Functionalization of PVC using a mussel-inspired coating strategy to target the polymicrobial nature of ventilator-associated pneumonia

Universidade do Minho Escola de Engenharia Tânia Raquel Rodrigues Grainha Functionalization of PVC using a mussel-inspired coating strategy to target the polymicrobial nature of ventilator-associated pneumonia maio de 2023 UMinho | 2023 Tânia Raquel Rodrigues Grainha Functionalization of PVC using a mussel-inspired coating strategy to target the polymicrobial nature of ventilator-associated pneumonia Tânia Raquel Rodrigues Grainha Functionalization of PVC using a musselinspired coating strategy to target the polymicrobial nature of ventilator-associated pneumonia Tese de Doutoramento Engenharia Química e Biológica Trabalho efetuado sob a orientação da Professora Doutora Maria Olívia Pereira E da Doutora Diana Filipa Barros Alves maio de 2023 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-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii Acknowledgments Ao terminar esta jornada, não poderia deixar de agradecer a todas as pessoas que me apoiaram e sem as quais a realização desta tese não seria possível. Em primeiro lugar deixo um agradecimento especial às minhas orientadoras, Professora Maria Olívia Pereira e Doutora Diana Alves, por me terem dado oportunidade de trabalhar com elas neste projeto de doutoramento. A sua orientação, apoio, incentivo e partilha de conhecimento foram, sem dúvida, fundamentais para a concretização deste trabalho. Agradeço às MOPas (Ana Margarida, Paula, Susana e Rosana) por todo o companheirismo, boa disposição e por estarem sempre prontas a ajudar. A todos os colegas e amigos com quem me cruzei no CEB, em especial Vânia, Daniela A., Daniela S., Nathalie, Susana Brás, Fernando, Graça e Luís, obrigada pelos momentos de convívio, mas também pelo apoio e ensinamentos sempre que foi preciso. Agradeço especialmente à Andreia e à Joana pela vossa amizade e por estarem sempre presentes. À minha família, em particular aos meus pais e à minha irmã, por estarem sempre por perto e dispostos a ajudar. Obrigada aos meus pais por confiarem em mim e me deixarem seguir os meus sonhos, sem isso não teria chegado aqui! A ti, Edgar, um agradecimento especial por estares sempre disposto a ouvir-me, por me apoiares e incentivares em todos os momentos. Obrigada por seres meu companheiro em todas as etapas da minha vida. E por fim, dedico esta tese ao meu querido Bernardo que tornou o final desta jornada tão doce e especial. Obrigada a todos os que direta e indiretamente estiveram presentes e contribuíram para que esta caminhada se tornasse mais leve e divertida. “O mundo está nas mãos daqueles que têm a coragem de sonhar e de correr o risco de viver os seus sonhos” Paulo Coelho iv This study was supported by the Portuguese Foundation for Science and Technology (FCT) through an individual PhD scholarship (SFRH/BD/136544/2018), the funded project PTDC/BTMSAL/29841/2017, under the scope of COMPETE2020 (POCI-01-0145-FEDER-029841), and the strategic fundings of UID/BIO/04469/2013, UID/BIO/04469/2019 and UID/BIO/04469/2020 unit. v 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. vi Abstract Ventilator-associated pneumonia (VAP) is a common nosocomial infection with high mortality and morbidity rates. The endotracheal tube (ETT) is a risk factor for developing VAP as they are prone to microbial adhesion and biofilm formation; hence, strategies to impart these devices with antimicrobial properties are in great need. As such, this PhD project aimed to engineer an antimicrobial coating for ETTs to prevent VAP occurrence. Since the polymicrobial (intraand interkingdom) nature of VAP is of particular concern, this work also aimed to understand the interactions within these consortia as well as to inspect their impact on the efficacy of the engineered surfaces . Through an in silico approach, experimental data on the molecular basis of P. aeruginosa – C. albicans interactions, two VAP-relevant pathogens, were systematically curated and deposited in the new InterSpecies CrossTalk Database (www.ceb.uminho.pt/ISCTD). Data reconstructed as networks revealed key entities regulating these interactions, potential therapeutic targets, and possible inhibitors, which helped in antimicrobial compound selection for the in vitro tasks. In parallel, polyvinyl chloride (PVC) was functionalized using an adhesive dopamine-based strategy, applying safe-by-design criteria, to prevent single, dual, and triple adhesion and biofilm formation of VAPrelevant pathogens: P. aeruginosa, Staphylococcus aureus , and C. albicans . The coating strategy was successful in immobilizing nine compounds (natural and synthetic) on PVC. Coatings containing ciprofloxacin (CIP) inhibited P. aeruginosa and S. aureus while those containing amphotericin B (AmB) prevented C. albicans single-species biofilms. Co-immobilization of these agents imparted PVC with broadspectrum activity, impairing the formation of single, dual, and triple-species biofilms up to 48 h, while also displaying biocompatibility. Longer application times showed reduced efficacy after 72 h against S. aureus and after 5 days against P. aeruginosa and S. aureus, but still demonstrated activity against C. albicans . Bacteria recovered from the surfaces after 5 days revealed enhanced CIP tolerance, probably due to CIP exposure and, in the case of S. aureus , such traits could be attributed to its interaction with P. aeruginosa . Still, the application of a single CIP dose at 48 h boosted triple consortia inhibition for up to 5 days. As such, this coating strategy holds great potential to be further explored in ETT design to fight VAP. Keywords: Antimicrobial coatings; endotracheal tube; polymicrobial biofilms; ventilator-associated pneumonia. vii Resumo A pneumonia associada à ventilação mecânica (PAV) é uma infeção nosocomial comum e apresenta elevadas taxas de mortalidade e morbilidade associadas. A presença do tubo endotraqueal (TET) é um fator de risco para o desenvolvimento da PAV pois é propenso à adesão microbiana e formação de biofilme; por isso, são necessárias estratégias para conferir propriedades antimicrobianas aos TETs. Como tal, este projeto de doutoramento teve como objetivo desenvolver um revestimento antimicrobiano para aplicar no TET e prevenir a ocorrência da PAV. Uma vez que a natureza polimicrobiana (intrae inter-reino) da PAV é de particular interesse, este trabalho também teve como objetivo contribuir para uma melhor compreensão das interações dentro destes consórcios bem como avaliar o seu impacto nos resultados antimicrobianos das superfícies desenvolvidas. Usando uma abordagem in silico , os dados experimentais sobre a base molecular das interações P. aeruginosa - C. albicans, dois agentes patogénicos importantes relacionados com a PAV, foram sistematicamente curados e depositados online . Os dados curados reconstruídos como redes revelaram entidades-chave que regulam estas interações, potenciais alvos terapêuticos e possíveis inibidores, o que ajudou na seleção dos compostos antimicrobianos para os estudos in vitro . Paralelamente, foi feita a funcionalização de policloreto de vinilo (PVC) usando uma estratégia de adesão baseada na dopamina, utilizando critérios de segurança, para prevenir a adesão única, dupla e tripla e a formação de biofilmes de agentes patogénicos importantes relacionados com a VAP: P. aeruginosa , Staphylococcus aureus e C. albicans . A estratégia foi aplicada com sucesso na imobilização de 9 compostos (naturais e sintéticos) em PVC. Os revestimentos que continham CIP apresentaram boa atividade inibitória contra biofilmes de espécies únicas de P. aeruginosa e S. aureus , enquanto que aqueles que continham AmB foram capazes de prevenir biofilmes de C. albicans . A co-imobilização destes agentes conferiu ao PVC atividade antimicrobiana de amplo espectro capaz de prevenir a formação de biofilmes de espécies únicas, duplas e triplas até 48 h, apresentando também características biocompatíveis. Para tempos mais longos, os revestimentos mostraram eficácia inferior após 72 h contra S. aureus e após 5 dias contra P. aeruginosa e S. aureus , mas ainda demonstraram atividade inibitória contra C. albicans . Por outro lado, a aplicação de uma única dose de CIP às 48 h aumentou o efeito antimicrobiano do PVC funcionalizado, garantindo a sua atividade de inibição tripla até 5 dias. Desta forma, esta estratégia de revestimento possui grande potencial para ser explorada no desenvolvimento de TET para combater a PAV. Palavras-chave: Pneumonia associada à ventilação mecânica; tubo endotraqueal; biofilmes polimicrobianos; revestimentos antimicrobianos. xiv SDB: Sabouraud dextrose broth spp.: Species TSA: Tryptic soy agar TSB: Tryptic soy broth US: United States V: Volume VAP: Ventilator-associated pneumonia VF: Virulence factors VM: Virulence mechanisms WHO: World Health Organization xv List of figures Figure 1. Schematic representation of the main goal of the present thesis ........................................... 4 Figure 2. Pathogenesis of ventilator-associated pneumonia.. .............................................................. 12 Figure 3. Schematic representation of biofilm formation on ETT surface……………………………………….13 Figure 4. (A and B) Photograph of a mussel and schematic illustration of the interfacial location of Mefp5. (C and D) Schematic representations of the molecular structure of Mefp-5. (E) Schematic representation of the molecular structure of dopamine. (F) Schematic representation of the molecular structure of DHI ................................................................................................................................ 40 Figure 5. Schematic illustration of the main steps to obtain a functional surface using a pDA-based strategy ............................................................................................................................................ 41 Figure 6. Searching within the ISCTD.. .............................................................................................. 73 Figure 7. Overview of the types of interactions and entities annotated for the effects of P. aeruginosa on C. albicans . ...................................................................................................................................... 75 Figure 8. Overview of the types of interactions and entities annotated for the effects of C. albicans on P. aeruginosa . ...................................................................................................................................... 76 Figure 9. Network of the effects of P. aeruginosa - C. albicans interactions on VF and VM .................... 81 Figure 10. Network of the effects of P. aeruginosa on C. albicans gene expression…………………………83 Figure 11. Network of the effects of P. aeruginosa on C. albicans protein expression ......................... 84 Figure 12. Network of the effects of C. albicans on P. aeruginosa gene expression…………………………84 Figure 13. Network of the effects of C. albicans on P. aeruginosa protein expression ......................... 85 Figure 14. Antimicrobial performance of PVC surfaces functionalized with AmB, CIP, and farnesol at different concentrations against C. albicans , S. aureus, and P. aeruginosa single-species biofilm, after a contact of 24 h.. ............................................................................................................................. 119 Figure 15. Antimicrobial performance of functionalized PVC surfaces using different combinations of antimicrobial concentrations against C. albicans , S. aureus, and P. aeruginosa single-species biofilm, after a contact of 24 h.. .................................................................................................................. 122 Figure 16. Surface morphology characterization.. ............................................................................ 124 Figure 17. Representative AFM images of unand modified PVC surfaces. ....................................... 125 Figure 18. Average roughness (Ra) of unand modified PVC surfaces .............................................. 126 Figure 19. Values of contact angles of unand modified PVC surfaces. ............................................ 128 Figure 20. Antimicrobial performance of PVC surfaces functionalized with AmB (0.1 mg/mL) and/or CIP (0.5 mg/mL) against mixed-species biofilms, after a contact of 24 h. .............................................. 131 xvi Figure 21. Representative fluorescent DAPI stained images of different consortia obtained after adhesion for 24 h on unand modified PVC surfaces ..................................................................................... 133 Figure 22. Viability of lung epithelial cells after indirect contact with unand modified PVC surfaces. 134 Figure 23. Antimicrobial performance of functionalized surfaces against mixed-specie biofilms after 5 days of incubation .................................................................................................................................. 153 Figure 24. Antimicrobial performance of the modified surfaces against triple-specie biofilms after a contact of 48 and 72 h. .............................................................................................................................. 155 Figure 25. Antimicrobial performance of co-functional PVC surfaces against triple-species biofilms after 5 days of incubation with an application of 0.5 ug/mL CIP at 48 h (for 24 h) ...................................... 158 xvii List of tables Table 1. Comparative overview of the main attributes of earlyvs. late-onset VAP. ................................ 9 Table 2. Antimicrobial surface modifications reported for ETT in last twenty years. ............................. 28 Table 3. Effect of AI on P. aeruginosa - C. albicans interactions. .......................................................... 78 Table 4. Contrasting P. aeruginosa > C. albicans interactions. ........................................................... 87 Table 5. Contrasting C. albicans > P. aeruginosa interactions. ........................................................... 91 Table 6. Antimicrobial compounds selected to be tested in this work. .............................................. 110 Table 7. Contact-killing and leaching properties of functionalized PVC with different antimicrobial compounds at different concentrations. ........................................................................................... 115 Table 8. EDS quantification of atomic compositions of PVC surfaces, before and after AmB and/or CIP immobilization. ............................................................................................................................... 127 Table 9. Quantification of AmB and CIP released from PVC surfaces after 24 and 48 h of initial incubation.. ...................................................................................................................................................... 128 Table 10. MIC and MBC values of AmB and CIP against fungal and bacterial cells, respectively………156 Table 11. MBC values of CIP against S. aureus and P. aeruginosa cells recovered after 5 days of exposure to different surfaces. MBC values are expressed in μg/mL. ............................................................. 157 xviii List of publications Part of the work described in this thesis has been published in international peer-reviewed journals and was presented in international scientific conferences. Papers in peer-reviewed journals Diana Alves; Tânia Grainha; Maria Olívia Pereira; Susana Patrícia Lopes. Antimicrobial materials for endotracheal tubes: A review on the last two decades of technological progress. Acta Biomaterialia, 158, 32-55, 2023. Grainha, Tânia; Jorge, Paula; Alves, Diana; Lopes, Susana Patrícia; Pereira, Maria Olívia. Unraveling Pseudomonas aeruginosa and Candida albicans Communication in Coinfection Scenarios: Insights Through Network Analysis. Frontiers in Cellular and Infection Microbiology 10(550505), 2020. Papers in preparation Grainha, Tânia; Alves, Diana; Neiva, Joana; Pereira, Maria O. Multi-functional PVC coating addressing the polymicrobial nature of Ventilator-associated Pneumonia. Grainha, Tânia; Alves, Diana; Pereira, Maria O. Polydopamine-based strategies to design anti-infective surface coatings in the fight against biomaterial-associated infections. Poster communication at international scientific conferences Grainha, Tânia; Alves, Diana F.; Pereira, Maria Olívia. A broad-spectrum antimicrobial coating targeting polymicrobial biofilms in ventilator-associated pneumonia. The 6th Stevens Conference on BacteriaMaterial Interactions. Hoboken, USA, May 31 – June 1, 2023. Grainha, Tânia; Alves, Diana F.; Nogueira, Eugénia; Pereira, Maria Olívia. Design of multi-functional PVC coating addressing the polymicrobial nature of Ventilator-Associated Pneumonia. ESB 2021 - 31st Annual Conference of the European Society for Biomaterials together with 43rd Annual Congress of the Iberian Society for Biomechanics and Biomaterials (SIBB). September 5-9, 2021, Online. CHAPTER 1 Introduction This chapter introduces the context and the motivation as well as the goals behind this work. The significance and the outline of this PhD project are also provided. Chapter 1 2 1.1 Context and Motivation The use of medical devices is undoubtedly crucial in current healthcare practices. In particular, the endotracheal tube (ETT) is essential to provide airway patency for patients under mechanical ventilation (MV). MV involves the use of a machine to help a person breathe when they are unable to do so on their own, in cases like surgeries using general anaesthesia, critical care situations, or a traumatically compromised airway [1]. During the coronavirus disease 2019 (COVID-19) pandemic, the usage of these devices was particularly high in order to mitigate the virus-induced lung injuries [2]. Although this technology can be lifesaving, it can also put patients at risk for developing infections, including ventilatorassociated pneumonia (VAP) [3]. VAP is a lung infection that develops after patients have been intubated and received MV. This infection occurs when bacteria, or other pathogens, enter the lungs through the ETT, usually by colonization and subsequent biofilm formation on the biomaterial itself [4]. Its rapid surface colonization and biofilm formation are critical events for VAP pathogenesis and relapses. Studies in this field have demonstrated a broad look at the diverse microbial communities in VAP, reporting distinct polymicrobial profiles and varied microbial causes [5–8]. Polymicrobial communities, encompassing inter-kingdom species, are frequently related to biofilm-associated infections, playing extensive ecological roles and substantially increasing the resistance and the burden of VAP infections [9,10]. VAP can be serious and even life-threatening, especially in patients who are already critically ill. There are several factors that can increase the risk of developing VAP, including prolonged MV, previous antibiotic use, and certain medical conditions, such as immunodeficiency [11]. Preventing VAP involves a combination of measures, such as minimizing ventilator exposure, oral care, aspiration of subglottic secretions, maintaining optimal positioning, and encouraging mobility [12]. Early diagnosis and treatment are also important in improving outcomes for patients with VAP. Despite the efforts made in clinical practices, up until now, there is no universal method able to prevent the occurrence of VAP. Therefore, it is urgent to find effective alternative therapeutic strategies targeting such infections that take into consideration their particularities, such as the polymicrobial aetiology of VAP, which is a critical issue in this infection but still often neglected. Indeed, the coexistence of a set of different microorganisms, including bacterial and fungal species, is frequent in nosocomial infections such as VAP [13–16]; however, the mechanisms of microbial interactions remain unclear or even contradictory. A better understanding of the social behaviour within the consortium and how microbes change their virulence as Chapter 1 3 a result of established interactions is also clinically relevant to better tackle this kind of infection. It is urgent to tailor effective therapeutic strategies aligned with the challenges faced in vivo . The development of materials that can resist or prevent microbial adhesion constitutes an emerging approach to deal with biomaterial-associated infections (BAI), such as VAP, and it has gained great attention over the years [17]. Modern biomaterial science has provided several modification strategies to impart biomaterials, such as ETTs, with anti-infective properties. However, most current strategies have raised some concerns, particularly their deficient antimicrobial efficacy, as most studies only address single-species adhesion, and their short-duration effectiveness [18]. Indeed, even though VAP is often mediated by biofilms, only a few studies address the effectiveness of ETT coatings towards biofilms and even less bear in mind its polymicrobial nature. Likewise, though significant work has been placed in combating bacterial pathogens, substantially less has been focused on combating ETT-related fungal infections. Additionally, some of the current studies involving the design of anti-infective materials do not evaluate the period of time that the antimicrobial activity lasts (the long-term stability). These abovementioned features should be taken into consideration in the search for a new strategy in order to boost the worth of the surface’s modification approaches. Thus, the key goal of this PhD project was to tailor a coating strategy with lasting and broad-spectrum antimicrobial activity to be applied to ETTs, addressing the biofilm-mediated and polymicrobial nature of VAP, features often overlooked in current research. 1.2 Research Aims The main goal of the present thesis was to engineer a functional broad-spectrum antimicrobial coating on polyvinyl chloride (PVC), the polymer constituting ETTs, based on the immobilization of antimicrobial compounds to impair the formation of polymicrobial inter-kingdom biofilms. This work was designed applying safe-by-design criteria appraising aspects such as the cytotoxicity of the coating, the development of antimicrobial tolerance, in addition to long-term antimicrobial activity. It was hypothesized that the combination of different antimicrobial agents with different microbial targets immobilized onto PVC would assist the development of a wide-spectrum antimicrobial surface. To establish such coating, it was therefore required to screen suitable antimicrobial combinations and respective concentrations to generate surfaces able to simultaneously prevent single, dual, and triple microbial adhesion and biofilm formation by relevant VAP-related pathogens ( Pseudomonas aeruginosa, Staphylococcus aureus , and Candida albicans ) and be non-cytotoxic to mammalian cells (Figure 1). Chapter 1 4 As the interactions underlying polymicrobial biofilms can alter the behaviour of the microbial players enrolled in it and account for the (in)success of the antimicrobial strategies, this work also intended to contribute to a better comprehension of the interactions and regulatory targets of the abovementioned VAP pathogens. As in-group efforts had already disclosed this information for the bacterial pair P. aeruginosa and S. aureus , this work focused on the still unexplored inter-kingdom duet of P. aeruginosa and C. albicans . To accomplish these purposes, the following specific aims were delineated: - In silico reconstruction of the inter-species communication network between P. aeruginosa and C. albicans in co-infection scenarios. - Immobilization of antimicrobial candidates, single and in combination, onto PVC using a polydopamine (pDA)-based approach for the intermediate coupling of the compounds. - Screening of the antimicrobial-modified PVC by assessing its contact-killing and leaching properties. - Examination of the antimicrobial performance of the functionalized PVC surfaces against single-, dual, and triple-species biofilms. - Evaluation of the antimicrobial stability overtime of the functionalized PVC. - Physicochemical characterization of the functionalized PVC surfaces. Figure 1. Schematic representation of the main goal of the present thesis: to tailor a wide-spectrum antimicrobial coating with both antiadhesive and anti-biofilm properties, while ensuring no toxicity towards mammalian cells. The ultimate goal of this project is that the present technology could be applied in ETT design. Chapter 1 5 1.3 Thesis Outline This thesis is organized into six chapters that cover the work performed for the research aims previously mentioned. Following this introductory chapter, Chapter 2 presents a literature review providing a general outline of the major aspects of the specific microbiological and clinical features of VAP, carefully emphasizing the presence of the ETT and biofilm development as key factors for VAP. Relevant information on C. albicans , S. aureus , P. aeruginosa , and their interactions is also given. A general outline of the different antimicrobial modifications that have already been applied to ETT is provided. Finally, the relevance of dopamine chemistry for the functionalization of biomaterials is enlightened. Chapter 3 presents the in silico approach used for the retrieval and analysis of experimental information from the scientific literature on the molecular basis of P. aeruginosa and C. albicans interactions its deposition on a public database, and its integration with other online resources to extrapolate candidate antimicrobials. Chapter 4 is dedicated to the optimization of a PVC coating to impair the development of a polymicrobial inter-kingdom biofilm model. After a preliminary screening of the compounds based on their antimicrobial release and contact-killing features, an optimization related to compound immobilization concerning their concentration was performed against single-species biofilms. The most promising antimicrobial compounds were further co-immobilized onto PVC at different concentrations to inspect their activity to combat single-, dual, and triple-species biofilms. In this chapter, a full physicochemical characterization of unand modified PVC surfaces was also performed. Chapter 5 presents an in-deep study regarding the antimicrobial performance of PVC-coated surfaces to be used for long-term applications. Finally, the main conclusions of the present work are presented in Chapter 6 and clues for future work are also suggested. 1.4 References [1] K. Crewdson, M. Rehn, D. Lockey, Airway management in pre-hospital critical care: A review of the evidence for a “top five” research priority, Scand J Trauma Resusc Emerg Med. 26 (2018) 1–6. https://doi.org/10.1186/S13049-018-0556-4. [2] M. Brioni, A. Meli, G. Grasselli, Mechanical Ventilation for COVID-19 Patients, Semin Respir Crit Care Med. 43 (2022) 405–416. https://doi.org/10.1055/S-0042-1744305. [3] I.A. Pneumatikos, C.K. Dragoumanis, D.E. Bouros, Ventilator-associated pneumonia or endotracheal tube-associated pneumonia? An approach to the pathogenesis and preventive strategies emphasizing the importance of endotracheal tube., Anesthesiology. 110 (2009) 673– 80. https://doi.org/10.1097/ALN.0b013e31819868e0. [4] P.S. Zolfaghari, D.L.A. Wyncoll, The tracheal tube: gateway to ventilator-associated pneumonia., Crit Care. 15 (2011) 310. https://doi.org/10.1186/cc10352. Chapter 2 12 alongside the formation of folds in the inflated cuff [52] leading to tracheobronchial colonization and VAP. Overall, the ETT surface provides conditions for microbial attachment and proliferation, ultimately resulting in microbial colonization over the whole external and internal ETT surface [45,53,54]. The pathogens involved in this process can arise from different sources including endogenous or exogenous origin. The exogenous sources of microbial pathogens responsible for VAP include aerosols of the contaminated air and medical devices (ventilatory circuit, catheter, bronchoscope, and humidifier) while the endogenous sources are mostly from the oral, pharyngeal and gastric flora of the patient [55,56] (Figure 2). Figure 2. Pathogenesis of ventilator-associated pneumonia. The colonization of the upper airway can be caused by the contaminated hands of healthcare workers, contamination of the ventilator equipment, a biofilm within the ETT, and from contaminated secretions from the nasopharynx, oropharynx, and trachea. The aspiration of colonized fluids from any of these sources into the lungs can result in pneumonia (adapted from [57]). Chapter 2 13 2.1.2 ETT biofilms An important feature of VAP pathogenesis is its association with biofilms. Microbial adhesion to medical devices including ETT and consequent biofilm formation remains one of the greatest current challenges in healthcare, becoming the focus of persistent infections. Biofilms are well-developed communities that represent the most dominant and active mode of microbial life in nature. They are matrix-enclosed communities harder to eradicate due to their inherent tolerance to antimicrobial therapies and the host immune system, which leads to the persistence and recurrence of infections [58]. It was previously reported that 80% of nosocomial infections are biofilm-mediated [59]. Biofilm development is a dynamic process that involves the following main stages: initial attachment; aggregation; maturation and dispersion (Figure 3). Following the initial attachment of planktonic cells on the surface, the microbial cells divide and proliferate to establish colonization. Cells within the consortia produce an extracellular matrix of polymeric substances (EPS) leading to a mature biofilm architecture. Microbial cells inside this mature biofilm can subsequently disperse, leading to the colonization and infection of other sites. During biofilm formation, attachment of different species can occur, leading to a multi-species biofilm [60]. Figure 3. Schematic representation of biofilm formation on ETT surface. (1) Free cells initiate attachment to the ETT surface; (2) reversible attachment of the planktonic cells is followed by the adhesion on the surface; (3) cells then form a monolayer and irreversibly attach by producing EPS; (4) microcolonies are formed and multilayers appear leading to a mature biofilm architecture; (5) the cells within a mature biofilm can subsequently detach and disperse, leading to the colonization of other sites. Chapter 2 14 Generally made of flexible materials such as PVC or polydimethylsiloxane (PDMS), the ETT acts as a reservoir for infecting microorganisms [61], thereby increasing the patient’s opportunity of getting pneumonia. The ETT provides an optimal surface for microbial colonization and biofilm proliferation which may occur in both its inner luminal and outer surface [62]. Microbial colonization of the ETT combined with the injured host-defense mechanisms of the patients significantly contributes to the development of the ETT biofilm and the consequent spread of the infection [63]. It was previously proposed by Vandecandelaere and Coenye (2015) a model elucidating how bacteria propagate and develop a multi-species biofilm at the distal end of the ETT. In this model, the first stage of biofilm formation results from the leak of nasopharyngeal secretions with non-pathogenic oral bacteria (mostly Streptococcus spp.) around the cuff of the ETT. After bacterial attachment and accumulation, the next step comprises streptococci co-aggregation with other commensal members of the subgingival flora such as Veilonella spp. and Actinomyces spp., followed by the recruitment of a variety of opportunistic oral species (e.g. Fusobacterium nucleatum and Prevotella spp.). This may allow further nosocomial pathogens such as P. aeruginosa or S. aureus , to consequently join and interact with oral bacterial species within the pre-formed biofilm, with harmful consequences leading to VAP development [63]. Other factors such as the supine patient positioning, alongside the shape, thickness, and permeability of the ETT cuff wall, together with variations in the cuff pressure, are sufficient to allow deposited contaminated secretions to migrate to the lower airways and easily lead to pneumonia [64–66]. Portions of the biofilm can also be easily dislodged during suctioning, bronchoscopy or simply by gravity, spreading into the lower airways, and invading the lungs [67]. The ETT poses, therefore, a crucial role in VAP pathogenesis, allowing microorganisms to colonize and form resilient biofilms on its surface, and likely shaping lung ecology during invasive MV at a microbiome scale [68]. The ETT biofilm colonization was early evidenced, first by culturing methods [69] and later, through advanced assessment techniques (e.g. scanning electron microscopy; quantitative polymerase chain) [70,71] . Increasing research has shown that microbial colonization and biofilm formation on ETT is an early and frequent event in intubated patients, contributing to the pathogenesis, lack of treatment and relapses of VAP [3,63,72–76]. When settled on the ETT surface, the biofilm becomes hard to treat due to its inherent recalcitrance nature [76]. Overall, bacteria in biofilms can require 500 to 5000 times more antibiotic doses than that necessary to kill planktonic counterparts [77]. Chapter 2 15 2.1.3 The polymicrobial nature of VAP Microbial pathogenesis research was originally focused on the analysis of infections as monomicrobial events. However, this idea has been refuted by mounting evidence which was supported by the presence of a distinct microbiome in several diseases, including VAP. Several studies have demonstrated that ETT biofilms generally harbour multiple microbial pathogens, which makes VAP a polymicrobial infection, with considerable inter-patient and intra-patient diversity [61]. Data have demonstrated a broad look at the diverse microbial communities in VAP, reporting distinct polymicrobial profiles and varied microbial causes [13,63,76,78]. A wide range of microorganisms is often involved in VAP, with bacteria being the most predominant. Bacteria from the ESKAPE ( Enterococcus faecium , S. aureus , Klebsiella pneumoniae , Acinetobacter baumannii , P. aeruginosa , and Enterobacter spp.) group are frequently found in nosocomial environments. Particularly P. aeruginosa , A . baumannii , K. pneumoniae , and S. aureus play a dominant role in VAP aetiology. Gram-negative bacterial species generally account for 80% of the total isolates [79], and Gram-positive bacteria represent 20-30% of VAP cases [80]. P. aeruginosa is the single-most common pathogen cause of VAP followed by Gram-positive organisms like S. aureus [81–84]. Alongside bacterial pathogens, there is an increasing recognition that a considerable part of nosocomial pneumonia may be also associated with viruses (e.g. Influenza and Cytomegalovirus) and fungi (e.g. Candida spp. and Aspergillus spp.) playing a more important role in immunocompromised patients [85,86]. For instance, Candida spp, especially C. albicans , has been found as part of the array of microorganisms isolated from the respiratory tract of patients diagnosed with VAP [81,83,84,87]. Polymicrobial communities, composed of inter-kingdom species, are often related to biofilm-associated infections playing extensive ecological roles, and substantially increasing the resistance and the burden of VAP infections [88,89]. For instance, it has already been demonstrated that C. albicans instillation in rats increased the probability to develop experimental P. aeruginosa and S. aureus pneumonia, fostering the production of lung inflammatory cytokines [90,91]. Understanding the impact of microorganisms in VAP and their interactions is essential to clearly unveil the infection pathogenesis. Co-infection studies involving bacterial and fungal species, such as P. aeruginosa and S. aureus with C. albicans , have emerged in the last decade [92–97]. The co-existence of these pathogens is devastating in a variety of human-associated infections, including lung infections [98,99], because of their propensity to develop biofilms highly resilient to conventional antimicrobial therapy [96,100–102]. In general, mixed bacterial and bacterial-fungal biofilms are more resistant to antibiotic treatment than the corresponding single-species biofilms [103]. Therefore, it is urgent to develop effective therapeutic strategies targeting this particular trait of infections. Chapter 2 16 2.1.3.1 P. aeruginosa P. aeruginosa is a motile Gram-negative bacterium ubiquitously found in human environments. It is an opportunistic pathogen with the ability to cause life-threatening acute and chronic infections, especially in hospitalized and immunocompromised individuals. P. aeruginosa is frequently associated with different types of infections, such as cystic fibrosis (CF) lung infection, burn wounds, otitis media, urinary tract infections, and those associated with biomaterials [104,105] such as VAP. There are essentially three key factors for P. aeruginosa ‘s pathogenic abilities: production of virulence factors (VF), resistance to antimicrobial agents, and biofilm formation [106]. P. aeruginosa is widely recognized for producing an arsenal of VF associated with diverse functions, such as motility (e.g. flagella, pilli), tissue invasion and damage to the host cells (e.g. proteases such as elastase, alkaline phosphatase, haemolysins, pyocyanin, siderophores, endotoxin A, lipopolysaccharides (LPS), exotoxin A), as well as surface adhesion and biofilm formation (e.g. alginate, Pel, Psl, lectins) [105,107,108]. In addition to these factors, P. aeruginosa is known for its remarkable ability to resist antibiotics. Actually, the World Health Organization (WHO) has listed carbapenem-resistant P. aeruginosa in the top three species for which the development of new treatments is in critical need [109]. The major resistance mechanisms of P. aeruginosa that allow it to withstand the action of antibiotics can be intrinsic or innate (e.g. restricted outer membrane permeability, efflux pumps, antibiotic-degrading enzymes), acquired by either horizontal transfer of resistance genes or by mutations, or adaptive (e.g. biofilm formation, persister cells) [110]. The ability of P. aeruginosa to form biofilms comprises an advantage in many infections [111]. In effect, it is considered the hallmark of chronic infections and the revealing of disease progression. The biofilm environment triggers the development of small colony variants (SCV) and persister cells that can resist higher doses of antibiotics [112,113]. 2.1.3.2 C. albicans C. albicans is a polymorphic fungus that has the ability to grow in several different morphological forms, namely yeast, hyphae, and pseudohyphae, depending on the environmental conditions [114]. This microorganism is the most common fungal species in the human microbiota colonizing the skin, oral cavity, gastrointestinal and reproductive tract, being harmless in the majority of cases [115]. However, C. albicans can take advantage of immunocompromised patients and cause several opportunistic infections [116]. It is the most frequent fungal species isolated from medical devices being one of the most common fungi found in the ETT microbiome [87]. Similarly to P. aeruginosa , there are three important features for Chapter 2 17 the pathogenesis of C. albicans : its ability to switch from yeast-to-filamentous growth and vice versa, secretion of VF, and biofilm formation [117]. Each morphology assumed by C. albicans provides different advantages in the development of infection. The filamentous forms (hyphae and pseudohyphae) are one of its most important virulence mechanisms (VM), being responsible for host cell invasion and macrophage destruction, thus having an important role in the establishment of an infection process [118,119]. In turn, the yeast form is believed to be important for dissemination through the bloodstream and adhesion to endothelial surfaces [120]. This phenotypic switch can be induced by temperature, pH, nutrient concentration, cell density, and human serum [121]. Besides this morphological plasticity, there are other VF contributing to C. albicans pathogenesis, such as the production of adhesins (biomolecules that enable binding to the host cells) and hydrolytic enzymes (e.g. aspartyl proteinases and phospholipases) [122,123]. It is now well acknowledged that biofilm formation is one of the main virulence features involved in the pathogenesis of C. albicans , as these organized communities confer protection against antimicrobial therapy and host defenses [124]. 2.1.3.3 S. aureus S. aureus is a Gram-positive commensal bacterium frequently found on mucous surfaces of the nose and respiratory tract, and on the skin [125,126]. This microorganism is also recognized as a human pathogen often associated with community-acquired and nosocomial infections. Indeed, S. aureus is responsible for a wide range of infections, from mild skin and soft-tissue infections to bacteremia, endocarditis, and osteomyelitis [127] displaying also the ability to adhere to medical devices as ETTs, resulting in biofilm formation and infection [128,129]. Its ability to evolve and adapt to multiple settings has led to the emergence of MRSA, a drug-resistant phenotype, being considered a high threat according to WHO [109]. Additionally, MRSA has been developing resistance to virtually all antibiotics classes that are used to tackle it, either by making use of its intrinsic resistance factors or by acquiring more through mutations or horizontal gene transfer [130]. The presence of MRSA seriously hinders the treatment of infections caused by this pathogen [131]. S. aureus biofilms have been implicated in serious acute chronic infections due to the presence of either MRSA or MDR [132,133]. Furthermore, the heterogeneous nature of biofilms comprising this bacterium increases the chances of plasmid horizontal transfer, leading to antibiotic resistance which can consequently culminate in ineffective treatment regimens and consequent recalcitrant infections [134]. S. aureus also possesses a set of VF which contributes to the success of this pathogen in a wide range of infections. These staphylococcal VF enable attachment to host cells, breaking down the host immune shield, tissue invasion, causing sepsis, and eliciting toxin-mediated Chapter 2 18 syndromes. These factors include the production of surface components such as cell wall proteins (e.g. collagen, fibronectin, protein A, and clumping factors) that are involved in attachment and evasion to the host-tissue. Extracellular toxins (e.g. haemolysin, leukotoxin, exfoliative toxin, enterotoxin, and toxic-shock syndrome toxin-1) and enzymes (e.g. proteases, lipases, and staphylokinase) are also secreted by S. aureus to help in tissue penetration and host invasion [135]. The phenotype SCV has also been strongly linked to pathogenic traits of S. aureus contributing to infection persistence [136]. 2.1.3.4 Quorum-sensing Microorganisms interact by means of a major process that allows them to coordinately sense and respond to the fluctuating conditions of the surrounding environment. This cell-to-cell communication is called quorum-sensing (QS) and is mediated by small diffusible signalling molecules, termed autoinducers (AI). AI regulate the expression of target genes, namely those related to virulence, pathogenicity, resistance, and competition when a threshold concentration is reached regarding population density [137–140]. AImediated signalling in QS allows microbial communication and the control of pivotal processes, namely VF production, biofilm formation, motility, sporulation, production of secondary metabolites, and stress adaptation through, for example, secretion systems [141]. The structure and functioning of the QS machinery deployed by P. aeruginosa have been well elucidated [142–144]. P. aeruginosa possesses four well-known QS systems: LasI/LasR, RhlI/RhlR, PqsABCDE/PqsR, and AmbBCDE/IqsR. Each of these systems produces an AI, namely 3-oxododecanoylL-homoserine lactone (3-oxo-C12-HSL), N-butanoyl homoserine lactone (C4-HSL), 2-heptyl-3-hydroxy-4quinolone (Pseudomonas Quinolone Signal - PQS), and 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (Integrated Quorum Sensing Signal - IQS), respectively [143]. The hierarchical regulation of P. aeruginosa QS systems allows the triggering of massive changes in genetic expression, namely in genes involved in motility, biofilm formation, iron sequestration, antibiotic resistance, and immune system evasion [145,146]. For example, the LasI/LasR system controls the production of multiple VF involved in acute infection and host cell damage, such as LasA and LasB elastases, exotoxin A, and alkaline protease [147– 149]. It also controls the expression of Pel, a major biofilm matrix component [150]. In turn, the excretion of bacterial biosurfactants, such as rhamnolipids, is regulated by the Rhl system [151], while the PqsABCDE/PqsR system controls biofilm formation and its structural stability through extracellular DNA (eDNA) and lectin production [144,152]. In the case of S. aureus , among the regulatory mechanisms described for this pathogen , its main system is called the accessory gene regulator (Agr) and ensures its adaptation to the environment and its Chapter 2 19 coordinated pathogenesis. This complex coordinated QS system encodes a signalling circuit that produces and senses the extracellular autoinducing peptide and the intracellular regulator RNA III [153]. Agr system is responsible for the increased expression of many toxins (e.g. haemolysins and enterotoxins) and degradative exoenzymes (e.g. proteases, lipases) [154] being also related to the decreased expression of several colonization factors. Accordingly, biofilm formation is strongly associated with Agr function, with down-regulation leading to excessive biofilm thickness and lack of structuring, while up-regulation induces biofilm dispersal [155,156]. The emergence of SCV in S. aureus is also associated with reduced Agr activity [157]. A second regulatory QS system in S. aureus , closely related to Agr, is the TRAP/RAP system. The RNAIII-activating peptide (RAP) is an AI that induces the phosphorylation of its target protein (TRAP). TRAP induces the production of adhesion proteins, stimulating biofilm formation, and activates the Agr system. The TRAP/RAP system is mainly active during the early/mid exponential growth phase, and it is followed by the Agr system, which is mainly active during the mid/late exponential phase [158]. Another QS regulator system, the staphylococcal accessory regulator (SarA) of S. aureus, regulates the expression of many VF, including biofilm formation, to mediate pathogenesis and evasion of the host immune system in the late phases of growth [154,159] Contrary to the enormous information available about QS in prokaryotes, QS in the fungal kingdom was somewhat concealed until the discovery of farnesol, a common sesquiterpene produced by C. albicans and similar in structure to bacterial 3-oxo-C12-HSL, in 2001 [160]. Although other molecules mediating QS in C. albicans have been identified since then, including farnesoic acid [161] and aromatic amino acid-derived alcohols like tyrosol [162], tryptophol, and phenylethanol [163,164], farnesol is undoubtedly the most explored AI. Regulation of fungal virulence by farnesol is thought to be particularly confined to inhibiting yeast-to-hypha transition [165], promoting reverse morphogenesis [166], and inhibiting biofilm development [167]. Farnesol is known to directly inhibit the fungal Ras1-cyr1-cAMP-protein kinase A (PKA) signaling pathway, ultimately blocking cAMP synthesis, and consequently suppressing hyphal development [168–170]. Following the same event cascade, the catalytic subunits of PKA, Tpk1 and Tpk2, are also stimulated. Tpk1 and Tpk2 share redundant functions in hyphal growth, adhesion, and biofilm formation, but also have distinct roles in stress responses and pathogenesis, respectively [171,172]. RAS1, CYR1 , and EFG1 (other important hypha-associated genes) have also been documented as important effector genes targeted by farnesol [173]. Apart from farnesol, the AI farnesoic acid, tyrosol, tryptophol, and phenylethanol may also affect important processes such as morphogenesis, biofilm development, limitation of cell population density, control of nutrient competition, and control of infection dissemination [174]. Tyrosol, for instance, stimulates C. albicans filamentation, biofilm Chapter 2 20 formation, and germ tube development [162,175], in contrast to farnesol. However, understanding their underlying regulatory mechanisms is yet to be fully elucidated. 2.1.3.5 P. aeruginosa and C. albicans interplay Bacteria and fungi often coexist in competitive ecological niches in a myriad of ways, communicating through the excretion of metabolic by-products, physical interactions, chemical signaling exchanges, and alterations in the environment [176]. P. aeruginosa and C. albicans are likely the best-studied models in the investigation of these inter-kingdom interactions since reports on their impressive interactions and communication have greatly evolved in the last decade [91,95,177–179]. Although P. aeruginosa-C. albicans interplay is fundamentally antagonistic, its interaction is rather complex, as synergistic, and antagonistic effects can occur simultaneously, mostly dictated by physical associations and by secreted factors via QS [97,180,181]. P. aeruginosa and C. albicans are frequently co-isolated in polymicrobial infections, such as those related to skin, lung, and medical devices [182]. Their genetic and phenotypic plasticity, along with their propensity to assemble as recalcitrant polymicrobial biofilms, places a considerable burden on infections in which they are involved, explaining the increasing interest and the bulk of research on this topic. For example, it has been well-documented that P. aeruginosa and C. albicans colonize the lungs of CF patients and readily form biofilms on ETT surfaces [116]. Interestingly, C. albicans is only implicated in airway colonization in critically ill (elderly, immunocompromised, and/or hospitalized) individuals undergoing invasive MV. However, despite their antagonistic relationship, those who display C. albicans tracheobronchial colonization are at increased risk of acquiring severe VAP infections due to P. aeruginosa [89]. The inhibitory effect of P. aeruginosa on C. albicans growth was first reported in the 1970s [183,184]. In 2002, Hogan and Kolter reported the killing of C. albicans hyphal cells by P. aeruginosa , showing, however, no such effect on fungal yeasts [185]. The deadly effect demonstrated by P. aeruginosa was further confirmed to be largely dependent on the distinct morphotypes exhibited by C. albicans [185– 187]. Often, a reversion of germ tube formation may occur in the presence of P. aeruginosa . The most common event elucidating the physical interaction between P. aeruginosa and C. albicans is likely the extensive bacterial attachment to fungal hyphae [97,188]. Physical association among P. aeruginosa and C. albicans occurs via cell wall-associated compounds in bacteria, such as type IV pili, lectin-carbohydrate interactions, and mannans [189]. The bacterial attachment to fungal hyphae is hypothesized to be caused by nutrient competition at the first hours of co-isolation, after which the bacterial-fungal interaction tends Chapter 2 21 to be of parasitism [178]. Secreted LPS, another bacterial cell-wall component, also functions on polymicrobial intertwining, by interfering with fungal filamentation, metabolism, and growth [190,191]. The P. aeruginosa QS signal 3-oxo-C12-HSL plays major roles in interaction, namely the bacterial binding to C. albicans filaments and in the inhibition of yeast to hyphae switch [187,188]. Because of the inhibition of yeast-to-hypha transformation, C. albicans ability to adhere or invade tissues is compromised [192]. The 2-heptyl-4-quinolone (HHQ), the immediate precursor of the PQS signal, has been shown to repress C. albicans biofilm formation [193]. PQS induces the expression of the VF phenazines, such as pyocyanin, which are toxic products that produce a deleterious effect in eukaryotic cells [194,195]. This effect may be associated with the generation of highly toxic reactive oxygen species [196,197]. Pyocyanin was shown to reduce cAMP [186], which is required for yeast-to-hyphae transition [94,198]. The effect of phenazines, including pyocyanin, in disturbing C. albicans biofilm formation and hyphal growth, has also been evidenced [199,200]. Certain phenazines have even shown the potential to act synergistically in concert with several azole agents against fungal infection [201]. Along with secreting inhibitory molecules, P. aeruginosa also produces substances such as the proteolytic enzyme elastase LasB, which increases the virulence of C. albicans [176]. C. albicans secreted factors, such as farnesol, tyrosol, ethanol, oxylipins, and eicosanoids, have been shown to affect P. aeruginosa growth and biofilm. C. albicans generally uses farnesol to resist oxidative stress and to induce the generation of ROS, thus providing a competitive advantage over bacteria [202]. Farnesol has a deleterious effect on P. aeruginosa , modulating PQS-controlled virulence in a dosedependent manner [203]. Farnesol is able to inhibit pyocyanin [203] and rhamnolipid-mediated swarming motility [204] through such modulation, while also inhibiting other virulence-related proteins. Tyrosol, another C. albicans AI, has been shown to inhibit the secretion of haemolysin and protease (toxins usually involved in tissue damage) by P. aeruginosa at high concentrations [205]. Ethanol is a common fermentation product produced by many bacteria and fungi known to influence P. aeruginosa in diverse polymicrobial settings. In the context of infections where P. aeruginosa coexists with C. albicans , exogenous fungal-produced ethanol may alter phenazine production and promote biofilm development on biotic and abiotic surfaces. In addition, ethanol enhances bacterial Pel matrix production and represses surface motility [206]. Its production is continuously stimulated by the enhanced P. aeruginosa biofilm formation and production of antifungal phenazines, in a positive feedback loop [207]. Eicosanoids (e.g. prostaglandin E2, PGE2) are often secreted by Candida spp., including C. albicans. Although the role of such fatty acid metabolites is still to be determined in the bacterial-fungal cross-talk, it has been suggested that they act as immunomodulatory mediators that affect the dynamics of mixed bacterial-fungal infections Chapter 2 28 Table 2. Antimicrobial surface modifications reported for ETT in last twenty years. Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Active Materials Metal-based coatings (Silver) Commercially available ETT Polyamide/AgNPs composite · A. bambini (including carbapenem-resistant) · C. albicans · Enterococcus faecalis · P. aeruginosa (including carbapenem-resistant) · K. pneumoniae (including extendedspectrum b-lactamase producing strain) · MRSA · S. aureus In vitro · Coated ETT resulted in a significant difference in reducing both planktonic growth and microbial adhesion of single and mixed-species cultures, compared with uncoated ETT. · A time-kill assay demonstrated rapid bactericidal effects of the coating on bacterial growth and cell adhesion to ETT surface. · Biofilm formation by P. aeruginosa and S. aureus was inhibited (4-6 log) after 72 h. · Broad-spectrum activity against Grampositive, and Gram-negative bacteria as well as C. albicans . · Prolonged antimicrobial activity (4 weeks). [54] Commercially available ETT Silver ions · P. aeruginosa In vivo (dog model) · Delay (1.4 days) in bacterial colonization of ETT and reduced number of attached bacteria. · Reduced lung inflammation. [259] PVC Silver ions · P. aeruginosa In vitro · Complete reduction of initial bacterial adhesion. · Reduced biofilm formation over a prolonged period of time (72 h). [260] Commercially available ETT Silver ions · P. aeruginosa In vitro · 2-log inhibition of bacterial attachment to the ETT, after overnight challenge. · Zone of inhibition, indicative of silver release from the ETT. [261] PVA hydrogel AgNPs · P. aeruginosa · S. aureus In vitro · Reduction of biofilm formation of both species, especially P. aeruginosa (about 1 log after 18 h). · No toxicity against lung epithelial cells. [262] Chapter 2 29 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Commercially available ETT AgNPs · P. aeruginosa · S. aureus In vitro · Inhibition of adhesion of both species to ETT (99,9%). · No toxicity against human lung epithelial cells. · Better efficacy than a commercially available silver-coated ETT. [263] Polyethylene ETT Silver-silicon dioxide · E. coli In vitro In vivo (rabbit and golden hamster model) · Antimicrobial activity reduced after contact (93,7%). · In rabbits, no pyrogenic effect was observed and haemolysis test showed biocompatibility with red blood cells. · In gold hamsters, no irritation of oral mucosa was found. [264] Commercially available ETT NMA of gold/silver/palladium (BIP ETT) · Enterococci spp. · Haemophilus parainfluenzae · Neisseria spp. · Staphylococci · Streptococcus In vivo (ICU patients) · The BIP ETT was well tolerated and presented good clinical short-term performance (5 h), while presenting low level of bacterial colonization. [256] Commercially available ETT NMA of gold/silver/palladium (BIP ETT) · Unknown In vivo (ICU patients) · No significant reduction in ICU length of stay, MV duration, tracheostomy rate and hospital mortality. · VAP incidence reduced by 5.1%. · Number of antibiotic days reduced by 6.6%. · Tracheal pathogen colonization reduced by 3.7% in the NMA-coated compared with non-coated subglottic suctioning ETTs. [257] Commercially available ETT Silver sulfadiazine · P. aeruginosa In vitro In vivo (sheep model) · No bacterial adhesion up to 72 h on ETT. · In sheep, after 24 h of ventilation, no bacterial growth was detected on the ETT, ventilator tubing or lower respiratory tract. [265] Chapter 2 30 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Commercially available ETT NMA of gold/silver/palladium (BIP ETT) · Unknown In vivo (ICU patients) · No significant reduction in ICU length of stay, MV duration, tracheostomy rate and hospital mortality. · VAP incidence reduced by 15.3%. · Mean MV duration reduced by 1.8 days. [258] Commercially available ETT NMA of gold/silver/palladium (BIP ETT) · Unknown In vivo (ICU patients) · Reduction in the volume of secretions, incidence of purulent secretions, fever, leukocytosis, culture positive, and the onset of VAP symptoms. · Incidence of VAP reduced from 26% to 18% (intervention vs control groups). [266] Commercially available ETT Nanosilver (AgNPs)- polyurethane polymer · E. coli · S. aureus In vivo (rat model) · Potent antimicrobial and antibiofilm proliferation properties. · Thickness and cell numbers of biofilm formed by catheterization 48 and 72 h after rat operation was significantly lower than that in the control group. [267] Commercially available ETT Silver ions · A. baumannii · Enterobacteriaceae · H. influenzae · P. aeruginosa · S. aureus · S. pneumoniae · Streptococcus viridans group · Other Nonfermentative Gramnegative bacilli In vivo (ICU patients) · Reduced colonization rates by patient (29%). · Delayed colonization on the inner tube surface (1.8 vs. 3.2 days in control device) and on the tube · Reduced bacterial burden in tracheal aspirates. [268] Chapter 2 31 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Metal-based coatings (ZnO) Commercially available ETT ZnO-NPs · S. aureus In vitro · Reduction of biofilm formation (55%) after 72 h. · After 24 h challenge, membrane’s compromise was observed after live/dead staining. [269] PVC taken from a commercially available ETT ZnO-NPs · S. aureus In vitro · Reduction (87%) of biofilm formation after 24 h. [270] Metal-based coatings (TiO2) Commercially available ETT TiO2-NPs (commercial and N-doped) · P. aeruginosa · S. aureus In vitro · Inhibition of bacterial growth under visible fluorescent light, after 24 h. · N-doped NPs more efficient against S. aureus . [271] PVC medical grade Iodine-modified TiO2 · E. coli In vitro In vivo (pig model) · Photocatalytic activity able to kill bacteria under visible light irradiation after 30 min. · Decrease of bacterial attachment and biofilm formation after 72 h. · Reduced inflammation of tracheal and lung tissues in pigs, after 72 h ventilation. [272] Metal-based coatings (Se) PVC medical grade SeNPs · S. aureus In vitro · Bacterial colonization was reduced by 80%. · Better antimicrobial efficacy than a silvercoated ETT. [273] Biocides impregnation Commercially available ETT Gardine and Gendine · A. baumannii · C. albicans · E. cloacae · K. pneumoniae · MRSA · P. aeruginosa In vitro · Complete inhibition of the adhesion of all species after 24h. · Antimicrobial activity was prolonged for 2 weeks against MRSA. · Better antimicrobial efficacy than a silvercoated ETT. [274] PVC Hexetidine · P. aeruginosa · S. aureus In vitro · Reduction of P. aeruginosa adhesion by 52%, after 7 days. · Reduction of S. aureus adhesion by 75%, after 7 days. [275] Chapter 2 32 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Commercially available ETT Gendine · MRSA · P. aeruginosa · E. coli · C. parapsilosis In vitro · Antimicrobial activity evidenced by an inhibition zone up to 3 weeks against all species. · Complete inhibition of MRSA, C. parapsilosis and E. coli after 24 h. · No toxicity against mouse fibroblast cells. [276] Commercially available ETT Poly(lauryl acrylate)- based nanocapsules encapsulating clove oil or eugenol · K. pneumoniae · MRSA In vitro · Both species were affected by the antibacterial loaded nanocapsules in a dose dependent manner, with >50% inhibition at a concentration of 0.625 mg mL−1 of eugenol or clove oil. · The observed surface-binding reduction of bacteria, biocompatibility and slow release of eugenol demonstrate the potential of this strategy for clinical applications. [277] PVC NO (SNAP as NO donor) · P. aeruginosa In vitro · NO release over a 7-day period without altering the mechanical properties of the ETT. · Reduction of P. aeruginosa adhesion by 92.72 ± 0.97% (1.5 log) compared with the control ETT, after 24 h. [278] Commercially available ETT Styrylbenzene-based (BCP3) · MSSA and MRSA · P. aeruginosa In vitro · A concentration-dependent release of BCP3 was observed for at least 31 days. · After 24 h, functionalized surfaces inhibited mainly the growth of MSSA (a maximum of 95%) and MRSA (a maximum of 80%) and to a smaller extent P. aeruginosa (maximum of 63%). · No cytotoxicity against L929 fibroblasts. [279] Chapter 2 33 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Bio-inspired antimicrobials (AMPs) Commercially available ETT Lasioglossin-III · S. epidermidis · S. pneumoniae · Pooled human microbiome samples In vitro · Prolonged, linear peptide release over 1 week. · Inhibition of planktonic bacterial growth and adhesion to tubes after 24 h. · No toxicity against laryngotracheal fibroblasts or lung epithelial cells. [280] Bio-inspired antimicrobials (Ceragenins) Commercially available ETT Ceragenin CSA-131 · C. albicans · C. auris · K. pneumoniae · MRSA · P. aeruginosa In vitro In vivo (porcine model) · Prevention of the colonization of ETT and biofilm formation for several days (from 4 to 16 days), depending on the species tested. · Prevention of mixed biofilms formation of MRSA/ P. aeruginosa and P. aeruginosa / C. auris for up to 2 and 3 days. · In pigs ventilated for 24 h, no abnormalities were found in the oropharyngeal area, trachea or lungs. · Histological analyses showed no inflammation and CSA-131 could not be detected in blood system. [281] Bio-inspired antimicrobials (Surfactants) Commercially available ETT Cholesterol and lecithin (different ratios) · P. aeruginosa · S. aureus In vitro · Reduction of bacterial adhesion by more than 90%, after 8h . [282] Commercially available ETT Sphingosine · A. baumannii · P. aeruginosa · S. aureus In vitro In vivo (mice) · Prevention of biofilm formation of all bacterial species after 24 h in vitro . · Prevention of bacterial colonization in vivo. · Coatings stable with no side effects on tracheal epithelial cells or inflammation. [283] Bio-inspired antimicrobials (Phages) Commercially available ETT Phage cocktail · P. aeruginosa In vitro · Antimicrobial action was strain dependent. · Phages-coated tubes did not promote substantial changes in metabolic activity. · Limited anti-biofilm effect after testing the tubes in artificial sputum medium up to 168 h. [284] Chapter 2 34 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Commercially available ETT Phages ΦJHS-PA1139 and ΦSMK-PA1139 · MDR P. aeruginosa In vitro · Phage-coated ETT segments of 12 -mm in length inhibited bacterial colonization by 1.2 log up to 3.2 log comparing with non-coated segments following 6 h coating. · Phage treatment of ETT segments yielded 1.0 log up to 1.6 log reductions in MDR biofilms for phage ΦJHS and 1.6 log up to 2.4 log reductions for phage ΦSMK. [285] Passive Materials Hydrophobic/ Hydrophilic surfaces PVC Oxygen plasma treatment · P. aeruginosa In vitro · Reduction of the number of adhered bacteria by 70%. [286] PVC Superhydrophobic surfaces · P. aeruginosa In vitro · No bacterial attachment in the first 6 h. · Reduction of biofilm formation after 24 h. [287] Nanomodified surfaces Commercially available ETT Nanoscale surface features created by a fungal lipase · S. aureus In vitro · Reduction of 1.5 log of the total number of adhered bacterial cells, after 24 h. [288] Commercially available ETT Nanoscale surface features created by a fungal lipase · P. aeruginosa In vitro · Reduction of 2.7 log of the total number of adhered bacterial cells, after 24 h. [289] Commercially available ETT Nanoscale surface features created by a fungal lipase · P. aeruginosa In vitro · Reduction of the number of bacterial cells adhered by 40%, after 24h. [290] Micropatterned surfaces Silicon wafers Engineered micropattern of surfaces using the Sharklet pattern design · A. baumannii · E. coli · K. pneumoniae · MRSA · P. aeruginosa In vitro · Reduction on the attachment of all species (from 1 to 3 log). · Reductions of MRSA and P. aeruginosa biofilms (67% and 58%). [291] Thermoplastic polyurethane ETT Engineered micro-pattern of surfaces using the Sharklet pattern design · P. aeruginosa In vitro · Reduction of biofilm accumulation by 71%. · Reduction of artificial mucus accumulation. [292] Chapter 2 35 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Combinatorial Materials Commercially available ETT CS-AgNPs@PAAmGelatin composite (CS=chitosan; PAAm=polyacrylamide) · P. aeruginosa · S. aureus In vitro (broncholung model) In vivo (porcine MV model) · Excellent antibacterial properties in both in vitro and in vivo models. · Antibiofouling property by decreasing lumen occlusion · Good biocompatibility (tested after 1, 4 and 7 days against fibroblasts). · Good stability, by keeping antimicrobial performance to up to 21 days. · Coated ETTs could decrease in vivo lumen occlusion by artificial mucus for up to 97%. [67] Commercially available ETT Chlorhexidine and silver carbonate · A. baumannii · Enterobacter aerogenes · MRSA · P. aeruginosa · S. aureus In vitro · Reduction of 4-6 log of bacterial colonization of ETT, after 5 days, using an airway model. [293] Commercially available ETT Nanoscale surface features created by a fungal lipase and Fructose · S. aureus In vitro · Fructose coating alone caused a reduction of 38% of bacterial adhesion. · Nanocoated surfaces alone caused a reduction of 45% of bacterial adhesion. [294] Commercially available ETT Copper and zeolite · MDR A. baumannii In vitro · A maximum reduction of immobilized cells of 14%, after 24 h. · Antimicrobial efficacy was lower than the silver coating. [295] Commercially available ETT Antimicrobial lipid (octadecylamine), mucolytic (Nacetylcysteine), and antibiotics (doxycycline and levofloxacin) No inoculation with exogenous bacteria In vivo (pig model) · Pigs ventilated with coated tubes were less hypoxic, had less bacterial colonization of the lungs and survived longer than pigs ventilated with uncoated tubes for 72 h. [255] Chapter 2 36 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Commercially available ETT Silver, zeolite and tyrosine · MDR A. baumannii In vitro · Combination of tyrosine to natural zeolite with silver increased the antimicrobial activity against immobilized cells (from 2.5 to 2.8 log achieving almost 100% reduction) after a contact of 24 h. · Combination of both strategies caused a higher reduction of 60% of bacterial adhesion, evidence of a synergistic effect. [296] Commercially available ETT Hydrogels of hydroxyethyl methacrylate (HEMA):methacrylic acid (MAA) entrapped with nebulized gentamicin · P. aeruginosa · S. aureus In vitro · No bacterial adherence occurred to gentamicin-containing HEMA:MAA copolymers. · 70:30 HEMA:MAA hydrogel exhibited >20 days persistence against S. aureus and P. aeruginosa. · Viable bacteria were not observed on the gentamicin-treated p(HEMA: MAA) copolymers, whereas growth was observed on gentamicin-treated p(HEMA). [297] Commercially available ETT Curcumin and photodynamic action · E. coli · P. aeruginosa · S. aureus In vitro · After 24 h, the combined effect of curcumin-functionalized ETT and light application caused a reduction of about 95%, 72% and 73% on the adhesion and biofilm formation of S. aureus , E. coli and P. aeruginosa , respectively. · Curcumin-ETT remains active after six photodynamic sessions every 24h up to 6 days with 23.76% of microbial reduction. [298] Chapter 2 37 Table 2. (continued) Type of designed material Approach(es) Source material Antimicrobial compound/feature Microorganism(s) tested Mode of growth/ Testing model Main Results/Remarks Ref. Commercially available ETT Combinations of silver, TiO2 and Degussa (metallic alloy) · P. aeruginosa · S. aureus In vitro · Combination of TiO2 and silver reduced P. aeruginosa growth after 24 h. · Combination of Degussa with TiO2 reduced P. aeruginosa growth up to 48 h. · No reduction was found against S. aureus up to 5 days. 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This comprises a competitive advantage, with interactions between different species altering host responses, antimicrobial effectiveness, microbial pathogenesis and virulence, usually augmenting the severity of the infection and contributing to the recalcitrance towards conventional therapy. P. aeruginosa and C. albicans are two opportunistic pathogens often co-isolated from infections, such as VAP. Despite the billions of years of coexistence, this pair of microorganisms is a great example of how little is known about cross-kingdom interactions, particularly within the context of coinfections. Given the described scenario, this study aimed to collect, curate, and analyse all published experimental information on the molecular basis of P. aeruginosa and C. albicans interactions in biofilms, in order to shed light into key mechanisms that may affect infection prognosis, increasing this area of knowledge. Publications were optimally retrieved from PubMed and Web of Science and classified as to their relevance. Data was then systematically and manually curated, analysed, and further reconstructed as networks. All annotated data was made publicly available at www.ceb.uminho.pt/ISCTD, a database already containing similar data for P. aeruginosa and S. aureus communication. Chapter 3 76 Figure 8. Overview of the types of interactions and entities annotated for the effects of C. albicans on P. aeruginosa . (A) Proportional data on the types of interactions; (B) Total number of interactions annotated for each source category; (C) Total number of interactions annotated for each target category. Legend: AI, autoinducer; VM, virulence mechanism; VF, virulence factor. 3.3.3 Effect of QS Molecular Players on P. aeruginosa - C. albicans Interactions Four different AI from P. aeruginosa were annotated for their effect on C. albicans . For instance, 3-oxoC12-HSL was annotated as inhibiting the hyphal form of the fungus [19,20], the secondary messenger cAMP [19], the cAMP synthesis-related enzyme Cyr1 [20], and biofilm formation, while stimulating C. albicans adhesion to mammalian cells [21] (Table 3). As previously mentioned, the morphogenesis of C. albicans is largely regulated through the cAMP/PKA pathway, meaning that 3-oxo-C12-HSL is able to inhibit yeast to hyphae transition by affecting it. Additionally, 3-oxo-C12-HSL was also shown to downregulate the expression of ECE1 , HWP1 , and SAP5 , all hyphae-specific genes (Table 3) [20]. As stated, hyphae development by C. albicans is one of its most important VM, having an important role in the establishment of the infection process, which is hindered by 3-oxo-C12-HSL. Although this effect may be apparently detrimental to the infection, the reversion or maintenance of the fungus in the yeast form may enhance its dissemination capabilities and spread the infection to other areas of the host. However, Chapter 3 77 it has also been shown that the presence of 3-oxo-C12-HSL, at sub-growth and sub-hyphal inhibitory concentrations, favourably affects C. albicans when challenged with fluconazole by upregulation of genes known to be associated with antimicrobial resistance (e.g. GAL102 , MDR2 , INO2 , ADA2 ) [22] (Table 3). Concerning other AI, C4-HSL, PQS, and its precursor, HHQ, were also annotated as interfering with biofilm formation on inert surfaces but to stimulate C. albicans adhesion to a cellular substratum [21] (Table 3). This switch from antagonistic to synergistic interactions is dependent on the host, which highlights the need for better understanding the role played by the latter in polymicrobial infections. PQS and HHQ have multifunctional roles in QS and iron uptake, playing a key role in coordinating virulence in P. aeruginosa [23]. Although the annotated effect of these molecules on C. albicans hyphae was null [24,25], they seem to interfere with biofilm formation (Table 3) [21,24]. Overall, there is a clear negative effect on the phenotypic switching and biofilm formation of C. albicans mediated through QS of P. aeruginosa when the host is not considered. Regarding the two annotated AI of C. albicans , farnesol and tyrosol, the former seems to affect the production of AI from P. aeruginosa , namely PQS [26,27], HHQ, and C4-HSL [27] (Table 3) . Farnesol is also involved in the inhibition of different VM and VF of this bacterium, such as adhesion, swarming motility, and haemolysin production [26,28,29] (Table 3). Distinct farnesol effects were also observed between planktonic and biofilm growth for pyocyanin production (Table 3). Tyrosol was also annotated as affecting the production of VF of P. aeruginosa , namely the inhibition of the production of haemolysin and proteases [29] (Table 3). These two exoenzymes greatly influence the pathogenicity of P. aeruginosa , contributing to infection establishment through elastin degradation and vascular permeability, respectively [30]. Overall, it is safe to say that QS in both pathogens is a key factor mediating their antagonistic relationship, with their capacity for infection establishment being one of their most affected traits. Chapter 3 78 Table 3. Effect of AI on P. aeruginosa - C. albicans interactions. Source AI Interaction Target Ref. P. aeruginosa 3-oxo-C12-HSL Inhibition Hyphae(p), cAMP(p), Cyr1(p), Biofilm(b) [19–21] Stimulation Adhesion(b) [21] Downregulation ECE1 (p), HWP1 (p), SAP5 (p), CDR1 (b) , MDR1 (b) , C6_02100W_A (b) , CRH11 (b) , FBA1 (b) , IFR2 (b), MNN12 (b) , PHHB (b) , SOD5 (b) , TEL1 (b) [19,20,22] Upregulation CDR2 (b), AAF1 (b), ADA2 (b), ADH3 (b), ALK2 (b), ALS7 (b), ARD (b), ATX1 (b), AXL1 (b), BCR1 (b), C1_01130W_A (b), C1_01510W_A (b), C1_03990W_A (b), C1_04010C_A (b), C1_09210C_A (b), C2_01750C_A (b), C2_02920W_A (b), C2_03690C_A (b), C2_09880C_A (b), C3_00360W_A (b), C3_02630C_A (b), C3_03460C_A (b), C3_04330C_A (b), C3_05450C_A (b), C4_02740W_A (b), C4_03020W_A (b), C5_04030W_A (b), C6_00110C_A (b), C6_00290W_A (b), C6_00920W_A (b), C7_00770W_A (b), C7_04090C_A (b), CDR4 (b), CR_00040C_A (b), CR_05860W_A (b), CR_06140W_A (b), CR_06960W_A (b), CR_07480W_A (b), CR_09100C_A (b), CR_10230W_A (b), CRZ2 (b), CSH1 (b), CUP9 (b), EFG1 (b), ERO1 (b), GAL102 (b), GOR1 (b), GRP2 (b), HAL9 (b), HSP104 (b), HSP78 (b), HSP90 (b), IFD6 (b), INO2 (b), ISA1 (b), LPG20 (b), MHP1 (b), MOH1 (b), NRG1 (b), OPT3 (b), PGA52 (b), RFG1 (b), RGS2 (b), RME1 (b), RPN4 (b), SIS1 (b), SNQ2 (b), SRR1 (b), STI1 (b), UGT51C1 (b), WOR4 (b), YIM1 (b), YOR1 (b), ZCF1 (b), ZCF39 (b) [22] C4-HSL Inhibition Biofilm(b) [21] Stimulation Adhesion(b) HHQ Inhibition Biofilm(b) [21,24] Stimulation Adhesion(b) [21] Null effect Hyphae(p), Adhesion(b) [24] PQS Inhibition Biofilm(b) [21] Stimulation Adhesion(b) Null effect Hyphae(p) [24,25] Chapter 3 79 Table 3. (continued) Source AI Interaction Target Ref. C. albicans Farnesol Inhibition PQS(p), Adhesion(b), Swarming motility(p), Pyocyanin(p), Haemolysin(p), Cell(p) [26,28,29] Downregulation pqsA (p), pqsR (p) [26] Null effect Cell(p);(v), pqsR (p), Proteases(p) [26,29,31] Upregulation pqsH (b) [27] Stimulation HHQ(b), PQS(b), C4-HSL(b), Pyocyanin(b) Tyrosol Inhibition Haemolysin(p), Proteases(p), Cell(p) [29] Legend: (p) planktonic; (b) biofilm; (v) in vivo . Chapter 3 80 3.3.4 Interaction Effects on Virulence Mechanisms and Virulence Factors The influence of inter-species interactions on the expression of VM and VF can greatly affect the severity of the polymicrobial infection. Given their importance, two networks were constructed regarding the effects of one species on the virulence of the other. Concerning the effect of P. aeruginosa on C. albicans , hyphal development was the most annotated VM (Figure 9), for which the majority of interactions (75%) were inhibitory. Concerning the most reported source entities affecting hyphae, most annotated interactions reported effects of bacteria as a whole (annotated as “cell”), meaning that no molecular entity was identified/tested. LPS, rhamnolipids, and 3-oxo-C12-HSL were also annotated as inhibiting hyphal growth [19,25,32], while HHQ and PQS had no effect [24,25]. Biofilm formation by C. albicans was the second most annotated affected VM as a result of P. aeruginosa interaction. In this case, almost all interactions were inhibitory (93%) and caused by different source entities (Figure 9). No VF were annotated for C. albicans . Regarding the effect of C. albicans on P. aeruginosa , swarming motility, adhesion, and biofilm formation were the most annotated VM (Figure 9). Swarming motility is inhibited in the presence of C. albicans due to farnesol [28] and ethanol [33]. The adhesion capability of P. aeruginosa was annotated as inhibited by farnesol [28] and stimulated by ethanol [33]. In the case of biofilm formation, 50% of interactions were of stimulation and the other 50% had a null effect. Concerning the source entities, these distinct biofilm effects were both annotated for the entity “cell”, while only stimulation was annotated for ethanol (Figure 9). Pyocyanin production by P. aeruginosa was the most annotated VF (Figure 9) and the effect of C. albicans on this molecule apparently depends on the mode of growth, with stimulation in biofilm growth and inhibition in planktonic growth. These differences are further explored in a later section. Other annotated VF include pyoverdine, proteases, rhamnolipids, and haemolysin (Figure 9). All interactions annotated for pyoverdine and rhamnolipids were stimulatory, with “cell” as the source entity and biofilm as the mode of growth for the first and both planktonic and biofilm as modes of growth for the latter. Both AI of C. albicans , farnesol and tyrosol, were annotated as inhibiting haemolysin in P. aeruginosa in planktonic cultures [29]. Regarding the production of proteases, tyrosol inhibited these enzymes, while a null effect was annotated for farnesol, in the planktonic mode of growth [29]. Chapter 3 81 Figure 9. Network of the effects of P. aeruginosa - C. albicans interactions on VF and VM. Teal nodes, P. aeruginosa ; orange nodes, C. albicans ; green arrows, stimulation; grey arrows, null effect; red arrows: inhibition; node and node label sizes are directly proportional to the number of related (outward and inward) edges (interactions). 3.3.5 Interaction Effects on Gene and Protein Expression Concerning the expression of genes and proteins as a result of microbial interaction, it was possible to annotate a total of 110 distinct genes differentially expressed in C. albicans due to the presence of P. aeruginosa , of which 83 were upregulated, 21 were downregulated, and 6 were both up and downregulated (Figure 10). These findings are correlated with the affected VM previously mentioned, especially hyphal growth. For instance, HWP1 and ECE1 , along with ALS3 , all hyphae-specific genes, were downregulated in the presence of 3-oxo-C12-HSL [20] and of P. aeruginosa “cell” [34] and its supernatant [35]. These results are in accordance with the well-known inhibitory effect annotated for hyphal growth. However, there are some cases where some contradictory effects were annotated for other source entities, namely LPS upregulation of the previously mentioned genes [32]. This and other contrasting annotations are discussed in the next section. With concern to protein expression, C. albicans differentially expressed 117 proteins, of which 59 were annotated as upregulated and 69 as downregulated (Figure 11). Some proteins are reported in more than one paper or even with different interaction types in the same paper; hence, there is a greater number of annotated interactions (Figure 11) in relation to the total number of different annotated proteins. Almost all interactions with proteins had “cell” as the source entity. Protein expression was also shown to vary depending on the time of maturation of the dual-species biofilm. For instance, proteins related with Chapter 3 82 adhesion and biofilm formation, namely Als1, Als2, Als3, and Pbr1, seem to be negatively affected by the presence of P. aeruginosa in a time dependent manner, being less expressed in later stages of biofilm development. The differences observed throughout time can be related with the interaction between both pathogens that probably is more pronounced with increased time of interaction, correlating with the lowering of the metabolic activity of C. albicans in the double consortia [36]. Three different proteins related to virulence in C. albicans , namely Tfp1, Ape2, and Bgl2, were annotated as upregulated in the presence of P. aeruginosa [37]. This upregulation could have a synergistic interaction with the VF of the bacterium, resulting in enhanced pathogenesis. All proteins of the cell wall were annotated as downregulated after interaction of the fungus with P. aeruginosa. This includes the cell wall proteins Crh11 and Ecm33, involved in cell wall assembly and regeneration, filamentation, and adherence to host cells, and also the hyphal cell wall proteins Rbt5, Hyr1, Ece1, and Rbe1 [36]. Regarding the effect of C. albicans on the gene expression of P. aeruginosa , 19 differentially expressed genes were annotated, of which 18 were downregulated and 1 was upregulated (Figure 12). It was very interesting to note that almost all interactions were of downregulation (90%). In fact, pqsH was the only gene of P. aeruginosa annotated as being upregulated in the presence of farnesol [26]. This QS-related gene is involved in the terminal step of the biosynthesis of quinolones by catalysing the hydroxylation of HHQ to PQS [38]. Most of the downregulated annotated genes belong to the pvd and pch gene families (Figure 12) in planktonic and in vivo conditions. Interestingly, the related protein PchD was upregulated in biofilm conditions [36,37]. These and other contrasting annotations are discussed in the next section. Concerning the expression of proteins, P. aeruginosa differentially expressed 147 proteins due to the presence of C. albicans. A total of 92 of these proteins were upregulated and 86 were downregulated (Figure 13). The majority of the annotated proteins from P. aeruginosa related to virulence were upregulated due to the interaction with C. albicans . For instance, proteins related to siderophore biosynthesis and/or transport, namely ChtA, FptA, FpvA, PchD, FpvB, PvdA, PvdH, PvdF, and PvdQ, were all upregulated in biofilm settings [36,37]. Other upregulated proteins, namely OpdO, OpdP OpmH, Opr86, OprC, OprE, and OprQ, are involved in the transport of small molecules and antibiotic resistance [37]. HasA and HasR, two proteins related to heme uptake, were also upregulated as well as PilQ and XcpQ, which are proteins responsible for motility and attachment of the bacterium [36,37]. Proteins involved in cell wall and LPS synthesis, namely, GlmU, RmlA, and WbpA, were also annotated as upregulated. These findings reinforce the notion that P. aeruginosa becomes more virulent as consequence of the interaction with C. albicans. Chapter 3 83 Figure 10. Network of the effects of P. aeruginosa on C. albicans gene expression. Legend: Teal nodes, P. aeruginosa ; orange nodes, C. albicans ; green arrows, upregulation; grey arrows, null effect; red arrows: downregulation; node and node label sizes are directly proportional to the number of related (outward and inward) edges (interactions). Chapter 3 84 Figure 11. Network of the effects of P. aeruginosa on C. albicans protein expression. Legend: Teal nodes, P. aeruginosa ; orange nodes, C. albicans ; green arrows, upregulation; grey arrows, null effect; red arrows: downregulation; node and node label sizes are directly proportional to the number of related (outward and inward) edges (interactions). Figure 12. Network of the effects of C. albicans on P. aeruginosa gene expression. Legend: Teal nodes, P. aeruginosa ; orange nodes, C. albicans ; green arrows, upregulation; grey arrows, null effect; red arrows: downregulation; node and node label sizes are directly proportional to the number of related (outward and inward) edges (interactions). Chapter 3 85 Figure 13. Network of the effects of C. albicans on P. aeruginosa protein expression. Legend: Teal nodes, P. aeruginosa ; orange nodes, C. albicans ; green arrows, upregulation; grey arrows, null effect; red arrows: downregulation; node and node label sizes are directly proportional to the number of related (outward and inward) edges (interactions). 3.3.6 Contrasting Annotations Illustrating the Complexity of Interspecies Study Upon the analysis of all the gathered information, it was possible to discern some seemingly opposite effects annotated from the literature regarding P. aeruginosa - C. albicans interactions. All of these annotated interactions are outlined in Tables 4 and 5 for P. aeruginosa > C. albicans and C. albicans > P. aeruginosa interactions, respectively. These tables contain not only information on the interactions but also on the experimental conditions in which they were observed, as these define their comparability. One of the more noticeable factors leading to contrasting observations is the period of time during which cells are co-cultivated or exposed to each other’s molecular factors (e.g. AI, supernatants). For example, Purschke and colleagues showed the time-dependent differential expression of several P. aeruginosa and C. albicans proteins when the two microorganisms are grown together as mixed biofilms [36]. Many of these proteins are upor downregulated dependent if cells are harvested at early or late time points (or vice-versa), which is corroborated by some similar observations in other publications (Table 4 and 5). Chapter 3 92 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA DnaN Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 48 YNBNP MALDI-TOF MS/MS SC5314 [36] ExaA Downregulation Cell Biofilm 24 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 48 FliC Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 4.5 YNBNP MALDI-TOF MS/MS SC5314 [36] Downregulation 1.5, 3, 24,48 FliD Downregulation Cell Biofilm 1.5, 6, 24, 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 3 FptA Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Chapter 3 93 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA fptA Downregulation Cell In vivo (n.a.) Virulence murine model RNA-Seq. PAO1 SC5314 [31] Planktonic 10 min GGP + YPD RT-qPCR Supernatant Planktonic 10 min GGP Supernatant proteins 10 min GGP FpvA Upregulation Cell Biofilm 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF CAI4 [37] fpvA Downregulation Cell In vivo (n.a.) Virulence murine model RNA-Seq. SC5314 [31] FusA1 Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 48 YNBNP MALDI-TOF MS/MS SC5314 [36] Chapter 3 94 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA GroEL Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 6 YNBNP MALDI-TOF MS/MS SC5314 [36] Downregulation 1.5, 24, 48 KatA Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Downregulation 1.5, 4.5, 6, 24, 48 YNBNP MALDI-TOF MS/MS SC5314 [36] Upregulation 3 LasB Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Downregulation 1.5, 4.5, 6 YNBNP MALDI-TOF MS/MS SC5314 [36] Upregulation 3, 24 Chapter 3 95 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA OprF Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 3 YNBNP MALDI-TOF MS/MS SC5314 [36] Downregulation 1.5, 4.5, 6, 24, 48 OprL Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 6 YNBNP MALDI-TOF MS/MS SC5314 [36] Downregulation 1.5, 4.5, 24, 48 PA0572 Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 3, 4.5, 6, 24, 48 YNBNP MALDI-TOF MS/MS SC5314 [36] Downregulation 1.5 PA0623 Downregulation Cell Biofilm 24, 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 3, 6 Chapter 3 96 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA PA1342 Downregulation Cell Biofilm 1.5, 4.5, 6, 24, 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 3 PA2453 Downregulation Cell Biofilm 24, 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 1.5, 4.5, 6 PA3313 Downregulation Cell Biofilm 24 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 48 PA3529 Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Downregulation 24 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 48 PA5339 Downregulation Cell Biofilm 1.5, 4.5, 6 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 48 PasP Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Upregulation 3, 24, 48 YNBNP MALDI-TOF MS/MS SC5314 [36] Downregulation 1.5, 4.5 Chapter 3 97 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA PchD Upregulation Cell Biofilm 1.5 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF CAI4 [37] pchD Downregulation In vivo (n.a.) Virulence murine model RNA-Seq. SC5314 [31] PilY1 Downregulation Cell Biofilm 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 24 Piv Downregulation Cell Biofilm 1.5, 3, 4.5, 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 6, 24 PnP (extracellular) Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] PnP (intracellular) Upregulation PQS Stimulation Farnesol Planktonic 14 LB agar TLC PA14, PA14Δ lasR , PA14Δ lasI (n.a.) [27] Chapter 3 98 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA PQS Stimulation Cell Planktonic 24, 96 (stirred batch) ASM Bioluminescence (reporter strain PAO1 ΔpqsA CTXlux::pqsA) PAO1 SC5314 [39] Inhibition Farnesol 6, 24 LB TLC PA14 (n.a.) [26] pqsH Upregulation Farnesol Planktonic 14 LB agar RT-qPCR PA14Δ lasR +pUCP22, PA14Δ lasR +pPQSHPA 14Δ pqsH +pUCP22, PA14Δ pqsH +pPQSH SC5314 [27] pqsA Downregulation Farnesol Planktonic 15 min LB RT-PCR PA14 (n.a.) [26] PvdF Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] pvdF Downregulation In vivo (n.a.) Virulence murine model RNA-Seq. SC5314 [31] PvdH Upregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Chapter 3 99 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA pvdH Downregulation Cell In vivo (n.a.) Virulence murine model RNA-Seq. PAO1 SC5314 [31] Planktonic 10 min GGP + YPD RT-qPCR Supernatant Planktonic 10 min GGP RT-qPCR Supernatant proteins Pyocyanin Stimulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine HPLC PAO1 CAI4 [37] Farnesol Planktonic 14 LB agar Chloroform extraction; Spectrophotometry PA14Δ lasR , PA14Δ lasI SC5314 [27] Inhibition Cell Planktonic 96 (aerobic batch and stirred batch) ASM Chloroform extraction; Spectrophotometry PAO1 SC5314 [39] Farnesol Planktonic 6, 24 LB PA14, PAO1 (n.a.) [26] SodB Downregulation Cell Biofilm 1.5, 4.5, 24, 48 YNBNP MALDI-TOF MS/MS PAO1 SC5314 [36] Upregulation 3 Chapter 3 100 Table 5. (continued) Target Interaction Source Mode of growth Experimental Conditions* Method Strains Ref. Time (h) Media PA CA TufA Downregulation Cell Biofilm 24 RPMI 1640 + L-glutamine + dextrose + uridine 2D-PAGE; MALDITOF PAO1 CAI4 [37] Downregulation 1.5 YNBNP MALDI-TOF MS/MS SC5314 [36] Upregulation 48 Legend: PA – P. aeruginosa ; CA – C. albicans ; MALDI-TOF - Matrix-Assisted Laser Desorption/Ionization - Time of Flight; MS/MS – tandem Mass Spectrometry; qPCR - quantitative Polymerase Chain Reaction; RNA-Seq. – RNA Sequencing; RT-PCR - Reverse Transcription Polymerase Chain Reaction; 2D-PAGE - two-Dimensional PolyAcrylamide Gel Electrophoresis; ASM - Artificial Sputum Medium; GGP - GlucoseGlycerol-Peptone; RPMI - Roswell Park Memorial Institute Medium; YNBNP - Yeast Nitrogen Base N-acetyl-D-glucosamine Phosphate; n.a. – not applicable. * - all experiments were conducted at 37 °C. Chapter 3 101 3.3.7 Alternative Approaches in P. aeruginosa - C. albicans Biofilm Control: QS Inhibition Biofilm is known as the preferred mode of growth of most microorganisms, including bacterial and fungal pathogens. These consortia are typically resilient with dynamic structures, giving their microbial constituents a broad range of advantages, as previously mentioned. In real infection scenarios, microorganisms are usually found in the biofilm form and these consortia can easily turn an infection into a chronic condition [40]. Thus, although planktonic testing is practical and informative, studies involving biofilms can better mimic a real-life infection scenario and allow a better comprehension of the microbial behaviour under these situations. Concerning the annotated information in this work, although the number of annotated interactions was higher for biofilms (83%), this only reflects the types of methods being used. Actually, the number of studies using planktonic or biofilm as the mode of growth was similar (18 vs 17, respectively), which shows that biofilm studies are still lacking in order to get a real perspective on these inter-species interactions. Given the biofilm problematic, along with the ever-rising antimicrobial resistance, the need for alternative therapies is in high demand. For example, antivirulence agents carry advantages like circumvention of antibiotic resistance, by targeting VF rather than bacterial growth [41], and a high number of putative virulent targets [42]. Considering that QS is the main regulator of virulence in both bacteria and fungi, the use of quorum quenching (QQ) compounds that inhibit specific QS mechanisms related to virulence can be a promising strategy to modulate it [43]. Additionally, QQ compounds are probably less likely to induce resistance in cases where their targets are located extracellularly [44]. Notwithstanding all the advantages, target selection in this approach is of critical importance given the existence of redundancy and alternative regulatory pathways, which may compensate for a given disturbance, and the complexity of the outcomes of inter-species interactions, which can make an apparently detrimental effect on the pathogen lead to an opposite desired effect in the infection as a whole. For example, the interference with the QS system of one pathogen can potentially facilitate the pathogenicity of the other co-infecting species. Moreover, effective antivirulence therapy would probably entail combinations with other agents, such as other antivirulence drugs or even antibiotics, to increase antimicrobial effectiveness in polymicrobial communities [17]. Other factors to take into account when designing anti-QS approaches are the negative impacts on the host and its microflora, the possibility of bacteremia/sepsis as a consequence of the biofilm disruption, altered immune and inflammatory responses, and resistance development for intracellular targets [45]. Chapter 4 108 4.1 Brief Introduction VAP is a common infection in ICU and in mechanically ventilated patients that arises after endotracheal intubation [1]. This nosocomial infection is particularly worrying because it is associated with prolonged hospitalization, high morbidity and mortality rates, as well as increased healthcare costs [2]. The use of the ETT is the main risk factor for VAP development [3], acting as an airway entry point for microorganisms through microbial adherence and multiplication on the trachea [1], giving rise to biofilms that provide a significant source of bacterial inoculation of the lungs. An important feature of VAP pathogenesis is their association with biofilms and several studies have demonstrated that most of them are composed of polymicrobial communities [4–6]. A wide range of microorganisms is often involved in VAP, with bacteria being the most predominant. P. aeruginosa and S. aureus are the most frequently reported Gram-negative and Gram-positive bacteria, respectively [7–10]. Additionally, there is an increasing recognition that a considerable part of nosocomial pneumonia may be also associated with viruses and fungi in immunocompromised patients [11,12]. For instance, Candida spp, especially C. albicans , has been found as part of the array of microorganisms isolated from the respiratory tract of patients diagnosed with VAP [7,9,10,13]. The co-existence of these pathogens is devastating in a variety of human-associated infections, including lung infections [14,15], because of their propensity to develop biofilms highly resilient to conventional antimicrobial therapy [16–20]. Therefore, it is urgent to develop effective therapeutic strategies targeting this particular trait of infections [21–23]. Understanding the impact of microorganisms in VAP and their interactions is essential to unveil infection pathogenesis. Co-infection studies involving bacterial and fungal species, such as P. aeruginosa and S. aureus with C. albicans , have emerged in the last two decades [17,24–28]. Given the recognized role of ETT in VAP development, one promising approach is to prevent ETT colonization through the modification of the surface materials used to design these medical devices [29]. Thus, the aim of the current study was, using dopamine chemistry to functionalize PVC, which has been widely used in the design of ETT and whose intrinsic properties are propitious to the establishment of infection, to leverage antimicrobial features towards P. aeruginosa , S. aureus and C. albicans . After identification of compounds retaining their antimicrobial activity after immobilization onto PVC, a subsequent screening testing different concentrations and antimicrobial combinations was performed to establish coating compositions giving rise to superior performance against all species without cytotoxicity to mammalian cells. Chapter 4 109 4.2 Materials and Methods 4.2.1 Microbial strains and culture conditions P. aeruginosa PAO1, S. aureus American Type Culture Collection (ATCC) 25923 and C. albicans SC5314 were used throughout this work. All strains were stored at -80 ± 2 °C in broth medium with 20% (v/v) glycerol. Before each assay, bacterial and fungal strains were subcultured from frozen stock preparations, onto Tryptic Soy Agar (TSA) or Sabouraud Dextrose Agar (SDA), respectively. TSA and SDA plates were prepared from Tryptic Soy Broth (TSB; Liofilchem) or Sabouraud Dextrose Broth (SDB; Liofilchem), respectively, supplemented with 2% (w/v) agar (Liofilchem). The agar plates were then incubated at 37 °C for 18-24 h. To prepare microbial suspensions, some colonies were retrieved from the agar plates and incubated in TBS or SDB at 37 C, overnight under agitation (120 rpm). Cells were then harvested by centrifugation (9000 g, 5 min) and washed in saline solution (0.9% w/v, NaCl) or phosphate-buffered saline (PBS, pH 7.4). The concentration of bacterial suspensions was finally adjusted by measuring the absorbance at 620 nm (EZ Read 800 Plus, Biochrom) and estimated by previously established standard curves while yeast cells were enumerated by microscopy using a Neubauer counting chamber. 4.2.2 Antimicrobials Several antimicrobial compounds, including potential alternatives to antibiotics, were tested (Table 6). These compounds were chosen since they have already been reported with antimicrobial effect against at least one of the species under study. The rationale was to characterize their effectiveness to be used further in the design of the antimicrobial PVC surfaces. Chapter 4 110 Table 6. Antimicrobial compounds selected to be tested in this work. Class Compound Natural compounds Carvacrol Chlorogenic acid Farnesol Linalool Salicylic acid Antimicrobial peptide Camel (KWKLFKKIGAVLKVL-NH2) Enzyme Acylase I Synthetic drugs Amphotericin B Ciprofloxacin Gentamicin All antimicrobial compounds were purchased from Sigma (Sigma-Aldrich, USA) with the exception of camel which was kindly provided by Doctor Wojciech Kamysz from the Faculty of Pharmacy, Medical University of Gdansk, Poland. For the functionalization assays, almost all of the compounds were directly dissolved in the work solutions. The only exception was AmB whose stock solutions were prepared using dimethyl sulfoxide (DMSO; Fisher Chemicals). Storage was performed according to the manufacturer’s instructions. To obtain the work solutions of carvacrol, farnesol, and linalool, DMSO was added to improve the solubility of these agents. 4.2.3 PVC preparation and functionalization Unplasticized sheets of PVC (Goodfellow GmbH, United Kingdom) with 1 mm thickness were cut into 1 cm × 1 cm squares. Before surface modification, PVC coupons were subjected to a cleaning process to remove all impurities and traces of grease. For that, samples were placed in a commercial ammonia solution (2%) and sonicated for 5 min, being then thoroughly washed with distilled water. The PVC samples were subsequently sterilized with ethanol (70%) for 30 min, washed with sterile distilled water, and afterwards exposed to ultraviolet irradiation for 1 h. Finally, they were dried at room temperature. PVC functionalization was performed using a pDA-based coating strategy [30] following a 1-step approach. Briefly, dopamine (Sigma, 2 mg/mL) was dissolved together with the antimicrobials in 10 mM bicine buffer (Sigma, pH 8.5) and the PVC coupons were immediately placed in this solution to obtain the different Chapter 4 111 coatings. After overnight incubation, at room temperature and constant agitation at approximately 60 rpm, the modified coupons were rinsed with sterile ultrapure water and air-dried before further utilization. 4.2.4 Antimicrobial performance of modified surfaces 4.2.4.1 Contact-killing and leaching properties The antimicrobial activity of the compounds was evaluated after immobilization onto PVC as previously described [30]. Briefly, microbial suspensions were adjusted to a concentration of approximately 106 colonyforming units (CFU) per mL and 20 μL of each were added on top of each surface and incubated under static conditions at 37 °C, until the drop was dried. Coupons were then placed on SDA or TSA plates (for C. albicans or bacterial strains, respectively), with the face exposed to microbial suspension in contact with the agar and incubated at 37 °C for 72 h. Microbial growth was then assessed and tabulated as “+” for growth and “-” for no visible growth. To evaluate the leaching of antimicrobials from the modified surfaces, a qualitative method previously described was used [30]. Surfaces were placed on top of TSA plates previously streaked with a microbial suspension adjusted to 108 CFU/mL and incubated for 72 h at 37 °C. Afterwards, TSA plates were inspected for the presence or absence of an inhibition zone as an indication of antimicrobial release from the surfaces. Two independent assays were performed in duplicate for each condition except for the cases where no inhibitory activity was detected in contact-killing and leaching assays. 4.2.4.2 Single-species biofilm inhibition activity The anti-biofilm potential of the modified surfaces was evaluated by enumerating the number of viable cells adhered to the surfaces. Microbial suspensions were prepared from overnight cultures adjusted to a final concentration of 106 CFU/mL in RPMI or TSB for C. albicans or bacteria, respectively. Unand modified PVC coupons were placed into the wells of a 24-well microtiter plate and covered with 1 mL of each microbial suspension. Plates were incubated at 37 °C, under static conditions and cells were allowed to adhere on the surfaces for 24 h. The coupons were then washed twice with 1 mL of PBS or NaCl for C. albicans or bacterial samples, respectively. They were placed in new wells filled with 1 mL of saline solution or PBS to detach the adhered cells from the surfaces using an ultrasonic bath (Sonicor SC-52, Sonicor Instruments) operating at 50 kHz, for 6 min (parameters previously optimized) or by scraping, in the case of C. albicans . Afterwards, Chapter 4 112 the resulting microbial solutions were collected, vortexed to disrupt possible cell aggregates, 10-fold serial diluted and plated into SDA or TSA plates that were incubated overnight at 37 °C under aerobic conditions before enumeration. Three independent assays with three replicates for each condition were performed. 4.2.4.3 Mixed-species biofilm inhibition activity The antimicrobial activity of the modified surfaces was also evaluated against polymicrobial consortia. Bacterial and fungal suspensions were prepared as previously described at a concentration of approximately 106 CFU/mL. For dual and triple-species adhesion, inoculums were mixed at proportions of 1:1 and 1:1:1, respectively, and then placed onto 24-well plates containing unand modified PVC surfaces. TSB was used for dual-species biofilms formed by S. aureus and P. aeruginosa while RPMI was used in all consortia encompassing C. albicans , including the triple one. Plates were incubated at 37 °C for 24 h, under static conditions. After that, the liquid content of the plates was discarded, and the PVC coupons plus 24 h-old biofilms were washed with saline solution or PBS according to the microorganism within the biofilms. The adhered cells were detached from the surfaces accordingly to the microorganisms involved following the sonication and scraping processes abovementioned . Serial 10-fold dilutions of the biofilm-cell suspensions were performed and plated onto agar plates with selective growth media. Pseudomonas isolation agar (PIA, Sigma), Mannitol Salt Agar (MSA, Liofilchem) and SDA supplemented with gentamicin (GM) (60 mg/L) were the selective media used to discriminate P. aeruginosa , S. aureus and C. albicans , respectively. TSA supplemented with AmB (10 mg/mL) was used to discriminate C. albicans in dual-species biofilms. The plates were incubated overnight at 37 °C under aerobic conditions before enumeration. Three independent assays with three replicates for each condition were performed. 4.2.4.3.1 Evaluation of mixed-species biofilms by fluorescence microscopy To obtain a more detailed investigation regarding the antimicrobial activity of the surfaces, the presence of cells adhered on PVC coupons was inspected by microscopy using DAPI staining (Invitrogen™, USA). After biofilm formation, the cells adhered to the coupons were washed and fixed as previously described [31]. Briefly, the coupons were left to dry for 1 h at 37 °C. Afterwards, 20 µL of methanol (Fisher Scientific, UK, 100%) was added on top of each surface for 10 min. After that time, the excess methanol was removed and 20 µL of paraformaldehyde (Sigma, 4%) was added on each surface and left to dry for 15 min. Then, the Chapter 4 113 excess of paraformaldehyde was removed and 20 µL of ethanol (Fisher Scientific, 50%) was added on top of each surface and left to dry for 15 min. Lastly, the excess of ethanol was removed, and the surfaces were allowed to completely dry before staining. For microscopy visualization, the cells were stained with 20 µL of DAPI (30 µg/mL) for 10 min. Then, the excess dye was removed and after drying, the cells were visualized under the epifluorescence microscope (Olympus, BX51). At least two independent assays were performed in duplicate for each consortium and several images per coupon were collected. 4.2.5 Surface characterization 4.2.5.1 Surface morphology, chemical composition, and roughness To have insights into the morphological aspects of the surfaces, unand modified PVC were analysed by scanning electron microscopy (SEM). Before observation, samples were covered with a very thin film of AuPd (80–20 weight %), 5 nm, in a high-resolution sputter coater and observed with an Ultra-high resolution Field Emission Gun Scanning Electron Microscopy (FEG-SEM), NOVA 200 Nano SEM FEI Company. Topographic images were performed with a secondary electron detector at an acceleration voltage of 10 kV. The chemical composition of the surfaces was also investigated through Energy Dispersive Spectroscopy (EDS), using an EDAX Si (Li) detector, with an acceleration voltage of 15 kV. Surface roughness was evaluated using atomic force microscopy (AFM). AFM measurements were performed at room temperature using a Nanoscope III Multimode Atomic Force Microscope (Digital Instruments) operating in tapping mode. Scan rates were set at 1 Hz and the scanning area per sample was fixed at 10 × 10 μm. Surface roughness analyses were conducted using Gwyddion software. 4.2.5.2 Surface hydrophobicity parameters The wettability parameters were determined using the sessile drop contact angle method. Contact angles were measured on an automated contact angle device (OCA 15 Plus, Dataphysics, Germany) that allows image acquisition and data analysis. Measurements were performed at room temperature, using 3 μL drops of water. Three independent assays with three replicates for each condition were performed. Chapter 4 114 4.2.5.3 AmB and CIP release profile To determine AmB and CIP release profiles, functionalized PVC coupons were placed into the wells of a 24well microtiter plate (Orange Scientific, USA) and covered with 1mL of PBS. The plate was then constantly agitated at 120 rpm and 37 °C. After 24 h, all PBS was collected and refreshed. The amount of released AmB or CIP was then determined by ultraviolet-visible spectroscopy (UV–Vis), measuring the absorbance at 335 or 270 nm in the case of AmB or CIP, respectively. The absorbance values were then converted to concentration values using calibration curves previously established. Two independent assays in triplicate were performed. 4.2.6 Cytotoxicity assay Cytotoxicity analysis of the coatings was performed using lung epithelial cells A549, according to ISO 109935:2006. Lung cells were grown in Dulbecco-modified eagle medium (DMEM, Gibco) supplemented with 10% of fetal bovine serum (FBS, Gibco) and 1% antibiotic (ZellShield™, Biochrom) at 37 °C and 5% CO2. Once achieved the confluence, cells were detached using trypsin (Sigma), and 1 mL of a cell suspension adjusted to approximately 1 × 105 cells/mL was added to each well of a 96-well microtiter plate. In parallel, unmodified and modified surfaces were inserted in 24-well plates filled with 1 mL of DMEM per well. Both plates were then incubated for 24 h at 37 °C and 5% CO2. Afterwards, the supernatant was removed and 100 μL of the medium, which was in contact with the surfaces, were added to the adhered cells. Fresh DMEM was also added to cells as a positive control. The plate was then incubated for further 24 h at 37 °C, 5% CO2, and after that period, 20 μL of MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2Htetrazolium) inner salt (Promega) were added in each well. After 1 h of incubation at 37 °C, 5% CO2 in the dark, the absorbance of the resulting solution was measured at 490 nm. The percentage of cell viability was calculated by the ratio between the cell metabolic activity in the presence of modified surfaces and the control (cell growth in DMEM). Two independent experiments in triplicate were performed. Chapter 4 115 4.2.7 Statistical analysis Results were presented as mean ± standard deviation (SD). Statistical analysis was performed by using the Prism software package (GraphPad Software version 9.0.0). One-way ANOVA tests were performed, and means were compared by applying Tukey’s multiple comparison test. In all the analyses performed, the confidence interval used was 95%. 4.3 Results 4.3.1 Antimicrobial performance of the modified surfaces 4.3.1.1 Contact-killing and leaching properties As in this study it was intended to tailor a coating able to prevent microbial attachment to PVC surfaces but also to the surrounding environment in the bulk phase, the contact-killing and antimicrobial-releasing features were analysed jointly to choose the best compounds. Table 7 summarizes the antimicrobial activity of the selected compounds after immobilization onto PVC, namely their ability to impede microbial growth, as well as the qualitative release of those antimicrobials from the surfaces. Table 7. Contact-killing and leaching properties of functionalized PVC with different antimicrobial compounds at different concentrations. Visible growth was used as an indication of antimicrobial activity being tabulated as “+” for microbial growth and “–” for no growth. The antimicrobial release was evaluated by the presence (P) or absence (A) of an inhibition zone. Some conditions that were not tested were listed as “NT”. CA: C. albicans ; SA: S. aureus ; PA: P. aeruginosa . Antimicrobial compound Concentration Microbial growth Inhibition zone CA SA PA CA SA PA Acylase I 1 mg/mL + + + A A A Amphotericin B 1 mg/mL − NT NT P NT NT 0.5 mg/mL − NT NT P NT NT 0.25 mg/mL − NT NT P NT NT Camel 1 mg/mL + + + A A A 1 mg/mL + + + A A A Chapter 4 116 Table 7. (continued) Antimicrobial compound Concentration Microbial growth Inhibition zone CA SA PA CA SA PA Carvacrol 10 µL/mL − − − P/A P/A A 5 µL/mL − −/+ − A A A Chlorogenic acid 2 mg/mL + + + A A A 1 mg/mL + + + A A A Ciprofloxacin 1 mg/mL + − − A P P 0.5 mg/mL + − − A P P 0.25 mg/mL + − − A P P Farnesol 5 mg/mL −/+ − −/+ A P A 1 mg/mL + − + A P A Gentamicin 2 mg/mL + − − A P P 1 mg/mL + −/+ − A P/A P Linalool 100 µL/mL − − − A A A 50 µL/mL − − −/+ A A A Salicylic acid 5 mg/mL + + + A A A 1 mg/mL + + + A A A This initial screening showed that the surfaces functionalized with acylase I, camel, chlorogenic acid and salicylic acid did not exhibit antimicrobial effects against C. albicans, S. aureus, and P. aeruginosa (Table 7). The PVC coupons functionalized with carvacrol showed antimicrobial activity against all species as evidenced by their contact-killing activity. However, this compound was not considered for further tests because solubility problems difficult to overcome were found with its use. Results showed that the immobilization of AmB did not compromise its antifungal activity, as shown by its contact-killing activity against C. albicans since no growth was noticed for any of the concentrations tested. Inhibition zones were also observed, indicating that AmB was released from the surfaces. Chapter 4 117 Functionalized surfaces with the antibiotic CIP exhibit antibacterial activity against both bacteria, whatever the concentration tested. Furthermore, the amount of CIP released from each of the PVC surfaces was also effective in inhibiting P. aeruginosa and S. aureus growth since inhibition zones were observed for all concentrations used in PVC functionalization. Regarding farnesol, this compound was efficient against S. aureus as evidenced by its contact-killing and leaching activities. Although some antimicrobial activity was detected against C. albicans and P. aeruginosa when using the highest concentration, no inhibition zone was observed in the leaching assay. GM maintained its antibacterial activity after immobilization on PVC surfaces, especially for the highest concentration tested, as evidenced by its contact-killing and leaching activities against S. aureus and P. aeruginosa . The bioactive compound linalool exhibited antimicrobial activity against all species under investigation when used to coat PVC surfaces as shown by contact-killing activity, nevertheless, no inhibition zone was observed in the leaching assay. Overall, these data determined the exclusion of some compounds (acylase I, camel, carvacrol, chlorogenic acid, linalool, and salicylic acid) and even of some concentrations of the remaining compounds for further investigation since not all exhibited the desired antimicrobial activities. GM was also excluded because, even though it is effective at the highest concentration, CIP, the other antibiotic, showed better performance against bacterial strains at lowest concentrations. Hence, as AmB, CIP, and farnesol exhibited promising contact-killing and leaching traits, they were selected to pursue the work. The highest concentrations of AmB and CIP (1 mg/mL) were also disregarded because the lower concentrations tested gave rise to similar antimicrobial activity. At this point, it is important to note that the concentrations presented in Table 7 are those that were initially used in the immobilization process, and the effective concentration retaining on the PVC surfaces will certainly be much lower. To facilitate the interpretation of the results, these initial concentrations will be shown in the following data presentations. The concentration that remains on the surface and that can be later released will be addressed and studied in more detail later in this work. Chapter 4 124 Figure 16. Surface morphology characterization. Representative images of SEM analysis of PVC surfaces before and after AmB and CIP immobilization. The scale bar in the upper and lower column indicates 10 and 1 μm, respectively. Chapter 4 125 Figure 17. Representative AFM images of unand modified PVC surfaces. The images on the right correspond to the 3D representation of the images on the left. The scale bar indicates 1 μm. Chapter 4 126 Figure 18. Average roughness (Ra) of unand modified PVC surfaces. Significant differences were found for (*) p < 0.05, compared to PVC. PVC surfaces were analysed in terms of chemical composition using EDS to confirm each modification step (Table 8). This polymer is composed of Carbon (C), Chlorine (Cl) and Hydrogen (H). Therefore, results showed high concentrations for C as well as Cl, whereas Oxygen (O) and Nitrogen (N) could not be detected. The deposition of pDA onto PVC could be confirmed by the decrease of Cl as well as the increase in N content. Functionalization of PVC surfaces using AmB, CIP, or both antimicrobial compounds together revealed similar patterns in terms of chemical composition. In all cases, an increase in the N content accompanied by a decrease of Cl, as compared to PVC untreated surfaces was also observed. A slight increase in the O content in all pDA-based coated surfaces was observed. Similar chemical structures can be explained by the sampling depth achieved by the EDS. Indeed, it is higher than 50 nm, which is the maximal thickness of pDA coatings [35], meaning thus that PVC is the major contributor to the chemical signature detected by EDS analysis. PVC pDA AmB CIP AmB+CIP 0 20 40 60 80 100 Ra / nm * Chapter 4 127 Table 8. EDS quantification of atomic compositions of PVC surfaces, before and after AmB and/or CIP immobilization. Surface C (%) O (%) Cl (%) N (%) PVC 73.27 2.06 24.67 0.00 pDA 73.42 3.47 21.33 1.77 AmB 73.88 3.41 22.01 0.70 CIP 72.35 3.34 21.66 2.64 AmB+CIP 73.52 3.13 21.87 1.47 4.3.2.2 Surface hydrophobicity parameters Surface hydrophobicity parameters of PVC and different modified surfaces were investigated by measuring the water contact angles (Figure 19). A significant decrease in the water contact angle values was observed for all coated surfaces when compared to bare PVC. Results showed that PVC is a hydrophobic surface, evidenced by the water contact angle of approximately 92.2°. pDA coating rendered the PVC surfaces with hydrophilic properties as evidenced by the decrease of water contact angle to 52.8°. This feature is attributed to dopamine since its polymerization introduces new hydrophilic functional groups, especially the catechol and amine groups, on the PVC surface. This observation on materials functionalized with pDA has been previously reported [30,36]. The surfaces functionalized with AmB and CIP separately and in combination also displayed this hydrophilic characteristic. Still, it is important to note that AmB immobilization on PVC rendered the most hydrophilic surfaces with values of water contact angle around 31.5° which may be attributed to the high roughness of that surfaces and the polyol subunit of AmB (which contains multiple −OH groups), which is capable of hydrogen bonding with water [37]. Additionally, co-functionalized surfaces with AmB and CIP reveal estimated values of contact angles between those determined for the compounds when used separately onto PVC surfaces, which suggests a contribution of both agents to the final wettability behaviour of these surfaces. Chapter 4 128 Figure 19. Values of contact angles of unand modified PVC surfaces. Significant differences were found for (*) p < 0.0001, compared to PVC. 4.3.2.3 AmB and CIP release profile Since the amount of effective agents retained on the surface is expected to be at a much lower concentration than those used initially in the immobilization solution, it was intended to assess the profile of AmB and CIP release from the surfaces after antimicrobial functionalization. For that, PVC surfaces were functionalized with AmB and CIP, separately, to detect and quantify each one of the antimicrobial compounds. Results, presented in Table 9, show that it was not possible to determine the amount of AmB both at 24 and 48 h, meaning that the amount of the antifungal released is probably lower than the limit detection of the apparatus. The amount of CIP released from the surfaces after 24 h was estimated at 1.68 µg/mL. After 48 h, no amount of CIP was detected probably due to the amount of released compound was under the limit detection of the apparatus. Table 9. Quantification of AmB and CIP released from PVC surfaces after 24 and 48 h of initial incubation. Results are expressed in [µg/mL]. ND: not detected. Antimicrobial Released amount 24 h 48 h AmB ND ND CIP 1.68 ± 0.19 ND PVC pDA AmB CIP AmB+CIP 0 20 40 60 80 100 Water contact angle / ° * * * * Chapter 4 129 4.3.3 Mixed-species biofilm inhibition activity The effectiveness of the co-functionalized surfaces with AmB and CIP (0.1 and 0.5 mg/mL, respectively) was further evaluated against mixed-species biofilms (i.e. dualand triple-species biofilms). Results demonstrated that, in general, the inhibitory effect of AmB and CIP previously observed for single species-biofilms was maintained when the compounds were tested individually and in combination against the dualand triplespecies 24 h-old biofilms (Figure 20). Concerning the dual-species biofilms comprising C. albicans and S. aureus , the number of CFU found for all tested conditions (control and functionalized surfaces) was similar to those obtained for single-species counterparts (Figure 14 and 15). There was even a slight decrease in the number of cells of both C. albicans and S. aureus adhered on the co-functionalized surfaces (AmB+CIP) when compared to the respective single-species biofilms. Therefore, it was possible to conclude that the eventual interactions established within the consortium did not affect the outcome of the antimicrobial performance of the surfaces when challenged by C. albicans and S. aureus simultaneously. Regarding the dual-species biofilms formed by C. albicans and P. aeruginosa , the number of CFU for all surfaces tested was also almost the same as compared to the respective parts in single-species biofilms. Results suggest that both species coexist without any effect observed on the growth between them for the time and surfaces tested. Additionally, the antimicrobial activity of the functionalized surfaces was also maintained. There was also a slight increase in the reduction of cells of both species adhered on the cofunctionalized surfaces (AmB+CIP), compared to respective single-species biofilms, being the CFU counts under the limit of detection. Regarding S. aureus and P. aeruginosa in dual-species biofilms, results showed that the capability of S. aureus to grow on bare PVC and surfaces coated with pDA and AmB was affected by the presence of P. aeruginosa. The number of S. aureus adhered cells on PVC control typically achieves more than 7 log CFU/mL, however, in the presence of P. aeruginosa , its growth decreased to 4.6 log CFU/mL under the same conditions. Also, for surfaces functionalized with pDA and AmB, its growth decreased by 1.8 log CFU/mL in both cases, when comparing dual versus single-species biofilms. In the case of P. aeruginosa , no differences in terms of CFU counts were observed, in the presence of S. aureus . Regarding the surfaces functionalized with CIP and cofunctionalized with AmB and CIP, the reductions observed for both bacteria in single-species biofilms were maintained when the surfaces were tested against both bacteria at the same time. The findings observed in Chapter 4 130 this consortium reflect some inhibitory effect of P. aeruginosa on S. aureus, which translates into their loss of ability to grow on solid media and consequently the decrease in CFU counts. To increase, even more, the complexity of our consortia, the co-adhesion of all the 3 microorganisms was also evaluated. The adhesion extent of C. albicans , S. aureus, and P. aeruginosa to PVC control and pDA surfaces was similar to single-species biofilms. The antimicrobial activity of the surfaces coated with AmB was preserved for C. albicans with no effect against S. aureus and P. aeruginosa. Interestingly, the loss of S. aureus capability to grow on PVC and surfaces functionalized with pDA and AmB, observed when this bacterium was grown with P. aeruginosa , was not detected when S. aureus integrated the triple-species biofilm. Concerning functional surfaces with CIP, the antimicrobial activity was maintained against S. aureus and P. aeruginosa without significant effect on fungal growth, as expected. Additionally, results showed that the surfaces co-functionalized with AmB and CIP were able to significantly inhibit all microorganisms in the triple-species biofilms. Overall, it was possible to conclude that the interactions established in this triple consortium did not affect the outcome of the antimicrobial performance of the surfaces when challenged by all species at the same time. Chapter 4 131 Figure 20. Antimicrobial performance of PVC surfaces functionalized with AmB (0.1 mg/mL) and/or CIP (0.5 mg/mL) against mixed-species biofilms, after a contact of 24 h. PVC was used as a control while pDA was included for comparison purposes. Significant differences were found for (*) p < 0.0001 and for (#) p < 0.05, when reductions were observed compared to cells adhered to PVC. The minimum value of the Y axis on the graphs was defined as 2 since it corresponds to the detection limit of CFU counting (log 2 CFU/mL). Chapter 4 132 To complement the previously established antimicrobial activity of the modified surfaces against mixedspecies biofilms, an in situ evaluation was performed by fluorescence microscopy to inspect the presence of adhered cells. DAPI is a cell-permeable fluorescent compound that can stain the DNA of eukaryotic and prokaryotic cells. Therefore, its application allowed the differentiation of each microorganism based on their distinctive morphological characteristics. C. albicans exhibit different morphotypes at different stages: yeast, pseudohyphae, and hyphae [38]. The presence of hyphae allows the identification of the fungus and even when C. albicans presents the oval form, it can be distinguished from the bacterial cells because of their size: around 3-4 µm in diameter. S. aureus is spherically shaped and exhibits approximately 0.5-1.5 µm in diameter [38], and P. aeruginosa exhibits a rod-shaped about 1–5 µm long and 0.5–1.0 µm wide [39]. The observation of the microscopic images (Figure 21) pointed out a significant reduction in the number of cells adhered to the modified surfaces, compared to the control surfaces (bare PVC). However, for some conditions, in which no cultivable cells were detected (Figure 20), some cells were observed under the microscope. Although this discrepancy does not predict the viability of cells, these results should be considered. In the case of C. albicans for all consortia tested, no cells were detected by CFU enumeration on surfaces functionalized with AmB alone or combined with CIP. However, some fungal cells could be observed for these conditions, when evaluated by fluorescence microscopy. Nevertheless, it is important to verify that these cells had lost their ability to form hyphae, as compared to control surfaces. Concerning S. aureus , some cells were observed adhered to all modified surfaces, even in dual-species biofilms with P. aeruginosa, in which no CFU had been detected. In the case of P. aeruginosa , this was not observed on surfaces functionalized with CIP in the dual consortia with C. albicans or in the triplespecies biofilm. Nevertheless, P. aeruginosa cells were visualized on surfaces modified with CIP when this bacterium was grown in the presence of S. aureus. In the case of surfaces co-functionalized with AmB and CIP, they were not able to completely prevent P. aeruginosa growth in all consortia studied. Chapter 4 133 Figure 21. Representative fluorescent DAPI stained images of different consortia obtained after adhesion for 24 h on unand modified PVC surfaces. The scale bars indicate 10 µm. CA: C. albicans ; SA: S. aureus ; PA: P. aeruginosa .