QUORUM QUENCHING ABILITY OF DIETARY SPICE CINNAMOMUM VERUM ON PATHOGENIC BACTERIA

QUORUM QUENCHING ABILITY OF DIETARY SPICE CINNAMOMUM VERUM ON PATHOGENIC BACTERIA

Y.Aparna ^*, Lalitha Narayanan and J. Sarada

Department of Microbiology, Bhavans Vivekananda College of Science, Humanities and Commerce, Sainikpuri, Secunderabad, India

ABSTRACT: Quorum sensing is a bacterial cell communication mechanism mediated   through signal molecules such as AHL’s. Many opportunistic pathogens use these quorum sensing circuits to coordinate their virulence gene expression. Wide range of naturally occurring substances particularly   plant extracts have been evaluated for their ability to modulate quorum sensing in Gram-negative bacteria. Preliminary screening of   popular food spices for QSI activity   revealed that Cinnamomum verum (Dalchini) had good QSI activity. Recognizing its medicinal value and dietary   consumption, an attempt was made to evaluate QSI activity of Cinnamomum verum on bacterial pathogens like Pseudomonas and Serratia and confirmed by using bioindicator organism Chromobacterium violaceum. Cinnamomum verum extract had more QSI activity than the antibacterial activity in Serratia.   In   Pseudomonas, the antibacterial activity was more while QSI activity was comparatively less. Interestingly spice extract could reduce swarming nature in both the organisms, which is one of the important virulent factor regulated by QS. The phytochemical components in the crude extract were analyzed by qualitative   methods. MS analysis of the treated samples revealed the fact that AHL molecule is subjected to lysis or degradation. Chemical characterization of bioactive components in the crude extract of Cinnamomum verum is under process.

Quorum quenching,Cinnamomum verum, Spice extracts, Dietary spices

INTRODUCTION: Quorum sensing is a bacterial cell communication mechanism which is mediated   through signal molecules such as AHL’s. Many pathogenic bacteria especially opportunistic pathogens use these quorum sensing circuits to coordinate their virulence gene expression in accordance with population density. Recent research has revealed that eukaryotes are capable of interfering with bacterial communication by the production of molecular signals that interact with the bacterial QS system.

Such Quorum quenchers have been intensively investigated as new class of antimicrobial agents. These compounds interfere in bacterial signaling processes¹.

Disruption of quorum sensing (Quorum Quenching) attenuates the pathogenicity   in bacteria without imposing resistance.  Hence   wide range of naturally occurring substances, particularly   plants extracts, have been evaluated for their ability to modulate quorum sensing in Gram-negative bacteria². Phytochemicals have enormous   structural diversity hence can be explored as   quorum quenchers.

Many research groups   are working on phytochemicals as quorum quenching agents by testing crude plant extracts. Vattem, D² had initiated the study on the QSI activity of   the dietary phytochemicals i.e secondary metabolites of plants against C. violaceum and P. aeruginosa.  Allison L. Adonizio³   reported inhibition of Quorum sensing controlled virulence factor production in P. aeruginosa by South Florida plant extracts. Bryant, S.⁴   published that various medicinal plants extracts from the Indian subcontinent had QSI activity against P. aeruginosa.  Maryam Zahin⁵, screened ethanolic extracts of 24 medicinal plants of Indian origin for QSI activity against Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PAO1.

Sandy Siew -Mian Yeo and Foong -Yee Tham⁶ screened twenty Traditional Chinese Medicine (TCM) plants commonly used in South-East Asia for QS inhibitors using two biomonitor strains, Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PAO1. These research papers proved that   phytochemical extracts could effectively inhibit virulence factors regulated by QS.

Food spices are an essential component to a nutritious lifestyle. Research indicates that they contribute to health just as much as fruits. Preliminary screening of   popular food spices for QSI activity was carried out in which Cinnamomum verum (Dalchini)hadgood QSI activity. Cinnamomum verum   belongs to the family Lauraceae. The dried bark of Cinnamomum is used as spice worldwide. Itis a very important food spice used in chocolate, apple pie, cinnamon buns, desserts, donuts and candies. It is also used to flavor hot cocoa, liquors and tea. It also comes in handy in flavoring cereals, sweet dishes and many more.

Cinnamon oils have   anti-microbial activity. They are effective in preventing the growth of fungi, bacteria and yeast. In fact, Cinnamon essential oils can take the place of traditional food preservative. If added to food as preservative, cinnamon oil does not just act like good preservative, but also help in bringing out the natural taste of food.    It was also known to exhibit antioxidant properties. Cinnamon is found to reduce type 2 diabetes in preclinical studies⁷. Recognizing its medicinal value and dietary   consumption, an attempt was made to evaluate QSI activity of Cinnamomum verum on bacterial pathogens like Pseudomonas, Serratia and Chromobacterium violaceum, a bioindicator organism for studying QSI activity.

 MATERIALS AND METHODS:

Microorganisms 

Serratia marcescenes: Isolate obtained from rhizosphere soil samples of college campus was characterized by morphological, biochemical   methods and by 16s rRNA analysis as Serratia marcescenes (16srRNA sequence was deposited in GenBank under Acc.no:JQ21730). It was named as Serratia sps YAJS and used for QSI activity study.

 Pseudomonas aeruginosa:This organism isolated from infected tomato plant   was used for QSI activity comparative studies after characterizing by standard microbiological, biochemical, enzymatic and 16s rRNA analysis.

 Chromobacterium violaceum 12472:A bio indicator for QSI activitywas obtained from Dr. Hammeda bee, Asst. Professor, Department of Microbiology, Osmania University.

 QSI activity analysis of bacterial cultures

As the study focused on use of dietary phytochemicals, the aqueous extract of spice Cinnamomum verum (Dalchini) was prepared for QSI activity analysis. The spice powder was dissolved in aqueous extracts to get a final concentration of 10mg/ml, filtered using 0.2 micrometer pore size filter and then used to test Quorum sensing inhibitory activity.

Quorum quenching activity on Serratia isolate

Serial dilution of the crude extract of food spice was made in nutrient broth and 0.1 ml of overnight grown culture of Serratia was added and incubated over night at 30^oC. After incubation, growth was monitored by recording absorbance at 600nm. Since the production of protease, prodigiosin were quorum sensing dependent, these parameters were monitored in the treated samples at all dilutions. Prodigiosin assay was done as described by Tomohiro Morohoshi⁸. Protease activity was determined by Mohsen Fathi Najafi⁹   method.

 Swarming activity was determined by Daniel B. Kearns ¹⁰ method. The swarm agar plates were prepared as recommended and 250 μL of spice extract was seeded with 5 mL of the agar and poured immediately on a 10 mL  pre-warmed agar plate as an overlay. Two hundred  microliters of the bacterial culture was inoculated at the center of the agar   surface and the plate was incubated for 16 hours at 37 °C.

Dnase activity was tested on DNA agar by inoculating the culture as a thick band at the center of the plate. The plate was incubated at 32^oC overnight. After incubation the plate was flooded with 1N HCl. Dnase production was identified as a halo zone of clearance (DNA degradation) around the culture streak¹¹.

 Quorum quenching activity on Pseudomonas:

Serial dilution of the spice extract was made in nutrient broth and 0.1 ml of overnight grown culture of Pseudomonas was added and   incubated overnight at 37^oC. After incubation, growth was monitored by recording absorbance at 600nm. Since the production of protease and pyocyanin were quorum sensing dependent, these parameters were monitored in the treated samples for all dilutions. Pyocyanin assay was done as described by Li Ying Tan¹².Protease activity was carried out by Mohsen Fathi Najafi⁹.

QSI activity on Chromobacterium violaceum:

QSI activity of crude extracts of spice was tested by violacein inhibition assay using Chromobacterium violaceum as bioindicator organism⁵. The nutrient agar plates were pre-seeded with overnight grown culture of Chromobacterium violaceum strain. Wells were made in the agar plate with a sterile cork borer of 6mm in diameter. The wells were loaded with 50µl   of the sample to be tested and incubated overnight at 30^oC. The QSI activity was calculated by measuring the diameter of colorless haloes created due to inhibition of violacein pigment but not the growth.

AHL extraction:

5ml of overnight grown culture was used for AHL extraction⁸. Cells were separated by centrifugation and supernatants were extracted twice with equal volumes of ethyl acetate. The extracts were evaporated to dryness. Later they were dissolved in 50-100 µl of ethyl acetate and used for further studies.

Mass spectroscopy:

The AHL samples   were   extracted as mentioned above and subjected for MS analysis. Samples treated with and without spice extract   were subjected to analysis for comparison. Mass spectral analysis was performed at IICT, Hyderabad. Mass spectra were recorded on Finnigan MAT 1020, mass spectrometer operating at 70 eV.

HPLC Analysis:

Aqueous crude extracts of Cinnamomum verum were subjected to HPLC analysis at central facilities for research and development (CFRD) at Osmania University, Hyderabad by standard methods¹³.

Statistical Analysis:

All experiments were performed in triplicates and standard deviation was calculated to all the samples in MS Excel. The data was also analyzed by ANOVA and one way variance using SPSS package. All the graphs were represented with error bars.

 RESULTS:

Quorum quenching activity on Serratia isolate:

Serratia is an opportunistic pathogen, which causes a variety of infections. Serratia marcescensinvolved in nosocomial outbreaks in intensive care units. The Serratia sps YAJS employed in present study was isolated and characterized as Serratia marcescens with 95% similarity by phylogenetic analysis ¹⁴.

Serratia produces N-Acetyl Homoserine lactone as quorum sensing signaling molecule which is used for regulation of genes for extracellular virulence factor like prodigiosin, swarming motility, secretion of enzymes like protease and   Dnase. These parameters were monitored in the treated samples.From the results represented in Figure 1, it was evident that there was difference in growth   of Serratia isolate   before and after treatment with this spice extract.  With increase in concentration of extract there was reduction in growth i.e. 8.9% reduction was noticed at 4mg/ml, which   further increased to 14.5% at higher concentration.  This result was in accordance with the   reported antibacterial activity of the spice.Interestingly, spice had quorum quenching ability as it effectively reduced the QS regulated virulent factors like prodigiosin, protease activity, and swarming nature. Relative prodigiosin values from figure 1 were observed to be influenced as there was 17% reduction at 2mg/ml concentration, which gradually   touched to 61.7% at higher concentration. The protease activity of Serratia was reduced by 41.1% at 2mg/ml concentration and increased to 54.9% at 8mg/ml. One of the important virulent characters like swarming nature of Serratia was also found effected with the treatment as there was 54 % reduction at 8mg/ml concentration in Figure 2. From these results it was evident that the phytochemical components of spice aqueous extract were effective in reducing the three virulent factors of Serratia tested. However, Dnase activity of Serratia was not affected by the treatment.

FIG 1: EFFECT OF CINNAMOMUM VERUM ON VIRULENCE CHARACTERS OF SERRATIA SPS YAJS

Reduction in swarming nature of Serratia.

A-Control   B- 2mg/ml concentration C- 8mg/ml concentration

Quorum quenching activity on Pseudomonas:

Pseudomonas aeruginosa is the second most prevalent organism in nosocomial infections, third in causing urinary tract infections, fifth in post-surgical infections. This was the major cause of mortality in patients suffering with cystic fibrosis. Continuous and improper use of antibiotics rendered this organism resistant to different antibiotics.

Pseudomonas aeruginosa produces N-Acetyl Homoserine lactone as quorum sensing signaling molecule which is used for regulation of genes for extracellular virulence factors like, phospholipase C, rhamnolipid, superoxide dismutase, HCN, exotoxin A, exoenzyme, pyocyanin, pyoverdin, LasA protease and LasB elastase. Many of these factors cause   reactions in the host leading to cell death and tissue necrosis.

An attempt was made to test QSI activity of this food spice against Pseudomonas as most of the research was reported against this organism. The effect of spice extract on pyocyanin production, protease activity, swarming nature and growth were assayed in the treated samples. Cinnamomum verum extract was observed from Figure 3a to have a concentration dependent reduction in growth. Maximum reduction was noticed at 8mg/ml by 44.61% proving its antibacterial activity.

 Pyocyanin is produced by strains of Pseudomonas aeruginosa as a water soluble blue-green pigment, which belongs to the Phenazine family. Pyocyanin enhances oxidative metabolism, which increases the formation of intracellular reactive oxygen species (ROS), which is advantageous for bacterial survival and thus the infection sustains. Chemicals which could reduce the pyocyanin production will definitely control the virulence behavior of the pathogen. Cinnamomum verum   extract recorded least pyocyanin production i.e. 11.64% and 6.86% respectivelyas showed in Figure 3b.

P. aeruginosa proteases are believed to play a major role in pathogenesis via host tissue degradation. In the present study effect of crude spice extract was tested on protease activity in P. aeruginosa. At 2mg/ml concentration, Cinnamomum verum extract had shown 25.7% reduction, and at higher concentration 45 % reduction was recorded in Figure 3c.

FIGURE 3A                                              FIGURE 3B                                                FIGURE 3C

*Error bars depicts standard deviation

Swarming motility is a quorum sensing regulated character which has been characterized as type of flagella-dependent movement on a semi-solid agar surface. In P. aeruginosa, the colonies display the formation of complex dendritic, fractal-like patterns. Overhage¹⁵ and coworkers reported that swarming cells exhibited up-regulation of genes associated with virulence character expression. In present study Cinnamomum verum extract   could influence swarming nature of Psuedomonas sps as depicted in Figure 4. As Pseudomonas sps do not produce extracellular Dnases, this factor could not be tested.

In Pseudomonas, Cinnamomum verum extract could reduce only the protease and swarming nature but could not influence pyocyanin production.

  A. BEFORE TREATMENT           B. AFTER TREATMENT

FIG 4: SWARMING NATURE OF PSEUDOMONAS

QSI activity on Chromobacterium violaceum

As most of the research papers to date on quorum quenching activity of plant extracts were tested against the bio-indicator organism Chromobacterium violaceum, present study was also extended to test spice extract on Chromobacterium violaceum (ATCC 12472).  This was performed by studying violacein inhibition assay, a plate based method.

Crude aqueous extract was loaded on to Luria Bertani (LB) plates spread with C. violaceum. Plates were incubated overnight at 30 °C, and QS inhibition was detected by a ring of colorless, but viable, cells around the well. Loss of purple pigment in C. violaceum is indicative of QS inhibition by spice aqueous extract.  Strong anti-QS activity was observed with aqueous extracts against distilled water as negative control.

The halos produced on lawns of the biomonitor strain could be the result of either (i) inhibition of cell growth or (ii) quenching of QS signals. Growth inhibition would produce a clear halo versus a turbid halo for the latter. To differentiate, the halo was examined under a higher magnification. The halo-effect is created by pigment less (QS interrupted) cells adjacent to the wells. Clear halos as seen in Figure 5 against violet background around the sample wells were measured.

FIG 5.  COLORLESS HALOES INDICATE THE QSI ACTIVITY

FIG 6:  EFFECT OF CINAMOMMUM VERUM EXTRACT ON CHROMOBACTERIUM VIOLACEUM

Interesting observations were made in this assay as both antibacterial activities were found associated with quorum quenching activity. Figure 6 showed antibacterial activity of Cinnamomum verum extract at 8mg/ml. However quorum quenching activity i.e. violacein inhibition activity was observed even at low levels of concentrations tested. These results indicate the presence of strong quorum quenching compounds in the aqueous crude extract of Cinnamomum verum.

To know the fate of AHLs for hypothesizing probable mechanism of quenching activity the treated samples were subjected to Mass spectroscopy. MS analysis revealed the cleavage of C₆AHL at the acyl side chain in Figure 7 which was evident from the fragmented peaks observed in MS analysis report at m/z 102. A peak at m/z 102 indicates the characteristic Homoserine lactone ring alone and the fragmented acyl side chain were indicated by small peaks. Dilara ¹⁶ reported the fragmentation of AHL molecule by Mass spectroscopy which stands as support to the present data obtained. MS analysis reveals the fact that AHL molecule is subjected to lysis or degradation. The inactivation of AHL could be by lysing the molecule or separating their lactone ring from the side chain.

FIG 7: SPECTRAL ANALYSIS OF AHL IN CINNAMOMUM VERUM (8mgl/ml) TREATED SAMPLE

8a. ALKALOIDS

8b. FLAVONOIDS

8c.PHENOLS

8d. TERPENOIDS

DISCUSSIONS: Cinnamomum verum showed a significant decrease in expression of QS regulated virulence factors tested. QSI activity of Cinnamomum verum   extract had   not been reported so far against virulence character expression in Serratia sps. In present study, spice aqueous extract could reduce pigment production by 61.7% and protease by 54.9% while it could affect growth only by 14.5%. These results suggest that Cinnamomum verum extract had more QSI activity than the antibacterial activity in Serratia. In Pseudomonas the antibacterial activity was more (44.61% reduction in growth) while QSI activity was comparatively less i.e. (6.86% in pigment production and 45 % reduction in protease activity). Interestingly spice extract could reduce swarming nature, one of the important virulent factors regulated by QS in both the organisms.

Antibacterial activity of Cinnamomum verum was observed at high concentration (8 and 10mg/ml) in Chromobacterium violaceum. However quorum quenching activity i.e. violacein inhibition activity was observed from 4 mg/ml concentration onwards. These results indicate the presence of strong quorum quenching compounds in the aqueous crude extract of Cinnamomum verum.  MS analysis of treated samples revealed the fact that AHL molecule is subjected to lysis or degradation. The inactivation of AHL could be by lysing the molecule or separating their lactone ring from the side chain. As the crude extract has mixture of phytochemicals, it could be difficult to pinpoint one component responsible for the AHL inactivation.

In order to figure out the bioactive component, qualitative analysis of phytochemicals in the crude extract was performed by Standard methods¹³. Figure 8a-d depicts HPLC analysis of aqueous extracts in which Flavonoids (8b), Phenolics, (8c) Alkaloids (8a) and Terpenoids (8d) were identified.

Cinnamaldehyde, a phenolic compound is a potent QS inhibitor. Studies by Niu¹⁷ reported that Cinnamaldehyde inhibited bioluminescence in two different reporter strains of Vibrio harveyi which are able to respond to both AHL and AI-2. This inhibition was mediated by modulation of Lux R activity.  Present study also is in agreement with the research findings that it could reduce the QS mediated gene expression. The same group have subsequently reported that Cinnamaldehyde is a potent inhibitor of 3 oxo C₆ HSL (OHHL) quorum sensing systems in reporter strain E. coli and was also found to inhibit QS in Vibrio harveyi by inhibiting the QS signaling molecule 3-hydroxy C₄ HSL at sub inhibitory concentrations.

The research findings in the above paper stated that cinnamaldehyde was able to bind to cognate receptors and interferes with binding of 3 hydroxy C4 HSL and 3 oxo C6 HSL   and inhibit the signaling circuits, but could not inhibit the LasR promoter activity induced by odDHL in an E. coli strain containing the Las R biosensor system. Studies by Brackmann¹⁸ proved that cinnamaldehyde was able to inhibit biofilm formation in Vibrio harveyi mediated by AI-2 system and reduce pigment and protease production in Vibrio anguillarum.

CONCLUSIONS: Aqueous extracts of dietary spice Cinnamomum verum had shown a strong Quorum Quenching activity against the three tested bacterial pathogens. The exact mode of action at molecular level has to be further explored with purified bioactive component from spice extract. Recognition of bioactive components in the crude extract of Cinnamomum verum is under process. The observation that dietary phytochemicals fromCinnamomum verum    can inhibit QS related processes opens up new scope for antimicrobial chemotherapy.

ACKNOWLEDGMENTS: We thank Prof. Y. Ashok, Principal and Dr. K. Anuradha, Head, Department of Microbiology, Bhavans Vivekananda College for their constant encouragement and support. We also thank Dr. Hameeda Bee, Assistant professor, Osmania University for providing standard strain of Chromobacterium violaceum 12472. We acknowledge the analytical support extended by IICT, Hyderabad and CFRD, Osmania University.

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    How to cite this article:

    AparnaY, Narayanan Land Sarada J: Quorum Quenching Ability of Dietary Spice Cinnamomum Verum on Pathogenic Bacteria.Int J Pharm Sci Res 2014; 5(12): 5216-23.doi: 10.13040/IJPSR.0975-8232.5 (12).5216-23.

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