8montano-high throughput-supplementary.pmd A.S.N.S. Ferrer et al 93 SCIENCE DILIMAN (JANUARY-JUNE 2017) 29:1, 93-97 High-throughput Screening for Quorum Sensing-inhibitory Compounds from Selected Phil ippine Marine Algae and Surface-associated Marine Microorganisms for Potential Anti-biofilm/biofoul ing Appl ications Supplementary Material Aira Sacha Nad ine S. Ferrer, Aljon Francis Koji P. Elegado, Mel iton R. Chiong III, Laude Karina G. Alcober, Dang Marviluz L. Espita and Marco Nemesio E. Montaño University of the Philippines Diliman FIELD COLLECTION OF SAMPLES AND IN-SITU DETECTION OF QUORUM SENSING INHIBITION Seaweed samples were obtained from 36 sites around Luzon, Visayas, and Mindanao (Table 1). Necessary permits were obtained from relevant government units in coordination with the regional off ices of the Bureau of Fisheries and Aquatic Resources (BFAR). Since a greater variety of seaweed species is observed during specif ic months, sampling visits accounted for the seasonality of seaweeds. Voucher specimens were submitted to the GT Velasquez Herbarium at the UP Marine Science Institute for proper identification and future reference. ISSN 0115-7809 Print / ISSN 2012-0818 Online Table 1. Field collection sites Site GPS Coord inates/Municipal ity Luzon Poblacion Dos, Calatagan, Batangas 13°49’47.0"N 120°37’02.9"E San Antonio, Zambales 14°54’50.6"N 120°00’29.7"E Minanga Weste, Buguey, Cagayan 18°16’5.69"N 121°52’1.77"E Minanga Weste, Buguey, Cagayan 18°16’9.82"N 121°51’39.12"E Gonzaga, Cagayan 18°22’48.73"N 122° 5’57.62"E Santa Ana, Cagayan 18°22’48.62"N 122° 5’57.97"E Taggat Sur, Claveria, Cagayan 18°36’44.00"N 121° 2’55.29"E Sitio Banwa, Balaoi, Pagudpud, Ilocos Norte 18°35’38.46"N 120°53’5.99"E Casa Teresita, Pagudpud, Ilocos Norte 18°36’29.84"N 120°46’42.55"E Paayas, Burgos, Ilocos Norte 18°29’56.31"N 120°34’11.04"E Currimao, Ilocos Sur 17°59’30.47"N 120°29’46.39"E Sinait, Ilocos Sur 17°51’53.33"N 120°26’25.30"E Santa Maria, Ilocos Sur 17°22’4.12"N 120°27’2.28"E Balaoan, La Union 16°48’12.89"N 120°19’42.38"E Rosario, Ilocos Sur 16°13’0.44"N 120°24’45.66"E Bolinao, Pangasinan 16°22’40.7"N 119°54’42.0"E High-throughput Screening for Quorum Sensing-inhibitory Compounds 94 Table 1. Field collection sites (Cont’n.) Visayas Marine Sanctuary, Luyang, Carmen, Cebu 10°35’53.88"N 124° 1’45.30"E Punta Engaño, Lapu-lapu, Cebu 10°19’51.81"N 124° 2’40.01"E Moalboal Beach Resort, Saavedra, Moalboal, Cebu 9°59’7.76"N 123°23’5.31"E Saavedra Fish Sanctuary, Moalboal, Cebu 9°59’51.36"N 123°22’46.79"E BCD’s Pace, Tan-awan, Oslob, Cebu 9°27’46.80"N 123°22’45.91"E Calaguyan Sur, Loon, Bohol 9°50’32.13"N 123°47’42.03"E Tambulian Shoal, Tubigon, Bohol 10° 3’20.16"N 123°56’34.17"E Ubay Island, Tubigon, Bohol 10° 1’21.26"N 123°58’0.57"E Poblacion, Bien Unido, Bohol 10° 8’10.16"N 124°22’51.06"E Larapan, Jagna, Bohol 9°39’10.39"N 124°22’51.31"E Doljo, Panglao, Bohol 9°35’10.27"N 123°43’27.94"E Mindanao Tibungco, Davao City, Davao del Sur 7°11’07.4"N 125°39’17.2"E Bunawan, Davao City, Davao del Sur 7°14’28.4"N 125°39’10.1"E Passig Islet, Digos, Davao del Sur 6°47’10.1"N 125°23’42.2"E Sarangani Island, Davao Occidental 5°25’30.5"N 125°27’44.1"E Moncado Poblacion, Samal Island, Davao del Norte 7°07’50.2"N 125°40’55.0"E Miranda Poblacion, Samal Island, Davao del Norte 7°08’19.1"N 125°40’59.8"E Tambo, Samal Island, Davao del Norte 7°08’57.9"N 125°41’07.7"E Kawas Beach, Alabel, Saranggani 6°04’00.2"N 125°16’37.2"E Bula Beach, General Santos City, Saranggani 6°05’58.7"N 125°11’55.7"E Site GPS Coord inates/Municipal ity Immediately after the f ield collection, preliminary qualitative high-throughput screening was performed with the seaweed fragments and microbial isolates using the method described by McLean et al. (2004). This determines whether QS inhibitory compounds are present on the seaweed surface and eff iciently tests all surface- associated microorganisms. The bacterial sensor Chromobacterium violaceum (CV 12472), which characteristically produces a water-insoluble purple pigment called violacein through the QS network, was used as an indicator strain. Quorum sensing inhibition activity would appear as zones around seaweed fragments or microbial isolates, indicating CV 12472 growths devoid of purple coloration. Seaweed fragments were cut into smaller size fragments (ca. 1 cm) aseptically and surface-sterilized. The fragments were subsequently washed in sterile seawater and placed on Luria broth-agar (LBA). Surface-associated microbial isolates were streaked on LBA and were incubated for 24 hours. Soft LBA inoculated with an overnight culture of CV 12472 was then poured over the plates with the seaweed fragments and isolates. The plates were observed after 24 hours of incubation. Results of the in-situ assays are depicted in Figure 1. Colorless zones around the seaweed fragment or microbial isolate indicate the presence of potential QS inhibitory compounds. A.S.N.S. Ferrer et al 95 CRUDE EXTRACTION AND AGAR WELL DIFFUSION A total of 51 crude methanol extracts from seaweed were obtained, and percent yields based on dry weight are presented in Figure 2. In order to test for QS inhibitory compounds in seaweed metabolites, all crude extracts were screened for QS inhibitory activity through agar well diffusion assay, similar to the method used by Rasmussen et al. (2005), but with CV12472 as reporter strain. Wells were bored on layers of LBA and soft LBA inoculated with an overnight culture of CV12472, in order to accommodate 50 ì L of extract. Plates were incubated for 24 hours at room temperature and observed. The absence of the purple pigmentation in areas surrounding the wells signif ies the inhibition of quorum sensing. Figure 3 depicts the inhibition of violacein production in CV12472 agar plates by QSI-positive extracts. Figure 3 shows the presence of zones of inhibition for the crude extract, as well as the hexane and ethyl acetate partitions. QUORUM SENSING INHIBITION ASSAY IN BROTH CULTURES The extract, together with an overnight culture of C. violaceum CV12472 in Luria broth, was incubated at room temperature for 24 hours without agitation. Violacein production and cell density were quantif ied by measuring the optical density (i.e. absorbance) at 585 nm and 600 nm, respectively. As illustrated in Figure 4, the crude extract of H. edulis has a lower absorbance reading for violacein compared to the negative control (methanol). Figure 1. Results of the in-situ assay. (a) Seaweed fragments of Chaetomorpha crassa exhibiting QS inhibitory activity (with replicates labelled as 1, 2, 3); (b) non- inhibitory seaweed fragments of Padina sp.; (c) microbial isolate showing the decolorization of Chromobacteria violaceum; and (d) a non-inhibitory microbial isolate. High-throughput Screening for Quorum Sensing-inhibitory Compounds 96 F ig u re 2 . P e rc e n t y ie ld s ( b a s e d o n d ry w e ig h t) o f c ru d e e x tr a c ts o f s e a w e e d s p e c ie s f ro m L u z o n ( b lu e ), V is a y a s ( o ra n g e ), a n d M in d a n a o ( g re e n ). Halymenia durvellei Chaetomorpha crassa Gracilaria sp. Halimeda opuntia Galaxaura oblongata Padina sp. Cheilosporum sagittatum Halimeda macroloba Halimeda opuntia Padina sp. Hormophysa cuneiformis Chaetomorpha crassa Mastophora rosea Turbinaria ornata Gelidiella acerosa Gracilaria edulis Gracilaria edulis Kappaphycus contonii Hydropuntia edulis Gracilaria sp. Laurencia sp. Halimeda macroloba Mastophora rosea Ulva reticulata Galaxaura oblongata Galaxaura fasciculata Turbinaria ornata Halimeda opuntia Amphiroa foliacea Laurencia cartilaginea Padina sp. Galaxaura oblongata Galaxaura apiculata Ulva reticulata Padina sp. Halimeda macroloba Turbinaria conoides Galaxaura fasciculata Halimeda opuntia Halimeda macroloba Turbinaria ornata Mastophora rosea Ulva reticulata Gracilaria sp. Gracilaria salicornia Caulerpa racemosa Halimeda sp. Laurencia tronoi Galaxaura fasciculata Ulva lactuca Bornetella sp. 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 A.S.N.S. Ferrer et al 97 Figure 4. Liquid assay of crude H. edulis extract (100 mg/ml), methanol, and cinnamaldehyde (75mM). Methanol and cinnamaldehyde served as the negative and positive controls, respectively. Data presented as mean ± SD (n=6). Figure 3. Agar well plate diffusion assay for the (a) crude extract, (b) hexane partition, (c) ethyl acetate partition, and (d) aqueous partition of Hydropuntia edulis. On each plate, the negative control (methanol, hexane, ethyl acetate, and water, repectively) is located on the lower right, while the positive control (75 mM cinnamaldehyde) was placed on the lower left. REFERENCES McLean RJ, Pierson LS, Fuqua C. 2004. A simple screening protocol for the identif ication of quorum signal antagonists. Journal of Microbiological Methods. 58:351–360. Rasmussen TB, Bjarnsholt T, Skindersoe ME, Hentzer M, Kristoffersen P, Kote M, Nielsen J, Eberl L, Givskov M. 2005. Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. Journal of Bacteriology. 187(5):1799–1814.