PHILIPP AGRIC SCIENTIST Vol. 94 No.

1, 14-22 March 2011

ISSN 0031-7454

Activity of the Ethanolic Extract of Propolis (EEP) as a Potential Inhibitor of Quorum Sensing-Mediated Pigment Production in Chromobacterium violaceum and Virulence Factor Production in Pseudomonas aeruginosa
Lisa E. Lamberte1, Esperanza C. Cabrera2 and Windell L. Rivera1,3,*
1 2

Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, Philippines Biology Department, De La Salle University-Manila, Manila, Philippines 3 Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City, Philippines * Author for correspondence; e-mail: wlrivera@science.upd.edu.ph; Fax: +63 2 920 5471
Bacteria are capable of the organized expression of specific sets of genes through a recently discovered phenomenon termed quorum sensing (QS). Researchers are beginning to focus their efforts into the discovery of potential QS inhibitors for the development of novel antipathogenic drugs. This study investigated the QS inhibitory potential of the ethanolic extract of propolis (EEP) in the test organism Chromobacterium violaceum ATCC 12472 and the opportunistic organism Pseudomonas aeruginosa PAO1. Results of this study showed EEP as a potential inhibitor of QSmediated violacein production in C. violaceum. EEP was thereby subjected to further testing on its ability to interfere with virulence factor production and biofilm formation in P. aeruginosa. It was found that EEP was able to significantly affect the LasA and LasB protease activities. In addition, changes in the protease activity were observed with no significant effects on the growth of the organism. This implies that changes in the enzyme activities are unrelated to bactericidal consequences. However, it was also found that EEP inhibited the biofilm formation of P. aeruginosa PAO1 at lower concentrations but not at higher concentrations. This suggests the need for further investigations to be made on the effect of EEP on the maturation and differentiation of biofilms.

Key Words: biofilm, Chromobacterium violaceum, propolis, Pseudomonas aeruginosa, quorum sensing, virulence Abbreviations: AHL acyl-homoserine lactone, DMSO dimethyl sulfoxide, ECR Elastin-Congo Red, EEP ethanolic extract of propolis, LB Luria-Bertani, OD optical density, PVC polyvinyl chloride, QS quorum sensing

INTRODUCTION
Quorum sensing (QS) is a phenomenon discovered by microbiologists wherein bacteria are shown to be capable of an organized expression of specific sets of genes. These genes are regulated as a response to bacterial population densities using a chemically-based, command language (Fuqua et al. 1994; Zhang and Pierson III 2001). In Gram-negative bacteria, most QS systems use N-acyl-homoserine lactones (AHL) as their command language. Pseudomonas aeruginosa is a common opportunistic Gram-negative pathogen found in immunocompromised patients (van Delden and Iglewski 1998). Among the immunocompromised patients who are particularly susceptible to this pathogen are those diagnosed with

cystic fibrosis. However, despite intensive antibiotic therapy, there is still a high rate of morbidity and mortality for these patients (Koch and Hiby 2000; Lyczak et al. 2002; Bjarnsholt et al. 2005). Therefore, alternatives to conventional antibiotic therapy are needed to combat such pathogens. Propolis, or bee glue, has been designated as the most important chemical weapon of bees (Bankova 2005). The evolutionary success of bees is largely due to propolis as a defense mechanism against pathogenic microorganisms. Propolis has also been used as a wound remedy in ancient and even modern medicine (Wollenweber et al. 1990). Although propolis was shown to have potential QS inhibitory (QSI) activity in the study conducted by Rasmussen et al. (2005), further investigations are

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The Philippine Agricultural Scientist Vol. 94 No.1 (March 2011)

Quorum Sensing Inhibitory Potential of the Ethanolic Extract of Propolis

Lisa E. Lamberte et al.

needed to verify such an activity in known indicator strains as well as in pathogenic organisms. Doing so would elucidate the potential of propolis in the development of future QSI drugs. This study therefore aimed to evaluate in vitro the activity of propolis on QSmediated pigment production in the Gram-negative test organism Chromobacterium violaceum ATCC 12472 and its effects on virulence factor production and biofilm formation in the opportunistic microorganism Pseudomonas aeruginosa PAO1.

MATERIALS AND METHODS

Propolis Material and Extracts The bee propolis used in the study was provided by Ilog Maria Bee Farm in Silang, Cavite, Philippines. Its age was 2 mo before it was extracted. One hundred grams of propolis were extracted three times with 800 mL of 96% ethanol for 5 d to yield ethanolic extract of propolis (EEP). The extracts were filtered and subsequently evaporated to dryness in a rotary vacuum evaporator to give a brown, resinous product. A stock solution of 28 mg mL-1 of extract was prepared in dimethyl sulfoxide (DMSO): Luria-Bertani broth (LB) (16:84). Microorganisms The bacterial strains which were used in the study were the wild type Chromobacterium violaceum ATCC 12472 (McClean et al. 1997), Staphylococcus aureus 1143 (ATCC 6538P) and Pseudomonas aeruginosa PAO1 (Latifi et al. 1995). C. violaceum ATCC 12472 in lyophilized form was kindly provided by Robert J.C. McLean of Texas State University-San Marcos, San Marcos, Texas, USA; S. aureus 1143 was kindly provided by the Microbiology Laboratory of the Institute of Biology, University of the Philippines-Diliman, Quezon City, Philippines; and P. aeruginosa PAO1, also in lyophilized form, was kindly provided by Roger C. Levesque of Universit Laval, Qubec, Canada. Cultures were revived and handled in the Laboratory of Applied Microbiology of the Institute of Biology, University of the Philippines-Diliman. Effect of Ethanolic Extract of Propolis (EEP) on Violacein Production in Chromobacterium violaceum ATCC 12472 Ten-millimeter wells were punched through the LB agar using a sterile cork borer and filled with 60 L of EEP. After an overnight incubation at 37 C, the absence of purple pigmentation of the bacterial lawn on the area surrounding the well indicated inhibition of violacein production by EEP. However, it is also possible that apparent QS inhibition was due to its antimicrobial activity. Such an event was checked through failure of

the indicator strains to grow in the immediate proximity of the well with EEP. An overnight culture of P. aeruginosa PAO1 was added to one of the wells as a positive control that inhibited the QS activity in C. violaceum ATCC 12472. P. aeruginosa PAO1 produces two signal molecules, 3oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL) and N-butanoyl homoserine lactone (C4-HSL), which competitively bind and inhibit the receptor for the cognate signal N-hexanoyl homoserine lactone (C6-HSL) in the C. violaceum ATCC strain (Fuqua et al. 2001; McLean et al. 2004). Thus, the presence of P. aeruginosa PAO1 resulted in the inhibition of pigment production by C. violaceum ATCC 12472. DMSO:LB (16:84) served as the negative control. Growth Curves of P. aeruginosa PAO1 Two milliliters of overnight cultures of the bacteria were diluted 100-fold in 500-mL Erlenmeyer flasks with 198 mL LB medium. An aliquot of 0.5 mL from this starting culture (time zero) was subjected to 10-fold dilutions, and each dilution was plated for viable count. The absorbance of the cultures at OD600 was also measured. The starting culture and the plates were incubated at 37 C. Viable counts and OD600 readings were taken every hour for 24 h. The logarithms of the viable plate counts were taken to facilitate the plotting of the graph. After establishing the growth curve of P. aeruginosa PAO1, EEP (in the highest concentration that was shown to be inhibitory to QS activity) as established in this study, was added to the mid-logarithmic phase culture. The OD600 and viable plate counts of the mixture were taken every hour. Dose-response Assay P. aeruginosa PAO1 was grown in LB medium for 16 18 h at 37 C. The culture was diluted 100-fold in LB medium, and allowed to grow to the mid-logarithmic phase which was found to be at 14 h of growth in this study. The culture was adjusted to an OD600 of 1.7, and was divided into 10 mL aliquots. EEP or DMSO:LB was added to the prepared cultures to the appropriate concentration of 0-28 mg mL-1 in two-fold dilutions. These dilutions were based on preliminary experiments which determined the range of concentrations that showed the best activity. The mixture was incubated at 37 C for 4 h to allow interaction of EEP with the culture. The supernatant after centrifugation was used for the different assays for virulence factor production described below. LasA Staphylolytic Assay LasA protease activity was determined by measuring the ability of the culture supernatants to lyse boiled S. aureus cells (Kong et al. 2005). A 30-mL volume of an

The Philippine Agricultural Scientist Vol. 94 No.1 (March 2011)

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