Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus

Abstract

Antibiotic tolerance is an underappreciated antibiotic escape strategy that is associated with recurrent and relapsing infections, as well as acting as a precursor to resistance. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting an elevated MIC. It is detected in time-kill assays as a lower rate of killing than a susceptible strain and can be quantified by the metric minimum duration for killing (MDK). The molecular mechanisms behind tolerance are varied, but activation of the stringent response (SR) via gene knockouts and/or chemical induction has long been associated with tolerance. More recently, two Gram-positive clinical isolates from persistent bacteremias were found to bear mutations in the SR controller, Rel, that caused elevated levels of the alarmone (p)ppGpp. Here, we show that introduction of either of these mutations into Staphylococcus aureus confers tolerance to five different classes of antibiotic as a result of (p)ppGpp-mediated growth defects (longer lag time and/or lower growth rate). The degree of tolerance is related to the severity of the growth defect and ranges from a 1.5- to 3.1-fold increase in MDK. Two classes of proposed SR inhibitor were unable to reverse or reduce this tolerance. Our findings reveal the significance of SR-activating mutations in terms of tolerance and clinical treatment failures. The panel of strains reported here provide a clinically relevant model of tolerance for further investigation of its link to resistance development, as well as potential validation of high-throughput tolerance screens.

Understanding tolerance to cell wall–active antibiotics

“…Accumulation of ppGpp results in massive transcriptional reprogramming of the cell, 176 and, most importantly, induces transient growth arrest 177–179 (this is the basis for the well‐established technique to isolate auxotrophic mutants using penicillin enrichment 180,181 ). Not surprisingly, the SR has been implicated in tolerance to CWA antibiotics in numerous studies with both Gram‐positive and ‐negative bacteria, 178,182–188 and in some cases, clinical mutants with enhanced tolerance due to background SR upregulation have been isolated 189 . In a particularly intriguing example, the ppGpp synthetases RelP and RelQ of S. aureus are induced by antibiotic exposure itself, resulting in high ampicillin and vancomycin tolerance 135 .…”

“…Not surprisingly, the SR has been implicated in tolerance to CWA antibiotics in numerous studies with both Gram‐positive and ‐negative bacteria, 178 , 182 , 183 , 184 , 185 , 186 , 187 , 188 and in some cases, clinical mutants with enhanced tolerance due to background SR upregulation have been isolated. 189 In a particularly intriguing example, the ppGpp synthetases RelP and RelQ of S. aureus are induced by antibiotic exposure itself, resulting in high ampicillin and vancomycin tolerance. 135 The more interesting question surrounding the SR and antibiotic tolerance, however, is whether or not growth arrest is the only effector of SR‐induced tolerance.…”

The alarmone (p)ppGpp confers tolerance to oxidative stress during the stationary phase by maintenance of redox and iron homeostasis in Staphylococcus aureus

“…In addition, (p)ppGpp conferred high level of beta lactam resistance in MRSA strains via increased expression of penicillin-binding proteins, encoded by mecA and pbpD [ 47 , 48 ]. Overproduction of (p)ppGpp due to rel mutations in clinical isolates resulted in increased tolerance to five different antibiotic classes [ 53 ].…”

Small Alarmone Synthetases RelP and RelQ of Staphylococcus aureus Are Involved in Biofilm Formation and Maintenance Under Cell Wall Stress Conditions

“…However, the persister assays were performed under relaxed conditions, and thus, the role of (p)ppGpp might have been missed. Nevertheless, (p)ppGpp synthesis was previously shown to contribute to antibiotic tolerance in S. aureus (Geiger et al, 2014;Bryson et al, 2020) and other pathogens (Nguyen et al, 2011;Bernier et al, 2013). Nguyen et al (2011) suggested that in Pseudomonas aeruginosa, the stringent response contributes to antimicrobial tolerance in biofilms by reducing oxidative stress.…”