International Journal of Antimicrobial Agents 32 (2008) 525–528
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents
journal homepage: http://www.elsevier.com/locate/ijantimicag
In vitro activity of daptomycin against Gram-positive cocci:
the first multicentre study in Greece
E. Mallia, I. Spiliopouloub, F. Kolonitsioub, D. Klapsac, E. Giannitsiotid, K. Pantelidic, A. Prattic,
M. Panopouloue, S. Grapsae, E. Alepopouloue, I. Neonakisf, F. Frantzidoug, S. Alexiou-Danielg,
D. Bakolah, C. Koutsia-Carouzoui, H. Malamou-Ladaj, L. Zervak, E. Vlahakil,
S. Kartali-Ktenidoue, E.D. Anastassioub, E. Petinakia,c,∗
aDepartment of Microbiology, University Hospital of Larissa, Larissa, Greece
bDepartment of Microbiology, School of Medicine, University of Patras, Patras, Greece
cDepartment of Molecular Microbiology, Institute of BioMedical Research and Technology, Larissa, Greece
d4th Department of Internal Medicine, ATTIKON University General Hospital, Athens, Greece
eDepartment of Microbiology, University Hospital of Alexandroupolis, Alexandroupolis, Greece
fDepartment of Microbiology, University Hospital of Heraklion, Crete, Greece
gDepartment of Microbiology, AHEPA University Hospital, Thessaloniki, Greece
hDepartment of Microbiology, General Hospital of Lamia, Lamia, Greece
iDepartment of Microbiology, General Hospital ‘Asclepeion’, Voula, Athens, Greece
jDepartment of Microbiology, General Hospital ‘Georgios Genimatas’, Athens, Greece
kDepartment of Microbiology, ATTIKON University General Hospital, Athens, Greece
lDepartment of Microbiology, General Hospital of Volos, Volos, Greece
a r t i c l e i n f o
Received 9 May 2008
Accepted 29 May 2008
a b s t r a c t
A total of 10420 Gram-positive cocci (including staphylococci, enterococci and various groups of strepto-
for their susceptibility to daptomycin. The minimum inhibitory concentration (MIC) was determined by
the broth microdilution method. Daptomycin demonstrated very high activity against Enterococcus fae-
tococcus pyogenes (MIC50=0.12mg/L and MIC90=0.50mg/L), Streptococcus agalactiae (MIC50=0.09mg/L
and MIC90=0.12mg/L), Streptococcus pneumoniae (MIC50=0.24mg/L and MIC90=0.5mg/L) and viridans
group streptococci (MIC50=0.50mg/L and MIC90=0.89mg/L). Resistance to linezolid and vancomycin for
enterococci and to penicillin for streptococci appears to be independent of reduced susceptibility to
daptomycin. On the other hand, daptomycin was also active against meticillin-resistant Staphylococcus
aureus (MIC50=0.44mg/L and MIC90=0.78mg/L) and meticillin-resistant coagulase-negative staphy-
lococci (MIC50=0.24mg/L and MIC90=0.44mg/L); however, 0.9% of the staphylococci tested had an
MIC>1mg/L, which is the Clinical and Laboratory Standards Institute breakpoint proposed for suscep-
tibility. For all tested organism groups, resistance to daptomycin was not associated with glycopeptide
© 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
The recent increase in infections caused by resistant Gram-
positive pathogens, such as meticillin-resistant staphylococci,
glycopeptide-intermediate-resistant staphylococci, vancomycin-
∗Corresponding author. Present address: Department of Microbiology, Medical
School, Papakyriazi 22, Larissa, Greece. Tel.: +30 2410 682517/682535;
fax: +30 2410 682517.
E-mail addresses: firstname.lastname@example.org, email@example.com (E. Petinaki).
resistant enterococci and penicillin-resistant pneumococci, has
prompted a search for new antimicrobials. The introduction of
quinupristin/dalfopristin and linezolid in clinical practice in 2003
was followed by the emergence of enterococci and staphylococci
resistant to these agents . Thus, the need for new treatment
by Streptomyces roseosporus that acts by binding in the presence of
calcium to bacterial membranes, causing depolarisation and thus
cell death. The agent has a unique chemical structure and has
shown activity against most Gram-positive bacterial species . A
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
E. Malli et al. / International Journal of Antimicrobial Agents 32 (2008) 525–528
once-daily dosing regimen makes daptomycin a promising alter-
native for the treatment of nosocomial infections caused by
multiresistant Gram-positive bacteria. Daptomycin has been
approved for the treatment of complicated skin and soft-tissue
infections and bacteraemia, including right-sided endocarditis;
since 2007, the agent has been occasionally used in Greek hospitals
for the treatment of serious infections caused by bacteria that are
resistant to other antimicrobial agents.
The purpose of this study was to evaluate the in vitro activity
of daptomycin against Gram-positive cocci in Greek hospitals and
to define a baseline for monitoring possible future emergence of
resistance to daptomycin in our clinical setting.
2. Materials and methods
2.1. Bacterial isolates and study design
From January 2006 to December 2007, a total of 10420
Gram-positive cocci, including 3600 Staphylococcus aureus (1500
meticillin-resistant and 2100 meticillin-sensitive), 2280 Enterococ-
cus faecalis (118 vancomycin-resistant and 20 linezolid-resistant),
1000 Enterococcus faecium (120 vancomycin-resistant and 25
linezolid-resistant), 1800 coagulase-negative staphylococci (CoNS)
(1320 meticillin-resistant and 480 meticillin-sensitive), 480 Strep-
tococcus pneumoniae (67 penicillin-non-susceptible isolates), 490
Streptococcus agalactiae, 600 Streptococcus pyogenes and 170 viri-
dans group streptococci, were tested for their susceptibility to
daptomycin. The isolates were recovered from clinically significant
specimens (blood, pus, pleural fluid, etc.) in ten Greek hospitals
located in different areas of the country (Northern, Central and
Southern Greece). Each participating institution was requested to
collect a minimum of 700 Gram-positive cocci, equally distributed
Microbiology of the University Hospital of Larissa, Greece, for sus-
ceptibility testing. A single isolate per patient was requested, along
with certain demographic (patient age, gender, location) and clin-
ical information (source of specimen, antibiotic therapy). Strains
were only included in the study if the additional information had
been provided by the referring institution. Isolates were identified
according to local protocols adapted by each referring institution
and were sent in transport swabs (Culturette®; Becton Dickinson
Microbiology Systems, Sparks, MD) to the co-ordinating laboratory
at the University Hospital of Larissa. Upon receipt, isolates were
subcultured onto 5% sheep blood agar to ensure purity. Identifi-
cation was confirmed using a combination of conventional and
molecular methods. Staphylococci were identified by amplifying
the tuf gene followed by restriction analysis, enterococci by ampli-
by agglutination tests using commercial kits (Pneumoslidex and
Streptoslidex; bioMérieux, La Balme les Grottes, France) . Iden-
tification of viridans group streptococci was performed using ID32
2.2. Susceptibility testing
Minimum inhibitory concentrations (MICs) to daptomycin were
determined by the broth microdilution method. Microtitre wells
were prepared in house using cation-adjusted Mueller–Hinton
broth (Difco Laboratories, Detroit, MI) with a final concentration
of 50mg/L calcium. Two-fold dilutions of daptomycin ranged from
0.008mg/L to 8mg/L [4,5]. For testing streptococci, 5% sheep blood
was also added. Microtitre broth MICs for daptomycin were com-
pared with the new Etest strips (AB BIODISK, Solna, Sweden),
which incorporate a constant and optimal level of Ca2+within the
daptomycin gradient and enable the use of standard Mueller–
Hinton agar plates . Etest MICs were rounded up to the next
higher log2dilution corresponding to broth microdilution MICs
for the purpose of comparison. Based on Clinical and Laboratory
Standards Institute (CLSI) criteria, the isolates were categorised
as susceptible or resistant; a daptomycin susceptible breakpoint
viridans group streptococci, whilst ≤4mg/L was used for the inter-
pretation of enterococci. In the absence of agreed CLSI breakpoints
for S. pneumoniae, the breakpoints for staphylococci were used.
All isolates were also tested against selected comparator agents
(ampicillin, penicillin, oxacillin, cefoxitin, vancomycin, teicoplanin,
linezolid, quinupristin/dalfopristin and erythromycin) by disk dif-
fusion method using CLSI criteria . Determination of MICs to
these agents was assessed by Etest according to the manufacturer’s
instructions. The following quality control organisms were tested
concurrently: E. faecalis ATCC 29212, S. aureus ATCC 29213 and S.
pneumoniae ATCC 49619.
2.3. Molecular methods
The presence of resistance genes vanABCDE, mecA and sat was
determined as markers for glycopeptide, ?-lactam and dalfopristin
resistance, respectively [3,8]. Clonality of isolates was assessed by
pulsed-field gel electrophoresis (PFGE) after digestion of chromo-
somal DNA with SmaI .
Of the 10420 isolates, 48% caused bloodstream infections, 24%
skin and soft-tissue infections, 12% pneumonia and 16% infections
of other sites. Of all the isolates tested, 99.52% were susceptible
and only 50 isolates (0.48%) were found to be resistant to dap-
tomycin. All the resistant isolates were correctly characterised by
Etest. On the other hand, 12 S. aureus isolates susceptible by the
microdilution method (MIC range 0.256–1mg/L) were falsely char-
acterised as resistant by Etest (MIC=1.5mg/L). Re-determination
of MICs by both methods revealed that the isolates were truly sus-
ceptible. The false-resistant Etest results were probably associated
with an inoculum effect.
Table 1 summarises the MICs at which 50% and 90% of the iso-
lates were inhibited (MIC50and MIC90, respectively) as well as the
MIC range. For meticillin-resistant S. aureus isolates, the MIC val-
ues of daptomycin ranged from 0.128mg/L to 2mg/L, with MIC50
and MIC90values 0.44mg/L and 0.78mg/L. Forty-six isolates col-
lected from different clinical settings had a MIC of 2mg/L. PFGE
Minimum inhibitory concentration (MIC) range and MIC at which 50% and 90% of
the isolates were inhibited (MIC50and MIC90, respectively) for a large collection of
Gram-positive cocci isolated in Greek hospitals
Viridans group streptococci
MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-sensitive S.
aureus; MRCoNS, meticillin-resistant coagulase-negative staphylococci; MSCoNS,
meticillin-sensitive coagulase-negative staphylococci.
E. Malli et al. / International Journal of Antimicrobial Agents 32 (2008) 525–528
analysis revealed that the daptomycin-resistant S. aureus strains
were genetically related to ST-80, ST-30 and ST-239 clones and
susceptible S. aureus isolates the MIC50and MIC90values were
Only three isolates had MICs of 2mg/L and were susceptible to
CoNS were 0.24mg/L and 0.44mg/L, respectively, with a MIC range
of 0.128–4mg/L. Among the isolates tested, 10% exhibited inter-
mediate resistance to both glycopeptides, without a significant
decrease in their daptomycin susceptibility. Only one Staphylo-
coccus epidermidis isolate had a MIC to daptomycin of 4mg/L.
This isolate had MICs to vancomycin and to teicoplanin of 8mg/L
and 16mg/L, respectively, and was isolated from a patient in
the orthopaedic ward of the University Hospital of Volos; the
susceptible CoNS demonstrated that they were a little more
susceptible than meticillin-resistant CoNS. As shown in Table 1,
MICs ranged from 0.032mg/L to 1mg/L, with MIC50and MIC90
values of 0.12mg/L and 0.24mg/L, respectively.
The MIC range ofdaptomycin
0.256–4mg/L and the MIC50and MIC90were 1mg/L and 1.36mg/L,
respectively. No differences were observed between vancomycin-
resistant and vancomycin-susceptible E. faecalis.
The MIC values for daptomycin against E. faecium ranged
between 0.512mg/L and 4mg/L, with MIC50and MIC90values of
1.36mg/L and 1.90mg/L, respectively. Among E. faecium, 12% of
isolates were resistant to vancomycin and teicoplanin carrying the
zolid and quinupristin/dalfopristin, respectively. No differences in
the MIC values were observed between enterococci with charac-
terised resistance determinants and the susceptible isolates.
The range of MIC values obtained for daptomycin against pneu-
mococcal isolates was 0.128–1mg/L. The MIC50and MIC90were
MIC90values were ca. two dilution tubes higher than the MIC50
and MIC90values obtained in previous studies . Penicillin resis-
tance and penicillin intermediate-resistance were observed in 3%
in 18% of isolates of our collection.
The MIC ranges for Group A, Group B and viridans group strep-
tococci were 0.128–1, 0.064–0.5 and 0.064–1mg/L, respectively.
Their MIC50and MIC90values are described in Table 1. Results
obtained were similar to those of previous studies, indicating
that the viridans group streptococci had MICs (MIC50=0.50mg/L
and MIC90=0.89mg/L) greater than the other streptococcal groups
(MIC50=0.12mg/L and MIC90=0.50mg/L for S. pyogenes and
MIC50=0.09mg/L and MIC90=0.12mg/L for S. agalactiae). Only the
group of viridans group streptococci exhibited 8% intermediate-
resistance and 3% resistance to penicillin.
susceptibility results between hospitals were observed. Since the
middle of 2007, the agent was occasionally used in three of the ten
participating hospitals, mainly in the Intensive Care Units.
This is the first multicentre study of the activity of dapto-
mycin against a large collection of Gram-positive cocci isolated
from clinically significant specimens from ten Greek medical cen-
European countries have demonstrated that the agent is active
A major problem in Greece is the nosocomial dissemina-
tion of vancomycin-resistant E. faecium; the use of linezolid for
the treatment of such infections has led to the emergence of
linezolid/vancomycin-resistant E. faecium . Thus, of particular
clinical importance is the fact that daptomycin was active against
enterococci resistant even to linezolid and vancomycin. On the
other hand, daptomycin also showed excellent activity against
?-haemolytic streptococci, pneumococci and viridans group strep-
tococci. However, in the present study, daptomycin was recognised
as a little less active against S. aureus and CoNS in comparison with
the other Gram-positive cocci; 0.9% of staphylococci (49 S. aureus
filing studies of S. aureus isolates from the USA and Europe have
shown that ca. 1% of S. aureus have a MIC of 2mg/L [6,12].
We noted that all daptomycin-resistant S. aureus in our col-
lection were susceptible to glycopeptides, whilst the unique
daptomycin-resistant S. epidermidis exhibited reduced suscep-
tibility to glycopeptides. Since a possible linkage of reduced
susceptibility to vancomycin and non-susceptibility to daptomycin
was suggested, the mechanism of the linkage remains unclear at
this time. There is evidence that thickened cell walls character-
istic of staphylococci with reduced susceptibility to vancomycin,
the metabolic changes that accompany the formation of thick-
ened cell walls and mutations in the mprF and yycG loci may
play a role in reduced susceptibility to daptomycin [13,14]. Clinical
microbiologists and infectious diseases clinicians should be aware
that staphylococci showing reduced susceptibility to vancomycin
may also demonstrate reduced susceptibility to daptomycin. Such
strains must be tested in the clinical microbiology laboratory for
their susceptibility to daptomycin by a reliable and easy to use
method. The new Etest strips give results comparable with the CLSI
reference method .
Our study provides current trends on daptomycin resistance in
Greece just less than 18 months post benchmark. Unfortunately, no
systematic data on daptomycin consumption in the Greek health-
care setting are currently available. It could only be speculated that
comial bacteraemia. Infections mostly treated with daptomycin in
our country are in line with current guidelines: bacteraemias and
skin and soft-tissue infections caused by Gram-positive bacteria.
In summary, this study confirms the good in vitro activity of
daptomycin against a range of Gram-positive pathogens recently
isolated in Greek hospitals. Treatment of Gram-positive pathogens
has become challenging for clinicians. Daptomycin appears to be
an excellent alternative therapeutic option for serious infections
caused by multidrug-resistant organisms. Daptomycin resistance
in staphylococci is still relatively low in our study. However, if
daptomycin is overused or misused in clinical practice, this might
promote emerging staphylococcal resistance. This novel antibiotic
enhanced within all healthcare settings in Greece towards the pru-
Funding: This study was funded by an educational/research
grant from the University of Thessalia, Greece, awarded to EP.
Competing interests: None declared.
Ethical approval: Not required.
Infections due to vancomycin-resistant Enterococcus faecium resistant to line-
zolid. Lancet 2001;357:1179.
528 Download full-text
E. Malli et al. / International Journal of Antimicrobial Agents 32 (2008) 525–528
 Canepari P, Boaretti M, Lleó MM, Satta G. Lipoteichoic acid as a new target for
activity of antibiotics: mode of action of daptomycin (LY146032). Antimicrob
Agents Chemother 1990;34:1220–6.
 Pratti A, Karanika M, Maniatis AN, Petinaki E, Spiliopoulou I, Kolonitsiou F, et al.
Activity of linezolid against Gram-positive cocci: a multicentre study in Greek
hospitals. Int J Antimicrob Agents 2007;29:604–5.
 Clinical and Laboratory Standards Institute. Performance standards for antimi-
crobial susceptibility testing. Approved standard. M100-S16. Wayne, PA: CLSI,
 National Committee for Clinical Laboratory Standards. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically. Approved
standard. M7-A6. Wayne, PA: NCCLS, 2003.
ing daptomycin-susceptible from non-daptomycin-susceptible Staphylococcus
aureus isolates. J Clin Microbiol 2006;44:3098–104.
 National Committee for Clinical Laboratory Standards. Performance standards
for antimicrobial disk susceptibility tests. Approved standard. M2-A8. Wayne,
PA: NCCLS, 2003.
 Karanika M, Prati A, Kiritsi M, Spiliopoulou I, Neonakis I, Anifantaki M, et
al. Reduced susceptibility to quinupristin/dalfopristin in Enterococcus fae-
cium in Greece without prior exposure to the agent. Int J Antimicrob Agents
 Fluit AC, Schmitz F-J, Verhoef J, Milatovic D. Daptomycin in vitro suscep-
tibility in European Gram-positive clinical isolates. Int J Antimicrob Agents
 Sader HS, Watters AA, Fritsche TR, Jones RN. Daptomycin antimicrobial activ-
ity tested against methicillin-resistant staphylococci and vancomycin-resistant
enterococci isolated in European medical centers (2005). BMC Infect Dis
 Jonhson AP, Mushtaq S, Warner M, Livermore DM. Activity of dapto-
mycin against multi-resistant Gram-positive bacteria including enterococci
and Staphylococcus aureus resistant to linezolid. Int J Antimicrob Agents
 Jevitt LA, Smith AJ, Williams PP, Raney PM, McGowan Jr JE, Tenover FC. In
vitro activities of daptomycin, linezolid and quinupristin–dalfopristin against
a challenge panel of staphylococci and enterococci, including vancomycin-
intermediate Staphylococcus aureus and vancomycin-resistant E. faecium.
Microb Drug Resist 2003;9:389–93.
 Cui L, Tominaga E, Neoh HM, Hiramatsu K. Correlation between reduced dapto-
mycin susceptibility and vancomycin resistance in vancomycin-intermediate
 Friedman L, Alder JD, Silverman JA. Genetic changes that correlate with
reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob
Agents Chemother 2006;50:2137–45.
 Fuchs PC, Barry AL, Brown SD. Evaluation of daptomycin susceptibility testing
by Etest and the effect of different batches of media. J Antimicrob Chemother