Enhanced Expression of dltABCD Is Associated with the Development of Daptomycin Nonsusceptibility in a Clinical Endocarditis Isolate of Staphylococcus aureus

Journal of Infectious Diseases, Dec 2009

Using isogenic clinical bloodstream Staphylococcus aureus strains from a patient with relapsing endocarditis, we investigated the transcriptional profiles of the mprF and dlt genes in the context of cell-surface charge and daptomycin nonsusceptibility. As in prior studies, a point mutation within mprF was observed in the daptomycin-nonsusceptible strain. However, neither the transcriptional profile of mprF nor the membrane phospholipid analyses were compatible with the anticipated mprF gain-in-function phenotype. In contrast, we demonstrated enhanced dlt expression coincident with increased positive surface charge and reduced daptomycin binding

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Enhanced Expression of dltABCD Is Associated with the Development of Daptomycin Nonsusceptibility in a Clinical Endocarditis Isolate of Staphylococcus aureus

Soo-Jin Yang 2 3 Barry N. Kreiswirth 2 4 5 George Sakoulas 1 2 Michael R. Yeaman 0 2 3 Yan Q. Xiong 0 2 3 Ayumi Sawa 2 3 Arnold S. Bayer 0 2 3 0 Geffen School of Medicine at UCLA , Los Angeles 1 Department of Medicine, Sharp Memorial Hospital , San Diego 2 Received 6 April 2009; accepted 28 July 2009; electronically published 9 November 2009. Potential conflicts of interest: G.S. has received research grant support from Cubist and Pfizer Pharmaceuticals ; consulting fees from Cubist, Ortho-McNeil, and Pfizer Pharmaceuticals ; and speaking fees from Cubist, Pfizer, and Wyeth Pharmaceuticals. All other authors report no potential conflicts. Financial support: National Institutes of Health (grant AI-39108 to A.S.B. and grant AI- 39001 to M.R.Y.) ; American Heart Association (grant 0465142Y to Y.Q.X.). UCLA, 1124 W Carson St, RB-2, Rm 230, Torrance, CA 90502 3 Los Angeles Biomedical Research Institute at Harbor-UCLA , Torrance 4 University of Medicine and Dentistry of New Jersey , Newark 5 Public Health Research Institute Using isogenic clinical bloodstream Staphylococcus aureus strains from a patient with relapsing endocarditis, we investigated the transcriptional profiles of the mprF and dlt genes in the context of cell-surface charge and daptomycin nonsusceptibility. As in prior studies, a point mutation within mprF was observed in the daptomycin-nonsusceptible strain. However, neither the transcriptional profile of mprF nor the membrane phospholipid analyses were compatible with the anticipated mprF gain-in-function phenotype. In contrast, we demonstrated enhanced dlt expression coincident with increased positive surface charge and reduced daptomycin binding. - a gain-in-function phenotype [1, 3]. There are 2 major functions of the mprF gene product in modifying organism net surface charge: (1) lysinylation of membrane phosphotidylglycerol (PG) to generate lysyl-PG (LPG) and (2) translocation of this positively charged phospholipid to the outer membrane leaflet. In addition to mprF, the dltABCD operon also contributes to the net positive surface charge by d-alanylating wall teichoic acids through distinct effector mechanisms [4]. Thus, greater net positive surface charge as mediated by mprF and/or dltABCD mechanisms would theoretically reduce the overall access of calcium-decorated daptomycin to its putative membrane target [4, 5]. In the present study, we examined an isogenic pair of clinical bloodstream S. aureus strains from a patient experiencing daptomycin therapy failure in which the relapse strain exhibited daptomycin nonsusceptibility in vitro. We focused our investigations on the transcriptional profiles of the mprF and dltABCD genes in the context of cell membrane surface charge and phospholipid profiles. These factors were hypothesized to affect interaction with and susceptibility to the calcium-decorated functional form of daptomycin, as well as susceptibility to innate cationic antimicrobial peptides (CAPs) involved in innate host defense [4, 6]. Methods. The S. aureus strains (BOY755 and BOY300) used in this study were methicillin-susceptible bloodstream isolates from a patient with prosthetic mitral valve endocarditis. BOY755 was the initial patient isolate, and BOY300 was subsequently isolated during daptomycin therapy and found to be nonsusceptible to daptomycin. Pulsed-field gel electrophoresis (PFGE) (with SmaI), agr sequencing (type 2), and spa typing (type 2) confirmed that these 2 isolates were clonal (data not shown). The minimum inhibitory concentrations (MICs) to daptomycin, as determined by standard micro-E-test, were 0.5 and 2 mg/mL for strains BOY755 and BOY300, respectively, using stationary-phase cells. The MICs to vancomycin were 1 mg/mL for both strains (the patient was not treated with vancomycin because of allergies to vancomycin and penicillin, although nafcillin therapy was eventually used after desensitization). Population analyses were performed with both daptomycin and vancomycin by standard protocols. Both strains exhibited equivalent growth curve kinetics and ultimate colony-forming unit (CFU) yields over a 24-h period (data not shown). S. aureus strains were grown in either tryptic soy broth (Difco Laboratories) or Mueller-Hinton broth (Difco Laboratories). To determine the impact of growth phasedependent dlt expression on daptomycin MICs, the above-described micro-Etest was used. For stationary-phase cells, overnight cultures were used. For exponential-phase cells, overnight cultures of S. aureus were diluted to an optical density of 0.1 (read at 600 nm) in fresh medium and incubated for 3 h at 37 C. All daptomycin (Cubist Pharmaceuticals) assays were done in the presence of 50 mg/mL calcium, as recommended by the manufacturer. Human neutrophil peptide 1 (hNP-1) from human polymorphonuclear leukocytes was purchased from Peptide International. The predominant CAP in mammalian platelets, thrombin-induced platelet microbicidal protein 1 (tPMP-1), was prepared (...truncated)


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Soo-Jin Yang, Barry N. Kreiswirth, George Sakoulas, Michael R. Yeaman, Yan Q. Xiong, Ayumi Sawa, Arnold S. Bayer. Enhanced Expression of dltABCD Is Associated with the Development of Daptomycin Nonsusceptibility in a Clinical Endocarditis Isolate of Staphylococcus aureus, Journal of Infectious Diseases, 2009, pp. 1916-1920, 200/12, DOI: 10.1086/648473