Dissecting Direct and Indirect DNA Damage Pathways in Non-Growing Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major clinical challenge because susceptibility to antibiotic treatment depends not only on genetic resistance, but also on bacterial physiological state. While many antibiotics are highly effective against actively dividing cells, non-growing or stationary-phase populations often display substantial tolerance and can survive treatment despite lacking new resistance mutations. These persistent populations are thought to contribute to chronic, relapsing, and difficult-to-eradicate infections, highlighting the need to better understand which antibacterial mechanisms remain effective when bacterial growth is limited. DNA damage represents a particularly important mechanism to study in this context, since some antibiotics interact more directly with DNA itself, whereas others generate damage more indirectly through disruption of growth-associated cellular processes. This thesis examines how mechanistically distinct DNA-damaging antibiotics behave in non-growing MRSA, with a focus on distinguishing direct from indirect pathways of DNA damage under growth-limited conditions. Using complementary reporter-based and viability-based assays, I tested whether antibiotic exposure in non-growing cells induces measurable DNA damage responses and whether those responses correspond to reduced bacterial survival. The results show that non-growing MRSA remains susceptible to antibiotic challenge, but that the magnitude of killing and the character of the DNA damage response vary substantially across drugs. Together, these findings indicate that replication-independent DNA damage can remain an effective route to antibacterial activity in growth-limited cells, while also showing that DNA damage signaling and bacterial killing are not equivalent outcomes. More broadly, this work supports a nuanced view of antibiotic action in non-growing bacteria, in which both the distinction between direct and indirect DNA damage and the specific type of direct DNA lesion influence whether antibiotic exposure is translated into loss of viability.