Whole-Genome Sequencing Reveals a Link Between β-Lactam Resistance and Synthetases of the Alarmone (p)ppGpp in Staphylococcus aureus

The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to beta-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance-often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 mg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 mu g/ml) was also present with a frequency of similar to 10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of beta-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.