Screening for antibacterial activity in plant extracts against pathogenic bacteria

Universidade do Minho Escola de Ciências Sara Martins Mendonça Screening for antibacterial activity in plant extracts against pathogenic bacteria janeiro de 2023 Screening for antibacterial activity in plant extracts against pathogenic bacteria Sara Mendonça UMinho | 2023 Universidade do Minho Escola de Ciências janeiro de 2023 Sara Martins Mendonça Screening for antibacterial activity in plant extracts against pathogenic bacteria Dissertação de Mestrado Mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas Trabalho efetuado sob a orientação do(a) Professor Doutor Rui Manuel Peixoto Tavares Doutora Ana Margarida Sousa ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição CC BY https://creativecommons.org/licenses/by/4.0/ iii ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisors Doctor Maria Ribeiro Rocha Soares Vicente and Doctor Ana Margarida Sousa, who granted me the opportunity of developing this work and guided me throughout this journey, sharing their scientific knowledge. I would like to thank all my colleagues at LTL and LIBRO. Thank you for all the support, the knowledge you shared with me and the patience you had. A special thanks to Eduarda, who followed up my work from close and helped me inside and outside the lab and Bianca, Joana and Sara, who always helped me, without any obligation. To my family, thank you for the support and for always believing in me. To my friends, thank you for the friendship, patience, encouragement, and for always sharing with me the best and the worst moments, especially Lili, Luís and Filipe. Without you none of this would be possible. iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v RESUMO Screening de atividade antibacteriana de extratos de plantas contra bactérias patogénicas O uso prolongado e intensivo de antibióticos contribuiu para o desenvolvimento de bactérias resistentes, o que culminou na perda de opções terapêuticas disponíveis. A Organização Mundial de Saúde publicou uma lista de agentes patogénicos resistentes a antibióticos que inclui Staphylococcus aureus e Pseudomonas aeruginosa . É por isso imperativo que sejam desenvolvidas estratégias alternativas aos antibióticos eficazes contra os agentes patogénicos e há evidências que demonstram a atividade antibacteriana dos produtos naturais contra bactérias multirresistentes; contudo ainda há muito por explorar. Assim, o objetivo deste estudo assenta na avaliação da atividade antibacteriana de plantas utilizadas no dia a dia, incluindo o alho, o gengibre e aa romã (casca, polpa e sumo) contra a S . aureus e a P. aeruginosa ; e a novidade é a determinação do impacto das condições de extração (o solvente, a duração e temperatura de extração) na atividade antibacteriana de cada planta e o estabelecer de uma reação dessa atividade com a atividade antioxidante e o seu teor fenólico. Os extratos foram obtidos a partir de diferentes solventes, etanol 96% e 70% e água destilada, em diferentes temperaturas (70 ºC durante 1h e overnight à temperatura ambiente). A avaliação do conteúdo fenólico foi feita pelo método Folin-Ciocalteu, a atividade antioxidante pelos métodos Ferric Reducing Antioxidant Power e 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), e a atividade antibacteriana pela determinação da MIC e da MBC. Os resultados demonstraram que os extratos etanólicos de plantas foram mais ativos contra as bactérias, particularmente a S. aureus . A sua atividade baseou-se principalmente na inativação do crescimento bacteriano, apesar de a erradicação também ter sido conseguida. Na maioria dos extratos, a atividade antibacteriana estava associada aos extratos com maior teor fenólico e capacidade antioxidante, extraídos com misturas etanol/água (EtOH (70%)). As condições de extração (70ºC durante 1h e overnight à temperatura ambiente) afetam a análise do teor fenólico e da atividade antioxidante, que estão relacionadas com a atividade antibacteriana. De todos os extratos etanólicos 70%, o extrato da polpa de romã foi o mais promissor contra ambas as bactérias, pois foi o extrato que inibiu o seu crescimento a concentrações mais baixas e foi também o único capaz de erradicar a bactéria gram-negativa; pelo que deve ser explorado no futuro. Palavras-chave: Extratos de plantas; Produtos naturais; Resistência a antibióticos; Pseudomonas aeruginosa; Staphylococcus aureus . vi ABSTRACT Screening for antibacterial activity in plant extracts against pathogenic bacteria The intensive and prolonged use of antibiotics contributed to the development of antibiotic resistance in bacteria, culminating in a loss of therapeutic options. World Health Organization list of antibiotic-resistant pathogens includes Staphylococcus aureus and Pseudomonas aeruginosa , reason that is imperative to evolve alternative non-antibiotic strategies that are effective against these infectious pathogens. A growing body of evidence have demonstrated the antimicrobial activity of natural products against multidrug-resistant bacteria and still much remains to be explored. Therefore, the aim of this work relies in the evaluation of the antibacterial activity of plants used in daily life, including as garlic, pomegranate (peel, pomace and juice) and ginger against S. aureus and P. aeruginosa . The novelty of this work is to determine the impact of extract conditions on the antibacterial activity of these plant extracts and to attempt to correlate with their antioxidant activity and total phenolic content. The extracts were obtained using different solvents, including 96% and 70% ethanol and distilled water, at different temperatures (70 ºC for 1h and overnight at room temperature). The evaluation of the total phenolic content was performed by the Folin-Ciocalteu method, the antioxidant activity by the Ferric Reducing Antioxidant Power and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) method, and antibacterial activity by MIC and MBC determination. Results demonstrated that ethanolic extracts of plants were the most active against bacteria, in particular S. aureus , than aqueous extracts. The activity of the ethanolic extracts was mainly based on inactivation of bacterial growth, but eradication was achieved in some cases. Frequently, this antibacterial activity was associated with the extracts with increased phenolic and antioxidant content, extracted with ethanol/water mixtures (EtOH (70%)). Extraction conditions (70 ºC for 1h and overnight at room temperature) affect total phenolic content and antioxidant activity analysis, which has correlation with antibacterial activity. Among ethanolic 70% extracts, extracts of pomegranate peel were the most promising non-antibiotic drugs against both S. aureus and P. aeruginosa eradication, since it was the ethanolic 70% extract that inhibited their growth at the lowest concentration and was the only one able to eradicate the gram-negative bacteria. In conclusion, pomegranate peel extracts exhibited promising potential as non-antibiotic drug to treat infections caused by S. aureus and should be further explored in near future. Keywords: Antibiotic resistance; Natural products; Plant extract; Pseudomonas aeruginosa; Staphylococcus aureus . vii LIST OF CONTENT DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS ........................... ii ACKNOWLEDGEMENTS ......................................................................................................................... iii STATEMENT OF INTEGRITY ................................................................................................................... iv RESUMO ................................................................................................................................................ v LIST OF CONTENT ................................................................................................................................ vii LIST OF ABBREVIATIONS ....................................................................................................................... ix LIST OF FIGURES .................................................................................................................................. xi LIST OF TABLES ................................................................................................................................... xiv 1. Introduction .................................................................................................................................... 1 1.1. Antibiotic Resistance ................................................................................................................... 1 1.1.1. Mechanisms of antibiotic resistance ....................................................................................... 2 1.1.2. Difficult-to-treat antibiotic resistant bacteria ............................................................................. 4 1.1.2.1. Staphylococcus aureus ........................................................................................................... 5 1.1.2.2. Pseudomonas aeruginosa ...................................................................................................... 7 1.2. Strategies to combat antibiotic resistance ................................................................................... 9 1.3. The role of natural products in the combat of antibiotic resistance .............................................12 1.4. Antimicrobial plant-derived products .........................................................................................14 1.4.1. Allium sativum (Garlic) .........................................................................................................17 1.4.2. Zingiber officinale (Ginger) ....................................................................................................18 1.4.3. Punica granatum (Pomegranate) ..........................................................................................18 1.5. Objectives ................................................................................................................................19 2. Materials and methods .................................................................................................................20 2.1. Plant material ...........................................................................................................................20 2.2. Preparation of plant extracts .....................................................................................................20 2.3. Determination of Total Phenolic Content (TPC) ..........................................................................21 2.4. Determination of antioxidant activity ..........................................................................................21 2.4.1. Ferric Reducing Antioxidant Power ........................................................................................21 2.4.2. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ...............................................................22 2.5. Determination of antibacterial activity ........................................................................................23 2.5.1. Bacterial species and growth conditions ................................................................................23 2.5.2. Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration 23 2.6. Statistical analysis ....................................................................................................................24 xiv LIST OF TABLES Table 1 Range of tested extract concentration obtained from different plants under study (mg/mL) Table 2 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of garlic extract obtained from different extraction conditions tested on S. aureus and P. aeruginosa Table 3 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of ginger extracts obtained from different extraction conditions tested on S. aureus and P. aeruginosa Table 4 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of pomegranate peel extracts obtained from different extraction conditions tested on S. aureus and P. aeruginosa Table 5 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of pomegranate pomace extracts obtained from different extraction conditions tested on S. aureus and P. aeruginosa Table 6 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of pomegranate juice tested on S. aureus and P. aeruginosa 1 1. Introduction 1.1. Antibiotic Resistance The administration of antibiotics was one of the most important medical interventions reductions of the human morbidity, mortality and increased life expectancy. Penicillin, discovered accidentally by Alexander Fleming in 1928, was the first natural antibiotic to be reported when the Penicillium fungus contaminated a culture plate in his laboratory, however, it was not developed for use until the late 1930s. Following the discovery of penicillin by Fleming, other scientists, including Rene Dubos and Selman Waksman, started a deliberate search for antibacterial agents among soil microorganisms, including bacteria and fungi.1,2 The next biggest discovery came about in 1943, resulting in identification of streptomycin produced by Streptomyces griseus , that marked the beginning of the golden age of antibiotic discovery and development (1940–1990).1,3 During the 1940s and early 1960s, antibiotic resistance to multiple antimicrobial agents was detected among enteric bacteria namely Staphylococcus aureus ( S. aureus ), Salmonella , Shigella , and Escherichia coli ( E. coli )for the very first time.2,4,5 In 2015, it was estimated that in Europe 25,000 people die per year as a result of multidrugresistant bacterial infections and it costs €1.5 billion annually to the European Union economy.6,7 Patients with antibiotic-resistant bacterial infections need to stay in the hospital for at least 13 days, adding an additional cost annually.2 Moreover, in the same year, MRSA killed more American people per year than HIV/AIDS, Parkinson’s disease, emphysema, and homicide combined.8 Given this scenario, The World Health Organization (WHO) declared that medicine entered in the post-antibiotic era in which the current antibiotics become less effective overtime and medical advances are insufficient to face antibiotic resistance. Moreover, WHO classified antibiotic resistance as one of the three most important public health threats of the 21st century of worldwide dimension.2,4,6,9 The clinical relevance of antibiotics goes beyond simply preventing death and illness due to infection, in fact antibiotics also have successfully prevented or treated infections that can occur in patients who are receiving cancer treatment by chemotherapy or radiation therapy, or patients who have had surgeries such as organ transplants or cardiac surgery, for example.10,11 There are several reasons that can also lead for that antibiotic resistance. The overuse and misuse of antibiotics are one of key factors attributed to antibiotic resistance.2,10 In 2015, 30% of the outpatient antibiotics prescribed were unnecessary, with acute respiratory infections holding the highest unnecessary use of antibiotics at 50%.2 Another factor is the extensive use of antibiotics on 2 agriculture for growth promotion and prevention of disease, not to eradicate a bacterial infection. Therefore, antibiotic resistant bacteria may reach people indirectly through food chain by consumption of contaminated food or derived products.11,12 1.1.1. Mechanisms of antibiotic resistance Antibiotics can be classified based on their structure and mode of action and at least seven major groups of antibiotics have been considered, including penicillin’s, β-lactams, cephalosporins, aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and glycopeptides.1 The most common target for antibiotics are metabolically active cells, and so antibiotics act preferably on inhibition of the cell wall synthesis, depolarization of the cell membrane, inhibition of protein synthesis, inhibition of nucleic acid synthesis, and inhibition of metabolic pathways in bacteria.13 However, bacteria are remarkably resourceful and, for that reason, they can easily adapt to a wide array of stressful conditions, including resist to antimicrobial agents. Various of these responsive mechanisms of resistance may have evolved in response to pressures applied by 'natural' antibiotics produced by other microorganisms with which these bacteria coexist. Therefore, most currently recognized antimicrobial resistance (AMR) mechanisms can be classified in three categories: altered target site for the antimicrobial agent; enzymatic inactivation of the antimicrobial agent; and decreased permeability of the bacterial envelope.14 Nevertheless, bacteria are not uniformly susceptible or resistant to antibiotic and thus they may exhibit different mechanisms for resistance: intrinsic resistance; acquired resistance and adaptive.2,6 The intrinsic antibiotic resistance refers to the innate ability of bacteria to resist to the action of an antibiotic as a result of their genome encoding inherent structural or functional properties independent of previous antibiotic exposure. This kind of antibiotic resistance explains why some antibiotics are more active against gram-negative than gram-positive bacteria and viceversa, due to their inherent distinct cell wall composition acting as barrier to the entrance of antibiotics into the cells.1,6,15 Intrinsic mechanisms confer low level antibiotic resistance in the original host, however the normal commensal flora or environmental bacteria containing intrinsic mechanisms can become opportunistic pathogens in immunocompromised patients.1 In addition to intrinsic resistance, bacteria can acquire resistance to antibiotics. This kind of resistance is the major cause of the global crisis of antibiotic resistance.6,15 It arises when bacteria becomes resistant through the acquisition and incorporation of new genetic material, such as plasmids, transposons, integrons or DNA from other microorganisms by horizontal gene transfer 3 or as a result of mutations of chromosomal genes. The acquisition may be temporary or permanent.6,9,16 Acquired resistance can be mediated by several mechanisms, which fall into three groups: (i) Those that minimize the intracellular concentrations of the antibiotic as a result of poor penetration into the bacterium or of antibiotic efflux (membrane proteins that export antibiotics from the cell and maintain their low intracellular concentrations).1,6,15,17 (ii) those that modify the antibiotic target. Those changes that may consist of point mutations in the genes encoding the target site, enzymatic alterations of the binding site, and/or replacement or bypass of the original target. Regardless of the type of change, the final effect is identical: a decreased affinity of the antibiotic for the target site.1,6,9,15,17 (iii) and those that inactivate the antibiotic by hydrolysis or modification. The enzyme catalyzed modification of antibiotics is a major mechanism of antibiotic resistance that has been relevant since the first use of antibiotics.1,6,15 Moreover, bacteria can also produce an alternative target (usually an enzyme) that is resistant to inhibition of antibiotic and at the same time produce a native target too, which is sensitive to antibiotics, allowing bacteria to survive by adopting the role of a native protein.17 Often, different mechanisms of resistance are combined, contributing to the expression of high levels of AMR.15 Furthermore, bacteria can develop other kind of resistance to antibiotics, which is called adaptive resistance. It can be define as a temporary increase in the ability of a bacterium to survive an antibiotic insult due to alterations in gene and/or protein expression as a result of exposure to an environmental trigger, such as pH, temperature, nutrient or oxygen limitation, ion densities and exposure to non-lethal doses of antibiotics.15,18–20 Unlike intrinsic and acquired resistance, which are stable and can be transmitted vertically to subsequent generations, adaptive resistance is unstable, transient and highly dependent on the presence of antibiotics. It cannot be vertically transmitted and usually reverts at the liminal of the inducing status.15,20 Because of its transient nature, adaptive resistance represents one of the biggest challenges in designing effective antimicrobial therapies, explaining the common differences found between in vitro and in vivo antibiotic susceptibilities exhibited by bacteria.15 There are several mechanisms of adaptive resistance, including epigenetic inheritance, population heterogeneity, mutability, gene amplification, efflux pumps and biofilm formation.15 Of all these mechanisms, biofilms represent one of the most effective antibiotic resistance strategies, as they have a 10 to 1000 times greater ability to resist the antibiotic than 4 planktonic cells. Moreover, they are responsible for approximately 80% of chronic and recurrent microbial infections in the human body.21 A biofilm can be defined as a community of cells attached to a substratum (biotic or abiotic), interface, or to each other that are embedded in a self-produced matrix of extracellular polymeric substance.22 Biofilms may cause inflammation, because they are protected from antibiotics and the body’s immune system.23 Slow or arrested cell growth deep in the biofilm is known to decrease antibiotic susceptibility, and metabolic responses to nutrient limitation may control antibiotic tolerance in growth-arrested cells under these conditions.24 Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. Combating this organization of cells usually requires high antibiotic doses for a prolonged time, and these approaches often fail, contributing to infection persistence.25 The structure and composition of the biofilm matrix can contribute to antibiotic resistance. Exopolysaccharide and extracellular DNA in the biofilm matrix can act as a barrier to diffusion, preventing drugs from reaching living cells. The effectiveness of this barrier varies between antibiotics — large molecules, positively charged aminoglycosides, and antimicrobial peptides diffuse poorly in biofilms, but quinolones and β-lactams appear to move freely. For antibiotics that can penetrate the matrix, inactivation by resistance enzymes can produce collective resistance.24 1.1.2. Difficult-to-treat antibiotic resistant bacteria In light of increasing antibiotic resistance, in February 2017, the WHO published a list of pathogens that includes the pathogens designated by the acronym ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species ) to which were given the highest “priority status” since they represent the great threat to humans. These pathogens have evolved into multidrug-resistant (MDR) forms subsequent to antibiotic use and can cause severe and often fatal infectious diseases such as bloodstream infections and pneumonia.3 Among gram-positive pathogens, a global pandemic of resistant S. aureus currently poses the biggest threat.11 In fact, this bacterium turned out to be one of the first causes of healthcareassociated infection, and in 1944, when S. aureus resistance was first identified, penicillin presented a solution. However, it offered only was only a short-term relief, because a few later (around 1950), Penicillin-Resistant Staphylococcus aureus appeared.26,27 Methicillin was then 5 produced around 1960, and a year after their clinical use there were already records of Methicillinresistant Staphylococcus aureu s (MRSA). Since then, MRSA infections have spread worldwide, appearing at a high incidence in several countries in Europe, America, and the Asia-Pacific region.11,15,27,28 For many years vancomycin has been considered a last-resort antibiotic against severe MRSA and other resistant gram-positive infections. However, by the late 1980s vancomycin resistance first appeared in enterococci (VRE) and later, in 1997, Vancomycin-Intermediate Staphylococcus aureus (VISA). In recent years in Vancomycin-resistant Staphylococcus aureus (VRSA), which also emerged from MRSA. Nowadays, they are also recognized as high priority pathogens since, without containment and effective therapeutic solutions, they can cause serious infections that are impossible to control.29 Regarding gram-negative pathogens, they are particularly worrisome because they are becoming resistant to nearly all the antibiotic drug options available, creating situations reminiscent of the pre-antibiotic era. The most serious gram-negative infections occur in health care settings are caused by Enterobacteriaceae, mostly Klebsiella pneumoniae ( K. pneumoniae ), Pseudomonas aeruginosa ( P. aeruginosa ) , and Acinetobacter baumannii. 11,15 Among these pathogens, P. aeruginosa infections are of particular importance due to the accumulation of resistance after exposure to nearly all antibiotics (including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems) and cross-resistance between agents, that may result in multidrug-resistant (MDR) P. aeruginosa .11,30 Understanding the resistance mechanisms of these bacteria is a key step towards the development of new antimicrobial strategies to tackle drug-resistant bacteria. Therefore, in the next sections it will be discussed the current state of antibiotic resistance in the most critical resistant gram-positive and gram-negative bacteria, S. aureus and P. aeruginosa , respectively, because WHO classification as critical threat to human health. 1.1.2.1. Staphylococcus aureus S. aureus is a gram-positive ubiquitous bacterial species and a member of the Micrococcaceae family, that can be found in the environment and in normal human flora, skin and mucous membranes of most healthy individuals (approximately 20–25% of individuals have become persistently colonized and 75–80% intermittently or never colonized).31–34 It is an opportunistic pathogen and the leading cause of a wide range of clinical infections, ranging from subclinical inflammation to severe infections causing pulmonary infections, pneumonia, endocarditis, 6 septicemia, skin and soft tissue infections, bacteremia, osteomyelitis, septic arthritis, gastroenteritis, meningitis, and urinary tract infections, bone, joints and infections associated with indwelling catheters or prosthetic devices.25,31,34–36 This pathogen is considered the most notorious superbug, which are microbes with higher morbidity and mortality rate increased due to several mutations being able to resist multiple classes of antibiotics, evading the majority of current therapies.2,3,15 The intrinsic resistance mechanism mainly includes three aspects: outer membrane permeability, because when the cell membrane permeability is lowered, the energy metabolism of the bacteria is affected, and the drug absorption is reduced, which leads to drug resistance; active efflux systems, that have the ability to efflux drugs (exists in MRSA); and excessive production of β-lactamase (that also exists in MRSA), through two mechanisms.26,37,38 One is the hydrolysis mechanism, where β-lactamase hydrolyses and inactivates β-lactam antibiotics and the other is the mechanism of pinching, where there is a large amount of β-lactamase binding to extracellular antibiotics, preventing the antibiotics from reaching the intracellular space, therefore the antibiotics are not able to reach the target site.38 Several mechanisms of acquired antibiotic resistance have been described, among which have been highlighted: resistance by mutations, there may be genetic mutations that alter the target DNA gyrase target or reduce outer membrane proteins, thereby reducing drug accumulation; acquisition of resistant genes, for example MRSA can obtain drug-resistant plasmids from Enterococcus; biofilm-mediated resistance, which allows bacteria to resist host immune responses and evade antibiotic killing; and persister cells, that can resist killing by reducing cell growth and metabolism, and even by becoming dormant and restart infection after antibiotic treatment.26 Treatment of S. aureus infections depends largely on the type of infection as well as the presence or absence of drug resistant strains. In general, penicillin remains the drug of choice if isolates are sensitive (MSSA, or methicillin sensitive S. aureus strains) and vancomycin in cause of MRSA infections.26,39 However, these are not the only drugs used. There are many others that are also described that can be used relatively effectively, despite their disadvantages such as norvancomycin, which is a glycopeptide antibiotic similar to vancomycin in its pharmacological effect; teicoplanin, clinically applicable when patients are allergic to β-lactam antibiotics; linezolid, mainly used to control systemic infection such as pneumonia; daptomycin, a cyclic peptide antibiotic with a fatty acid side chain that bind to the bacterial cytoplasmic membrane in the presence of calcium ions; tigecycline, which is specially applicable against gram-positive bacteria; quinupristin/dalfopristin, which has 7 comparable to or stronger than vancomycin; and ceftobiprole, used to treat skin and soft tissue infections and medical care related pneumonia.26,29,40 In some cases, alternative therapy is necessary for addition to antimicrobial therapy, such as quorum sensing inhibition, that can inhibit the expression of bacterial virulence genes without affecting the growth and proliferation of bacteria, which makes the bacteria unable to develop resistance due to growth stress; lectin inhibition; iron chelation, which causes a lack of ions necessary for the growth and metabolic activity of pathogenic bacteria; nanoparticles; and phage therapy.26,41 Most of bacteriophages utilize lysis systems through phage endolysins to hydrolyze the peptidoglycan of the infected bacteria and thereby destroy its cell wall.41 Due to continuous increasing rate of MRSA infection, there is an urgent interest in agents that treat such infections. 1.1.2.2. Pseudomonas aeruginosa Pseudomonas aeruginosa ( P. aeruginosa ) is a gram-negative, rod-shaped bacteria that belongs to the family Pseudomonadaceae.42 It is a non-lactose fermenting oxidase-positive opportunistic bacterium that causes a range of infections including acute and chronic infection that can persist for years43,44, some of them in patients with compromised immune systems and/or disrupted epithelial barriers. It is consistently ranked among the most frequent pathogens found in nosocomial infections particularly in critically ill patients, such as pneumonia, urinary tract infections, and surgical site/soft tissue and blood infections.42,45,46 This pathogen is widespread in nature, inhabiting soil, water, plants and animals (including humans). It rarely causes disease in healthy people, but can multiply easily in immunocompromised patients.47 It’s actually the major cause of serious infection in many patients, particularly those who lack white cells as a result of hematologic malignancy or chemotherapy.16 Cystic fibrosis (CF) airway infections is an example of a P. aeruginosa chronic infection resistant to antibiotic treatments (mono and combinatorial therapy) resulting declined respiratory function and death of patients.43,48,49 This opportunistic bacterial pathogen is the most prevalent pathogen and accounts for most of the morbidity and mortality in CF patients.50 According to the survey conducted by the US National Healthcare Safety, P. aeruginosa was found to be the sixth most repeatedly occurring pathogen, the second most frequent cause of ventilator-associated pneumonia and the seventh commonest cause of catheter-related bloodstream infection.45,42 The treatment of P. aeruginosa infections has become a great challenge due to the ability of this bacterium to resist a variety of antibiotics, including aminoglycosides, quinolones and β-lactams.51 8 The mains intrinsic resistance mechanisms of this bacteria are over-expression of efflux pumps, that expel antibiotics out of the cell; decreasing outer membrane permeability; and the production of antibiotic-inactivating enzymes such as β-lactamases.29,52–54 Its acquired resistance can be achieved by horizontal transfer of resistance genes from other organisms via plasmids, transposons and bacteriophages or by mutational changes, that encode for proteins that control the passive diffusion of antibiotics across de outer membrane (in DNA gyrases and type IV topoisomerases, e.g.).52,53,55,56 The adaptive resistance of P. aeruginosa involves formation of biofilm, e.g. in the lungs of CF patients where the biofilm serves as a diffusion barrier to limit antibiotic access to the bacterial cells.52,56 Moreover, persister cells are survive antibiotic attack, being responsible for prolonged and recurrent infections in CF patients.52 Current therapeutic options for P. aeruginosa treatment are the use of different antibiotic combinations and development of new antibiotics.52 Polymyxins; carbapenems, such as doripenem which is a new carbapenem antibiotic with broad spectrum activity against bacteria, through inhibition of bacterial cell wall synthesis by binding to penicillin-binding proteins; antipseudomonal β-lactams; aminoglycosides, such as semisynthetic aminoglycoside antibiotic synthetically derived from the natural product sisomicin; and fosfomycin are currently available antimicrobials for the treatment of MDR P. aeruginosa infections.52,55 Unfortunately, carbapenem-resistant P. aeruginosa and other resistant variants were detected and WHO has recently listed this resistant variant as critically human health threatening.52 Recent studies have reported several novel non-antibiotic therapeutic approaches that are highly effective in killing antibiotic-resistant P. aeruginosa strains. These approaches include inhibition of quorum sensing and bacterial lectins, use of iron chelation, phage therapy, vaccine strategy, nanoparticles, antimicrobial peptides and electrochemical scaffolds. These therapeutic approaches can be used as either an alternative to or in combination with conventional antibiotic treatments.52 Regardless the mechanisms involved, the prevalence of MDR P. aeruginosa is increasing worldwide over the last few decades. Furthermore, a significant proportion of MDR further restricts the treatment options available and, to date, few of these newer approaches cannot be used due to high cost, side effects and safety concerns.52,55 So, there is an urgent need for the development of new strategies to treat these infections, with less side effects and costs and increased safety. 9 1.2. Strategies to combat antibiotic resistance The rapid increase in resistance, along with the emergence of microbial pathogens resistant to broad-spectrum antibiotics, which include antibacterial agents such as ampicillin, amoxicillin, streptomycin, chloramphenicol, and tetracycline, as well as the slow discovery of new antibiotics, threatens to undermine future options for antibiotic therapy.57,58 Moreover, development and dissemination of resistant strains against carbapenems has a devastating impact on the healthcare system across the globe, because these antibiotics are employed in last resource to treat multidrug-resistant bacterial infections.58 Therefore innovative strategies are urgently required to treat the development and dissemination of multidrug-resistant pathogens. Drug combination therapies have become a powerful approach to fight against complex diseases in recent years.59 The administration of multiple licensed therapeutic agents has been employed as an alternative strategy to treat microbial diseases that do not respond to conventional drugs.58 Clinical trials show higher synergy outcomes for proper combinations, such as higher efficacy and less toxicity, and many approaches neglect the toxicity and efficacy of drug combinations.59,60 Moreover, this method is advantageous, as different drugs are directed against different therapeutic targets simultaneously. A single drug typically targets a single protein or pathway, so traditional therapies need to go beyond the ‘one disease, one drug, one target’ paradigm, thus, combination therapy is more efficient and, for that reason, is becoming more regular.58,59 This therapy is a strategy for preventing infections caused by MDR gram-negative pathogens. New combinations are increasingly proposed as a therapeutic option. The combinations include antibiotics plus drugs without antibiotic activity, or antibiotics plus other antibiotics.61 With the recent advances, Food and Drug Administration (FDA) approved new drug therapies. For example, combination of dolutegravir and lamivudine blocks the HIV-1 multiplication, treating HIV1 infection and neutralizing emerging drug-resistant HIV strains.58,59 In addition, this therapy has been utilized in the treatment of fungal diseases, e.g., by combining fluconazole and dexamethasone, replication of drug-resistant Candida albicans has been inhibited; and drug resistant tubercolosis, with the activity of moxifloxacin and linezolid, because the anti-efflux pump molecules timcodar and verapamil destabilize the Mycobacterium tuberculosis cell wall.58 It has also been evaluated as a therapeutic method to treat and regulate the spread of malaria, by using tafenoquine and chloroquine along with six artemisinin drugs.58 Moreover, plazomicin can be used in combination with tazobactam/piperacillin or ceftazidime against multidrug-resistant (to β-lactam 16 (x) induction of coagulation of cytoplasmic constituents.87 Besides the mentioned examples, other examples of plants with compounds that present antimicrobial activity can be reported, such as piperine isolated from Piper nigrum that has shown to enhance antimicrobial activity of mupirocin against S. aureus strains including MASA through the inhibition of efflux of ethidium bromide76; ethanol extract of Momordica charantia L. (bittermelon) which displayed the antibiotic activity against MRSA strain76; the ethanol extract of Hypericum perforatum L . (St. John's wort) that exerts strong antimicrobial activity against Streptococcus mutans , Streptococcus sobrinus , Lactobacillus plantarum and Enterococcus faecalis , as well as its water extracts that display strong antibacterial activity against Streptococcus sobrinus and Lactobacillus plantarum 76; coumarins, which can be extracted from Melilotus albus (honey-clover) and whose extracts with ethanol, acetone, and ethyl acetate have been shown to be active against Bacillus subtilis and S. aureus 93; flavonoid-rich water-ethanol (70%) extract of Equisetum arvense L . (common horsetail) had antibacterial activity against S. aureus 93; quercetin and hydroxycinnamic derivatives from 70% ethanol extract of Urtica dioica (stinging nettle) showed activity against MSSA and MRSA93. Many more examples could be included here. More specifically with regard to phenolics compounds and plant extracts rich in these substances, it is important to mention that these can be excellent inhibitors of bacteria. Some examples are mentioned below. For example, bergamot peel has been found to be effective against gramnegative foodborne pathogens E. coli and Salmonella enterica and Bacillus subtilis ; quince peel against E. coli , P. aeruginosa and S. aureus ; mango kernel against E. coli . Other fruits such as jackfruit, papaya, plum, guava, and tamarind and their seed and many more examples that could me mentioned, have also shown antimicrobial activity against both gram-positive and gramnegative bacteria.94 In addition to all that has already been mentioned, it is also important to note that plants also have high antioxidant activity, which, although it is not yet fully understood, may be connected to antimicrobial activity.88 It perhaps can be attributed to their capacity to chelate iron, vital for the survival of almost all bacteria and due to their capacity to eliminate free radicals.88,95 Antioxidants, such as phenolic compounds existing in plants mentioned before, are responsible for inhibiting oxidation at several points, depriving cells of energy, which may result in interrupting nucleic acid synthesis.88 Moreover, their hydroxyl (–OH) groups are thought to cause inhibitory action by interacting with the cell membrane of bacteria to disrupt disrupting microbial membranes or impairing cellular metabolism.88,94 Gram-negative and gram-positive bacterial cell walls play a very 17 important role in osmotic protection of cell and many researchers have demonstrated that the interaction of phenols and polyphenols with bacterial cell wall is different for gram-negative and gram-positive bacteria, because their cell wall composition differs significantly.88,95 Nowadays, plant phenols and polyphenols enjoy an ever-increasing recognition not only by the scientific community but also, and most remarkably, by the general public because of their presence and abundance in fruits, seeds, vegetables, and derived foodstuffs, whose regular consumption has been claimed to be beneficial for human health.96 So the focus of this study relies on plants with antioxidant properties used daily, as is the case of garlic, pomegranate and ginger. 1.4.1. Allium sativum (Garlic) Allium sativum , more known as garlic, is among the oldest cultivated plants and one of the most important bulb vegetables. It has been used as a spice and flavoring agent, and in folklore medicine for over 4000 years, and consequently is a widely researched medicinal plant.97,98 It has been used and investigated for diverse medicinal properties, such as anticancer, anti-inflammatory, antifungal, antiviral and antioxidant properties, and in 1858, Louis Pasteur reported its antibacterial properties.97–102 More recently, garlic has been proven to be effective against gram-positive and gramnegative bacteria, including P. aeruginosa , S. aureus , E. coli , Salmonella enterica , Klebsiella aerogenes and Mycobacterium .98,103 Most of the health benefits of garlic are attributed to a myriad of cysteine-derived sulfur-containing organic compounds present in garlic, mainly alliin and its crushing converts it into allicin.103–106 Allicin is a highly reactive, very unstable with low bioavailability compound, that degrades and rearranges itself into different sulfides or ajoene.102,105 The extraction procedure results in concentrating a particular compound rather than providing a pure compound and the extraction of garlic with water or ethanol followed by the concentrating of the extract will provide an allicin-rich product and it was noticed that yield with ethanol is better compared to water.98 An in vitro study with allicin vapors showed that they were able to exhibit bactericidal activity against MDR lung pathogenic bacteria such as P. aeruginosa and Streptococcus pyogenes .105 Nevertheless, it was also described that aqueous extract showed antibacterial activity against S. aureus , K. pneumoniae and Bacillus subtilis 98, which means that garlic has effectively a large potential as an antibacterial agent. Moreover, ethanolic extract of garlic revealed that it contains various thoisulfinates, being the major one also allicin, and exhibited some degree of antibacterial activity against test enteropathogenic bacterial strains.105 18 1.4.2. Zingiber officinale (Ginger) Ginger, the rhizome of Zingiber officinale , is a member of the Zingiberaceae family that has been used as a spice globally for over 2000 years because of its characteristic spicy aroma and taste.107– 110 It is a perennial herb originated South-East Asia (today’s northeast India) and now cultivated in many different countries.107,111,112 Chemical analysis of ginger shows that it contains over 400 different compounds, being carbohydrates (50–70%), lipids (3–8%), terpenes, and phenolic compounds e its pharmacological activity is mainly attributed to its active phytocompounds 6-gingerol and 6shogaol, beside other phenolics and flavonoids.108,109,113,114 The rhizomes have been used in many oxidative stress related medical conditions, but in recent years, it has also been described as a potential antibacterial agent.107,109,112,115–118 Ginger antimicrobial activity is due to its phenolic compounds insoluble in water and, thus, its aqueous extracts exhibit lower antimicrobial activity than organic extracts, because ginger hydrophobic compounds interact with the lipophilic part of the membrane and isolated mitochondria, promoting its integrity and function disrupt.108,113 Actually, it was showed that ethanolic ginger extract has antimicrobial activity against E. coli, Salmonella typhi , Bacillus subtilis, Candida albicans 113,119,120 and also can inhibit the growth of a multidrug-resistant strain of P. aeruginosa , by affecting membrane integrity and inhibit biofilm formation.117 1.4.3. Punica granatum (Pomegranate) Pomegranate ( Punica granatum L. ) is a plant of Punicaceae family cultivated and naturalized over the whole Mediterranean region since ancient times that has prominent medical history and possesses remarkable medicinal properties.121–123 The antioxidant activity of the pomegranate peel extract (PPE) is attributed to the bioactive phenolic compounds ranging from simple phenolic acids, such as hydroxybenzoate to complex polyphenols, such as tannins and water-soluble polyphenolic compounds, such as ellagitannins). 124,125 Tannins may be toxic to the microorganisms, since their hydrophilic parts may interact with the polar region of membrane whereas the hydrophobic part is immersed in the non-polar inner region of the bacterial membrane, causing instability of the membrane.126 Nevertheless, it is important to note that the content of the tannins can have large variations between different pomegranate cultivars. For instance, it has been described that in pomegranate fruits of Egyptian origin, the punicalagin (an ellagitannin, a type of phenolic compound) concentration in aqueous methanol extracts of peels 19 was reported to be 98.02 mg/g, while extracts from pomegranates from Israel presented a considerably higher content, at about 612.8 mg/g.127Moreover, chlorogenic acid, one of the major compounds found in the PPE can also interact with the bacterial outer membrane, and rupture the cell membrane, deplete intracellular content and release macromolecules from the cytoplasm, leading to bacterial death.128 In general, phenolic compounds can inhibit the activity of essential proteins by interacting with the sulfhydryl groups.128 The literature has widely discussed the efficacy of PPE at inhibiting or reducing the growth of a wide range of microorganisms, such as S. aureus , Staphylococcus epidermidis, Lactobacillus acidophilus, Streptococcus mutans and Streptococcus salivarius .128–130 The Pomegranate pomace (PP) is rich in carbohydrates and fibers and has high water-absorption capacity. Its active phenolic compounds belong to three groups: ellagitannins, ellagic acid derivatives, and gallic acid derivates and they represent the reason why PP has such a high antioxidant activity and has the capacity to inhibit the growth of pathogenic bacteria such as P. aeruginosa and K. pneumonia .131,132 The pomegranate juice (PJ) contains considerable amounts of total soluble solids, total sugars, reducing sugars, anthocyanins, phenolics, ascorbic acid and proteins and has also been reported to be a rich source of antioxidants.133 Its antioxidant activity is mainly attributed to their flavonoid content, such as anthocyanins ,catechins, and tannins, that together account for 92% of their antioxidant activities.123,126,134–136 PJ was tested against 60 clinical strains of S. epidermidis isolated from ocular infections and resistant to ampicillin and it completely inhibited the growth of all 60 strains. In this study, ampicillin was used as a control and various bacteria shown to be resistant to the antibiotic and sensitive to PJ.126 In general, pomegranate’s extracts exhibited bactericidal activity against various pathogenic bacteria including S. aureus, P. aeruginosa , E. coli , K. pneumoniae, Streptococcus pneumoniae and Candida albicans .124,130,136,137 Although it has been described that P. aeruginosa was sensitive to the pomegranate extracts, in general, various studies reported that gram-positive bacteria were more sensitive than gram-negative.130,137 1.5. Objectives Antibiotic resistance is a global public health threat and it is urgent to solve it or at least minimize it. One of the strategies is to replace or complement antibiotic treatment with natural products that typically exhibited antioxidant properties that perhaps can be correlated with their antibacterial 20 properties so the aim of this study is to investigate the impact of the extract conditions, specifically the solvent, the duration and temperature on the antibacterial activity of garlic, ginger and pomegranate (peel, pomace and juice) against P. aeruginosa and S. aureus . Moreover, it is aimed to correlate the enhanced antibacterial activity of plant extracts with antioxidant activity and total phenolic content in order to understand the underlying mechanisms of action and possibly to accelerate the screening of bioactive potential of other plants for antibacterial purposes. 2. Materials and methods 2.1. Plant material All edible biomasses used in this work were provided by a local supermarket: Allium sativum (garlic); Zingiber officinale (ginger) and Punica granatum (pomegranate). Garlic, ginger and pomegranate were peeled, cut into small pieces and ground with a coffee mill. Garlic was then centrifuged for 10 minutes, 200 rpm (RS LAB, HIGUGE-GJ6). In the case of ginger, as a very wet sample was obtained, it was dried in an oven at 60 ºC, until a powder was obtained. The pomegranates were divided into three fractions: the peel, the pomace and the juice, which were then frozen in sample tubes and lyophilized. All samples were then stored at -18 ºC, until further use. 2.2. Preparation of plant extracts The extractions were performed at two different temperatures (overnight at room temperature and at 70 ºC for 1 hour) with different water/ethanol mixtures as solvents: EtOH (96%), EtOH (70%) and H2O(d). 1 mg of each biomass was mixed with 20 mL of solvent, in duplicate, both for the overnight at room temperature extraction and for the extraction at 70 ºC for 1 hour. Then, for the first one, the samples were placed on a tray covered with aluminum foil (no shaking in this process); and for the second, they were placed in a water bath with shaker at 70 ºC. Subsequently, the extracts were filtered and stored at -18 ºC, until further use. To assess the extraction yield, the solid content of each extract was determined as follows: 1 mL of each extract was placed in a pre-weighted aluminum crucible, which were taken to the oven at 105 ºC overnight, until constant weight. The analysis was performed in triplicate, and the yield value was obtained using the following formula: Extraction yield = (dried sample concentration x volume of extraction / mass of matrix) x 100 (eq. 1) 21 2.3. Determination of Total Phenolic Content (TPC) Phenolic content was determined using the Folin–Ciocalteu assay. The Folin-Ciocalteu method is an electron transfer-based assay, and assesses the reducing capacity which is expressed as phenolic content (that is highly dependent of extraction yield and solvent).138 The calibration curve was done with different concentrations of gallic acid (0.200, 0.150, 0.100, 0.075, 0.050, 0.025, 0.010 and 0.005 mg/mL) and distilled water was used as a blank. The solvent used in each extraction (EtOH (96%), EtOH (70%) and H2O (d)) was taken as the blank for each sample. Sample solutions were added to microplate with 100 µL of Folin-Ciocalteu reagent (1:10 in H2O). 80 µL of Na2CO3 were also added to each well and the reaction was incubated at 42 ºC, protected from light, for 30 minutes. The absorbance was measured with a microplate reader at 750 nm (Thermo Fisher Scientific, Lisboa, Portugal). The total phenolic content was calculated as gallic acid equivalent (mg/mL) by using gallic acid calibration curve. 2.4. Determination of antioxidant activity Antioxidant potential of extracts was assed using two different methods: Ferric Reducing Antioxidant Power (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method. 2.4.1. Ferric Reducing Antioxidant Power It has been 27 years since the Ferric Reducing Antioxidant Power (FRAP) assay method was first described, by Benzie & Strain. This method monitors the reaction of Fe2+ with 2,4,6Tripyridyl-sTriazine (TPTZ) to form a violet-blue color with an absorbance maximum at 593 nm.139,140 Sample or standard Trolox solutions (20 μL) were added directly to the 96-well microplate followed by 280 μL of FRAP working solution. The mixtures were shaken, incubated at 37 ºC protected from light, for 30 minutes. The absorbance was read at 593 nm using a microplate reader (Thermo Fisher Scientific, Lisboa, Portugal). The extracts from garlic, ginger and pomegranate (peel, pomace and juice) were used. Some dilutions were done in order to obtain absorbances within the linear range of the calibration curve, allowing the equivalent Trolox concentration (mg/mL) calculus. The Trolox Equivalent Antioxidant Capacity (TEAC) was calculated using the pre-determined calibration curve, using Trolox as 22 standard in concentrations (1.250, 0.9, 0.625, 0.313, 0.156, 0.078 and 0.039 mM) and Methanol/Water (70:30, v/v) as the blank control: abs = 2,47 x [TEAC] - 8,01x103 (eq. 2) The solvent used in each extraction (EtOH (96%), EtOH (70%) and H2O(d)) was taken as the blank for each sample. 2.4.2. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid The ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, was first described by Miller et al. (1997). It is based on the ABTS radical (ABTS•+) that absorbs at 743 nm, formed by the loss of an electron by the nitrogen atom of ABTS. The ABTS•+ radical is strongly colored (blue-green color), but ABTS is colorless. When Trolox is present, the nitrogen atom quenches the hydrogen atom, the ABTS•+ declines and the solution decolorize, which causes the absorbance at 743 nm to decrease and allows the evaluation of compounds antioxidant capacity.140,141 As with the FRAP assay, the extracts from garlic, pomegranate (peel, pomace and juice) and ginger were used with some dilutions in order to obtain absorbances within the linear range of the calibration curve, which was obtained using Trolox as standard in concentrations (0.200, 0.140, 0.098, 0.069, 0.048 and 0.034 mM) and Methanol/Water (70:30, v/v) as the blank control. The solvent used in each extraction (EtOH (96%), EtOH (70%) and H2O (d)) was taken as the control for each sample. In a 96-well plate, 180 μl ABTS working solution and 20 μl sample or control solution were added, shook well, and protected from light for 30 min. The absorbance was measured in a microplate reader at 734 nm (Thermo Fisher Scientific, Lisboa, Portugal). The Trolox equivalent concentration was calculated using the pre-determined calibration curve and the percentage of inhibition indicating the ABTS radical scavenging capacity was calculated as follows: % Inhibition SAMPLE = 100 x (Abs734 ABTS BLANK - Abs734 SAMPLE)/Abs734ABTS BLANK (eq. 3) Where Abs734sample is the absorbance of each sample; The TEAC (Trolox Equivalent Antioxidant Capacity) of samples is calculated as follows: 23 TEAC SAMPLE (mg/mL) = (% inhibition – 1.33)/102 (eq. 4) 2.5. Determination of antibacterial activity 2.5.1. Bacterial species and growth conditions In this study Pseudomonas aeruginosa clinical isolate U147016-1 and Staphylococcus aureus ATCC 25923 were used. Bacteria were routinely cultured on Tryptic Soy Broth (TSB, Liofilchem) or Tryptic Soy Agar (TSA, Liofilchem) at 37 ºC. All strains were preserved in cryovials (Nalgene) with TSB supplemented with 20% glycerol at -80 ± 2 ºC to minimize putative adaptation to the laboratory environment. Prior to each experiment, bacterial cells were grown on TSA plates overnight at 37 ºC. 2.5.2. Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration The antimicrobial activity of the extracts was established by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) using the microdilution method following the recommendations of the Clinical and Laboratory Standards Institute.142 Before the experiment, ethanolic extracts were prepared to antibacterial activity analysis with 5% aqueous DMSO (aqueous extracts suffer no alteration). MIC and MBC were assayed using a 96-well plate with different concentrations of plant extract described in Table 1. All extracts were prepared in Mueller Hinton Broth (MHB). Bacteria were added to the wells to obtain a final concentration of 5x105 CFU/mL (Colony Formation Unit) and incubated at 37 ºC, 120 rpm for 18 to 21 hours. Afterward, cultures were plated onto Mueller Hinton Agar. MIC was defined as the lowest concentration of an extract that inhibited the 99% bacterial growth determined by optical density reading at 620 nm using a microplate reader (Biochrom EZ Read 800 Plus, Cambridge, UK). The lowest concentration of crude extracts with the absence of growth on solid medium after overnight incubation at 37 ºC was considered as MBC. All tests were performed at least in duplicate. Table 1 Range of tested extract concentration obtained from different plants under study (mg/mL) 24 Plant Solvent Extraction Conditions Range of tested [extract] (mg/mL) Garlic EtOH (96%) 70 ºC ≈ 1H 0.002 - 1.1 Overnight 0.001 - 0.4 EtOH (70%) 70 ºC ≈ 1H 0.013 - 6.8 Overnight 0.010 - 5.1 H2O (d) 70 ºC ≈ 1H 0.015 - 7.9 Overnight 0.014 - 7.3 Ginger EtOH (96%) 70 ºC ≈ 1H 0.004 - 1.8 Overnight 0.002 - 1.2 EtOH (70%) 70 ºC ≈ 1H 0.009 - 4.7 Overnight 0.005 - 2.5 H2O (d) 70 ºC ≈ 1H 0.012 - 6.3 Overnight 0.016 - 8.0 Pomegranate Peel (PPE) EtOH (96%) 70 ºC ≈ 1H 0.010 - 5.1 Overnight 0.009 - 4.7 EtOH (70%) 70 ºC ≈ 1H 0.011 - 6.0 Overnight 0.010 - 5.3 H2O (d) 70 ºC ≈ 1H 0.011 - 5.6 Overnight 0.010 - 5.3 Pomegranate Pomace (PP) EtOH (96%) 70 ºC ≈ 1H 0.008 - 4.0 Overnight 0.007 - 3.5 EtOH (70%) 70 ºC ≈ 1H 0.008 - 4.2 Overnight 0.007 - 3.8 H2O (d) 70 ºC ≈ 1H 0.007 - 3.7 Overnight 0.008 - 4.1 Pomegranate Juice (PJ) - - 0.117 - 90.4 2.6. Statistical analysis All data were analyzed using GraphPad Prism 6 software. Data were compared by two-way analysis of variance (ANOVA) followed by Turkey multiple comparisons test. 3. Results and Discussion 3.1. Plant extraction yield The extraction of active compounds can be performed using various solvents with distinct polarities resulting in the solubilization of distinct bioactive compounds and with the extraction efficiencies.143 25 Water, methanol, ethanol, and acetone are amongst the most used solvents for the extraction of bioactive compounds from plants143, and in this study ethanol (96% and 70%) and water were selected to evaluate their impact on the antibacterial activity and their correlation with antioxidant activity and phenolic content In garlic extracts, the extraction yield was higher on aqueous extracts (0.32 ± 2.12x10-4 and 0.29 ± 2.12x10-3 at 70 ºC and overnight extraction, respectively) followed by the ethanolic extracts. It was noted that as ethanol concentration increased, the lower the extraction yield was as expected (Figure 1(a)).143–145 One of the factors that may account for the high yield of the aqueous extracts may be the high percentage of carbohydrates (approximately 30%) in its composition.146 Nevertheless, this does not implies that these will be the most active extracts, as the bioactivity in garlic is not commonly associated with its composition in carbohydrates. Regarding the extraction yield of ginger extracts, the same trend was verified, as the higher the percentage of ethanol in the solvent, the lower the yield (0.25 ± 1.84x10-3 mg/mg and 0.32 ± 6.36x10-4 mg/mg in 70 ºC and overnight extractions, respectively) (Figure 1(b)). This is justified by the fact that carbohydrates represent the vast majority of the constitution of ginger (50-70%), as mentioned above, since carbohydrates are highly soluble in water, given the presence of -OH groups.147 However, this trend was not verified in several studies. Figure 1 Extraction yield of extracts obtained from plants under study. The result is presented in mg of extract per mg of matrix of garlic (a) and ginger (b) extracts. Data presented represent the mean ± standard deviation of three assays in triplicate. The lowercase letter represents the significant comparison of the extraction solvents, considering the same extract and the same extraction conditions. The uppercase letter represents the significant comparison of the extract conditions, considering the same extract and the same solvent. The analysis starts with the letter “a” and “A” representing the highest value. 0.0 0.1 0.2 0.3 0.4 Garlic 70ºC ~1H Overnight Yield of extraction (mg/mg) bA cA aA bB aA aA 0.0 0.1 0.2 0.3 0.4 Ginger 70ºC ~1H Overnight Yield of extraction (mg/mg) aA aA aA aA bA bA (a) (b) 32 Data presented represent the mean ± standard deviation of three assays in triplicate and each replicate included several ginger rhizomes. The lowercase letter represents the significant comparison of the extraction solvents, considering the same extract and the same extraction conditions. The uppercase letter represents the significant comparison of the extract conditions, considering the same extract and the same solvent. The analysis starts with the letter “a” and “A” representing the highest value. In PPE extracts (Figure 7(a)), the Fe3+-TPTZ complex was most strongly reduced to Fe2+ by ethanolic (70%) overnight extract (1.66 ± 0.12 mmol/g) and aqueous extract, 70 ºC for 1 hour (1.615 ± 0.15 mmol/g), with no significative difference. On the other hand, the lowest value was obtained by aqueous overnight extract (1.09 ± 0.6 mmol/g). In Figure 7(b), the results were in line with what was expected, ethanolic (70%) extracts showed higher antioxidant capacity in both 70 ºC and overnight extraction according to the ABTS method, (1.41 ± 0.08 mmol/g and 1.316 ± 0.09 mmol/g, respectively). As was the case in the evaluation of the phenolic compounds (Figure 4(a)), generically the highest antioxidant activity was also obtained when the extraction solvent used was EtOH (70%), which may mean that the phenolic compounds are responsible for the antioxidant activity. This becomes even more evident, knowing that this extract contains a wide range of phenolic compounds, some soluble in water and others mostly insoluble, that make a mixture of solvents more effective, as described in literature.159 (a) 0.0 0.5 1.0 1.5 2.0 TEAC (mmol / g extract) 70ºC ~1H Overnight bA bB aA bA aA cB (b) 0.0 0.5 1.0 1.5 2.0 TEAC (mmol / g extract) 70ºC ~1H Overnight EtOH (96%) EtOH (70%) H2O (d) aA aA aA aA bA bB Figure 7 Trolox equivalent concentration, mmol Trolox Equivalent per g of extract (mmol/g), by FRAP analysis (a) and by ABTS analysis (b), of pomegranate peel extracts. Data presented represent the mean ± standard deviation of three assays in triplicate and each replicate included several pomegranates. The lowercase letter represents the significant comparison of the extraction solvents, considering the same extract and the same extraction 33 conditions. The uppercase letter represents the significant comparison of the extract conditions, considering the same extract and the same solvent. The analysis starts with the letter “a” and “A” representing the highest value. As described above for PPE, the same is true for PP, since the compounds that compose that shows its antioxidant activity are mostly ellagitannins, ellagic acid derivatives and gallic acid, where ellagic acid and gallic acid are more soluble in ethanol than in water160,161 and ellagitannins are soluble in water162, so it was indeed expected that a mixture of solvents would perform better antioxidant activity. In Figure 8 is possible to verify that both FRAP (a) and ABTS (b) analysis, the highest antioxidant activity was performed by ethanolic (70%) extract, but in FRAP analysis it happened in overnight extraction (0.34 ± 0.07 mmol/g), with no significative difference for the one extracted at 70 ºC; and in ABTS in 70 ºC extraction (0.29 ± 0.01 mmol/g), also with no significative difference with the overnight extract. It is also important to note that at 70 ºC, the aqueous extracts present an antioxidant activity close to the ethanolic (70%) extracts, while overnight, this similarity is obtained between the ethanolic extracts (96%) and (70%). However, the same was verified in the analysis of phenolic compounds (Figure 4(b)), which may be another indicator that the activity of PP is due to its phenolic content. (a) 0.0 0.1 0.2 0.3 0.4 0.5 TEAC (mmol / g extract) 70ºC ~1H Overnight bB aA aA bA aA cB (b) 0.0 0.1 0.2 0.3 0.4 0.5 TEAC (mmol / g extract) 70ºC ~1H Overnight cB aA bA bA aA cB EtOH (96%) EtOH (70%) H2O (d) Figure 8 Trolox equivalent concentration, mmol Trolox Equivalent per g of extract (mmol/g), by FRAP analysis (a) and by ABTS analysis (b), of pomegranate pomace extracts. Data presented represent the mean ± standard deviation of three assays in triplicate and each replicate included several pomegranates. The lowercase letter represents the significant comparison of the extraction solvents, considering the same extract and the same extraction conditions. The uppercase letter represents the significant comparison of the extract conditions, 34 considering the same extract and the same solvent. The analysis starts with the letter “a” and “A” representing the highest value. Although the antioxidant activity of PJ is mainly attributed to compounds that also exist in PPE and PP, such as anthocyanins, catechins, and tannins, this extract has a lower antioxidant activity per g extract than PPE and PP, both in FRAP (0.10 ± 0.01 mmol/g) and ABTS analysis (0.14 ± 0.01 mmol/g) (Figure 9). 0.00 0.05 0.10 0.15 0.20 TEAC (mmol / g extract) FRAP ABTS Figure 9 Trolox equivalent concentration, mmol Trolox Equivalent per g of extract (mmol/g), by FRAP and ABTS analysis, of pomegranate juice extracts. Data presented represent the mean ± standard deviation of three assays in triplicate and each replicate included several pomegranates. 3.4. Determination of antibacterial activity Natural biomasses as plants are composed by panoply of compounds with different bioactive activities. The use of various solvents affects the solubility of different phytochemicals and, consequently, the antimicrobial properties of each extract can be distinct.143 In this study, different extraction conditions were used in order to evaluate their impact on antibacterial activity of the selected plant biomasses, garlic, ginger and pomegranate peel, pomace and juice, against P. aeruginosa and S. aureus . Antibacterial activity was stipulated according to the MIC and MBC values. Analyzing the results (Table 2), only the ethanolic extract 96% overnight inhibited S. aureus growth but it was not able to eradicate bacteria and o activity against P. aeruginosa was recorded. Its activity against gram-positive bacteria rather than gram-negative was reported previously in literature.163 This difference resulted from the distinct cell wall structure between these major classes of bacteria, since gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, 35 which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the gram-negatives.164,165 Considering the previous results, the antibacterial activity of the ethanolic (96%) extracts of garlic against S. aureus might be related with the antioxidant activity and the total phenolic content (TPC). The ethanolic (96%) extract was the one with highest antioxidant activity (Figure 5) and with significant TPC (Figure 3(a)). Being allicin described as one of the compounds responsible for antioxidant activity of garlic102, as mentioned above, and being this a compound more easily solubilized in ethanol, this bioactivity may be due to its presence. This is also in agreement with a study done with garlic extract, in which it was found that the higher the allicin content in the extract, the higher its antibacterial activity, and when the formation of allicin was inhibited during extraction, the extract lost its activity.166 It is also described that phenolics can play an important role in antibacterial activity, although, as mentioned earlier, the main contributors are organosulfur compounds (such as allicin).167 Moreover, it is important to emphasize that being the same compound the primarily responsible for both antioxidant and antibacterial properties of garlic, there may be effectively a relationship between the two bioactivities. Few studies reported that aqueous extracts of garlic can be antibacterial98 but in this work no inhibition or eradication was observed for both species. Mozaffari Nejad et al described the minimum concentration of aqueous garlic extract that prevents the growth of gram-positive microorganisms is between 15.6 and 48.3 mg/mL and gram-negative microorganisms is between 14.9 and 37.2 mg/mL168, which are much higher extract concentrations than those that were tested in the present work. Furthermore, it is important to emphasize that the only extract capable of inhibit the growth of S. aureus was the extract obtained overnight, which was in line with the literature, since it is described that temperature may degrade important garlic bioactive compounds.153 36 Table 2 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of garlic extract obtained from different extraction conditions tested on S. aureus and P. aeruginosa Bacteria Solvent Extraction conditions MIC99 (mg/mL) MBC (mg/mL) Maximum concentration tested (mg/mL) S. aureus EtOH 96% 70 ºC ~1H > 1.1 > 1.1 1.1 EtOH 70% > 6.8 > 6.8 6.8 H2O (d) > 7.9 > 7.9 7.9 EtOH 96% Overnight 0.4 > 0.4 0.4 EtOH 70% > 5.1 > 5.1 5.1 H2O (d) > 1.8 > 7.3 7.3 P. aeruginosa EtOH 96% 70 ºC ~1H > 1.1 > 1.1 1.1 EtOH 70% > 6.8 > 6.8 6.8 H2O (d) > 7.9 > 7.9 7.9 EtOH 96% Overnight > 0.4 > 0.4 0.4 EtOH 70% > 5.1 > 5.1 5.1 H2O (d) > 7.3 > 7.3 7.3 Among the ginger extracts tested, only ethanolic extract (96%, at 70 ºC, 1h) was able to inhibit the growth of S. aureus (Table 3). Although in both antioxidant analyses it showed slightly less activity than ethanolic (96%) extract overnight (Figure 6), with regard to the phenolic content analysis, the ethanolic extracts were also the ginger extracts that showed the highest phenolic content, especially the extract obtained at 70 ºC (Figure 3(b)), which could perhaps justify the fact that this extract showed higher antibacterial activity, which is usually associated with compounds such as 6-gingerol and 6-shogaol, as already mentioned. According to the literature, ginger extracts were expected to also inhibit the growth of P. aeruginosa 169, although this did not occur at the extract concentrations tested in this study. It is also important to note that no extracts obtained from ginger demonstrated bactericidal activity. 37 Table 3 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of ginger extracts obtained from different extraction conditions tested on S. aureus and P. aeruginosa Bacteria Solvent Extraction conditions MIC99 (mg/mL) MBC (mg/mL) Maximum concentration tested (mg/mL) S. aureus EtOH 96% 70 ºC ~1H 0.9 0.9 1.8 EtOH 70% >4.7 >4.7 4.7 H2O (d) >6.3 >6.3 6.3 EtOH 96% Overnight >1.2 >1.2 1.2 EtOH 70% >2.5 >2.5 2.5 H2O (d) >8.0 >8.0 8.0 P. aeruginosa EtOH 96% 70 ºC ~1H >1.8 >1.8 1.8 EtOH 70% >4.7 >4.7 4.7 H2O (d) >6.3 >6.3 6.3 EtOH 96% Overnight >1.2 >1.2 1.2 EtOH 70% >2.5 >2.5 2.5 H2O (d) >8.0 >8.0 8.0 PPE extracts revealed to be the most active against S. aureus and P. aeruginosa (Table 4). The extracts with the highest ability to cause growth inhibition at lower concentrations were the extracts obtained with EtOH (70%), that corresponded to PPE extracts with highest total phenolic content (Figure 4(a)) and antioxidant activity (Figure 7). This can be justified by the fact the active bioactive compounds in PPE are more soluble in solvent mixtures, as already mentioned. However, this mixture between ethanol and water seems to be more efficient in the 70:30 (v/v), since it was found that in ethanol (80%) extracts tested, the MIC was between 15.62 and 19.5 mg/mL170 and that in ethanolic (50%) extracts the MIC was around 10 mg/mL.171 This may indicate that the percentage of ethanol in the solvent has high influence in the extracted compounds. However, studies with a wider range of ethanol concentrations used as an extraction solvent would be needed to validate this information. Aqueous overnight extracts were effective against S. aureus using the microdilution method, but these results cannot be compared, because no studies were found about aqueous extracts obtained with the same extraction method at similar conditions or against the same microorganisms. Both overnight and 70 ºC and overnight ethanolic (70%) extracts were effective against both bacteria. However, it is possible to verify that overnight extracts were effective at lower 38 concentrations than extracts obtained at 70 ºC, indicating that the temperature may have affected the extraction of some compounds responsible for antibacterial activity. It is also important to mention that S. aureus is more susceptible than P. aeruginosa , since there was no bactericidal activity against the gram-negative bacteria, which is also described in literature.128 Table 4 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of pomegranate peel extracts obtained from different extraction conditions tested on S. aureus and P. aeruginosa Bacteria Solvent Extraction conditions MIC99 (mg/mL) MBC (mg/mL) Maximum concentration tested (mg/mL) S. aureus EtOH 96% 70 ºC ~1H >5.1 >5.1 5.1 EtOH 70% 1.5 - 2.9 2.9 6.0 H2O (d) ≥5.6 ≥5.6 5.6 EtOH 96% Overnight ≥4.7 ≥4.7 4.7 EtOH 70% 0.7-2.6 2.6 5.3 H2O (d) 2.7-5.3 5.3 5.3 P. aeruginosa EtOH 96% 70 ºC ~1H ≥5.1 ≥5.1 5.1 EtOH 70% 1.5-2.9 ≥5.8 6.0 H2O (d) ≥5.6 ≥5.6 5.6 EtOH 96% Overnight >4.7 >4.7 4.7 EtOH 70% 0.7-2.6 >5.3 5.3 H2O (d) ≥5.3 ≥5.3 5.3 Analyzing Table 5, it is possible to verify that the extracts of PP with increased antibacterial activity were the ethanolic (70%) extracts, corresponding to the extract with highest total phenolic content (Table 2(c)) and the antioxidant activity (Figure 3). Furthermore, it is important to note that the extract obtained overnight was able to inhibit the growth of S. aureus at a lower concentration than the extract at 70 ºC, which is in agreement with the result obtained in the FRAP analysis (Figure 3(a)), although in the ABTS analysis the opposite was expected (Figure 3(b)). It is important to note that the aqueous extract obtained at 70 °C showed inhibition of the growth of P. aeruginosa , which is noteworthy because this extract showed high antioxidant activity and a high content of phenolic compounds. This also helps validate the fact that the antibacterial activity of PP can result from its phenolic compounds. This study is the first one reporting the antibacterial activity of PP for S. aureus and P. aeruginosa. None of the extracts showed bactericidal activity at the concentrations tested against both species. 39 Table 5 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of pomegranate pomace extracts obtained from different extraction conditions tested on S. aureus and P. aeruginosa Bacteria Solvent Extraction conditions MIC99 (mg/mL) MBC (mg/mL) Maximum concentration tested (mg/mL) S. aureus EtOH 96% 70 ºC ~1H >4.0 >4.0 4.0 EtOH 70% ≥4.2 >4.2 4.2 H2O (d) >3.7 >3.7 3.7 EtOH 96% Overnight >3.5 >3.5 3-5 EtOH 70% ≥3.8 >3.8 3.8 H2O (d) >4.1 >4.1 4.1 P. aeruginosa EtOH 96% 70 ºC ~1H >4.0 >4.0 4.0 EtOH 70% >4.2 >4.2 4.2 H2O (d) ≥3.7 ≥3.7 3.7 EtOH 96% Overnight >3.5 >3.5 3-5 EtOH 70% ≥3.8 ≥3.8 3.8 H2O (d) >4.1 >4.1 4.1 PJ is one of the extracts with lower content of total phenolics (Figure 4(c)) and, when compared to PPE and PP extracts, it is possible to verify that it also presents lower antioxidant activity (Table 6). However, it is possible to register the growth inhibition of both bacteria, although maybe this inhibition has to do with the higher concentration of the extract used and not exactly with the extract itself. The values obtained for S. aureus are not very different from those found in the literature, although the extracts in the study in question refer to juice extracted with EtOH (50%), which in this case were MIC = 25 µg/µL, equivalent to 25 mg/mL and MBC = 40 µg/µL, equivalent to 40 mg/mL.171 Being PJ rich in sugars133, as mentioned before, and being sugars growth enhancers for bacteria172, it would be interesting to perform an ethanolic extraction of PJ in order to extract more phenolic compounds, which are mainly attributed to antibacterial activity, so as to possibly obtain an extract with even more activity. Nevertheless, it is important to note that the tested extracts demonstrated bactericidal activity against both bacteria. 40 Table 6 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of pomegranate juice tested on S. aureus and P. aeruginosa Bacteria MIC99 (mg/mL) MBC (mg/mL) Maximum concentration tested (mg/mL) S. aureus P. aeruginosa 22.6 45.2 90.4 90.4 90.4 90.4 It is important to note that PPE was also able to inhibit the growth of both bacteria (although only with some solvents, as mentioned earlier), but it was able to do so at considerably lower extract concentrations than those tested with the juice, suggesting that PPE not only has more potential as an antioxidant, but also has more potential as an antibacterial. 4. Conclusion and future perspectives The aim of this study was to identify non-antibiotic alternatives with potential to address, or at least minimize, the problem of antibiotic resistance, one of the biggest crises in the world. It has been described that natural products, and in particular plants, have a high potential as antibacterial drugs, often associated with their high antioxidant activity. In this study, total phenolic content, antioxidant and antibacterial properties of extracts from five different biomasses, garlic, pomegranate (peel, pomace and juice) and ginger, were evaluated. Regarding the extraction yields, no optimal solvent was found for the three biomasses used. Deionized water demonstrated to produce higher yield of extracts of garlic and ginger, while ethanol (70%) was the best solvent for the pomegranate extraction (both peel and pomace). In contrast, in most the extractions 70 ºC for 1 hour results in increased yield than overnight extraction at room temperature, because mass transfer rates and target’s compounds solubility are generally higher at higher temperatures. Further, temperature may also have a positive effect in damaging the plant structure, thus facilitating extraction. In the analysis of total phenolic content, most of the ethanolic extracts showed more phenolic content than the aqueous extracts and, ethanol (70%) showed to solubilize more content of phenolic compounds than ethanol (96%). In aqueous extracts, the extraction performed at 70 ºC always promoted a higher extraction of phenolic content, although in less quantity than the ethanolic ones, than the overnight extraction. The extracts that showed higher phenolic content were the PPE extracts obtained with ethanol (70%), at 70 ºC and overnight. 41 Analyzing the antioxidant capacity of the different extracts, it was concluded that the ethanolic solvents produced extracts with higher antioxidant activity than the aqueous extracts. The concentration of ethanol used (70 and 96%) did not seem to influence the antioxidant activity of the extracts. Furthermore, it was possible to conclude that in the aqueous extracts the antioxidant activity was higher when the extraction was done at 70 ºC than when it was performed overnight. On the other hand, in most of the ethanolic extracts, higher antioxidant activity was obtained in the overnight extracts than in the extractions done at 70 ºC, which allows the conclusion that for this specific type of evaluation, the extraction at 70 ºC for is not the best option. Eventually, the more active fractions are also more thermolabile and their functionality was impaired at higher temperatures. Therefore, temperature is an important operational parameter to control and optimize when dealing with the extraction of functional fractions from natural biomasses, to achieve a reasonable balance between process feasibility (including extraction yield) and expressed bioactivity. Finally, regarding the antibacterial activity, in most extracts, it is associated with the extracts with higher phenolic and higher antioxidant content. Among ethanolic extracts, PPE extracts were the most promising non-antibiotic drugs against S. aureus and P. aeruginosa eradication and must be deeply explored in near future. As future work, it would be interesting to complement this work with a phytochemical analysis of plant extracts in order to validate some assumptions made throughout this work. Moreover, investigation of the activity of plant extracts on antibiotic resistant isolates, multi-resistant and extensively resistant isolates to evaluate its true potential and eventually against biofilms could be included. Moreover, it could be investigated the synergistic activity of the plant extracts with antibiotics on antibiotic resistant isolates and multi-resistant and extensively resistant isolates. 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