J Antimicrob Chemother 2016; 71: 438 – 448
doi:10.1093/jac/dkv371 Advance Access publication 20 November 2015
Staphylococcus aureus develops increased resistance to antibiotics
by forming dynamic small colony variants during chronic osteomyelitis
L. Tuchscherr1*†, C. A. Kreis2†, V. Hoerr1,3†, L. Flint4, M. Hachmeister4, J. Geraci1, S. Bremer-Streck5,
M. Kiehntopf5, E. Medina6, M. Kribus7, M. Raschke2, M. Pletz8, G. Peters4 and B. Löffler1,9
1
Institute of Medical Microbiology, Jena University Hospital, Jena, Germany; 2Department of Trauma, Hand and Reconstructive Surgery,
University Hospital of Münster, Münster, Germany; 3Department for Clinical Radiology, University Hospital of Münster, Münster, Germany;
4
Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany; 5Department of Clinical Chemistry and Laboratory
Medicine, Jena University Hospital, Jena, Germany; 6Helmholtz Center for Infection Research, Braunschweig, Germany; 7Department of
Trauma, Hand and Reconstructive Surgery, Jena University Hospital, Jena, Germany; 8Center for Infectious Diseases and Infection Control,
Jena University Hospital, Jena, Germany; 9Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany
*Corresponding author. Tel: +49-03641-9-393628; Fax: +49-03641-9-393502; E-mail: [email protected]
†Contributed equally.
Received 29 July 2015; returned 14 September 2015; revised 23 September 2015; accepted 4 October 2015
Objectives: Staphylococcus aureus osteomyelitis often develops to chronicity despite antimicrobial treatments that
have been found to be susceptible in in vitro tests. The complex infection strategies of S. aureus, including host cell
invasion and intracellular persistence via the formation of dynamic small colony variant (SCV) phenotypes, could be
responsible for therapy-refractory infection courses.
Methods: To analyse the efficacy of antibiotics in the acute and chronic stage of bone infections, we established
long-term in vitro and in vivo osteomyelitis models. Antibiotics that were tested include b-lactams, fluoroquinolones,
vancomycin, linezolid, daptomycin, fosfomycin, gentamicin, rifampicin and clindamycin.
Results: Cell culture infection experiments revealed that all tested antibiotics reduced bacterial numbers within
infected osteoblasts when treatment was started immediately, whereas some antibiotics lost their activity against
intracellular persisting bacteria. Only rifampicin almost cleared infected osteoblasts in the acute and chronic stages.
Furthermore, we detected that low concentrations of gentamicin, moxifloxacin and clindamycin enhanced the
formation of SCVs, and these could promote chronic infections. Next, we treated a murine osteomyelitis model
in the acute and chronic stages. Only rifampicin significantly reduced the bacterial load of bones in the acute
phase, whereas cefuroxime and gentamicin were less effective and gentamicin strongly induced SCV formation.
During chronicity none of the antimicrobial compounds tested showed a beneficial effect on bone deformation
or reduced the numbers of persisting bacteria.
Conclusions: In all infection models rifampicin was most effective at reducing bacterial loads. In the chronic
stage, particularly in the in vivo model, many tested compounds lost activity against persisting bacteria and
some antibiotics even induced SCV formation.
Introduction
Chronic and relapsing infections are severe clinical problems, e.g.
in orthopaedic surgery. In particular, bacterial infections of bone
tissue tend to evolve into chronicity and become extremely difficult to treat.1 According to the route used by the infecting bacteria
to gain access to the bone tissue, different types of osteomyelitis
can be categorized, such as haematogenous osteomyelitis2 or
osteomyelitis due to continuous spread from a local infection
after trauma3 or due to infected foot ulcers.4 All different types
of osteomyelitis can develop chronic and recurrent courses that
remain symptomatic several weeks to months after infection.
Even though the involved pathogens have been found to be susceptible to antimicrobial compounds in in vitro tests, combined
and prolonged antibiotic treatments often fail to clear the infection. Consequently, most cases of chronic infections require additional surgical interventions for debridement of infected and
devitalized bone tissue or prosthetic material, which puts patients
at risk of disability or amputation.1,5,6
One of the most frequent causative pathogen of all different
types of osteomyelitis is Staphylococcus aureus.1,5 To establish an
infection S. aureus uses a wide variety of virulence factors that
enable different infection strategies, including host cell invasion
and intracellular persistence.7 During the last several decades
# The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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438
JAC
S. aureus SCVs induce antibiotic resistance
chronic and therapy-refractory infections have been highly associated with an altered bacterial phenotype, the so-called small colony variants (SCVs).8 Studies with stable or site-directed SCV
mutants (e.g. hemB mutants9) revealed that SCVs grow slowly,
form only tiny colonies on agar plates and have a reduced rate of
metabolism. Due to the altered metabolism some stable SCVs have
been found to be less susceptible to antibiotics, e.g. gentamicin and
b-lactams.8,10,11 The mechanisms of SCV formation during chronic
infections are largely unknown, but there are many indications that
SCVs are a very heterogeneous bacterial population.12,13 Only
recently we could demonstrate that S. aureus SCVs not only originate from gene mutations, but form continuously in a highly dynamical manner during the infection process.14,15 The precise signals
and conditions that mediate SCV formation and/or selection are
most likely multifactorial, but are only marginally understood.16
Previous work suggests that both the intracellular milieu14,17 and
subinhibitory concentrations of gentamicin16,18 strongly promote
the appearance of SCVs.
Antibiotics that are frequently used to treat S. aureus osteomyelitis include b-lactams, clindamycin and fluoroquinolones.
Vancomycin, linezolid, daptomycin and fosfomycin are applied
against resistant strains, such as MRSA.19 – 21 Although rifampicin
should never be used in monotherapy, several infection models
and clinical studies have shown that it improves treatment outcomes when used in combination with other antibiotics.22,23
Despite the availability of many different antimicrobial compounds, treatment of chronic bacterial bone infections remains
a big clinical challenge, which could be due to several reasons.
(i) Pathogens like S. aureus can invade host cells, including osteoblasts.24,25 Although many antibiotics accumulate intracellularly,
their activity can be lower within host cells.26,27 (ii) As many antimicrobial compounds require metabolically active pathogens for effectiveness, reduced susceptibility can be suspected for bacteria that have
switched to low-metabolic SCV phenotypes.8 (iii) Particularly during
chronic infections, bone tissue is altered by destructive and remodelling processes that could provide poorly perfused areas for bacterial
persistence. (iv) Finally, low concentrations of antibiotics might even
promote the formation of SCVs. SCV-promoting/-selecting activity
has already been reported for gentamicin and for antifolate
agents.10,28 – 30
To investigate these hypotheses we developed in vitro and in vivo
long-term osteomyelitis models in which the bacteria have to
adapt to their host. In our work we particularly addressed the problems of dynamic SCV development during persistence and the
consequences for antimicrobial susceptibility. We measured antimicrobial activity within the intracellular location and within chronically infected host tissue and the effects of antimicrobial
compounds in inducing SCV formation. Detection of the deficiencies
and pitfalls of antimicrobial treatment strategies in osteomyelitis is
a prerequisite to optimize and develop novel treatment options.
Materials and methods
Bacterial strains and preparation of bacterial inoculum
For the infection experiments, overnight cultures of the S. aureus strains
685028 and SH100031 and two clinical osteomyelitis strains (clinical isolates 1 and 2 in this study) were prepared in 30 mL of brain heart infusion
(BHI) broth and incubated at 378C and 160 rpm (for each strain). After two
washing steps with PBS and centrifugation at 5000 rpm, the concentration
of each strain was adjusted to an OD of 1 at 578 nm. The inoculum was
re-suspended in tryptic soy broth (TSB) +20% glycerin in order to freeze
and preserve the sample until the experiment was done. To control the
concentration of the inoculum, the number of cfu/mL was determined
by plating on blood agar plates. The strains were susceptible to all antibiotics according to EUCAST susceptibility testing guidelines (Table S1,
available as Supplementary data at JAC Online).
Preparation and culture of primary human
osteoblasts (pHOBs)
pHOBs were isolated from human bone as described before.14 The use of
human tissue was approved by the local ethics committee (EthikKommission der Ärztekammer Westfalen-Lippe und der Medizinischen
Wilhelms-Universität Münster) and written informed consent was obtained
(Az. 2010-155-f-S). Isolated osteoblasts were frozen in liquid nitrogen and
were cultivated 3 days in advance of each experiment. Osteoblasts were
cultured at a density of 6×104/mL in culture medium consisting of MEM
Alpha Modification, FCS, penicillin/streptomycin, L-ascorbit-acid-2-phosphate,
b-glycerolphosphate and dexamethasone. Osteoblasts were incubated at
378C and 5% CO2. All experiments were performed with osteoblasts at
passage 4 or 5.
Preparation, concentrations and use of antibiotics
in the infection experiments
The selection of the unbound antibiotic concentrations in vitro was guided by
the unbound peak concentrations at clinically relevant doses, as these concentrations may be most relevant for rapidly killing antibiotics. The maximal
concentrations in human serum were summarized only recently32 and were
the basis for the concentrations used in cell culture experiments.
Concentrations used for cefuroxime and gentamicin were based on further
references:33,34 40 mg/L cefuroxime, 50 mg/L vancomycin, 20 mg/L rifampicin, 10 mg/L moxifloxacin, 30 mg/L flucloxacillin, 20 mg/L linezolid, 60 mg/L
daptomycin and 20 mg/L clindamycin. As human serum concentrations
of fosfomycin can vary between 250 and 500 mg/L, both concentrations
were tested.35,36 Gentamicin was also used at two different concentrations: 20 mg/L, to simulate the human serum concentration, and
140 mg/L, to simulate local concentrations, that can be measured in
bone and soft tissue after local application of gentamicin, e.g. in bone
cement. Local concentrations of gentamicin were measured to be
7-fold higher than the serum concentration.37,38
The compounds were diluted in lithium water (Merck Millipore catalogue number 11533) in order to keep the correct pH and purity and
were then added to culture medium without penicillin/streptomycin. The
MICs measured for all antibiotics are described in Table S1. Gentamicin
was added only to control cells, at 0.3 mg/L, to prevent overgrowth with
bacteria that were released by the infected host cells.
Cell culture infection experiments with human
osteoblasts
To test bacterial susceptibility to antibiotics inside host cells, pHOBs were
infected with S. aureus and were treated in the acute or chronic stage of infection with different antibiotics for 48 h. pHOBs were isolated and prepared as
described above. pHOBs were infected using invasion medium consisting of
culture medium, 1% human serum albumin (HSA) and 10 mM HEPES.
Infection with S. aureus 6850 or SH1000 was performed at an moi of 50.
To determine the moi required for achieving maximum infection without affecting cell viability, osteoblasts were infected at mois of 10, 20, 30,
40, 50, 60, 70, 80 and 100. At 24, 48, 72 and 96 h after infection, cell viability was monitored by Trypan blue staining (automated cell counter
TC-20; Bio-Rad Laboratories) and the proportion of infected cells was
439
Tuchscherr et al.
determined by lysing the cells and plating. The best results were obtained
at the moi of 50, where between 80% and 90% of the cells were found to
be infected, with a cell viability of .80% at the 96 h timepoint. Whereas
cell viability was retained at the lower mois, the percentage of infected
cells was greatly reduced. In contrast, although .90% of infected cells
were obtained at mois of 70 and 80, there was a significant reduction in
the number of viable cells. Consequently, an moi of 50 was adopted for our
cell culture model (Figure S1B).
After invasion for 3 h at 378C and 5% CO2, infected pHOBs were washed
with PBS then treated with lysostaphin (20 mg/L) for 30 min to eliminate
all adherent or extracellular bacteria. It is known that lysostaphin does not
enter host cells.17,39 To analyse the susceptibility of S. aureus to lysostaphin, a protocol to measure the MIC of lysostaphin was performed40 and
the MIC was measured as 0.0313 mg/L (Figure S2).
After washing with PBS, the infected pHOBs were treated either directly
(acute, day 0) or after 7 days (chronic, day 7) with antibiotics. For direct
treatments all antibiotics mentioned above were added to the infected
host cells for 48 h. For treatment after 7 days culture medium was added
to the infected cells and the lysostaphin step and medium exchange
were repeated every second day until the start of antibiotic treatment
after 7 days. To detect the numbers of surviving bacteria, host cells were
lysed with 1 mL of H2O (acute group) or 500 mL of PBS/collagenase type I
(Sigma-Aldrich, 1 mg/mL, chronic group) 48 h after antibiotic treatment.
Before host cell lysis a lysostaphin treatment was performed as mentioned
above to eliminate all extracellular adherent bacteria. Enumeration of cfu
was performed by serial dilution of cell lysates and plating on blood agar
plates. The relative number of cfu (%) is shown in relation to the corresponding control cells (control group ¼ 100%, cells infected but not treated).
Additionally, the percentages of big WTand SCV-like colonies of the intracellular surviving bacteria in the acute and the chronic group were determined
by a colony counter (Biocount 5000, BioSys GmbH). All colonies with a diameter ,0.6 mm were considered as SCVs. Due to the slow formation of SCVs,
the final values of the amount of SCVs on agar were determined after 72 h
of incubation.
To test the stability of all antibiotics during our experiment, the cell
culture medium was sampled and tested on Mueller–Hinton (MH) plates
on days 0 and 2 (acute phase) and on days 7 and 9 (chronic phase). With
all compounds tested we found activity after 2 days within cell culture
medium (Figure S3), but cefuroxime lost activity during longer incubation
periods (Figure S4).
In vitro incubation of bacteria with antibiotics
To investigate the activity of various antimicrobial compounds (flucloxacillin,
cefuroxime, vancomycin, daptomycin, rifampicin, moxifloxacin, linezolid,
gentamicin, fosfomycin, clindamycin) in inducing SCV formation, we incubated bacteria without shaking with increasing concentrations of antibiotics. After being prepared in an overnight culture, S. aureus 6850 (OD 0.05,
578 nm) was incubated in medium consisting of 20% MH and 80% PBS in
order to get slow bacterial growth over a period of 10 days. The different
antibiotics were added to the incubation medium (20% MH+PBS) to obtain
increasing MICs (MIC, 2×MIC, 5×MIC). For clindamycin only, 10% serum
was added to activate the antibiotic41 and cell culture medium had to be
used instead of incubation medium (20% MH +PBS). Bacterial growth in
the incubation medium was measured daily by measuring OD (578 nm)
and by plating. For daptomycin, measurements were only done by plating
due to turbidity of the daptomycin-containing medium. Following CLSI
guidelines, fosfomycin was used with addition of 50 mg of glu-6-phosphate
per 200 mg of drug.40 Daptomycin was used in vitro in the presence of
1.05 mM CaCl2 (same concentration of CaCl2 as in cell culture medium
Gibco DMEM F12). The numbers of SCVs were determined every day from
day 1 until day 10 by plating 100 mL of the incubated medium on agar
plates. Agar plates were incubated for 24 h before counting and investigating the percentage of SCVs. To test the stability of antibiotic-induced SCVs,
440
the recovered SCVs were subcultured on blood agar daily for 10 days.
Cultures were observed daily to detect a switch to the WT phenotype
and the percentage of stable SCVs was determined after 10 days.
To check the stability of antibiotics, all tested compounds were prepared in 20% MH+PBS at increasing MICs (1×, 10×, 50× and 10×MIC) in
the absence of bacteria. Cefuroxime was the only compound for which we
detected reduced activity after 10 days (Figure S4).
Animal experiments
Pathogen-free female C57BL/6 mice aged 10 weeks were obtained from
Harlan-Winkelmann (Borchen, Germany). All in vivo experiments were performed according to the guidelines of the European Regulations for Animal
Welfare. The animals were maintained in individually ventilated cages and
were given food and water ad libitum. All experiments were approved by the
North Rhine-Westphalia Agency for Nature, Environment, and Consumer
Protection (Landesamt für Natur, Umwelt und Verbraucherschutz
Nordrhein-Westfalen-LANUV; permit number 84-02.04.2012.A293).
Treatment in the murine haematogenous
osteomyelitis model
To further investigate the efficiency of antibiotics in vivo, a chronic osteomyelitis model in mice that closely reproduces the features of human
osteomyelitis (bacterial persistence in bone tissue, bone deformation
and remodelling, formation of sequestra) was developed and used as
already described.42 Mice were infected with S. aureus 6850 at a concentration of 1×106 cfu/200 mL via the tail vein. The infected mice were
divided into an acute (treatment 5 days post-infection) and a chronic
(treatment 5 weeks post-infection) group. In each group treatment was
performed for 6 days by subcutaneous daily injection of 100 mL of antibiotics at the following concentrations: 850 mg/L rifampicin; 4300 mg/L cefuroxime; and 600 mg/L gentamicin. The group size for each antibiotic
compound tested in the acute and chronic groups was n¼6. MRI monitoring was performed before and after antibiotic treatment in each group. After
treatment the mice were sacrificed and the kidneys and bones of the lower
extremity of each mouse were homogenized in PBS and plated on blood
agar plates in serial dilution to determine the bacterial load in tissues.
To analyse whether treatment of the animals resulted in serum concentrations comparable to those known for humans, we treated three uninfected
animals per group for 3 days with rifampicin, cefuroxime or gentamicin as
described. On day 4 we took blood by cardiac puncture 1 h after application
of the antibiotics. The resulting serum concentrations were determined
by MS and are given in Table 1. Tandem MS (API 4000, AB Sciex) was
performed as previously described with minor modifications.43 – 45
Table 1. Antimicrobial peak levels reached in serum of C57BL/6 mice after
subcutaneous treatment with rifampicin, gentamicin or cefuroxime
Serum concentrations in C57BL/6 mice 1 h
after 4 days of treatment (mg/L)
Rifampicin
Gentamicin
Cefuroxime
25.6+1.96
8.2+3.1
1.54+0.7
C57BL/6 mice were treated subcutaneously with rifampicin, gentamicin
or cefuroxime once a day for 3 days. On day 4 whole blood was sampled
by cardiac puncture and antibiotic concentrations in the serum samples
were measured by MS. The measured values reached levels comparable
to peak serum levels reported for humans (rifampicin, 10 mg/L; gentamicin,
10 – 20 mg/L; cefuroxime, 1 – 4 mg/L for oral use and up to 100 mg/L for
intravenous use).32 – 34
JAC
S. aureus SCVs induce antibiotic resistance
(a)
(b) 150
Day 0
Day 7
%cfu ± SD
100
50
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pi
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100
80
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(d) 120
*
6850
SH1000
Clinical isolate #1
Clinical isolate #2
*
100
%cfu ± SD
Ri
6850
SH1000
Clinical isolate #1
Clinical isolate #2
(c) 120
20
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%cfu ± SD
*
*
Figure 1. Intracellular activity of antibiotics against S. aureus persisting in host cells. (a) pHOBs were infected with S. aureus 6850 and electron
micrographs were taken directly after infection. (b) Osteoblasts were infected with S. aureus 6850 followed by treatment with antibiotics for 48 h
directly after infection or at day 7 post-infection. The numbers of surviving bacteria were determined by plating and are shown in relation to control
(untreated, 100%; 4.36×108+8×106 cfu/mL) cells. Statistical analysis was performed by ANOVA comparing bacterial numbers in untreated control
cells with those in treated cells at the two timepoints (n ≥ 3; +SD). *P ≤0.05. (c and d) Treatment of osteoblasts was performed with rifampicin or
cefuroxime in cells infected with S. aureus 6850, SH1000 or two different clinical osteomyelitis strains. Statistical analysis was performed with the
unpaired t-test comparing bacterial numbers in untreated control cells with those in treated cells at the two timepoints (n ≥3; +SD). *P≤ 0.05.
441
Tuchscherr et al.
Statistical analysis
Data analysis was performed with GraphPad Prism 5.0 (GraphPad
Software). One-way ANOVA was used to compare multiple groups by the
Dunnett post test. The unpaired Student’s t-test was used for comparison
between any two individual groups. P values of ≤0.05 were considered to
be statistically significant. Errors are expressed as the standard deviations
of the means.
Gentamicin, fosfomycin and clindamycin induced
a rapid formation of SCVs inside osteoblasts
The intracellular location is known to be a strong stimulus to induce
the formation of SCVs.14,28 Accordingly, we detected a high rate of
(a) 100
40
(b) 70
60
50
40
30
20
10
0
7
Da
y
0
0
*
*
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Some antibiotics lost activity against persisting bacteria
inside osteoblasts; only rifampicin almost cleared
infected host cells
442
60
20
Results
In recent decades S. aureus has been increasingly recognized as a
facultative intracellular pathogen, as the bacteria can invade different types of host cells, including osteoblasts, and persist within
cells24,46 (Figure 1a). To find out how antibiotics act on bacteria
that persist within the intracellular milieu, we infected cultured
human osteoblasts with S. aureus 6850 and treated the infected
host cells directly (acute stage, 0 days) or 7 days (chronic stage)
post-infection (Figure 1b and Table S2). The viability of the cells
was checked every day (Figure S1B). We did not observe that
the intracellular bacteria grew over time (Figure S1A). By treating
host cells we found that rifampicin, gentamicin, moxifloxacin,
linezolid and vancomycin were efficient in reducing intracellular
loads of bacteria in the acute as well as in the chronic stage,
whereas the other compounds (daptomycin, fosfomycin, cefuroxime, flucloxacillin and clindamycin) lost their effect on persisting
bacteria. Here, treatment after 7 days of persistence did not
reduce the number of intracellular bacteria within host cells compared with untreated but infected control cells.
To exclude loss of activity of antibiotics during osteoblast treatment, the cell culture medium with each antibiotic was analysed
at 0, 2, 7 and 9 days after infection. In all cases, the drugs did not
*
80
Da
y
Therapeutic treatment in the acute and chronic phases of S. aureus-induced
osteomyelitis was followed up by in vivo MRI. Within this frame baseline
scans were performed on day 5 and 5 weeks after bacterial administration
and were compared with the results obtained after 5 days of antibiotic treatment, measured on day 11 and 6 weeks post-infection. Four groups of mice
(n¼11) were investigated and were either treated with rifampicin, gentamicin or cefuroxime or were used as controls without any treatment. For comparison, additional scans were performed on healthy mice (n¼4). Images
were acquired at 9.4 T on a Bruker BioSpec 94/20 (Ettlingen, Germany)
equipped with a 0.7 T/m gradient system. Lesions and structures of bones
were imaged by a 3D FLASH sequence using the following parameters:
matrix size, 512×256×102; field of view, 4.5×2.5×2.0 cm3; echo time
(TE), 3.1 ms; repetition time (TR), 20.0 ms; flip angle (FA), 108. During MRI
measurements, animals were anaesthetized with 2% isoflurane and were
monitored for core body temperature and respiration rate using an
MRI-compatible monitoring system (SA Instruments, Stony Brook, NY,
USA). To reduce motion artefacts, scans were acquired with respiratory triggering. In MRI images inflamed regions and tibia volume were segmented
using Amira software tools (version 5.4.0) for volumetric data analysis.
%SCVs ± SD
MRI and reconstruction and quantification of the
inflammatory focus in murine bone tissue
show loss of activity (Figure S3). To exclude a strain-dependent
effect of rifampicin, we performed analogous experiments using
the S. aureus strain SH1000 (laboratory strain) and two clinical
osteomyelitis isolates. Here, we also obtained nearly complete
bacterial clearance following rifampicin treatment (Figure 1c),
whereas for cefuroxime we detected a lack of activity on intracellular persisting bacteria (Figure 1d).
%SCVs ± SD
HPLC separation was performed using a Luna Phenyl-Hexyl column
(Phenomenex, Germany). Multiple Reaction Monitoring (MRM) transition
has been optimized for each antibiotic drug for the API 4000.
(c)
SCV
WT
Figure 2. Appearance of SCVs in infected osteoblasts following treatment
with antibiotics. After infection and treatment of the pHOBs (see Figure 1)
the percentages of big and small colonies of intracellular surviving bacteria
were determined by plating. (a) The percentage of SCVs was determined
directly or after 7 days of intracellular persistence in primary osteoblasts
without treatment (control). *P ≤ 0.05 (unpaired t-test). (b) The
percentage of SCVs was determined after infection and direct treatment
with the different antibiotics for 48 h (n ¼ 3; +SD). *P ≤ 0.05 [ANOVA
comparing the percentages of SCVs in treated cells with those in
untreated control cells (where the osteoblasts were infected but not
treated with antibiotic)]. (c) Photograph of recovered phenotypes after
plating and incubating (24 h) cell lysates from infected osteoblasts
without treatment after 7 days.
JAC
S. aureus SCVs induce antibiotic resistance
SCVs in vitro non-inducers
Cefuroxime
3 × 108
0.8
Control
2 × 108
MIC
0.6
2 × 108
2 × MIC
0.4
5 × MIC
1 × 108
0.2
5 × 107
0.0
0
0 3 6 9 12
Time (days)
cfu/mL
(b)
SCVs in vitro inducers
Gentamicin
Moxifloxacin
IC
5M
IC
cfu/mL
1 × 108
5 × 107
*
0
3 6 9 12
Time (days)
*
Co
nt
ro
l
M
IC
2×
M
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5× C
M
IC
OD578
5×
0
3 6 9 12
Time (days)
Fosfomycin
OD578
0.6
Control
MIC
2 × MIC
5 × MIC
0.4
0.2
0.0
0
3 6 9 12
Time (days)
%SCVs
(c)
120
100
80
60
40
20
0
*
*
*
*
cfu/mL
4 × 106
3 × 106
2 × 106
1 × 106
0
*
*
*
Control
MIC
2 × MIC
5 × MIC
**
C
Ge ont
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ic
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Figure 3. Ability of various antimicrobial compounds to induce the
development of SCVs after long-term incubation. (a and b) S. aureus 6850
was incubated in 20% MH medium and 80% PBS, which allowed slow
bacterial growth over the time period of 10 days. The different antibiotic
compounds were added to the incubation medium at increasing MICs
(MIC, 2×MIC, 5×MIC). For daptomycin the OD could not be determined
due to the turbidity of the daptomycin-containing medium and for
clindamycin 10% serum had to be added to the medium to induce activity
of the antimicrobial compound. Daptomycin was tested in vitro with 1.05 mM
CaCl2 (same concentration as in the cell culture medium in which we
observed high activity). According to the recommendations of CLSI
fosfomycin was used with 50 mg of glu-6-phosphate per 200 mg of drug.
Bacterial growth in the incubation medium was measured daily by
determining OD578 (n¼5; +SD) and by plating and counting the cell lysates
after 10 days. (c) The rates of SCV formation were determined on day 10 by
plating 100 mL of medium on agar plates and counting the percentage of
SCVs after 72 h of incubation. Statistical analysis was performed with the
absolute numbers, using ANOVA. *P≤ 0.05. (d) Photographs of recovered
phenotypes after plating and incubating bacteria treated with gentamicin,
moxifloxacin and clindamycin for 1 day or 10 days.
3 × 108
2 × 108
2 × 108
1 × 108
5 × 107
0
Co
nt
ro
l
M
IC
2×
M
I
5× C
M
IC
5×
M
IC
IC
Control
MIC
2 × MIC
5 × MIC
Co
nt
ro
l
M
IC
2×
M
I
5× C
M
IC
*
*
1.0
0.8
0.6
0.4
0.2
0.0
cfu/mL
OD578
*
M
12
2×
3
6
9
Time (days)
IC
0
M
0.0
2 × 108
2 × 108
1 × 108
5 × 107
0
Co
nt
ro
l
0.8
0.4
3 × 108
cfu/mL
OD578
Control
MIC
2 × MIC
5 × MIC
1.2
0
2 × 108
Linezolid
Clindamycin
1.6
Control
MIC
2 × MIC
5 × MIC
M
IC
*
0.8
0.6
0.4
0.2
0.0
IC
*
0
12
M
3
6
9
Time (days)
2×
0
5 × 107
IC
0.0
1 × 108
M
0.4
2 × 108
Co
nt
ro
l
0.8
*
Flucloxacillin
Control
MIC
2 × MIC
5 × MIC
cfu/mL
OD578
1.2
*
*
M
Co
nt
ro
l
IC
IC
M
M
5×
12
*
cfu/mL
*
2×
3
6
9
Time (days)
1 × 108
8 × 107
6 × 107
4 × 107
2 × 107
0
5 × 108
4 × 108
3 × 108
2 × 108
1 × 108
0
0
l
0
5 × 107
IC
0.0
3 6 9 12
Time (days)
Daptomycin
1 × 108
Co
nt
ro
0.4
0
2 × 108
M
Control
MIC
2 × MIC
5 × MIC
cfu/mL
OD578
0.8
Control
MIC
2 × MIC
5 × MIC
Co
nt
ro
l
M
IC
2×
M
I
5× C
M
IC
1.2
1.0
0.8
0.6
0.4
0.2
0.0
cfu/mL
To analyse the direct effect of the different antibiotics on SCV formation, we performed in vitro incubation experiments in dilutions of MH
OD578
Vancomycin
Low concentrations of gentamicin, moxifloxacin
and clindamycin induced SCV formation in vitro
(a)
*
Co
nt
ro
l
M
IC
2×
M
I
5× C
M
IC
OD578
SCVs after 7 days of intracellular persistence that had developed in
untreated host cells (Figure 2a). In the treatment experiments
described in Figure 1 we obtained indications that some antibiotics
can further enhance the rapid formation of SCVs in the first 2 days
after infection (Figure 2b). This phenomenon is well known for gentamicin.28 In our cell culture experiments we observed an increased
rate of SCVs after 48 h of treatment with gentamicin, fosfomycin
(500 mg/L) or clindamycin (Figure 2b and c).
Figure 3. Continued.
443
Tuchscherr et al.
(d)
Day 1
Day 10
Gentamicin
Moxifloxacin
Clindamycin
+10% serum
Figure 3. Continued.
medium. Firstly, we identified a nutrition-poor medium (20% MH in
PBS) in which the bacteria slowly grew to the stationary phase for up
to 10 days (Figure 3a and b). Then, we performed experiments in
this medium by adding antibiotics at increasing concentrations
(MIC, 2×MIC, 5×MIC) and obtained a concentration-dependent killing effect by measuring the OD and the bacterial counts daily
(Figure 3a and b). Under these conditions, rifampicin induced a
resistant population after 24 h of incubation, which rendered this
compound ineffective (Figure S5).
To exclude a lack of killing effect due to non-stability of the
drugs used, all the antibiotics were incubated for up to 10 days
in 20% MH medium (Figure S4). Almost all antibiotics showed
equal activity at the beginning and after 10 days of incubation,
except cefuroxime, which lost activity after 10 days of incubation.
We determined the rates of SCV formation after 1 day and
10 days (Figure 3c and d). We found that gentamicin, moxifloxacin
and clindamycin already induced the formation of some SCVs after
1 day of incubation, but strongly enhanced SCV formation after
10 days compared with control bacteria incubated without antibiotics. The other antibiotics tested did not induce the development of
SCVs in vitro (Figure 3c).
The formation mechanism of SCVs is only marginally understood and most likely multifactorial. Our recently developed longterm infection models revealed that dynamic SCVs develop during
any chronic infection course and can rapidly revert to the fully
aggressive WT phenotype when subcultured in rich medium.14,15,42
To test whether gentamicin, moxifloxacin and clindamycin induce
stable or dynamic SCVs, we subcultured the obtained SCVs
(Figure 3c and d) on blood agar plates daily for 10 days. Even
after 10 days we still found high rates of SCVs, ranging from 40%
(for gentamicin-induced SCVs) to 80% (for moxifloxacin-induced
SCVs). Only the clindamycin-induced SCVs were less stable and all
of them rapidly reverted to the WT phenotype upon subculturing.
Furthermore, all the SCVs analysed did not show auxotrophism
for menadione, haemin or thymidine.8
In a chronic haematogenous murine osteomyelitis model
all tested antibiotics did not reduce the bacterial load in
the chronic stage
Finally, we aimed to verify the findings of the in vitro experiments
in vivo. For this, we performed antimicrobial treatments in a
haematogenous osteomyelitis model in mice. We chose three
444
representative antibiotics according to our results from the
in vitro tests: rifampicin, which was the only compound to almost
clear the infected host cells; gentamicin, which acts on intracellular
persisting bacteria, but is also a strong SCV inducer; and cefuroxime, which lost activity against intracellular persisting bacteria,
but had no activity on SCV development. One group of infected
mice was treated in the acute stage of the disease (5 days postinfection) and another group was treated when the bone infection
had developed to chronicity (5 weeks post-infection). The antimicrobial compounds were injected once daily subcutaneously
for 5 days. The measured levels of all antibiotics reached concentrations that were comparable to those published for treated
patients (Table 1). The efficacy of treatment was evaluated by plating bone tissue and counting the bacterial loads (Figure 4a) and by
quantifying bone thickness and inflammatory regions in chronically
infected bones by MRI (Figure 4c and Table 2). Only rifampicin
reduced the bacterial load in the acute stage compared with
untreated animals. As in the in vitro experiments, gentamicin
induced a high rate of SCV development (Figure 4b). In the chronic
stage of infection many bones had tremendously increased in volume due to continuous inflammation, which was quantified by MRI
measurements (Table 2). Our analysis revealed that none of the
tested antibiotics, not even rifampicin, had a beneficial effect on
the bacterial loads or disease development in the chronic stage
(Figure 4a and Table 2). However, in the chronic stage we obtained
a high rate of SCV formation, which was indicative of bacterial
adaptation (Figure 4b).
Discussion
Osteomyelitis caused by S. aureus is a severe clinical problem, as it
frequently develops a chronic and therapy-refractory course despite
antimicrobial treatments. Although the involved pathogens have
been tested to be susceptible to antibiotics in vitro, they can nevertheless persist in host tissue and cause a relapsing infection.1,5 From
clinical studies it is well known that chronic infections are associated
with the bacterial SCV phenotype.8,47 Subsequently, stable SCVs
have been tested for their susceptibility to different antibiotics.10
Cell culture models provide additional information on the intracellular activity of compounds and have shown bactericidal effects for
most antibiotics (except for vancomycin and daptomycin) directly
after infection when used at tissue concentrations.48 In general,
JAC
S. aureus SCVs induce antibiotic resistance
Chronic
(a) Acute
*
8
8
Log cfu/mL ± SD
6
4
2
0
2
0
Day 5
Before
treatment
n
Ce
fu
ro
xi
m
e
Ge
nt
am
ic
in
ci
Ri
Rifampicin
Day 11
pi
ci
n
Ce
fu
ro
xi
m
e
Ge
nt
am
ic
in
Ri
Co
nt
ro
l
Chronic
70
60
50
40
30
20
10
0
pi
%SCVs
im
e
Ge
nt
am
ic
in
Ce
fu
ro
x
n
ci
pi
fa
m
Ri
Co
nt
ro
l
%SCVs
*
Control
fa
m
Co
nt
ro
l
ci
n
Ce
fu
ro
xi
m
e
Ge
nt
am
ic
in
pi
fa
m
Ri
(b) Acute
70
60
50
40
30
20
10
0
(c)
4
–2
Co
nt
ro
l
–2
6
fa
m
Log cfu/mL ± SD
10
Gentamicin
After
treatment
Before
treatment
Cefuroxime
After
treatment
Before
treatment
After
treatment
Acute
Chronic
Figure 4. Treatment of a haematogenous osteomyelitis model in mice with rifampicin, gentamicin and cefuroxime. C57BL/6 mice were infected via the
tail vein with S. aureus 6850 and subsequently developed osteomyelitis in the acute (after 5 days) and chronic (after 5 weeks) stages. Treatment with
rifampicin, gentamicin or cefuroxime was performed subcutaneously every 24 h for 5 days. (a) The bacterial loads within the tibiae were analysed after
treatments in the acute and chronic stages of infection by plating host tissue and counting the numbers of recovered colonies. n ¼10–12 mice; +SD.
Statistical analysis was performed with ANOVA comparing the bacterial load in tibiae of treated animals with that of untreated animals. *P≤ 0.05. (b) The
percentage of SCVs among the recovered colonies was evaluated for each antibiotic in the acute and chronic stages of the infection. n ¼10–12 mice;
+SD. Statistical analysis was performed with ANOVA comparing the rate of SCVs in tibiae of treated animals with that of untreated animals. *P≤ 0.05. (c)
Sequential MRI showing the progression of osteomyelitis in the tibiae during the acute and chronic phases with and without therapeutic treatment with
rifampicin, gentamicin or cefuroxime. The images show representative three-dimensional reconstructions of the tibiae and inflammatory lesions after
segmentation of MRI images. Inflammatory lesions are coloured red and brown (right) or orange and yellow (left) according to the inflammatory depth
in the bones. Non-inflamed area (area of bone without signs of inflammation) of the left leg is shown in magenta and the non-inflamed area of the right
leg is shown in green.
445
Tuchscherr et al.
Table 2. Calculation of the volume of inflammatory lesions in tibiae of chronically infected treated and untreated animals
Control before infectiona
Chronic infection without treatment at week 5b
Chronic infection without treatment at week 6b
Chronic infection after rifampicin treatment at week 6b
Chronic infection after gentamicin treatment at week 6b
Chronic infection after cefuroxime treatment at week 6b
Inflammatory volume (mm3)
Total bone volume (mm3)
0
20+21
19+18
19+17
22+13
16+13
26+1
84+58
84+52
77+48
103+99
67+29
a
n ¼4.
n ¼11.
b
almost all antibiotics exhibit higher activity against normal than
against SCV phenotypes.11 Furthermore, some animal models
have been developed to investigate the persistence of SCVs and
their resistance to antibiotics. All these results are summarized in
a recently published review10 and suggest that SCVs can better
spread and persist within the body and are more tolerant to antimicrobial treatments.
The described studies have been mainly performed with stable,
well-characterized mutants that have defects in the electron transport system (e.g. hemB mutants). However, recent work indicates
that clinical SCVs are a very heterogeneous population that is
more complex than defined electron transport chain-interrupting
mutants.13 Little is known about the formation mechanisms of
SCVs. SCVs can appear constitutively during bacterial replication16
and/or the development of SCVs can be induced by various stress
conditions, such as the intracellular milieu or selective antibiotic
pressure.17,18 By using different long-term infection models we
demonstrated that SCVs always and continuously appear during
chronic infection courses in a very dynamic manner that allows
rapid reversion to the WT phenotype.14 This dynamic switching
mechanism, which is mediated by regulatory processes rather
than by gene mutations,14,49 is most likely an early bacterial adaptation process that takes place in any kind of infection and paves
the way for long-term bacterial persistence.
In our work, we investigated the influence of this dynamic
switching mechanism on antibiotic tolerance/resistance. We
used complex in vitro and in vivo infection models that closely
mimic the human situation and force the bacteria to adapt to
their host.14,42 As these early bacterial adaptation mechanisms
are highly reversible, all antibiotic resistance tests had to be performed within the models to avoid a bacterial subculturing step.
In the long-term cell culture infection models we found that some
antimicrobial compounds (b-lactams, daptomycin, fosfomycin
and clindamycin) lost activity against chronically infecting bacteria. b-Lactams, daptomycin and fosfomycin are cell-wall-active
antibiotics. These compounds are highly effective bactericidal
antibiotics against fast-growing bacteria, but apparently rapidly
lose activity when the bacteria slow their growth rate, which
could limit their efficacy during chronic infections and against
SCVs (adaptive antibiotic resistance).50 Additionally, clindamycin,
which inhibits protein biosynthesis, lost activity against persisting
bacteria, which can be explained by a reduced metabolism in persisting bacteria and the bacteriostatic mode of action. By contrast,
other protein biosynthesis inhibitors (gentamicin and rifampicin)
were still active against persisting bacteria; this might be due to
446
their bactericidal effect, which is apparently still effective in
SCVs. The bactericidal and/or bacteriolytic effects of moxifloxacin
and vancomycin also resulted in high activity against persisting
bacteria at the serum concentrations tested.
In our study, we further included a haematogenous osteomyelitis model that develops to chronicity and closely mimics human
infection.42 The subcutaneous application of defined antibiotics
(rifampicin, gentamicin, cefuroxime) for 5 days resulted in serum
levels that were similar to the concentrations reached in humans.
To evaluate the success of treatment we determined the bacterial
load in the bones and applied MRI to quantify the areas of inflammation and bone deformation in the chronic stage. In the acute
phase of infection we detected a significant reduction in bacteria
after treatment with rifampicin, whereas during chronic infection
none of the tested antibiotics exhibited a beneficial effect.
Consequently, our data reveal that bacterial resistance during
chronic tissue infection is much more complex than is reflected by
cell culture experiments. In the in vivo situation additional mechanisms must be considered, as the host tissue is altered by destructive
and remodelling processes, including the formation of dead bone
fragments and newly built woven bone.1,42,51 Particularly in poorly
or non-perfused tissue areas, effective concentrations of antibiotics
are most likely not reached, leaving bacterial reservoirs within the
host tissue.52
Additionally, our results demonstrate that low concentrations
of some antibiotics even promote SCV formation and could
support the development of a chronic infection course. An
SCV-promoting/-selecting activity has already been observed for
gentamicin and for antifolate agents. 10,28 – 30 Our systematic
in vitro analysis revealed that further compounds—moxifloxacin
and clindamycin—induce SCVs with increased stability. This phenomenon has not been described before. Consequently, the application of these compounds for chronic infections should be
investigated in appropriate models and in clinical studies to
exclude the possibility that they promote SCV formation and bacterial persistence in host tissue.
Although gentamicin was effective in diminishing the bacterial
load of infected host cells, it failed to have a beneficial effect in the
acute and chronic stages of the haematogenous osteomyelitis
model in mice. As described before, we identified gentamicin as
a strong SCV inducer.18,28 Not only in the in vitro systems but
also in the in vivo model we recovered a high percentage of
SCVs at an early stage of infection and treatment. Particularly in
the in vivo model, rapidly formed SCVs apparently help the bacteria
to survive within the host in high numbers. The SCV-inducing activity
JAC
S. aureus SCVs induce antibiotic resistance
of gentamicin must be considered, as this compound is frequently
used locally, e.g. as a coating for implants or antibiotic-loaded
cement.53 For local uses, gentamicin is applied at higher doses to
archive a bactericidal effect,37,38 but within host cells or in the surrounding tissue it must be suspected that much lower doses could
exert SCV-promoting activity.
Rifampicin was the only compound to completely clear infected
host cells in the acute and chronic stages of infection, even when
different strains were tested. For rifampicin a high intracellular
accumulation has been described,32 but pharmacodynamic studies revealed that the intracellular activities are independent of
the level of drug accumulation.27,32 Although rifampicin failed to
demonstrate significant intracellular bactericidal effects in an
infection model of macrophages,32 it was the most effective antimicrobial compound in our osteoblast cell culture system. The high
antimicrobial activity of rifampicin in bone cells is in line with the
successful treatment of our haematogenous osteomyelitis model
in the acute stage and with many clinical studies that encourage
the adjuvant use of rifampicin for the treatment of S. aureus
bone infections.22,23
Taking the results of this study together, we developed in vitro
and in vivo long-term osteomyelitis models to investigate the antimicrobial tolerance/resistance that is associated with pathogenic
adaptation mechanisms. The early formation of dynamic SCVs
provides increased resistance to antibiotics that interfere with
cell wall synthesis or act in a bacteriostatic way. A further problematic phenomenon is that low doses of defined antimicrobial
compounds (gentamicin, moxifloxacin and clindamycin) induce
the formation of SCVs, which could even promote the development of a chronic infection course. Rifampicin showed the highest
activity against intracellular persisting bacteria and was further
effective in decreasing the bacterial load in the acute stage of
the murine osteomyelitis model. In the chronic stage of the in
vivo model, none of the tested antibiotics reduced the bacterial
load or degree of inflammation and bone deformation, indicating
that many different mechanisms impede the action of the commonly used antibiotics.
Acknowledgements
We thank B. Schuhen, K. Broschwig and C. Pilz for excellent technical
assistance. We thank Dr Adriana Perez for statistical support.
Funding
This work was supported by DFG grants of the Transregional Collaborative
Research Centre 34 (C12), by the Bundesministerium für Bildung und
Forschung (BMBF; grant 0315830B) and by the Center for Sepsis Control
and Care (CSCC) of the Federal Ministry of Education and Research
(BMBF: SKZ01EO1502).
Transparency declarations
None to declare.
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