Mussel adhesive-inspired surface modification to design bi-functional antibacterial coatings

PhD in Chemical and Biological EngineeringEven though the introduction of biomaterials in modern medicine has been crucial in restoring body function and quality of life, all biomaterials are prone to be colonised by microorganisms, representing, therefore, niches for infection in vivo. These biomaterial-associated infections (BAI) are often associated to the biofilm mode of growth, in which bacteria encase themselves in a selfproduced hydrated matrix of extracellular polymeric substances (EPS), conferring them protection against the host immune system and antibiotic treatment. Since bacterial adhesion to the surface of a biomaterial is a crucial step in BAI pathogenesis, surface modification of biomaterials to impart them with the ability to resist bacterial colonisation represents the most potential approach to fight these infections. Considerable advances in the field of antibacterial coatings have been occurred, but few biomaterials have been designed that effectively reduce the incidence of BAI. Therefore, the key goal of this thesis was to propose an effective coating strategy to impart biomaterials with the ability to prevent bacterial adhesion and simultaneously kill the adherent ones, with low propensity for developing bacterial resistance and with absence of adverse effects on the interaction with mammalian cells. Antimicrobial peptides (AMP) and enzymes targeting different EPS were the compounds chosen as antimicrobials alternatives to be immobilized onto biomaterial surfaces. Compounds immobilization was performed using a facile mussel-inspired adhesive coating strategy in which materials were immersed in a solution containing dopamine and the compounds together (1-step approach immobilization), or materials were immersed in an alkaline solution of dopamine to form a thin layer of polydopamine (pDA) and then transferred into a solution containing the AMP and/or enzymes (2-step approach immobilization). Mono and bi-functional coatings were physically characterized in what concerns their morphology, wettability, surface composition and roughness. Scanning electron microscopy and atomic force microscopy showed that the presence of pDA increased the surface roughness of both polydimethylsiloxane (PDMS) and polycarbonate materials, while the measuring of water contact angles showed a decrease on the hydrophobicity characteristic of these materials. Further functionalization with AMP or enzymes yielded surfaces with similar morphology or a more homogeneous coating, when a 2-step or 1-step approach immobilization was performed, respectively. Their antimicrobial and anti-adhesive performance as well their cytotoxicity were also evaluated. A screening with several AMP more traditional and natural such as polymyxins B and E, as well as analogues peptides more active and stable such as Palm and Camel was performed. AMP proved to be good alternatives to antibiotics as they were able to compromise biofilm formation at similar range concentrations to inhibit planktonic growth. Polymyxins B and E were more effective against Pseudomonas aeruginosa while Camel and Palm were more promising against Staphylococcus aureus. Polymyxin E potential was further demonstrated after its physical adsorption onto polystyrene surfaces as it proved to impair biofilm formation and increase P. aeruginosa biofilms susceptibility to antimicrobial treatment. Peptides immobilization was afterwards optimized using the pDA-based approaches. Immobilization of polymyxins B and E onto PDMS rendered the surfaces with antimicrobial activity towards the Gram-negative bacteria P. aeruginosa and showed great potential to overcome some concerns associated to bacterial resistance and toxicity reported in the past for these compounds when in solution. Palm was, however, the AMP chosen to design bi-functional coatings as its immobilization rendered PDMS with effective antimicrobial activity against both Gram-negative and Gram-positive bacteria, especially against the Gram-positive ones, the most commonly found associated to BAI. The immobilization of different enzymes (alginate lyase, lysozyme, proteinase K and DNase I) was afterwards optimized and results showed that catechol chemistry allowed their grafting without compromising their catalytic activity. DNase I was the enzyme chosen for further investigations because exhibited the best anti-adhesive features against a wider spectrum of bacterial strains. Once established the AMP and enzyme with most promising features, their co-immobilization was optimized in order to impart PDMS surfaces with potent antimicrobial and anti-adhesive properties against the adhesion of several strains of P. aeruginosa, S. aureus and Staphylococcus epidermidis as single and dual-species, with excellent stability and no cytotoxicity. To better discriminate co-adhesion of both species on modified surfaces, PNA FISH (Fluorescence in situ hybridization using peptide nucleic acid probes) was also employed, and results showed that P. aeruginosa was the dominant organism, with S. aureus adhering afterwards on P. aeruginosa agglomerates. The fate of bacteria that managed to adhere to the proposed bi-functional coatings was also investigated and results showed that bacteria were more susceptible to antibiotic treatment and to macrophages phagocytosis, without developing bacterial resistance towards the immobilized AMP. In conclusion, a facile and non-toxic mussel-inspired adhesive coating strategy was applied to coimmobilize Palm and DNase I onto biomaterial surfaces without compromise their activity and rendering the surfaces with good antimicrobial, anti-adhesive and anti-biofilm features together with no cytotoxicity and no propensity for developing bacterial resistance. This coating strategy holds, therefore, great potential to be further explored in the design of biomaterial implants and devices to combat BAI.Ainda que a introdução de biomateriais na medicina atual tenha sido fundamental para recuperar funções do corpo humano comprometidas e melhorar a qualidade de vida em geral, todos eles são propensos a ser colonizados por microrganismos, constituindo, desta forma, nichos para infeção in vivo. As infeções associadas a biomateriais (BAI) estão frequentemente associadas a biofilmes, estruturas biológicas nas quais as bactérias se envolvem numa matriz hidratada de substâncias poliméricas extracelulares (EPS) por elas produzida, que lhes confere proteção contra o sistema imunitário do hospedeiro e tratamentos com antibióticos. Uma vez que a adesão bacteriana à superfície de um biomaterial desempenha um papel crucial na patogénese de BAI, a modificação de superfícies para as dotar de capacidade de resistir à colonização bacteriana representa a abordagem mais promissora para combater estas infeções. Nos últimos anos tem-se assistido a grandes avanços na área dos revestimentos antibacterianos, contudo, são ainda poucos os biomateriais concebidos que efetivamente reduzem a incidência de BAI. A presente tese teve como objetivo principal propor uma estratégia de revestimento capaz de eficazmente dotar os biomateriais com características de prevenção da adesão bacteriana e, simultaneamente, com capacidade para matar bactérias que eventualmente consigam aderir, e sem potencial para desenvolver resistência bacteriana ou citotoxicidade. Os compostos selecionados como alternativos aos antibióticos para serem imobilizados em biomateriais foram péptidos antimicrobianos (AMP) e enzimas que atuam em diferentes EPS. A imobilização dos compostos foi efetuada recorrendo a uma estratégia de adesão inspirada em mexilhões, seguindo duas abordagens: numa, os materiais foram colocados numa solução contendo simultaneamente dopamina e os compostos a imobilizar (abordagem de imobilização num passo); noutra, os materiais foram incubados primeiramente numa solução alcalina de dopamina, para formar um filme fino de polidopamina (pDA), e depois transferidos para uma solução contendo AMP e/ou enzimas (abordagem de imobilização em 2 passos). Os revestimentos mono e bi-funcionais foram caracterizados fisicamente no que diz respeito à sua morfologia, molhabilidade, composição atómica da superfície e rugosidade. A microscopia eletrónica de varrimento e microscopia de força atómica demonstraram que a presença de pDA aumentou a rugosidade da superfície do polidimetilsiloxano (PDMS) e do policarbonato, enquanto a medição dos ângulos de contacto da água demonstrou uma diminuição da hidrofobicidade característica destes materiais. A funcionalização posterior com AMP ou enzimas gerou superfícies com morfologia semelhante ou com um revestimento mais homogéneo mediante a realização de uma abordagem de imobilização em 2 ou num passo, respetivamente. Os materiais funcionalizados foram também avaliados em termos do seu desempenho antibacteriano e citotoxicidade. Um primeiro estudo para averiguar o potencial antimicrobiano de uma série de AMP mais tradicionais e naturais como as polimixinas B e E, bem como péptidos análogos mais estáveis e potentes, como o Palm e Camel, demonstrou que estes constituem uma alternativa aos antibióticos uma vez que foram capazes de comprometer a formação de biofilme quando utilizados em concentrações semelhantes às necessárias para inibir o crescimento planctónico. As polimixinas B e E foram mais eficazes contra Pseudomonas aeruginosa enquanto o Camel e o Palm foram mais promissores contra Staphylococcus aureus. O potencial da polimixina E foi, ainda, demonstrada após a sua adsorção física em superfícies de polistireno, ao comprometer a formação de biofilme de P. aeruginosa e ainda promover a sua suscetibilidade a tratamentos antimicrobianos posteriores. A imobilização dos AMP foi posteriormente otimizada usando estratégias baseadas na pDA. A imobilização de polimixinas B e E em PDMS conferiu a esta superfície atividade antimicrobiana contra a bactéria Gram-negativa P. aeruginosa e evidenciou o carácter promissor da sua utilização, uma vez que foram superados problemas relacionados com o desenvolvimento de resistência e toxicidade associados a estes AMP quando usados em solução. Palm foi, contudo, o AMP selecionada para manufaturar os revestimentos bi-funcionais dado que a sua imobilização conferiu ao PDMS atividade antimicrobiana contra bactérias Gram-positivas e Gram-negativas, sendo mais relevante contra as Gram-positivas, as mais frequentemente associadas a BAI. A imobilização de várias enzimas (alginato liase, lisozima, proteinase K e Dnase I) foi também otimizada, tendo-se demonstrado que a imobilização baseada na pDA não comprometeu a sua atividade catalítica. A DNase I foi a enzima que exibiu melhores propriedades anti-adesivas contra um espectro mais alargado de estirpes bacterianas, tendo por isso sido selecionada para a investigação de revestimentos bi-funcionais. Uma vez estabelecido o AMP e a enzima com as caraterísticas mais promissoras, otimizou-se a sua co-imobilização de modo a conferir às superfícies de PDMS atividades antimicrobianas, anti-adesivas e anti-biofilme contra a adesão de várias estirpes de P. aeruginosa, S. aureus and Staphylococcus epidermidis, de forma isolada ou em consórcios de duas espécies, com excelente estabilidade e sem citotoxicidade. A hibridação fluorescente in situ combinada com moléculas de ácido péptido-nucléico (PNA FISH) foi ainda utilizada para a discriminação dos microorganismos nos consórcios polimicrobianos. Foi possível observar que a P. aeruginosa foi o organismo dominante no cosnsórcio, com S. aureus a aderir aos aglomerados de P. aeruginosa. A suscetibilidade das bactérias que eventualmente consigam aderir aos revestimentos propostos foi também investigada tendo-se mostrado que estas bactérias foram mais sensíveis ao tratamento com antibióticos e à fagocitose levada a cabo por macrófagos, sem desenvolverem resistência bacteriana em relação ao AMP imobilizado. Em conclusão, a estratégia de adesão inspirada em mexilhões aplicada para, de forma simples e não-tóxica, co-imobilizar um AMP e uma enzima em biomateriais não comprometeu a sua atividade e dotou as superfícies de PDMS com propriedades antibacterianas relevantes e sem indícios de desenvolvimento de citotoxicidade e de resistência bacteriana. Estes revestimentos apresentam um grande potencial para o desenvolvimento de biomateriais capazes de resistir efetivamente a BAI.Fundação para a Ciência e Tecnologia (FCT) e Fundo Europeu de Desenvolvimento Regional (FEDER), através do Programa COMPETE por suportar financeiramente este projeto (SFRH/BD/78063/2011) e o projecto AntiPep PTDC/SAU-SAP/113196/2009 (FCOMP-01- 0124-FEDER-016012)

Influence of a quaternary ammonium compound on the cell structure of bacteria using atomic force microscopy

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)Bacterial adhesion and subsequent biofilm formation remains a serious concern, especially in clinical applications, since bacteria associated with biofilms are more resistant to antibiotic treatment and to the host immune system. A potential approach to deal with this drawback may rely on the use of antimicrobial coatings comprising quaternary ammonium compounds (QACs). The main purpose of the present thesis was to study the efficacy of a QAC against staphylococci when compared with two other antimicrobial compounds, an antibiotic (Gentamicin) and an antimicrobial peptide (Gramicidin S) using atomic force microscopy (AFM). After assessing the antimicrobial activity of the compounds against planktonic cultures by determining their minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC), adhering staphylococcal cells were exposed to the antimicrobial compounds and their cell surfaces analyzed with AFM. The number of bacteria removed by the AFM tip was determined and taken as an indication of cell surface damage. The antimicrobial action of the compounds on staphylococcal biofilms was evaluated by the determination of the metabolic activity of biofilm through an 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay. Finally, in order to get some insights about QAC mode of action against bacterial cells, its antimicrobial activity in planktonic cultures, as well as their effects on adhering staphylococcal cells by AFM were performed in the presence of calcium ions. All the antimicrobial compounds proved to exhibit antimicrobial activity against staphylococcal planktonic cultures. AFM measurements allowed the analysis in situ of the bacterial cells behaviour when exposed to antimicrobial compounds. Bacterial cell surface wrinkled upon exposure to the antimicrobials until bacteria disappear from the surface. Gentamicin yielded faster wrinkling and removal of bacteria from the surface as compared to QAC and Gramicidin S which yielded similar results. As the staphylococci outside the continuously scanned area did not seem affected by the compounds it was suggested that the pressure of the AFM tip assisted the incorporation of antimicrobials in the membrane, enhancing their bactericidal efficacy. Staphylococci biofilm proved to be susceptible to all the antimicrobials, with QAC being the most effective one, causing a complete loss of metabolic activity at 2xMBC. The antimicrobial action of the QAC was compromised by the presence of calcium ions both in planktonic cultures and in adhering staphylococci which suggests that its mode of action relies on its ability to exchange with calcium ions that are responsible for membrane stability.A adesão bacteriana e consequente formação de biofilmes constitui um problema sério, sobretudo na área clínica, visto que as bactérias associadas em biofilmes são menos susceptíveis à terapia antibiótica e à acção do sistema imunitário. Uma potencial estratégia para lidar com este problema consiste no uso de revestimentos antimicrobianos constituídos por compostos quaternários de amónio (QACs). A presente tese teve como principal objectivo o estudo da eficácia de um QAC sobre uma estirpe bacteriana do género estafilococos quando comparado com outros dois compostos antimicrobianos, um antibiótico (Gentamicina) e um péptido antimicrobiano (Gramicidina S). Depois de avaliada a actividade antimicrobiana dos compostos em culturas planctónicas através da determinação da concentração mínima inibitória (MIC) e da concentração mínima bactericida (MBC), as bactérias aderidas a uma superfície foram expostas aos compostos e a sua superfície celular foi analisada por AFM. O número de bactérias removidas pela ponta do AFM foi determinado e considerado como uma indicação dos danos causados na superfície celular. A susceptibilidade dos biofilmes aos compostos foi avaliada pela determinação da actividade metabólica do biofilme através de um ensaio de 3-(4,5-dimetiltiazol-2-il)-2-5 difeniltatrazólio de brometo (MTT). Finalmente, com o intuito de investigar o modo de acção do QAC sobre as células bacterianas, a sua actividade antimicrobiana, bem como os seus efeitos nas células aderidas e analisados por AFM foram realizados na presença de iões de cálcio. Todos os compostos exibiram actividade antimicrobiana no estado planctónico. O efeito dos compostos sobre as células bacterianas foi analisado in situ com o AFM. A superfície celular apresentou-se mais enrugada depois de exposta aos compostos até as bactérias serem removidas da superfície. A Gentamicina resultou numa remoção mais rápida que o QAC e a Gramicidina S que exibiram resultados semelhantes. Como as bactérias localizadas fora da área continuamente analisada pelo AFM não foram igualmente influenciadas pelos compostos foi colocada a hipótese de que a pressão da ponta do AFM auxiliou a incorporação dos compostos na membrana, melhorando assim a sua eficácia bactericida. O biofilme formado pela estirpe estudada na presente tese apresentou-se susceptível a todos os compostos antimicrobianos, sendo o QAC o mais eficaz ao provocar uma redução completa da actividade metabólica do biofilme a 2xMBC. A acção antimicrobiana do QAC foi comprometida pela presença de iões de cálcio o que sugere que o seu modo de acção depende da sua capacidade de substituir estes iões na membrana

Terpinen-4-ol combined with colistin effectively impairs Pseudomonas aeruginosa biofilm formation

Due to widespread and indiscriminate use of conventional antibiotics, bacteria have acquired a resistant phenotype in response to those antimicrobials pressure. Therefore, microbial infections associated with biofilms have become hard to treat with conventional therapy. The development of microbial drug resistance and drugrelated toxicity has promoted the search for new alternatives, to control, mostly, healthcare-associated infections. Terpinen-4-ol is the major component of tea tree essential oil and has shown strong antimicrobial properties as a single agent against planktonic cultures. Colistin is an antimicrobial peptide with great antimicrobial activity, essentially against Gram-negative bacteria, such as P. aeruginosa. The aim of this study was to explore the synergism between terpinen-4-ol and colistin, using low doses of each natural antimicrobial, to control the establishment of P. aeruginosa biofilms. Biofilms were formed in the presence of both antmicrobials, alone or in combination, being after characterized by total biomass, through crystal violet, and number of cultivable bacterial cells (log CFU/cm2). Data related with the individual antimicrobial activity of terpinen-4-ol revealed that the biomass of P.aeruginosa biofilms was significantly reduced for 0.19 % (v/v), though it not affected the viability of cells even for the highest concentration tested (0.38 % (v/v)). On the other hand, colistin promoted a significantly reduction of the biofilm mass but only for concentrations higher than 1 μg ml-1. The number of viable cells entrapped within the biofilms was only affect for colistin doses higher for 4 μg ml-1. The association of terpinen-4-ol (0.19 % (v/v)) with colistin revealed to be a very efficient prophylactic strategy, as it impaired significantly biofilm formation. In fact, it was observed biofilm mass reductions closed to 100 % and significant decreases of the numbers of viable biofilm-cells (3-5 log of reduction) even for the lowest colistin concentration tested (0.5 μg ml-1). These data highlighted the promising antibiofilm activity of association of natural compounds, as antimicrobial peptides and secondary metabolites of essential oils, suggesting that this combination may have prophylactic potential for the prevention of P. aeruginosa biofilm-associated infections

The fate of adhering bacteria on antimicrobial surfaces: transcriptomic analysis of resistance associated genes and macrophage-mediated phagocytosis

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio

Combination of Posaconazole and Amphotericin B in the treatment of Candida glabrata biofilms

Candidemia cases have been increasing, especially among immunosuppressed patients. Candida glabrata is one of the most resistant Candida species, especially to the azole drugs, resulting in a high demand for therapeutic alternatives. The minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and minimum biofilm eradication concentration (MBEC) were determined for posaconazole (Pcz) and amphotericin B (AmB). The drug combinations of both drugs were evaluated on pre-formed biofilms of C. glabrata ATCC 2001, through XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay, colony forming units (CFU), crystal violet, and the fractional inhibitory concentration index (FICI). C. glabrata revealed higher susceptibility and biofilm reduction in the presence of AmB alone, but both drugs revealed a good capacity in the biomass elimination. In the majority of the tested combinations, the interactions were defined as indifferent (FICI 4). The combination of the two drugs does not seem to bring a clear advantage in the treatment of biofilms of C. glabrata.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and Célia F. Rodrigues’ (SFRH/BD/93078/2013) PhD grant.info:eu-repo/semantics/publishedVersio

Blocking of Pseudomonas aeruginosa biofilm formation by a colistin coating

Bacterial colonisation of indwelling devices followed by biofilm formation remains a serious threat in clinical field as it is commonly associated to persistent infections. Once adhered to a surface, bacteria embed themselves in a self-produced matrix mainly composed of extracellular polymeric substances which confers them protection against antimicrobial agents and the host immune system. Early bacterial adhesion is a crucial step in biomaterial associated infections pathogenesis, representing therefore a promising target for the development of biofilm preventive measures. Several strategies have been developed to prevent bacterial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of anti-adhesive, antiseptic and antibiotic coatings. Although some of these coatings have been shown efficient in the prevention of biofilm formation, an important drawback associated to them is the development of microbial resistance that limits the usefulness of classical antimicrobials. A promising solution to overcome this problem may rely on the use of new alternatives, as antimicrobial peptides (AMPs) that are unlikely to induce any resistance because of their evolutionary path. The aim of this work was to evaluate the potential role of colistin, a traditional AMP, as an antimicrobial coating for biomaterials. Based on the observation that the presence of colistin as a biofilm growth media complement was able to significantly impair Pseudomonas aeruginosa biofilm formation at concentrations below its MBC, polystyrene (PS) surfaces were coated with this AMP and its ability to prevent biofilm formation was assessed. A P. aeruginosa reference strain (ATCC 10145) and a P. aeruginosa clinical isolate (U147016) were used as biofilm producers. PS surfaces were pre-coated with several concentrations of colistin and the biofilms formed on conditioned and clean surfaces were then characterized in terms of biomass (CV), respiratory activity (XTT) and number of viable cells (CFU). The susceptibility of biofilms formed on colistin-conditioned surfaces to ciprofloxacin (CIP) treatment was further investigated. Results showed that the clinical isolate produces biofilms with more activity, less biomass and similar number of cells than the reference strain. Random deposition of colistin residues on the adhesion surfaces significantly reduced biofilm activity and mass accumulated in a dose-dependent manner for both strains. Regarding biofilm entrapped cells, the conditioning film proved to be less efficient, causing significant reductions for the highest concentrations tested. Concerning the combined application of colistin surface conditioning and biofilm treatment with CIP, it was observed that biofilms formed on colistin-conditioned surfaces were more susceptible to CIP treatment in terms of biofilm-entrapped cells. The presence of colistin during biofilm formation may have interfered in the transition from reversible to irreversible interactions during the early steps of bacterial adhesion to PS, disturbing and delaying the mature biofilm development. Biomaterial associated infections remains a major drawback to the long-term use of medical devices. This study demonstrates the potential of the AMP colistin as an excellent candidate for biomaterials coating limiting biofilm formation on their surfaces

Evaluation of potential resistance development by cells adhered to antimicrobial surfaces

Publicado em "Biofilms7: microbial works of art"Immobilization of antimicrobials onto a surface has been proposed as a promising approach to fight biomaterial-associated infections (BAI). In this study, three antimicrobials, currently under investigation for use in medical devices, were evaluated for the risk of inducing bacterial resistance after their immobilization. An antibiotic, a quaternary ammonium compound (QAC) and an antimicrobial peptide (AMP) were immobilized onto polydimethylsiloxane (PDMS) using a mussel-inspired coating strategy. Results showed that antimicrobial surfaces exhibited contact-killing activity and were able to impair biofilm establishment. However, and similar to previously reported studies, a complete biofilm eradication was not achieved. The potential development of resistance towards these antimicrobials immobilized were then evaluated by continuously recovering the cells adhered to these antimicrobial surfaces and allowing them to adhere to new modified surfaces for a total of 10 passages. As a control, the same procedure was performed for unmodified PDMS. After 10 days, the cells recovered from the un- and modified surfaces were used to determine the MIC and MBC of antimicrobials. No propensity for developing bacterial resistance was found for immobilized QAC or AMP as the same susceptibility pattern was obtained for cells recovered from unmodified or modified surfaces. Cells recovered from the surfaces modified with antibiotic, exhibited a higher MBC as compared to cells recovered from unmodified PDMS. This study highlighted the risk associated to the immobilization of antibiotics and the promising potential of QAC and AMP to be used in the design of materials able to prevent BAI

Modulating an antimicrobial release approach by dopamine chemistryto fight infections associated to orthopedic implants

Alongside with orthopedic implants contribution for modern healthcare improvements, theres the risk associated to their microbial colonization and biofilm formation, compromising the performance of the implant itself and representing niches for infection. This study aimed to engineer an antimicrobial release coating for stainless steel surfaces (SS) to empower them with the ability to prevent Staphylococci colonization. Surface modification was based on dopamine chemistry, which self-polymerization results in the deposition of a thin, adhered film called polydopamine (pDA). Chlorohexidine (CHX) was chosen to confer the antimicrobial features. Its immobilization was performed through a 2-step approach, including pDA formation and immersion in CHX solution, and 1-step strategy, in which dopamine and CHX were dissolved together and SS coupons were immersed in this solution. An additional layer of pDA was also performed for both strategies. SEM and AFM confirmed pDA coating by the presence of self-polymerized pDA particles without altering the roughness of SS surfaces. Immobilization of CHX using a 1-step approach yielded surfaces with a more homogenous coating than the 2-step approach. Different pDA-based strategies yielded different CHX release profiles: the amount of CHX released was higher for the 2-step approach and the addition of another pDA layer reduced the amount of CHX released. The antimicrobial performance of the modified surfaces was evaluated against S. aureus and S. epidermidis and the results showed that all the strategies caused a significant reduction (more than 3 LOG) in the number of cells adhered to the surfaces and in suspension, after 24 h. The 2-step approach was able to impart SS surfaces with antimicrobial activity even after 10 days of exposure. In conclusion, dopamine chemistry can modulate CHX release from the surfaces to obtain an antimicrobial coating strategy with great potential to fight infections associated with orthopedic implants.info:eu-repo/semantics/publishedVersio

Antimicrobial materials for endotracheal tubes: a review on the last two decades of technological progress

Ventilator-associated pneumonia (VAP) is an unresolved problem in nosocomial settings, remaining consistently associated with a lack of treatment, high mortality, and prolonged hospital stay in mechanically ventilated patients. The endotracheal tube (ETT) is the major culprit for VAP development owing to its early surface microbial colonization and biofilm formation by multiple pathogens, both critical events for VAP pathogenesis and relapses. To combat this matter, gradual research on antimicrobial ETT surface coating/modification approaches has been made. This review provides an overview of the relevance and implications of the ETT bioburden for VAP pathogenesis and how technological research on antimicrobial materials for ETTs has evolved. Firstly, certain main VAP attributes (definition/categorization; outcomes; economic impact) were outlined, highlighting the issues in defining/diagnosing VAP that often difficult VAP early- and late-onset differentiation, and that generate misinterpretations in VAP surveillance and discrepant outcomes. The central role of the ETT microbial colonization and subsequent biofilm formation as fundamental contributors to VAP pathogenesis was then underscored, in parallel with the uncovering of the polymicrobial ecosystem of VAP-related infections. Secondly, the latest technological developments (reported since 2002) on materials able to endow the ETT surface with active antimicrobial and/or passive antifouling properties were annotated, being further subject to critical scrutiny concerning their potentialities and/or constraints in reducing ETT bioburden and the risk of VAP while retaining/improving the safety of use. Taking those gaps/challenges into consideration, we discussed potential avenues that may assist upcoming advances in the field to tackle VAP rampant rates and improve patient care. Statement of significance : The use of the endotracheal tube (ETT) in patients requiring mechanical ventilation is associated with the development of ventilator-associated pneumonia (VAP). Its rapid surface colonization and biofilm formation are critical events for VAP pathogenesis and relapses. This review provides a comprehensive overview on the relevance/implications of the ETT biofilm in VAP, and on how research on antimicrobial ETT surface coating/modification technology has evolved over the last two decades. Despite significant technological advances, the limited number of gathered reports (46), highlights difficulty in overcoming certain hurdles associated with VAP (e.g., persistent colonization/biofilm formation; mechanical ventilation duration; hospital length of stay; VAP occurrence), which makes this an evolving, complex, and challenging matter. Challenges and opportunities in the field are discussed.This research was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalização (COMPETE2020) and by national funds, through FCT for the POLY-PrevEnTT project (PTDC/BTMSAL/29841/2017- POCI-01-0145-FEDER029841) and the Ph.D. Grant of Tânia Grainha (SFRH/BD/136544/2018).info:eu-repo/semantics/publishedVersio

Colistin heteroresistance in Klebsiella pneumonia and its association with slow-growing subpopulations within biofilms

The emergence of multidrug-resistant strains of Klebsiella pneumoniae is a growing clinical concern that is leading to the re-introduction of the old and toxic colistin as a salvage therapy. However, several cases of heteroresistance to this antimicrobial have been recently reported in planktonic studies. Therefore, the understanding of the conditions that trigger heteroresistance is attracting considerable research interest. In this scope, this work aimed to more comprehensively study the response of K. pneumoniae biofilms to colistin and to inspect the occurrence of heteroresistance in biofilm-cells.K. pneumoniae presented susceptibility to colistin in its planktonic form, though biofilms presented an enhanced resistance. The population analysis profiles pointed out the existence of a slow-growing sub-population resistant to colistin within a K. pneumoniae strain that seemed to be exclusively associated with biofilms. This resistant sub-population is characterized by a small colony morphology (diameter around 5 mm), which remains unchangeable, and a completely different response to colistin compared to the observed in the wild-type morphotype. Colistin was ineffective in this small colony variant since it was never achieved any reduction in biofilm-cells viability. These findings suggest that heteroresistance is linked to biofilm formation and to a morphological distinct sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance from an apparently susceptible strain