Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection

Abstract

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.

Author Summary

The treatment of serious infections caused by Staphylococcus aureus is complicated by the development of antibiotic resistance, and in some cases the appearance of more persistent bacteria that have a reduced growth rate resulting in small colony variants (SCV). Here we have shown using whole genome sequencing and gene replacement experiments on sequential S. aureus isolates obtained from a patient with a serious bloodstream infection, how S. aureus evolved into a multi-antibiotic resistant, persistent and almost untreatable SCV. Specifically we show that a minor DNA change in a S. aureus gene encoding an enzyme called RelA causes an accumulation of a small signalling molecule called (p)ppGpp, which in turn leads to persistent activation of the important bacterial stress response known as the stringent response. This is the first report of the involvement of the stringent response in S. aureus SCV formation and its association with persistent infection. Additionally, we have uncovered a novel mechanism of resistance to the new antimicrobial linezolid, caused by a mutation in a gene encoding a 23S rRNA methyltransferase. This study highlights the exquisite adaptability of this important pathogen in the face of antimicrobial treatment.