Clinical treatment options for daptomycin (DAP)-resistant (DAP-R), methicillin-resistant Staphylococcus aureus (MRSA) infections are relatively limited. Current therapeutic strategies often take advantage of potential synergistic activity of DAP plus β-lactams; however, the mechanisms underlying their combinatorial efficacy are likely complex and remain incompletely understood. We recently showed that in vitro β-lactam passaging can resensitize DAP-R strains to a DAP-susceptible (DAP-S) phenotype. To further investigate the implications of selected β-lactam pretreatments on DAP plus β-lactam combination efficacy, we utilized DAP-R strain D712. We studied six such combinations, featuring β-lactams with a broad range of penicillin-binding protein-targeting profiles (PBP-1 to -4), using DAP-R strain D712. Of note, preconditioning with each β-lactam antibiotic (sequential exposures), followed by DAP exposure, yielded significantly enhanced in vitro activity compared to either DAP treatment alone or simultaneous exposures to both antibiotics. To explore the underpinnings of these outcomes, proteomic analyses were performed, with or without β-lactam preconditioning. Relative proteomic quantitation comparing β-lactam pretreatments (versus untreated controls) identified differential modulation of several well-known metabolic, cellular, and biosynthetic processes, i.e., the autolytic and riboflavin biosynthetic pathways. Moreover, these differential proteomic readouts with β-lactam preconditioning were not PBP target specific. Taken together, these studies suggest that the cellular response to β-lactam preconditioning in DAP-R MRSA leads to distinct and complex changes in the proteome that appear to resensitize such strains to DAP-mediated killing.

Objectives Daptomycin non-susceptibility in Staphylococcus aureus can emerge via the accumulation of single or multiple mutations, each resulting in a slight increase in the daptomycin MIC. The daptomycin-non-susceptible phenotype may include other features such as daptomycin tolerance. This study identifies S. aureus genomic regions that frequently develop mutations following prolonged daptomycin exposure but have not been previously associated with daptomycin non-susceptibility. Methods Sequence variations in the same eight loci independently observed following 28 day parallel serial passages of S. aureus J01 in daptomycin were introduced in isolation into S. aureus J01. MICs were determined by microbroth dilution. Daptomycin killing and tolerance were determined by kill curve analysis. Results Single mutations in snoF, hmp1, sspA, rimP, hepT, rsh, map1 and amaP had only a modest impact on the daptomycin MIC (≤2-fold). In contrast, individual mutation in several of these regions resulted in pronounced changes to daptomycin tolerance. Conclusions This study demonstrates that less characterized mutations in S. aureus following daptomycin exposure do not result in significant daptomycin susceptibility changes, but rather allow for enhanced survival characteristics during treatment. This sheds new light on genetic adaptations that may play a role in persistent infection. Further studies are needed to elucidate the prevalence of these mutations in clinical isolates.

Abstract Background Clinicians can employ suppressive antimicrobial therapy in patients with persistent or relapsing bacteremia. However, bacteria with favorable susceptibility profiles may exhibit antimicrobial tolerance wherein bacteria cannot proliferate yet can survive in high concentrations of antibiotics. The antimicrobial tolerance phenotype can thwart efforts to prevent bacteremia recurrence with prolonged exposure to antimicrobials and may contribute to breakthrough bacteremias while the patient is receiving active therapy. Here we present a patient case consisting of multiple episodes of breakthrough Staphylococcus aureus bacteremia over several years in the setting of appropriately dosed antimicrobial suppressive therapy and describe organism mutations that developed during therapy. Methods Six clinical bloodstream isolates were recovered from the patient during distinct episodes of MSSA bacteremia over a 5-year period. The identified source for each bacteremia was a central line infection (CLABSI). Isolates recovered were susceptible to the individual therapies received, which included oxacillin, daptomycin, and dalbavancin. Bacterial whole genome sequence data were collected using Illumina technology. Results The first two isolates (USA600) and the last four isolates (USA800) represent distinct populations and suggest that a distinct MSSA strain displaced the previous population between bacteremia episodes 2 and 3. Of note, all of these strains were able to survive and establish breakthrough bacteremias despite favorable susceptibility profiles to the agents used as suppressive therapy. Although the MICs remain low and in the susceptible range to oxacillin, daptomycin, and dalbavancin, these isolates progressively developed significant antimicrobial tolerance phenotypes, which coincided with mutations in walK (yycG), htrA2, ftsW, ebh and iarS that may be advantageous to survival under antibiotic pressure. Conclusion These genetic, phenotypic and patient case data identify important changes that can occur in bacterial populations over time that are distinct from antibiotic susceptibility. These findings point to factors that may result in breakthrough bacteremia, limiting the clinical utility of antimicrobial suppressive therapy. Disclosures W. Rose, Theravance: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Speaker honorarium; Merck: Grant Investigator, Research grant; The Medicines Company: Speaker’s Bureau, Speaker honorarium; Visante, Inc: Consultant, Consulting fee.