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Abstract
Staphylococcus aureus, a normal commensal bacterium of the human skin and nares, is also an invasive pathogen, frequently causing acute skin and soft tissue infections, septic shock and various chronic infections such as osteomyelitis and lung infections in cystic fibrosis patients. Due to its capacity to survive on abiotic surfaces, S. aureus can be easily transmitted, making it a leading cause of nosocomial infections. This is particularly worrisome as many clinical isolates are resistant against commonly used antibiotics. These strains are collectively referred to as methicillin-resistant S. aureus or MRSA, for which the annual survival rate in infected patients is less than 50%. The multifactorial process that contributes to whether S. aureus causes mild, acute infections or life-threatening chronic infections is an ongoing research question. It has been proposed that one element contributing to the ability of S. aureus to persist in chronic infections is hypermutation, a phenomenon through which bacteria experience an increased mutation rate, often due to defects in DNA repair enzymes. Bacterial hypermutators are thought to increase the rate at which beneficial mutations are selected in the host microenvironment, thereby leading to more rapid pathoadaptation.
S. aureus has two main methods of DNA repair, their methyl-mismatch repair (MMR) and oxidized guanine (GO) systems, which scan hemi-methylated DNA for mismatches and remove oxidized guanine lesions from DNA, respectively. Our lab has recently characterized a set of S. aureus hypermutators, deficient in enzymes in both MMR and GO pathways. Here, we chose to focus on the MMR pathway, as mutants in this system are most frequently isolated from chronic infections in the clinical setting. We examined the ability of these hypermutator strains to survive more efficiently in the face of various selective pressures S. aureus might encounter in the host, such as antibiotic pressure, biofilm formation, exposure to competing bacteria, intracellular survival and reactive oxygen species (ROS). The most striking differences in survival between wild-type and hypermutators was in long-term co-culture with an immortalized lung epithelial cell line, which included increased recovery of colony forming units and the production of a population of heterogeneous colony morphologies of hypermutators. This could indicate the importance of studying hypermutators in the context of extended exposure to chronic conditions and emphasizes the need to carefully select appropriate in vitro models.
Upon examining the ability of hypermutators to withstand ROS using photodynamically induced oxidative species, we noted no differences in survival between wild-type and hypermutator strains. However, we did find an increase in surviving S. aureus colonies upon repeated exposure of the cells to antimicrobial photodynamic inactivation (PDI). PDI, most classically used as a tumor therapy, is a form of treatment that employs a photosensitive chemical (known as a photosensitizer), light, and molecular oxygen to create ROS to damage and ultimately kill targeted cells. It was very surprising to find a developing resistance to PDI upon repeat exposure, as this has been recorded in the literature as a phenomenon that does not occur. We have used RNA sequencing to explore potential mechanisms of resistance of S. aureus to PDI. Our findings highlight the need for a greater understanding of the underlying mechanisms of resistance to novel therapeutics against S. aureus, a very formidable pathogen.
