Development of Reduced Vancomycin Susceptibility in Methicillin-Susceptible Staphylococcus aureus
Development of Reduced Vancomycin Susceptibility in Methicillin-Susceptible Staphylococcus aureus
Satish K. Pillai () 0 1 2
Christine Wennersten 1 2
Lata Venkataraman 1 2
George M. Eliopoulos 0 1 2
Robert C. Moellering 1
Jr 0 1 2
Adolf W. Karchmer 0 1 2
0 Harvard Medical School , Boston, Massachusetts
1 Received 2 February 2009; accepted 28 May 2009; electronically published 21 September 2009. Center, Div of Infectious Diseases , 110 Francis St, Suite GB, Boston, MA 02215
2 Division of Infectious Diseases , Beth Israel Deaconess Medical Center
Background. Most cases of reduced vancomycin susceptibility in Staphylococcus aureus reported in the literature have been in methicillin-resistant strains. We report the development of reduced vancomycin susceptibility in a series of clonally related, methicillin-susceptible S. aureus (MSSA) clinical isolates. This isogenic series permitted us to determine whether the evolution of reduced vancomycin susceptibility in MSSA is similar to that seen in MRSA. Methods. Differences in vancomycin population analysis profiles; chemical autolysis; vancomycin, oxacillin, and daptomycin minimum inhibitory concentrations; and bactericidal activities were examined. Results. Progressive vancomycin resistance correlated with increasing daptomycin nonsusceptibility. Chemical autolysis and the bactericidal activity of vancomycin, oxacillin, and daptomycin were reduced in the final, vancomycin-intermediate S. aureus isolate, compared with the vancomycin-susceptible MSSA progenitor. Conclusions. Clinicians should recognize that reduced vancomycin susceptibility can occur in S. aureus irrespective of background methicillin susceptibility and that development of intermediate vancomycin susceptibility in MSSA may result in increased tolerance to several classes of anti-staphylococcal antibiotics.
Methicillin-resistant Staphylococcus aureus (MRSA) is a
major cause of community and nosocomial infections.
Treatment with vancomycin has been the standard of
care for serious MRSA infection for 140 years. However,
over the past decade there have been reports of
vancomycin treatment failures for MRSA infection [1–3].
True vancomycin-resistant S. aureus (VRSA; defined as
a minimum inhibitory concentration [MIC] of 16
mg/mL ), which is associated with the incorporation
of the enterococcal vanA cassette into S. aureus, has
been reported only sporadically . In addition to
VRSA, reduced vancomycin susceptibility (RVS) is seen
among vancomycin-intermediate S. aureus (VISA; MIC
of 4–8 mg/mL) and hetero-VISA (susceptible to
vancomycin by routine susceptibility testing, but
containing resistant subpopulations with MICs 4 mg/mL) [4,
6]. Development of hetero-VISA and/or VISA does not
involve incorporation of the vanA cassette, is associated
with altered expression of multiple regulatory genetic
elements, and is phenotypically characterized by
increased cell wall thickness, altered cell wall
penicillinbinding protein profiles, and reduced rates of cell wall
autolysis [7–13]. Decreasing vancomycin susceptibility
is a predictor of failing vancomycin therapy [2, 3, 14].
Most reported cases of hetero-VISA and VISA have
arisen from MRSA progenitors. There are few detailed
reports of RVS in methicillin-susceptible isolates [15–
17]. In fact, in some of these cases the
methicillinsusceptible S. aureus (MSSA) with RVS may have arisen
from MRSA backgrounds. For instance, in 1 report of
MSSA-RVS, molecular investigation revealed the
presence of the genetic element conveying
methicillin-resistance, mecA, and the authors demonstrated an
inverse correlation between oxacillin and vancomycin
MICs in the series of S. aureus isolates recovered from
the index patient . A similar inverse correlation
between oxacillin and vancomycin MICs has been
reported in other mecA-positive S. aureus isolates .
In another case, the source patient harbored genetically
related methicillin-resistant and methicillin-susceptible
Table 1. Antibiotic Susceptibility Results (Agar and Macro-Broth Dilution) for Study
Minimum inhibitory concentration, mg/mL
VISA, and the authors postulated that the
methicillin-susceptible VISA arose from deletion of the mecA element .
BobinDebruex et al  described the clinical recovery of a
mecAnegative, methicillin-susceptible hetero-VISA isolate; however,
there was no vancomycin-susceptible progenitor to serve as a
In this study, we investigated a series of clinical MSSA isolates
recovered from a patient who experienced vancomycin therapy
failure (case 12 in Table 1 from reference ). Briefly, a patient
with an infected foot ulcer initially completed a 2-week course
of antibiotic therapy with oxacilllin for MSSA bacteremia
without evidence of remote sites of infection. The patient,
complaining of back pain, returned 3 weeks after the original
bacteremia and was found to have vertebral osteomyelitis, for
which oxacillin was reinitiated. The development of abnormal
liver function, presumed to represent oxacillin hepatotoxicity,
prompted a treatment change to intravenous vancomycin. Nine
weeks after the initial bacteremia, because of ongoing back pain,
debridement of the infected vertebral bodies was undertaken
with implantation of spinal hardware. Operative bone cultures
were positive for MSSA, which was susceptible to vancomycin
in the clinical microbiology laboratory. The patient continued
receiving vancomycin with trough concentrations ranging from
15 to 20 mg/mL. Three weeks after the operative debridement
and 2 months into vancomycin therapy, the patient developed
increasing back pain and intermittent fevers. Blood cultures
again yielded MSSA, for which the vancomycin MIC was
reported to be 4 mg/mL by the clinical microbiology laboratory.
Therapy with nafcillin was subsequently initiated for the
breakthrough bacteremia. Liver enzyme levels remained stable, and
then rifampin was added. Following 2 months of combination
intravenous therapy and a prolonged course of oral levofloxacin
plus rifampin, antibiotics were discontinued without
recrudescence of infection.
Because of the development of VISA in this series of MSSA
isolates, the following study was undertaken to characterize the
natural history of RVS development in a methicillin-susceptible
background and to evaluate the potential ramifications of this
on the susceptibility to other anti-staphylococcal agents.
The isolates described in this study are listed in Table 1.
Vancomycin MIC, pulsed field gel electrophoresis, and delta-lysin
activity were assessed for 2 isolates in this series, S. aureus A9635
and S. aureus A9639, which have been described elsewhere as
part of an unrelated study . Vancomycin (Sigma-Aldrich)
and oxacillin (Sigma-Aldrich) susceptibilities were assessed in
our laboratory by agar dilution as described by the Clinical
Laboratory Standards Institute , with a slight modification
in that incremental concentrations of vancomycin were used
to assess for subtle changes in MIC. Daptomycin (Cubist
Pharmaceuticals) susceptibility testing was performed using
standard broth macrodilution technique, with a starting inoculum
of ∼5 105 colony forming units (CFU)/mL prepared by direct
colony suspension, in Mueller-Hinton broth (Becton
Dickinson) supplemented to a final calcium concentration of 50–55
mg/L. Vancomycin population analysis was performed for S.
aureus A9635-A9637, S. aureus A9639, a
vancomycin-susceptible control (ATCC 29213), and a known VISA (PC-3) .
Serial 1:10 dilutions of an approximately ∼1 108 CFU/mL
inoculum were plated on brain-heart infusion agar (Becton
Dickinson) containing varying concentrations of vancomycin
and incubated at 35 C for 48 h, as described elsewhere . The
lower limit of detection for this assay was 1.6 log CFU/mL.
Pulsed-field gel electrophoresis with SmaI-digested genomic
DNA of S. aureus A9635-A9639 was performed using published
methods [21, 22]. In addition, polymerase chain reaction was
performed using previously published mecA primers  and
genomic DNA isolated from S. aureus A9635, MRSA 32
(mecApositive control ), S. aureus ATCC 29213 (mecA-negative
control) (GenElute Bacterial Genomic DNA kit;
Sigma-Aldrich). A method published elsewhere  for determining
delta-hemolysin activity was used to assess agr function.
Chemical-induced autolysis of the vancomycin-susceptible
progenitor, S. aureus A9635, and the final VISA isolate, S. aureus
A9639, was assessed as follows. Overnight cultures of the 2
isolates were diluted in brain-heart infusion broth and
incubated at 35 C with shaking at 250 rpm. At mid-exponential
growth (optical density measured at 600 nm [OD600]), ∼0.50),
the cultures were centrifuged, the resulting pellets were washed
once in sterile normal saline, centrifuged again, and the pellets
were suspended in 0.05% Triton X-100 in 0.05 mol/L Tris buffer
(pH, 7.2) to achieve an OD600 of 0.25–0.35. The samples were
subsequently incubated at 35 C with shaking at 250 rpm, and
the OD600 was measured every 30 min. Autolysis was defined
as (OD600 at time x)/(OD600 at time 0). Autolysis experiments
were performed in triplicate.
Bactericidal activities of vancomycin, oxacillin, and
daptomcyin against S. aureus A9635 and VISA A9639 were determined.
Overnight broth cultures were diluted 1:10 in either brain-heart
infusion broth (for vancomycin and oxacillin) or
Mueller-Hinton II broth (Becton Dickinson) supplemented to a final
concentration of 50–55 mg/L calcium (for daptomycin) to achieve
a starting inoculum of 6.5–7.0 log CFU/mL. The antibiotic
concentrations, chosen to simulate clinically achievable
concentrations, included oxacillin (20 mg/mL), vancomycin (16 mg/
mL), and daptomycin (8 mg/mL). Cultures were incubated at
35 C and sampled at 0, 4, and 24 h. For each antibiotic
condition, 5 separate time kill experiments were performed.
Subsequently, antibiotic killing of S. aureus A9635 and VISA A9639
at 4 h and at 24 h was compared by t test (Excel; Microsoft).
For the series of S. aureus isolates described in this study,
vancomycin MICs, as assessed by agar dilution, demonstrated a
progressive increase over time (Table 1), with S. aureus A9638
and S. aureus A9639 being vancomycin nonsusceptible
according current Clinical Laboratory Standards Institute break points
(susceptible defined as an MIC 2 mg/mL) . This increase
in vancomycin MIC was accompanied by an increase in
daptomycin MICs, and the final 2 isolates in the series were
daptomycin nonsusceptible (MIC, 11 mg/mL) . All isolates were
oxacillin susceptible. Figure 1 demonstrates that the
population profile of S. aureus A9635 paralleled that of the MSSA
control strain ATCC 29213. Neither S. aureus A9635 nor S. aureus
ATCC 29213 grew on the brain-heart infusion agar with 4-mg/
mL vancomycin. Although S. aureus A9636 was vancomycin
susceptible by agar dilution, it demonstrated growth on
brainheart infusion agar containing 4 mg/ml of vancomycin, which
is consistent with hetero-VISA [25, 6]. The population profiles
of S. aureus A9637 and S. aureus A9639 revealed a continued
rightward shift and increasingly resembled the profile of S.
aureus PC-3, a known VISA (vancomycin MIC, 8 mg/mL) .
Polymerase chain reaction failed to identify the mecA genetic
element in S. aureus A9635. All study isolates (S. aureus
A9635A9639) were indistinguishable by pulsed field gel
electrophoresis and lacked delta-lysin activity, suggesting impaired agr
function (data not shown).
Triton X-100–induced autolysis was reduced in S. aureus
A9639, compared with S. aureus A9535 (Figure 2). In addition,
killing of VISA A9639 was attenuated, compared with S. aureus
A9635, at one or both of the time points in the daptomycin,
vancomycin, and oxacillin time kill experiments. At the fixed,
uniform drug concentrations used in these studies, significantly
reduced killing of S. aureus A9639 relative to S. aureus A9635
was observed for daptomycin at 4 h, for vancomycin at 24 h,
and for oxacillin at both at 4 h and 24 h (Table 2).
The majority of cases of RVS in S. aureus reported in the
literature have involved MRSA, with few well described cases
of RVS occurring in MSSA [15–17]. It is possible that RVS may
remain undetected in MSSA with hetero-VISA characteristics,
because these isolates appear to be vancomycin susceptible by
current testing guidelines [4, 6]. Because hetero-VISA appears
to be an intermediary step toward the development of
fullfledged VISA  in the absence of vancomycin selective
pressure, it is possible that the true burden of RVS among MSSA
is higher than is currently appreciated. The data presented in
this study reaffirms the concept that RVS (hetero-VISA and
VISA) can arise in MSSA. Unlike in other reports [15, 16],
evolution of RVS occurred in a mecA-negative background.
Figure 2. Triton X-100–induced autolysis (mean and standard
deviations for 3 experiments) for vancomycin-susceptible Staphylococcus
aureus A9635 and VISA A9639. OD600 (Tx/T0), optical density at 600 nm at
time x was divided by that at time 0.
Table 2. Bacterial Killing of Staphylococcus aureus A9635 and S. aureus
A9639 following 4 h and 24 h Exposure To Vancomycin, Oxacillin, and
Bacterial killing, log CFU/mL
Antibiotic, duration of exposure
S. aureus A9635
S. aureus A9639a
NOTE. Bacterial killing was calculated as the log colony forming units (CFU)/mL at time
0 minus the log CFU/mL at time x. Five experiments were performed for each set of conditions.
The P value was determined by t test comparing the killing of S. aureus A9635 to that of VISA
A9639. SD, standard deviation.
a Vancomycin-intermediate S. aureus.
Furthermore, this series of clinical isolates permitted us to
establish certain similarities between the development of RVS
in MSSA and in MRSA. Multiple genetic events have been
associated with the development of hetero-VISA and VISA
among MRSA, including impaired function of the global
regulator, agr . In all of the study isolates, agr function was
altered (whether through mutation or down-regulation),
potentially providing 1 of the necessary genetic characteristics
required for the subsequent emergence of RVS following
Both the loss of agr function and development of VISA have
been associated with decreased autolysis [26, 13]. Autolysins
have been implicated in penicillin- and vancomycin-mediated
staphylococcal killing. Therefore, the reduced rates of Triton
X-100–induced autolysis observed in S. aureus A9639 (VISA),
relative to S. aureus A9635, raised the possibility that
vancomycin and b-lactam killing would also be decreased.
The significantly attenuated vancomycin killing seen in the
VISA isolate at 24 h, relative to the vancomycin-susceptible
progenitor, is not unexpected given its elevated vancomycin
MIC. In addition, this observation is supported by clinical data
noting reduced vancomycin efficacy for the treatment of RVS
S. aureus infections [2, 3, 14]. Despite the subtly lower oxacillin
MIC observed in S. aureus A9639 (compared with S. aureus
A9635), there was significantly reduced killing by oxacillin at
both 4 h and 24 h in our time kill experiments. Of note,
Sieradzki and Tomasz  reported that the development of
VISA in a clinical series of MRSA isolates exposed to
vancomycin was associated with reduction in both autolysis and the
cell wall content of penicillin binding protein 4 (PBP4).
Interestingly, loss of PBP4 activity has been associated with decreased
methicillin MIC in S. aureus . We hypothesized that the
development of VISA A9639 may have involved multiple
changes to the cell wall, including increased cell wall thickening
(resulting in increased vancomycin MIC), decreased expression
of PBP4 (resulting in decreased oxacillin MIC), and reduction
in autolysin activity (resulting in attenuation of Triton X-100
autolysis and vancomycin and oxacillin killing). The rising
daptomycin MIC that developed in these isolates during
vancomycin therapy, despite the lack of daptomycin exposure, is
consistent with findings of other investigators studying MRSA
[28, 29]. Despite the reported positive correlation between RVS
and daptomycin nonsusceptibility, other groups have reported
that daptomycin retains bactericidal activity against
heteroVISA and VISA in vitro [30–32], and against VISA in a rabbit
endocarditis model . In this study, the bactericidal activity
of daptomycin was significantly greater at 4 h for the
vancomycin- and daptomycin-susceptible MSSA progenitor A9635,
compared with VISA A9639; at 24 h, the mean daptomycin
killing was higher for S. aureus A9635 than for VISA A9639,
but the difference was not statistically significant (P p .08).
Whereas daptomycin is thought to exert its antibacterial effect
through cell membrane depolarization , recently it has been
shown that daptomycin also induces the S. aureus cell wall
stimulon, suggesting that it may also inhibit peptidoglycan
synthesis . Thus, the finding of reduced daptomycin,
vancomycin, and oxacillin killing (at 4 h, 24 h, or both time points)
in the VISA isolate relative to its MSSA and VSSA progenitor
may relate to 1 common targets involved in peptidoglycan
In conclusion, clinicians should be aware that, in settings in
which MSSA infections are treated with vancomycin, failure of
antibiotic therapy may be attributable to development of RVS.
Indeed, because hetero-VISA is not readily detected by routine
susceptibility testing, the true burden of RVS (in either MSSA
or MRSA backgrounds) remains unknown. Similar to the
observations seen in RVS-MRSA, absence of agr function was
noted in this series of MSSA isolates. In this series, development
of VISA was associated with reduced autolysis and attenuated
killing following in vitro exposure to several antibiotics of
Potential conflicts of interest. G.M.E. has received research funding
from and has served as a consultant to Cubist Pharmaceuticals, Pfizer, and
Astellas; R.C.M. has served as a consultant to Pfizer, Cubist, Astellas, and
Wyeth; A.W.K. has received research funding from Cubist Pharmaceuticals,
Pfizer, and Merck and has served as a consultant to Pfizer, Cubist, and
Astellas. All other authors: no conflicts.
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