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

Abstract

The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to β-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 μg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 μg/ml) was also present with a frequency of ∼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 β-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.

Two Small (p)ppGpp Synthases in Staphylococcus aureus Mediate Tolerance against Cell Envelope Stress Conditions

“…This subclone had a growth defect that was presumably due to (p)ppGpp overexpression. When screened for repressor mutations that recovered normal growth and antibiotic-sensitive phenotypes, a single point mutation in relQ was revealed (30). These results demonstrate that (p)ppGpp molecules play an important role in the high-level resistance toward methicillin in MRSA strains that is most likely linked to the slow-growth phenotype imposed by (p)ppGpp.…”

“…(p)ppGpp molecules, as effectors of the stringent response, are molecules that contribute to the survival of bacteria under nonfavorable growth conditions. Previous studies revealed a contribution of (p)ppGpp to increased ␤-lactam resistance in E. coli and S. aureus (29,30). To further show this contribution, we analyzed the growth and survival of strains lacking rsh Syn , relP, and/or relQ under cell envelope stress conditions employed by vancomycin and ampicillin.…”

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

“…The genome of S. aureus also encodes two other monofunctional synthetases, RelP and RelQ, and transcription of these genes increases when cells are exposed to cell wall-targeting antimicrobials (20,21). Recent work on S. aureus has shown that the ability to switch on the stringent response is essential for its virulence and is required for the organism to cause chronic infections (22)(23)(24)(25).…”

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

“…For example, whole-genome sequencing analysis of bacterial isolates from a patient with recurrent S. aureus infections identified a single-nucleotide substitution in the rel gene that affected hydrolase activity and caused accumulation of (p)ppGpp (98). Similar observations have been made with laboratory strains, as increased (p)ppGpp production due to spontaneous point mutations in rel were observed in S. aureus populations that survived lethal doses of methicillin (87,88).…”

“…4). In particular, different laboratories have systematically shown that increased (p)ppGpp levels parallel observed increases in persistence (85)(86)(87)(88)(89). By coupling microfluidics with fluorescent reporter gene fusions in E. coli, the Gerdes group demonstrated that (p)ppGpp levels vary stochastically in exponentially growing cultures, and these investigators confirmed that persistence and (p)ppGpp levels are positively correlated at the single-cell level (89).…”