Daptomycin is a cyclic lipopeptide antibiotic with activity against Gram-positive organisms that received approval from the United States Food and Drug Administration in September, 2003 [
1]. It is a concentration-dependent bactericidal antibiotic that acts by binding to and inserting into the bacterial cytoplasmic membrane resulting in rapid depolarization and deregulation of several cell functions such as DNA, RNA and protein synthesis [
2‐
4]. Daptomycin susceptibility in
Staphylococcus aureus is defined as a minimum inhibitory concentration (MIC) of ≤1 mg/L and any strain with an MIC >1 mg/L is considered daptomycin non-susceptible (DNS) [
5].
The development of DNS in
S. aureus laboratory studies, clinical trials, and post-marketing surveillance has been relatively low. Spontaneous mutagenesis in
S. aureus for DNS appears at a rate of less than 10
10 [
6].
Staphylococcus aureus with DNS can be obtained via extended serial passage with increasing daptomycin concentrations and via chemical mutagenesis. An in vitro model evaluated standard vancomycin and daptomycin dosing regimens against 5 clinical strains of
S. aureus that developed DNS in vivo [
7]. The DNS could only be replicated in vitro in 1/5 of these strains and with vancomycin but not daptomycin exposure. Interestingly, the DNS in this
S. aureus strain was unstable and reverted back to susceptible upon passage on antibiotic free media. Only 7 of 120 patients in the phase III trial for
S. aureus bacteremia and infective endocarditis trial developed isolates with DNS [
8]. Evaluation of 22,858
S. aureus isolated in North America from 2005 to 2010 revealed only 14 strains with a daptomycin MIC of ≥2 mg/L, and no trend indicating increasing MICs was noted [
9].
Daptomycin non-susceptibility in
S. aureus does not appear to be an all or nothing phenomenon, but instead a series of incremental changes that increase the MIC [
10‐
15]. To date, four main genetic changes (
mprF,
yycG,
rpoB/rpoC,
dltABCD) have been associated with increased MIC and DNS in
S. aureus. Mutations in or overexpression of the
mprF gene is commonly found in both laboratory derived and clinical DNS isolates [
11‐
14]. Increased quantities of the MprF protein, a lysylphosphatidylglycerol synthase, is thought to contribute to DNS by affecting the composition of the cell membrane via translocation of positively charged phospholipids to the outer side of the cytoplasmic membrane and by lysinylation of membrane phosphatidylglycerol (PG) to generate lysyl-PG (LPG) thereby increasing positive surface charge [
14‐
16]. Recently mutations in the phospholipid biosynthesis genes cardiolipin synthase (
cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3 phosphatidyltransferase (
pgsA) have been found in clinical DNS strains [
17]. Another altered protein sometimes found in DNS strains is YycG, which is one of two components of a response regulator system involved in the metabolism of the cytoplasmic membrane and cell wall [
11]. The proteins RpoB and RpoC, which comprise the β and β′ subunit of RNA polymerase, have also been found with amino acid substitutions in DNS
S. aureus strains [
11]. Recently, a single nucleotide polymorphism in
rpoB from a laboratory derived DNS
S. aureus was associated with decreased negative surface charge, increased cell wall thickness, and both vancomycin and daptomycin heteroresistance [
18]. Additionally, increased expression of the
dltABCD operon increases
d-alanylation of cell wall teichoic acids contributing to an increase in positive surface charge [
13]. Recent work has also suggested membrane proteins may augment the bactericidal effects of daptomycin, and alteration or loss of these proteins may contribute to DNS [
15]. It has also been proposed that changes in carotenoid biosynthesis in
S. aureus can increase membrane rigidity and contribute to increases in daptomycin MIC values [
19]. Overall, DNS
S. aureus strains show altered membrane potential, changes in membrane fluidity, increased positive membrane surface charge, and decreased membrane depolarization [
10‐
15]. It is hypothesized that the increase in cytoplasmic membrane surface charge repels the active daptomycin-Ca
2+ complex and therefore impedes interaction of daptomycin with the membrane [
10,
20]. There are likely other genetic changes that contribute to DNS in
S. aureus as strains exhibiting elevated MICs often have only some of the changes mentioned above [
21‐
24]. There is still much room for discovery of novel cell membrane and genetic changes in DNS strains of
S. aureus.
We have observed that some of the
S. aureus strains identified as DNS by the clinical microbiology laboratory at our institution using Microscan® (Dade Behring, Deerfield, IL, USA) were actually susceptible via broth microdilution following passage on antibiotic free agar or time being stored at −80 °C. This observation led us to question the stability of these isolates. Additionally, previous in vitro work we have done with DNS strains has demonstrated variable activity of daptomycin [
25,
26]. In some cases, daptomycin regimens of 10 mg/kg per day maintain antibacterial activity and led us to hypothesize that some
S. aureus strains with DNS may still be treatable with daptomycin. The objectives of this study were to evaluate the stability of DNS strains from the clinical microbiology laboratory and to evaluate the activity of daptomycin regimens against DNS
S. aureus strains with differing daptomycin population profiles.