8montano-high throughput-shortened.pmd A.S.N.S. Ferrer et al. 87 SCIENCE DILIMAN (JANUARY-JUNE 2017) 29:1, 87-92 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 Aira Sacha Nadine S. Ferrer University of the Philippines Diliman Aljon Francis Koji P. Elegado University of the Philippines Diliman Mel iton R. Chiong III University of the Philippines Diliman Laude Karina G. Alcober University of the Philippines Diliman Dang Marviluz L. Espita University of the Philippines Diliman Marco Nemesio E. Montaño* University of the Philippines Diliman _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online One of the main global problems that cause significant losses in mariculture, medical, and industrial f ields is biof ilm formation and biofouling (Bixler and Bhushan 2012). Biof ilms, which are composed of thick layers of cells embedded in an intricate exopolysaccharide matrix, are often the preferred mode of growth for most bacteria. Biof ilms manifest as the slime often found attached to surfaces in aquatic or marine environments, or even in medical polymers such as catheters and implants. Biof ilm growth provides a strategic niche for planktonic bacteria to thrive in environments prone to mechanical and chemical disruptions. In the complex domain of the extracellular carbohydrate matrix of biof ilms, bacterial aggregates become much less susceptible to treatment with the most diverse chemical biocides and antibiotics than the planktonic cells (Joint et al. 2007). It has been shown that bacteria and biof ilms are able to act together concertedly through the metabolism of compounds formed within the intact community of bacterial biof ilm cells. These compounds were then later associated with quorum sensing (QS) (Rasmussen and High-throughput Screening for Quorum Sensing-inhibitory Compounds 88 Givskov 2006). The formation of biof ilms, which lead to biofouling or the development of complex biological communities on surfaces, such as ship hulls and aquaculture substrates, results in massive material and economic loss (Lehaitre et al. 2008). In different ecological environments, nature has given advantage to organisms that are able to diminish or eradicate detrimental biofouling. Quorum sensing is def ined as a community genetic regulation mechanism that controls microbiological functions of medical, agricultural, and industrial importance in response to population density (Zhang and Dong 2004). Concomitant to an increase in bacterial population, small, freely diffusible signal molecules excreted by the organisms will accumulate. A critical threshold concentration is necessary to initiate a response in the whole population and to activate target genes essential in the synchronization of gene expression and functional coordination among bacteria (Swift et al. 2001; Zhang and Dong 2004). Studies show that some human and plant pathogens, such as Pseudomonas aeruginosa and Erwinia carotovora, regulate virulence through quorum signaling. Quorum sensing has been suggested as an ideal target for treatment of both Gram-negative and Gram-positive bacterial infections often associated with the formation of biof ilms (Kim et al. 2007). Interception of this communication pathway thus implies a potential for QS inhibition to prevent diseases and adverse environmental problems, such as biof ilm growth and biofouling. QS-inhibitory molecules that were initially identif ied include triclosan, furanone, 3-oxo-C12-(2aminocyclohexanone), furanylhydrazides and macrolides, 2-O-glycerol- alpha-D-galactopyranoside (floridoside), betoncine, isethionic acid, and several other molecules (Zhang and Dong 2004). The red alga Delisea pulchra was shown to produce a class of halogenated furanones that accelerate the turnover of the N-acyl homoserine lactone (AHL)-responsive regulatory protein of the LuxR family and inhibit bacterial quorum sensing and biof ilm formation (Manef ield et al. 2002). On the other hand, the green alga Ulva lactuca was demonstrated to rely on the epiphytic bacterium Pseudoalteromonas tunicata to inhibit AHL-dependent transcriptional control by means of synthesizing pigmented substances (Egan et al. 2002). There are numerous studies worldwide reporting the bioactivity of marine-sourced organisms, and these provide impetus to utilize the rich biodiversity of the relatively untapped seaweed resources of the Philippines. This work utilized a high-throughput screening method for quorum sensing-inhibitory molecules from selected Philippine marine algae and associated marine microorganisms. This will subsequently aid in the systematic fast identif ication, extraction, and upscale A.S.N.S. Ferrer et al. 89 production of the bioactive molecules for applications in the prevention of biof ilm formation and biofouling, especially in mariculture systems. Seaweed samples were obtained from 36 sites around Luzon, Visayas, and Mindanao. Out of 86 seaweed fragments collected from the respective sampling sites, six yielded positive results for QSI. Of the 179 microbial isolates that were assayed, nine exhibited QS inhibitory activity. A total of 51 crude methanol extracts from seaweed were obtained. A total of 25 out of 51 crude seaweed extracts tested positive for QS inhibitory activity (Table 1). Solvent fractions for the crude methanol extracts of Hydropuntia edulis, Halymenia durvillei, Chaetomorpha crassa, and Halimeda macroloba were also tested using agar well diffusion assay (Table 2). The putative QS inhibitor(s) in H. edulis possesses non-polar to partially non-polar characteristics, similar to that of H . d u r v i l l e i . Comparison of the size of the pigment inhibition in H. edulis shows more pronounced QSI activity in the hexane partition compared to the ethyl acetate partition. The putative QS inhibitors in both C. crassa and H. macroloba are partially polar. Seaweed Site Collection Period Halymenia durvillei Bolinao, Pangasinan October 2015 Chaetomorpha crassa Balaoan, La Union June 2014 Gracilaria sp. Buguey, Cagayan June 2014 Halimeda opuntia Santa Ana, Cagayan June 2014 Tricleocarpa fragilis (=Galaxaura oblongata) Santa Ana, Cagayan June 2014 Padina sp. Santa Ana, Cagayan June 2014 Jania sagittata (=Cheilosporum sagittatum) Santa Ana, Cagayan June 2014 Halimeda macroloba Gonzaga, Cagayan June 2014 Halimeda opuntia Gonzaga, Cagayan June 2014 Hormohysa cuneiformis Gonzaga, Cagayan June 2014 Kappaphycus cottonii Calatagan, Batangas April 2015 Hydropuntia edulis Calatagan, Batangas October 2015 Mastophora rosea Lapu-lapu, Cebu September 2014 Ulva reticulata Lapu-lapu, Cebu September 2014 Turbinaria ornata Moalboal, Cebu September 2014 Amphiroa foliacea Moalboal, Cebu September 2014 Chondrophycus cartilagineus (=Laurencia cartilaginea) Oslob, Cebu September 2014 Padina sp. Oslob, Cebu September 2014 Padina sp. Panglao, Bohol September 2014 Halimeda macroloba Panglao, Bohol September 2014 Galaxaura divaricata (=Galaxaura fasciculata) Panglao, Bohol September 2014 Halimeda macroloba Loon, Bohol September 2014 Mastophora rosea Loon, Bohol September 2014 Ulva reticulata Jagna, Bohol September 2014 Chondrophycus tronoi (=Laurencia tronoi) Sarangani Island, Davao Occidental February 2016 Table 1. Seaweed extract positive for QSI High-throughput Screening for Quorum Sensing-inhibitory Compounds 90 The method described by Chaudhari et al. (2014) and Choo et al. (2006) quantif ies changes in violacein production and cell density according to the addition of seaweed and microbial extracts. The results obtained using this method provide more def initive data for QS inhibitory activity. Inhibition of purple pigmentation with constant cell density indicated QSI. On the other hand, a decrease in optical density with diminished pigmentation is attributed to cell death, which often implies antibacterial activity and not QSI. The crude extract of H. edulis has a lower absorbance reading for violacein compared to the negative control (methanol). There is a 75.6% decrease in the violacein absorbance in the crude H. edulis extract compared to the negative control. The cell density remained relatively constant, with a decrease of only 14.5%, similar to the results obtained using the positive control cinnamaldehyde. The decrease in QS- regulated violacein production without manifestations of bacterial death conf irms the QS-inhibitory activity of the crude H. ed ulis extract. A Tukey post-hoc test conf irmed that , while the difference in violacein between methanol and crude H. edulis extract is signif icant (p= 5.801E-09), the difference in cell density is not (p=.011). The in-situ method provides a rapid and systematic means of screening for QS inhibition exhibited by seaweeds and associated marine microorganisms in the f ield. The agar well diffusion assay, although quick and convenient, has several limiting factors in terms of miscibility, volume, and molecule size of the possible QS inhibitors. On the other hand, the broth assay provides more quantitative information on the relative absorbance range of potential bioactive compounds. This further facilitates the isolation and characterization process, and validates the results obtained in the agar well diffusion assay. Overall, the three assays described provide high throughput methods to maximize the screening of available QS inhibitory compounds from marine seaweeds and associated microorganisms. Chaetomorpha crassa + - + - Hydropuntia edulis + + + - Halimeda macroloba + - + - Halymenia durvillei + + + - Seaweed Species Crude extract Partition Hexane Ethyl Acetate Aqueous Table 2. QSI activity of seaweed partitions A.S.N.S. Ferrer et al. 91 SUPPLEMENTARY MATERIAL E x p e r i m e n t a l d e t a i l s r e l a t i n g t o t h i s p a p e r a r e a v a i l a b l e o n l i n e : h t t p : / / www.journals.upd.edu.ph/index.php/sciencediliman/article/view/5626/5046 ACKNOWLEDGMENTS This research was funded by the Philippine Department of Agriculture – Bureau of Agricultural Research (DA-BAR). The authors would like to acknowledge the staff of the Seaweed Chemistry Laboratory for their assistance, the Seaweed Taxonomy and Ecology Laboratory headed by Dr. Edna Ganzon-Fortes for identifying the seaweed specimens, and Dr. T ilman Harder of the University of New South Wales for generously providing the indicator strain used in this study. REFERENCES B i x l e r G D a n d B h u s h a n B . 2 0 1 2 . B i o f o u l i n g : Le s s o n s f r o m n a t u r e . P h i l o s o p h i c a l Transactions of the Royal Society. 370:2381-2417. 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Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. Journal of Bacteriology. 187(5):1799–1814. Rasmussen TB, Givskov M. 2006. Quorum sensing inhibitors: A bargain of effects. Microbiology. 152:895-904. Swift S, Downie JA , Whithead N, Barnard AML, Salmond GPC, Williams P. 2001. Quorum sensing population density dependent determination of bacterial physiology. Advances in Microbial Physiology. 45:199270. Z h a n g L H a n d D o n g Y H . 2 0 0 4 . Q u o r u m s e n s i n g a n d s i g n a l i n t e r f e r e n c e : d i v e r s e implications. Molecular Microbiology. 53:1 563-1571. _____________ Aira Sacha Ferrer obtained her undergraduate degree from the Institute of Chemistry, University of the Philippines Diliman and is currently a research associate, as well as a graduate student, at the Institute of Environmental Science and Meteorology, University of the Philippines Diliman. Her work now centers on marine and aquatic pollution studies and health risk assessment. Aljon Francis Koji Elegado is a graduate student in the Marine Science Institute, University of Philippines. His research interests include Marine Microbiology, Organic Geochemistry and Metagenomics. Mel iton R. Chiong III is a graduate student of Materials Science and Engineering in the College of Science, University of the Philippines Diliman. He received his Bachelor of Science in Chemistry degree from the Institute of Chemistry, University of the Philippines Diliman in 2015. He is currently pursuing his M.S. in Materials Science and Engineering degree under the Structure and Dynamics Laboratory, National Institute of Physics, University of the Philippines Diliman. His research i n te r e s t s i n c l u d e s o l i d s t a te c h e m i s t r y, b i o m a te r i a l s , n a n o m a te r i a l s , a n d computational chemistry. Marco Nemesio E. Montaño is a retired professor at the Marine Science Institute, University of the Philippines Diliman. He has devoted his career working on seaweeds since 1977 and is known for his extensive work on Philippine marine algal chemistry research. He obtained his Ph.D. in Biological Chemistry from Griff ith University in Australia where he worked on isolation and characterization of secondary metabolites from marine organisms.