Avaliação dos mecanismos de interação entre hospedeiro e fago em biofilmes

Dissertação de mestrado em BioengenhariaPseudomonas aeruginosa e Acinetobacter baumannii são duas bactérias gram-negativas patogénicas altamente problemáticas em infeções hospitalares muito devido à sua capacidade de aderir a variadíssimas superfícies e formar biofilmes que contribuem bastante para o aumento da resistência aos agentes antimicrobianos, o que consequentemente potencia infeções graves em pacientes. Apesar da terapia fágica ter vindo a ser referida como uma estratégia alternativa no controlo de biofilmes, ainda há poucos estudos sobre a interação de fagos com as respetivas bactérias hospedeiras em biofilme mistos. Este trabalho descreve o efeito de fagos em biofilmes simples de A. baumannii AB1 e biofilmes mistos formados por A. baumannii AB1 e P. aeruginosa PAO1. Para este fim, foram desenvolvidos métodos de diferenciação das duas espécies no biofilme recorrendo à técnica de FISH (do inglês, Fluorescent in situ hibridization). Para uma deteção multiplex de bactérias e fagos foram desenhadas “in silico” sondas com base num novo ácido nucleico LNA (do inglês Locked Nucleic Acid), contudo só a sonda específica para a bactéria P. aeruginosa foi sintetizada e aplicada ao estudo. O método de LNA FISH para a identificação desta bactéria foi otimizado e testes laboratoriais em amostras de subespécies de Pseudomonas confirmaram os valores teóricos previstos de especificidade e sensibilidade. Os ensaios de infeção fágica contra biofilme simples de A. baumannii, mostraram que os fagos são eficientes, mesmo usando uma razão de 1 fago para 1 célula hospedeira. Com efeito, verificou-se uma redução de aproximadamente 2 log de células viáveis em biofilmes de 24 h após 6 h de contacto com o fago. Contudo a eficácia dos fagos mostrou-se significativamente superior contra células do biofilme raspado o que indica que a matriz do biofilme constitui uma barreira para a eficiente atuação do fago. Os biofilmes mistos mostraram uma predominância de P. aeruginosa em relação a A. baumannii, mesmo quando P.aeruginosa é adicionada após colonização inicial por A.baumannii. A aplicação de um cocktail formado por fagos específicos contra estas duas espécies presentes em biofilme misto revelou uma redução de 2 log de P. aeruginosa e de aproximadamente 1,5 log de A. baumannii após 6 h. No entanto, após 24 h de infeção, foi possível observar, em todos os biofilmes estudados, um aumento de células viáveis podendo este facto estar relacionado com a aquisição de resistência das bactérias aos respetivos fagos. A técnica de LNA FISH demostrou ser eficiente na discriminação clara das populações de biofilme misto, sendo um complemento na deteção / compreensão na interação dos fagos com os seus hospedeiros em estruturas complexas como os biofilmes. Em suma, este trabalho demonstrou o valor potencial dos fagos no controlo de biofilmes, tornando-se no entanto necessário progredir na presente investigação, para se definirem as condições ótimas de atuação dos fagos e as razões da diminuição da eficácia dos fagos quando em contacto com o biofilme por períodos superiores a 6 h.Pseudomonas aeruginosa and Acinetobacter baumannii are two pathogenic gram-negative bacteria that are highly prevalent in hospital infections due to their natural ability to adhere to surfaces and form biofilms. Cells in biofilms are more tolerant to antimicrobial agents and this enhances serious infections in patients. Phage therapy has been reported as an alternative strategy for biofilm control but the interaction of phages with the respective bacteria in mixed biofilm in poorly understood. The work described herein focuses on the effect of phages in single species of A. baumannii biofilms and in mixed biofilms formed by A. baumannii AB1 and P. aeruginosa PAO1. To this end, methods were developed for the differentiation of the two species in biofilm using the FISH (Fluorescent in situ hibridization) technique. For multiplex detection of bacteria and phage probes based on a novel nucleic acid LNA (Locked Nucleic Acid) were designed "in-silic", but only the probe specific for the bacteria P. aeruginosa was synthesized and applied to the study. The LNA FISH method was optimized, and laboratory testing on representative strains from the Pseudomonas genus subspecies confirmed the predicted theoretical values of specificity and sensitivity. Phage infection of A. baumannii single biofilms shows that phages were efficient even when a ratio of 1 phage to 1 host cell is used. Indeed, there was a reduction of 2 log in viable cells of 24 h biofilms, after 6 h of contact with the phage. Furthermore, the efficiency of phage was significantly higher against biofilm scraped cells indicating that the biofilm matrix is hindering the efficient performance of the phage. The composition of mixed biofilms revealed a predominance of P. aeruginosa compared to that of A. baumannii... The application of a phage cocktail resulted in a reduction of 2 log of P. aeruginosa and 1,5 log of A. baumannii after 6 h. Nevertheless, after 24 hours of infection it was observed the increase of viable cells in all biofilms studied. This result can be explained by the acquisition of resistance of the bacterial hosts towards the respective phages. The LNA FISH technique demonstrated to be efficient in the clear discrimination of mixed biofilm populations, being a complement the detection / understanding the interaction of phages with their hosts in complex structures such as biofilms. Briefly, this study demonstrated the potential use of phages for biofilm control, however become necessary to progress in this research, to define the optimal conditions of phage infections and the reasons for the decreased effectiveness of phages when in contact with the biofilm for periods longer than 6 h

Antimicrobial assessment of phage therapy using a porcine model of biofilm infection

Antibiotic resistant bacterial communities persist in many types of wounds, chronic wounds in particular, in the form of biofilms. Biofilm formation is a major cause of severe infections and the main reason for a negative treatment outcome and slow healing progression. Chronic wounds are a silent epidemic essentially affecting people with co-morbid conditions such as diabetes and obesity and elderly persons particularly those with movement limitations. The development of complementary and alternative effective strategies to antibiotics for the treatment of chronic wounds is highly desired. Phage therapy constitutes a very promising approach in the control of topical microbial populations. In this work newly isolated phages were tested for their efficacy to control bacterial species that predominate in chronic wounds. Phage effectiveness was studied on 24-h old biofilms formed in polystyrene microplates and in porcine skin explants using two treatment approaches: individual phage and a cocktail of phages against four main pathogens commonly isolated from chronic wounds. The two models produced variations in the surface colonization ability, assessed by viable bacterial counts and microscopy visualization after using peptide nucleic acid (PNA) or locked nucleic acid probes (LNA) and 2-O-methyl (2-OMe) in fluorescence in situ hybridization (FISH), and in the phage-host interactions. Phages alone and combined caused greater reductions in the number of viable cells when biofilms had been formed on porcine skins and with greater variations detected at 4 h and 24 h of sampling. These results suggest that porcine skin models should be preferentially used to assess the use of phages and phage cocktails intended for topical use in order to understand the fate, throughout treatment time, of the population when dealing with biofilm-related infections.This work was supported by Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020(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 the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). CM ac- knowledges the Portuguese Foundation for Science and Technology (FCT) grant SFRH/BD/94434/2013. SS is an Investigador FCT (IF/ 01413/2013).info:eu-repo/semantics/publishedVersio

Challenging mono and dual-species biofilms with phages and antibiotics

Pseudomonas aeruginosa is a relevant opportunistic pathogen, and worryingly it frequently shows a low antibiotic susceptibility. This bacterium is responsible for 65% of mortality in hospitals all over the world. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the results from several years of investigation using P. aeruginosa phages alone and combined with antibiotics or other phages against single and mixedspecies biofilms. The mixed species biofilms of P. aeruginosa reported herein were formed with fungi (Candida albicans) since these two microorganisms co-inhabit a wide variety of environments and the interactions between them can result in huge medical and economic impacts; and with Acinetobacter baumannii also an opportunistic pathogen associated with nosocomial infections. In both mixed species biofilms there was observed an inhibitory effect of P. aeruginosa since the levels of C. albicans and A. baumannii were highly reduced in the presence of P. aeruginosa. In P. aeruginosa - C. albicans biofilms, the Pseudomonas phages were able to attack their host population; however, as soon as the phages had killed P. aeruginosa, the numbers of viable C. albicans cells increased rapidly. Furthermore, C. albicans’ morphology and virulence were significantly affected in the presence of P. aeruginosa. In P. aeruginosa – A. baumannii biofilms, phages applied only to one of the hosts decreased, as expected, that specific population already after 6 h. Nevertheless, while after treatment of the mixed species biofilms with P. aeruginosa phages we observed a growth of A. baumannii, the same did not occur when the biofilms were only treated with the A. baumannii phage. The use of both phages was effective and reduced significantly the numbers of viable cells of the mixed population biofilm. Despite the potential of the phages used in this work as antimicrobial agents, it is well known that bacteria can quickly adapt and create new survival strategies and the emergence of phage-resistant phenotypes is inevitable. Indeed, we observed the rapid appearance of Pseudomonas aeruginosa resistant phenotypes following 24h of biofilm contact with phages and those phenotypes exhibited altered LPS structures. Thus, the combination therapy of phages and 4 commonly used antibiotics in the treatment of P. aeruginosa infections was also evaluated. The results obtained proved that certain antibiotics and phages have potentially more benefits and even act synergistically compared to just phages or antibiotics alone

Phage therapy as an alternative or complementary strategy to prevent and control biofilm-related infections

The complex heterogeneous structure of biofilms confers to bacteria an important survival strategy. Biofilms are frequently involved in many chronic infections in consequence of their low susceptibility to antibiotics as well as resistance to host defences. The increasing need of novel and effective treatments to target these complex structures has led to a growing interest on bacteriophages (phages) as a strategy for biofilm control and prevention. Phages can be used alone, as a cocktail to broaden the spectra of activity, or in combination with other antimicrobials to improve their efficacy. Here, we summarize the studies involving the use of phages for the treatment or prevention of bacterial biofilms, highlighting the biofilm features that can be tackled with phages or combined therapy approaches.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project PTDC/BBB-BSS/6471/2014, the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). This work was also supported by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

The MNN2 gene knockout modulates the antifungal resistance of biofilms of Candida glabrata

Candida glabrata biofilms are recognized to have high resistance to antifungals. In order to understand the effect of mannans in the resistance profile of C. glabrata mature biofilms, C. glabrata mnn2 was evaluated. Biofilm cell walls were analysed by confocal laser scanning microscopy (CLSM) and their susceptibility was assessed for fluconazole, amphotericin B, caspofungin, and micafungin. Crystal violet and Alcian Blue methods were performed to quantify the biomass and the mannans concentration in the biofilm cells and matrices, respectively. The concentration of -1,3 glucans was also measured. No visible differences were detected among cell walls of the strains, but the mutant had a high biomass reduction, after a drug stress. When compared with the reference strain, it was detected a decrease in the susceptibility of the biofilm cells and an increase of -1,3 glucans in the C. glabrata mnn2. The deletion of the MNN2 gene in C. glabrata induces biofilm matrix and cell wall variabilities that increase the resistance to the antifungal drug treatments. The rise of -1,3 glucans appears to have a role in this effect.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

Candida tropicalis biofilm and human epithelium invasion is highly influenced by environmental pH

Objective: The main goal of this study was to investigate the role of pH on Candida tropicalis virulence determinants, namely the ability to form biofilms and to colonize/invade reconstituted human vaginal epithelia. Methods: Biofilm formation was evaluated by enumeration of cultivable cells, total biomass quantification and structural analysis by scanning electron microscopy and confocal laser scanning microscopy. Candida tropicalis human vaginal epithelium colonization and invasiveness were examined qualitatively by epifluorescence microscopy and quantitatively by a novel quantitative real-time PCR protocol for Candida quantification in tissues. Results: The results revealed that environmental pH influences C. tropicalis biofilm formation as well as the colonization and potential to invade human epithelium with intensification at neutral and alkaline conditions compared to acidic conditions. Conclusions: For the first time, we have demonstrated that C. tropicalis biofilm formation and invasion is highly influenced by environmental pH.We would like to thank Mrs Lucília Goreti Pinto for processing and sectioning tissue samples. The authors acknowledge Fundação para a Ciência e Tecnologia (FCT), Portugal, for supporting Sonia Silva (SFRH/BPDT/111645/2015). This study was also supported by the Programa Operacional, Fatores de competitividade– COMPETE and by national funds through FCT –Fundação para a Ciência e a Tecnologia on the scope of the projects FCT PTDC/EBB-EBI/120495/2010 and RECI/EBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462).info:eu-repo/semantics/publishedVersio

Candida tropicalis biofilm is highly influenced by the environmental human pH

In the last decades, the increase of candidiasis has been accompanied by an intensification of infections caused by Candida tropicalis.Indeed, C. tropicalis has been described as able to colonize and infect several anatomically distinct sites, including the skin, gastrointestinal, genitourinary and respiratory tracts. Adaptation to diverse pH that exists in each human niche has been shown to be critical for virulence in many commensal pathogens, but there are no reports concerning C. tropicalis. Biofilm formation ability is one of the most important virulence factors that have important clinical repercussions due to its increased resistance to antifungal therapy. Thus, the aim of the current study was to characterize the influence of pH on C. tropicalis biofilm formation, structure and composition. The effect of pH (3, 4, 7 and 8) on C. tropicalis biofilms was evaluated by enumeration of culturable cells, total biomass quantification and matrix composition. Biofilm structure and the morphology of its cells were analysed through scanning electron microscopic and confocal laser microscopy. The resultsrevealed an intensification of C. tropicalis capacity to form biofilm at neutral and alkaline conditions, with an increased on number of culturable cells and total biomass and in its structural complexity, comparatively to acid conditions. For the first time, we have demonstrated that C. tropicalis biofilm formation is highly influenced by the environmental human pH, which has an important clinical impact, which may partly explain the increase incidence of candidiasis

Disclosing the complexity involved in phage-biofilm interaction: the case study of a Sep1virus phage infecting S. epidermidis

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms high tolerance to antibiotics, virulent bacteriophages have been suggested as novel anti-biofilm therapeutic agents. In this study, we used the S. epidermidis-specific phage phiIBB-SEP1 (SEP1) [1] and evaluated its activity against biofilms. Despite its broad host spectrum and high activity against exponential phase cells, the same was not observed for cells encased in a biofilm structure. To understand the underlying factors impairing SEP1 inefficacy against biofilms, we tested this phage against distinct bacterial populations. Interestingly, SEP1 was able to infect late stationary-phase (dormant), persister and biofilm-released cells, suggesting that the inefficacy for biofilm control resulted from the biofilm structure. To demonstrate this hypothesis, SEP1 activity was tested against clusters of cells from scraped biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells, after six hours of infection. Additionally, LIVE/DEAD staining allowed the observation that stationaryphase cells responded to phage addition, as determined by the increase in SYBR medium fluorescence intensity, which can be related with an increase on the cell metabolic activity. [...]info:eu-repo/semantics/publishedVersio

Microscopy techniques for the study of biofilm

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Bacteriophage biodistribution and infectivity from honeybee to bee larvae

Viruses of Microbes 2018 (EMBO Workshop) - Abstract Bookinfo:eu-repo/semantics/publishedVersio