Antimicrobial Activity of Selected Phytochemicals against Escherichia Coli and Staphylococcus Aureus Cells and Biofilms

! ! ! ! Integrated Master in Bioengineering ! Antimicrobial activity of selected phytochemicals against Escherichia coli and Staphylococcus aureus cells and biofilms Dissertation for Master Degree in Bioengineering – Specialization in Biological Engineering Joana Isabel Carvalho Monte Supervisor: Manuel José Vieira Simões (PhD) ! June 2013 ! ! ! The present thesis was developed for the obtention of Master degree in Bioengineering, in the Faculty of Engineering of University of Porto. The work was carried out at LEPAE during 6 months. The main objective of the thesis was the evaluation of the efficacy of phytochemicals against Escherichia coli and Staphylococcus aureus planktonic cells and biofilms. The judge that approved the present document was composed by three elements: Luís de Melo (Cathedratic Professor), Maria da Conceição Fernandes (PhD), Manuel Simões (PhD). The author ____________________________________________________ (Joana Isabel Carvalho Monte) The supervisor ____________________________________________________ (Manuel Simões) ! ! ! “Try to learn something about everything, and everything about something.” Thomas H. Huxley ! !Master!Thesis! ! i! Acknowlegments Em primeiro lugar, gostaria de agradecer ao meu orientador, o Professor Doutor Manuel Simões, por toda a dedicação, disponibilidade e empenho ao longo da duração deste projeto. Gostaria também de agradecer à Ana Abreu, pela incansável ajuda e auxílio na realização deste trabalho. Estou também grata à Anabela e à Joana pelo apoio que me deram nalguns dos ensaios realizados no laboratório. Quero agradecer ainda a todos do laboratório E007, Carla, Luciana, Ritas, Catarina, Paula, Joanas, Madalena e Renato pela disponibilidade e simpatia. A todos os meus amigos, em especial às minhas colegas de laboratório Inês, Helena e Carolina pelos ótimos momentos proporcionados e pela sua companhia. Quero agradecer ainda ao João Manuel, ao Francisco, à Catarina e ao João Paulo, por estarem incondicionalmente do meu lado durante os últimos cinco anos. Um especial obrigada ao Nelson pela companhia, apoio e gargalhadas que me proporcionou. À Ana e à Cató por serem as minhas amigas, companheiras e confidentes e por sempre estarem do meu lado. Finalmente, de modo muito especial, um obrigada à minha família, mãe, pai, Helena e Marta, pelo incentivo, paciência e apoio incondicional durante a realização deste projeto. !Master!Thesis! ! ! ! ii! ! ! ! !Master!Thesis! ! iii! Abstract Antimicrobial resistance is one of the biggest problems facing global public health. The effectiveness of antimicrobial drugs has been lost due to the evolution of pathogen resistance. Plants are considered the greatest source to obtain new antimicrobials. They produce secondary metabolites, phytochemicals, which protect the plant against pathogens. The aim of this study was to assess the antimicrobial activity of four phytochemicals - 7-hydroxycoumarin (7-HC), indole-3-carbinol (I3C), salycilic acid (SA) and saponin (SP) – against Escherichia coli and Staphylococcus aureus and also understand their ability to control biofilm formation. Several experiments were carried out in order to: i) test the ability of phytochemicals to control planktonic bacteria growth through the measurement of minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC); ii) evaluate the phytochemicals action in the control of biofilms; iii) understand aspects of the phytochemicals mode of action against the bacteria. Results have shown that MIC values were higher for E. coli than for S. aureus. The 7-HC and I3C were the most effective, with MICs of 200 and 400 µg/mL for S. aureus, respectively, and 800 µg/mL against E. coli. Regarding MBC, 1600 and 5000 µg/mL were obtained for I3C and SA, respectively. It was also observed that 7-HC and SP has no significative effect in surface charge of E. coli; in contrast, I3C and SA make the membrane more and less negative, respectively. S. aureus surface charge was changed in contact with SA and SP. It was observed that phytochemical concentration did not affect the biofilm removal for both bacteria. E. coli biofilms are more susceptible to phytochemicals comparing to S. aureus biofilms. SA and SP promoted the increase and decrease of hydrophilic properties of E. coli, respectively. S. aureus became less hydrophilic in contact with 7-HC and SA. E. coli showed the highest motility and also an increasing in swimming and swarming motility over time. Motility was mostly affected when I3C was added. Swimming and sliding motilities were completely inhibited and swarming motility was not affected by I3C. The quorum-sensing results indicated that inhibition of violacein production was detectable with 7-HC, I3C and SA, with halos ranging from 5 to 19 mm. I3C was also the most effective phytochemical. The increasing of concentration resulted in an !Master!Thesis! ! ! ! x! !Master!Thesis! ! xi! Tables List Table 1. MIC for E. coli and S. aureus. ...................................................................... 23 Table 2. MBC values for E. coli and S. aureus. .......................................................... 24 Table 3. Percentages of biofilm removal and inactivation by the selected phytochemicals against E. coli and S. aureus. ..................................................... 26 Table 4. Zeta potential (mV) results of suspensions of E. coli and S. aureus in contact with phytochemicals at the MIC. ......................................................................... 39 Table 5. Hydrophobicity (∆𝐆𝐓𝐎𝐓), and apolar (γLW) and polar (γAB) components of the surface tension of untreated and treated cells. The means ± SDs are illustrated. 40 Table 6. Free energy of adhesion (∆𝐆𝐓𝐎𝐓bws) of bacterial cells to polystyrene, treated and untreated with phytochemicals. ..................................................................... 42 Table 7. Motility results for bacteria with and with phytochemicals. The drop baseline was 6mm which was subtracted from the results presented. ................. 44 Table 9. Antimicrobial activity of antibiotics. The means (mm) ± standard deviation for at least three replicates are illustrated. ........................................................... 51 Table 10. Classification of the effect of dual combinations of phytochemicals and antibiotics. ............................................................................................................ 52 Table A.1. Physico-chemical characterization of polystyrene (PS). ............................. I !Master!Thesis! ! ! ! xii! ! ! !Master!Thesis! ! xiii! Glossary ! Indexes and parameters: AB – Lewis acid-base component B – Bacteria B - α-bromonaphtalene F – Formamide LW – Lifshitz-van der Waals component S – Bubstratum TOT – Total W – Water swsBetween teo entities od a given surface when immersed in water bws – Between one bacteria and a substratum that are immersed or dissolved in water bwb – Between two bacterial surfaces, when immersed in water bw – Between bacteria and substratum sw – Between substratum and bacteria TOT – Total ∆G – Free energy of interaction (mJ/m2) γ - Surface free energy (mJ/m2) θ - Contact angle (°) + - Electron acceptor paramenter of Lewis acid-base component - - Electron donor parameter of Lewis acid-base component Abreviations: AHL - N-acyl-homoserines AI – Autoinducers CIP – Ciprofloxacin CV – Crystal Violet DMSO – Dimethyl sulfoxide EPS – Extracellular polymeric substances ERY - Erythromycin I3C – Indole-3-carbinol MIC – Minimum Inhibitory Concentration !Master!Thesis! ! ! ! xiv! MBC – Minimum Bactericidal Concentration MDR – Multi-drug resistant OD – Optical Density ODC – Cut-off optical density OMPs – Outer membrane proteins p – Statistical significance level PS – Polystyrene QS – Quorum-sensing QSI – Quorum-sensing inhibition RMAs – Resistance-modifying agents SA – Salycilic Acid SDS-PAGE – Sodium dodecyl sulfate polyacrylamide gel electrophoresis SP – Saponin SPSS - Statistical Package for the Social Sciences TET - Tetracycline WHO – World Health Organization 7-HC – 7-hidroxycoumarin !Master!Thesis! ! ! 1! Chapter 1 Work Outline 1.1. Background and Project Presentation Since the discovery of the first antibiotic, penicillin, the employment of any novel antibiotic has been followed by the appearance of bacterial resistance to that antibiotic in as little time as a few years. Antibiotics have the ability to kill bacteria or inhibit their growth. Resistance to antibiotics is one of the biggest problems that global public health is facing. Antimicrobial resistance is a natural consequence of the adaption of pathogens to the exposure to antimicrobials used in medicine, food, crop production and to disinfectants in farms and households. Resistant organisms cause infections that are more difficult to treat and more expensive; some strains have become resistant to all available antimicrobial agents (Byarugaba, 2004). Resistant infections affect treatment costs, disease spread and duration of illness (Okeke et al., 2005) !Master!Thesis! ! ! ! 2! In order to find novel antimicrobial agents with new modes of action, plants have been explored as sources for the identification of new and effective antibacterials. An endless number of plant species have been reported to act against several bacteria in vitro, and many medicinal plants produce secondary metabolites (phytochemicals) capable of inhibiting the growth of a wide range of microorganisms including fungi, yeasts and bacteria. Phytochemicals have been studied for the treatment of microbial infections since 1990, due to the increasing inefficacy of conventional antibiotics (Simões et al., 2009). 1.2 Main objectives The main aim of this work was to assess the antimicrobial efficacy of selected phytochemicals against Escherichia coli and Staphylococcus aureus planktonic cells and also to evaluate them on biofilm control. In the present study, four different phytochemicals – 7-hydroxycoumarin (7HC), indole-3-carbinol (I3C), salicylic acid (SA) and saponin (SP) – were tested against E. coli and S. aureus in both planktonic and sessile states. The strains tested are considered the most clinical significant bacteria due to their capacity to resist against several antibiotics (Simões et al., 2008; Xu et al., 2006). To evaluate the antimicrobial activity of the several phytochemicals two experiments were performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The biofilm control was also performed to understand the efficacy of phytochemicals to remove biomass and inactivate biofilm cells. The biofilm control assay was studied in 24 h aged biofilms after 1 h in contact with the phytochemicals. The biomass removal and metabolic inactivation were calculated through the optical density (OD) and fluorescence measurements. Several aspects of planktonic cells were evaluated to understand the mode of action of the selected phytochemicals. The surface charge of bacteria was studied through the measurement of Zeta potential and the hydrophobicity of cells was also assessed. To evaluate the potential activity of the phytochemicals to prevent E. coli and S. aureus adhesion to polystyrene (PS), the prediction of theoretical adhesion !Master!Thesis! ! ! 3! through the measurement of contact angles was performed. The phytochemicals were also studied on the ability to interfere with bacterial motility and quorum-sensing (QS), two microbial aspects involved in biofilm formation. The OMPs expression of E. coli was studied in contact with phytochemicals through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in order to detect the possible expression of resistance proteins. Regarding S. aureus resistant strains, dual combinations of phytochemicals and antibiotics – tetracycline (TET), ciprofloxacin (CIP) and erythromycin (ERY) - were tested to understand their ability to act in efflux pumps inhibition. 1.3 Thesis Organization In chapter 1, the context, motivations and main goals for the development of this thesis are explained. This chapter is also a guideline to the overall work, composed by 5 chapters. Chapter 2 includes the literature review about the main subjects of this work. In this chapter, the main problems associated with the appearance of bacterial resistance are highlighted. Plant products, especially secondary metabolites, are introduced as one of the solution for the antimicrobial resistance. The mode of action of phytochemicals is also developed in this chapter. Finally, it is reported the problem of the higher resistance associated to bacterial growing in biofilms and quorumsensing inhibition (QSI) is presented as one possible solution for the prevention of biofilm formation. In the third chapter are described the results of the activity of phytochemicals as antimicrobial agents against E. coli and S. aureus. The MIC and MBC of the selected phytochemicals are presented. In this chapter are also studied the phytochemicals in the control of biofilm, showing the biofilm removal and metabolic inactivation for each one of the phytochemicals at the MIC and 5 × MIC. Chapter 4 describes the study of surface charge and hydrophobicity characteristics of E. coli and S. aureus when exposed to phytochemicals. The influences of phytochemicals in motility and QS are also presented in this chapter. To finalize the chapter, OMPs expression of E. coli are studied when exposed to !Master!Thesis! ! ! ! 4! phytochemicals; and dual combination of phytochemicals and antibiotics are described in order to study the antimicrobial activity and their synergistic effects in S. aureus efflux pump inhibition. Finally, in chapter 5 the main conclusions of the work are referred and some recomendations for future research are given. !Master!Thesis! ! ! 5! Chapter 2 ! ! Literature Review 2.1 Antimicrobial resistance and phytochemicals There is a continuous search for new drugs and antibiotics in order to heal the main infectious diseases. However, the microorganisms have become resistant to most of the antibiotics. The microorganisms are successful when facing adverse conditions, because they seem to sense and respond to the external environment and modulate gene expression accordingly. Antimicrobial resistance is one of the biggest problems threatening global public health (Byarugaba, 2004; Okeke et al., 2005). This problem is a natural consequence of the adaption of infectious agents to antimicrobials used in several areas, including medicine, food animals, crop production and disinfectants in farms, hospitals and households (Bloomfield, 2002; McEwen and Fedorka-Cray, 2002; Vidaver, 2002; Wise and Soulsby, 2002). Resistance allows microorganisms to survive in the presence of toxic conditions. The effectiveness of many antimicrobial drugs has been lost due to the evolution of pathogen resistance. Many of the microorganisms are no longer susceptible to most of the existing antibiotics and therapeutic agents (Byarugaba, 2004). Bacteria generally !Master!Thesis! ! ! ! 12! The alkaloids are heterocyclic nitrogenous compounds that present analgesic, antispasmodic and bactericidal action (Ciocan and Bara, 2007; Das et al., 2010; Stary, 1996). 2.4. Mode of Action of Phytochemicals Bacterial growth can be inhibited by phytochemicals through several mechanisms. These plant products can act on various biochemical targets on the bacterial cells. The mode of action of phytochemicals is not completely understood, neither the phytochemical antibacterial specificity (Simões et al., 2009). Some experiments have been done to study the mode of action of several phytochemicals. The chemical structure and properties influence the site of action of phytochemicals. The mechanism of action of essential oils against bacteria involves membrane disruption through the lipophilic structure (Griffin et al., 1999; Mendoza et al., 1997). Alkaloids, such as berberine and piperine, interact with bacterial cytoplasmic membrane, intercalate with DNA or inhibit efflux pumps in S. aureus (Khan et al., 2006). Phenols act by interruption of energy production due to enzyme inhibition by the oxidized products, which react with sulfhydyl groups or non-specific interaction with proteins (Mason and Wasserman, 1987). In the case of flavonoids, they inhibit the synthesis of nucleic acids of Gram-negative and Gram-positive bacteria (Cushnie and Lamb, 2005; Mori et al., 1987). Other authors, shown that glycoside saponins are able to induce pore-like structures that change the membrane permeability; they can also interfere with energy metabolism (Mandal et al., 2005; Melzig et al., 2001; Sinha Babu et al., 1997). ! ! 2.5. Biofilms and phytochemicals: Natural products have been isolated from plants for usage in biodeterioration control. Biodeterioration is the chemical and physical alteration resulting from !Master!Thesis! ! ! 13! biological activity. The microorganisms associated to biodeterioration growth as biofilms that adhere to substrates. The treatment for this problem involved the use of biocides, however these are chemical agents and most of them are cytotoxic. So, natural compounds from plants with biocidal activity have emerged, and they are promising alternative for the control of biodeterioration without negative impacts of the environment (Guiamet et al., 2006). The chemical biocides are toxic and difficult to degrade, being persistent in the environment, causing chemical contaminations and the spread of resistance. In contrast, natural biocides, extracted from plants, are biodegradable and environmental friendly (Guiamet and Saravia, 2005). The phytochemicals can act as control agents on the bacterial biofilm formation and development (Simões et al., 2009). Guiamet and co-workers (2006) performed a study with Cichorium intybus, Arctium lappa and Centaurea cyanus from Asteraceae family to test their ability to be used as antimicrobial agents against different microorganisms associated with biodeterioration. These plants showed moderate activity against two species of Pseudomonas and no activity against Bacillus cereus. Rosmarinus officinalis L. extracts act as an antimicrobial agent against S. aureus. Allium sativum produce allicin, which is one of the most effective antimicrobial products isolated from garlic (Abreu et al., 2012). Biofilm formation is a feature closely related to pathogenicity (Ren et al., 2005). A biofilm is formed by planktonic bacteria that adhere to a surface and initiate the development of sessile microcolonies surrounded by an extracellular matrix (Otto, 2009) (Figure 2). Bacteria form complex surface-attached communities, also called biofilms (Hentzer and Givskov, 2003). Biofilms develop structures that are morphologically and physiologically differentiated from free-living bacteria (Davies et al., 1998). The process of biofilm formation includes several steps: preconditioning of the adhesion surface; planktonic cells are transferred from the bulk liquid to the surface; adsorption of cells at the surface; desorption of reversible adsorbed cells; the bacterial cells are adsorbed irreversibly at the surface; transport of substrates to the biofilm; substrate metabolism by the biofilm cells and transport of products out of the biofilm; finally, biofilm is removed by detachment or sloughing (Simões, 2005). The formation of biofilm is dependent of several parameters. Regarding to the surfaces, the attachment is easier on rough, hydrophobic and coated surfaces (Donlan, 2002; !Master!Thesis! ! ! ! 14! Pereira, 2001). Parameters like flow velocity, water temperature and nutrient concentration also influence the biofilm attachment (Pereira, 2001; Vieira, 1995). ! ! Biofilms are an example of physiological modifications and they also increase the tolerance to antimicrobial therapies and to the host immune response (Hentzer and Givskov, 2003; Simões et al., 2009). Most of the bacterial infections detected in human body involve the formation of biofilms. The biofilm mode of growth permits an increased bacterial survival in hostile conditions, such as in the presence of antibiotics and disinfectants (Hentzer and Givskov, 2003; Trentin et al., 2011). There is an increasing interest in preventing, controlling and eradicating biofilms. Biofilms by bacterial cells are thought to be regulated by autoinducer molecules; in a process called quorum-sensing (Ren et al., 2002). The impairment of bacterial adhesion and biofilm formation by a pathway that does not affect the bacterial death is an important feature of the new concept in antivirulence therapies (Trentin et al., 2011). This alternative should maintain the cells in a planktonic state, switching off the virulence expression and attenuating the pathogen, making the microorganisms more susceptible to antimicrobial agents and immune system (Macedo and Abraham, 2009; Martin et al., 2008). Bacteria in biofilms present a reduced susceptibility to antimicrobial agents caused by a variety of factors, such as: nutrient depletion within the biofilm, reduced access to cells in the biofilm, production of degradative enzymes and neutralizing Figure%2.%Model%of%biofilm%development.%Planktonic%cells%contact%with%surface%resulting%in%the% formation%of%microcolonies.%Cells%in%the%biofilm%can%return%to%a%planktonic%lifestyle%to% complete%the%cycle%of%biofilm%development.% !Master!Thesis! ! ! 15! chemicals, between others (Brown and Gilbert, 1993). Biofilms have been reported to be 100-1000 more protectors to bacteria than populations of planktonic cells (Gilbert et al., 2002; Mah and O’Toole, 2001; Stewart and Costerton, 2001). The main difference between planktonic cells and biofilms is the presence of a polysaccharide matrix, delaying the diffusion of antimicrobials into the biofilm (Brooun et al., 2000). Traditional treatment of infectious diseases is related with compounds that inhibit the growth of bacteria. But, it has been observed that they develop resistance to antimicrobial compounds. So, quorum-sensing seems to be the next opportunity to improve bacterial infection. Quorum-sensing influences bacterial biofilm growth and biofilm development that is related with cell-cell interactions (Simões et al., 2009). Quorum-sensing inhibitory compounds are the new line of antimicrobial agents and can be applied in several areas: medicine, agriculture and aquaculture (Hentzer and Givskov, 2003). Several biotechnology companies have already tried to develope some strategies to interrupt the bacterial quorum-sensing, such as: inhibition of N-acylhomoserines (AHL) signal recognition, signal dissemination and signal reception (Hentzer and Givskov, 2003). By interfering with cells communication, it is possible to interfere also in the resistance of biofilms and their ability to form resistant structures, causing cell dispersion. Quorum-sensing systems are involved in a wide range of microbial activities: extracellular enzyme biosynthesis, biofilm development, antibiotic biosynthesis, biosurfactant production, extracellular polymeric substances (EPS) synthesis and production of extracellular virulence factors (Chatterjee et al., 1995; Davies et al., 1998; Daniels et al., 2004; Fux et al., 2005; McGowan et al., 1995; Passador et al., 1993; Pearson et al., 1995; von Bodman and Farrand, 1995). Autoinducers (AI) are molecules to perceive the size of bacterial population and AHL are the major AI molecules. Several Gram-negative bacteria use AHL signals to coordinate the behaviour of cells in a population. An important achievement was the discovery of molecules produced by plants that mimic AHL signals, affecting quorum-sensing behavior. Several plants of medicinal use demonstrated potential to inhibit quorum-sensing (Hentzer and Givskov, 2003; Waters and Bassler, 2005). Studying the grapefruit and its furanocoumarins as inhibitors of biofilm formation, it has been shown that dihydroxybergamottin and bergamottin exhibit strong inhibition of both AI-1 and AI-2 activities even at concentrations as low as 1 !Master!Thesis! ! ! ! 16! µg/mL (Girennavar et al., 2008). Furanocoumarins are able to interfere with cell-cell signalling and also inhibit biofilm formation. However, the mechanisms of action are not completely understood (Girennavar et al., 2008). Delissea pulchra, an Australian macroalga, produces halogenated furanones, which are inhibitors of AHL, inhibiting bacterial quorum-sensing and biofilm formation (McLean et al., 2004). Auraptene and lacinartin, two compounds belonging to coumarin family, are promising natural compounds that can be used to prevent and treat periodontal diseases. They were also evaluated on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of Porphyromonas gingivalis. Lacinartin was able to inhibit biofilm formation and to cause desorption of a pre-formed biofilm of P. gingivalis. This suggests that coumarins may contribute to reducing tissue destruction (Marquis et al., 2012). Some information about the structure-activity relationship of coumarins showed that the group on position 5 and position 2’/3’ of the isoamylene chain can affect the antifouling activities against both Balanus albicostatus and Bugula neritina (Wang et al., 2013). Therefore, phytochemicals are not only important for the antimicrobial response and the substitution of antibiotics because of bacterial resistance, but also for the control of biofilms formation. Natural products are important sources of bioactive compounds and the medicinal plants used in folk medicine can facilitate the search of new agents. ! ! ! ! ! ! !Master!Thesis! ! ! 17! Chapter 3 ! Activity of Selected Phytochemical Products as Antimicrobials and in Biofilm Control ! ! ! ! 3.1 Introduction Microbiologists have learned to assess antibiotic effects in vivo by evaluating the MIC and MBC in vitro. These methods assess the influence of antibiotics against planktonic microorganisms in the exponencial phase of growth and predict the efficacy of antibiotcs against bacteria in infections (Ceri et al., 1999; Fux et al., 2005). Staphylococcus aureus and Escherichia coli are two human pathogens that can cause a variety of infections. The main charactheristic of these infections is the formation of biofilms (Beenken et al., 2004). In order to prevent biofilm formation, several studies have been performed to find antimicrobial agents that affect the viability of bacteria in biofilms. Natural products from plants have been shown to influence microbial biofilm formation (Rasooli et al., 2008). Plants are an important source of disinfection compounds as they produce a wide range of phytochemicals with antimicrobial properties, most of them against microorganisms, insects, nematodes and other plants (Abreu et el., 2013a). Phytochemicals are able to inhibit peptidoglycan synthesis, damage microbial !Master!Thesis! ! ! ! 18! membrane structures, modify bacterial membrane surface hydrophobicity and also modulate quorum-sensing (Rasooli et al., 2008). These processes can also inhibit or affect biofilm formation. To reach high killing rates and to avoid resistance and adaption, disinfectants are used at very high concentrations relative to their MICs (Abreu et al., 2013a). So, after the detection of MIC and MBC for the selected phytochemicals, they were tested at MIC and 5×MIC. The effects of phytochemicals on E. coli and S. aureus biofilms were evaluated through biomass prevention and metabolic inactivation. The quantification of biofilm removal takes into account both live and dead cells assessed by the Crystal Violet method (CV). CV is a dye wich binds to negatively charged surfaces in the extracellular matrix (Extremina et al., 2011; Peeters et al., 2008). Resazurin is used to quantify the viability of cells, based on the live cells. This compound is a blue redox indicator and it reduces to pink by contact with viable bacteria in the biofilm (Extremina et al., 2011). Pathogens are increasing their capacity to survive after contact with antimicrobials and disinfectants. Many of the existing antibiotics are ineffective due to their extensive and inappropriate use (Abreu et al., 2013a). Plants are important sources for the development of antimicrobials and strategies to control growth and biofilm formation (Abreu et al., 2013a). The purpose of this study was to assess the antimicrobial efficacy of selected phytochemicals (7-HC, I3C, SA and SP) against E. coli and S. aureus planktonic cells. Moreover, the effects of these phytochemicals were assessed on biofilm control. ! ! !Master!Thesis! ! ! 19! 3.2 Matherial and methods 3.2.1. Bacterial Strains The bacteria used in this study were obtained from the Spanish Type Culture Collection (CECT): the Gram-negative bacterium Escherichia coli (CECT 434) and the Gram-positive bacterium Staphylococcus aureus (CECT 976). The bacteria were distributed over the surface of Plate Count Agar (PCA – Merck) and incubated for 24 h at 30 ± 3 ºC. 3.2.2. Phytochemicals The phytochemicals used were: 7-hidroxicoumarin (7-HC), indol-3-carbinol (I3C), salicylic acid (SA) and saponin (SP). These compounds were obtained from Sigma-Aldrich (Portugal) and prepared in dimethyl sulfoxide (DMSO, Sigma). 3.2.3. Determination of Minimum Inhibitory Concentration The MIC is considered the lowest concentration of an antimicrobial that will maintain or reduce the growth of a microorganism after 24 h incubation (Andrews, 2001). MIC of phytochemicals was determined by microdilution method in sterile 96wells microtiter plates (McBain et al., 2004). The cell suspensions of S. aureus and E. coli were obtained by overnight cell cultures in Mueller-Hinton broth (MHB) (Fluka, Portugal)!and were adjusted to a OD620nm at 0.1±0.02 (corresponding to approximately 1×106 cells/mL) in the spectrophotometer (VWR V-1200). The suspension cells were added to sterile 96-well polystyrene microtiter plates (Orange Scientific) with different phytochemicals in several concentrations (25, 50, 100, 200, 400, 800, 1600, 3200 µg/mL) in a final volume of 200 µL. The volume of 7-HC, I3C, SA and SP added to each well was 10 µL. DMSO was used as a negative controls. No antimicrobial activity was detected by DMSO at this concentration (data not shown). After 24 h at 30 °C, the MIC of each sample was determined by measuring the optical density in the spectrophotometer (620 nm) (SpectraMax M2E, Molecular devices). MIC corresponds to the concentration in which the final OD is inferior or equal to the initial OD. !Master!Thesis! ! ! ! 20! 3.2.4. Determination of Minimum Bactericidal Concentration The MBC is defined as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media (Andrews, 2001). MBC of phytochemicals was determined by the drop method. After measuring the MIC, the wells corresponding to the phytochemicals concentrations equal and above the MIC were added (10 µL) to PCA plates. The drops were drained along the plate. After 24 h at 30 ºC, the plates were analysed and the MBC of each phytochemical corresponding to the concentration which inhibited the growth of the bacteria. 3.2.5. Biofilm formation and control in sterile 96-well polystyrene microtiter plates Biofilms were developed according to the modified microtiter plate test proposed by Stepanović et al. (2000). For both bacteria, at least 6 wells of a 96-well polysytrene microtiter plate were filled with 200 µL of overnight batch cultures in MHB (OD620nm= 0.04 ± 0.02). The plates were incubated overnight at 30 ºC and 150 rpm. The negative control wells were also placed on the plates, being sterile water and medium. Plates were incubated for 24 h at 30 ºC and agitated at 150 rpm. In order to test the effects of phytochemicals in several steps of the process there are various treatments that can be applied. However, in this case, the treatment applied consisted in incubating overnight the strains without phytochemicals. The biofilms were formed in microtiter plates for 24 h, and subsequently, were incubated with phytochemicals for 1 h. After biofilm development, the content of wells was removed and the wells were washed three times with 200 µL of NaCl (8.5 g/L) to remove reversibly adherent bacteria. The phytochemicals were added to the wells at the MIC and 5 × MIC. The microtiter plates were incubated for 1 hour. The remaining attached bacteria were analysed by using crystal violet and resazurin. !Master!Thesis! ! ! 21! 3.2.6. Biofilm analysis 3.2.6.1 Crystal Violet method Before phytochemicals application, the inoculum in the walls was removed and the wells were washed with 200 µL of sterile water. Later, 250 µL of ethanol were loaded for 15 minute to promote biofilm fixation. The supernatant was removed and the plates were air-dried. Subsequently, 200 µL of CV solution (Gram color staining set for microscopy, Merck) was added for 10 minutes to stain the fixed bacteria. After washing in water, the plates were dried and finally, the wells were loaded with 200 µL of aceditic acid 33% (v/v) (Merck) to release and dissolve the stain. To analyse the biofilm, the OD of the solutions was measured at 570 nm using a microtiter plates reader (SpectraMax M2E, Molecular Devices). After obtaining the values of absorbance, the percentage of biomass removal is obtained according equation 1: %!𝐵𝑖𝑜𝑚𝑎𝑠𝑠!𝑟𝑒𝑚𝑜𝑣𝑎𝑙 =!!"!!"#$%"&!"#!!"!!!!"#$!!"#$%&!"# !"!!"#$%"&!"# ×100 (1) where OD control570 represents the optical density of the control at 570 nm, and OD phytochemicals570 is the optical density of the phytochemical. 3.2.6.2 Resazurin Method In this method, a commercially available resazurin solution (Sigm) was used. The plates were loaded with 190 µL of sterile MH medium and 10 µL of resazurin solution. After 20 minutes of incubation at room temperature, fluorescence (λex:!570! nm!and!λem:!590!nm) was measured using the microtiter plates reader. After measuring the fluorescence, it is possible to calculate the percentage of metabolic inactivation: %!𝑀𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑐!𝑖𝑛𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 =!!"#$!!"#$%"&!!"#$!!!!"#$!!"#$%& !"#$!"#$%"& ×100 (2) where FLUOcontrol represents the fluorescence intensity of biofilms not exposed to phytochemicals and FLUOphytochemical represents the fluorescence intensity value for biofilms exposed to phytochemicals. ! !Master!Thesis! ! ! ! 28! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !Master!Thesis! ! ! 29! Chapter 4 ! Aspects Underlying the Antibacterial and Biofilm Control Action of Phytochemicals ! 4.1 Introduction The use of dual combinations of antimicrobial drugs with positive in vitro interactions has become an important parameter to evaluate in clinical applications. The appearance of this practice starts to prevent the emergence and widespread multidrug resistant (MDR) infections (Sopirala et al., 2010). Several antibiotics have been analysed for their action as resistance-modifying agents (RMAs), i.e. compounds able to modify or inhibit the bacterial resistance, so the antibiotics kill efficiently the resistant bacteria (Abreu et al., 2013b). The clinical effects and synergism of dual combinations have to be tested in vivo. Determination of synergy in vitro might not be reflected in vivo, due to the achievement of synergic levels of drugs in the tissues, differences in plasma protein binding and drug metabolism (Kalan and Wright, 2011). Dual combinations of phytochemicals and antibiotics were tested to understand their ability to act in efflux pumps of S. aureus. !Master!Thesis! ! ! ! 30! The elucidation of the molecular details of drug resistance is a crucial area of research that crosses many subjects since an understanding of the mechanisms by which drug resistance develops leads to enhancements in extending the efficacy of current antibiotics. Cell envelope is the main target for drug-resistant mechanisms of various pathogens because many drugs need to rapidly diffuse into a cell to meet their targets (Nikaido, 1994; Savage, 2001; Zhang et al., 2001). Biofilms constitute a threat in the clinical environment by acting as pools of multidrug resistant pathogenic bacteria. Biofilm may be formed in a variety of surfaces including living tissues, indwelling medical devices, portable water system piping or natural water system piping (Kokare et al., 2009). Diverse mechanisms allow microorganisms to come into closer contact with a surface, attach to it, promote cell-cell interactions and grow as a 3-D structure (Bryers, 2000). There are several mechanisms influencing the attachment of biofilm. The properties of the surface of attachment influence the process. In the case of a rougher, more hydrophobic, and coated surface, the attachment will occur efficiently (Donlan, 2002; Pereira, 2001). Other variable can affect the attachment: flow velocity, water temperature or nutrient concentration (Pereira, 2001; Vieira, 1995). Maximum attachment depends upon high surface free energy or wettability of surfaces. Surfaces with high surface free energies are more hydrophilic and generally show greater bacterial attachment than hydrophobic surfaces. Quorum sensing, related with cell-cell signalling, play a role in cell attachment and detachment from biofilms (Simões, 2005). This mechanism induces and increased in the intrinsic antimicrobial resistance of biofilm (Brooun et al., 2000). QS regulates a wide number of physiological activities, such as motility, conjugation, competence, sporulation, virulence and biofilm formation. The signal of QS may alter distribution of bacterial species in the biofilm, alter protein expression, introduce new genetic trait and incorporate bacteria in biofilm. The properties of cells, including cell surface hydrophobicity, presence of fimbriae and flagella, and production of EPS influence the attachment of microbial cell (Watnick and Kolter, 2000). One key element of the adaptability of bacterial cells is their ability to be in a niche where they can propagate. Various mechanisms of motility have been described for bacteria. The most common is flagellar motility, but other mechanisms can be highlighted: twitching, gliding, darting, sliding, swimming and swarming (Davey and O’Toole, 2000). !Master!Thesis! ! ! 31! The aim of this study was to assess aspects of the mode of action of the selected phytochemicals on planktonic cells and in the early stages of biofilm formation. The physicochemical properties of bacterial surface (zeta potential and hydrophobicity) were performed with and without phytochemicals. The evaluation of phytochemicals to inhibit efflux pumps was studied with S. aureus while the effects of these products were tested on E. coli outer membrane proteins (OMPs) expression. The free energy of adhesion, quorum-sensing inhibition and bacterial motility were assessed in order ascertain the role of phytochemicals on the early stages of biofilm development. ! ! ! ! ! ! ! ! !Master!Thesis! ! ! ! 32! 4.2 Materials and methods 4.2.1. Bacterial Strains Escherichia coli (CECT 434) Staphylococcus aureus (CECT 976) were used according to section 3.2.1.. The Chromobacterium violaceum (ATCC 12472) were distributed over the surface of Luria–Bertani Agar (Merck) and incubated for 24 h at 30 ± 3 ºC. S. aureus RN4220 containing plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein; S. aureus SA1199B, which overexpresses the NorA MDR efflux protein and S. aureus XU212, which possesses the TetK efflux pump and is also a MRSA strain, were kindly provided by S. Gibbons (University College London, UCL) (Gibbons et al., 2003; Oluwatuyi et al., 2004; Gibbons and Udo; 2000; Smith et al., 2007). Prior to use, these strains at -80ºC were transferred onto Mueller-Hinton (Merck, Germany) agar plate, grown overnight, and inoculated into MH broth at 30 ºC and under agitation (150 rpm).! 4.2.2. Phytochemicals and antibiotics The phytochemicals used were the same referred before and prepared as explained in section 3.2.2. Ciprofloxacin, erythromycin and tetracycline were obtained from Sigma (Portugal). Every antibiotic were prepared in DMSO, which was filtrated before utilization to avoid contamination. After preparation, antibiotics were frozen. 4.2.3. Determination of Zeta Potential The overnight cultures of E. coli and S. aureus were centrifuged (Eppendorf centrifuge 5810R) at 3777 g for 10 min and washed twice with sterile water. The OD620 nm of strains was adjusted to 0.2 ± 0.02 and samples were incubated with phytochemicals for 30 minutes at 30 ºC. Phytochemical concentration used was the MIC. Cells suspensions without phytochemicals were used as control. The zeta potential experiments were performed using a Malvern Zetasizer instrument (Nano Zetasizer, Malvern instruments, UK). All experiments were carried out in triplicate at room temperature and were repeated at least at three different occasions. !Master!Thesis! ! ! 33! 4.2.4. Physico-chemical charecterization of bacterial surface The physico-chemical properties were measured using the sessile drop contact angle method. The bacteria cultures were grown overnight in MH medium. The cells suspensions were washed with NaCl (8.5 g/L) and centrifuged (10 minutes at 3777 g) twice. The OD620nm was adjusted to 0.4 ± 0.02. After that, the biocides were applied (at MIC concentration) during 30 minutes. The solutions were filtrated (0.45 µm, Whatman) and placed in microscope slides. The contact angle was measured with 3 different liquids: water, formamide (polar) and α-bromonaphtalene (nonpolar) (Sigma, Portugal). The measurement of contact angles was performed using a model OCA 15 Plus (Dataphysics, Germany) video based optical contact angle measuring instrument, allowing image acquisition and data analysis. The degree of hidrophobicity of a surface is expressed as the free energy of interaction between entities of that surface, when immersed in water. ΔGsws (mJ/m2) can be positive or negative according of the interaction between the surfaces. In the case of ΔGsws >0, the material is considered hydrophilic, because the interaction between the two surfaces is weaker than the interaction of each entity with water. In contrast, when ΔGsws <0, the interaction between the surfaces is stronger than the interaction of each entity with water and the material is hydrophobic. Hydrophobicity was evaluated after contact angles measurements, following the van Oss approach (van Oss et al., 1987; 1988; 1989). The degree of hidrophobicity can be calculated through the surface tension components of interacting entities, according to: !!!!!!!!!!(3)! Where, γLW is the Lifshitz-van der Waals component of the surface free energy and γ+ and γare the electron acceptor and donor, respectively, of the Lewis acid-base parameter (γAB), being . The analysis was performed at room temperature using the three liquids referred before. The surface tension components of liquids were obtained from literature (Janczuk et al., 1993). Subsequently, three equations can be solved: !!!!!!!!!!!!!!!!!!!!!!!!!!!(4)! Where θ is the contact angle and . ()() −+−++−−+−−++−−=Δwwsswsws 2 LW w LW ssws 42G γγγγγγγγγγ −+ = γγγ AB 1+cos θ ( ) γ 1 Tot =2 γ s LW γ w LW + γ s + γ w −+ γ s − γ w + ( ) ABLWTOT γγγ += !Master!Thesis! ! ! ! 34! These measurements were performed at least with 12 determinations for each liquid and microorganism. ! 4.2.5. Free energy of adhesion The free energy of adhesion between the bacterial cells and polystyrene surfaces was calculated through the surface tension components of the entities involved in the process using the Dupré equation and the procedure described by Simões et al. (2010). The total interaction energy (∆G!"# !"!) is studied by the interaction between one bacteria (b) and a substratum (s) that are immersed or dissolved in water (w) and is expressed by the interfacial tension components: ∆G!"# !"! =!𝛾!" −𝛾!" −𝛾!"! (5) The thermodynamic theory of the interfacial tension of one system of interaction (for example, bacteria/surface - γbs) can be defined by the following equations: 𝛾!" =!𝛾!" !" +!𝛾!" !"! (6) 𝛾!" !" =𝛾! !" +𝛾! !" −!2×𝛾! !"×𝛾! !"! (7) !𝛾!" !" =2×!𝛾! !×!𝛾! !+!𝛾! !×!𝛾! !−!𝛾! !×!𝛾! !−!𝛾! !×!𝛾! !! (8) The two other interfacial tension components, γbw and γsw, were calculated in the same way, which permits the assessment of thermodynamic energy of adhesion. The bacterial adhesion to the substratum can be favorable or is not expected to occur, according to the values of ∆G!"# !"! if are negative or positive, respectively (Simões et al., 2010). ! 4.2.6. Motility Plates containing 1% tryptone (Merck), 0.25% NaCl (Merck) and 0.25% or 0.7% (w/v) agar (Merck) were prepared for swimming/sliding or swarming motilities, !Master!Thesis! ! ! 35! respectively (Butler et al., 2010; Stickland et al., 2010). Phytochemicals at MIC concentration were incorporated in the growth medium after sterilize and cooling the medium, to avoid the deterioration. Overnight cultures of E. coli and S. aureus grown on LB broth (Merck, Germany) were used to determine bacterial motility. A volume of 15 µL of cell suspension (OD620nm= 0.4 ± 0.02) was placed in the center of the plates. Then, plates were incubated at 30 ºC and the diameter (mm) of the bacterial motility halos were measured at 24, 48 and 72 h (Borges et al., 2012). All experiments were carried out in triplicate. The negative control was performed with DMSO. 4.2.7. Detection of quorum-sensing inhibition The culture of Chromobacterium violaceum (CV12472) was grown overnight in Luria-Bertani (LB) broth at 30 ºC. The OD620nm of the strain was adjusted to 0.1 ± 0.02 (1.4 × 108 CFU/mL). For each phytochemical a sterile 96-well polystyrene microtiter plates were filled with cells (180 µL) and phytochemicals (20 µL). As a negative controls were used cell suspension with DMSO and without phytochemicals. The microtiter plates were incubated during 24 h at 30 ºC in an orbital shaker (150 rpm). Subsequently, the absorbance at 620 nm was measured using a Microplate Reader (Spectramax M2e, Molecular Devices, Inc.). MIC values were determined using the microdilution method, explained above. All tests were performed in triplicate. All the further experiments were performed at sub-MIC concentrations of phytochemicals (Packiavathy et al., 2012). After these experiments, C. violaceum was used to perform the detection of quorum-sensing inhibition (QSI) by the disc diffusion method. The detection of QS activity of phytochemicals was performed at the range of sub-MIC. LB agar (LBA) plates were spread with 100 µL (OD620nm of 0.1 ± 0.02) (1.4 × 108 CFU/mL) of overnight culture of C. violaceum CV12472. Sterile paper disks (6 mm diameter) were placed in the plates and impregnated with varios concentrations of each phytochemical (15 µL). DMSO was used as a negative control. The plates were incubated at 30 ºC for 24 h to check the inhibition of pigment production around the disc. The growth inhibtion was also recorded. The zones of QS inhibition were measured from the disks to the edges (Adonizio et al., 2006; Khan et al. 2009). Bacterial growth inhibition by the phytochemicals was measured as radius (r1) in mm while phytochemicals showing both growth and pigment inhibition was measured as !Master!Thesis! ! ! ! 36! radius (r2) in mm. The pigment inhibition (QS) was determined by subtracting bacterial growth inhibition radius (r1) from the total radius (r2) thus QS inhibition = (r2-r1) in mm (Zahin et al. 2010). 4.2.8. Outer Membrane Proteins 4.2.8.1. Extraction The Outer Membrane Protein extraction was performed for E. coli. The bacterial culture was grown overnight at 30 ºC in an orbital shaker (120 rpm). The inoculum was centrifuged at 3777 g for 15 minutes and washed with twice NaCl (8.5 g/L). After that, the OD was measured and adjusted to 0.4 ± 0.02 (λ = 620 nm). The cell suspension was incubated for 1h with the different phytochemicals at MIC. After that, the cells suspension were centrifuged and the pellet was ressuspended in 25 mM Tris and 1 mM MgCl2 (Merck) buffer (pH 7.4), twice. Behind this process, each sample was sonicated for 5 times (20 seconds) (Vibracell, 60 W) on ice to promote cell lysis. After sonication the solution was centrifuged (7000 g, 10 min, 4ºC) (Beckman Avanti J25 centrifuge) in order to remove non-lysed cells. The supernatant was collect and 1 mL of Sarcoisine (Sigma) solution was added to obtain a final concentration of 2% (w/v), in order to solubilize the inner membrane proteins. The samples were left on ice for 20 minutes. After incubation time, 25 mL of TrisHCl 25 mM and 1mM MgCl2 buffer. The solution was centrifuged (13000 g, 1 h, 4 ºC) (Avantis J-25) two times, to recover the pellet containing the OMP. The pellet was ressupended in 300 µL of 25 mM Tris-HCl buffer (pH 7.4) and stored at -20 ºC until required. 4.2.8.2. OMP Analysis The protein content of OMP samples was determined using Bicinchoninic Acid Protein Assay Kit (BCA) (BCA-PIERCE Cat. No. 23225) with BSA as standard. This procedure was applied in order to insert in the gel cassettes the same OMP concentration for the several samples (3 µg protein in each well). The OMP samples obtained were subjected to SDS-PAGE, as reported by Laemmli (1970) with 12% (w/v) acrylamide (Bio-rad). Electrophoresis was !Master!Thesis! ! ! 37! performed at a constant current of 10 mA. After electrophoresis, the gel was stained with Coomassie blue (Bio-rad) for protein profile detection. 4.2.9. Antibiotic-Phytochemical Dual Combinations Assay – Efflux Pumps Inhibition To study the antimicrobial effects of phytochemicals conjugated with antibiotics, they were inserted in MH agar medium (at MIC). The phytochemical was inserted after sterilize and cooling the medium, to avoid the deterioration. Colonies of bacteria were picked from overnight PCA cultures (log phase cultures) in solid medium (Merck, Portugal). The suspension of bacteria was prepared with 0.9% NaCl, which was adjusted to match to 0.5 McFarland turbidity standards. The suspension was spread with a sterile cotton swap into Petri dish (90 mm of diameter) containing 20 ml of Mueller-Hinton Agar. Sterile filter paper discs (with 6 mm in diameter), impregnated with 15 µL of antibiotics, were placed on the agar plate seeded with the respective bacteria. Discs of ciprofloxacin, erythromycin and tetracycline were used as positive controls and discs impregnated with DMSO were used as negative controls. The concentration of antibiotics used was according to Clinical and Laboratory Standards Institute (2005): Performance Standards for Antimicrobial Susceptibility Testing (Fifteen Informational Supplement): ciprofloxacin – 5 µg/disc; erythromycin – 15 µg/disc; and tetracycline – 30 µg/disc. The plates were incubated at 30 °C for 24 hours. After incubation, zones of growth inhibition were measured. All tests were performed in triplicate and the antibacterial activity was expressed as the mean of inhibition diameters (mm). 4.2.9.1. Classification of dual combinations The effect of dual combinations of antibiotics and phytochemicals can be classified according Saavedra et al. (2010): § Antagonism (-) – [inhibition halo – (antibiotic inhibition halo + phytochemical inhibition halo)/2] < 0 § Indifference (+) – 0 ≤ [inhibition halo – (antibiotic inhibition halo + phytochemical inhibition halo)/2] < antibiotic inhibition halo or phytochemical inhibition halo !Master!Thesis! ! ! ! 44! Table%7.%%Motility%results%for%bacteria%with%and%with%phytochemicals.%The%drop%baseline%was% 6mm%which%was%subtracted%from%the%results%presented.% Time/ Phytochemical E. coli S. aureus Swimming (mm) Swarming (mm) Sliding (mm) 24h Control 79.0 ± 1.2 8.7 ± 0.6 7.0 ± 0.0 7-HC 7.0 ± 1.0 7.7 ± 1.5 5.0 ± 0.0 I3C 4.7 ± 0.6 7.7 ± 2.9 0.0 ± 0.0 SA 3.3 ± 0.9 2.0 ± 0.9 7.7 ± 0.6 SP 80.0 ± 0.0 56.0 ± 2.0 84.0 ± 0.0 48h Control 84.7 ± 0.6 13.7 ± 3.8 8.0 ± 1.0 7-HC 43.3 ± 2.9 8.7 ± 1.2 8.3 ± 0.6 I3C 0.0 ± 0.0 10.0 ± 7.8 0.0 ± 0.0 SA 0.0 ± 0.0 55.0 ± 8.7 0.0 ± 0.0 SP 84.0 ± 0.0 61.7 ± 9.1 56.7 ± 5.8 72h Control 84.0 ± 0.0 64.3 ± 7.6 7.7 ± 0.6 7-HC 51.3 ± 2.3 8.3 ± 0.6 8.3 ± 0.6 I3C 0.0 ± 0.0 8.7 ± 5.5 0.0 ± 0.0 SA 0.0 ± 0.0 54.3 ± 6.4 2.0 ± 0.6 SP 84.0 ± 0.0 13.3 ± 3.2 55.0 ± 8.7 Swimming and swarming motilities have been documented as two forms of surface motility for E. coli, and sliding for S. aureus (Borges et al., 2012; Harshey, 2003; Pratt and Kolter, 1998). E. coli exhibits flagella; in contrast, S. aureus is a nonflagelated bacterium with a motility phenomenum defined as colony spreading (Borges et al., 2012). The Gram-negative bacteria increased their growth over the time and the Gram-positive bacteria maintained its growth, without the addiction of phytochemicals. In this study, it was verified that E. coli presented the highest motility and also showed an increasing in swimming and swarming motility, without the addiction of phytochemicals. However, S. aureus had more capacity to adhere to PS, concluding that motility does not regulate adhesion. Regarding the several phytochemicals performed, motility was mostly affected when I3C is added. In the case of swimming and sliding, the motility was completely inhibited with this phytochemical (p<0.05). However, I3C did not influence swarming motility. SA was also able to stop swimming and sliding motilities. Swarming motility was very low in the first 24h, and in the last 48h, increased. Probably, E. coli is able to adapt to SA after a long period of exposure. Swimming motilily increased with the addiction of SP after 72h of exposure (p<0.05); however, with swarming and !Master!Thesis! ! ! 45! sliding motility, the bacteria showed an increase in the first hours, but after a long period of exposure, the motility decreased. Finally, 7-HC influenced swarming and swimming motility and it was not able to change sliding motility. In the case of swimming motility, the value was very low in the first 24h, after that, it started to increase. Borges et al. (2012) also studied E. coli and S. aureus motilities in contact with ferrulic and gallic acids. They concluded that both compounds show potential to inhibit cell motility. Swarming was completely inhibited in E. coli, after 24h; Swimming motility of E. coli was reduced by the addition of both compounds. S. aureus motility was also inhibited by ferrulic acid. Although the compounds tested are not the same, it is possible to conclude that the motility can be changed. The motility is apparently related with the cell state of the bacteria. The changes in the motility are explained by cell stimuli to alter the funtions of its motility machinery to improve or decrease its chances of migrating to a better location (Jarrell and McBride, 2008). Morphological differentiation in bacteria, which distinguishes them from their planktonic state, occurs as a response of motility (Julkowska et al., 2004). A relationship between cells surface motility and biofilm formation has been reported, especially in the case of swarming motility. Both processed, biofilm formation and swarming, require production of flagella and surface polyssacharides (Borges et al., 2012). Several authors have been reported mutants with altered swarming motility that present difficulties in biofilm formation, concluding that they can play a role in biofilm development (Pratt and Kolter 1998; Shrout et al., 2006). 4.3.4. Quorum-sensing assays QS is a mechanism by which a bacterial population senses its cell density (Khan et al., 2009). This mechanism influences bacterial biofilm growth and development and it is related to cell-cell interactions (Simões et al., 2009). This cellcell communication is dependent of several factors: synthesis, exchange and perception of small signal molecules between bacteria (Khan et al., 2009). The 4 phytochemicals was tested as QS inhibitors, at several concentrations. The Table 8 shows the results obtained. !Master!Thesis! ! ! ! 46! The MIC of the phytochemicals tested against C. violaceum CV12472 ranged from 25 to 3200 µg/mL. This means that phytochemicals are able to inhibit the bacteria growth. QS results show the effect of the phytochemicals in the bacteria growth (inhibition halo) and also the effect of phytochemical in quorum-sensing, through the detection of pigment inhibition (QS halo). Inhibition of pigment production was detected with some phytochemicals at different concentrations. Figure 4 shows several agar plates with different results. Of the 4 phytochemicals tested, inhibition of pigment production was detected with 7-HC, I3C and SA with zones of pigment inhibition ranging 5 to 19 mm. No effect on the pigment was observed with SA at the concentrations tested. Figure%4.%%Examples%of%results%obtained%with%quorum^sensing%assay:%(a)%no%bioactivity%(SA% at%5000%µg/mL);%(b)%antibacterial%and%QSI%halos%are%observed%(I3C%at%5000%µ g/mL)% with%addition%of%phytochemicals.% !Master!Thesis! ! ! 47! !Master!Thesis! ! ! ! 48! The I3C is the most effective to inhibit production of pigment. At 500 µg/mL, the inhibition is low, but by increasing the concentration the zone of pigment inhibition is also increasing. Regarding 7-HC and SA, at low concentrations there is no inhibition of pigment production; but from 1000 and 1500 µg/mL of 7-HC and SA, respectively, the QSI halo is detectable. Although, SA shows antimicrobial activity, it was not possible to observe an effect on pigment inhibition at the concentrations tested. Regarding QS halos, the concentration of I3C, 7-HC and SA added influences significantly the quantity of pigment production. So, QS activity of phytochemicals is concentration dependent, as reported by other authors (Khan et al., 2009; Zahin et al., 2010). The same authors identified one compound (clove oil) able to inhibit pigment production with 19 mm of pigment inhibition zone against C. violaceum (CV12472). Also cinnamon, peppermint and lavender present zones of pigment inhibition against the same bacteria (Khan et al., 2009). Al-Hussaini and Mahasneh (2009) reported T. capensis, Sonchus oleraceus, Pityriasis alba, Pinus nigra, Jasminum sambac, Rosmarinus officinalis, Lavandula angustifolia and Laurus nobilis as great sources of microbial growth and QS inhibitors.! The phytochemicals and other compounds that affect QS can interfere at different levels: inhibition of signal biosynthesis or inhibition of activity of AHLproducing enzymes, enzymatic signal degradation and inhibition of reception signal molecules (Khan et al., 2009). 4.3.5. Characterization of cell membranes In order to characterize both bacterial cell membranes, two different techniques were performed: outer membrane protein extraction and analysis for E. coli, and study of efflux pumps for S. aureus. 4.3.5.1. Outer Membrane Proteins The OMPs are key molecules that are the interface between the cell and the environment. E. coli comprises three different layers, as explained before. The external layer consists of lipids, polysaccharides and proteins. Some bacterial proteins are expressed in high copy number, which aid their detection and characterization. These include porins (OmpC and OmpF). Porins are a protein family of OMPs that !Master!Thesis! ! ! 49! form a hydrophilic channel, permiting nonspecific diffusion of small molecules across the outer membrane (Molloy et al., 2000). Due to their location, OMPs are important candidate antigens for the development of strategies to protect against bacterial patogens (Kawahara et al., 1994a; Kawahara et al., 1994b; Negm and Pistole, 1998; Pages et al., 1987). The dominant OMPs of E. coli are OmpA, OmpX, OmpF and OmpC (Molloy et al., 2000). The planktonic cells of E. coli were characterized phenotypically in terms of OMPs. The OMPs of E. coli strains as planktonic cells were isolated and analysed by SDS-PAGE. The OMP profiles obtained with different phytochemicals (at the MIC) are presented in Figure 5. According to Figure 3, the OMP profiles obtained with cells and phytochemicals do not differ considerably from the ones obtained with E. coli without phytochemicals (well 2). The application of several phytochemicals does not seem to affect OMP expression since, for all lanes, the protein expression is similar. Nevertheless, the outer membrane of cells with SA exhibits a higher intensity than the other OMP. The profile of OMPs shows different proteins. According to the results the phytochemicals applied do not act on membrane proteins. Figure%5.%OMPs%profiles%of%E.#coli.%The%profile%of%molecular%weight%standards%(1),% control%(2),%7^HC%(3),%I3C%(4),%SA%(5),%SP%(6)%are%presented.% !Master!Thesis! ! ! ! 50! Regarding the OMPs analysed, there are three proteins that were isolated with all the phytochemicals and also at the control. The molecular weights of them are 55, 38 and 35 kDa. The 35 kDa OMP it was identififed as OmpA (Hellman et al., 2000; Molloy et al., 2000), one of the most important OMPs, as referred before. Other studies have been done to study the OMPs of E. coli. Proteins with 38 and 55 kDa were identified as being OmpC and ATP synthase alfa subunit (Molloy et al., 2000; Xu et al., 2006) OMPs have a significant role in the context of biofilm eradication. The bacterial proteins form an adaptative barrier to the external environment, protecting cells from damaging substances, such as biocides and antimicrobial agents, when procedures of desinfections are applied. This permits the selective uptake of nutrients (Simões, 2005). Due to their location, OMPs are important candidates antigens for the development of strategies to protect bacterial cells against pathogens (Molloy et al., 2000). 4.3.5.1. Efflux pumps Efflux mechanisms are implicated in antimicrobial. Efflux pumps contribute to the resistance of bacteria by pumping out a wide variety of products: dyes, detergents and antibiotics (Simões, 2005). However, efflux pumps have been studied as membrane components in all cell types, from prokaryotes to eukaryotes (van Bambeke et al., 2007). The role of efflux pumps in bacteria has been related to the elimination of metabolites that are poisonous to the cell and in cell stress responses (Costa et al., 2013). The association of antibiotics with phytochemicals can create a synergistic effect against resistant bacteria, creating new choices for the treatment of infectious diseases. S. aureus genome reveals high potential multidrug efflux-pump-encoding genes (Huet et al., 2008). Several efflux resistance mechanisms have been described for S. aureus such as QacA and NorA, which are multidrug transporters, and the more specific MsrA and TetK transport proteins (Gibbons et al., 2003). In this study, the antimicrobial activity of several phytochemicals was tested in combination with three antibiotics. For this experiments 4 different strains of S. aureus were tested. Table 9 shows the antimicrobial activity of antibiotics against S. aureus strains. !Master!Thesis! ! ! 51! According to the Clinical and Laboratory Standards Institute (2005), the organisms are susceptible, intermediate or resistant to the agents. S. aureus CECT 976 is considered susceptible to all antibiotics tested. S. aureus XU212, S. aureus RN4220 and S. aureus SA1199B present resistance to tetracycline, erythromycin and ciprofloxacin, respectively. The negative control performed with DMSO in the preparation of phytochemical solutions presented no effects on bacterial growth. Table%9.%Antimicrobial%activity%of%antibiotics.%The%means%(mm)%±%standard%deviation%for%at% least%three%replicates%are%illustrated.% Diameter of inhibition zone (mm) S. aureus CECT 976 S. aureus XU212 S. aureus RN4220 S. aureus SA1199B TET 41.5±9.2 16.0±4.2 42.5±0.7 46.5±2.1 ERY 37.5±3.5 24.5±4.9 22.0±4.2 35.5±3.5 CIP 40.5±0.7 26.0±2.8 31.5±2.1 18.0±1.4 The antibiotics (tetracycline, erythromycin and ciprofloxacin) had antimicrobial effect against the bacteria tested. Tetracycline was the most effective against all the bacteria, while erythromycin has the lowest antimicrobial activity (p<0.05). Regarding to phytochemicals, I3C and SA showed antimicrobial activity against S. aureus, with inhibition halos of 20 and 14 mm, respectively. In contrast, 7HC and SP do not demonstrate antimicrobial properties against the same strain. The most effective phytochemicals was I3C (p<0.05). Dual combinations of antibiotic-phytochemicals were performed. Table 10 shows that the combined application of tetracycline, erythromycin and ciprofloxacin had both negative and positive antimicrobial activities compared to the single application of phytochemicals and antibiotics. The classification presented in Table 10 was done according Saavedra et al. (2010). The combination of bioactive compounds is expected to exert a synergistic effect or to reduce possible adverse side effects. The development of active compounds, such as phytochemicals, in conjunction with antibiotics could avoid the emergence of resistant variants that might otherwise arise during treatment (Abreu et al., 2013b). !Master!Thesis! ! ! ! 52! Table%10.%Classification%of%the%effect%of%dual%combinations%of%phytochemicals%and% antibiotics.% 7-HC I3C SA SP S. aureus CECT 976 TET + +++ + - ERY - +++ + ++ CIP + +++ + - S. aureus XU212 TET ++ +++ +++ +++ S. aureus RN4220 ERY - +++ +++ +++ S. aureus SA1199B CIP + +++ +++ +++ (-) – Antagonist; (+) – Indifference; (++) – Additive; (+++) - Synergistic The results present dual combinations able to improve the antimicrobial activity against the resistant strains. The combined application of ERY with 7-HC, TET or CIP with SP against S. aureus CECT 976 was antagonist. Combination of SA with all antibiotics tested showed to be indifferent against S. aureus CECT 976. Regarding 7-HC, the combination of phytochemicals with TET and CIP is also indifferent in the antimicrobial activity when compared with single antibiotic and phytochemicals activities against S. aureus CECT 976. The dual combination of ERY and SP showed an additive effect against the growth of S. aureus CECT 976. Regarding the resistant strains of S. aureus, combined application of TET and 7-HC against S. aureus XU212 had an additive effect. The remaining combinations produced a synergistic effect. Antibiotic synergism occurs when the effects of combination of antimicrobials is greater than the sum of the effects of individual antimicrobials (Saavedra et al., 2010). An additive effect of phytochemical combined with antibiotic may occur due to a double attack of both agents at different target sites of bacteria (Adwan and Mhanna, 2008). The identification of effective efflux pumps inhibitors of S. aureus could restore the clinical utility of pump substrates. Efflux pumps inhibitors could extend the useful lifetime of antibiotics by improving therapeutic efficacy, suppressing the emergence of resistant variants, reducing the effective dose of antibiotics to reduce the adverse toxic effects (Gibbons et al., 2003; Saavedra et al., 2010). Combination therapy with two or more antimicrobials is used to prevent the emergence of resistant strains, to treat emergency cases and to take advantage of antimicrobial products synergy (Hemaiswarya and Doble, 2009). !Master!Thesis! ! ! 53! Chapter 5 Concluding remarks and perspectives for further research ! ! ! ! 5.1. Conclusions To find new antimicrobial agents, plant products have been studied as substituints of antibiotics for which bacteria already acquired resistance. So, in this work, the antimicrobial effect of four phytochemicals - 7-HC, I3C, SA and SP – was evaluated. After the development of the experiments, it was possible to conclude about the antimicrobial activity of the selected phytochemicals and their ability to control biofilms of two important pathogens – E. coli and S. aureus. The 7-HC was one of the most effective phytochemicals testedagainst E. coli and S. aureus. The values of MIC obtained were 800 and 200 µg/mL for E. coli and S. aureus, respectively. 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