Investigating the Antimicrobial, Anti-Biofilm and Anti-Quorum Sensing Potential of South African Seaweed-Associated Bacteria
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INVESTIGATING THE ANTIMICROBIAL, ANTI-BIOFILM AND ANTI-QUORUM SENSING POTENTIAL OF SOUTH AFRICAN SEAWEED-ASSOCIATED BACTERIA Prinsloo Xolisa Phiri Thesis presented in partial fulfillment of the degree of Master of Science at University of KwaZulu-Natal (Westville Campus) Supervisor: Dr. Hafizah Y. Chenia Co-supervisor: Dr Mary-Jane Chimuka January 2017 College of Agriculture, Engineering and Science DECLARATION - PLAGIARISM I, Prinsloo Xolisa Phiri, declare that: 1. The research reported in this thesis, except where otherwise indicated, is my original research. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed ……………… i DECLARATION I, the undersigned, hereby declare that the work contained in this thesis is my own, unaided work. It has been submitted for the degree Master of Science to the College of Agriculture, Engineering and Science, School of Life Sciences, Discipline of Microbiology at the University of KwaZulu-Natal, Durban, South Africa. It has not been submitted previously, in its entirety or in part, at any other university. Signature: __________________ Date: __________________ Supervisor: _________________ Date: _30/01/17_________________ Co-supervisor: _ Date: _30/01/17_________ ii ACKNOWLEDGEMENTS Above all I would like to thank the Lord all mighty for granting me the strength to endure this study; My sincere gratitude to my supervisor Dr H.Y. Chenia for her support, patience and help throughout this study; The entire Mabuza and Phiri family for their joint encouragement and parenting thank you for always believing in me; Special thanks goes to my colleagues in the Microbiology lab for their technical assistance and constant encouragement; Agricultural Research Council (ARC) and National Research Foundation (NRF) for their financial assistance; and Lastly, to my friends for their love, patience and moral support. iii ABSTRACT Marine substrata are colonized by a variety of marine microorganisms, which are capable of producing novel compounds due to their diverse and often extreme environmental conditions as well as interactions with their associated eukaryotic host organisms. Seaweed serve as an important host for bacteria that synthesize bioactive natural substances. Seaweed-associated bacteria represent an inexhaustible reservoir of bioactives with potential application in pharmaceutical, medicine and food industries. The antibacterial, anti-biofilm and anti-quorum sensing potential of bacteria associated with ten South African seaweeds was therefore assessed. Cultivable seaweed-associated bacteria (n=96) were screened for antibacterial activity against five resistant clinical and six aquaculture indicator bacteria utilizing the primary cross streak screening assay. Following shake flask fermentations and ethyl acetate extractions extracts, from 30 selected isolates were screened using the agar-well diffusion assay and minimum inhibitory concentrations (MIC) were determined for 14 extracts. Selected bacteria were identified by 16S rRNA gene amplification and sequencing. Active crude extract of MAB24-SW1 was then analysed using nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). Extracts capable of inhibiting initial adhesion and detachment of mature biofilm of clinical and aquaculture pathogens were identified using the crystal violet microtitre plate assay. The anti-quorum sensing inhibitory potential was assessed utilizing Staphylococcus aureus agr and Enterococcus faecalis fsr inhibition assays. Primary screening indicated that 38% (37/96) and 73% (71/96) of isolates displayed varying antimicrobial activity against clinical and aquaculture indicator strains, respectively. Isolate MAB24-SW1 was found to be active against clinical and aquaculture indicators with MICs as low as 0.39 mg/ml. The isolate identified as Bacillus velezensis, produced a potential analogue of surfactin, which was identified through NMR following partial purification of the crude extract. Crude extracts also demonstrated anti-biofilm potential. Inhibition of adhesion was most effective at 5 mg/ml for Staphylococcus aureus, Yersinia ruckeri, Vibrio parahaemolyticus, Pseudomonas aeruginosa and Aeromonas hydrophila. Extracts were most effective in the dispersal of mature biofilms of clinical and aquaculture strains, with the best results been observed against S. aureus, V. parahaemolyticus A. hydrophila, P. aeruginosa, and Edwardsiella tarda. The total range of biofilm inhibition by extracts was between 0.08% - 113.84%. Variation was observed in the efficacy of extracts against initial adherence of indicator strains, with ≥ 90% reduction potential being demonstrated by MAB10B-SW1 iv (Bacillus sp.), MAB24-SW1 (B. velezensis), AB4-SW2 and AB3-SW6 against Pseudomonas aeruginosa. Anti-quorum sensing inhibitory potential against Staphylococcus aureus was assessed utilizing luminescence and green protein fluorescence (GFP) reduction in the Staphylococcus agr assay, crude extracts of MAB6-SW1 (Rhodococcus fascians), MAB10B- SW1 (Bacillus sp.), MAB24-SW1 (B. velezensis), AB7-SW8 (Streptomyces sp.), AB1-SW9 (Microbacterium maritypicum) and AB4-SW10 (Strepyomyces labedae) were capable of quorum sensing inhibition without causing cell death, with some isolates demonstrating ≥ 50% inhibition of GFP. When the anti- quorum sensing inhibitory potential of extracts were tested against Enterococcus virulence factor under the control of the fsr quorum sensing system (gelatinase production), isolate AB2-SW8 (Streptomyces sp.) demonstrated inhibitory potential against both fsr and agr systems without causing cell death. Decreased efficiency and resistance of pathogens to antibiotics has necessitated the development of new therapeutic alternatives. Based on their anti-biofilm and anti-quorum sensing potential, South African seaweed-associated bacteria with their secondary metabolites may provide viable alternatives to antimicrobial therapy in the form of anti-virulence compounds. v LIST OF FIGURES Figure 1.1: The seaweed holobiont and factors predicted to influence bacterial colonization on macroalgal hosts (Egan et al., 2013)…………………………………………………….........11 Figure 1.2: Graphical representation of green seaweed, existing of bacterial and fungal communities on their surface and potential bioactive (Singh et al., 2014)…………….……...12 Figure 1.3: Schematic overview of Staphylococcus aureus accessory gene regulatory (agr) system. The RNAII transcript is an operon of four genes, agrBDCA that encode factors required to synthesize AIP and activate the regulatory cascade (Quave and Horswill, 2014)………………………………………………………………………………………….19 Figure 1.4: The Fsr QS system of Enterococcus faecalis. FsrD, the gelatinase biosynthesis activating pheromone (GBAP) precursor, is processed to a cyclical peptide during secretion by FsrB. Mature GBAP pheromone interacts with the FsrC sensor kinase on the surface of surrounding cells causing phosphorylation of the DNA-binding response regulator, FsrA. Phosphorylated FsrA binds to promoters, including those of fsrB and gelE/ sprE, and upregulates gene expression (Cook and Federle, 2014)………………………………………20 Figure 1.5: The agr quorum sensing gene regulatory system. Quorom sensing inhibitory targets are indicated for inhibitory compounds savrin and solonamide B (Painter et al., 2014).…………………………………………………………………………………………21 Figure 1.6: Biofilm formation is a process in which bacteria adhere to surfaces, through the production of pili, fimbriae, and exopolysaccharides. After initial attachment, biofilm development starts with the building of microcolonies. Biofilm maturation, is dependent on matrix production, which ensures cohesion and structure of mature biofilms. The final step in biofilm formation is cellular detachment or dispersion, by which bacteria regain the planktonic lifestyle to colonize other surfaces. Microbial interferences can inhibit biofilm formation or enhance biofilm dispersion through different mechanisms and strategies at different stages of their development (Rendueles and Ghigo, 2012)…………………………………..………...28 Figure 2.1: Brown, green and red seaweed collected along the coast of Durban, South Africa for isolation of associated bacteria (A) Amphiroa bowerbankii Harvey (B) Cheilosporum cultratum Areschoug (Harvey), (C) Codium duthieae (P. Silva), (D) Codium spp., (E) Gelidium pteridifolium (R.E, Norris, Hommersand & Fredericq), (F) Jania verrucosa (Lamourous), (G) Laurencia brongiartii (J. Agarah) and (H) Ulva rigida (C. Agarah)…….……………………58 Figure 2.2: Primary screening of (A) MAB4-SW1, (B) MAB11-SW1, (C) MAB19B-SW1 and (D) AB3-SW8 against (1) E. coli ATCC 35218 (2) P. aeruginosa ATCC 27853 (3) C. violaceum ATCC 12472 (4) K. pneumoniae ATCC 700603 (5) E. faecalis ATCC 51299 and