Integração de Fagos em Sensores para a Determinação Rápida do Perfil de Infeções Nosocomiais do Trato Respiratório e Genes de Resistência Antimicrobiana

We intend to develop the next generation of rapid bacteriophages (phages) based and culture-independent diagnostic tools to identify the bacteria responsible for respiratory infection. We propose a new multi-detection biosensor, combining bacteria identification, quantification, and antimicrobial resistance gene analysis. To achieve this objective, it is necessary to be able to overcome a whole series of goals. The first objective is to find suitable phage mixtures for the bacteria found in clinical samples from the study population. The German group will provide phage cocktails optimized for bacteria based on the local population, but it may eventually be necessary to make some adjustments to their composition to be able to apply to more general clinical samples. The Portuguese hospital Pneumonology Department will provide patient samples, permitting optimize a more generic phage cocktail or, if intended, a specific cocktail for each patient case. The next objective is to immobilize the phages on magnetic particles and optimize the capture conditions. The major challenge in immobilizing phages on particles is to achieve their oriented immobilization, with the tail accessible to bind to bacteria, and the surface blocked to prevent non-specific capture of other bacteria or compounds. The next goal is developing a rapid screening test for various respiratory bacteria in sputum samples, combining phagomagnetic capture with solid-phase DNA isothermal amplification by RPA on disposable screen-printed gold electrodes. This first device should allow simultaneous detection of K. pneumoniae, P. aeruginosa and, A. baumannii. Once the pathogenic bacteria have been identified, it is worth identifying antimicrobial resistance genes. The strategy will be the same as the followed for identifying bacteria but including primers designed for the target multi-drug resistance sequence genes. Finally, a system could be designed in which identification and analysis of resistance genes can be carried out simultaneously. However, it is a gain of time but could be a waste of resources since it is unknown which bacteria are present in the sample. The final version of the developed biosensor must be an easy-to-use kit with minimal sample manipulation.