Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons
et al. (2014) Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-
Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons. PLoS ONE 9(9): e107426. doi:10.1371/journal.pone.0107426
Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons
Nagendra N. Mishra 0
Arnold S. Bayer 0
Christopher Weidenmaier 0
Timo Grau 0
Stefanie Wanner 0
Stefania Stefani 0
Viviana Cafiso 0
Taschia Bertuccio 0
Michael R. Yeaman 0
Cynthia C. Nast 0
Soo-Jin Yang 0
Franklin D. Lowy, Columbia University, College of Physicians and Surgeons, United States of America
0 1 Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, United States of America, 2 The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 3 Interfaculty Institute of Microbiology and Infection Medicine, University of Tu bingen , Tu bingen, Germany , 4 German Center for Infection Research (DZIF) , Tu bingen, Germany , 5 Department of Biomedical Sciences-Microbiology, University of Catania , Catania , Italy , 6 Division of Molecular Medicine, Harbor-UCLA Medical Center, Torrance, California, United States of America, 7 Cedars-Sinai Medical Center , Los Angeles, California , United States of America
Development of in vivo daptomycin resistance (DAP-R) among Staphylococcus aureus clinical isolates, in association with clinical treatment failures, has become a major therapeutic problem. This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three wellcharacterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases (i.e., dysregulation of dltA and mprF), (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains (i.e., increased fluidity) may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This research was supported in-part by grants from National Institutes of Health (AI-39108-15 to A.S.B and AI-111611 to MRY), the U.S. Department of
Defense (W81XWH-12-2-0101 to MRY), the American Heart Association (12BGIA11780035 to SJY), and the German Research Foundation (TR-SFB34 and SFB766 to
CW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Daptomycin (DAP) is a calcium-dependent lipopeptide
antibiotic with potent bactericidal effects against most Gram-positive
pathogens [1,2]. Since its release for use in 2003 for skin and soft
tissue infections, followed by approval for Staphylococcus aureus
bacteremia and right-sided endocarditis in 2006, DAP has become
a key antibiotic for invasive staphylococcal infections. This is
especially relevant to methicillin-resistant S. aureus (MRSA)
infections in the era of rising vancomycin MICs associated with
clinical treatment failures , as well as vancomycin
(VAN)intermediate S. aureus (VISA)-related infections [2,5,6]. Recently,
there have been an alarming number of reports of both S. aureus
and enterococcal DAP-resistant (DAP-R) strains emerging during
DAP treatment failures . The mechanisms by which such
organisms develop resistance to the microbicidal effects of DAP
are likely multi-modal and organism-dependent [9,1318]. Thus,
integrative genotypic and phenotypic profiles among DAP-R S.
aureus strains differ substantially from those defined among
DAPR enterococci [9,10,13,14,1618]. Moreover, although a principle
mechanism of DAP action appears to be perturbation of the
bacterial cell membrane (CM), key impacts upon cell wall (CW)
turnover processes seem to play a cardinal role in this regard [19
21]. Since DAP unambiguously requires calcium complexing to
execute its CM-targeting and subsequent bactericidal effects, this
agent acts similarly to cationic antimicrobial peptides involved in
innate host defenses (e.g., from PMNs and platelets [22,23]). The
range of potential phenotypic adaptations that may be associated
with staphylococcal DAP-R include: i) increased positive surface
charge (charge-repulsion hypothesis) [9,17,18,24]; ii) altered CM
fatty acid composition resulting in altered CM fluidity [14,25,26]
(membrane order hypothesis); iii) increased CM carotenoid
pigment content yielding very rigid CMs ; iv) a combination
of factors; for example, enhanced CM content of
positivelycharged phospholipids, as well as increased D-alanylation of CW
teichoic acid, resulting in reduced affinity of DAP to the CM target
; and v) augmented synthesis of CW teichoic acid, creating a
thickened CW phenotype and a putative mechanical barrier to
DAP penetration to reach its CM target [28,29].
The above phenotypic correlates of DAP-R in S. aureus point
to specific genes as potentially involved in this resistance process.
In this regard, the mprF operon (involved in lysinylation of CM
phosphatidylglycerol [PG] to synthesize lysyl-PG [L-PG], as well
as to translocate this latter positively-charged species to the outer
CM) and the dlt operon (responsible for the D-alanylation of CW
teichoic acid) have received particular attention [17,24,2833].
However, studies have varied widely in ascribing causality to these
two loci in DAP-R S. aureus strains, since a broad range of
genotypic correlates with the DAP-R phenotype have emerged,
including: i) acquisition of gain-in-function single nucleotide
polymorphisms (SNPs) in the mprF ORF [9,11,24,25,34]; ii)
over-expression of dlt genes ; and iii) dysregulation of both
mprF and dlt expression profiles [17,24,29]. Recently, Cafiso et al
 studied three clinical MRSA strain-sets in which DAP-R
evolved during treatment, and were able to pinpoint dltA
overexpression (especially in strains acquiring a concomitant
SNP in mprF) as potentially causal in the DAP-R phenotype.
This intriguing investigation was, however, somewhat limited, in
that within the DAP-S and DAP-R strain-sets: i) there was no
growth phase-dependent gene expression profiling performed; ii)
there were no phenotypic correlates of dlt and mprF
overexpression studied, especially surface charge measurements; iii)
specific phenotypic readouts of dlt and mprF over-expression were
not quantified (i.e., CW D-alanylation and CM L-PG synthesis
and/or translocation, respectively [25,28,32]); iv) other
parameters of CM physiology previously associated with DAP-R (e.g., CM
fluidity and fatty acid contents) were not queried [9,27,35]; and v)
assessment of DAP-R isolates for in vitro cross-resistance with
several prototypical host defense cationic peptides (HDPs)
involved in staphylococcal pathogenesis was not performed
[9,18,25,35]. The latter cross-resistance phenomenon has been
well-chronicled for other DAP-R S. aureus strains . The
current study, employing the same previously published DAP-S/
DAP-R strain-pairs , was designed to address these limitations,
and to further characterize potential mechanisms of DAP-R in
Methods and Materials
The three clinically-derived DAP-S/DAP-R MRSA strain-pairs
used in the current study (Table 1) have been described in detail
before . Briefly, the epidemiologically unrelated stran-pairs
were isolated from different patients, were from different clinical
infection types (skin and soft tissue; bacteremia), and were from
three different Italian hospitals. All three patients had received and
failed therapy with the glycopeptides antibiotic, teicoplanin, and
were subsequently treated with DAP . Each strain-pair
included an initial pre-DAP therapy DAP-S MRSA strain and
an isogenic DAP-R isolate obtained during DAP therapy.
Isogenicity of each strain-pair was confirmed by identical
outcomes of several discriminative genotypic assays within each
DAP-S and DAP-R strain-pair, including: pulse field gel
electrophoresis typing (PFGE), agr typing, multi-locus sequence typing
and staphylococcal casette chromosome (SCCmec) typing. The
three strain-pairs were previously characterized for mutations in
the mprF genes. Two of the three DAP-R variants within each
strain-pair exhibited a non-synonomous SNP in the mprF ORF
(Table 1), while the third strain-set did not. Of note, these two
SNPs occurred in previously described hotspots within the mprF
locus associated with MprF gains-in-function and the DAP-R
phenotype [9,32,34]. The above genotypic data for these three
strain-pairs have been previously published .
Oxacillin, vancomycin and DAP MICs were performed
according to CLSI guidelines . These data have been
previously reported (Table 1) .
The cytochrome c binding assay was performed as a surrogate
measure of the relative net positive surface charge of the
strainpairs as described previously [17,18,37]. Briefly, cells were grown
overnight in TSB media, washed with 20 mM MOPS buffer
(pH 7.0) three times and resuspended in the same buffer at
OD578 = 1.0. Cells were incubated with 0.5 mg/ml cytochrome c
for 10 minutes and the amount of cytochrome c remaining in the
supernatant was determined spectrophotometrically at OD530 nm.
The more unbound cytochrome c that was detected in the
supernatant, the more net positively charged the bacterial surface.
Data were converted and expressed as mean (6 SD) amount of
bound cytochrome c. At least three independent runs were
performed on separate days.
Wall teichoic acid (WTA) isolation and purification
S. aureus CW and WTA were specifically isolated as described
in detail before [39,40]. In brief, bacteria were grown overnight in
B-Medium (1% peptone, 0.5% yeast extract, 0.1% glucose, 0.5%
NaCl and 0.1% K2HPO4) containing 0.25% (wt/vol) glucose,
washed twice in sodium acetate buffer (20 mM, pH 4.7) and
disrupted in the same buffer with glass beads for 1 h on ice in a cell
disruptor (Euler). Protein-free CW was isolated, and WTA was
released by treatment with 5% trichloroacetic acid in sodium
acetate buffer for 4 h at 60uC. The CWs were removed by
centrifugation and WTA was quantified by determining its
inorganic phosphate (Pi) content as described before . The
isolation was performed in triplicate for each strain, and assayed in
triplicate for their respective Pi content.
Quantification of D-alanylated WTA content
The D-alanylation of the WTA polymers was assayed and
quantified as described before . In brief, D-alanine esters were
hydrolyzed by a mild alkaline hydrolysis carried out at 37uC for
1 h in 0.1 M NaOH. The supernatant was neutralized, dried
under vacuum, and used for pre-column derivatization with
Marfeys reagent (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide;
Sigma). Amino acid derivates (detection at 340 nm) were then
I I II II I I
separated as described before and analyzed with the ChemStation
software. Data were expressed as percent of WTA (6 SD) that was
D-alanylated. A minimum of three independent runs was
Cell wall (CW) thickness
The CW thickness of study strains were measured by
transmission electron microscopy (TEM; [25,26]). The mean
CW thickness (nm 6 SD) of 100 cells was determined for the
strains at a constant magnification of 190,0006 (JEOL, Model#
100CX, Tokyo, Japan) using digital image capture and
morphometric measurement (Advanced Microscopy Techniques v54,
Host defense peptides (HDPs)
Thrombin-induced platelet microbicidal proteins (tPMPs) were
obtained from thrombin-stimulated rabbit platelets as previously
described . This preparation contains several tPMPs, but
predominantly tPMP-1. The bioequivalency (activity in mg/ml) of
the tPMP preparation was determined as detailed before, using a
Bacillus subtilis bioassay . Purified human neutrophil
defensin1 (hNP-1) and LL-37 (prevalent in neutrophils and skin epithelium
[22,44,45]) were purchased from Peptides International
(Louisville, KY). RP-1 (a synthetic congener of the microbicidal domain
of the platelet factor-4 family of kinocidins) was synthesized as
previously detailed .
Susceptibilities to HDPs
For tPMPs and RP-1, a microtiter bactericidal assay was carried
out in minimal liquid nutrient medium (Eagles minimal essential
media [MEM]) in appropriate buffers ; the hNP-1 and LL-37
killing assays were performed in 1% BHI +10 mM potassium
phosphate buffer (PPB). A final bacterial inoculum of 103
stationary phase CFU was employed. The peptide concentrations
used in the 2 h killing assays were: 1.5 or 2.0 mg/ml bioactivity
equivalent for tPMPs; 5 or 10 for hNP-1; and 0.5 and 1 mg/ml for
RP-1 and LL-37. After extensive pilot studies, these peptide
concentrations were selected based on: (i) sub-lethality, with ,
50% reductions in counts of the parental DAP-susceptible (DAP-S)
strain; and (ii) encompassing peptide concentrations used in prior
investigations of HDP:S. aureus interactions . After 2 h
peptide exposure, samples were obtained and processed for
quantitative culture to evaluate the extent of killing by each
HDP condition. Final data were expressed as mean (6 SD)
percent survival rate. Since there is no bona fide resistance
breakpoint for HDPs, the mean percent survival (6 SD) was
statistically evaluated for potential correlates of HDP and DAP
susceptibility profiles. Data included a minimum of three
experiments performed on separate days.
CM phospholipid (PL) and amino-PL translocation
To investigate potential correlates between mprF
polymorphisms and CM features, PLs were extracted from study strains
under specific test conditions as described . The major CM
PLs of S. aureus (PG; L-PG and cardiolipin [CL]) were separated
by two-dimensional thin-layer chromatography (2-D TLC) using
Silica 60 F254 HPTLC plates (Merck). Fluorescamine labeling (a
fluorophore which binds only to positively charged PLs, such as
LPG, and which does not penetrate the outer CM leaflet), combined
with ninhydrin staining localization, was used within the 2-D TLC
plate assay to assess the translocation of L-PG between the
innerto-outer CM bilayer [13,17,26]. First-dimension
chloroformmethanol25% ammonium hydroxide (65:25:6, by volume) in the
vertical orientation and second-dimension
chloroform:water:methanol:glacial acetic acid:acetone (45:4:8:9:16, by volume) in
the horizontal orientation were used for the separation of the PLs
for further quantitation by phosphate estimation. PL identity was
confirmed using known PL standards. For quantitative analysis,
resulting isolated PLs (identified by iodine staining) were digested
at 180uC for 3 h with 0.3 ml 70% perchloric acid and the oxidized
derivatives quantified spectrophotometrically at OD660.
CM fatty acid composition
Given the impact of fatty acid composition on CM adaptability
to stress, the comparative fatty acid profiles of the DAP-S/DAP-R
strain-pairs were determined. Approximately 20 mg of bacterial
cells were harvested from late log phase growth preparations, and
then saponified, methylated, and fatty acid esters extracted into
hexane as described previously [17,26]. The resulting methyl ester
mixtures were separated by an Agilent 5890 dual-tower gas
chromatograph. Fatty acids were identified and quantified by a
microbial identification system (Sherlock 4.5; courtesy of
Microbial ID Inc., Newark, DE) [17,26].
CM fluidity was determined by fluorescence polarization
spectrofluorometry as detailed previously [9,25,26] using the
fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). An inverse
relationship exists between polarization indices and the degree of
CM order (i.e., lower polarization indices [PI value] denotes a
greater CM fluidity) [9,25,26]. To address day-to-day biological
variability inherent to CM dynamics, these assays were performed
a minimum of six times for each strain on separate days.
DNA isolation and targeted mprF, dltA, and dltB
Genomic DNA was isolated from S. aureus using the method of
Dyer and Iandolo . PCR amplification of the mprF ORF was
performed as we have previously described, using the primers,
(59-CCCGGATCCAATTAGAATTGATGTGAAAAAATG-39) and mprF-R-sph
PCR amplification of the dltA and dltB ORFs were performed as
we have described previously with primer pairs dltA-ORF-F
(59CAGTGGCGACACACACAATA-39) and dltA-ORF-R
(59TGGAACAATTGCCATTGACTT-39) and dltB-ORF-R
(59TCCAACTGTTTGGAAAGA ATCA-39), respectively . DNA
sequencing of the mprF and dlt genes was kindly performed at City of
Hope, Duarte, CA.
RNA isolation and qRT-PCR analysis for mprF and dltA
For RNA isolation, fresh overnight cultures of S. aureus strains
were used to inoculate NZY broth to an optical density at 600 nm
(OD600) of 0.1. Cells were harvested during both exponential
growth (2.5 h; OD = 0.5) and stationary phase (12 h). Total RNA
was isolated from the cell pellets by using the RNeasy kit (Qiagen,
Valencia, CA) and the FASTPREP FP120 instrument (BIO 101,
Vista, CA), according to the manufacturers recommended
Quantitative real time PCR assay was carried out as detailed
previously [29,48]. Briefly, 1 mg of DNase-treated RNA was
reverse transcribed using the SuperScript III first-strand synthesis
kit (Invitrogen) according to the manufacturers protocols.
Quantification of cDNA levels was performed following the
instructions of the Power SYBR green master mix kit (Applied
Biosystems) on an ABI PRISM 7000 sequence detection system
(Applied Biosystems) or on a LightCycler using the Quanti Fast
SYBR green real-time (RT)-PCR kit (Qiagen). The mprF, dltA,
and gyrB genes were detected using specific primers and
computational methods as described before [29,48].
HDP susceptibility profiles
As anticipated, the peptides exerted distinct efficacies against the
study strains (Table 2). RP-1 exhibited the greatest
anti-staphylococcal activity, including against stain 2C which exhibited high
level resistance to other peptides (Table 2). There was a general
trend for all HDPs tested, showing that the DAP-R isolate in each
strain-pair was significantly more resistant to peptide-mediated
killing than its DAP-S parental strain. This difference in HDP
killing profiles between DAP-S/DAP-R strain-pairs was most
dramatic for tPMPs and RP-1 of platelet origin, and LL-37 from
leukocytes, while being the least notable for hNP-1 (by far the least
active of the peptides against these study strains).
Surface charge (Figure 1)
Outcomes of relative positive surface charge comparisons
between DAP-S and DAP-R isolates varied substantially between
BCFAs = Branched chain fatty acids; UFAs = unsaturated fatty acids;
SFAs = Saturated fatty acids.
*P,0.05 vs. respective parental strain.
FA species (% of fatty acid composition SD)
% of total phospholipids SD
Abbreviations: LPG, lysyl-phosphatydylglycerol; PG, phosphatidylglycerol; CL, cardiolipin.
**P,0.01 vs respective DAP-S parental strains.
strain-pairs. For example, for strain-pair 1, the DAP-R isolate (1C)
exhibited a significantly more relative positive surface charge than
its parental DAP-S isolate (1A). In contrast, the 3A/3B strain pair
showed the exact opposite surface charge relationships; the DAP-S
strain 3A bound more cytochrome C than did strain 3C. The 2A/
2C strain set had virtually identical surface charge readouts.
Wall teichoic acid (WTA) content (Figure 2)
A significant increase in the amount of WTA in the CW of the
DAP-S/DAP-R strain-pair 2A/2C was detected, with the DAP-R
strain producing significantly more WTA than the respective
DAP-S strain (P,0.05). In contrast, in the strain-pairs 1A/1C and
3A/3B, no difference in WTA amount in the CWs of the
respective DAP-R strains was detected.
WTA D-alanylation (Figure 3)
In addition to the significant increase in overall WTA content in
the DAP-R strain 2C, there was also a substantial difference in the
proportion of WTA that was D-alanylated when comparing strain
2C to its isogenic DAP-S strain 2A. Interestingly, the percentage of
D-alanine contained within the WTA (nmol D-alanine/nmol Pi) of
the DAP-R strains that did not exhibit an increase in WTA
content (1C and 3B) was also significantly higher than that
observed in their respective DAP-S parental strains (1A and 3A).
These latter data speak to a clear-cut gain-in-function of the DltA
CM fatty acid profiles
Fatty acid compositional data for the three strain-pairs are
shown in Table 3 (a detailed Table enumerating each specific
fatty acid species is included as Table S1). In two of the three
strain-pairs (1A1C and 2A2C), fatty acid composition exhibited
a rather similar overall profile, including proportionality of
isoand ante-iso branched chain fatty acids, unsaturated fatty acids
(UFAs), and acyl chain-length profiles. Thus, the increases in CM
fluidity observed in the above two DAP-R S. aureus strains could
not be directly correlated to changes in the proportion of key fatty
acids known to impact CM fluidity; i.e, iso- vs anteiso branch
chain fatty acids, and saturated vs unsaturated fatty acids). In
contrast, in one DAP-R strain (3B), fatty acid analyses
demonstrated a significant increase in the proportion of anteiso-branch
chain species (vs iso-branch chain species) and decrease in
saturated fatty acids as compared to its respective parental
DAPS strain (3A) (P,0.05). In this strain-pair, the total proportions of
unsaturated fatty acids were not altered, although an additional
fatty acid species (18:2v6, 9c) was present in DAP-R strain 3B.
This increase in anteiso-branch chain species appeared to
correspond with a significant reduction in the major iso-branched
chain and saturated fatty acid (SFAs) species, respectively, in this
DAP-R strain (P,0.05). These proportionality perturbations in
this latter DAP-R strain provided a potential explanation for the
observed increment in CM fluidity in this latter strain (see below).
CM phospholipid (PL) content
Since mprF regulates the lysinylation of PG to yield L-PG, as
well as its subsequent outer CM translocation, we compared these
parameters in the three DAP-S/DAP-R strain pairs. As noted in
Table 4, in strain pairs 1A/1C and 2A/2C, the DAP-R isolates
contained significantly more L-PG than their respective DAP-S
parental strains. This outcome was associated with a significant
reduction in PG content in both DAP-R isolates. For the third
strain-pair (3A/3B), an opposite readout was observed, with the
DAP-R strain containing less L-PG than its DAP-S parental strain.
Of interest, the outer CM flipping profiles for L-PG did not differ
substantially in comparing any DAP-S strain to their respective
DAP-R isolates. Thus, the net CM L-PG profile of our study
strains reflected the rate of L-PG synthesis, rather than a difference
in the extent of CM PL flipping.
CM fluidity (Figure 4)
The polarization indices (PI values) for all three DAP-R isolates
were substantially lower than that of their respective DAP-S
parental strains, indicating that the CMs of these DAP-R isolates
were more fluid than their DAP-S counterparts. These differences
reached statistical significance for strain-pairs 2A2C and 3A3B.
mprF expression profiles
Figures 5A and 5B demonstrate the relative expression
profiles for mprF during exponential vs stationary growth phases
in these strain-pairs. During exponential growth, the DAP-R strain
1C showed ,4-fold increase in mprF expression vs its DAP-S
parental strain. Expression profiles for the other two strain pairs
did not demonstrate such increases among the DAP-R isolates. In
contrast, at stationary growth (when mprF expression is generally
minimal ), the two other DAP-R isolates (2C and 3B) exhibited
,3 and ,6-fold increases, respectively, in mprF expression as
compared to their DAP-S parental strains. Of note, this stationary
phase dysregulation of mprF expression in DAP-R strain, 3B,
dltA expression profiles
As shown in Figures 6A and 6B, growth phase-dependent
dltA expression profiles for the DAP-S vs DAP-R strain-pairs
showed remarkably similar metrics as for the mprF profiles above.
Thus, dltA expression was increased in DAP-R isolates as
compared to their DAP-S parental strain at either exponential
or stationary phases of growth.
dltA and dltB sequencing
Although a few nucleotide sequence polymorphisms were
observed within the dltA and dltB among the 3 pairs of the
strains, only the DAP-R 3B strain revealed one non-synonymous
SNP (T-to-C at 763) in the dltA gene as compared to its DAP-S
parental strain. This mutation yields a phenylalanine-to-leucine
(F255L) amino acid substitution (data not shown). Sequence
analyses of dltB in the three pairs revealed no differences between
the DAP-S and DAP-R pairs.
Recently, Cafiso et al  have provided further evidence for
the potential role of both the CW, and specifically the dlt operon
in the DAP-R phenotype. These investigators documented the
exponential growth-phase enhancement of dlt expression in three
DAP-R MRSA strains which emerged during DAP therapy, in the
presence or absence of mprF hot spot SNPs. These authors
proposed that dlt over-expression is a common pathway of
DAPR. Of note, their data regarding the potential role of dlt
overexpression and DAP-R mirrors recent findings from our
own laboratories [28,29]. However, the investigation of Cafiso et
al did not elucidate potential phenotypic or genotypic
mechanism(s) by which dlt over-expression could be causal in DAP-R.
The current study, utilizing these same DAP-S/DAP-R
strainpairs sought to extend and adjudicate the studies of Cafiso et al
. A number of interesting observations emerged from this
investigation. First, as has been seen previously in selected DAP-R
MSSA and MRSA strains [17,28,29,51], dltA expression is
frequently dysregulated, either by enhanced exponential phase
or increased stationary phase transcription profiles. Of interest,
dltA expression is generally quite minimal at stationary phase in
DAP-S S. aureus strains . Furthermore, despite these notable
differences in dltA transcription, only one of the three DAP-R
isolates exhibited a non-synonymous SNP in dltA. Phenotypically,
all three DAP-R strains demonstrated a significant increase in
their WTA D-alanylation content, reflecting DltA gains in
funtionality. Of interest, concomitant increases in CW thickness
were not observed in any of the three DAP-R strains (data not
Second, mprF expression profiles were remarkably similar to
those of dltA above in all three strain-pairs, whether or not hot
spot non-synonymous mprF SNPs were identified in the DAP-R
isolates. The observation that mprF transcriptional dysregulation
occurred despite the absence of such SNPs supports the notion
that regulatory genes outside of mprF can affect its expression
profile. As expected, phenotypically, in the two DAP-R strains
with mprF SNPs, enhanced amounts of L-PG synthesis were
detected as compared to their respective DAP-S parental isolates.
In the remaining DAP-R strain, despite overt mprF dysregulation,
L-PG synthesis was not increased. This finding is reminiscent of
data in selected MSSA DAP-R isolates [9,24].
Third, one prevailing concept that has been put forward to
explain the DAP-R phenotype in S. aureus has been perturbations
in relative cell surface positive charge, creating a charge
repulsion scenario against calcium complexed-DAP. However,
despite the increases in dltA transcription, D-alanylation of WTA
and L-PG synthesis among the three DAP-R strains, this did not
translate into consistent enhancement of surface positive charge in
these isolates. These data refute charge repulsion as a principal or
comprehensive explanation for the DAP-R phenotype in these
Fourth, all three DAP-R strains demonstrated substantial
increases in their CM fluidity properties as compared to their
respective DAP-S parental strains. Altered CM fluidity has been
closely linked to resistance to killing by cationic antimicrobial
peptides in general [9,19,25]. Such outcomes are presumed to
relate to perturbations in the ability of such peptides to interact
with and/or insert within target CMs. It is hypothesized that there
are biophysical and/or biochemical optima in the CM
microenvironments of S. aureus strains, making them more or less
susceptible to cognate host defense peptides (HDPs) with specific
structure-activity signatures. Thus, CMs that exhibit high degrees
of rigidity or fluidity are relatively resistant to interactions with
specific HDPs that likely rely most upon more intermediate
degrees of CM order [25,27]. The global impact of altered fluidity
in the current DAP-R vs DAP-S strains is underscored by the in
vitro cross-resistance of the DAP-R strains to cationic HDPs
which are structurally distinct from DAP, including prototypical
peptides from mammalian platelets and leukocytes. Such
crossresistance between DAP and HDPs has been well-chronicled in
recent studies amongst both staphylococci and enterococci
[16,25,34,52,53]. The mechanism(s) of enhanced fluidity in the
current three DAP-R strains is not entirely clear, although in one
DAP-R isolate the ratio of anteiso-branch chain fatty acids
(BCFAs) was significantly increased as compared to its DAP-S
parental strain. Such iso-to-anteiso-BCFA shifts have been
previously associated with increased CM fluidity properties in
Bacillus subtilis in response to cold shock .
Thus, in summary, the ultimate mechanism(s) of DAP-R in S.
aureus is highly likely to be multi-factorial and strain-specific.
Although over-expression and dysregulation of dltA transcription
may well be identifiable in selected DAP-R S. aureus strains and
correlated with increased WTA D-alanylation, its subsequent
potential phenotypic consequences (i.e., changes in relative surface
charge) were not sufficient to entirely explain the DAP-R
phenotype in these strains.
Distinct fatty acid (FA) compositions of strain pairs.
We thank Kuan-Tsen and Steven N. Ellison for excellent technical
assistance with peptide susceptibility testing and dlt operon sequencing; and
Danya Alvarez for cell membrane fluidity assays.
Conceived and designed the experiments: NNM ASB CW SJY. Performed
the experiments: NNM CW CCN SJY SW. Analyzed the data: NNM ASB
MRY CCN SJY. Contributed reagents/materials/analysis tools: SS VC
TB TG. Contributed to the writing of the manuscript: NNM ASB CW SS
1. Alborn WE Jr, Allen NE , Preston DA ( 1991 ) Daptomycin disrupts membrane potential in growing Staphylococcus aureus . Antimicrob Agents Chemother 35 : 2282 - 2287 .
2. Schriever CA , Fernandez C , Rodvold KA , Danziger LH ( 2005 ) Daptomycin: a novel cyclic lipopeptide antimicrobial . Am J Health Syst Pharm 62 : 1145 - 1158 .
3. Boucher HW , Sakoulas G ( 2007 ) Antimicrobial resistance: perspectives on daptomycin resistance, with emphasis on resistance in Staphylococcus aureus . Clin Infect Dis 45 : 601 - 608 .
4. Joson J , Grover C , Downer C , Pujar T , Heidari A ( 2011 ) Successful treatment of methicillin-resistant Staphylococcus aureus mitral valve endocarditis with sequential linezolid and telavancin monotherapy following daptomycin failure . J Antimicrob Chemother 66 : 2186 - 2188 .
5. Marco F , Garcia de la Maria C , Armero Y , Amat E , Soy D , et al. ( 2008 ) Daptomycin is effective in treatment of experimental endocarditis due to methicillin-resistant and glycopeptide-intermediate Staphylococcus aureus . Antimicrob Agents Chemother 52 : 2538 - 2543 .
6. Sakoulas G ( 2009 ) Clinical outcomes with daptomycin: a post-marketing, realworld evaluation . Clin Microbiol Infect Suppl : 11 - 16 .
7. Arias CA , Torres HA , Singh KV , Panesso D , Moore J , et al. ( 2007 ) Failure of daptomycin monotherapy for endocarditis caused by an Enterococcus faecium strain with vancomycin-resistant and vancomycin-susceptible subpopulations and evidence of in vivo loss of the vanA gene cluster . Clin Infect Dis 45 : 1343 - 1346 .
8. Hayden MK , Rezai K , Hayes RA , Lolans K , Quinn JP , et al. ( 2005 ) Development of daptomycin resistance in vivo in methicillin-resistant Staphylococcus aureus . J Clin Microbiol 43 : 5285 - 5287 .
9. Jones T , Yeaman MR , Sakoulas G , Yang SJ , Proctor RA , et al. ( 2008 ) Failures in clinical treatment of Staphylococcus aureus infection with daptomycin are associated with alterations in surface charge, membrane phospholipid asymmetry, and drug binding . Antimicrob Agents Chemother 52 : 269 - 278 .
10. Kelesidis T , Tewhey R , Humphries RM ( 2013 ) Evolution of high-level daptomycin resistance in Enterococcus faecium during daptomycin therapy is associated with limited mutations in the bacterial genome . J Antimicrob Chemother 68 : 1926 - 1928 .
11. Murthy MH , Olson ME , Wickert RE , Fey PD , Jalali Z ( 2008 ) Daptomycin nonsusceptible methicillin-resistant Staphylococcus aureus USA 300 isolate . J Medical Microbiol 57 : 1036 - 1038 .
12. Skiest DJ ( 2006 ) Treatment failure resulting from resistance of Staphylococcus aureus to daptomycin . J Clin Microbiol 44 : 655 - 656 .
13. Arias CA , Panesso D , McGrath DM , Qin X , Mojica MF , et al. ( 2011 ) Genetic basis for in vivo daptomycin resistance in enterococci . N Engl J Med 365 : 892 - 900 .
14. Mishra NN , Bayer AS ( 2013 ) Correlation of cell membrane lipid profiles with daptomycin resistance in methicillin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 57 : 1082 - 1085 .
15. Silverman JA , Perlmutter NG , Shapiro HM ( 2003 ) Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus . Antimicrob Agents Chemother 47 : 2538 - 2544 .
16. Tran TT , Panesso D , Mishra NN , Mileykovskaya E , Guan Z , et al. ( 2013 ) Daptomycin-resistant Enterococcus faecalis diverts the antibiotic molecule from the division septum and remodels cell membrane phospholipids . mBio 4.
17. Yang SJ , Kreiswirth BN , Sakoulas G , Yeaman MR , Xiong YQ , et al. ( 2009 ) Enhanced expression of dltABCD is associated with development of daptomycin nonsusceptibility in a clinical endocarditis isolate of Staphylococcus aureus . J Infect Dis 200 : 1916 - 1920 .
18. Yang S-J , Nast CC , Mishra NN , Yeaman MR , Fey PD , et al. ( 2010 ) Cell wall thickening is not a universal accompaniment of the daptomycin nonsusceptibility phenotype in Staphylococcus aureus: evidence for multiple resistance mechanisms . Antimicrob Agents Chemother 54 : 3079 - 3085 .
19. Bayer AS , Schneider T , Sahl H-G ( 2013 ) Mechanisms of daptomycin resistance in Staphylococcus aureus: role of the cell membrane and cell wall . Ann N Y Acad Sci 1277 : 139 - 158 .
20. Humphries RM , Pollett S , Sakoulas G ( 2013 ) A current perspective on daptomycin for the clinical microbiologist . Clin Microbiol Rev 26 : 759 - 780 .
21. Kaatz GW , Lundstrom TS , Seo SM ( 2006 ) Mechanisms of daptomycin resistance in Staphylococcus aureus . Int J Antimicrob Agents 28 : 280 - 287 .
22. Ganz T , Selsted ME , Lehrer RI ( 1990 ) Defensins. Eur J Haematol 44 : 1 - 8 .
23. Yeaman MR , Yount NY ( 2003 ) Mechanisms of antimicrobial peptide action and resistance . Pharmacol Rev 55 : 27 - 55 .
24. Yang SJ , Xiong YQ , Dunman PM , Schrenzel J , Francois P , et al. ( 2009 ) Regulation of mprF in daptomycin-nonsusceptible Staphylococcus aureus . Antimicrob Agents Chemother 53 : 2636 - 2637 .
25. Mishra NN , McKinnell J , Yeaman MR , Rubio A , Nast CC , et al. ( 2011 ) In vitro cross-resistance to daptomycin and host defense cationic antimicrobial peptides in clinical methicillin-resistant Staphylococcus aureus isolates . Antimicrob Agents Chemother 55 : 4012 - 4018 .
26. Mishra NN , Yang SJ , Sawa A , Rubio A , Nast CC , et al. ( 2009 ) Analysis of cell membrane characteristics of in vitro-selected daptomycin-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) . Antimicrob Agents Chemother 53 : 2312 - 2318 .
27. Mishra NN , Liu GY , Yeaman MR , Nast CC , Proctor RA , et al. ( 2011 ) Carotenoid-related alteration of cell membrane fluidity impacts Staphylococcus aureus susceptibility to host defense peptides . Antimicrob Agents Chemother 55 : 526 - 531 .
28. Bertsche U , Yang S-J , Kuehner D , Wanner S , Mishra NN , et al. ( 2013 ) Increased cell wall teichoic acid production and D-alanylation are common phenotypes among daptomycin-resistant methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates . PLoS ONE 8 : e67398 .
29. Bertsche U , Weidenmaier C , Kuehner D , Yang S-J , Baur S , et al. ( 2011 ) Correlation of daptomycin resistance in a clinical Staphylococcus aureus strain with increased cell wall teichoic acid production and D-alanylation . Antimicrob Agents Chemother 55 : 3922 - 3928 .
30. Baba T , Takeuchi F , Kuroda M , Yuzawa H , Aoki K-i , et al. ( 2002 ) Genome and virulence determinants of high virulence community-acquired MRSA . The Lancet 359 : 1819 - 1827 .
31. Cafiso V , Bertuccio T , Purrello S , Campanile F , Mammina C , et al. ( 2014 ) dltA overexpression: A strain-independent keystone of daptomycin resistance in methicillin-resistant Staphylococcus aureus . Int J Antimicrob Agents 43 : 26 - 31 .
32. Ernst C , Staubitz P , Mishra NN , Yang SJ , Hornig G , et al. ( 2009 ) The bacterial defensin resistance protein MprF consists of separable domains for lipid lysinylation and antimicrobial peptide repulsion . PLos Pathogens 5.
33. Weidenmaier C , Peschel A , Kempf VAJ , Lucindo N , Yeaman MR , et al. ( 2005 ) DltABCD- and MprF-mediated cell envelope modifications of Staphylococcus aureus confer resistance to platelet microbicidal proteins and contribute to virulence in a rabbit endocarditis model . Infect Immun 73 : 8033 - 8038 .
34. Yang S-J , Mishra NN , Rubio A , Bayer AS ( 2013 ) Causal role of single nucleotide polymorphisms within the mprF gene of Staphylococcus aureus in daptomycin resistance . Antimicrob Agents Chemother 57 : 5658 - 5664 .
35. Mishra NN , Yang S-J , Chen L , Muller C , Saleh-Mghir A , et al. ( 2013 ) Emergence of daptomycin resistance in daptomycin-nave rabbits with methicillin-resistant Staphylococcus aureus prosthetic joint infection is associated with resistance to host defense cationic peptides and mprF polymorphisms . PLoS One 8 : e71151 .
36. Clinical and Laboratory Standards Institute ( 2012 ) Performance standards for antimicrobial susceptibility testing; 20th informational supplement, M100-S22 . CLSI, Wayne, PA.
37. Yang S-J , Bayer AS , Mishra NN , Meehl M , Ledala N , et al. ( 2012 ) The Staphylococcus aureus two-component regulatory system, GraRS, senses and confers resistance to selected cationic antimicrobial peptides . Infect Immun 80 : 74 - 81 .
38. Keynan Y , Rubinstein E ( 2013 ) Staphylococcus aureus bacteremia, risk factors, complications, and management . Critical Care Clinics 29 : 547 - 562 .
39. Peschel A , Otto M , Jack RW , Kalbacher H , Jung G , et al. ( 1999 ) Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides . J Biol Chem 274 : 8405 - 8410 .
40. Weidenmaier C , Kokai-Kun JF , Kristian SA , Chanturiya T , Kalbacher H , et al. ( 2004 ) Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections . Nat Med 10 : 243 - 245 .
41. Kristian SA , Datta V , Weidenmaier C , Kansal R , Fedtke I , et al. ( 2005 ) Dalanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion . J Bacteriol 187 : 6719 - 6725 .
42. Cui L , Tominaga E , Neoh H-m, Hiramatsu K ( 2006 ) Correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycinintermediate Staphylococcus aureus . Antimicrob Agents Chemother 50 : 1079 - 1082 .
43. Yeaman MR , Puentes SM , Norman DC , Bayer AS ( 1992 ) Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein . Infect Immun 60 : 1202 - 1209 .
44. Sieprawska-Lupa M , Mydel P , Krawczyk K , Wojcik K , Puklo M , et al. ( 2004 ) Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureusderived proteinases . Antimicrob Agents Chemother 48 : 4673 - 4679 .
45. Yeaman MR , Bayer AS , Koo S-P , Foss W , Sullam PM ( 1998 ) Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action . J Clin Invest 101 : 178 - 187 .
46. Yeaman MR , Gank KD , Bayer AS , Brass EP ( 2002 ) Synthetic peptides that exert antimicrobial activities in whole blood and blood-derived matrices . Antimicrob Agents Chemother 46 : 3883 - 3891 .
47. Dyer DW , Iandolo JJ ( 1983 ) Rapid isolation of DNA from Staphylococcus aureus . Appl Environ Microbiol 46 : 283 - 285 .
48. Yang S-J , Xiong YQ , Yeaman MR , Bayles KW , Abdelhady W , et al. ( 2013 ) Role of the LytSR two-component regulatory system in adaptation to cationic antimicrobial peptides in Staphylococcus aureus . Antimicrob Agents Chemother 57 : 3875 - 3882 .
49. Kilelee E , Pokorny A , Yeaman MR , Bayer AS ( 2010 ) Lysyl-phosphatidylglycerol attenuates membrane perturbation rather than surface association of the cationic antimicrobial peptide 6W-RP-1 in a model membrane system: implications for daptomycin resistance . Antimicrob Agents Chemother 54 : 4476 - 4479 .
50. Pillai SK , Gold HS , Sakoulas G , Wennersten C , Moellering RC Jr, et al. ( 2007 ) Daptomycin nonsusceptibility in Staphylococcus aureus with reduced vancomycin susceptibility is independent of alterations in MprF . Antimicrob Agents Chemother 51 : 2223 - 2225 .
51. Center for Disease Control and Prevention ( 1999 ) Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus-Minnesota and North Dakota , 1997 - 1999 . Morb Wkly Rep 48 : 707 - 710 .
52. Mishra NN , Bayer AS , Moise PA , Yeaman MR , Sakoulas G ( 2012 ) Reduced susceptibility to host-defense cationic peptides and daptomycin coemerge in methicillin-resistant Staphylococcus aureus from daptomycin-naive bacteremic patients . J Infect Dis 206 : 1160 - 1167 .
53. Mishra NN , Bayer AS , Tran TT , Shamoo Y , Mileykovskaya E , et al. ( 2012 ) Daptomycin resistance in enterococci Is associated with distinct alterations of cell membrane phospholipid content . PLoS ONE 7 : e43958 .
54. Beranova J, Jemioa-Rzemin ska M , Elhottova D, Strzaka K , Konopasek I ( 2008 ) Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation . Biochim Biophys Acta 1778 : 445 - 453 .