Sean Kelly


INTRODUCTION - Mycobacterium abscessus (MAB) and Staphylococcus aureus (SA) are bacterial pathogens capable of causing a range of infections in humans, and pulmonary infections are significant amongst them. During pulmonary infection, alveolar macrophages (AMs), as sentinel immune cells of the airways, are key cells for the recognition of these invaders, and they are central to activating and orchestrating the host immune responses aimed at eliminating these pathogens. Consequently, these cells are also fundamental mediators of the exaggerated inflammatory responses that characterise infection-associated lung disease.MAB and SA are renowned for their multi-drug resistance and they both have an ability to cause aggressive and debilitating pulmonary disease. Both these pathogens have the ability to survive within host cells, and have evolved mechanisms to survive within macrophages by resisting killing mechanisms; therefore, in the lungs, the sentinel AMs are a probable target for infection. The ability of MAB and SA to reside intracellularly helps them to avoid elimination by the host immune system, and confers a barrier to effective antimicrobial treatment. Therefore, the development of novel antimicrobial treatments, which show efficacy towards inhibition of bacteria residing intracellularly within AMs, would be beneficial for treatment of their associated lung diseases. METHODS - In this project, an AM-like cell model called Max Planck Institute cells (MPIs) was used to investigate MAB-macrophage interactions in vitro. MPI cells were infected with phenotypically different strains of MAB. The intracellular survival and replication potential of the different strains was investigated, by lysing infected macrophages and estimating numbers of viable bacteria over time. Moreover, the inflammatory cytokine levels induced by the strains were measured and compared. The MPI cell model was also used to assess antimicrobial treatments against MAB and SA, namely for their ability to reduce the number of intracellular bacteria, by comparing viable bacteria recovered from the lysis of infected cells over time. The novel β-lactam/β-lactamase inhibitor combination amoxicillin/relebactam (AMX/REL) was tested against MAB, and the antimicrobial peptide epidermicin NI01 (NI01) against SA. RESULTS - In this project, MAB strains with different phenotypes, rough (R-MAB) and smooth (S-MAB), were both found to be associated with an increasing number of viable bacteria recovered from infected MPIs over time, but the R-MAB numbers were significantly greater across the infection duration. This study also showed that both phenotypes were able to induce the expression of pro-inflammatory cytokines, such as IL-6, IL-1β and MIP-1α, which underpin the characteristic inflammation of infection-associated disease, and that the levels of cytokines produced were generally more exaggerated following R-MAB infection. It was also shown that treatment of infected MPIs with novel antimicrobial therapies was able to reduce the number of viable bacteria recovered from the infected cells: AMX/REL treatment reducing MAB, and NI01 reducing SA. However, it was found that the concentration required to achieve intracellular inhibition was notably higher than that capable of inhibiting free bacteria in culture, and it is likely that the barrier to elimination imparted by intracellular localisation is integral to this effect. MAJOR FINDINGS - 1) Replication within macrophages has mainly been considered a feature of strains with the R-MAB phenotype. This project found that both R-MAB and S-MAB strains are capable of survival and replication within AM-like MPIs during in vitro infection, however, R-MAB strains appear to grow more aggressively. 2) There were differences with respect to intracellular replication and cytokine responses when MPI cells were infected with different S-MAB strains. Therefore, it is likely that there are differences not related to mechanisms of rough/smooth phenotype generation that influence MAB infection of macrophages. 3) Treatments that exhibit activity against MAB and SA during sensitivity testing show potential for reducing the intracellular numbers of these bacteria within infected AM-like MPIs. However, the concentration required to kill internalised bacteria is likely to be considerably higher. In addition, with respect to MAB, the effectiveness of such treatments appear to be phenotype/strain-dependent.

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