Land and Food Systems, Faculty of
DSpace
UBCV
Li, Shenmiao
Master of Science - MSc
University of British Columbia
Quorum sensing is a process of bacterial intercellular communication that relies on the production and perception of chemical signals called autoinducers. Via quorum sensing, bacteria can modulate a range of behaviours in response to the change of population density for better fitness. Quorum sensing mediated biofilm formation is a vital survival strategy for many bacteria. At a high population density, bacteria can aggregate and attach to a surface by producing and secreting extracellular polymeric substances (EPS). The surrounding EPS protects the encased cells from the unfavourable conditions. Campylobacter jejuni is a major bacterial cause of human diarrhoeal diseases worldwide. Despite its sensitivity to environmental stresses, C. jejuni ubiquitously distributes throughout the poultry production chain. Autoinducer-2 (AI-2) mediated biofilm formation is known to be critical to the environmental survival of C. jejuni. C. jejuni possesses LuxS, the enzyme involved in the production of AI-2. In this dissertation, two natural fatty acids, namely decanoic acid and lauric acid, were identified as AI-2 inhibitors of C. jejuni. Decanoic acid and lauric acid at 100 ppm inhibited ~90% AI-2 activity of C. jejuni without inactivation of the bacterial cells. Anti-biofilm effect of decanoic acid and lauric acid was investigated. C. jejuni culture was incubated with or without the treatment by fatty acids for 72 h. Biofilm biomass was determined by crystal violet assay, and the numbers of viable planktonic and biofilm-associated cells were separately determined by the conventional plating assay. Except one C. jejuni strain increased biofilm biomass at the treatment groups, the fatty acids reduced 10-50% of the biofilm biomass in the other strains. Lauric acid at 100 ppm achieved ~3-log and ~2-log reduction of C. jejuni biofilm-associated and planktonic cells, respectively. In addition, both fatty acids effectively reduced C. jejuni motility. This study identified the AI-2 inhibitors of C. jejuni and investigated their inhibitory effect on biofilm formation and motility. The findings of this study can aid in the development of alternative C. jejuni control strategies.
IDENTIFICATION OF NATURAL COMPOUNDS TO QUENCH QUORUM SENSING IN CAMPYLOBACTER JEJUNI FOR THE REDUCTION OF BIOFILM FORMATION by Shenmiao Li B.Sc., The University of British Columbia, 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Food Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2020 © Shenmiao Li, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Identification of natural compounds to quench quorum sensing in Campylobacter jejuni for the reduction of biofilm formation submitted by Shenmiao Li in partial fulfillment of the requirements for the degree of Master of Science in Food Science Examining Committee: Dr. Xiaonan Lu, Associate Professor, Food Science, UBC Supervisor Dr. Bruce A. Vallance, Principle Investigator, BC Children's Hospital Supervisory Committee Member Dr. Keng C. Chou, Professor, Chemistry, UBC Additional Examiner iii Abstract Quorum sensing is a process of bacterial intercellular communication that relies on the production and perception of chemical signals called autoinducers. Via quorum sensing, bacteria can modulate a range of behaviours in response to the change of population density for better fitness. Quorum sensing mediated biofilm formation is a vital survival strategy for many bacteria. At a high population density, bacteria can aggregate and attach to a surface by producing and secreting extracellular polymeric substances (EPS). The surrounding EPS protects the encased cells from the unfavourable conditions. Campylobacter jejuni is a major bacterial cause of human diarrhoeal diseases worldwide. Despite its sensitivity to environmental stresses, C. jejuni ubiquitously distributes throughout the poultry production chain. Autoinducer-2 (AI-2) mediated biofilm formation is known to be critical to the environmental survival of C. jejuni. C. jejuni possesses LuxS, the enzyme involved in the production of AI-2. In this dissertation, two natural fatty acids, namely decanoic acid and lauric acid, were identified as AI-2 inhibitors of C. jejuni. Decanoic acid and lauric acid at 100 ppm inhibited ~90% AI-2 activity of C. jejuni without inactivation of the bacterial cells. Anti-biofilm effect of decanoic acid and lauric acid was investigated. C. jejuni culture was incubated with or without the treatment by fatty acids for 72 h. Biofilm biomass was determined by crystal violet assay, and the numbers of viable planktonic and biofilm-associated cells were separately determined by the conventional plating assay. Except one C. jejuni strain increased biofilm biomass at the treatment groups, the fatty acids reduced 10-50% of the biofilm biomass in the other strains. Lauric acid at 100 ppm achieved ~3-log and ~2-log reduction of C. jejuni iv biofilm-associated and planktonic cells, respectively. In addition, both fatty acids effectively reduced C. jejuni motility. This study identified the AI-2 inhibitors of C. jejuni and investigated their inhibitory effect on biofilm formation and motility. The findings of this study can aid in the development of alternative C. jejuni control strategies. v Lay Summary Bacteria can communicate with each other via chemical signals produced by themselves, known as quorum sensing. Quorum sensing allows groups of bacteria to cooperate and coordinate together to form a community, called biofilm, typically sticking onto a surface. Compared with free-swimming bacteria, those living inside biofilms are more resilient and adaptive to many unfavourable conditions. Campylobacter jejuni is a common foodborne pathogen that forms biofilms in food processing environments, causing persistent contaminations to the agri-food chain. Quorum sensing is believed to be crucial to biofilm formation. This study identified two natural fatty acids that can repress quorum sensing and potentially interrupt the biofilm formation of C. jejuni. This project provides the possibility of targeting quorum sensing as a novel approach to control C. jejuni in agri-food systems. vi Preface This dissertation is original, unpublished, independent work by the author, Shenmiao Li, under the guidance of Dr. Xiaonan Lu. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction and literature review ...........................................................................1 1.1 Epidemiology of Campylobacter jejuni .......................................................................... 1 1.2 Prevalence and survival of C. jejuni in food-related conditions ..................................... 2 1.3 Survival mechanisms of C. jejuni ................................................................................... 3 Stress response ............................................................................................................ 3 1.3.1.1 Cold stress ........................................................................................................... 5 1.3.1.2 Oxidative stress ................................................................................................... 6 1.3.1.3 Starvation ............................................................................................................ 6 Viable but non-culturable state ................................................................................... 7 Biofilm formation ....................................................................................................... 7 1.3.3.1 Overview of bacterial biofilm ............................................................................. 7 1.3.3.2 Effect of bacterial motility on biofilm .............................................................. 12 viii 1.3.3.3 Biofilm formation of C. jejuni .......................................................................... 13 1.4 Quorum sensing of C. jejuni ......................................................................................... 14 Overview of bacterial quorum sensing ..................................................................... 14 Quorum sensing in C. jejuni ..................................................................................... 20 AI-2 detection ........................................................................................................... 21 Quorum quenching .................................................................................................... 23 1.4.4.1 Enzymatic degradation ...................................................................................... 23 1.4.4.2 Antagonistic analogue ....................................................................................... 24 1.4.4.3 Quorum quenching as an alternative strategy ................................................... 25 1.5 Hypotheses .................................................................................................................... 26 1.6 Objectives ..................................................................................................................... 26 Chapter 2: Materials and methods .............................................................................................27 2.1 Chemicals and reagents ................................................................................................. 27 2.2 Media, bacterial strains, and growth conditions ........................................................... 27 2.3 Preparation of treatment stock solutions ....................................................................... 29 2.4 Determination of subinhibitory concentrations of the selected compounds ................. 29 2.5 Biofilm cultivation ........................................................................................................ 30 2.6 Quantification of autoinducer-2 activity ....................................................................... 31 Screening of AI-2 inhibitors ..................................................................................... 31 Quorum sensing inhibitory effect during biofilm formation .................................... 32 Direct reaction between AI-2 inhibitor and cell-free-supernatant ............................ 32 V. harveyi autoinducer-2 bioassay ............................................................................ 33 2.7 Quantification of biofilm biomass ................................................................................ 34 ix 2.8 Quantification of C. jejuni cells in supernatant and biofilm ......................................... 34 Quantification of C. jejuni planktonic cells in the supernatant ................................. 34 Quantification of C. jejuni biofilm-associated cells in biofilms ............................... 35 2.9 Motility assay ................................................................................................................ 36 2.10 Statistical analysis ......................................................................................................... 37 Chapter 3: Results........................................................................................................................38 3.1 Subinhibitory concentrations of selected compounds................................................... 38 3.2 Inhibition of C. jejuni autoinducer-2 activity by the selected compounds ................... 40 Selection of quorum quenching compounds ............................................................. 40 Inhibition of quorum sensing of C. jejuni with decanoic acid and lauric acid during biofilm formation .................................................................................................................. 41 Interaction of C. jejuni cell-free-supernatant with decanoic acid and lauric acid .... 44 3.3 Effect of fatty acids on C. jejuni biofilm biomass ........................................................ 46 3.4 Effect of fatty acids on C. jejuni survival in biofilm and supernatant .......................... 48 3.5 Effect of fatty acids on C. jejuni cell motility ............................................................... 52 Chapter 4: Discussion ..................................................................................................................55 Chapter 5: Conclusion .................................................................................................................65 5.1 Study summary ............................................................................................................. 65 5.2 Future directions ........................................................................................................... 66 References .....................................................................................................................................68 Appendices ....................................................................................................................................84 x List of Tables Table 1.1 Summary of common stress response genes that absent in C. jejuni but present in E. coli. (Park, 2002)............................................................................................................................. 4 Table 1.2 Identified quorum sensing-mediated phenotypic traits in various bacteria. (West et al., 2012) ............................................................................................................................................. 18 Table 1.3 Summary of different AI-2/AI-2 precursor detection and quantification methods. (Zhao et al., 2018) ................................................................................................................................... 22 Table 2.1 Bacterial strains used in this study. ............................................................................... 28 Table 2.2 Concentration of each compound used in the screening of quorum sensing inhibitory effects. ........................................................................................................................................... 32 Table 3.1 The highest SICs of the selected compounds against four C. jejuni isolates. ............... 39 xi List of Figures Figure 1.1 Characterization of channels within B. subtilis biofilms. (A) Biofilm is grown on the surface of an agar gel containing water and nutrients. The biofilm increases in height to hundreds of micrometres spreads to reach a diameter of several centimetres and forms macroscopic wrinkles. (B) Series of microscopy images of a region near the center of the biofilm. Injection of an aqueous dye reveals a network of channels beneath the wrinkles. (C) Microscopy image of a biofilm after injection of an aqueous solution containing a mixture of fluorescent beads reveals the connectivity of the channels. (D) SEM image of a wrinkle cross-section. (E) SEM image of the underside of a biofilm reveals well-defined channels. (Inset) SEM image of the microstructure of the biofilm. (F) Side view of a biofilm wrinkle reconstructed from profiles of plastic moulds of the upper and lower surfaces of the biofilm and the surface of the agar. (Wilking et al., 2013) ...................................................................................................................... 9 Figure 1.2 Schematic illustration of a biofilm formation process. The formation of biofilms is a cyclic process that occurs in a stage-specific and progressive manner. The process is initiated following surface contact by single planktonic cells. Several developmental steps are discernible as a reversible attachment (step 1), irreversible attachment (step 2), microcolony formation (step 3), macrocolony formation (step 4), biofilms dispersion (step 5). The figure is adapted from the work by Rumbaugh & Sauer (2020). ............................................................................................ 12 Figure 1.3 Schemes of different quorum-sensing systems (a-i). Autoinducers (chemical structure), corresponding synthases (blue), and receptors (green and pink ovals – transcription factor; orange schemes – transmembrane receptors). (Papenfort & Bassler, 2016) ..................... 16 xii Figure 1.4 Interconverting of AI-2 molecules. The precursor molecule, DPD, undergoes various rearrangements and additional reactions to form distinct biologically active AI-2 molecules. The figure is adapted from the work by Miller et al. (2004). ............................................................... 18 Figure 1.5 Possible linkage degraded by quorum quenching enzymes in quorum sensing molecule N-acyl homoserine lactone (A) and corresponding degradation mechanism of quorum quenching enzymes (B). (Chen et al., 2013) ................................................................................. 24 Figure 2.1 Schematic illustration of a typical plate layout of biofilm cultivation in a polystyrene 96-well plate.................................................................................................................................. 30 Figure 2.2 Scheme of experiment flow. CFS: cell-free supernatant; AI: autoinducer. ................ 36 Figure 3.1 Screening of C. jejuni autoinducer-2 inhibitor. Bioluminescence activity of C. jejuni ATCC 33560 treated with 12 different compounds was compared to the ethanol control. Each bar represents the average of three biological replicates with standard deviation. MHB is shown as broth control and EtOH is shown as negative control. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered as statistically significant). ........ 41 Figure 3.2 Inhibition of C. jejuni AI-2 activity by decanoic acid (DA) and lauric acid (LA). C. jejuni strain F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D). Bioluminescence emission of V. harveyi BB170 with the addition of cell-free supernatant collected from biofilm supernatant of four C. jejuni strains incubated with or without the treatment for 72 h at 37°C under microaerobic condition was separately determined. Measurements were performed at 4.5 h xiii after the addition of cell-free supernatant. MHB is shown as broth control and EtOH is shown as negative control. Bioluminescence level of the negative control (EtOH) was set at 100%. Bioluminescence emission of each treatment was normalized to the control. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). ...................................................................... 43 Figure 3.3 Influence of decanoic acid (DA) and lauric acid (LA) on AI-2 activity in C. jejuni cell-free-supernatant (CFS). C. jejuni strain F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D). CFS from each C. jejuni culture grown in MHB was incubated with treatment for 1 h, respectively. Bioluminescence emission of V. harveyi BB170 with the addition of each CFS was detected 4.5 h after the addition of CFS. MHB is shown as broth control and EtOH is shown as negative control. Bioluminescence level of the negative control (EtOH) was set at 100%. Bioluminescence emission of each treatment was normalized to the control. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). ........................................... 45 Figure 3.4 Effect of decanoic acid (DA) and lauric acid (LA) on biofilm formation of C. jejuni strains. C. jejuni strain F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D). Total biomass of the formed biofilms was quantified by crystal violet staining at OD595 nm after 72 h incubation with or without the treatment at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control xiv and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered as statistically significant). ...................................... 47 Figure 3.5 Survival of C. jejuni F38011 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). ....................................................................................... 49 Figure 3.6 Survival of C. jejuni 81-116 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). ....................................................................................... 50 Figure 3.7 Survival of C. jejuni NCTC 11168 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the xv treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). ...................................................................... 51 Figure 3.8 Survival of C. jejuni ATCC 33560 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). ...................................................................... 52 Figure 3.9 Inhibition of C. jejuni cell motility by lauric acid (LA) and decanoic acid (DA). Swarming of C. jejuni F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D) in soft agar (MHB+0.4% agar) was recorded in the absence and presence of specific concentrations of fatty acids after 72 h incubation at 37°C in a microaerobic condition. Soft agar containing 0.5% carrier solvent, ethanol (EtOH) is served as the negative control. ................................................ 54 Figure A.1 Effect of 0.1% trypsin on AI-2 Activity on C. jejuni cell-free-supernatants (CFSs). CFS from each C. jejuni culture grown in MHB was incubated with the 0.1% trypsin for 1 hour, respectively. The bioluminescence emission of the V. harveyi BB170 with the addition of each xvi CFS was detected 4.5 h after the addition of the CFS. Three biological replicates were performed. Each bar represents the average of three biological replicates with standard deviation........................................................................................................................................................ 84 Figure A.2 AI-2 Activity of C. jejuni cell-free-supernatants (CFSs) at different incubation periods. The C. jejuni overnight culture was adjusted to OD600nm = 0.3 and further diluted to ~2 × 108 CFU/mL. The time right after the dilution was taken as 0 h. The bioluminescence emission of the V. harveyi BB170 with the addition of cell-free supernatant collected from C. jejuni incubated for 0, 24, 48, and 72 h at 37°C under microaerobic condition for 72 hours. Measurements were performed 4.5 h after the addition of the cell-free supernatant. Three biological replicates were performed. Each bar represents the average of three biological replicates with standard deviation. ................................................................................................ 85 Figure A.3 AI-2 activity detected from C. jejuni biofilm and biofilm supernatant. The bioluminescence emission of the V. harveyi BB170 with the addition of cell-free supernatant collected from the biofilm and supernatant phases of C. jejuni strains that incubated at 37°C under microaerobic condition for 72 hours. Measurements were performed 4.5 h after the addition of the cell-free supernatant. Three biological replicates were performed. Each bar represents the average of three biological replicates with standard deviation. ............................. 86 xvii List of Abbreviations AB autoinducer bioassay AHLs N-acyl-homoserine lactones AHQ 2-alkyl-4-quinolones AI-2 autoinducer-2 AIPs autoinducing peptides AIs autoinducers CAI-1 cholera autoinducer-1 CFS cell-free-supernatant CFU colony-forming unit CSP competence-stimulating peptide DA decanoic acid DPD 4,5-dihydroxy-2,3-pentanedione EPS extracellular polymeric substances EtOH ethanol FSIS Food Safety and Inspection Service h hour(s) LA lauric acid M.W. molecular weight MH Mueller-Hinton min minute(s) OD optical density PBS phosphate-buffered saline ppm one part per million ROS reactive oxygen species RUL relative light units SIC subinhibitory concentration VBNC viable but non-culturable xviii Acknowledgements I would like to express my deep and sincere gratitude to my supervisor, Dr. Xiaonan Lu, for offering the opportunity to join the group as well as all the support, encouragement and patience through my master’s study. I would like to express my appreciation to Dr. Tao Huan, Dr. Bruce A. Vallance, and Dr. Keng C. Chou for serving my supervisory and examining committees and for all the help on my thesis. A special thanks goes to Dr. Jinsong Feng, for his advice on my experimental design and generous sharing of experiences. I also want to express my heartfelt appreciation to Dr. Lina Ma, who taught me the techniques to carry out the experiments. My thanks also extend to everyone in Lu’s lab for their continuous support, friendship, and encouragement. I am grateful for the financial support from NSERC, UBC Faculty of Land and Food Systems, and scholarship donors. A great gratitude goes to my parents. I would not be who I am today without your love and support. Finally, I would like to thank my husband. You have been continuously supportive of my graduate education. Thank you for being my rock when all I have known is rock bottom. xix Dedication To my beloved parents, Mr. & Mrs. Li. and To Vancouver, such a lovely place. 1 Chapter 1: Introduction and literature review 1.1 Epidemiology of Campylobacter jejuni Campylobacter jejuni has a widespread occurrence in the environment and is recognized as one of the most common bacterial causes of diarrhoeal diseases worldwide (Kaakoush et al., 2015). The global burden of Campylobacter infection had been comprehensively assessed by the Foodborne Disease Burden Epidemiology Reference Group (Havelaar et al., 2015). In 2010, about 166 million diarrheal illnesses were attributed to Campylobacter, and an estimated 37,600 deaths were caused by the infections or the post-infectious sequelae (Kirk et al., 2015). Moreover, the epidemiological evidence collected from FoodNet showed that Campylobacter-associated illnesses increased by 12% in 2018 compared to that in the period of 2015-2017 (Tack et al., 2019). The infectious dose of Campylobacter genus for human infections varies widely depending on bacterial species and route entry (Teunis et al., 2018). Among the 32 officially identified species and 9 subspecies, C. jejuni is responsible for the vast majority of Campylobacter infections that may be caused by a dose of as low as ca. 500 cells (Black et al., 1988; Costa & Iraola, 2019; Teunis et al., 2018). The typical clinical symptoms of Campylobacter infection, namely Campylobacteriosis, include diarrhea (frequently bloody), fever, abdominal pain, and nausea (Brunette, 2017). Although most Campylobacteriosis cases are self-limited, the prolonged infections have been suggested as predisposing factors of inflammatory bowel diseases, inflammation of digestive organs, reactive arthritis, and Guillain–Barré syndrome (GBS) (Kaakoush et al., 2015). Campylobacteriosis has also been recognized as the most common 2 antecedent infection of Guillain–Barré syndrome, an autoimmune disorder that leads to neuromuscular inflammation and paralysis (Acheson & Allos, 2001; Kaakoush et al., 2015). Overall, Campylobacter-associated illnesses caused a loss of approximately 7.54 million years of full health among the global population in 2010, outnumbering the burden caused by Shigella, Cholera, and enterotoxigenic E. coli (Murray et al., 2012). The majority of human Campylobacter infections can be ascribed to the consumption of contaminated poultry products (Acheson & Allos, 2001). Campylobacter species are highly prevalent in avian hosts, especially in broiler flocks, and avian hosts can carry this microorganism asymptomatically throughout the entire lifespan (Sahin et al., 2015). Poor hygiene practice and inadequate cleaning and sanitation could result in cross-contamination during food processing as well as food preparation (Skarp et al., 2016). 1.2 Prevalence and survival of C. jejuni in food-related conditions C. jejuni is nutritionally fastidious and sensitive to food production-induced stresses (Mihaljevic et al., 2007). The growth of C. jejuni can be inhibited by atmospheric levels of oxygen, low temperature, or hyperosmotic stress (Cameron et al., 2012; Mihaljevic et al., 2007). Despite its fastidious and sensitive nature, C. jejuni was identified to withstand food processing-induced stresses and survive well in many food products (Man, 2011; Mihaljevic et al., 2007). At the farm level, C. jejuni has been isolated from various food-producing animals and environmental sources, including poultry, cattle, swine, surface water, sewage, animal feeds, and animal litters (Hussain et al., 2007; Jones, 2001; Møller Nielsen et al., 1997; Stanley & Jones, 2003). A survey of C. jejuni prevalence conducted by the European Food Safety Authority showed that the prevalence of C. jejuni at poultry farm level could go up to 100% (European Food Safety 3 Authority, 2011). Moreover, the high prevalence of C. jejuni in poultry farms may increase the presence of C. jejuni in the downstream products (Skarp et al., 2016). Although most Campylobacter infections are sporadic and can be attributed to the consumption of undercooked poultry products, several Campylobacteriosis outbreaks were directly linked to the consumption of contaminated water (Lind et al., 1996; O'Reilly et al., 2007; Vogt et al., 1982). C. jejuni can survive and remain infective in various food matrices (raw chicken, beef, milk, and fresh produces) during food transportation and storage, ultimately reaching to consumers (Hussain et al., 2007; Johnsen et al., 2006). 1.3 Survival mechanisms of C. jejuni The survival mechanisms of C. jejuni have been explored by many studies since the whole-genome of C. jejuni NCTC 11168 was sequenced and published in 2000 (Parkhill et al., 2000). Although the underlying mechanisms of the environmental survival of C. jejuni have not yet been fully understood, stress responses, biofilm formation and viable but non-culturable (VBNC) state are believed to be the three critical factors to the survival of this microbe (García-Sánchez et al., 2018; Svensson et al., 2008). Stress response C. jejuni has a relatively small genome (ranging from 1.5 Mb to 2.1 Mb) compared with those of other enteric bacteria, such as E. coli (4.5 Mb to 5.5 Mb) and Salmonella enterica serotype Typhimurium (~5 Mb) (Allard et al., 2013; Bergthorsson & Ochman, 1998; Parkhill et al., 2000). Many hallmark stress response genes in Gam-negative bacteria are identified as absent from 4 complete sequenced C. jejuni genomes (Table 1.1). However, many alternative mechanisms were identified to be critical for C. jejuni stress response. Table 1.1 Summary of common stress response genes that absent in C. jejuni but present in E. coli. (Park, 2002) Genes Functions C. jejuni E. coli Oxidative stress soxRS Positive regulators of the response to superoxide stress - + oxyR Positive regulator of the response to peroxide stress - + SodA Manganese cofactored superoxide dismutase - + KatG HPI, catalase - + Osmoregulation proU or opuC High-affinity osmoregulatory uptake of compatible solutes - + otsAB Osmoregulatory trehalose synthesis - + betAB or gbsAB Osmoregulatory choline–glycine betaine synthesis pathway - + Stationary phase/starvation 5 Genes Functions C. jejuni E. coli rpoS General stress/stationary phase sigma factor in Gram-negative bacteria - + Heat and cold shock rpoH Alternative sigma factor regulating the heat shock response - + cspA Major cold shock protein - + Global regulation lrp Global regulator of metabolism - + “+”: present; “-“: absent. 1.3.1.1 Cold stress Refrigeration and freezing are common challenges that C. jejuni might encounter during food processing and preservation. C. jejuni cannot grow at a temperature below 30°C, which may be related to the lack of cold-shock proteins (Murphy et al., 2006; Parkhill et al., 2000). Nonetheless, C. jejuni is able to maintain viability at refrigeration and freezing temperature, and the survival time in the food matrix was extended even up to 7 months (Lázaro et al., 1999; Sampers et al., 2010). Although the mechanisms of cold-shock responses of C. jejuni have not been understood clearly, the transcription studies of C. jejuni at refrigeration temperature revealed that genes involved in energy metabolism, motility, and chemotaxis might be associated 6 with the adaption of C. jejuni to a cold environment (Moen et al., 2005; Stintzi & Whitworth, 2003). 1.3.1.2 Oxidative stress Another noticeable trait of C. jejuni is its tolerance to oxidative stresses. The reactive oxygen species (ROSs) are inevitably generated during the aerobic metabolism in bacteria, and ROSs are deleterious to many intercellular biomacromolecules, such as proteins, lipids, and nucleic acids (Imlay, 2003). Thus, C. jejuni requires redox enzymes to survive due to its microaerophilic nature (Jang et al., 2014). Several enzymes have been identified as critical components of the C. jejuni oxidative defence system, including superoxide dismutase, catalase, and alkyl hydroperoxide reductase (Kim et al., 2015). In addition, oxidative stress adaptation could provide cross-protection for C. jejuni against acid stress in food processing. The stress responses induced in the aerobic condition conferred acid tolerance to C. jejuni and aerobic adapted C. jejuni showed 500-fold increased in survival at pH 4.5 (Murphy et al., 2003). Environmental stresses (e.g., starvation, aerobic condition) can promote biofilm formation of C. jejuni (Reeser et al., 2007; Reuter et al., 2010). 1.3.1.3 Starvation Foodborne pathogens may be subject to nutrient-limiting conditions at any stage of food processing and transportation. A series of physiological changes in bacterial cells can be triggered by starvation, such as growth arrest and low metabolic activity (D. Nguyen et al., 2011). The slowdown of growth could increase the resistance of bacteria to other environmental stresses, including acid stress, oxidative stress, heat shock, and osmotic stress (Rees et al., 1995). 7 In most Gram-negative bacteria, the starvation response is regulated by rpoS, a stationary phase sigma factor (Park, 2002). However, C. jejuni does not elicit RpoS-mediated responses under the starvation condition due to the lack of RpoS regulon (Park, 2002; Parkhill et al., 2000). Therefore, the susceptibility of C. jejuni to acid, heat, and oxidative stress was increased under the starvation condition (Kelly, 2001). Interestingly, C. jejuni responded to starvation stress via a RpoS-independent pathway that involves in spoT, a general regulator, and ppk1, the polyphosphate kinase 1 (Candon et al., 2007; Gaynor et al., 2005). Viable but non-culturable state Entering the VBNC state is considered as one of the long-term survival strategies of C. jejuni when exposed to prolonged challenges. VBNC C. jejuni cells are morphologically distinguishable from normal culturable C. jejuni cells. C. jejuni cells change from the original spiral form to a coccoid form upon entering the VBNC state (Rollins & Colwell, 1986). C. jejuni VBNC cells can resuscitate and become infective under appropriate conditions. Although the resuscitation-promoting factors of VBNC C. jejuni have not been determined, in vivo revival of C. jejuni VBNC cells had been confirmed in mouse and chicken models (Baffone et al., 2006; Cappelier et al., 1999). These aforementioned findings demonstrate that VBNC C. jejuni induced during food processing could survive for a prolonged time and pose a risk to public health. Biofilm formation 1.3.3.1 Overview of bacterial biofilm Biofilms can be broadly defined as a slimy layer of a community of microorganisms enclosed in a self-produced polymeric matrix and stick to either inert or living surfaces (Nobile & Mitchell, 8 2007). The extracellular polymeric substances (EPS) matrix compose of various polysaccharides, lipids, proteins, as well as extracellular DNA (Flemming & Wingender, 2010). This polymeric matrix can protect its encased bacterial cells away from the external stresses. A bacterial biofilm can be made up of one or multiple species, and the multi-species biofilms are the predominant form in the natural ecological niches (O'Toole et al., 2000). The three-dimensional structure of fully hydrated bacterial biofilms was first observed by Lawrence and co-authors in 1991 via scanning confocal laser microscopy (Lawrence et al., 1991). It was observed that bacterial biofilm had open water channels, allowing the diffusion of nutrients and waste, and it was also identified that biofilm structure varied depending on the embedded bacteria species. The structure and function architectures of biofilm have been further explored during the past decade via more advanced techniques. For example, a mature Bacillus subtilis biofilm was identified to have well-organized water channels to ensure water and nutrient transportation (Figure 1.1). 9 Figure 1.1 Characterization of channels within B. subtilis biofilms. (A) Biofilm is grown on the surface of an agar gel containing water and nutrients. The biofilm increases in height to hundreds of micrometres spreads to reach a diameter of several centimetres and forms macroscopic wrinkles. (B) Series of microscopy images of a region near the center of the biofilm. Injection of an aqueous dye reveals a network of channels beneath the wrinkles. (C) Microscopy image of a biofilm after injection of an aqueous solution containing a mixture of fluorescent beads reveals the connectivity of the channels. (D) SEM image of a wrinkle cross-section. (E) SEM image of the underside of a biofilm reveals well-defined channels. (Inset) SEM image of the microstructure of the biofilm. (F) Side view of a biofilm wrinkle reconstructed from profiles of plastic moulds of the upper and lower surfaces of the biofilm and the surface of the agar. (Wilking et al., 2013) 10 Biofilm formation is a developmental process that is commonly described in five demarcated stages (Figure 1.2). (1) Reversible attachment: planktonic cells first attach to the surface reversibly via weak interactions, such as electrostatic force (Srey et al., 2013). The attached cells at this stage can either detach from the surface or build a stronger association with the surface by secreting EPS (Chmielewski & Frank, 2003). (2) Irreversible attachment: A subgroup of attached cells immobilized on the surface and irreversibly associated with the surface (Monds & O’Toole, 2009). The irreversible attachment may require the change of transcriptional regulation in the attached cells to promote the initial formation of a mono-layer biofilm (Hinsa et al., 2003). (3) Microcolony formation: The irreversibly attached cells start to aggregate and multiply, forming discrete cell clusters. The continuous secretion of EPS results in the enlargement of biofilm. (4) Macrocolony formation: Along with the maturation of biofilm, more complicated structures of biofilm starts to develop, including open channels, voids, and architectures (Stoodley et al., 2002). The arrangement and redistribution of the enclosed bacterial cells are influenced by many physiological factors of bacteria, including motility, cell-to-cell communication, growth rate, and membrane proteins (Stoodley et al., 2002). Besides the genetic determinants, the development of 11 biofilm structure is affected by many environmental factors, such as fluid shear, gravity, oxygen concentration, and temperature (Kim et al., 2013; Stoodley et al., 1999; Toyofuku et al., 2016). (5) Dispersal: Inside a mature biofilm, the increase of cell density could trigger the production of matrix-degrading enzymes. These enzymes can dissolve EPS and disperse the enclosed bacterial cells (Winzer et al., 2002). The dispersed cells will then grow to a more planktonic mode and ready for a new cycle of biofilm formation (Rumbaugh & Sauer, 2020). 12 Figure 1.2 Schematic illustration of a biofilm formation process. The formation of biofilms is a cyclic process that occurs in a stage-specific and progressive manner. The process is initiated following surface contact by single planktonic cells. Several developmental steps are discernible as a reversible attachment (step 1), irreversible attachment (step 2), microcolony formation (step 3), macrocolony formation (step 4), biofilms dispersion (step 5). The figure is adapted from the work by Rumbaugh & Sauer (2020). 1.3.3.2 Effect of bacterial motility on biofilm Bacterial motility can influence the initial attachment and structure development of bacterial biofilm. Pseudomonas aeruginosa could form a mushroom-like biofilm if the motility of a subgroup of bacterial cells inside the biofilm was repressed. In contrast, a flat and homogenous biofilm was formed when the motility repression force was absent (Klausen, Aaes-Jørgensen, et al., 2003; Klausen, Heydorn, et al., 2003). During the initial attachment, motility might be essential for bacterial cells to overcome the electrostatic repulsion of the surface (Pratt & Kolter, 1998; Walker et al., 2004). However, the relationship between motility and biofilm maturation is 13 controversial. The loss of biofilm formation capability was reported in a flagellum-minus Listeria monocytogenes mutant (Lemon et al., 2007). With the deletion of the flagellin gene, the mutant strain was not able to initiate attachment, let alone to form a mature biofilm. In contrast, the flagellum-deficient L. monocytogenes mutant was identified to form more biofilm with a higher density compared to the wildtype, although the initial attachment was slowed down (Todhanakasem & Young, 2008). 1.3.3.3 Biofilm formation of C. jejuni Biofilm formation is another important strategy for C. jejuni to survive in challenging conditions. C. jejuni can either form mono-species biofilms on a range of abiotic surfaces or colonize into pre-formed biofilms to establish multi-species biofilm (Teh et al., 2014). In comparison to planktonic cells, cells inside a biofilm have a higher stress tolerance due to metabolism changes and protection from the biofilm matrix (Svensson et al., 2008). Compared with that for planktonic cells, the survival in drinking water for biofilm-enclosed C. jejuni cells was significantly extended up to several weeks (Buswell et al., 1998; Lehtola et al., 2006). The waterborne transmission of C. jejuni may explain the prevalence of C. jejuni in poultry farms and slaughterhouse houses where there is a high cross-contamination risk among water sources and animal feces (Trigui et al., 2015). Environmental factors (e.g., temperature, osmotic press, and oxygen level) can affect biofilm formation of C. jejuni. An increase in C. jejuni biofilm formation was observed under aerobic condition (Reuter et al., 2010). The lack of nutrient and elevated temperature (>42°C) could 14 inhibit C. jejuni from forming biofilms (Reeser et al., 2007; Teh et al., 2016). Further, surface hydrophobicity may affect the stability of a formed biofilm (V. T. Nguyen et al., 2011). Biofilm formation of C. jejuni is also influenced by genetic determinants. For example, C. jejuni RM1221 is not able to form a biofilm (Brown et al., 2015). This strain can produce DNase that degrades eDNA to prevent biofilm formation (Brown et al., 2015). In addition, the existing C. jejuni biofilm can be detached by the DNase released by C. jejuni RM1221 (Brown et al., 2015). The level of biofilm formation also varied, depending on strains with different levels of motility (Turonova et al., 2015). 1.4 Quorum sensing of C. jejuni Overview of bacterial quorum sensing Quorum sensing is a population-dependent signalling system used by bacteria. This system enables bacteria to coordinate their physiological functions to adapt to population changes and environmental stimuli (Bassler & Losick, 2006). A wide variety of quorum sensing circuits are utilized by different bacterial species. The associated signal molecules, termed autoinducers (AIs), are highly diverse among different species (Figure 1.3). Most of the identified quorum sensing molecules in the early studies can be classified into three categories, namely N-acyl-homoserine lactones (AHLs), autoinducing peptides (AIPs), and autoinducer-2 (AI-2). AHLs, AIPs, and AI-2 are mainly used by Gram-negative bacteria, Gram-positive bacteria, and both, respectively (Reading & Sperandio, 2006). This convergence of discoveries laid the groundwork for future exploration of bacterial quorum sensing. The molecular structure of AHLs in different bacterial species have been widely studied since the first identification of AHLs in Vibrio 15 fischeri (Nealson & Hastings, 1979). This group of molecules contain a conserved N-acylated homoserine-lactone and a carbon acyl chain varying in length (C4 – C8) with side chains (Galloway et al., 2011). The variations in AHL structures ensure the species-specific recognition of signalling molecules by different transcriptional activator proteins. The LuxR-type proteins are the most extensively studied AHLs-mediated transcription factors in Gram-negative bacteria (Patankar & González, 2009). This type of protein works as AHLs receptors, activating the transcription of the certain operon(s) upon the binding of AHLs. In Gram-positive bacteria, AIPs are produced and recognized for intraspecies communication. In brief, AIPs precursors are synthesized inside bacterial cells and further processed and exported via AIP-binding cassette transporter. At sufficient concentrations, the extracellular AIPs are then detected by transmembrane two-component histidine sensor kinases. The kinases further activate the response regulator that enables the changes in gene expression (Mukherjee & Bassler, 2019). 16 Figure 1.3 Schemes of different quorum-sensing systems (a-i). Autoinducers (chemical structure), corresponding synthases (blue), and receptors (green and pink ovals – transcription factor; orange schemes – transmembrane receptors). (Papenfort & Bassler, 2016) 17 Besides intraspecies communication, many species are known to communicate cross species. The AI-2/luxS mediated quorum sensing system has been identified in both Gram-negative and Gram-positive bacteria (Pereira et al., 2013). AI-2 consists of a group of interconverting molecules derived from a common precursor, 4,5-dihydroxy-2,3-pentanedione (DPD), and two types of AI-2 molecules have been characterized (Miller et al., 2004). In the AI-2 molecule converting environment, the increase or decrease in tetrahydroxyborate concentration could shift the equilibrium toward S-THMF-borate or R-THMF (Figure 1.4) (Miller et al., 2004). These results may explain the difference between identified AI-2 structures in V. harveyi and S. Typhimurium. V. harveyi is a marine bacterium living in an environment usually high in boron so that it produces and utilizes boron-contained AI-2 molecule. S. Typhimurium usually lives in environments that lack boron, and thus its AI-2 structure is tetrahydroxy furan that contains no boron (Priya et al., 2013). To date, luxS, the gene coding the DPD synthase, has been identified in more than 500 bacterial species and has been recognized as the most common quorum sensing related genes in bacteria (Priya et al., 2013). 18 Figure 1.4 Interconverting of AI-2 molecules. The precursor molecule, DPD, undergoes various rearrangements and additional reactions to form distinct biologically active AI-2 molecules. The figure is adapted from the work by Miller et al. (2004). Quorum sensing serves as global regulatory factors for cellular growth, stress responses, biofilm formation, and virulence factors in many Gram-positive and Gram-negative bacteria (Table 1.2). The connections between quorum sensing and pathogenic traits (e.g., biofilm, virulence, host colonization) have brought a great interest in studying quorum sensing towards a novel pathogen control intervention. Table 1.2 Identified quorum sensing-mediated phenotypic traits in various bacteria. (West et al., 2012) Trait Example organisms Type of autoinducer Aggregation Yersinia pseudotuberculosis Rhodobacter sphaeroides AHL AHL Antibiotics Chromobacterium violaceum Erwinia carotovora Serratia spp. ATCC 39006 AHL AHL AHL Bacteriocins, lantibiotics Lactococcus spp. Lactobacillus spp. Carnobacterium piscicola Bacillus subtilis Enterococcus faecium Streptococcus thermophilus Streptococcus mutans AIP AIP AIP AIP AIP AIP AIP Biofilm formation and maturation Aeromonas hydrophila Burkholderia cenocepacia Pseudomonas putida Serratia liquefaciens Enterococcus faecalis Staphylococcus aureus Vibrio cholerae AHL AHL AHL AHL AIP AIP CAI-1, AI-2 19 Trait Example organisms Type of autoinducer Bioluminescence Vibrio fischeri Vibrio harveyi AHL AHL, AI-2 Biosurfactant production (aids motility) Serratia liquefaciens Serratia marcescens Pseudomonas aeruginosa AHL AHL AHL Competence Streptococcus pneumoniae CSP Conjugation Enterococcus faecalis AIP Exoproducts (products excreted from cells with a variety of functions) Erwinia carotovora Erwinia chrysanthemi Burkholderia cenocepacia Pseudomonas aeruginosa Aeromonas salmonicida Pseudomonas aureofaciens AHL AHL AHL AHL, AHQ AHL AHL Exopolysaccharides (polymers which protect from environmental stresses and can be a component of biofilms) Pantoea stewartii Pseudomonas syringae Vibrio cholerae AHL AHL CAI-1, AI-2 Membrane vesicles (packaging and trafficking of toxins and small molecules) Pseudomonas aeruginosa AHQ Motility (e.g., swimming, twitching, or swarming) Yersinia pseudotuberculosis Burkholderia cenocepacia Pseudomonas aeruginosa Pseudomonas syringae Staphylococcus aureus Yersinia enterocolitica AHL AHL AHL AHL AIP AHL Nodulation/symbiosis (nitrogen fixation which benefits host plant) Rhizobium leguminosarum AHL Sporulation Bacillus subtilis Clostridium perfringens Clostridium botulinum Clostridium acetobutylicum Clostridium sporogenes AIP AIP AIP AIP AIP Virulence Burkholderia mallei Burkholderia pseudomallei Pseudomonas aeruginosa Staphylococcus aureus Agrobacterium tumefaciens AHL AHL AHL, AHQ AIP AHL 20 Trait Example organisms Type of autoinducer Bacillus thuringiensis Bacillus anthracis Clostridium perfringens Clostridium botulinum Enterococcus faecalis Listeria monocytogenes AIP AIP AIP AIP AIP AIP Abbreviations: AHL, N-acylhomoserine lactone; AIP, autoinducing peptide (can be linear or cyclic); AI-2, autoinducer-2; AHQ, 2-alkyl-4-quinolone; CAI-1, (S)-3-hydroxytridecan-4-one; CSP, competence stimulating peptide Quorum sensing in C. jejuni C. jejuni is known to have AI-2/luxS mediated quorum sensing system (Fouts et al., 2005; Parkhill et al., 2000). A novel exporter of C. jejuni AI-2, CjNC110, was speculated to be involved in AI-2 transportation, motility, and in vivo colonization (Kreuder et al., 2020). The increase of intracellular AI-2 and decrease of extracellular AI-2 were observed in the CjNC110-deficient mutant compared with that in the wildtype. However, neither the exact structure of the AI-2 molecule nor the receptor protein has been identified in C. jejuni. Investigation of the role of AI-2-mediated quorum sensing in C. jejuni was primarily relied on luxS-deficient mutant (Plummer, 2012). AI-2 production in ΔluxS mutant can be completely repressed, and AI-2 activity was restored by genetic complementation (Plummer et al., 2011). The change in phenotypic traits of ΔluxS C. jejuni has been extensively studied, but the result varied among studies depending on strains and experimental settings (Plummer, 2012). The decrease of motility in ΔluxS C. jejuni mutant was widely reported (Adler et al., 2014; Elvers & Park, 2002; Plummer et al., 2011; Quiñones et al., 2009; Šimunović et al., 2020), whereas a few studies pointed out that the decrease of motility in ΔluxS mutant was only observed at 37°C but 21 not 42°C (He et al., 2008; Holmes et al., 2009). The ΔluxS C. jejuni M129 showed reduced biofilm formation on polystyrene surface, and the reduction was partially restored by adding the cell-free-supernatant collected from the wildtype culture (Reeser et al., 2007). Similarly, when incubated in a 96-well polystyrene plate, luxS-deficient C. jejuni NCTC 11168 had a 1.47-log reduction on cell adhesion compared with that of the wildtype (Šimunović et al., 2020). Nevertheless, no significant difference was observed in biofilm formation between ΔluxS C. jejuni NCTC 11168 and the wildtype on stainless steel surface (Bezek et al., 2016). AI-2 detection The discovery of AI-2 triggers the development of AI-2 detection and quantification methods. Vibrio harveyi bioassay was first developed by Bassler (1994) and is still widely used in AI-2 activity detection and rapid screening of AI-2 inhibitors (Bassler et al., 1993). Bioluminescence production in V. harveyi BB170 can be stimulated by exogenous AI-2 molecules, and the intensity of stimulated bioluminescence is positively related to AI-2 concentration. This method measures AI-2 activity indirectly as a bioluminescence signal but not directly quantifies the actual AI-2 concentration presented in the sample. More AI-2 detection methods have been developed, achieving quantification with a higher accuracy (Table 1.3). 22 Table 1.3 Summary of different AI-2/AI-2 precursor detection and quantification methods. (Zhao et al., 2018) Detection methods Target Location Signal Range engineered V. harveyi -based biosensor strains V. harveyi BB170 DPD/BAI-2 in vivo luminescence 0.08 -0.8 μM V. harveyi MM32 BAI-2 luminescence – engineered E. coli-based biosensor strains E. coli CT104 harbouring the plasmids pCT6 and pET200/DsRed AI-2 (without boron) fluorescence ≤8 μM E. coli strain KX1446 AI-2 (without boron) β-galactosidase activity – engineered S. Typhimurium-based biosensor strain S. Typhimurium MET844 AI-2 (without boron) β-galactosidase activity – FRET-based biosensing systems fusion protein CLPY BAI-2 in vitro FRET ratio ≥1 μM fusion protein LuxP-EGFP with a fluorophore MDCC BAI-2/ AI-2 (without boron) fluorescence ≥0.1 nM Fluorescent biosensors LuxP/LsrB modified with fluorescent dyes BAI-2/ AI-2 (without boron) fluorescence 1-20 μM / 20-2000 μM chromatography methods LC-Tandem MS DPD – 230 pM-23.5 μM HPLC-Tandem MS DPD – 1-1000 ng/mL HPLC–FLD DPD – 10–14000 ng/mL GC–MS DPD/ BAI-2/ AI-2 (without boron) – 5.3-16.0 nM colorimetric method colorimetric method BAI-2/ AI-2 (without boron) absorbance – BAI-2: S-THMF-borate; AI-2: R-THMF 23 Quorum quenching Quorum quenching refers to interference with quorum sensing through pathway inhibition or signal degradation. Quorum sensing circuits involve a range of enzymes and receptors so that there are multiple potential targets for quorum quenching. The production of signalling molecules can be inhibited by repression/inactivation of enzymes required in AIs synthesis. Chemical or enzymatic degradation of AIs can prevent the accumulation of active AIs. Besides, quorum sensing can be inhibited at the perception stage by blocking the receptors (Hentzer & Givskov, 2003). 1.4.4.1 Enzymatic degradation Over the past decade, enzymatic degradation of AIs is the most studied strategy for quorum quenching. Four potential degradation sites have been found for AHLs (Figure 1.5). After the identification of AHLs in P. Pseudomonas, AHL lactonases and AHL acylase were identified to enable cleaving the ester bond and amide bond of AHLs, respectively. The change in molecular structure resulted in reduced bindings between AHLs and the receptors (Dong et al., 2001). The oxidoreductase was later found to be able to substitute the oxo-group at C3 with the hydroxyl group and result in AHLs degradation (Uroz et al., 2005). The other two presumptive enzymes have yet to be discovered. 24 Figure 1.5 Possible linkage degraded by quorum quenching enzymes in quorum sensing molecule N-acyl homoserine lactone (A) and corresponding degradation mechanism of quorum quenching enzymes (B). (Chen et al., 2013) 1.4.4.2 Antagonistic analogue The DPD synthase, LuxS, is the most common target to inhibit AI-2 production. Several synthetic LuxS inhibitors have been developed and examined (Alfaro et al., 2004; Wnuk et al., 2009; Zhu & Li, 2012). The naturally formed halogenated furanones and synthetic Alkyl-DPD analogues were found to be a potent AI-2 inhibitor for S. Typhimurium and V. harveyi (Defoirdt 25 et al., 2007; Lowery et al., 2009). Cinnamaldehyde was also reported as a general inhibitor for Vibrio quorum sensing (Brackman, Celen, et al., 2011; Niu et al., 2006). This group of analogues targets the receptor proteins to prevent the binding between regulator protein and promoter sequence. 1.4.4.3 Quorum quenching as an alternative strategy Antimicrobial resistance (AMR) has been considered a huge threat to humans. Each year, there are about 2.9 million antibiotic-resistant infection cases in the U.S., among which 35,900 incidents resulted in death (Centers for Disease Control and Prevention, 2019). A report issued by the UK Government indicated that the death caused by AMR would rise from 700,000 per year to 10 million per year by 2050 without more effective interventions (O’Neill, 2018). Indiscriminate use of antibiotics in food-producing animals is worsening the situation to a great extent (Tang et al., 2017). Although its fundamental mechanisms have not been fully determined, quorum quenching is commonly considered as a progressive and promising solution for pathogen control, especially for antimicrobial-resistant pathogens (Whiteley et al., 2017). Quorum quenching does not directly kill pathogens, but inhibits the expression of virulence factors and increases stress susceptibility (Allen et al., 2014). Therefore, this approach minimizes the selective force for the development of resistant bacterial strains. 26 1.5 Hypotheses Hypothesis 1: The natural compounds that inhibit quorum sensing in Campylobacter jejuni could be identified. Hypothesis 2: The identified quorum quenching compound(s) can effectively inhibit biofilm formation of C. jejuni. 1.6 Objectives Objective 1: To screen and identify natural compounds that are able to reduce quorum sensing activities in C. jejuni. Objective 2: To evaluate the effects of identified quorum quenching compounds on C. jejuni biofilm formation. Objective 3: To evaluate the effects of identified quorum quenching compounds on C. jejuni motility (a phenotypic trait associated with biofilm formation). 27 Chapter 2: Materials and methods 2.1 Chemicals and reagents Hexanoic acid (>98% purity), octanoic acid (>98% purity), decanoic acid (>99% purity) and L-arginine (>98% purity) were purchased from Alfa Aesar (Ward Hill, MA, USA). Lauric acid was obtained from Acros Organics (Fair Lawn, NJ, USA). Crystal violet (1% w/v aqueous solution in deionized water) was purchased from Ricca Chemical (Arlington, TX, USA). Pre-measured phosphate-buffered saline (PBS) tablets (pH 7.3-7.5), potassium phosphate monobasic (>99% purity), and di-potassium hydrogen phosphate (>99% purity) were purchased from VWR International (Mississauga, ON, Canada). Sodium chloride (≥99% purity), magnesium sulfate (>97% purity), 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (≥98% purity), 5-Ethyl-4-hydroxy-2-methyl-3(2H)-furanone (≥96% purity), 2,5-Dimethyl-3-oxo-(2H)-fur-4-yl butyrate (≥93% purity), crotonic acid (≥98% purity), trans-ferulic acid, quercetin (≥95% purity), trans-cinnamaldehyde (≥99% purity), and naringenin (≥95% purity) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Casamino acid (Difco, vitamin-free) was purchased from BD Diagnostic Systems (Sparks, MD, USA). Glycerol was obtained from Fisher Scientific (Ottawa, ON, Canada). Trypsin solution (2.5% w/v) was purchased from Life Technologies (Burlington, ON, Canada). 2.2 Media, bacterial strains, and growth conditions Bacterial strains used in this study are listed in Table 2.1. C. jejuni strains were preserved at -80°C in Mueller-Hinton (MH) broth (BD Difco™) containing 25% glycerol and 20% (v/v) defibrinated sheep blood. C. jejuni strains were routinely cultivated either on Mueller Hinton II agar (BD BBL™) plates supplemented with 5% defibrinated sheep blood (MHB agar) or in MH 28 broth at 37°C under microaerobic condition (85% N2, 10% CO2, 5% O2) with constant shaking at 175 rpm. V. harveyi strains were preserved at -80°C in Marine broth 2216 (BD Difco™) containing 25% glycerol and were routinely cultivated either on Marine agar 2216 (BD Difco™) plates or in Marine broth at 30°C under aerobic condition with constant shaking at 175 rpm. Autoinducer bioassay (AB) medium was prepared in the laboratory according to the recipe described by Greenberg and colleagues (Greenberg et al., 1979) with some minor modifications. Briefly, 17.5 g NaCl, 6.02 g MgSO4, and 2.0 g vitamin-free casamino acids were added into 960 mL of deionized water and then adjusted the pH to 7.5 with KOH. The medium was autoclaved at 121ºC for 40 min. After the medium was completely cooled down, 10 mL of 1 M sterile potassium phosphate buffer (pH 7.0), 10 mL of 0.1 M L-arginine (filtrated through a 0.22-µm PES syringe filter), and 20 mL of 50% pre-sterilized glycerol were added into the autoclaved medium. The final volume of the medium was made up to 1 L. Table 2.1 Bacterial strains used in this study. Species Strain Description Source C. jejuni F38011 Human clinical isolate Laboratory stock 81-116 Human clinical isolate Laboratory stock NCTC 11168 Human clinical isolate Laboratory stock ATCC 33560 Bovine feces isolate ATCC V. harveyi BB152 luxM::Tn5 (AI-1-, 2+) ATCC BB170 luxN::Tn5 (sensor 1-, sensor 2+ ) 29 2.3 Preparation of treatment stock solutions The stock solutions of hexanoic acid, octanoic acid, decanoic acid, lauric acid, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 5-ethyl-4-hydroxy-2-methyl-2(2H)-furanone, 2,5-dimethyl-3-oxo-2(H)-fur-4-yl butyrate, crotonic acid, and trans-ferulic acid were prepared by separately dissolving each compound in absolute ethanol to achieve a concentration of 20,000 ppm. The stock solution of each compound was further diluted to working solutions as 400, 300, 200, 150, 100, 50, and 25 ppm with MH broth upon the test. The stock solutions of quercetin, trans-cinnamaldehyde, and naringenin were prepared by separately dissolving each compound in absolute ethanol to achieve a concentration of 5,000 ppm. The stock solution of each compound was further diluted to working solution as 100, 50, 25, 12.5, 6.25, and 3.125 ppm with MH broth upon the test. MH broth with 1% and 0.5% ethanol were used as the control groups. We verified that the addition of ethanol at a concentration of 1% or below did not affect C. jejuni cell viability and growth (data not shown). 2.4 Determination of subinhibitory concentrations of the selected compounds The highest subinhibitory concentration (SIC) of each selected compound was determined using a microtiter broth dilution method, as described in a previous study (Duarte et al., 2016). In brief, overnight C. jejuni culture of each strain was diluted to ~108 CFU/mL in MH broth. One hundred microliters of the working solution of each compound were added to each well of a sterile 96-well polystyrene plate (Corning, untreated, float-bottom), followed by inoculation of 100 µL of C. jejuni culture (~108 CFU/mL). Then, the inoculated 96-well plates were covered by low evaporation lids and incubated at 37°C under the microaerobic condition for 48 h. The highest 30 concentration of the compound that did not inhibit the visible growth of C. jejuni culture was regarded as SIC of the strain. 2.5 Biofilm cultivation C. jejuni biofilm was cultivated according to a previously published method with some modifications (Müsken et al., 2010). In brief, overnight C. jejuni culture was adjusted to OD600nm = 0.3 and further diluted to ~2 × 108 CFU/mL with fresh MH broth. One hundred microliters of C. jejuni culture (~2 × 108 CFU/mL) were added to each well of a sterile polystyrene 96-well plate, followed by the addition of 100 µL of decanoic acid and lauric acid working solutions, respectively. MH both and MH broth with 0.5% ethanol were served as broth control and ethanol control. The outer wells of 96-well plates were filled with sterile deionized water to avoid evaporation caused by edge-effect (Shukla & Rao, 2017). A typical plate layout is shown in Figure 2.1. The plates were covered with low evaporation lids and incubated at 37°C under the microaerobic condition for 72 h. Figure 2.1 Schematic illustration of a typical plate layout of biofilm cultivation in a polystyrene 96-well plate. 31 2.6 Quantification of autoinducer-2 activity Screening of AI-2 inhibitors Based on the preliminary data, C. jejuni cultivated in a static condition in a 96-well plate has a higher AI-2 activity at 48 h than that either at 24 h or 72 h (Figure A.2). In addition, C. jejuni ATCC 33560 forms relatively less biofilm than other strains. The AI-2 activity ins biofilm was confirmed by detaching and suspending biofilm with 0.1% trypsin solution. The trypsin solution did not inhibit AI-1 activity (Figure A.1). A larger portion of AI-2 molecules was identified in biofilm supernatant than biofilm of ATCC 33560 (Figure A.3). Based on these preliminary results, we selected C. jejuni ATCC 33560 as the representative strain and 48 h as the treatment time for AI-2 inhibitor screening. Quorum sensing inhibition capabilities of 12 compounds against C. jejuni ATCC 33560 cells were evaluated at the concentration summarized in Table 2.2. One hundred microliters of the working solution of each compound were added to each well of sterile 96-well polystyrene plates, followed by inoculation of 100 µL of C. jejuni ATCC 33560 overnight culture (~108 CFU/mL). For the broth control and ethanol control, the treatment solutions were replaced with sterile MH broth and MH broth with 1% ethanol. Then, the microtiter plates were covered with low evaporation lids and incubated at 37°C under the microaerobic condition for 48 h. The supernatant from each well was collected separately after incubation, and then centrifuged at 8,000 ×g for 10 min and passed through 0.22-µm PES filters. Cell-free supernatants (CFSs) were stored at -20°C until further usage. 32 Table 2.2 Concentration of each compound used in the screening of quorum sensing inhibitory effects. Compound Concentration (ppm) Hexanoic acid 200 Octanoic acid 100 Decanoic acid 100 Lauric acid 100 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 200 5-Ethyl-4-hydroxy-2-methyl-2(2H)-furanone 200 2,5-Dimethyl-3-oxo-2(H)-fur-4-yl butyrate 200 Crotonic acid 200 Trans-Ferulic acid 200 Quercetin 25 Trans-cinnamaldehyde 12.5 Naringenin 25 Quorum sensing inhibitory effect during biofilm formation C. jejuni biofilm supernatants (the top portion containing spent media and planktonic cells) of treatment groups and control groups in each well of the 96-well plates were collected separately after 72 h incubation (Figure 2.2A). V. harveyi BB152 (positive control strain) was grown in AB medium for 16 h at 30°C under the aerobic condition with constant shaking (175 rpm). The CFSs of C. jejuni biofilm supernatants and V. harveyi BB152 were obtained by centrifuging the culture supernatant at 8,000 ×g for 10 min and passed through 0.22-µm PES filters. The CFSs were stored at -20°C for further usage. Direct reaction between AI-2 inhibitor and cell-free-supernatant AI-2 activity in biofilm supernatant was initially tested to determine quorum sensing inhibitory effect during biofilm formation (section 2.6.1.2). Additional experiments were conducted to determine whether there could be a direct interaction between C. jejuni AI-2 molecule and the 33 inhibitors. C. jejuni strains were grown in MH broth and MH broth supplemented with 0.5% ethanol overnight at 37°C under the microaerobic condition. CFSs were collected by centrifugation and filtration, as aforementioned. One hundred microliters of the collected CFS were mixed with 100 µL of 200, 100, and 50 ppm decanoic acid and lauric acid working solutions, separately, and incubated for 1 h at 37°C. V. harveyi autoinducer-2 bioassay V. harveyi autoinducer-2 (AI-2) assay was conducted following a previously published method with modifications (Bassler et al., 1997). The reporter strain V. harveyi BB170 was grown in AB medium for 16 h at 30°C under the aerobic condition with constant shaking at 175 rpm and then diluted 5,000-fold with fresh AB medium. One hundred and eighty microliters of diluted V. harveyi BB170 culture were added to each well of a sterile white 96-well plate (opaque, flat bottom). Twenty microliters of collected C. jejuni and V. harveyi BB152 CFSs were separately added to each well that contained diluted reporter suspension. One hundred and eighty microliters of diluted reporter suspensions with 20 µL of uninoculated AB medium were served as the negative control. In addition, 20 µL of MH broth was served as broth control, and 20 µL of MH broth containing 0.5% ethanol was used as ethanol control. The 96-well plate was covered by a low evaporation lid and incubated at 30°C with aeration (169 rpm). The bioluminescence signal of each well was measured after 4.5 h incubation using a Tecan microplate reader (Infinite 200 Pro; Tecan Life Sciences). The bioluminescence signals of ethanol control were subtracted from the treatment groups as baselines, and the signals of broth control were subtracted from non-treated groups. 34 2.7 Quantification of biofilm biomass After biofilm cultivation, the biofilm supernatant was collected for the preparation of CFSs and enumeration of planktonic cells. Crystal violet staining assay was used to quantify the total biomass of the formed biofilm in a polystyrene 96-well plate according to a previously published protocol with minor modifications (Feng et al., 2018). Each well of the polystyrene plate was gently washed two times with PBS and air-dried for 30 min. One hundred and fifty microliters of 1% (w/v) crystal violet solution was added into each well to stain the attached biofilm for 15 min (Figure 2.2D). Then, the crystal violet solution was removed from each well, followed by washing three times with sterile deionized water to remove any unbounded stain residuals. The plates were air-dried for 30 min, followed by addition of 200 µL of 95% ethanol (v/v) to dissolve the biofilm-associated crystal violet for 10 min. The absorbance level of the dissolved dye in each well was measured by using a Tecan microplate reader at 595 nm (Infinite 200 Pro; Tecan Life Sciences). Sterile MH broth as well as MH broth with each selected fatty acid were stained using the same method to serve as broth control and treatment controls for non-treated and treated groups. The absorbance of each broth control and treatment controls was subtracted from non-treated and treated groups, respectively, as the baselines. 2.8 Quantification of C. jejuni cells in supernatant and biofilm Quantification of C. jejuni planktonic cells in the supernatant The biofilm supernatants of the treatment groups and control groups in the 96-well plates were collected separately after 72 h incubation (Figure 2.2B). C. jejuni cells in the supernatants were then quantified using the plating assay. In brief, the collected supernatants were serially diluted 35 with PBS and spread onto MHB agar plates. The plates were enumerated after 48 h incubated at 37°C under the microaerobic condition. Quantification of C. jejuni biofilm-associated cells in biofilms After 72-h biofilm cultivation, the broth containing planktonic cells was aseptically removed and collected as aforementioned in section 2.8.1. The 96-well plates were then gently washed two times with PBS to remove any remaining planktonic cells. Biofilms were detached as described in a previous study (Feng et al., 2018) with minor modifications. Briefly, 200 µL of 0.1% trypsin solution was added to each well and incubated for 30 min, followed by pipetting up and down 10 times to suspend the loosened biofilm in the solution. The detached biofilm suspensions were then collected separately, and C. jejuni cells in the suspensions were quantified using the plating assay (Figure 2.2C). Treatment with 0.1% trypsin did not affect C. jejuni cell viability during biofilm detachment (data not shown). 36 Figure 2.2 Scheme of experiment flow. CFS: cell-free supernatant; AI: autoinducer. 2.9 Motility assay The effect of the selected treatment on C. jejuni cell motility was assessed using the soft agar plate assay described in the previous studies (Kalmokoff et al., 2006; Upadhyay et al., 2017) with modifications. Briefly, the soft agar plates contained 25 mL of MH broth supplemented with 0.4% agar with or without the addition of selected compounds at subinhibitory or a lower concentration. The soft agar plates that contained 0.5% ethanol were used as negative control. C. jejuni overnight culture was adjusted 108 CFU/mL, and 2 µL of the adjusted culture was stab inoculated at the centre of the soft agar plates. The inoculated plates were then incubated at 37°C for 48 h under the microaerobic condition. The motility of C. jejuni cells was then determined by measuring the distance of bacterial migrating from the inoculation site. 37 2.10 Statistical analysis All of the experiments were conducted at least in three biological replicates. Results were presented as the mean ± standard deviation. Data analysis and visualization were performed using Origin software (version OriginPro 2020, OriginLab Corporation, USA). One-way ANOVA followed by appropriate posthoc test was used to determine if the difference was statistically significant (P < 0.05 was considered as statistically significant). 38 Chapter 3: Results 3.1 Subinhibitory concentrations of selected compounds The subinhibitory concentration of an antimicrobial is the concentration below the minimum inhibitory concentration of that substance. An antimicrobial at its subinhibitory concentration does not inhibit visible growth of a microbe. Exposing microorganisms to the subinhibitory concentration of an antimicrobial allows the observation of its effects on cellular processes without disturbing cell viability. Quorum sensing is a cell-to-cell communication process underpinned by various biological processes (Papenfort & Bassler, 2016). Using subinhibitory concentration to study quorum sensing quenching effect can largely avoid bias caused by bactericidal effect. The highest subinhibitory concentrations of the tested compounds against four C. jejuni isolates are shown in Table 3.1. Among 12 tested compounds, hexanoic acid, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 5-ethyl-4-hydroxy-2-methyl-2(2H)-furanone, 2,5-dimethyl-3-oxo-2(H)-fur-4-yl butyrate, crotonic acid, and trans-ferulic acid did not show growth inhibitory effect on all four strains at the concentration of 200 ppm, which was the highest tested concentration. For the four fatty acids (i.e., hexanoic acid, octanoic acid, decanoic acid, and lauric acid), the inhibitory concentrations decreased along with the increase of alkyl chain length. Susceptibility of the four tested C. jejuni strains to octanoic acid, lauric acid, quercetin, and naringenin was slightly different. For example, C. jejuni ATCC 33560 was more susceptible to octanoic acid than the other tested strains, and C. jejuni 81-116 showed higher tolerance to lauric acid treatment than the other three C. jejuni strains. 39 Table 3.1 The highest SICs of the selected compounds against four C. jejuni isolates. Compound Chemical Structure M.W. (g/mol) Strain SIC (ppm) Hexanoic acid 116.16 F38011 No inhibition at conc. ≤ 200 81-116 11168 33560 Octanoic acid 144.21 F38011 No inhibition at conc. ≤ 200 81-116 11168 33560 150 Decanoic acid 172.26 F38011 150 81-116 11168 33560 Lauric acid 200.32 F38011 100 81-116 150 11168 100 33560 100 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 128.13 F38011 No inhibition at conc. ≤ 200 81-116 11168 33560 5-Ethyl-4-hydroxy-2-methyl-2(2H)-furanone 142.15 F38011 No inhibition at conc. ≤ 200 81-116 11168 33560 2,5-Dimethyl-3-oxo-2(H)-fur-4-yl butyrate 198.22 F38011 No inhibition at conc. ≤ 200 81-116 11168 33560 Crotonic acid 86.09 F38011 No inhibition at conc. ≤ 200 81-116 11168 33560 Trans-Ferulic acid 194.18 F38011 No inhibition at conc. ≤ 200 81-116 40 11168 33560 Quercetin 302.23 F38011 25 81-116 50 11168 50 33560 25 Trans-cinnamaldehyde 132.16 F38011 12.5 81-116 12.5 11168 12.5 33560 12.5 Naringenin 272.25 F38011 50 81-116 50 11168 25 33560 25 3.2 Inhibition of C. jejuni autoinducer-2 activity by the selected compounds Selection of quorum quenching compounds Twelve compounds were screened and assessed for their ability to inhibit AI-2 activities of C. jejuni ATCC 33560 at the subinhibitory concentrations. The inhibitory effects of AI-2 mediated quorum sensing activity in C. jejuni by the compounds were determined as the intensity of bioluminescence signal emitted by the reporter strain V. harveyi BB170. Among the 12 compounds, both decanoic acid and lauric acid significantly inhibited AI-2 activities of C. jejuni, whereas hexanoic acid, three furanones, crotonic acid, and trans-cinnamaldehyde increased the AI-2-induced bioluminescence (Figure 3.1). Octanoic acid, trans-ferulic acid, quercetin, and naringenin had no significant effect on AI-2 activity of C. jejuni (P > 0.05). Therefore, decanoic acid and lauric acid might be able to quench AI-2 mediated quorum sensing activities in C. jejuni. In addition, there was no significant difference in bioluminescence activities between MH 41 broth control and ethanol control (P > 0.05). Thus, the effect of carrier ethanol on C. jejuni AI-2 activity at 0.5% was negligible. Figure 3.1 Screening of C. jejuni autoinducer-2 inhibitor. Bioluminescence activity of C. jejuni ATCC 33560 treated with 12 different compounds was compared to the ethanol control. Each bar represents the average of three biological replicates with standard deviation. MHB is shown as broth control and EtOH is shown as negative control. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered as statistically significant). Inhibition of quorum sensing of C. jejuni with decanoic acid and lauric acid during biofilm formation Based on the results obtained from the screening study described in section 3.2.1, decanoic acid and lauric acid were selected as the potential quorum sensing inhibitors for further experiments. Inhibitory effects of C. jejuni quorum sensing activity by decanoic acid and lauric acid during biofilm formation were evaluated using V. harveyi AI-2 assay. AI-2 activities of all four C. jejuni strains were significantly reduced (P < 0.05) by both decanoic acid and lauric acid at all the MHB EtOH A B C D E F G H I J K L0.05.0x1031.0x1041.5x1042.0x1042.5x104A: Hexanoic acidB: Octanoic acidC: Decanoic acidD: Lauric acidE: 4-Hydroxy-2,5-dimethyl-3(2H)-furanoneF: 5-Ethyl-4-hydroxy-2-methyl-2(2H)-furanoneG: 2,5-Dimethyl-3-oxo-2(H)-fur-4-yl butyrateH: Crotonic acidI: Trans-Ferulic acidJ: QuercetinK: Trans-cinnamaldehydeL: NaringeninInduced bioluminescence in RLU ** ******42 tested subinhibitory concentrations (100 ppm, 50 ppm, 25 ppm) compared to the ethanol control (Figure 3.2). Addition of different concentrations of either decanoic acid or lauric acid demonstrated a concentration-dependent inhibitory effect on C. jejuni AI-2 activity. In general, lauric acid at 100 ppm showed the most substantial quorum sensing quenching effect on all C. jejuni strains used in the current study. However, the inhibitory effects of decanoic acid and lauric acid varied among different C. jejuni strains. For example, AI-2 activity of C. jejuni 81-116 was reduced >95% by lauric acid at all three concentrations after 3-day incubation (Figure 3.2B). In contrast, lauric acid at 25 ppm only decreased 65% of AI-2 activity of C. jejuni NCTC 11168 (Figure 3.2C). 43 Figure 3.2 Inhibition of C. jejuni AI-2 activity by decanoic acid (DA) and lauric acid (LA). C. jejuni strain F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D). Bioluminescence emission of V. harveyi BB170 with the addition of cell-free supernatant collected from biofilm supernatant of four C. jejuni strains incubated with or without the treatment for 72 h at 37°C under microaerobic condition was separately determined. Measurements were performed at 4.5 h after the addition of cell-free supernatant. MHB is shown as broth control and EtOH is shown as negative control. Bioluminescence level of the negative control (EtOH) was set at 100%. Bioluminescence emission of each treatment was normalized to the control. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of EtOH)**** **AMHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120MHB: MH brothEtOH: MHB + 0.5% EtOHDA1: DA at 100 ppmDA2: DA at 50 ppmDA3: DA at 25 ppmLA1: LA at 100 ppmLA2: LA at 50 ppmLA3: LA at 25 ppmBioluminescence emission (% of EtOH)* *** * *BMHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of EtOH)** ****CMHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of EtOH)** *** *D44 Interaction of C. jejuni cell-free-supernatant with decanoic acid and lauric acid To investigate whether there was a direct interaction between fatty acids and C. jejuni AI-2 molecules, CFSs collected from C. jejuni overnight culture were directly reacted with decanoic acid and lauric acid at the same concentration as that used in section 3.2.2. The inhibitory effect was significant (P < 0.05) in all the treatment groups except decanoic acid at 25 ppm on C. jejuni NCTC 11168 and ATCC 33560 (Figure 3.3C, D). The results of direct reaction study were similar to the results shown in section 3.2.2. Concentration-dependent inhibitory effect of decanoic acid and lauric acid was also observed. Lauric acid at 100 ppm showed the most potent inhibition against C. jejuni AI-2 activity. 45 Figure 3.3 Influence of decanoic acid (DA) and lauric acid (LA) on AI-2 activity in C. jejuni cell-free-supernatant (CFS). C. jejuni strain F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D). CFS from each C. jejuni culture grown in MHB was incubated with treatment for 1 h, respectively. Bioluminescence emission of V. harveyi BB170 with the addition of each CFS was detected 4.5 h after the addition of CFS. MHB is shown as broth control and EtOH is shown as negative control. Bioluminescence level of the negative control (EtOH) was set at 100%. Bioluminescence emission of each treatment was normalized to the control. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of control)******AMHB: MH brothEtOH: MHB + 0.5% EtOHDA1: DA at 100 ppmDA2: DA at 50 ppmDA3: DA at 25 ppmLA1: LA at 100 ppmLA2: LA at 50 ppmLA3: LA at 25 ppmMHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of control)******BMHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of control)*****CMHB EtOH DA1 DA2 DA3 LA1 LA2 LA3020406080100120Bioluminescence emission (% of control)**** *D46 3.3 Effect of fatty acids on C. jejuni biofilm biomass The effects of decanoic acid and lauric acid at different subinhibitory concentrations on C. jejuni biofilm formation are presented in Figure 3.4. In general, there was a considerable discrepancy between the inhibitory effect on quorum sensing and biofilm formation. Although decanoic acid and lauric acid were potent on inhibiting AI-2 activity of C. jejuni, it was ineffective in inhibiting C. jejuni biofilm formation (Figure 3.4). Among the four tested C. jejuni strains, decanoic acid at 100 ppm reduced the total biofilm biomass of C. jejuni F38011 and NCTC 11168 by 35% and 10%, respectively (Figure 3.4A, B). Lauric acid at 100 ppm reduced biofilm formation of three C. jejuni strains (F38011, NCTC 11168, and ATCC 33560), ranging from 50% for NCTC 11168 to 10% for F38011 and ATCC 33560. In contrast, biofilm formation of C. jejuni 81-116 was significantly stimulated by decanoic acid and lauric acid by 200% compared to that of negative control. Interestingly, the concentration-dependent effect was also observed on the stimulating effect on C. jejuni 81-116 biofilm formation. Both decanoic acid and lauric acid at the highest concentration had the strongest biofilm stimulating effect. In addition, stimulating effect on C. jejuni NCTC 11168 biofilm formation was also observed at 50 ppm decanoic acid, 25 ppm decanoic acid, and 25 ppm lauric acid treated groups. Lauric acid at 100 ppm reduced ~50% of C. jejuni NCTC 11168 biofilm, whereas lauric acid at 25 ppm stimulated C. jejuni NCTC 11168 biofilm formation to 225% compared to that of negative control. 47 Figure 3.4 Effect of decanoic acid (DA) and lauric acid (LA) on biofilm formation of C. jejuni strains. C. jejuni strain F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D). Total biomass of the formed biofilms was quantified by crystal violet staining at OD595 nm after 72 h incubation with or without the treatment at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered as statistically significant). MHB EtOH DA1 DA2 DA3 LA1 LA2 LA30.00.10.20.30.40.50.60.70.8Mean absorbance at OD595 nm* *** * *AMHB EtOH DA1 DA2 DA3 LA1 LA2 LA30.00.10.20.30.40.50.60.70.8Mean absorbance at OD595 nm** *** *BMHB EtOH DA1 DA2 DA3 LA1 LA2 LA30.00.10.20.30.40.50.60.70.8Mean absorbance at OD595 nm* ***CMHB EtOH DA1 DA2 DA3 LA1 LA2 LA30.00.10.20.30.40.50.60.70.8Mean absorbance at OD595 nm* *DMHB: MH brothEtOH: MHB + 0.5% EtOHDA1: DA at 100 ppmDA2: DA at 50 ppmDA3: DA at 25 ppmLA1: LA at 100 ppmLA2: LA at 50 ppmLA3: LA at 25 ppm48 3.4 Effect of fatty acids on C. jejuni survival in biofilm and supernatant A large portion of total biomass of the formed biofilm is EPS rather than bacterial cells. The mature bacterial biofilm is able to release the encased bacterial cells from the biofilm and disperse the planktonic cells to the environment nearby (Flemming et al., 2016). Both planktonic cells in the supernatant and biofilm-associated cells were quantified so as to evaluate the effect of quorum sensing quenching fatty acids on the survival of C. jejuni cells in both biofilm and supernatant. For C. jejuni F38011, all treatments significantly reduced viable cells inside the biofilm, but the reduction of viable planktonic cells was only observed for lauric acid treatment group (Figure 3.5). Lauric acid at 100 ppm achieved a 3.5-log reduction on planktonic cells and a 3-log reduction on biofilm-associated cells. 49 Figure 3.5 Survival of C. jejuni F38011 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). Similar observations were identified for C. jejuni 81-116. Most decanoic acid and lauric acid treatments reduced viable biofilm-associated cells, except decanoic acid at 100 ppm that had no MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3 MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3Planktonic Biofilm012345678910Log CFU/mL Planktonic Biofilm** ** **** *50 significant difference from negative control group (Figure 3.6). Lauric acid significantly reduced viable planktonic cells of C. jejuni 81-116 in supernatant after 3-day incubation (P < 0.05). Figure 3.6 Survival of C. jejuni 81-116 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3 MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3Planktonic Biofilm012345678910Log CFU/mL Planktonic Biofilm** *** * *51 Decanoic acid was relatively ineffective in the reduction of viable C. jejuni NCTC 11168 and ATCC 33560 cells. No significant reduction of viable cells was identified either in the supernatant or biofilm with decanoic acid or lauric acid treatment. However, lauric acid at 100 ppm reduced ~2-log viable planktonic and biofilm-associated cells of C. jejuni NCTC 11168 and ATCC 33560 after 3-day incubation. Figure 3.7 Survival of C. jejuni NCTC 11168 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3 MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3Planktonic Biofilm012345678910Log CFU/mL Planktonic Biofilm****52 Figure 3.8 Survival of C. jejuni ATCC 33560 planktonic cells in supernatant and biofilm-associated cells in biofilms after fatty acid treatment. Plating assay was applied to quantify the survived planktonic cells (left) and biofilm-associated cells (right) incubated with or without the treatment for 72 h at 37°C under microaerobic condition. MH broth and MH broth containing 0.5% EtOH are served as broth control and negative control, respectively. Three biological replicates were performed. * Statistical significance between negative control and treated samples was determined by one-way ANOVA coupled with Dunnett's multiple comparisons test (P < 0.05 was considered statistically significant). 3.5 Effect of fatty acids on C. jejuni cell motility Flagella-driven motility has been validated to be one of the virulence factors of most C. jejuni strains because it is critical for its colonization (Malik-Kale et al., 2007; Plummer, 2012). This MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3 MHB EtOH DA1 DA2 DA3 LA1 LA2 LA3Planktonic Biofilm012345678910Log CFU/mL Planktonic Biofilm** **53 bacterial process has been associated with AI-2 mediated system and also plays an important role in biofilm formation (Guerry, 2007). Both decanoic acid and lauric acid at 50 ppm and 25 ppm inhibited C. jejuni migration among all tested trains. C. jejuni motility level varied among different strains. C. jejuni 81-116 and NCTC 11168 are more motile than F38011 and ATCC 33560. The inhibition of C. jejuni migration was more obvious at a high concentration of the treatment. 54 Figure 3.9 Inhibition of C. jejuni cell motility by lauric acid (LA) and decanoic acid (DA). Swarming of C. jejuni F38011 (A), 81-116 (B), NCTC 11168 (C), and ATCC 33560 (D) in soft agar (MHB+0.4% agar) was recorded in the absence and presence of specific concentrations of fatty acids after 72 h incubation at 37°C in a microaerobic condition. Soft agar containing 0.5% carrier solvent, ethanol (EtOH) is served as the negative control. 55 Chapter 4: Discussion Despite the ongoing attempts to control Campylobacter-associated foodborne illnesses, Campylobacteriosis remains the leading cause of human gastroenteritis in the world (Marder et al., 2017). In 2016, the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS) updated food safety standards with lower tolerances of Campylobacter in raw poultry products so as to reduce the related illnesses (FSIS, 2016). The more stringent regulations of Campylobacter in poultry production have brought challenges to food industry because of the high prevalence of Campylobacter-positive flocks and widespread occurrence of Campylobacter in the agri-food chain (Wagenaar et al., 2013). Therefore, developing more effective control measures is highly necessary to reduce the prevalence of Campylobacter in the agroecosystem. C. jejuni is the most commonly reported Campylobacter species that cause human infections (Havelaar et al., 2015). C. jejuni could form biofilm on a range of surfaces, such as glass (Joshua et al., 2006), polystyrene plastic (Gunther IV & Chen, 2009), nitrocellulose membrane (Kalmokoff et al., 2006), and stainless steel (Sanders et al., 2007). A considerable amount of studies have examined the importance of biofilm formation in the survival of C. jejuni in the unfavourable conditions (Feng et al., 2018; Oh et al., 2016; Reuter et al., 2010). Since the gene related to the production of quorum sensing molecule, luxS, has been identified in C. jejuni, many studies have investigated the role of AI-2 mediated quorum sensing system in this microbe. AI-2 signalling system plays critical roles in many biological processes of C. jejuni, including biofilm formation, motility, and chemotaxis. The luxS-deficient mutants showed reduced biofilm formation and motility in some C. jejuni strains (Holmes et al., 2009; Plummer 56 et al., 2011; Šimunović et al., 2020). Interfering bacterial communication is regarded as a novel approach for pathogen control. Compounds that can inhibit quorum sensing process of C. jejuni might be able to inhibit biofilm formation and reduce the survival of C. jejuni inside biofilm. The main strategies for AI-2 inhibition are to use synthetic/natural substances to either inhibit the enzymes required for signaling molecule production or block the transducers along the signal transmission pathway (Mina & Chbib, 2019; Zhao et al., 2018). Several chemically synthesized or modified luxS inhibitors have been reported to enable inhibiting the activity of LuxS enzyme and AI-2 mediated processes (Alfaro et al., 2004; Malladi et al., 2011; Zang et al., 2009). However, the synthesis of these compounds involves complicated chemical reactions. In addition, the toxicities of these compounds to humans are not comprehensively evaluated yet, so that their application in agri-food industry is still limited. In recent years, plant-based quorum sensing inhibitors have been gaining more attention due to high consumer acceptability and safety (Baptista et al., 2019; Meireles et al., 2016). Many plant-based compounds have been investigated for quorum sensing inhibition in P. Pseudomonas (Sarkar et al., 2014; Zhang et al., 2014), E. coli (Priya et al., 2013), and S. Typhimurium (Girennavar et al., 2008), but only a few studies have been conducted using natural compounds to quench quorum sensing in C. jejuni (Bezek et al., 2016; Castillo et al., 2014; Castillo et al., 2015). In the current study, the inhibitory effects of several natural-origin compounds on C. jejuni AI-2 activity were investigated. Two medium-chain fatty acids, namely decanoic acid and lauric acid, were able to significantly reduce AI-2 activity in four C. jejuni strains at subinhibitory concentrations (Figure 3.2). Inhibition of AI-2 activity can be achieved by different mechanisms. Some quorum sensing inhibitors target AI-2 production-related enzymes and result 57 in the reduction of AI-2 synthesis (Lee et al., 2004). Quorum sensing antagonists are another type of inhibitors that hinder the perception of signalling molecules (Ni et al., 2008). Besides, enzyme degradation and chemical denaturation of signalling molecules were also validated to be effective strategies to reduce quorum sensing activity (Liu et al., 2010; Yates et al., 2002). The mechanisms of quorum sensing inhibition in V. harveyi, Pseudomonas spp., and E. coli have been explored to unravel their molecular biological fundamentals (Papenfort & Bassler, 2016). However, AI-2 signalling pathway and its chemical structure of C. jejuni remain to be studied. The mechanisms of quorum sensing inhibition by decanoic acid and lauric acid has not been elucidated due to a significant knowledge gap. Inhibition of AI-2 activity was observed when we directly incubated decanoic acid and lauric acid with AI-2 containing C. jejuni CFSs (Figure 3.3). A previous study reported that several long-chain fatty acids identified from poultry meat wash were able to reduce the activity of in vitro synthesized AI-2. The authors speculated that the inhibition could be caused by either direct interaction with AI-2 molecules or interference of AI-2 receptor (Widmer et al., 2007). Similarly, another study demonstrated that chicken juice inhibited AI-2 activity in C. jejuni NCTC 11168, and there was possible direct interaction between food matrix and AI-2 molecules (Gölz et al., 2012). In our preliminary experiment, we confirmed that the treatment of decanoic acid and lauric acid at 200 ppm did not affect the viability of V. harveyi BB170. Thus, we speculated that C. jejuni AI-2 molecules might interact with decanoic acid and lauric acid and resulted in the loss of activities. 58 C. jejuni biofilm formation arguably has been recognized as one of AI-2 mediated bacterial social behaviours influenced by quorum sensing (Plummer, 2012; Teren et al., 2018). Reeser and co-authors reported that biofilm formation level was lower in luxS-deficient C. jejuni mutant compared to that of wildtype, and biofilm formation ability of the mutant was partially restored by adding AI-2 containing C. jejuni CFS (Reeser et al., 2007). However, another study indicated that the phenotypes of luxS-deficient C. jejuni mutants varied along with the changes of mutagenesis, strains, and cultivation conditions (Adler et al., 2014). Moreover, luxS protein is identified to be essential for s-adenosyl-l-methionine recycle via the activated methyl cycle, and this biological process generates an important methyl donor for DNA methylation (Vendeville et al., 2005). Deletion of luxS could affect not only AI-2 production but also DNA methylation-regulated gene expression. Therefore, biofilm formation ability of ∆luxS C. jejuni mutant may not reflect the quorum sensing inhibitory effect of AI-2 inhibitors (Plummer, 2012; Winzer et al., 2002). Besides, biofilm formation potential of different C. jejuni strains is different due to the variations in genetic features. Brown and co-authors demonstrated that C. jejuni NCTC 11168 could form slightly more biofilm than C. jejuni 81-116 on polystyrene surface under the microaerobic condition (Brown et al., 2014). Similarly, the difference in biofilm formation potential of C. jejuni 81-176 and C. jejuni RM 1221 was described by Gunther and Chen. They indicated that C. jejuni 81-176 was able to form biofilm on multiple surfaces, but C. jejuni RM 1221 could not form biofilm (Gunther IV & Chen, 2009). Because of the potential difference, we used four C. jejuni isolates from different origins in the current study to ensure the results to be representative. 59 We observed that biofilm formation capabilities of four C. jejuni strains varied (Figure 3.4). C. jejuni 81-116 and NCTC 11168 formed relatively more biofilm than F38011, and C. jejuni ATCC 33560 had the lowest biofilm formation among four strains. This finding was in good agreement with some other previous studies (Brown et al., 2014; Teh et al., 2010). For anti-biofilm activity of decanoic acid and lauric acid, we identified that there were large disparities among different C. jejuni strains. For C. jejuni F38011, both decanoic acid and lauric acid significantly reduced biofilm formation at all tested subinhibitory concentrations, and the inhibitory effects increased along with the increase in concentration of the fatty acids used (Figure 3.4A). Biofilm formation reduction was also observed for both C. jejuni NCTC 11168 and ATCC 33560 treated by lauric acid at 100 ppm. Decanoic acid and lauric acid could reduce biofilm formation of C. jejuni NCTC 11168 by 12% to 50%, and ~10% biofilm formation of C. jejuni ATCC 33560 was reduced by fatty acids. These results were inconsistent with the previous findings of citrus extracts that reduced AI-2 activity and biofilm formation of C. jejuni (Castillo et al., 2014). The authors indicated that citrus extracts could inhibit biofilm formation of C. jejuni NCTC 11168 by 60% to 75% in a 72-h incubation period. AI-2 inhibitory effect of citrus extracts was determined only by direct mixing the extracts with C. jejuni CFSs, but the inhibitory effects during biofilm formation were not tested. Another difference worth noting is that biofilm cultivation in the study by Castillo and co-authors was on a stainless coupon at 42°C instead of a polystyrene microtiter plate at 37°C used in the current study. Biofilm formation capabilities of C. jejuni were validated to be largely affected by environmental conditions and adhesion surface properties (Teh et al., 2016; Wu et al., 2016). In sum, anti-biofilm activity of quorum sensing inhibitors can vary depending on the variations in temperature and surface materials. 60 Contrary to the findings of other natural AI-2 inhibitors, biofilm formation of C. jejuni 81-116 was significantly enhanced by decanoic acid and lauric acid and showed a concentration-dependent manner (Figure 3.4B). Similarly, lauric acid increased C. jejuni NCTC 11168 biofilm formation at 25 ppm but suppressed biofilm formation at 100 ppm (Figure 3.4C). To the best of our knowledge, this was the first study to investigate quorum sensing quenching of C. jejuni 81-116 using medium-chain fatty acids and test the antibiofilm capabilities of fatty acids. C. jejuni 81-116 showed distinctive characteristics on the relationship between quorum sensing quenching and biofilm formation compared to other tested strains. Although suppression of C. jejuni biofilm formation by inhibiting AI-2 activity is a common theme, the inconsistent phenotypes of luxS-deficient mutants derived from different C. jejuni strains indicate different AI-2 responses within Campylobacter species (Plummer, 2012; Teh et al., 2010). A previous study reported that the deletion of luxS in Helicobacter pylori resulted in a twice amount of biofilm formed compared to that by wildtype strain (Cole et al., 2004). Coincidentally, biofilm formation of Bacillus cereus was significantly increased at early stage by adding synthesized AI-2 molecules (Auger et al., 2006). In another study, formation of P. Pseudomonas biofilm was identified to be affected by AI-2 in a concentration-dependent manner (Li et al., 2015). Based on these, we speculated that different C. jejuni strains might respond to different equilibrium concentrations of AI-2, and the same quorum sensing inhibition level could affect different strains in a variety of manners. Biofilm formation quantified by crystal violet staining reflects the total mass of the formed biofilm, including EPS (Wilson et al., 2017). EPS matrix contains various polysaccharides, proteins, lipids, as well as cell appendages from lysed bacterial cells (Hori & Matsumoto, 2010). 61 A mature biofilm can not only protect its encased bacterial members but also facilitate cell-to-cell communication and disseminate the planktonic cells into the environment (Flemming et al., 2007; Reuter et al., 2010). Viable counts of C. jejuni cells in biofilm and biofilm supernatant aid in determining biofilm formation level. By enumerating viable cells in the biofilm and biofilm supernatant, we identified that lauric acid was more effective than decanoic acid in reducing viable cells in biofilm supernatant for all tested C. jejuni strains. Decanoic acid treatment at all concentrations did not reduce viability of C. jejuni strains in biofilm supernatant. In contrast, significant reductions in viable cell counts in biofilm supernatant were identified for three C. jejuni strains (F38011, 81-116, ATCC 33560) with the treatment of 25 ppm or higher concentrations of lauric acid (Figure 3.5, Figure 3.6, and Figure 3.8). Although lauric acid at 50 ppm and 25 ppm did not affect the viability of C. jejuni NCTC 11168 planktonic cells, lauric acid at 100 ppm could reduce 2-log C. jejuni NCTC 11168 in the biofilm supernatant (Figure 3.7). Survival of C. jejuni inside biofilm treated with decanoic acid varied among different strains. Decanoic acid decreased viable cells in C. jejuni F38011 and 81-116 biofilms, while it did not significantly affect viable cells inside C. jejuni NCTC 11168 and ATCC 33560 biofilms (Figure 3.5–Figure 3.8). The results of C. jejuni survival were in line with quorum sensing inhibition studies, indicating that lauric acid was more potent on quorum sensing quenching. In addition, the increase of C. jejuni 81-116 biofilm biomass induced by fatty acids did not imply a higher number of viable cells inside the biofilm. Neither decanoic acid at 100 ppm nor lauric acid at 100 ppm affect viability of encased C. jejuni 81-116 cells in biofilm, despite 200% increase of total biofilm biomass was determined for these two treatment groups. Furthermore, lauric acid also reduced viable cells in biofilm supernatant of C. jejuni 81-116. Similar results were also observed in the treatment groups of lauric acid at 25 ppm and 50 ppm against C. jejuni 62 81-116. The biomass of C. jejuni 81-116 biofilm treated by lauric acid was at least 100% higher compared to that of the negative control (Figure 3.4B), but viable cell numbers in C. jejuni 81-116 biofilm were either decreased or not affected by lauric acid (Figure 3.6). Hence, we speculated that lauric acid might reduce the shedding of C. jejuni 81-116 cells from the biofilm so that viable cells in biofilm supernatant were decreased. On the other hand, a decrease in cell dispersion might cause accumulation of cells inside biofilm. Accelerated depletion of nutrients and accumulation of wastes in biofilm could affect cell viability. Besides EPSs amount, three-dimensional structure of biofilm is also vital to bacteria survival inside the biofilm (Hardie & Heurlier, 2008). C. jejuni motility mediated by AI-2 was determined to be associated with the development of biofilm structure and survival of C. jejuni (Reeser et al., 2007; Turonova et al., 2015). For example, C. jejuni 81-176 was validated to form a thicker biofilm and enclose a higher number of cells than that by C. jejuni NCTC 11168. C. jejuni 81-176 biofilm had pores and channels; in contrast, C. jejuni NCTC 11168 had a thin and compact structure (Turonova et al., 2015). The porous open ultrastructure of C. jejuni 81-176 biofilm allows more efficient oxygen penetration and waste diffusion. Most C. jejuni strains are aerotactic and not able to maintain viability under anaerobic condition for a prolonged period (Marchant et al., 2002). Therefore, sessile C. jejuni cells in biofilm would encounter unfavourable stressful conditions due to the limited access of oxygen and nutrients (Joshua et al., 2006). In addition, inhibition of AI-2 activity has been proposed as a strategy to reduce motility mediated by quorum sensing (Girennavar et al., 2008; Vikram et al., 2011). Herein, we evaluated the effects of decanoic acid and lauric acid on the motility of C. jejuni and identified that both decanoic acid and lauric acid were able to reduce the motility for all tested strains (Figure 3.9). 63 Although the connection between AI-2 activity and motility of C. jejuni has not been identified, substantial efforts were applied to discover the role of quorum sensing in regulating C. jejuni motility. A previous study carried out by Elvers and Parked (2002) illustrated that the growth halo of ΔluxS mutant on soft agar plate was significantly smaller than that of the wildtype strain. Since then, other studies also used soft agar method to exam the motility change of luxS null mutant, and a consistent conclusion was reached that deletion of luxS resulted in reduction of C. jejuni motility (Holmes et al., 2009; Plummer et al., 2011; Quiñones et al., 2009). In addition, Jeon and co-authors (Jeon et al., 2003) attempted to explain the change of motility via molecular techniques. They determined the expression change of flaA and flaB, two flagellin genes identified in C. jejuni 81-116. The flaA was downregulated by over 50% in ΔluxS mutant compared to that of wildtype strain, although flagellar structure of the mutant remained the same as that of wildtype strain. In more recent studies, 2(5H)-furanone, epigallocatechin gallate and citrus extracts were identified as potential AI-2 inhibitors against C. jejuni and could reduce C. jejuni motility at the concentration below the minimal bactericidal concentration (Castillo et al., 2014; Castillo et al., 2015). The results in the current study were in agreement with these previous findings. As potential AI-2 inhibitors, both decanoic acid and lauric acid could reduce C. jejuni motility. Because of current knowledge gap of C. jejuni quorum sensing at molecular level, further study is thus required to unravel the quorum quenching mechanism and exploit potential candidates of quorum sensing inhibitors. Antimicrobial effects of organic acids have been extensively investigated and used for foodborne pathogen control (Ricke, 2003). However, quorum sensing quenching effect of fatty acids has barely been studied yet. Both decanoic acid and lauric acid at 5000 ppm have been validated to 64 inactive C. jejuni in an acidic environment (Molatová et al., 2010). Some short-chain and medium-chain fatty acids can also be used as sanitizers for food contact surfaces with pH <4.0 (Gaulin et al., 2011; Marriott et al., 2018). Nevertheless, carboxylic acid at high concentration and low pH are corrosive to equipment, and long-term use of acidic solution may create niches for biofilm development due to equipment tear and wear (Schmidt, 1997). Using decanoic acid and lauric acid as quorum sensing inhibitors, in contrast, would reduce AI-2 activity at neutral pH and much lower concentration (≤100 ppm). Many studies demonstrated the great potential of quorum sensing inhibitors in controlling bacterial infections because it could increase the susceptibility of pathogens to antimicrobials and physical treatments as well as decrease the virulence of pathogens (Brackman, Cos, et al., 2011; Chen et al., 2018; Jiang et al., 2019). Our current results provide a new insight into a broader use of fatty acids for C. jejuni control in the environment and agri-food chain. 65 Chapter 5: Conclusion 5.1 Study summary This thesis project identified two natural-origin AI-2 inhibitors, namely decanoic acid and lauric acid, and investigated their effect on biofilm formation, survival, and motility of four C. jejuni strains. The hypothesis of this study was that effective AI-2 inhibitors could reduce C. jejuni quorum sensing without affecting cell viability, and reduction of AI-2 activity could suppress the quorum sensing-mediated biofilm formation of C. jejuni. Firstly, we determined the subinhibitory concentrations of twelve candidates and examined their quorum sensing inhibitory effect at the subinhibitory concentrations. Two medium-chain fatty acids, namely decanoic acid and lauric acid, are the most potent AI-2 inhibitors among the twelve natural compounds. AI-2 activity was quantified in C. jejuni biofilm supernatant cultivated with and without decanoic acid and lauric acid. AI-2 activity was reduced by over 50% for treated groups after 72-h incubation. Direct addition of either decanoic acid or lauric acid to C. jejuni CFS also decreased AI-2 activity in a concentration-dependent manner. After confirmed the quorum sensing quenching capability of two fatty acids, we investigated their anti-biofilm effect on C. jejuni. Both decanoic acid and lauric acid reduced C. jejuni F38011 biofilm formation at all the tested concentrations (25 ppm, 50 ppm, and 100 ppm), whereas reduction of biofilm formation of C. jejuni ATCC 33560 was only achieved by decanoic acid and lauric acid at 100 ppm. Addition of decanoic acid and lauric acid resulted in increased biofilm formation of C. jejuni 81-116 and NCTC 11168. In contrast, viable cells in biofilm and spent supernatant were either lower or no different compared to the control for all C. jejuni strains, including the strains that developed increased biofilm biomass. 66 Furthermore, the motility of four C. jejuni strains was impaired by either decanoic acid or lauric acid. Taken together, the effect of decanoic acid and lauric acid on C. jejuni biofilm formation might be determined by a complicated working mechanism, including AI-2 activity, motility, and the survival of the cells. 5.2 Future directions This study demonstrates the capability of decanoic acid and lauric acid in inhibiting AI-2 activity and reducing the motility of C. jejuni. A plethora of further work can be performed to explore the underlying mechanisms and test the real-world applications. The initial attachment is crucial for C. jejuni biofilm formation, and biofilm morphology is another important determinant of its stability and survival (Klančnik et al., 2020). The expression of surface proteins, motility, and change of phenotypic traits are affected by quorum sensing, and those factors are vital to cell adhesion and organization (Kalmokoff et al., 2006; Teh et al., 2016). The effect of AI-2 inhibitors on initial attachment and morphology changes of biofilm can be studied using a microfluidic platform coupled with confocal reflection microscopy (Feng et al., 2018; Yawata et al., 2010). This can help understand how AI-2 inhibitors affect the adhesion rate and structural development of biofilm. In real-world applications, this knowledge can help determine the appropriate cleaning and sanitation procedures to achieve the maximum inhibitory effect. The target and transportation of AI-2 inhibitors are rarely uncovered. In earlier studies, attempts were made to identify C. jejuni AI-2 signaling pathway. AI-2 molecule is not internalized by C. jejuni, and there is no AI-2 receptor encoded gene that has been identified in C. jejuni genome (Adler et al., 2015; Teren et al., 2018). A recent study detected E. coli K-12 AI-2 molecules by 67 high-performance liquid chromatography-tandem mass spectrometry. However, the method used 4,5-dihydroxy 2,3-pentanedione (a precursor of AI-2) internal standard, which may have a different structure of C. jejuni AI-2 molecule (Xu et al., 2017). Thus, the optimization and validation will be challenging due to the lack of AI-2 standard for C. jejuni. In addition, it will be challenging to determine if there is a direct interaction between AI-2 molecule and the inhibitor. Considering the applications in food industry, fatty acid-based quorum sensing inhibitors can be used as sanitizers in combination with other ingredients. Fatty acids belong to the Generally Recognized as Safe food additives (https://www.fda.gov/food/food-additives-petitions/food-additive-status-list#ftnF), and they are allowed to be applied onto food contact surfaces as no-rinse sanitizers when the concentration is below 200 ppm (Parker, 2007). 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CFS from each C. jejuni culture grown in MHB was incubated with the 0.1% trypsin for 1 hour, respectively. The bioluminescence emission of the V. harveyi BB170 with the addition of each CFS was detected 4.5 h after the addition of the CFS. Three biological replicates were performed. Each bar represents the average of three biological replicates with standard deviation. 050001000015000 NC TrypsinInduced Bioluminescence (RLU)85 Figure A.2 AI-2 Activity of C. jejuni cell-free-supernatants (CFSs) at different incubation periods. The C. jejuni overnight culture was adjusted to OD600nm = 0.3 and further diluted to ~2 × 108 CFU/mL. The time right after the dilution was taken as 0 h. The bioluminescence emission of the V. harveyi BB170 with the addition of cell-free supernatant collected from C. jejuni incubated for 0, 24, 48, and 72 h at 37°C under microaerobic condition for 72 hours. Measurements were performed 4.5 h after the addition of the cell-free supernatant. Three biological replicates were performed. Each bar represents the average of three biological replicates with standard deviation. 0 24 48 720200040006000800010000Induced bioluminescence (RLU) Time (h) F38011 81-116 11168 3356086 F38011 81116 11168 335600100020003000400050006000Induced Bioluminescence (RLU) Planktonic Sessile Figure A.3 AI-2 activity detected from C. jejuni biofilm and biofilm supernatant. The bioluminescence emission of the V. harveyi BB170 with the addition of cell-free supernatant collected from the biofilm and supernatant phases of C. jejuni strains that incubated at 37°C under microaerobic condition for 72 hours. Measurements were performed 4.5 h after the addition of the cell-free supernatant. Three biological replicates were performed. Each bar represents the average of three biological replicates with standard deviation. he averages of three biological replicates with standard deviation.
Thesis/Dissertation
eng
Food Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Identification of natural compounds to quench quorum sensing in Campylobacter jejuni for the reduction of biofilm formation
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