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)