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The shortage of antibiotics against drug-resistant Staphylococcus aureushas led to the development of new drugs targeting the elongation cycle of fatty acid (FA) synthesis that are progressing toward the clinic. An objection to the use of FA synthesis inhibitors is that S. aureuscan utilize exogenous FAs to construct its membrane, suggesting that the bacterium would bypass these therapeutics by utilizing host FAs instead. We developed a mass spectrometry workflow to determine the composition of the S. aureusmembrane at the infection site to directly address how S. aureususes host FAs. S. aureusstrains that cannot acquire host FAs are as effective in establishing an infection as the wild type, but strains that require the utilization of host FAs for growth were attenuated in the mouse thigh infection model. We find that S. aureusdoes utilize host FAs to construct its membrane, but host FAs do not replace the requirement for pentadecanoic acid, a branched-chain FA derived from isoleucine (or leucine) that predominantly occupies the 2 position of S. aureusphospholipids. The membrane phospholipid structure of S. aureusmutants that cannot utilize host FAs indicates the isoleucine is a scarce resource at the infection site. This reliance on the de novosynthesis of predominantly pentadecanoic acid that cannot be obtained from the host is one reason why drugs that target fatty acid synthesis are effective in treating S. aureusinfections.

ABSTRACTSerious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium(VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relAmutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relAmutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state.IMPORTANCEThe increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faeciumthat failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relAgene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci.