Genetic tools derived from staphylococcus aureus for biotechnological applications in gram-positive bacteria

Zusammenfassung

Staphylococcus aureus is a model organism whose ubiquity and adaptative capacity have prompted us to develop new genetic tools based on staphylococci for applications in Gram-positive bacteria. One of the main virulence factors Staphylococcus aureus possesses is its elevated biofilm formation capacity. Inside biofilms, bacteria exhibit spatial heterogeneity and superior tolerance to physicochemical insults and harsh reaction conditions, when compared to their planktonic counterparts. These properties that might have deleterious consequences in clinical settings, can be of great appeal in environmental biotechnology. Bacteria from the Rhodococcus genus present diverse metabolic activities of industrial interest, ranging from degradation of recalcitrant compounds to the synthesis of high-value metabolites. In Chapter I, as a proof of principle, we decided to promote biofilm formation in the environmental bacteria Rhodococcus erythropolis IGTS8, and, as a reporter of the metabolic activity, we chose the dibenzothiophene desulfurization process. The performance of the biofilm-forming strain was compared to that of the planktonic clone, obtaining increased dibenzothiophene desulfurization rates when Rhodococcus was structured as a biofilm. Engineered Rhododoccus cells showed a strong biofilm formation capacity under flow conditions which opens the possibility of exploring their use in the development of new industrial reactor systems such as fluidized bed reactors. Plasmids are main agents in the spread of antibiotic resistance genes. However little is known about the mechanisms governing plasmid maintenance and adaptation in the Staphylococcus genus. In Chapter II, we decided to explore the adaptation process of a clinically relevant strain to new plasmids carrying multiple resistance determinants. To easily manipulate plasmid content from a cell, a CRISPR-Cas9-based tool was generated and used to cure a plasmid-bearing isolate, which was then transformed with three plasmids from different origins. After fitness evaluation, the new plasmid-host combinations were subjected to experimental evolution and, following 35 days of uninterrupted growth, the evolved clones of the initial costly unstable derivatives were analysed for compensatory mutations. The results obtained showed the importance of insertion sequences in mediating plasmid rearrangements and fragment loss for mitigating plasmid cost. In the past few decades, extensive, and often indiscriminate, use of antimicrobial agents has led to a dramatic increase in the incidence of nosocomial infections caused by strains of Staphylococcus aureus that are resistant to multiple antibiotics. It does not only involve an increased economic burden to hospital setting but it is also a major health issue. With the aim to contribute to solving this problem, in Chapter III, we propose an alternative to the sole use of antibiotics for the treatment of Staphylococcus aureus-mediated infections. For that, we modified a native Staphylococcus aureus pathogenicity island to carry a CRISPR-Cas9 system targeting the chromosome of the bacteria. Several combinations of these synthetic elements with currently used antibiotics showed synergistic effects proving their potential use as versatile and effective anti-staphylococcal therapeutic agents.