Reduced Susceptibility to Host-Defense Cationic Peptides and Daptomycin Coemerge in Methicillin-Resistant Staphylococcus aureus From Daptomycin-Naive Bacteremic Patients
Reduced Susceptibility to Host-Defense Cationic Peptides and Daptomycin Coemerge in Methicillin-Resistant Staphylococcus aureus From Daptomycin-Naive Bacteremic Patients
Nagendra N. Mishra 2
Arnold S. Bayer 1 2 5
Pamela A. Moise () 3
Michael R. Yeaman 0 1 2 5
George Sakoulas 4
0 Division of Molecular Medicine, Harbor-UCLA Medical Center , Torrance
1 Department of Medicine, Division of Infectious Diseases
2 Los Angeles Biomedical Research Institute
3 Department of Medical Affairs, Cubist Pharmaceuticals , Lexington, Massachusetts
4 UCSD School of Medicine , San Diego, California
5 Geffen School of Medicine at UCLA , Los Angeles
(See the editorial commentary by Kelley et al on pages 1153-6.) Background. We hypothesized that, for methicillin-resistant Staphylococcus aureus (MRSA), in vitro daptomycin susceptibility could be influenced by exposures to endogenous host defense peptides (HDPs) prior to clinical exposure to daptomycin. Methods. Two endovascular HDPs were used: thrombin-induced platelet microbicidal protein (tPMP) and human neutrophil defensin-1 (hNP-1) from neutrophils. Forty-seven unique MRSA isolates obtained from bacteremic patients in multicenter prospective clinical trials were studied. Clinical characteristics, microbiologic parameters, prior vancomycin therapy, and susceptibilities to tPMP, hNP-1, and daptomycin were compared using univariate and multivariate analyses. Results. All strains were daptomycin susceptible. Daptomycin minimum inhibitory concentrations (MICs) were inversely related to in vitro tPMP (but not hNP-1) killing. Strains with a daptomycin MIC of 1 mg/L exhibited significantly less killing by tPMP, compared with strains with an MIC of ≤ 0.5 mg/L. Prior vancomycin therapy did not influence this relationship. Regression tree modeling confirmed that reduced tPMP-induced killing in vitro was the strongest predictor of higher daptomycin MICs within the daptomycin-susceptible range. Conclusions. Among daptomycin-susceptible MRSA isolates from patients who had never received daptomycin, higher daptomycin MICs tracked with increased resistance to killing by platelet-derived but not neutrophilderived HDPs. These findings support the notion that endogenous exposure of MRSA to specific HDPs may play a role in selecting strains with an intrinsically higher daptomycin MIC phenotype.
Staphylococcus aureus presents significant clinical
challenges because of its rising prevalence of
antimicrobial resistance and its wide repertoire of virulence
factors that have coevolved with the human immune
]. The progressive development of
antimicrobial resistance has traditionally been thought
to be driven by the selection pressure of exposure of
microorganisms to exogenously administered
]. However, little attention has been paid to the
potential “priming” role(s) of exposures of such
organisms to endogenous cationic host defense peptide
(HDP) molecules in this regard. Recent data from our
laboratories have shown that methicillin-resistant
Staphylococcus aureus (MRSA) strains that, after failed
daptomycin therapy, developed in vitro resistance to
calcium-daptomycin, a cationic antimicrobial,
frequently exhibit “cross-resistance” to killing by prototypical
HDPs from platelets and neutrophils [
importance, preliminary data suggest that MRSA isolates
passaged through animal models with localized infections may
also demonstrate such cross-resistance in the absence of prior
daptomycin therapy [
In the current investigation, we examined the relationship
of in vitro susceptibilities to vancomycin and daptomycin in
the presence of 2 prototypical HDPs, one derived from
platelets and the other derived from neutrophils, that are thought
to be important in the innate immune response to S. aureus
endovascular infections [
]. We focused on: MRSA strains
from patients without endocarditis; in vitro susceptibility
profiles of daptomycin and HDP in strains from patients obtained
before daptomycin exposures; and potential impacts of recent
prior vancomycin treatments on daptomycin-HDP
susceptibility outcomes, given the putative link between prior
vancomycin exposure in vivo and in vitro and HDP resistance in
vitro . By using a previously well-characterized group of
MRSA isolates from bacteremic patients without endocarditis
who had never received daptomycin [
], we provide the
first evidence that endogenous exposures to cationic HDPs
may promote a “minimum inhibitory concentration (MIC)
creep” to calcium-daptomycin among bacteremic MRSA
isolates prior to daptomycin exposures.
Forty-seven well-characterized MRSA isolates were obtained
from bacteremic patients in 4 multicenter clinical trials
performed during 1998–2002 at multiple US hospitals, as
previously described [
]. For each study, appropriate ethical
regulations were followed and approval from the ethics
committee or institutional review board at each participating
institution was received. None of these clinical trials involved the
preclinical evaluation of daptomycin, and, given that all the
cases were obtained prior to the US Food and Drug
Administration approval of daptomycin in 2003, none of the patients
had ever received this agent. The strains studied represented
initial unique bloodstream isolates from different patients. To
maintain relative clinical homogeneity, patients with clinical
evidence of endocarditis, determined on the basis of standard
clinical and echocardiographic parameters, were excluded. The
strains were randomly selected for study by an investigator (G. S.)
who was blinded to clinical and microbiological data. The
clinical and demographic characteristics of the patients from
whom the MRSA isolated were obtained have been detailed
Prior studies from our laboratory and from other
laboratories have suggested a relationship between the genotype and
function of the accessory gene regulator (agr) locus and
outcomes of antibiotic therapy, particularly vancomycin [
Thus, for the current bacteremia isolates, multiplex
polymerase chain reaction (PCR) was used to determine agr
genotypes, as described previously [
]. In addition, a
semiquantitative δ-hemolysin–production assay was
performed to assess agr function on a scale of 0 to 4, relative to
production by agr group II prototype strain RN6607. A score
of 2 represented a δ-hemolysin phenotype comparable to that
of RN6607, while a score of 0 denoted no δ-hemolysin
activity, which is comparable to that of agr knockout RN9120. A
score of 1 denoted minimal δ-hemolysin production, a score
of 3 denoted production higher than that of RN6607, and a
score of 4 denoted production markedly higher than that of
RN6607 . Scoring of δ-hemolysin assays was performed by
one of the authors (G. S.), who was blinded to strain
identifications. Staphylococcal cassette chromosome mec (SCCmec)
genotyping was also performed using multiplex PCR.
Vancomycin and daptomycin MICs were determined in duplicate on
different days by broth microdilution, using Clinical and
Laboratory Standards Institute (CLSI) methods [
chloride supplementation (50 mg/L) was routinely used for
determining daptomycin MICs. Isolates were also screened for
the presence of heterogeneous vancomycin-intermediate
S. aureus (hVISA), using glycopeptide-resistance detection
Etest (AB BioMerieux).
HDP In Vitro Susceptibilities
Two prototypical HDPs felt to be involved in innate host
defense against S. aureus infections were selected for analysis.
These HDPs differ in source (neutrophils vs platelets), size,
structure, charge, and putative mechanisms of action [
The α-defensin human neutrophil defensin-1 (hNP-1) was
purchased from Peptide International (Louisville, KY). The
thrombin-induced platelet microbicidal protein (tPMP)
preparation was isolated from freshly collected, thrombin-stimulated
rabbit platelets, and its bioactive equivalency was determined
as described previously [
]. For HDP killing assays, each
MRSA isolate was grown to stationary phase (16–20 hours) in
Luria-Bertani broth, pelleted, and then washed in assay buffer
( phosphate-buffered saline for tPMP assays and protein
binding buffer for hNP-1 assays). The final HDP
concentrations chosen for study were 1 and 2 mg/L of tPMP and 10 and
20 mg/L of hNP-1. These concentrations were selected because
they caused <50% killing over the 2-hour assay period when
tested against several of the MRSA study isolates in extensive
pilot investigations. The initial bacterial inoculum was 103
colony-forming units/mL, as described previously . The
percentage of surviving bacteria (±SD) after 2 hours of
incubation at 37°C with HDPs of interest was calculated by
quantitative culture plating on blood agar plates. Results represent 3
separate experiments performed in duplicate on separate days.
Clinical and microbiological characteristics associated with
tPMP and hNP-1 susceptibility profiles were compared using
univariate and multivariate analyses. Continuous and ordinal
variables were compared using Kruskal-Wallis analysis of
variance and Mann-Whitney U tests. Categorical variables were
compared using χ2 tests or Fisher exact tests, when
appropriate. Correlation analysis was used to examine the relationships
between tPMP and hNP-1 susceptibilities. Multivariate
analyses to detect potential correlations among HDP and
daptomycin in vitro susceptibility patterns were performed using
classification and regression tree modeling and logistic
regression. Differences were considered to be statistically significant
at P values of < .05. All analyses were performed using Systat
11 software (Systat Software).
Patient Demographic Characteristics
Forty-seven initial MRSA isolates obtained before therapy
from 47 unique patients with MRSA bacteremia were studied.
Patient ages ranged from 24 to 87 years, with a median age of
70 years; 57% were male, and 43% were in an intensive care
unit at the onset of their infection. The source of bacteremia
was vascular-catheter related in 12 patients. In the remaining
patients, the bacteremia source was a soft-tissue abscess (for
17 patients), the lower respiratory tract (for 6), an
osteomyelitic site (for 3), a device (for 1), and an intra-abdominal site
(for 1); sources for 7 patients were unknown. Twenty-five
patients (53%) had received intravenous vancomycin therapy
≤30 days before the documented MRSA bacteremic episode.
In Vitro Susceptibilities to Vancomycin and Daptomycin
All isolates were susceptible to vancomycin and daptomycin,
with vancomycin MICs of 0.5 mg/L (for 10 isolates), 1.0 mg/L
(for 33), and 2.0 mg/L (for 4) and daptomycin MICs of 0.25
mg/L (for 4), 0.5 mg/L (for 31), and 1.0 mg/L (for 12). The
MICs obtained in the duplicate, separate-day experiments
were identical for each isolate. In total, only 6% of the isolates
(3 of 47) were categorized as hVISA by the
glycopeptideresistance detection Etest.
In Vitro Susceptibilities to HDPs
Table 1 shows the tPMP and hNP-1 susceptibility profiles in
the context of the presence or absence of an intravascular
catheter at the time of bacteremia detection and of recent
prior use of vancomycin. No significant relationship was
noted between the source of bacteremia or previous
vancomycin use with tPMP or hNP-1 susceptibility profiles.
A statistically significant relationship was noted between
tPMP and hNP-1 susceptibilities for the overall MRSA strain
]. Thus, bacterial survival in the in vitro assays for the 2
HDPs tracked together when 1 mg/L tPMP and 10 mg/L
hNP-1 were compared (P < .001) (Figure 1). Similar results
were seen when bacterial survival in 2 mg/L tPMP was
compared with survival in 20 mg/L hNP-1 (data not shown). The
relationship of tPMP and hNP-1 susceptibility profiles with 2
genotypic markers (agr type and SCCmec type) are displayed
in Table 2. The majority of strains (89%) were SCCmec type
II, with no statistically significant relationship between HDP
All Strains, No. (%) (n = 47)
susceptibility profiles and this genotypic marker. In contrast,
although representing a minority of isolates, agr type III
strains were significantly more resistant to killing by tPMP at
1 and 2 mg/L and by hNP-1 at 10 mg/L, compared with agr
type I or II isolates, when comparing mean survival data.
Virtually identical data were observed when comparing median
survival data sets (for 1 mg/L tPMP: 70% for group III, 39%
for group I, and 16% for group II [P = .002]; for 2 mg/L
tPMP: 67% for group III, 22% for group I, and 8% for group
II [P = .001]; and for 10 mg/L hNP-1: 53% for group III, 36%
for group I, and 34% for group II [P = .044]).
Relationship Between HDP Susceptibilities (tPMP and hNP-1) and Daptomycin MICs
Table 2 also shows that daptomycin MICs were significantly
related to tPMP susceptibility profiles (P = .007 for 1 mg/L
tPMP and P = .019 for 2 mg/L tPMP). Because tPMP killing
assays at 1 mg/L and 2 mg/L were highly correlated (correlation
coefficient, 0.920), only data from tPMP tested at 1 mg/L were
included in the multivariable model. By classification and
regression tree modeling, relative resistance to tPMP killing (1 mg/L)
was the strongest predictor of MRSA isolates with the highest
daptomycin MICs (1.0 mg/L), compared with strains with
daptomycin MICs of ≤ 0.5 mg/L (P < .001). Thus, when analyzed as
specific “groups” of strains with daptomycin MICs of 1.0 mg/L
as compared to those with daptomycin MICs of ≤0.5 mg/L, the
daptomycin MIC was significantly associated with tPMP-related
survival profiles (P = .009 for 1 mg/L tPMP and P = .033 for
2 mg/L tPMP) (Table 2 and Figure 2A). Higher daptomycin
MICs tracked with reduced killing by tPMPs, irrespective of the
δ-hemolysin profile (ie, agr functionality) (Figure 2B and 2C).
In contrast, there were no statistically significant relationships
identified between hNP-1 susceptibility profiles and daptomycin
MICs detected in these studies (eg, median survival in hNP-1
was 34% and 47% for strains with daptomycin MICs of ≤ 0.5
mg/L and 1.0 mg/L, respectively; P = .168).
Relationship Between HDP Susceptibilities and agr Function
As noted above, MRSA with agr group III genotype exhibited
decreased HDP susceptibility as compared to group I or II
isolates. The interrelationships among agr function, daptomycin
MICs, and tPMP susceptibilities were assessed by classification
and regression tree modeling. Among the 12 strains in which
agr function was preserved or enhanced as assessed by
δ-hemolysin +assay, higher daptomycin MICs tracked with
reduced tPMP-mediated (but not hNP-1–induced) killing
(P = .012; Figure 2C). A similar trend was noted for strains
with reduced or no agr function, although this outcome did
not quite reach statistical significance (P = .057). Thus, there
was no clear-cut impact or relationship of agr functionality
with daptomycin MICs or tPMP susceptibility profiles.
The virulence of S. aureus in a wide spectrum of clinical
settings is attributable to its elegant host-invasion machinery
plus its ability to evade the host’s innate defenses [
major component of the host’s innate defense system is the
repertoire of cationic HDPs contained within or elaborated by
a number of cell types, including neutrophils and platelets [
]. These latter HDPs are particularly relevant to
bloodstream infections. Our laboratories have previously
demonstrated in a number of experimental models and clinical
studies that S. aureus strains that exhibit reduced killing by
platelet-derived HDPs (eg, tPMPs) have a distinct survival
advantage in the context of bloodstream infections, including
catheter sepsis and endocarditis [
]. Genetic changes
accompanying in vitro– or in vivo–derived daptomycin
resistance in S. aureus have been implicated in resistance
to HDP-mediated killing [
]. In addition, our group
recently showed that MRSA strains from a broad range
of infectious syndromes in which in vitro daptomycin
resistance developed during such treatments were statistically
more likely to also exhibit “cross-resistance” to platelet- and
neutrophil-derived HDPs [
]. The mechanisms involved in
this cross-resistance phenotype have not been completely
delineated but include the apparent ability of the organism to
adapt to daptomycin exposures in vivo by increasing its
membrane fluidity, a key parameter previously associated with
resistance to killing by tPMPs [
]; by substantially
thickening its cell wall, a phenotype that may impede the transit
of cationic HDPs to the cell membrane by mechanical
interference or affinity trapping [
]; and by increasing
the relative surface positive charge, potentially reducing the
affinity of cationic molecules by a “charge-repulsion”
Similar to many endogenous HDPs, daptomycin contains a
significant peptide moiety that can be positively charged by
calcium decoration during in vivo use. Therefore, one
potential driver of such HDP-daptomycin cross-resistance
phenotypes may be the capacity of innate HDPs to impact
organisms before daptomycin therapy, facilitating increased
daptomycin MICs on subsequent daptomycin exposure. To
study this, we required a cadre of daptomycin-susceptible
strains that were relatively homogeneous in terms of their
recent clinical characterizations, microbiologic phenotype (ie,
MRSA only), clinical syndrome (ie, bacteremia without
endocarditis), and isolation from patients who had never received
prior daptomycin therapy (ie, daptomycin-naive individuals).
A number of interesting findings emerged from this study.
First, as expected, a substantial proportion of isolates came
from patients with indwelling intravascular catheters. Of
interest, there were no differences in HDP susceptibility profiles
between patients with and patients without intravascular
catheters. These data are consistent with prior studies from
our laboratories, in which the greatest differences in HDP
killing profiles were detected between S. aureus isolates from
patients with endocarditis versus those from patients with
either catheter-associated or soft tissue abscess–associated
]. This relationship is felt to reflect the capacity
of staphylococcal strains with reduced susceptibility to HDP
killing to seed, persist, and proliferate at sites of endovascular
damage, especially abnormal cardiac valves .
Second, a number of in vitro and clinical studies have
identified prior vancomycin exposures as an important potential
risk factor for development of increased daptomycin MICs
]. The mechanism by which this occurs is not well-defined
but may include increased cell wall thickness and/or reduced
autolysis phenotypes, as commonly exhibited by strains with
reduced susceptibility to vancomycin [
]. In the current
study, we detected no relationships between prior vancomycin
treatment and increased resistance to in vitro killing by the
prototypic HDPs tested. This inability to detect a vancomycin
impact may reflect the following differences between the past
and current investigations: the intervals between vancomycin
therapy and MRSA bacteremia, the duration of prior
vancomycin use, and/or the clinical syndrome studied (eg,
endocarditis vs nonendocarditis).
Third, the major finding from this study was that, even in
the absence of prior daptomycin therapy, modestly increased
daptomycin MICs in the study strain set were significantly
correlated with reduced capacity to kill the organism in vitro
by selected HDPs. Of note, although the tPMP and hNP-1
susceptibility profiles for individual organisms tracked
together, only the tPMP susceptibilities correlated with increases in
daptomycin MICs (ie, comparing isolates with daptomycin
MICs of 0.25–0.5 mg/L vs 1 mg/L). We hypothesize that the
principle HDPs to which these bacteremic organisms were
exposed were platelet derived, resulting in an adaptive
enhancement of resistance to tPMP killing. If so, these findings
would also suggest that platelet-mediated defenses are
critically important in protection against staphylococcal bacteremia
]. Alternatively, one could speculate that a certain
proportion of this bacteremic patient population was initially
colonized and infected by a cohort of isolates intrinsically more
resistant to tPMP-mediated killing. However, the studies
presented herein were not designed to determine such potential
cause-and-effect relationships. Nonetheless, it is remarkable
that HDP-daptomycin cross-resistance appeared to be
relatively HDP selective. In contrast, in our prior HDP-daptomycin
cross-resistance study , tPMP and hPN-1 resistance
statistically tracked together with increases in daptomycin MICs.
This latter difference may well be related to the presumed
manner in which the increased daptomycin MICs were
induced (ie, by exposure to HDPs in daptomycin-naive
patients in the current study vs prior exposures to daptomycin
in the previous study [
]). Another potential explanation for
the differences in outcomes between platelet-derived tPMPs
and neutrophil-derived hNP-1 may be the well-known
differences in their primary, secondary, and tertiary structures and/
or their distinct mechanisms of action [
Fourth, clinical correlations of agr genotype and function
with reduced susceptibilities to both cationic calcium–
daptomycin and HDPs have been inconsistent. In our recent
analysis of daptomycin-HDP cross-resistances, 7/10 MRSA
strains showing the cross-resistant phenotype were agr type
]. In another recent investigation from our laboratories of
36 MRSA strains [
], the vast majority were either agr types I
or II; there was no apparent linkage of agr type II with HDP in
vitro susceptibility profiles in this latter study. In contrast, we
recently found that tPMP resistance in vitro tracked with agr
type III and reduced agr functionality [
]. In the present study,
we also found a significant relationship between agr type III
and reduced killing by tPMP. In this regard, the recent finding
that S. aureus isolates of the clonal complex 30 genetic
background are highly associated with endocarditis is of particular
]. This clonal complex generally falls within the agr
group III genotype [
]. Given the link between tPMP
resistance and endocarditis [
], these data suggest a genotypic
predisposition of these strains to establish endovascular
infection. However, there was no clear interrelationship between
HDP profiles and agr functional status in the present
investigation. Thus, the relationship among agr types, agr function, and
HDP susceptibilities may well be strictly specific to strain or
clonal complex type; moreover, specific agr types and/or
functionality may merely be “biomarkers” for daptomycin-HDP
cross-resistance and not causal in this phenotype .
Last, although all MRSA strains in this study were within
the “daptomycin-susceptible” range, the significant correlation
of reduced in vitro killing by prototypical HDPs with a
modest daptomycin “MIC creep” (0.25–0.5 mg/L vs 1 mg/L) is
quite reminiscent of the “vancomycin creep” scenario. In this
latter circumstance, a 2-fold increase in vancomycin MICs
within the “susceptible” range (≤1 vs 2 mg/L) appears to
identify a S. aureus strain cohort (both methicillin-susceptible and
MRSA) with increased likelihood of suboptimal clinical
]. Whether MRSA strains with modestly
increased daptomycin MICs and reduced capacities to be killed
by HDPs (as in the current study) are associated with less
favorable treatment outcomes remains to be determined.
The results of our investigations have interpretive
limitations. For example, the in vitro HDP susceptibility testing was
performed in austere media, in the absence of host factors (eg,
serum proteins). Moreover, both neutrophils and platelets
contain a large cohort of HDPs that were not individually
tested in this study. Additional nonbloodstream HDPs (eg,
HDPs of cutaneous origin) were not explored, but future
investigations are planned. In addition, it is likely that
bloodstream bacteria are simultaneously exposed to multiple HDPs.
Finally, the concentrations of HDPs used in our in vitro assays
are undoubtedly lower than those encountered physiologically
by organisms in vivo and were chosen for their ability to
discriminate different populations of MRSA in our cohort.
Present investigations are in progress to further address the
phenomenon of daptomycin-HDP “cross-resistance” and to
adjudicate several of the above limitations. However, on the
basis of our current data, the development of cationic
antimicrobial peptides as novel therapeutics, designed on
endogenous antimicrobial templates, should be screened against
well-characterized cohorts of “antibiotic-naive” bloodstream
isolates for the potential endogenous priming phenomena.
Financial support. This work was supported by grants from the
National Institutes of Health (grant AI-39108 to A. S. B. and grants
AI39001 and AI-48031 to M. R. Y.). Journal publication charges were paid
by Cubist Pharmaceuticals.
Potential conflicts of interest. A. S. B. has received grant support from
Cubist, Astellas, Morphotek, and Lytix Pharmaceuticals. P. A. M. is an
employee and shareholder of Cubist Pharmaceuticals. M. R. Y. has
participated in research supported by Cubist and Pfizer. He is cofounder of
NovaDigm Therapeutics and ImmunoTx, both of which are involved in
development of novel anti-infective vaccines and therapeutics. G. S. has
received research grant support from Cubist Pharmaceuticals; speaking
honoraria from Cubist, Pfizer, Forest, and Astellas Pharmaceuticals; and
consulting fees from Cubist and Pfizer Pharmaceuticals. N. N. M. certifies
no potential conflicts of interest.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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