration (MIC)
Plant essential oils (common name) MIC against CV12472 (%v/v) MIC against CVO26 (%v/v) MIC against PAO1 (%v/v)Anti-QS activity against CV12472 (inhibition zone size in mm)Anti-QS activity against CVO26 in presence of natural C6-AHL (inhibition zone size in mm)
  1. + Indicates the presence of anti-QS activity; − indicates the absence of anti-QS activity; MIC value is ranged from 0·05% to ≥6·4% (v/v)

Apium graveolens (celery)>6·4>6·4>6·4
Cinnamomum verum (cinnamon)0·20·23·2+ (12)+ (11)
Citrus limon (lemon)1·40·6>6·4
Citrus paradisi (grape)>6·4>6·4>6·4
Citrus sinensis (orange)3·0>6·4>6·4
Cymbopogon citratus (lemongrass)0·40·4>6·4
Cymbopogon martini (palmarosa)0·40·2>6·4
Eucalyptus sp. (eucalyptus)3·02·2>6·4
Foeniculum vulgare (sweet fennel)0·2>6·4>6·4
Lavandula angustifolia (lavender)0·40·4>6·4+ (11)+ (10)
Mentha piperita (peppermint)0·21·0 >6·4+ (11)+ (10)
Myristica fragrans (nutmeg)>6·4>6·4>6·4
Olea europaea (olive)>6·4>6·4>6·4
Petroselinum crispum (parsley)>6·4>6·4>6·4
Rosmarinus officinalis (rosemary)1·8>6·4>6·4
Santalum album (sandalwood)>6·4>6·4>6·4
Syzygium aromaticum (clove)0·20·23·2+ (19)+ (17)
Thymus vulgaris (thyme)0·20·2>6·4
Trachyspermum ammi (ajowan)0·20·2>6·4
Zea mays (corn)>6·4>6·4>6·4
Zingiber officinale (ginger)>6·4>6·4>6·4
Figure 1.

 (a) Anti-quorum sensing (anti-QS) activity by clove oil against bioreporter strain CV12472, using disc diffusion method (a) 12 μl (b) 8 μl (c) 4 μl and; (b) no bioactivity by clove oil at lower concentration (2 μl); (c) antibacterial activity is observed in addition to anti-QS by clove oil at higher concentration (20 μl).

Figure 2.

 Inhibition of violacein production in CVO26 by clove oil (a) control, violacein synthesis by CVO26 in the presence of 100 μl natural N-acyl-homoserine lactones (AHL) (b) reduction in violacein synthesis in the presence of 100 μl natural AHL + 5 μl of oil (c) further reduction in violacein synthesis in the presence of 100 μl natural AHL + 10 μl of oil. Final volume of well was adjusted to 150 μl by adding DMSO.

Table 3.   Concentration-dependent reduction in swarming motility by clove oil in PAO1
Oil concentration (%v/v)Diameter of swarm (mm)Decrease in swarming motility over control (%)
  1. All the experiments were performed in triplicate, and data are presented as mean values.

Control74 
0·24440
0·43356
0·82468
1·61678
Figure 3.

 Reduction in swarming motility in PAO1 by clove oil (a) control (b) 0·8% clove oil treatment.

To ensure that inhibition of violacein production was not because of the antibiotic effect, extraction of violacein in clove-oil-treated and untreated culture of CV12472 was also performed. Violacein production was inhibited by 48·0%, 58·0% and 78·4% at 0·04%, 0·08% and 0·12% oil treatment, respectively, with little or no significant growth inhibition. However, 0·16% oil treatment was found to be inhibitory with 92·6% inhibition in violacein production. Reduction in viable count at 0·16% was significant with untreated culture (Table 2). Therefore, it seems that up to 0·12% treatment, only anti-QS action was detectable and that at 0·16% treatment, growth inhibition was evident.

Table 2.   Concentration-dependent inhibition of violacein by clove oil in CV12472
Oil concentration (%v/v)Pigment productionOD of violacein at 585 nmReduction in the absorbance of violacein (%)Cell viability assay (log CFU ml−1 at 105 dilution)
  1. All the experiments were performed in triplicate, and data are presented as mean at 95% confidence level.

  2. + Indicates visualization of pigment color; ± indicates partial visualization of pigment color.

Control+0·342 8·14
0·04+0·17548·08·12
0·08+0·14158·08·05
0·12+0·07478·48·02
0·16±0·02692·37·29
0·20nilnilnilnil

Major ingredients of clove oil as revealed by GC–MS analysis (Table 4) is eugenol (74·32%), and other constituents identified were α-caryophyllene (4·05%), iso-caryophyllene (5·96%), caryophyllene oxide (2·41%), β-caryophyllene (4·92%), napthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methyl ethyl) (7·04%) and 1,6-Octadiene-ol-,3,7-dimethyl acetate (1·28%). When pure eugenol was tested for anti-QS activity against violacein production (C. violaceum) and on swarming growth (PAO1), no activity was detected.

Table 4.   Components of clove oil as identified by Gas chromatography–mass spectrometry
Peak no.ComponentsRetention timeArea %
1Eugenol6·2874·32
2β-Caryophyllene7·03 4·92
3Iso-caryophyllene7·09 5·96
4Napthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methyl ethyl)7·14 7·04
51,6-Octadiene-ol-,3,7-dimethyl acetate7·34 1·28
6α-Caryophyllene7·40 4·05
7Caryophyllene oxide8·61 2·41

Discussion

A number of bacterial traits including virulence and pathogenicity in many Gram-negative bacteria are QS regulated, the interruption of QS is one example of antipathogenic effects and can render bacteria nonpathogenic (Zhang and Dong 2004). Because of continued emergence and spread of multidrug-resistant bacteria, antipathogenic strategy to combat bacterial infections has received increased attention in recent years. In the present study, the plant essential oils that are commonly used both in modern and in traditional systems of medicine are subjected to anti-QS testing. The screening result of 21 essential oils indicated that clove, cinnamon, peppermint and lavender oils have potential anti-QS activity on C. violaceum pigment production. This is the first report on anti-QS activity of the tested essential oils. However, other workers have reported anti-QS activity of medicinal plant extracts and certain phytocompounds (Vattem et al. 2007). Similarly, anti-QS activity of cinnamaldehyde at sub-MIC value is reported by Niu et al. (2006). Remaining 17 essential oils have not shown any effect on pigment production and such essential oils may be subjected with other bioassays to find possible antipathogenic effect on bacterial strains. As eugenol is a major active compound in clove oil and is well known for its antimicrobial action, we tested eugenol for anti-QS property with bioreporter strains. However, inhibition of violacein production could not be detected, indicating that anti-QS activity is probably because of the other ingredients, like α-caryophyllene and β-caryophyllene, of clove oil either alone or in combination with other ingredients. Further, effect on QS-regulated and nonregulated bacterial virulence and pathogenicity should be tested for therapeutic productive outcome.

Quantitative assessment of pigment inhibition has clearly indicated that anti-QS activity of clove oil is concentration dependent. Because many plant products have antimicrobial action, testing antipathogenic and anti-QS activity at sub-MIC would be a good basis for selection of concentration. Swarming phenomenon in bacteria is considered to be a virulence factor as it is involved in biofilm formation because of mass translocation of cells, and this relies on expression of biosurfactants molecules, the expression of which is under QS control in PAO1 (Daniels et al. 2004). Hence, any compound inhibiting the swarming motility in PAO1 is expected to interfere QS and its regulated traits. In our study, inhibition of swarming motility by clove oil has further strengthened its anti-QS behaviour. Plant essential oils contain a mixture of various active compounds. Majority of essential oils have one or few major constituents and a variety of other minor compounds. Thus, it is difficult to comment on the exact mode of action on QS system. However, neither major constituent like eugenol nor minor constituents like α-caryophyllene and β-caryophyllene share structural similarity with AHLs or known QS inhibitors like halogenated furanones. Common mechanism of QS interference include (i) inhibition of signal biosynthesis or inhibition of activity of AHL-producing enzymes, (ii) enzymatic signal degradation, (iii) inhibition of reception signal molecules. Recent reports have indicated the presence of anti-QS activity in natural products including plants (Vattem et al. 2007; Allison et al. 2008), although the mechanism of action is to be investigated. It is possible that plant essential oils exhibiting anti-QS activity might influence bacterial QS-controlled phenotypes through inhibiting AHL synthesis or through some indirect mechanism but not through degradation of AHL.

It is also possible that the end effect on inhibition of particular QS-linked traits may be the result of multitarget action of various ingredients of essential oils on bacterial QS system. Therefore, the immediate concern should be to identify the compound(s) with anti-QS activity assessing its mode of action and its antipathogenic efficacy in experimental animal model.

Acknowledgements

We gratefully acknowledge the financial support from University Grant Commission, New Delhi, and Sophisticated Analytical Instrument Facility at Indian Institute of Technology, Bombay, India, for conducting GC–MS analysis. We are also thankful to Professor Robert J.C. McLean, Texas State University, USA, for providing QS bioreporter strains.

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