Emergence of Daptomycin Resistance in Daptomycin-Naïve Rabbits with Methicillin-Resistant Staphylococcus aureus Prosthetic Joint Infection Is Associated with Resistance to Host Defense Cationic Peptides and mprF Polymorphisms
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(8): e71151. doi:10.1371/journal.pone.0071151
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
Nagendra N. Mishra 0
Soo-Jin Yang mailto: 0
Liang Chen 0
Claudette Muller 0
Azzam Saleh-Mghir 0
Sebastian Kuhn 0
Andreas Peschel 0
Michael R. Yeaman 0
Cynthia C. Nast 0
Barry N. Kreiswirth 0
Anne-Claude Cre mieux 0
Arnold S. Bayer 0
Karsten Becker, University Hospital Mu nster, Germany
0 1 Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-University of California at Los Angeles Medical Center, Torrance, California, United States of America, 2 David Geffen School of Medicine at University of California at Los Angeles , Los Angeles , California, United State of America, 3 Public Health Research Institute Tuberculosis Center , Newark , New Jersey, United States of America, 4 Bichat-Claude Bernard Hospital , Paris, France, 5 EA 3647 Versailles St-Quentin Univ. , R- Poincare Hospital , Garches , France , 6 Interfaculty Institute of Microbiology and Infection Medicine, University of Tuebingen , Tuebingen, Germany , 7 Division of Molecular Medicine, Harbor- University of California at Los Angeles Medical Center, Torrance, California, United States of America, 8 Cedars-Sinai Medical Center , Los Angeles, California , United States of America
Background: Previous studies of both clinically-derived and in vitro passage-derived daptomycin-resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R). Methods: In the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles. Results: In comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P,0.05); (ii) thicker cell walls (P,0.05); (iii) increased synthesis of CM lysyl-phosphatidylglycerol (L-PG); (iv) reduced content of CM phosphatidylglycerol (PG); and (v) SNPs within the mprF locus No significant differences were observed between parental or variant strains in outer CM content of L-PG, CM fluidity, CM fatty acid contents, surface charge, mprF expression profiles or MprF protein content. An isolate which underwent identical in vivo passage, but without evolving increased DAP MICs, retained parental phenotypes and genotype. Conclusions: These results suggest: i) DAP MIC increases may occur in the absence of DAP exposures in vivo and may be triggered by organism exposure to endogenous HDPs: and ii) gain-in-function SNPs in mprF may contribute to such HDPDAP cross-resistance phenotypes, although the mechanism of this relationship remains to be defined.
Funding: This research was supported by grants from the National Institutes of Health: AI-39108 (to ASB) and AI-39001 (to MRY); and from the German Research
Council: SFB766 (to AP). 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.
S. aureus is a prominent human pathogen which can cause severe
infections including endocarditis, septicemia, and osteomyelitis [1
3]. DAP is a lipopeptide antibiotic with potent activity against
Gram-positive bacteria, including multidrug-resistant S. aureus.
Evolution of DAP-R in Staphylococcus aureus during therapy is a
growing concern, especially in patients with bone and joint or
endovascular infections, when treated by DAP or vancomycin .
The emergence of DAP-R strains was also recently observed in a
post infection (days)
rabbit MRSA prothetic joint infection model , which closely
parallels similar post-operative infections in human . DAP-R
strains emerged in 6/10, and 3/12 rabbits treated with DAP or
vancomycin monotherapy, respectively [5,7]. Interestingly,
spontaneous emergence of MRSA with decreased susceptibility to DAP
was detected in 2/9 untreated control animals with joint infection.
This observation suggested that endogenous host factors
interacting with the microorganism during the course of infection could
promote the DAP-R phenotype.
We have previously documented in vitro cross-resistance
between DAP and cationic host defense peptides (HDPs) from
neutrophils and platelets in S. aureus isolates obtained from patients
failing DAP therapy [8,9]. This same cross-resistance
phenomenon has been seen in MRSA strains developing DAP-R following
serial in vitro passage in this agent . This concept was supported
by the observation that among 47 DAP-susceptible (S) MRSA
bloodstream isolates from DAP-nave patients, higher DAP MICs
(although still in the susceptible range) tracked with reduced
susceptibility in vitro to sublethal concentrations of platelet-derived,
but not PMN-derived HDPs . The mechanism(s) involved in
the emergence of such DAP-HDP cross-resistance are not
welldefined. The above MRSA strain-set isolated from rabbits with
prosthetic joint infections in the presence or absence of DAP
therapy provided a unique opportunity to study the influence of
endogenous exposures of S. aureus strains to specific HDPs, with
respect to in vivo evolution of the DAP-R phenotype.
Moreover, the current study included specific evaluation of
potential correlates of mprF point mutations with DAP-HDP
crossresistance phenotypes in MRSA strains from the above animal
model, focusing on: i) alterations in cell membrane (CM)
physiology and metabolism (surface charge; fatty acid content;
phospholipid profiles and phospholipic asymmetry; and fluidity);
ii) cell wall thickness; and iii) expression profiles, point mutations
and synthetic functions of mprF. The mprF gene product is of
particular relevance in this regard, as it is intimately involved in
maintaining bacterial cell surface charge, and has been previously
implicated in the DAP-R phenotype [4,810].
Although the official term for reduced susceptibility to
daptomycin is daptomycin nonsusceptibility, we will employ the
term daptomycin-resistance (DAP-R) for ease of presentation.
This work was presented in part at the 51st Interscience
Conference on Antimicrobial Agents and Chemotherapy,
Chicago, IL; USA, Sept 1720, 2011; abstract #C1-1775  .
Materials and Methods
The bacterial strains used in this study are listed in Table 1
(including their antibioitic susceptibility profiles, their mprF
mutations, if present, and their animal isolation descriptions).
Four MRSA strains were primarily used in this study, including: i)
a parental DAP-S strain used to induce prosthetic joint infection in
rabbits (L-271); and ii) three strains with increased DAP MICs
isolated from either DAP-treated or DAP-untreated rabbits (L-8
and L56; and L-16, respectively) . The two strains with
increased DAP MICs obtained from DAP-treated animals (L-8
and L-56) were isolated at 17 d post-infection, following 7 d of
DAP therapy . The one strain with increased DAP MICs in the
absence of DAP therapy was also obtained at sacrifice after 17d of
infection. The details of the animal model, including induction of
infection, DAP treatment regimens and therapeutic outcomes
have been recently detailed . For selected studies (especially in
which phenotypic or genotypic metrics differed between parental
L-271 vs L-8, L-16 and L-56 strains), an additional strain (L-76)
was employed. This isolate, obtained from bone cultures of a
DAP-untreated animal with prosthetic joint infection at 30 d
postinfection, maintained a near parental-level DAP MIC (0.38 mg/
Isogenicity of the above five study isolates was investigated by
profiling: i) PFGE patterns; ii) agr types; iii) spa types; iv) SCCmec
types; v) presence of pvl and acme genes; and vi) clonal complex
types (inferred from spa types). All assays were performed by
standard methodologies  .
The minimal inhibitory concentrations (MICs) of the strains to
DAP, vancomycin (VAN) and oxacillin (OX) were determined by
standard Etest (AB Biodisk, Dalvagen, Sweden) on Mueller
Hinton agar (MHA) plates, following the manufacturers protocol
[Difco Laboratories, Detroit, MI]. DAP-R was defined as an Etest
MIC of $2 mg/ml .
We performed DAP population analyses of the selected
strainset by standard protocols . Briefly, the study strains were grown
up overnight and the cells were washed with the PBS (phosphate
buffer saline), adjusted to OD600 nm at 1.00 (,109 CFU/ml).
Then, 10 ml of the bacterial suspension was added on
MuellerHilton broth agar plates containing DAP, ranging from 0.062 to
16 mg/ml to encompass the range of sublethal-to-lethal drug
levels. Colonies counts were quantified after 24 hour incubation at
37uC and the viable count plotted against DAP concentrations.
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 defensin-1
(hNP-1) was purchased from Peptides International (Louisville,
For tPMPs, a microtiter bactericidal assay was carried out in
minimal liquid nutrient medium (Eagles minimal essential media
[MEM]) in appropriate buffers ; the hNP-1 killing assay was
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 equivalent for tPMPs; and 5 or
10 mg/ml for hNP-1. After extensive pilot studies, these peptide
concentrations were selected based on: (i) sub-lethality, with
,50% reductions in counts of the parental DAP-susceptible
(DAPS) 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) per
cent surviving CFU/ml. 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.
tPMPs and hNP-1 passage study
For the tPMP passage study, the parental strain (271) was
cultured overnight in BHI medium. The initial inoculum of
,105 CFU/ml was exposed to 0.5 mg/ml equivalence of tPMPs in
MEM, and the strain passaged 15 times on successive days.
Surviving colonies after each days passage were stored at 280uC,
then used as the starting inoculum for the ensuing passage in
tPMP. After 15 d of such passage, the initial inoculum and tPMP
levels were both increased (,109 CFU/ml [OD600 = 1.00]; and
1 mg/ml equivalence of tPMPs, respectively) to increase selective
pressure for peptide-resistant clones. An additional 15 d serial
passage was then carried out. An identical protocol for hNP-1
passage was performed, employing this peptide at 10 mg/ml for
the entire passage period.
After the 30d passage periods, the comparative susceptibility
profiles for the pre-passage and post-passage isolate for tPMPs,
hNP-1 and DAP were performed. For tPMPs and hNP-1, the 2 hr
killing assay in MEM was used as detailed above . For DAP in
vitro susceptibility, the standard E-test MICs were determined as
To investigate the stability of any peptide-resistant phenotypes
that emerged during the 30 d passage period, the post-passage
strain was again passaged, but in antibiotic-free BHI medium; the
DAP, tPMPs and hNP-1 susceptibility profiles reassessed.
CM phospholipids (PLs) and aminophospholipid
Because of its role in lysyl-phosphotidylglycerol (L-PG) synthesis
and translocations, the functionality of the mprF locus has a major
impact on the relative proportions of the principle PLs contained
within the CM of S. aureus . To investigate potential correlates
between mprF polymorphisms and CM features, PLs were
extracted from study strains under all test conditions as described
[8,10]. 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 does not penetrate
the outer CM), combined with ninhydrin staining localization, was
used within the 2-D TLC plate assay to assess the translocation of
L-PG between the inner-to-outer CM bilayer [8,10].
Firstdimension chloroform-methanol25% ammonium hydroxide
(65:25:6, by volume) in the vertical orientation and
seconddimension 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. For quantitative analysis, isolated PLs were digested at
180uC for 3 h with 0.3 ml 70% perchloric acid and quantified
spectrophotometrically at OD660.
As a validation for the 2D-TLC assay above, we used an
adaptaption of our previously described annexin V-Ca++ assay
which measures binding to phosphatidyl serine accessible on the
outer CM [flipped] . This assay has been traditionally
utilized in eukaryotic systems to identify apoptotic reactions, due
to the ability of this fluorophore to bind to and detect outer CM
translocation of phosphotidylserine (a negatively-charged PL
species not present on the outer CM leaflet of S. aureus). We
modified this assay as an indirect measure of the relative content of
outer CM-translocated, positively-charged L-PG; i.e., the more
positively-charged L-PG that is translocated to the outer CM
leaflet, the less negatively-charged PL species are available for
annexin V-Ca++ binding . Briefly, S. aureus cells were
grown overnight in BHI broth. After centrifugation, the cell pellet
was washed twice and resuspended in binding buffer to
OD600 = 0.5 (,108 CFU/ml). Next, 5 ml of APC annexin V
(purchased from BD Biosciences; San Jose, CA) was added to the
cells, with gentle vortexing followed by incubated at room
temperature for 15 min in the darkness. Fluorescence was then
acquired for 10,000 cells by flow cytometry (FACScalibur) and
analyzed for surface-bound Annexin-V (excitation and emission
wavelengths = 650 nm and 660 nm, respectively). Data are
expressed in relative fluorescent units (parental strain set at 100%).
CM fatty acid composition
Given the impact of fatty acid composition on CM adaptability
to stress, the fatty acid profile of the parental vs animal passage
strains was 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 [8,10]. The resulting methyl ester mixtures
were separated by an Agilent 5890 dual-tower gas chromatograph.
Fatty acids were identified by a microbial identification system
(Sherlock 4.5; courtesy of Microbial ID Inc., Newark, DE) [8,10].
CM fluidity was determined by fluorescence polarization
spectrofluorometry as detailed previously  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 . To address biological variability
inherent to membrane dynamics, these assays were performed a
minimum of six times for each strain on separate days.
DNA isolation and targeted mprF sequencing
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
(59-CCCGCATGCAGCGCTTCAGG CATAACTGT-39) . DNA sequencing
of the mprF ORFs was kindly performed at City of Hope, Duarte,
RNA isolation and qRT-PCR analysis for mprF
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) 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 (q-RT-PCR) assay was carried out
as detailed previously [24,25]. 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 as described before
[24,25]. The dltA gene was included in these analyses as it also
contributes to surface charge maintenance in S. aureus .
Determination of mprF transcript half-lives
To determine mRNA stability, transcript synthesis was arrested
by the addition of 200 mg/ml rifampicin (Sigma; St. Louis, MO).
Aliquots were removed at 0, 1, 2, 3, and 5 min post-transcriptional
arrest and total RNA samples were isolated as described before
[26,27] Quantitative real time PCR (qRT-PCR) assays were
carried out as described above in triplicate, with 16S rRNA  as
an internal control. RNA half-lives were determined by linear
regression analysis of percent RNA remaining versus time.
Whole genome sequencing
As a complement to the targeted mprF sequencing above, it was
also important to obtain a more global comparative genomics
profile of the parental vs animal passage strains. Briefly, genomic
DNA was extracted using a Wizard genomic DNA purification kit
(Promega, Madison, WI), following treatment with 20 mg/ml
lysostaphin (Sigma-Aldrich, St. Louis, MO). The Genomic DNA
Sample Preparation Kit (Illumina, San Diego, CA) was used to
generate paired-end libraries. Fragments of the library were
sequenced using Genome Analyzer II (Illumina, San Diego, CA).
After trimming the reads for low quality bases, Illumina-sequence
reads were mapped against S. aureus USA300-FPR3757 genome
 with the Burrows-Wheeler Alignment Tool (BWA) . The
SAM file of BWA outputs was then converted to BAM file using
SAMtools . Single nucleotide polymorphisms (SNP) and short
indels (insertions and/or deletions) were called using the default
parameters for SAMtools mpileup utility, followed by bcftools and
the vcfutils.pl varFilter script
(samtools.sourceforge.net/mpileup.shtml) [31,32]. SNPs and InDels were annotated by an
inhouse Perlscript using USA300-FPR3757 genome as the reference
. The SNPs and indels were classified as coding region,
intergenic region and RNA sequences according to the positions.
SNPs in the coding sequences were annotated as synonymous or
non-synonymous amino acid substitutions. The sequences
reported in this study have been deposited in the National Center for
Biotechnology Information (NCBI) Sequence Read and Archive
(SRA) database (accession no. SRP025984).
MprF protein content
To correlate mprF gene polymorphisms with quantity of MprF
protein, we determined the relative amounts of MprF protein
produced by the parental or in vivo passage variants in relation to
changes in DAP MICs. Flow cytometry employing a translational
plasmid-based green fluorescent protein (GFP) reporter system was
used. The plasmid employed was pCX-mprF-sfGFP, a derivative
of pCX19 , containing the gene coding for super-folder (sf)
GFP with staphylococcal codon optimization  fused in-frame
to the 39 end of mprF under control of the xylose-inducible xyl
promoter. A 2558-bp DNA fragment including the mprF
ribosomal binding site and coding region was amplified from
plasmid pRB474-mprF  by PCR with primers replacing the
stop codon with a PvuI restriction site in a way that allowed an
inframe fusion with sfGFP gene (forward primer:
59-CATCGAATTATAGGAATAGAGCAAA CAAGC-39; reverse primer:
59GGCCGATCGTTTGTGACGTATTACACGCATTACTTTAG-39). The resulting plasmid was used to transform S. aureus
271, L8, and L16, and sfGFP-mediated fluorescence intensities
were compared. In brief, for the flow cytometry assays, S. aureus
strains containing the plasmid construct expressing the
xyloseinducible MprF-GFP fusion protein were grown overnight in
Mueller-Hinton broth (MHB) and cultures were diluted to OD600
of 0.1 into 5 ml of fresh media which was either xylose-free or
containing 0.25% xylose (wt/vol) and incubated with agitation
(200 rpm at 37uC for 24 hr). Fluorescence was quantified for
10,000 cell using excitation and emission parameters of 485 nm
and 525625 nm respectively. Mean channel fluorescence units (6
SD) were calculated from three separate analyses performed
The relative positive surface charge of the staphylococcal
envelope has been shown to correlate with susceptibility to killing
by a variety of cationic HDPs . Of note, two of our target
genesof-interest queried for expression profiles in this investigation
(mprF; dlt see below) significantly contribute to surface positive
charge maintenance [35,36]. The cytochrome c binding assay was
performed as surrogate measure of the relative net positive surface
charge of the strain-set as described previously [36,37]. Briefly,
cells were grown overnight in BHI media, washed with 20 mM
MOPS buffer (pH 7.0) 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 positively charged the bacterial surface.
Data were converted and expressed as mean (6 SD) amount of
unbound cytochrome c. At least three independent runs were
performed on separate days.
Cell wall thickness
Resistance in vitro to DAP in S. aureus is frequently correlated
with a thickened cell wall phenotype reminiscent of VISA strains
. Therefore, cell wall thickness of study strains were, compared
by transmission electron microscopy [TEM; 9,10]. The mean
thickness (nm 6 SD) of 100 cells was determined for the strain-set
at a constant magnification of 190,0006 (JEOL, Model# 100CX,
Tokyo, Japan) using digital image capture and morphometric
measurement (Advanced Microscopy Techniques v54, Danvers,
The two-tailed Student T-test was used for statistical analysis of
all quantitative data. P values of #0.05 were considered
DAP, VAN and OX MICs are shown in Table 1. For the two
in vivo-derived isolates with increased DAP MICs following DAP
therapy (L8 and L56), DAP MICs increased 16-fold as compared
to the parental strain (271), reaching the DAP-R breakpoint of
2 mg/ml. Of note, the DAP MIC of the post-infection isolate from
the animal unexposed to DAP therapy (L16) increased 6-fold as
compared to the parental strain, although not reaching the DAP-R
breakpoint above. Interestingly, a reduction in OX MICs from 2
6-fold was observed in all three post-infection strains, representing
the so-called OX-DAP see-saw effect . All VAN MICs
were in the VISA range . A 30d post-infection,
DAPunexposed control isolate (L76) maintained parental-equivalent
MICs to all study antibiotics.
Comparative genotyping confirmed that the parental strain
(L271), the three in vivo animal passage variant strains with
increased DAP MICs (L8, L16, L56), as well as the control animal
passage isolate (L76) were of identical spa type (YHGFMBQBLO;
type 1), inferred clonal complex type , SCCmec type IV and agr
type I, and were both pvl- and acme-negative. All isolates were
PFGE-identical (data not shown). These data strongly suggested
isogenicity among the strain-set.
As noted in Figure 1, the DAP population curves of the two
variant strains with increased DAP MICs paralleled these strains
differences in MICs. Thus, for both the L8 and L16 isolates, there
was a notable shift of the population analysis curves to the right,
with the L8 strain curve being substantially more shifted than that
for the L16 strain. Of note, there were no DAP hetero-resistant
subpopulations detected for either the L8 or L16 isolates.
Among the study strains, both DAP-treated and DAP-untreated
in vivo strains with increased DAP MICs exhibited reduced
susceptibility to killing by HDPs as compared to the parental strain
(Table 2). These differences were most substantive in comparing
the two DAP-treated isolates with the parental strain. Of note, the
strain that maintained near parental-level DAP MICs post-in vivo
passage (L76) remained highly susceptible to killing by study HDPs
(data not shown). All HDP susceptibility profiles were stable on
multiple passages in nutrient media in vitro (data not shown).
HDP passage and stability studies
After 30d passage of the parental DAP-S isolate (271) in
sublethal concentration of tPMPs, the DAP MIC had increased
substantially into the DAP-R range (2 mg/ml) (Table 3). Of
% survival of a 103 inoculum after 2 hr peptide exposures
*P -value , 0.05 vs parental strain.
Mean % survival ( SD) after 2-h exposure to:
Post-passage in tPMP- free medium
*P,0.05 vs 271 parental strain pre-passage data; ND not determined.
interest, passage in sublethal hNP-1 did not induce a similar
increase in DAP MICs. Further, as shown in Table 3, passage in
sublethal tPMPs resulted in significant increases in survivability of
post-passage isolates to killing by both tPMPs and
higherconcentration (but not lower-concentration) hNP-1. Importantly,
after serial passage in drug-free media for 5 consecutive days, both
the tPMP-passage and hNP-1 passage isolates had reverted to the
parental phenotypes in terms of their DAP MICs and HDP
survival profiles (data not shown), indicating that these post-HDP
passage phenotypes were unstable.
CM PL composition and aminophospholipid asymmetry
Negatively-charged PG was the predominant CM PL in all four
principle study strains (Table 4). The proportions of the
negatively-charged species, CL, were low, and similar among the
strains. The proportions of PG were significantly lower in both
DAP-treated and DAP-untreated in vivo passaged strains exhibiting
increased DAP MICs, as compared to the parental strain
(P,0.00001). Interestingly, the reduction in PG proportionality
was related to a relative increase in amounts of the
positivelycharged species, L-PG, in these latter isolates. Thus, total L-PG
levels were more than 2-fold higher in these DAP-treated and
DAP-untreated strains as compared to the parental strain
(P,0.000001), suggesting adaptive gains-in-function via the mprF
locus (i.e., enhanced L-PG synthesis; [810,35]). Of note, the
proportion of L-PG that was accessible on the outer CM leaflet
was approximately 47% of total L-PG in all four strains,
suggesting similar levels of MprF translocase (flippase) activity
among the strains (Table 4). This theme is supported by the
annexinV flow cytometry analyses, in which annexinV-Ca++
binding was similar in all four study strains (% relative fluorescent
units ranging from 57.2560.47 to 63.5963.24). Importantly, the
L-PG profile (i.e., overall proportionality; translocation; etc) of the
CM fatty acid composition; CM fluidity
The parental strain, as well as the three in vivo passaged strains
with increased DAP MICs showed a similar CM fatty acid pattern
in terms of iso- and anteiso- (branched chain) fatty acids (BCFA),
as well as straight-chain saturated and unsaturated fatty acids
(SCFA; UFA, respectively) (data not shown).
Prior investigations of DAP-R S. aureus strains indicated frequent
alterations in CM fluidity amongst such isolates . In the
present study, however, no statistically significant fluidity
differences were observed among the three animal passage strains with
increased DAP MICs as compared to the parental strain (PI values
ranging from 0.35760.04 to 0.37760.05).
mprF gene point mutations. All three strains (DAP-treated
and DAP-untreated) with increased DAP MICs and reduced HDP
killing following in vivo passage exhibited non-identical single
nucleotide polymorphisms (SNPs) within the mprF ORF (Table 1).
Each of the three SNPs were noted to occur in previously reported
hot spots within the mprF ORF, mostly in the 8th12th
transmembrane segments of the protein, which bridges synthase
and flipping domains (central bifunctional domain) . Of
note, the in vivo-passaged strain which maintained near
parentallevel DAP MICs and HDP susceptibility profiles exhibited a
parental mprF gene sequence. Furthermore, the above-described
parental isolate which exhibited unstable increases in its DAP MIC
following passage in sublethal tPMPs also retained the parental
mprF and dltA expression profiles; surface charge
Exponential phase mprF and dltA expression profiles did not
reveal significant differences in comparing the in vivo-passaged
% of total CM phospholipid composition (mean SD)
*P -value,0.000001 vs parental strain;
**P -value,0.00001 vs parental strain.
Figure 2. qRT-PCR analyses of mprF (A) and dltA (B) expression in the study strains. RNA samples were isolated from exponential-phase
cultures of the strains and were subjected to qRT-PCR to detect relative transcription levels of mprF and dltA. Expression levels of mprF and dlt genes
in the parental strain 271 were normalized to 1.
strains with the parental strain (Figure 2). Stationary phase
expression of both genes was very low in comparison to
exponential phase profiles, and did not indicate any differences
in expression amongst the strain-set (data not shown). Paralleling
the expression data, all four principle study strains exhibited
equivalent levels of relative surface positive charge (.90%
repulsion of cytochrome c; data not shown).
mprF mRNA half-lives
All mprF mRNA half-lives were ,2 min. Thus, analysis of
mRNA half-lives revealed no substantive differences among the
four study strains in terms of mprF transcript stabilities.
Whole genome sequencing
Whole genome sequencing revealed no significant
recombination or excision genomic events (e.g. recombination or excision of
sequences) in comparing the three in vivo isolates vs. the parental
(271) strain. Whole genome sequencing also confirmed the
presence of the SNPs identified above within the mprF genes in
L8, L16, and L56 strains by selected sequence analysis. Of interest,
there were relatively few other SNPs identified amongst the in vivo
passage isolates. Interestingly, several SNPs were identified within
oppB (oligopeptide ABC transporter permease), deoC
(deoxyribosephosphate aldolase), dut (dUTP diphosphatase), chs
(chemotaxisinhibiting proteins), and int (integrase) genes in one or two of the
three passage strains (Table 5). Of note, all the three passage
strains had identical SNPs within two previously uncharacterized
genes, SAUSA300_0039 (a hypothetical protein) and
SAUSA300_0070 (a putative lysophospholipase). However, no
SNPs were found within rpoB/C, yycFG, vraRS, or cls1 or cls2 genes
in any of three passage strains as compared to the parental strains.
These latter loci, along with mprF and dlt genes, have been variably
described to be associated with the DAP-R phenotype in other
MprF protein content
Since SNPs found in genes other than mprF might have an
impact on stability of MprF, we compared fluorescence intensities
of MprF-GFP fusion proteins in the parental and two of the
mutant strains. Flow cytometric analyses showed that the parental
and animal passage strain, L8, had similar levels of MprF
produced when protein expression was induced with equal
oligopeptide ABC transporter permease
*SNPs identified in all three passage strains;
amounts of xylose (Figure 3). Of interest, the L16 strain exhibited
a substantially lower mean level of MprF protein signal produced
as compared to either the parental or L8 isolate. MprF protein was
undetectable in control studies performed in the absence of xylose.
Cell wall thickness
All three isolates obtained following in vivo passage exhibited
significantly thicker cell walls than the parental strain
(20.5562.24 nm). For example, the cell wall thickness of the
DAP-untreated strain, L16, was 21.3762.67 nm (P,0.05 vs
parental strain), while those for the two DAP-treated isolates of
parental strain 271 (L8 and L-16) were 24.9862.94 nm and
25.5362.97, respectively; (both P,0.0001 vs the parental strain).
The control in vivo passage isolate which retained near-parental
DAP MICs did not exhibit thickened cell walls as compared to the
parental strain (data not shown).
DAP and most cationic HDPs initially target the bacterial CM
as a key early part of their lethal mechanisms . This
commonality has led to the notion that resistance to killing by
one peptide class (e.g., DAP) might be associated with a reduced
bactericidal effects among other electrostatically-similar but
structurally-unrelated peptide classes (e.g., HDPs). In this regard,
it was noted in several studies of individual, clinically-derived, as
well as in vitro passage-derived DAP-R S. aureus strains, that DAP-R
tended to track with relative cross-resistance to killing by HDPs
(HDP-R) . We recently extended these observations in a
more detailed analysis of this DAP-HDP cross-resistance
phenomenon in 10 DAP-S/DAP-R isogenic MRSA bloodstream isolate
pairs . In this latter investigation, two HDPs, selected for their
importance in defense against endovascular infections, were
queried , including one group from platelets (tPMPs) and one
from neutrophils (hNP-1). Of note, DAP-R tracked again with
HDP-R in the majority of strains. One major limitation of this
prior study was that the DAP-R strains were pre-selected for this
resistance phenotype a priori. This pre-selection, thus, did not
address whether S. aureus strains can adapt to reduced HDP and
DAP susceptibility phenotypes independently of one another.
Moreover, as antibiotic pressure is considered as a leading cause of
emergence of antimicrobial resistance, the relative contributions of
DAP therapy in relation to endogenous HDP exposures in
facilitating development of the cross-resistance phenotypes could
not be discerned.
Two recent observations from our laboratories provided further
insights into the above questions [9,11]. Among a collection of 47
MRSA bloodstream isolates obtained from patients who had never
received DAP (i.e., DAP-nave), 12 isolates had relatively high
DAP MICs (1 mg/ml), while the remaining 35 isolates had median
MICs of 0.25 mg/ml. When comparing the in vitro HDP
susceptibility profiles of these two MIC groups, increased
resistance to killing by tPMPs (but not hNP-1) tracked with the
higher DAP MIC group. These data suggested that bloodstream
exposure of these isolates (presumably including exposure to
platelet-derived HDPs) could prime for or select out
populations of infecting S. aureus strains with higher DAP MICs.
Prior vancomycin exposures in these patients did not impact the
categorization of strains into the lower-versus-higher DAP MIC
groups. Moreover, in a recent experimental prosthetic joint
infection model, DAP-R MRSA were isolated from both
DAPuntreated, as well as DAP-treated animals infected with a DAP-S
MRSA parental strain . This study supported the hypothesis
that DAP-R could be naturally selected in vivo, and that this
natural selection pressure could be amplified by specific antibiotic
treatment. However, it remained to be determined whether the
phenotypic and/or genotypic perturbations documented in
natural DAP-R strains paralled those emerging after selective
The present investigations were designed to assess concomitant
perturbations in HDP and DAP susceptibility phenotypes, as well
as relevant genotypes thereof, that arose in the course of a
controlled in vivo infection, especially focused on events occurring
in DAP-nave scenarios. We took advantage of isolates obtained
from the above well-characterized animal model of sub-acute,
localized and non-bacteremic staphylococcal infection (prosthetic
joint osteomyelitis), with or without DAP exposures . It was
reasoned that the major HDPs which the osteomyelitic organisms
likely encounter in this prosthetic joint model would be: i)
neutrophil-derived (related to localized bone-joint abscess
formations); and ii) to a lesser extent, platelet-derived (related to
prosthetic device-induced trauma). These hypotheses provided an
important context in which to select specific HDPs for study, as
well as in the interpretation of any cross-resistance phenomena.
Several compelling themes emerged from the study data. First,
three of the aforementioned isolates with increased post-infection
or post-therapy DAP MICs were tested for their concomitant
susceptibility profiles to the prototypical neutrophil HDP, hNP-1
. These three isolates were prioritized for investigation
based on exhibiting the greatest increases in DAP MICs vs the
parental strain (ranging from 616-fold). Following in vivo passage,
all three of these isolates exhibited significantly reduced killing by
hNP-1, especially those also exposed to DAP therapy. In contrast,
a control in vivo passage isolate which maintained near
parentallevel DAP MICs (L-76) did not exhibit such hNP-1
crossresistance. The susceptibility profiles of the three strains passaged
in animals were also determined against a prototypical platelet
tPMP preparation to evaluate potential cross-resistance between
hNP-1 and the platelet HDP mixture . Importantly, even
though tPMPs and hNP-1 differ in structure, charge and specific
mechanisms of action , reduced hNP-1 killing among the
animal passage strains tracked closely with reduced tPMP killing of
the same isolates. This relationship likely indicates that such
peptides share S. aureus CM targeting (e.g. initial electrostatic
affinity) as a common step in their otherwise distinghishable
mechanisms of action. This parallel tracking of hNP-1 and tPMP
cross-resistance is reminiscent of several other recent studies in this
arena . These outcomes, along with those from the recent
clinical study noted above , underscore the concept that
endogenous exposures of S. aureus to one or more HDPs under
sublethal conditions may prime such strain populations for
selection of either pre-existing or adaptive strains with co-reduced
susceptibilities to HDPs and DAP.
Second, we attempted to recapitulate the potential mechanism(s)
by which the in vivo passage isolates adaptively increased DAP
MICs in the absence of DAP exposures. Thus, we serially exposed
the parental strain to sublethal concentrations of either hNP-1 or
tPMPs over a similar 30d time-course, as carried out in the formal
animal model studies. Of note, serial passage in a very low and
sublethal concentration of tPMPs did induce a substantive increase
in DAP MICs, as well as reduced killing profiles for both HDPs
above. However, none of these resistance phenotypes was durable
following passage in drug-free media, and there were no mprF
SNPs detected post-HDP passage. Therefore, it seems clear that if
in vivo exposures to HDPs within a localized infection (as in the
current animal model) are playing a role in the emergence of
DAP-R, it likely requires exposure of the infecting strain to either:
i) multiple HDPs from neutrophils and/or platelets; ii)
combinations of HDPs; iii) higher peptide concentrations reflecting those
likely to exist in vivo; and/or iv) additional host factors.
Third, the mechanism(s) of in vitro adaptive co-resistance to
killing by DAP and sublethal levels of HDPs in S. aureus remains to
be delineated. One prevailing theory in this regard has been the
capacity of this organism to modulate its surface charge towards a
more relatively positive charge phenotype, potentially creating a
charge-repulsive surface milieu [8,35]. However, there were no
differences in relative surface charge detected among the parental
versus in vivo-passage isolates in this investigation, despite the
emergence of SNPs within the mprF operon during passage in vivo
. An additional pathway by which S. aureus may avoid killing by
cationic molecules such as calcium-DAP and HDPs is to alter its
relative CM order towards either a much more fluid or more rigid
configuration [810,46]. This is generally accomplished in
Grampositive bacteria by modifying the relative fatty acid saturation
indices and/or proportionality of anteiso-branched chain fatty
acids in its CM [46,47]. However, there were no significant
differences in either the CM fluidity index or fatty acid
composition among the parental vs animal-passage strains in this
study. Other possible mechanisms that have been co-associated
with HDP-R and DAP-R include changes in transmembrane
potential, carotenoid content, peptide-induced CM permeability,
or adaptive responses involving stress response and similar gene
pathways. Such mechanisms were not addressed in this
Fourth, all three principle isolates emerging as DAP-R during in
vivo passage had substantially thicker cell walls than the parental
strain. Such thick cell wall phenotypes have been a common, albeit
not universal, feature of DAP-R strains . Although
controversial, the thickened cell wall phenotype in S. aureus has been
postulated to be an important contributor to DAP-R either as a
mechanical barrier for peptide penetration or via an affinity
trapping mechanism [9,10,48,38]. The relative impact of the
thickened cell wall upon HDP-induced killing of S. aureus has not
Fifth, studies from our laboratories and others have suggested
that cationic HDPs or DAP may employ negatively-charged lipid
domains as putative docking sites during their initial interaction
with the target CM . Such events would imply that the
proportional CM composition of PG and CL (negatively-charged)
vs L-PG (positively-charged) would be expected to influence the
amount of cationic peptide eventually binding to the CM. In this
respect, it is noteworthy that the relative amount of the
negativelycharged PL species, PG, in the in vivo-passaged strains with
increased DAP MICs was significantly reduced as compared to the
parental strain as a reflection of the enhanced synthesis of L-PG
[10,35,48]. Consistent with this finding, the mprF SNPs identified
among strains isolated post-in vivo passage were localized to the
central bifunctional domain of the MprF protein, putatively
involved in both L-PG synthesis and/or translocation . These
results support an L-PG synthesis gain-in-function phenotype
among the current study strains with increased DAP MICs. A
genetic basis for such a potential gain-in-function remains to be
defined, as the observed mprF mRNA expression profiles, mRNA
half-lives and regulation of MprF protein expression did not differ
substantially between the parent and in vivo-passaged isolates.
Moreover, recent unpublished data document that the relative
distribution of the MprF protein within the free CM and septal
CM regions are equivalent amongst our study strains (Kuhn S et al;
personal communication). Collectively, these results suggest that the
observed mprF mutations seen in the in vivo passage strains with
increased DAP MICs may affect either MprF structural (e.g.,
protein conformation) and/or functional characteristics.
Lastly, our whole genome sequencing investigations were
important in the context of identifying a relatively limited cardre
of genes among in vivo passage strains within which point
mutations emerged. Such data should provide an important
framework to further pursue the genetic basis of in vivo emergence
of the DAP-R phenotype. Of particular interest was the
observation of SNPs occurring within the oppB gene (oligopeptide
ABC transporter permease) in one of the isolates post-in vivo
passage (L8). The opp loci were previously identified in
signaturetagged mutagenesis library studies as important virulence genes in
multiple animal models, including rabbit endocarditis .
We recognize that our current investigations have methodologic
challenges which somewhat limit interpretation. For example, the
in vitro HDP susceptibility testing was performed in rather austere
media, in the absence of host factors (e.g., serum or complement
proteins, etc.). Moreover, both neutrophils and platelets contain a
large cohort of HDPs which were not tested either individually or
in combination in this study. Also, the concentrations of HDPs
utilized in our in vitro assays are undoubtedly lower than bacteria
encounter in vivo. Moreover, both the host and pathogen are
constantly adapting to changing contexts of infection, from the
point of initial inoculation to potential long-term persistence, or to
host clearance. These, and other factors, alone and in combination
are likely critical to selective pressures for immune avoidance
placed on S. aureus during the course of infection. Further, only a
single clonal lineage genotype strain was assessed in the present
investigation. It has been well-chronicled that different S. aureus
clonal lineages are associated with rather distinct clinical outcomes
. Finally, there are likely other genes or genetic pathways
outside of mprF or dlt that may be involved in the evolution of
increased DAP MICs following in vivo passage. However, it was
impressive that on comparative whole genome sequencing, only a
limited cadre of SNPs were identified among the animal passage
strain, primarily in ORFs of unknown functions. It will be pivotal
to characterize these loci further through genetic manipulations
(e.g. generation of knock-out/overexpression strains) in the context
of DAP-R. Moreover, comparative and quantitative gene
expression profiles of the parental vs passage isolates may provide
additional insights into mechanisms of increasing DAP MICs in
the latter strains.
Based on current study, DAP-nave MRSA strains exposed to
HDPs in vivo may increase their DAP MICs prior to DAP
exposures. In addition, it has also been previousl shown that
vancomycin exposures either in vitro or in vivo can independently
lay the foundation for both DAP-R and HDP-R phenotypes (e.g.
to tPMPs) . Therefore, we propose the caution against the use
of vancomycin in treating DAP-nave MRSA strains with higher
DAP MICs, even if within the susceptible range (11.5 mg/ml).
In summary, S. aureus strains isolated from a localized in vivo
infection model exhibited a cross-resistance phenotype to
unrelated HDPs from platelets and PMNs, as well as increased DAP
MICs, even in the absence of DAP exposures. These findings are
consistent with the concept that encountering specific HDPs
during infection may select for surviving strains which are
crossresistant to DAP, and/or prime surviving organisms for
subsequent and parallel adaptations to both HDPs and DAP.
Whether the mechanism(s) of resistance to killing by such diverse
cationic peptides among S. aureus strains is a single shared pathway
or represents multiple distinct pathways remains to be determined.
We thank Drs. Wenqi Yu and Friedrich Gotz for the sfGFP-containing
plasmid vector. We also thank Kuan-Tsen Chen, Steven N. Ellison, and
Danya Alvarez for excellent technical assistance and Apurva Narechania
(Sackler Institute for Comparative Genetics, American Museum of Natural
History, New York) for sequencing analyses.
Conceived and designed the experiments: NNM SJY BNK AP ACC ASB.
Performed the experiments: NNM SJY LC CM ASM SK CCN. Analyzed
the data: NNM SJY LC CM ASM SK AP MRY CCN BNK ACC ASB.
Contributed reagents/materials/analysis tools: CM SK AP ACC. Wrote
the paper: NNM SJY MRY BNK ACC ASB.
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