M. lunu-ankenda was obtained from a local market located in Selangor (Malaysia). A voucher specimen of M. lunu-ankenda was deposited at the University Malaya Herbarium (Voucher Number: 047697). The plant samples were washed twice with sterile distilled water followed by a final rinse with 70% (v/v) ethanol. Plant samples were dried in an oven at 45 °C for 72 hours. The dried plant samples were ground to a fine powder and submerged sequentially in hexane, chloroform and methanol (ratio 1:10 w/v) for 72 hours. The extracts were filtered through Whatman No.1 paper and concentrated under vacuum using a rotary evaporator. Plant extracts of 10 mg/mL (w/v in 100% DMSO) were diluted with sterile distilled water to 1, 2, 3, 4 and 5 mg/mL prior to use.

Bacterial strains used in this study are listed in Table 1. Bacteria were grown in Luria-Bertani (LB) medium (1% w/v NaCl, 1% w/v tryptone, 0.5% w/v yeast extract) with shaking (220 rpm). Chromobacterium violaceum CV026 was cultured in 28 °C, while P. aeruginosa strains at 37 °C. C. violaceum CV026 growth medium was supplemented with kanamycin (30 μg/mL) and chloramphenicol (30 μg/mL).

C. violaceum CV026 assay was performed as described by Renee and Gray [21] with modification. Overnight grown C. violaceum CV026 cells (15 mL) were added into 200 mL of molten LB agar that has been supplemented with N-hexanoylhomoserine lactone (C6-HSL, 0.25 μg/mL). C. violaceum CV026 agar suspension was poured into Petri dishes and allowed to solidify, wells were then made using sterile pipette tips. Plant extract (30 μL) was placed in each well and the extract solvent (DMSO, 50% v/v) served as the negative control. The plates were incubated at 28 °C for 24 hours. Halo formation on a purple background suggested that the plant extracts exhibited anti-QS.

Violacein quantification assay was performed in a 96-well plate [22]. Optical density (OD_600nm) of overnight culture of C. violaceum CV026, supplemented with C6-HSL (0.125 μg/mL), was adjusted to 1.2 prior to use. C. violaceum CV026 cells (90 μL) were added to each well followed by the addition of 10 μL of plant crude extract. The 96-well plate was incubated at 28 °C in a shaking incubator. After 16 hours, the mixtures in the 96-well plate were completely dried at 60 °C. DMSO (100 μL) was added onto each well and the microplate was placed in a shaker until all the violacein was solubilized. The absorbance of each well was read at 590 nm using DYNEX MRX Elisa reader (Chantilly, VA, USA).

Bioluminescence expression was quantified using a Tecan luminometer (Infinite M200, Männerdorf, Switzerland). Briefly, overnight culture of E. coli biosensors cells was diluted to an OD_600nm of 0.1. Then, 230 μL of E. coli biosensors cells and 20 μL of plant extract were added into the well of 96-well microtitre plate. The bioluminescence and OD_495nm were determined every 30 min for 24 hours by the luminometer [19]. Expression of bioluminescence was given as relative light unit (RLU)/OD_495nm against time [19]. Reduction of bioluminescence in E. coli [pSB401] and E. coli [pSB1075] suggested anti-QS properties of the plant extracts.

Pyocyanin quantification assay was performed as described by Essar et al. [23] with slight modification. Briefly, overnight culture of P. aeruginosa PA01 was adjusted to an OD_600nm of 0.2. Then, 250 μL of plant extract was added and mixed well with P. aeruginosa PA01 cells (4.75 mL) in a polypropylene tube and incubated at 37 °C for 24 hours. The 5 mL culture was extracted with 3 mL of chloroform, followed by mixing the chloroform layer with 1 mL of 0.2 M HCl. The absorbance of the pink extracted organic layer was then measured at 520 nm using the UV-visible spectrophotometer (UV1601, Shidmazu, Kyoto, Japan).

P. aeruginosa PA01 lecA expression was quantified using a Tecan luminometer (Infinite M200). Briefly, overnight culture of P. aeruginosa PA01 lecA::lux was diluted to an OD_600nm of 0.1. Then, 230 μL of P. aeruginosa PA01 lecA::lux and 20 μL of plant extract were added into the well of 96-well microtitre plate. The bioluminescence and OD_495nm were determined every 30 min for 24 hours by the Tecan luminometer. P. aeruginosa PA01 lecA::lux expression was given as relative light unit (RLU)/OD_495nm against time [19]. Reduction in the bioluminescence of P. aeruginosa PA01 lecA::lux suggested anti-QS properties of the plant extracts.

Swarming agar was prepared by the following compositions: glucose (1% w/v), Bacto agar (0.5% w/v), Bacto peptone (0.5% w/v) and yeast extract (0.2% w/v). Solidified swarming agar (10 mL) was overlaid with 4.75 mL of swarming agar supplemented with 250 μL of plant extract. Overnight culture of P. aeruginosa PA01 (2 μL) was inoculated in the centre of the agar and incubated for 16 hours at 37 °C. Reduced swarming motility of P. aeruginosa PA01 suggested anti-QS properties of the plant extracts.

All assays were performed on triplicate basis and the significance of the data was tested using ANOVA test (P < 0.05) using GraphPad Prism software.

Formation of a visible halo zone indicates anti-QS action exerted by the plant extract. In Figure 1, a halo zone was formed when the extract was applied at 1 mg/mL and the size of the halo zone increased in parallel with the concentration of the M. lunu-ankenda hexane extract (Figure 1).

This suggests stronger anti-QS action of the extract corresponds to its increasing concentration. In contrast, M. lunu-ankenda chloroform extract showed weaker anti-QS properties as it only started to exert its anti-QS effect at a concentration of 2 mg/mL (Figure 1). As compared to the M. lunu-ankenda hexane extract, the methanolic extract showed weaker anti-QS activity (Figure 1). DMSO (50% v/v) served as the negative control which showed no bactericidal or anti-QS effects (data not shown).

Anti-QS activity of M. lunu-ankenda extracts (applied at 1, 2, 3 and 4 mg/mL) were analyzed using violacein quantitative assay and DMSO (10%, 20%, 30% and 40% v/v) served as the negative controls at each corresponding concentrations. By using the statistical ANOVA test, it was found that M. lunu-ankenda chloroform and methanol extracts of 4 mg/mL causes significant inhibition of violacein as compared to the control (Figure 2). In general, there was reduction in violacein production as the concentration of the extracts increases. However, these reductions were not significantly different at P < 0.05.

C. violaceum is a Gram negative bacterium which synthesizes the purple pigment violacein, a QS-mediated trait regulated by C6-HSL. C. violaceum CV026, on the other hand, is a transposon mutant strain of C. violaceum that is unable to synthesize C6-HSL. Thus, C. violaceum CV026 can only produce violacein in the presence of exogenous short chain AHLs [14,18]. In C. violaceum CV026 plate assay, formation of halo zone indicates the plant samples is either inhibiting the C6-HSL competitively from binding to its transcriptional regulator, cviR; degrading the C6-HSL enzymatically, or removing the C6-HSL via active transport [24–26]. Inhibition on violacein production was quantified and from the results obtained in this study, it was proven that M. lunu-ankenda reduced violacein production significantly. In agreement to this finding, other plant extracts such as vanilla, Tremella fuciformis, Conocarpus erectus, Quercus virgiana, pea seedlings and other various higher plants have been found to possess anti-QS activity against biosensor strain C. violaceum CV026 [10,14,24,27,28].

All three solvents extracts of Melicope lunu-ankenda showed significant inhibition against E. coli [pSB401], but not E. coli [pSB1075] (data not shown). E. coli [pSB1075] produces optimum luminescence in the presence of exogenous N-3-dodecanoylhomoserine lactone (3-oxo-C12-HSL) [20]. Plant extracts that reduced bioluminescence of E. coli [pSB401] and E. coli [pSB1075] suggest the presence of anti-QS activity.

Pyocyanin is one of the exoproducts produced by P. aeruginosa. It causes extensive cellular damage in the lungs of cystic fibrosis patients. Chloroform and hexane extracts of M. lunu-ankenda showed significant reduction in pyocyanin production (Figure 3(a,b)). The methanolic extract only showed significant reduction of pyocyanin at 3 mg/mL (Figure 3(c)).

Pyocyanin can be found in great quantity in the sputum of cystic fibrosis patients and it is also highly permeable to the biological membranes. P. aeruginosa causes developing loss of pulmonary function which leads to premature death in the majority of cystic fibrosis patient [29,30]. Mutations in lasR-lasI, rhlR-rhlI and the mvfR-haq QS systems caused loss in pyocyanin production [31,32]. These QS systems are also involved in production of among others, rhamnolipids, proteases and elastase [30]. Pyocyanin formation involved a multifactorial system and though extracts of M. lunu-ankenda could significantly inhibit its production in this study, it is possible that the compounds from M. lunu-ankenda influence directly the virulence factor production in a QS independent manner. A study found that cyclic disulphides and trisulphides obtained from garlic have no antibiotic properties but instead suppress the expression of LuxR and LuxR based QSI in P. aeruginosa [33,34].

P. aeruginosa PA01 lecA::lux was constructed by the cloning of luxCDABE (from Photorhabdus luminescens) into the lecA gene region of P. aeruginosa [19,20]. The hexane and chloroform extracts of M. lunu-ankenda caused a significant reduction in PA01 lecA expression (Figure 4). However, the methanolic extract did not show any observable results (data not shown). lecA is the structural gene of PA-IL, which is a cytotoxic lectin. Synthesis of lectin is directly related to the rhl locus as it has been demonstrated that in the lasR mutant, lectin synthesis was delayed but not prevented totally [19,35,36]. Reduction of P. aeruginosa PA01 lecA::lux expression by M. lunu-ankenda extracts indicates that these extracts were exerting theirs activity more on the rhl system which also controls the expression of elastase and biofilm. Similarly, rosmarinic acid reduces the expression of proteases, elastases and biofilm in P. aeruginosa has been reported [37].

Swarming refers to the bacterial surface translocation which is QS-dependent and it requires flagella and pili [38,39]. Of the three extracts of M. lunu-ankenda, only the chloroform extract showed inhibition against the swarming of P. aeruginosa. Swarming, swimming, gliding, twitching, sliding and darting are modes of surface translocation used by bacteria [38]. In this study, we investigated the anti-swarming ability of M. lunu-ankenda extract against P. aeruginosa PAO1. Generally, there are 3 stages in the swarming process, firstly, differentiation of vegetative cells into swarmer cells, followed by migration of swarmer cell populations and finally consolidation [40,41]. The extract of M. lunu-ankenda were seeded into the swarming agar and as shown in Figure 5, the chloroform extract showed observable inhibition against the swarming of P. aeruginosa PAO1. The extract of M. lunu-ankenda might be exerting its inhibition of swarming during the migration of swarmer cells or causing biofilm dispersal. Similar observation has also been observed in malabaricone C, a compound purified from nut meg extract [42]. Subsequently, as the migration process is a QS-regulated trait, further studies can be done by using the mutants of PA01 which is deficient in QS.