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DEVELOPMENT OF PHAGE COCKTAILS AGAINST BACTERIA ASSOCIATED TO HOSPITAL-ACQUIRED PNEUMONIA
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A pneumonia bacteriana hospitalar é uma infecção pulmonar aguda muito frequente em doentes internados nas unidades de cuidados intensivos e está associada a uma elevada mortalidade. O tratamento comum para estas infeções são os antibióticos; no entanto, as opções terapêuticas para as bactérias multirresistentes são cada vez mais escassas, especialmente para as Gram-negativas como Pseudomonas aeruginosa e Klebsiella pneumoniae. Por conseguinte, a procura de tratamentos alternativos é urgente e a terapia bacteriofágica tem vindo a despertar interesse como uma opção válida. Os bacteriófagos são vírus que infectam e se replicam exclusivamente em bactérias e têm demonstrado potencial terapêutico para o tratamento de infeções bacterianas.
O objetivo desta tese foi desenvolver duas novas preparações ou “cocktails” de bacteriófagos capazes de eliminar estirpes clinicamente relevantes de P. aeruginosa e K. pneumoniae, incluindo clones de elevado risco tais como os ST27 e ST235 (P. aeruginosa), e os ST11 e ST258 (K. pneumoniae). O trabalho envolveu o isolamento de centenas de bacteriófagos a partir de amostras ambientais, seguido de uma estratégia de selecção que incorporou resultados de estudos de gama de hospedeiros e de caracterização genómica dos fagos mais promissores. A atividade lítica dos bacteriófagos individuais e de cocktails foi avaliada in vitro contra células em crescimento planctónico. Os cocktails bacteriofágicos foram mais eficientes em retardar o crescimento de bactérias resistentes aos fagos, quando comparados com a ação individual dos bacteriófagos. A eficácia e citotoxicidade das suspensões fágicas administradas por aerossois foram avaliadas num modelo ex vivo de “precision cut lung slices” após infeção por P. aeruginosa ou K. pneumoniae. Os cocktails de bacteriófagos de P. aeruginosa e de K. pneumoniae demonstraram ter uma forte capacidade de redução da carga bacteriológica, bem como aptidão para recuperar a viabilidade das células de pulmão humano, após a infeção bacteriana.
Hospital-acquired bacterial pneumonia is an acute pulmonary infection highly frequent in intensive care unit settings and associated with increased mortality rates. The standard of care for these infections are antibiotics alone or in combinations; however, the therapeutic options for the multidrug-resistant bacteria are increasingly scarce, especially for Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Therefore, a search for alternative treatments is urgently necessary and bacteriophage therapy has been gaining interest as a valid option. Bacteriophages are viruses that infect exclusively bacteria and have demonstrated their potential for the treatment of bacterial infections. The purpose of this thesis was to develop two new phage cocktails capable of eliminating clinically relevant strains of P. aeruginosa and K. pneumoniae, including high-risk clones, such as ST27 and ST235 (P. aeruginosa), ST11 and ST258 (K. pneumoniae). The work involved the isolation of hundreds of phages from environmental samples, followed by a selection strategy that incorporated data from host-range studies and genome characterization of the most promising phages. The killing activity of the elaborated cocktails and of the individual phages composing them was evaluated in vitro against P. aeruginosa and K. pneumoniae strains under planktonic growth, together with the profiling of cells resistant to infection that emerged in each experiment. The cocktails were most effective in delaying the emergence of phage-resistant bacteria. The efficacy and safety of the phage cocktails delivered by aerosolization was assessed in an ex vivo model of P. aeruginosa or K. pneumoniae infection employing precision cut lung slices. The triple-phage cocktails developed against the two model bacteria demonstrated effectiveness in reducing the bacteriological load, and the ability to rescue human lung cell viability, after bacterial infection.
Hospital-acquired bacterial pneumonia is an acute pulmonary infection highly frequent in intensive care unit settings and associated with increased mortality rates. The standard of care for these infections are antibiotics alone or in combinations; however, the therapeutic options for the multidrug-resistant bacteria are increasingly scarce, especially for Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Therefore, a search for alternative treatments is urgently necessary and bacteriophage therapy has been gaining interest as a valid option. Bacteriophages are viruses that infect exclusively bacteria and have demonstrated their potential for the treatment of bacterial infections. The purpose of this thesis was to develop two new phage cocktails capable of eliminating clinically relevant strains of P. aeruginosa and K. pneumoniae, including high-risk clones, such as ST27 and ST235 (P. aeruginosa), ST11 and ST258 (K. pneumoniae). The work involved the isolation of hundreds of phages from environmental samples, followed by a selection strategy that incorporated data from host-range studies and genome characterization of the most promising phages. The killing activity of the elaborated cocktails and of the individual phages composing them was evaluated in vitro against P. aeruginosa and K. pneumoniae strains under planktonic growth, together with the profiling of cells resistant to infection that emerged in each experiment. The cocktails were most effective in delaying the emergence of phage-resistant bacteria. The efficacy and safety of the phage cocktails delivered by aerosolization was assessed in an ex vivo model of P. aeruginosa or K. pneumoniae infection employing precision cut lung slices. The triple-phage cocktails developed against the two model bacteria demonstrated effectiveness in reducing the bacteriological load, and the ability to rescue human lung cell viability, after bacterial infection.
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Hospital-acquired bacterial pneumonia Pseudomonas aeruginosa Klebsiella pneumoniae bacteriophage therapy