Drug Resistance Updates 17 (2014) 1–12

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Drug Resistance Updates

journal homepage: www.elsevier.com/locate/drup

Review

Linezolid update: Stable in vitro activity following more than a decade

of clinical use and summary of associated resistance mechanisms
Rodrigo E. Mendes a,∗ , Lalitagauri M. Deshpande a , Ronald N. Jones a,b
a
JMI Laboratories, North Liberty, IA 52317, USA
b
Tufts University School of Medicine, Boston, MA 02111, USA

a r t i c l e i n f o a b s t r a c t

Keywords: Linezolid, approved for clinical use since 2000, has become an important addition to the anti-Gram-
Linezolid positive infection armamentarium. This oxazolidinone drug has in vitro and in vivo activity against
Surveillance essentially all Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA)
Oxazolidinones
and vancomycin-resistant enterococci (VRE). The in vitro activity of linezolid was well documented
Resistance
prior to its clinical application, and several ongoing surveillance studies demonstrated consistent and
LEADER
ZAAPS potent results during the subsequent years of clinical use. Emergence of resistance has been lim-
ited and associated with invasive procedures, deep organ involvement, presence of foreign material
and mainly prolonged therapy. Non-susceptible organisms usually demonstrate alterations in the 23S
rRNA target, which remain the main resistance mechanism observed in enterococci; although a few
reports have described the detection of cfr-mediated resistance in Enterococcus faecalis. S. aureus isolates
non-susceptible to linezolid remain rare in large surveillance studies. Most isolates harbour 23S rRNA
mutations; however, cfr-carrying MRSA isolates have been observed in the United States and elsewhere.
It is still uncertain whether the occurrences of such isolates are becoming more prevalent. Coagulase-
negative isolates (CoNS) resistant to linezolid were uncommon following clinical approval. Surveillance
data have indicated that CoNS isolates, mainly Staphylococcus epidermidis, currently account for the major-
ity of Gram-positive organisms displaying elevated MIC results to linezolid. In addition, these isolates
frequently demonstrate complex and numerous resistance mechanisms, such as alterations in the ribo-
somal proteins L3 and/or L4 and/or presence of cfr and/or modifications in 23S rRNA. The knowledge
acquired during the past decades on this initially used oxazolidinone has been utilized for developing
new candidate agents, such as tedizolid and radezolid, and as linezolid patents soon begin to expire,
generic brands will certainly become available. These events will likely establish a new chapter for this
successful class of antimicrobial agents.
© 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Antimicrobial spectrum and activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Pre-FDA approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Post-FDA approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Resistance development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. Resistance mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1. Mutations in 23S rRNA and ribosomal proteins L3 and L4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.2. Acquired resistance mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.3. Resistance mechanisms other than target site modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

∗ Corresponding author at: JMI Laboratories, 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317, USA. Tel.: +1 319 665 3370; fax: +1 319 655 3371.
E-mail address: rodrigo-mendes@jmilabs.com (R.E. Mendes).

http://dx.doi.org/10.1016/j.drup.2014.04.002
1368-7646/© 2014 Elsevier Ltd. All rights reserved.
2 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
1. Introduction
During the last decades, only a few new antimicrobial agents
have been approved for clinical use by the United States (USA)
Food and Drug Administration (FDA), European Medicines Agency
(EMA) and other regulatory agencies (Gould and Bal, 2013). Among
those agents with Gram-positive organism coverage, ceftaroline
and telavancin are improved analogues of known scaffolds, while
daptomycin and linezolid are its sole representative molecules
of entirely new classes of antimicrobials (i.e. lipopeptides and
oxazolidinones, respectively). Linezolid received a priority review
by the FDA, which approved this drug for clinical use in March
of 2000, and linezolid has become an important addition to the
anti-Gram-positive infection armamentarium. Indications consist
of treatment for uncomplicated and complicated skin and skin
structure infections (cSSSI) and hospital- and community-acquired
pneumonia caused by Gram-positive pathogens (Zyvox, 2010).
Linezolid is also indicated for the treatment of vancomycin-
resistant Enterococcus faecium (VRE) infections (including cases
with concurrent bacteremia).
Since the ribosome represents the core for protein synthesis in
all living cells, numerous antimicrobial agents targeting the pep-
tidyltransferase centre (PTC) of the large ribosomal subunit have
been developed. Targeting the PTC remains one of the main advan-
tages of the oxazolidinones due to the number of rRNA genes, which
minimizes the emergence of resistance (Toh et al., 2007). Oxazolidi-
nones were initially known to affect protein synthesis during the
initiation phase of translation (Lin et al., 1997; Shinabarger et al.,
1997). A later study offered additional insights and demonstrated
that linezolid interacts with the 23S rRNA (A2602; Escherichia coli
numbering used throughout) and prevents binding or proper place-
ment of aminoacyl-tRNA in the PTC site (Leach et al., 2007).
However, linezolid also seems to interact with ribosomal protein
L27, ribosomal-associated protein LepA and tRNA (Colca et al.,
2003). These interactions are still unknown, but could be associated
with ribosome formation and fidelity of translation (Colca et al.,
2003).
Linezolid is available in intravenous and oral formulations,
which have provided this agent as an attractive alternative for
treating numerous infection types, including respiratory tract
infection caused by methicillin-resistant Staphylococcus aureus
(MRSA) and other serious multidrug-resistant (MDR) infections due
to vancomycin-resistant enterococci (VRE). The clinical and com-
mercial success of linezolid has prompted many pharmaceutical
companies to investigate and develop oxazolidinone-like com-
pounds (Michalska et al., 2013). Several molecules have been devel-
oped as candidates, but only tedizolid and radezolid have advanced
into clinical trials (Shaw and Barbachyn, 2011). This review pro-
vides a summary update for linezolid with regard to the pre-
and post-FDA approval study results related to in vitro antimicro-
bial activity and spectrum, and development and dissemination of
resistance mechanisms. A thorough review on the activity in vitro of
other oxazolidinones tested against Gram-positive isolates, includ-
ing linezolid-resistant strains, was recently published and this topic
will not be addressed this topic (Shaw and Barbachyn, 2011).
2. Antimicrobial spectrum and activity
2.1. Pre-FDA approval
Investigational studies performed during the development of
linezolid documented a broad and potent antimicrobial activ-
ity against Gram-positive organisms. Linezolid displayed in vitro
inhibitory activities against numerous clinically relevant bacte-
rial species, including staphylococci (methicillin-susceptible and
-resistant), enterococci (vancomycin-susceptible and -resistant),
streptococci, Corynebacterium spp., Mycobacterium tuberculosis and
some species of anaerobic bacteria (Zurenko et al., 1996).
Table 1 summarizes the MIC50 and MIC90 values for line-
zolid when tested against Gram-positive clinical isolates obtained
from studies performed prior to the FDA approval. Overall, these
studies reported linezolid MIC results between 0.25 and 4 mg/L,
and non-susceptible results were only initially obtained against
laboratory-derived mutants after numerous daily passaging exper-
iments in drug containing media. During the pre-FDA approval era,
linezolid demonstrated a normal MIC distribution (i.e. Gauss curve)
when tested against clinical Gram-positive pathogens, which was
confirmed by several local and multicentre in vitro studies con-
ducted by investigators on several continents (Zurenko et al.,
1996; Jones et al., 1996; Wise et al., 1998; von Eiff and Peters,
1999; Noskin et al., 1999; Johnson et al., 2000; Rybak et al., 1998,
2000).
Overall, studies evaluating the in vitro activity and spectrum
of linezolid have reported MIC50 values of 2–4, 2–4 and 1–2 mg/L
when tested against staphylococci, enterococci and streptococci,
respectively. However, Wise et al. (1998) published linezolid MIC
results when tested against staphylococci and enterococci lower
than other authors, with highest linezolid values at 1 mg/L (see
Table 1). In addition, Jones et al. (1996) published linezolid MIC50
values (1 mg/L) against enterococci similar to those reported by
Wise et al. (1998), which were, in general, lower than those from
other publications.
The variations observed in the linezolid MIC results in these
studies can, perhaps, be explained by the different guidelines
(EUCAST versus CLSI) and methods (broth microdilution and agar
dilution versus Etest). Also, some studies utilized agar dilution
techniques and IsoSensititre media (Wise et al., 1998; von Eiff
and Peters, 1999; Johnson et al., 2000), while other investigators
made use of broth microdilution methods and the Mueller-Hinton
medium (Zurenko et al., 1996; Jones et al., 1996; Noskin et al.,
1999; Rybak et al., 1998, 2000). Regardless, the different guidelines,
methods and media utilized only indicate the presence of several
uncontrolled variables among early studies and imply methodolog-
ical differences, and there was no correlation of the method applied
with the MIC results obtained. The MIC endpoint reading itself
can be another potential element causing variability. Trailing is a
common phenomenon when testing linezolid, requiring additional
expertise for determining the correct endpoint value as currently
stated in some international standardized methods (Biedenbach
and Jones, 1997, 2001, 2003; Poppe et al., 2006; Worth et al., 1996;
Tenover et al., 2007).
These technical difficulties along with the lack of reading stan-
dards when testing such drug for susceptibility provide additional
challenges for clinical microbiologists and research personnel. Dur-
ing the January 2006 Clinical and Laboratory Standards Institute
(CLSI) meeting (Miami, FL), the Staphylococci Working Group pro-
posed additional language in the M02, M07 and M100 documents to
provide information detail on how to read MIC results and zones of
inhibition in the presence of so-called “trailing endpoints” (Clinical
and Laboratory Standards Institute, 2006). These changes were
proposed to contain the following recommendation: “For some
antimicrobial agents (such as for chloramphenicol, clindamycin,
erythromycin, linezolid and tetracycline), trailing growth can make
endpoint determination difficult. In such cases, the MIC should be
read at the first well that shows a prominent reduction in growth.
Tiny buttons of growth should be ignored”. However, these pro-
posed languages have not been incorporated into the M02, M07
or M100 documents by the time this review article was written.
According to the summary minutes of the 2012 CLSI meeting, this
recommendation will be published in the M07-10 in 2015 (Clinical
and Laboratory Standards Institute, 2012a, 2014).
 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12 3

Summary of linezolid MIC50 and MIC90 values when tested against Gram-positive isolates obtained from investigational studies published prior to FDA approval and those obtained from the most recently reported post-marketing
2.2. Post-FDA approval

Mendes et al.
(2014b)

0.5/1
The activity and spectrum of linezolid has been continually

1/2
1/1

1/2
1/2
1/1
1/1
1/1
monitored via international surveillance programmes, which were
initiated several years prior to product launch with multiple
Zyvox® Antimicrobial Potency Study (ZAPS) (Ballow et al., 2002a,

Mendes et al.
2002b; Bell et al., 2003; Bolmstrom et al., 2002; Jones et al., 2001)
and other programmes (Mutnick et al., 2002). These surveillance
(2014a)

0.5/1
1/2
1/1

1/2
1/2
1/1
1/1
1/1
studies were followed by the Zyvox® Annual Appraisal of Potency
and Spectrum (ZAAPS) Programme developed as a postmarket-
ing risk management during regulatory compliance tool, which
remains active. ZAAPS initially consisted of a worldwide interna-
tional and multicentre surveillance study (Anderegg et al., 2005;
Rybak et al.

Ross et al., 2005). In 2004, the number of sites in the USA was
(2000)

expanded and the USA sampling was separated from ZAAPS and
4/4
2/4
2/4
2/4
4/4

designated as the Linezolid Experience and Accurate Determination

–
–
–
of Resistance (LEADER) Programme (Draghi et al., 2005; Jones et al.,
2007a, 2008; Farrell et al., 2009, 2011; Flamm et al., 2012a, 2013a;
Johnson et al.

Mendes et al., 2014a). Participating sites from ex-USA remained

within the ZAAPS Programme (Mendes et al., 2014b; Jones et al.,
(2000)

2/2
2/2

4/4
4/4
1/2
2/2
2/2

2006, 2007b, 2009, 2010; Ross et al., 2007a, 2009; Flamm et al.,
–b

2012b, 2013b).
The ZAAPS and LEADER surveillance programmes have sys-
tematically surveyed for linezolid resistance since 2002 and 2004,
Noskin et al.

respectively; applying reference and centralized broth microdilu-

(1999)

tion MIC testing, following CLSI methods (Clinical and Laboratory

2/4
2/4
2/4
2/4
2/4
1/1

Standards Institute, 2012b) and CLSI and EUCAST MIC interpre-

–
–

tative criteria (Clinical and Laboratory Standards Institute, 2013;

EUCAST, 2013). The linezolid MIC distributions obtained during the
Peters (1999)

last three surveillance years for LEADER and ZAAPS are displayed
von Eiff and

in Tables 2 and 3, respectively. The postmarketing MIC results for

both programmes are very comparable, which remain very similar
2/2
2/2
1/1

to those described prior to FDA approval by Wise et al. (1998) and

–
–
–
–
–

Jones et al. (1996). Also, Tables 2 and 3 exhibit consistent modal

MIC results (1 mg/L) across all species or groups of organisms and
geography, except for CoNS (modal MIC and MIC50 of 0.5 mg/L for
both programmes).
Rybak et al.
(1998)

2/4
2/4
1/2
2/4
2/2

3. Resistance development
–
–
–
Linezolid MIC50 /MIC90 values in mg/L by reference/method:

The linezolid molecule possesses several advantageous char-

MSSA = methicillin-susceptible S. aureus; MRSA = methicillin-resistant S. aureus.

acteristics, including its synthetic nature, bacterial redundancy of

0.25/0.5
Wise et al.

target site (PTC at the 23S rRNA) and absence of cross-resistance

(1998)

1/1
1/1

1/1
1/1
2/2
1/1
1/1

(more on this later). The synthetic nature of this agent implies a low
probability for naturally-occurring resistance mechanisms, which
explains the in vitro study data described above (Tables 2 and 3)
where the linezolid normal and narrow MIC distribution persist
when tested in large surveys of naive clinical Gram-positive iso-
Jones et al.

lates. In fact, studies pre-FDA approval demonstrated essentially

complete antimicrobial coverage against tested Gram-positive
(1996)

2/2
2/2
1/2
1/1
1/2
1/1
2/4
1/2

pathogens (Ballow et al., 2002a, 2002b; Bolmstrom et al., 2002;

Mutnick et al., 2002).
Serial passaging analyses (selection of spontaneous mutations)
Zurenko et al.

performed during drug development demonstrated that resistance

occurred rarely and at a frequency of <1 resistant mutant per
surveillance programmes in 2012.

(1996)

8 × 1011 CFU of S. aureus ATCC 29213, and with even more diffi-
4/4
4/4
2/2
4/4
2/2
1/1
1/2

culty for enterococci (Zurenko et al., 1996). During Phase 3 clinical

–

trials, resistance to linezolid developed in six patients infected with

Group A streptococci
Group B streptococci

E. faecium (four patients received 200 mg every 12 h, lower than the

Data not available.
Resistance subsets
Organism/Groupa

recommended dose; and two patients received 600 mg every 12 h)

S. pneumoniae

(Zyvox, 2010). In a compassionate use programme, resistance to

S. epidermidis

linezolid developed in eight patients with E. faecium and in one

E. faecium
E. faecalis

patient with Enterococcus faecalis.

MRSA
MSSA
Table 1

After FDA-approval, case reports describing emergence of resis-

a

tance among clinical isolates were published (Gales et al., 2006;

4 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12

Table 2
Linezolid MIC distribution when tested against species and groups of Gram-positive cocci isolated from USA hospitals during the LEADER Programme for the 2010–2012
period.

Organisma (no. tested) Number (cumulative %) of isolates inhibited at linezolid MIC in mg/L of:b MIC (mg/L)

≤0.12 0.25 0.5 1 2 4 8 >8 MIC50 MIC90

S. aureus (9110) 1 (<0.1) 12 (0.1) 573 (6.4) 7602 (89.9) 910 (99.9) 6 (>99.9) 5 (>99.9) 1 (100.0) 1 2
Oxacillin-susceptible (4584) 0 (0.0) 5 (0.1) 251 (5.6) 3778 (88.0) 549 (>99.9) 0 (>99.9) 0 (>99.9) 1 (100.0) 1 2
Oxacillin-resistant (4526) 1 (<0.1) 7 (0.2) 322 (7.3) 3824 (91.8) 361 (99.8) 6 (99.9) 5 (100.0) – 1 1
CoNS (2458) 6 (0.2) 222 (9.3) 1645 (76.2) 544 (98.3) 10 (98.7) 1 (98.8) 0 (98.8) 30 (100.0) 0.5 1
Enterococcus spp. (3031) 2 (<0.1) 13 (0.5) 302 (10.5) 2398 (89.6) 298 (99.4) 6 (99.6) 9 (99.9) 3 (100.0) 1 2
E. faecalis (1970) 1 (0.2) 10 (0.6) 206 (11.0) 1557 (90.1) 192 (99.8) 2 (99.9) 1 (>99.9) 1 (100.0) 1 1
VRE (71) 0 (0.0) 0 (0.0) 9 (12.7) 60 (97.2) 0 (97.2) 1 (98.6) 1 (>99.9) 1 (100.0) 1 1
E. faecium (942) 0 (0.0) 3 (0.3) 75 (8.3) 757 (88.6) 93 (98.5) 4 (98.9) 8 (99.8) 2 (100.0) 1 2
VRE (716) 0 (0.0) 0 (0.0) 62 (8.7) 596 (91.9) 48 (98.6) 3 (99.0) 6 (99.9) 1 (100.0) 1 1
S. pneumoniae (3019) 13 (0.4) 74 (2.9) 974 (35.1) 1904 (98.2) 53 (>99.9) 1 (100.0) – – 1 1
VGS (1456) 19 (1.3) 56 (5.2) 566 (44.0) 787 (98.1) 27 (>99.9) 0 (>99.9) 0 (>99.9) 1 (100.0) 1 1
BHS (2459) 2 (<0.1) 8 (0.4) 629 (26.0) 1815 (99.8) 5 (100.0) – – – 1 1
a
CoNS = coagulase-negative staphylococci; VRE = vancomycin-resistant enterococci (MIC, ≥8 mg/L); VGS = viridans group streptococci; and BHS = ␤-haemolytic strepto-
cocci.
b
Linezolid MIC distribution for the LEADER surveillance sampling years of 2010, 2011 and 2012 were adapted from Flamm et al. (2012a, 2013) and Mendes et al. (2014b).

Meka et al., 2004; Tsiodras et al., 2001; Wilson et al., 2003; Johnson
et al., 2002; Auckland et al., 2002). These clinical isolates had alter-
ations in the linezolid target site (i.e. PTC at 23S rRNA) and these
sporadic reports corroborate the in vitro early experiments suggest-
ing that selection for resistance occurs rarely (Zurenko et al., 1996).
These earlier cases of resistance development to linezolid occurred
during prolonged therapy (>21 days) (Peeters and Sarria, 2005),
as practice contrary to product indications. In addition, these cases
were associated with invasive procedures, deep organ involvement,
and presence of foreign material. Moreover, all patients infected
by isolates that developed resistance during the Phase 3 trials for
linezolid described above had either unremoved prosthetic devices
or undrained abscesses (Zyvox, 2010). Therefore, caution should
be exercised during linezolid therapy, especially during extended
treatment courses to monitor for development of resistance.
The rare clinical occurrence of linezolid resistance development
also coincide with the LEADER Programme results, which reported
non-susceptibility rates between 0.03% and 1.83% when tested
against more than 42,419 clinical isolates of staphylococci and ente-
rococci across nine consecutive surveillance years (2004–2012)
in the USA (Mendes et al., 2014a). The ZAAPS Programme has
documented even lower linezolid non-susceptibility rates (≤1.2%)
among staphylococci and enterococci from non-USA hospitals
(Mendes et al., 2014b) (Fig. 1). More specifically, non-susceptibility
rates against S. aureus noted in LEADER increased from 2004
to 2009, remaining stable in the latter period (2010–2012). The
linezolid non-susceptibility rates against CoNS exhibited an overall
slight increased trend for non-susceptibility during ZAAPS Pro-
grammes, while increasing rates were observed during LEADER
from 2004 to 2008, and decreasing in the following years. Increased
non-susceptibility rates against enterococci reached a peak in
2006 for both the ZAAPS and LEADER Programmes, and fluctu-
ated between 0.4–0.7% and 0.3–1.0%, respectively, in the following
period.
These reported low non-susceptibility rates for linezolid are
likely due to the number of bacterial 23S rRNA target sites alle-
les (4–6 copies) present in Gram-positive isolates and resistance
development requires alteration in several alleles. Further studies
demonstrated that the level of decreased linezolid susceptibility
is associated with the number of alleles mutated (Marshall et al.,
2002). However, these alterations in the 23S rRNA cause a consider-
able bacterial fitness cost, mainly when several alleles are mutated.
Interestingly, it has been reported that isolates containing alter-
ations in 23S rRNA revert to a wildtype genotype and susceptible
phenotype, once removed from selective drug pressure; although
not completely, since a single allele may remain mutated, which
provides rapid selection to resistance if selective pressure returns
(Tsakris et al., 2007).
Most of these study results were obtained from staphylo-
cocci and enterococci and selection for resistance seems to be
extremely rare among streptococci. Although published reports
have described the in vitro selection of laboratory strains of

Table 3
Linezolid MIC distributions when tested against species and groups of Gram-positive cocci isolated from hospitals located in four continents during the ZAAPS Programme
for the 2010–2012 period.

Organisma (no. tested) Number (cumulative %) of isolates inhibited at linezolid MIC in mg/L ofb MIC (mg/L)

≤0.12 0.25 0.5 1 2 4 8 >8 MIC50 MIC90

S. aureus (10836) 2 (<0.1) 26 (0.3) 781 (7.5) 8229 (83.4) 1793 (>99.9) 2 (>99.9) 3 (100.0) – 1 2
Oxacillin-susceptible (7191) 2 (<0.1) 9 (0.2) 374 (5.4) 5408 (80.6) 1397 (>99.9) 1 (100.0) – – 1 2
Oxacillin-resistant (3645) 0 (0.0) 17 (0.5) 407 (11.6) 2821 (89.0) 396 (>99.9) 1 (>99.9) 3 (100.0) – 1 2
CoNS (2718) 5 (0.2) 160 (6.1) 1750 (70.5) 754 (98.2) 19 (98.9) 3 (99.0) 6 (99.2) 21 (100.0) 0.5 1
Enterococcus spp. (2344) 0 (0.0) 8 (0.3) 247 (10.9) 1813 (88.2) 263 (99.4) 4 (99.6) 7 (99.9) 2 (100.0) 1 2
E. faecalis (1376) 0 (0.0) 7 (0.5) 141 (10.8) 1056 (87.5) 166 (99.6) 3 (99.8) 2 (>99.9) 1 (100.0) 1 2
VRE (16) 0 (0.0) 0 (0.0) 2 (12.5) 13 (93.8) 1 (100.0) – – – 1 1
E. faecium (870) 0 (0.0) 1 (0.1) 87 (10.1) 687 (89.1) 89 (99.3) 0 (99.3) 5 (99.9) 1 (100.0) 1 2
VRE (249) 0 (0.0) 0 (0.0) 28 (11.2) 188 (86.7) 29 (98.4) 0 (98.4) 3 (99.6) 1 (100.0) 1 2
S. pneumoniae (3343) 9 (0.3) 100 (3.3) 1239 (40.3) 1942 (98.4) 53 (100.0) – – – 1 1
VGS (1255) 7 (0.6) 50 (4.5) 479 (42.7) 692 (97.8) 27 (100.0) – – – 1 1
BHS (1840) 0 (0.0) 0 (0.0) 342 (18.6) 1491 (99.6) 7 (100.0) – – – 1 1
a
CoNS = coagulase-negative staphylococci; VRE = vancomycin-resistant enterococci (MIC, ≥8 mg/L); VGS = viridans group streptococci; and BHS = ␤-haemolytic strepto-
cocci.
b
Linezolid MIC distribution for the ZAAPS surveillance sampling years of 2010, 2011 and 2012 were adapted from Flamm et al. (2012b, 2013) and Mendes et al. (2014b).
 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
Fig. 1. Summary of the linezolid susceptibility rates, for a 98.0–100.0% range (CLSI
breakpoint criteria) documented during the last nine-years of LEADER and ZAAPS
surveillance programmes (2004–2012). Graph A., B. and C. represent the linezolid
susceptibility rates against S. aureus, CoNS and enterococci, respectively. Graphs
adapted from Mendes et al., 2014 (42, 43). All streptococcal isolates were susceptible
to linezolid, except for one S. pneumoniae isolate (MIC, 4 ␮g/mL) and one S. sanguinis
(MIC, 32 ␮g/mL) included in the LEADER Programme for 2010 and 2011, respectively
(40, 68). Polynomial trendlines are also shown.
streptococci showing decreased susceptibility to linezolid due to
mutations in the PTC (Bozdogan and Appelbaum, 2004; Long and
Vester, 2012), just a few clinical cases of linezolid-resistant strepto-
coccal isolates (Streptococcus pneumoniae and Streptococcus oralis)
have been described to date (Flamm et al., 2012a, 2013a; Wolter
et al., 2005; Mutnick et al., 2003). Those reports demonstrated
that these clinical and laboratory isolates had mutations in the 23S
rRNA or amino acid substitutions in L4. In addition, we recently
described a case of linezolid-resistant Streptococcus sanguinis dis-
playing a linezolid MIC result of 32 mg/L, also associated with a
prolonged therapy course (79 days) (Mendes et al., 2013a). This
isolate demonstrated several mutations in the 23S rRNA (T2211C,
5
T2406C, G2576T, C2610T) and the L22 protein (N56D), and in con-
trast to other results, this isolate did not revert into a wildtype
phenotype/genotype, despite numerous drug-free daily passages
and high fitness cost associated with several ribosomal mutations.
Another linezolid characteristic has been the absence of cross
resistance, which was demonstrated by an early investigation
utilizing several isolates possessing a variety of resistance mech-
anisms (Fines and Leclercq, 2000). Even isolates resistant to other
ribosomal target agents possessing overlapping target sites with
linezolid, remained susceptible to this drug (Long et al., 2010).
Miller et al. (2008) also subsequently reported that mutant isolates
selected with the pleuromutilin, tiamulin, did not exhibited resis-
tance to linezolid, while those selected with linezolid were resistant
to both linezolid and tiamulin. These findings were explained by the
development of specific resistance mechanisms. Isolates selected
with tiamulin developed L3 alterations, while linezolid promoted
the development of 23S rRNA mutations. Isolates carrying the lat-
ter, more specifically at positions G2447, A2503, T2504, G2505 and
G2576 exhibit linezolid and chloramphenicol resistance, but no
correlation was observed for linezolid, clindamycin and valnemulin
(Long et al., 2010).
These previous studies indicate that the presence of cross resis-
tance remained unidirectional, and isolates resistance to some
ribosomal targeting agents remained susceptible to linezolid. This
phenomenon was most likely due to the unique interactions of the
oxazolidinone with the ribosome PTC cavity, which are specific
and allow drug activity (Lin et al., 1997; Leach et al., 2007; Colca
et al., 2003; Shinabarger et al., 1997b). The lack of cross resistance
remained valid for several years until a new mechanism (cfr) was
detected in staphylococcal isolates initially recovered from animals
(Schwarz et al., 2000). Additional details on cfr are described in the
next section.
4. Resistance mechanisms
4.1. Mutations in 23S rRNA and ribosomal proteins L3 and L4
It has been recognized since the pre-FDA approval of linezolid
that alterations in the 23S rRNA are responsible for resis-
tance development. The PTC binding pocket comprises conserved
nucleotides, which interact directly with linezolid. In addition,
other nucleotides located more distally, also confer decreased
levels of susceptibility. Several previous literature reviews cover-
ing linezolid have specifically described the 23S rRNA alterations
associated with linezolid resistance (Shaw and Barbachyn, 2011;
Bozdogan and Appelbaum, 2004; Long and Vester, 2012; Stefani
et al., 2010; Meka and Gold, 2004). Therefore, these mutations
will not be detailed here. However, of note, alterations in the 23S
rRNA are the most common resistance mechanism detected among
staphylococcal and enterococcal clinical organisms, and mutation
at the G2576 position is often the only mechanism detected among
enterococci (see Table 4).
In addition to alteration in the 23S rRNA, it has been proposed
more recently that mutations in the L3 and L4 ribosomal proteins
can cause decreased susceptibility to linezolid. In fact, alterations
in L3 have long been associated with resistance to pleuromutilins
(tiamulin, valnemulin and retapamulin) (Bosling et al., 2003; Bock
et al., 1982) and several studies have reported similar findings
(Miller et al., 2008; Gentry et al., 2007; Kosowska-Shick et al., 2006;
Pringle et al., 2004). During an in vitro selection study of mutants
under high concentrations of linezolid and tedizolid, several L3
and L4 mutations were detected and these alterations were sug-
gested to confer decreased susceptibility to linezolid (Locke et al.,
2009a, 2009b). However, few studies have confirmed the associa-
tion of each of L3 and/or L4 alterations with the levels of linezolid
6 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12

Table 4
Summary of clinical isolates displaying elevated linezolid MIC results (≥4 mg/L) that were screened for resistance mechanisms during ZAPS, ZAAPS, LEADER and other
surveillance programmes.

Country Year Organism (no.) Resistance mechanism

23S rRNAa Ribosomal proteins Methyltransferase

L3 L4 cfr

Belgium 2006 S. aureus (1) WT S145 deletion WT +

Brazil 2005 S. aureus (1) G2576T WT WT −

2012 S. aureus (1) WT WT V142I +
2006/2010–2012 S. epidermidis (8) G2576T WT WT −
2011 S. hominis (1) G2576T F147I/M156T S77T −
2011 S. lugdunensis (1) G2576T WT WT −
2011 S. haemolyticus (1) G2576T WT WT −
2007/2011 E. faecalis (2) G2576T WT WT −
2008/2010 E. faecium (2) G2576T WT WT −

France 2010 S. aureus (1) WT WT WT +

2008 S. epidermidis (1) G2576T G137V WT −
2011 S. epidermidis (1) G2576T WT WT +
2011 S. epidermidis (1) WT WT WT +

Germany 2010 S. hominis (1) G2576T M156T WT −

2006–2012 E. faecium (11) G2576T WT WT −

Greece 2003 S. aureus (1) G2576T WT WT −

2011 S. capitis (1) G2576T T83A WT −

Hong Kong 2012 S. aureus (1) G2447T WT WT −

Ireland 2007 S. aureus (1) G2576T WT WT −

2011 E. faecium (1) G2576T WT WT −

Italy 2012 S. aureus (1) G2576T (1) WT WT −

2012 S. aureus (1) WT Q136H/H146 deletion G69R/T70P/G71S −
2006–2007 S. epidermidis (8) WT F147L/A157R WT −
2007 S. epidermidis (1) WT F147L/A157R K68R −
2008–2009 S. epidermidis (1) WT F147L/A157R WT +
2008 S. epidermidis (1) G2447T WT WT −
2010/2012 S. epidermidis (3) G2576T WT WT −
2011 S. epidermidis (1) G2576T F147I WT −
2011–2012 S. epidermidis (2) WT F147L WT +
2012 S. epidermidis (1) WT H146Q/V154L/A157R 71G72 insert −
2012 S. epidermidis (1) C2319T H146Q/V154L 71G72 insert −

Korea 2009 E. faecium (1) G2576T WT WT −

Mexico 2009 S. epidermidis (2) WT S158Y/D159Y WT +

2010 S. epidermidis (2) T2504/C2534T G152D/D159Y WT −
2011 S. epidermidis (1) WT WT WT +
2009/2012 S. cohnii (2) WT S158F/D159Y A133T/V155I +
2009 S. haemolyticus (1) WT WT WT +
2011 S. haemolyticus (1) WT M156T WT +
2010 S. capitis (1) WT G152D N158S −

PR China 2006–2010 E. faecalis (8) WT WT WT −

Panama 2011 E. faecalis (2) WT WT WT +

Poland 2012 E. avium (1) WT WT P171S −

2012 E. faecalis (1) G2576T WT WT −

Romania 2010 S. cohnii (1) WT D108E/S158Y/D159Y A133T/V155I +

2010 S. simulans (1) WT A143R/S144F/D145Y WT −
2011 S. simulans (1) WT N130D/F147S/G152A/A157R WT −

Spain 2010 S. epidermidis (1) G2576T WT WT +

2010 S. epidermidis (1) G2576T WT WT −
2011 S. epidermidis (1) WT G152S WT +

Sweden 2002/2008 E. faecalis (2) WT WT WT −

Taiwan 2012 E. faecalis (1) WT WT WT −

Thailand 2010 E. faecalis (1) WT WT WT +

Turkey 2011 E. faecium (1) G2576T WT WT −

Wales 2008 E. faecalis (1) G2576T WT WT −

USA 2002 S. aureus (1) G2576T WT A118V −

2002/2006–2011 S. aureus (9) G2576T WT WT −
2007/2009–2012 S. aureus (12) WT WT WT +
2008 S. aureus (1) WT G152D WT −
2009/2011 S. aureus (2) WT S145 deletion WT −
 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12 7

Table 4 (Continued)

Country Year Organism (no.) Resistance mechanism

23S rRNAa Ribosomal proteins Methyltransferase

L3 L4 cfr

2010 S. aureus (1) WT G155D/S145 deletion WT −

2011 S. aureus (1) G2576T WT WT +
2012 S. aureus (1) G2576T S145 deletion G69A/T70P/G71S −
2002 S. epidermidis (1) C2534T/G2576T WT WT −
2005–2006/2008–2010 S. epidermidis (10) G2576T H146R/M156T 71G72 insert −
2005/2008 S. epidermidis (2) G2576T M156T WT −
2006 S. epidermidis (1) WT H146Q/F147I WT −
2006/2008 S. epidermidis (2) G2447T WT WT −
2006/2010 S. epidermidis (3) WT A157R WT −
2006–2007/2009/2011 S. epidermidis (12) G2576T WT WT −
2006–2008/2011–2012 S. epidermidis (5) G2576T H146R/V154L/M156T 71G72 insert −
2006–2008 S. epidermidis (3) G2576T F147I WT −
2006 S. epidermidis (1) G2447T A157R WT −
2007 S. epidermidis (1) G2576T H146P/Q136L WT −
2007 S. epidermidis (1) G2447T G152D/D159E WT −
2007 S. epidermidis (1) C2534T H146Q/V154L/A157R 71G72 insert +
2008 S. epidermidis (3) G2576T F147L/M156T 71G72 insert −
2008 S. epidermidis (1) T2504A WT WT −
2008 S. epidermidis (1) G2576T WT N130K −
2008 S. epidermidis (1) WT H146deletion/P151Q WT −
2008/2010–2011 S. epidermidis (3) WT H146Q/V154L/A157R 71G72 insert +
2008 S. epidermidis (1) G2576T G137S/H146P 71G72 insert −
2008–2009/2012 S. epidermidis (4) G2576T G137S/H146P/M156T 71G72 insert −
2008 S. epidermidis (1) G2576T F147L/M156T WT −
2009/2012 S. epidermidis (2) WT V154L/A157R 71G72 insert −
2009 S. epidermidis (1) WT H146Q WT −
2009 S. epidermidis (1) WT V154L/A157R P171S −
2009 S. epidermidis (1) WT H146Q/A157R 71G72 insert +
2009 S. epidermidis (1) WT F147L/M157R WT −
2009 S. epidermidis (1) WT H146Q 71G72 insert −
2009–2012 S. epidermidis (4) G2576T G137D/H146R/V154L/M156T 71G72 insert −
2010 S. epidermidis (1) G2576T V154L/M156T WT −
2010 S. epidermidis (1) G2576T G137D/H146P/M156R G69R/71G72 insert −
2010 S. epidermidis (1) G2576T H146P/M156T WT −
2010 S. epidermidis (1) WT A157R WT +
2010 S. epidermidis (1) WT V154L/A157R WT +
2011 S. epidermidis (1) G2576T Q136L/G137S/H146P 71G72 insert −
2011 S. epidermidis (1) G2576T G137S/H146P/M156T WT −
2011 S. epidermidis (2) G2576T H146R/V154L/M156T WT −
2011 S. epidermidis (1) WT A143R/H146Q/V154L 71G72 insert −
2011–2012 S. epidermidis (2) G2576T G137S/H146P/F147Y/M156T 71G72 insert −
2012 S. epidermidis (1) T2531A G152D/D159K WT −
2012 S. epidermidis (1) C2561T H146Q/V154L/A157R 71G72 insert −
2012 S. epidermidis (1) WT H146Q/V154L/A157R 71G72 insert −
2005–2008 S. haemolyticus (4) G2576T M156T WT −
2006 S. capitis (1) G2576T T83A WT −
2007 S. capitis (1) G2576T WT WT −
2009 S. capitis (1) WT WT WT +
2007 S. simulans (1) G2576T WT WT −
2007–2008 S. hominis (2) G2576T WT WT −
2001–2006 E. faecalis (15) G2576T WT WT −
2008/2010–2012 E. faecalis (5) G2576T WT WT −
2001–2012 E. faecium (91) G2576T WT WT −
2012 E. faecium (1) G2576T WT WT +
2003 E. faecium (2) G2576T WT N130K −
2003 E. faecium (1) WT WT N130K −
2002 S. oralis (1) G2576T WT WT −
2010 S. pneumoniae (1) WT WT Q67K/G69V −
2011 S. sanguinisb (1) G2576T/C2610T WT WT −

WT = wildtype nucleotide or amino acid sequencing.

a
E. coli numbering.
b
Alterations also observed in L22 (N56D, I29V and V110A).

resistance. Wolter et al. demonstrated that deletions in L4 (65WR66
and 68KG69 deletions) were responsible for a four-fold increased
in the linezolid MIC result (Wolter et al., 2005).
Since the recognized association of L3 and L4 with linezolid
decreased susceptibility, several studies have reported the pres-
ence of L3 and/or L4 mutations with or without 23S rRNA or the
acquired cfr gene. The vast majority of these publications described
alterations among staphylococcal clinical isolates within the amino
acids 127 and 174 of L3 protein and amino acids 65 and 72 of L4 pro-
tein (Kosowska-Shick et al., 2010; Locke et al., 2010; Mendes et al.,
2010a, 2010b, 2012, 2013b; Endimiani et al., 2011; Beckert et al.,
2012; LaMarre et al., 2013; Pournaras et al., 2013; Cui et al., 2013;
de Almeida et al., 2013; Baos et al., 2013). Table 4 describes the
linezolid resistance mechanisms observed among clinical isolates
included in several surveillance programmes, including L3 and/or
L4 alterations. Mutations in ribosomal proteins, mainly L3, were
8 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
detected in staphylococci collected as early as 2005 in the USA, and
2006 in Belgium and Italy. A few isolates screened in earlier years
(i.e. 2002, 2003 and 2005–2006 in USA, Greece and Brazil) did not
show ribosomal protein alterations, except for one S. aureus (A118V
in L4) collected in 2002 from the USA. It is not certain whether
this L4 modification alone confers decreased linezolid susceptibil-
ity. However, these results do indicate that alterations in L3 and L4
as a resistance mechanism appeared later in time and the complex-
ity and number of such alterations certainly increased in CoNS in
the last six to eight years.
It is interesting to note that mutations in L3 and L4 proteins
are commonly detected among CoNS clinical isolates, while a small
number of S. aureus and enterococci have demonstrated such muta-
tions. Moreover, L3 proteins show amino acid sequences with a
diversity greater than L4, likely due to the fact that L3 residues
are in close proximity to the PTC (Locke et al., 2009a, 2009b). Other
studies have suggested that L3 alterations may decrease the fitness-
cost associate with 23S rRNA mutations, when the latter is present.
More specifically, Billal et al. (2011) reported that a mutation at
position Y137 in S. pneumoniae L3 (which corresponds to amino
acid F147 in Staphylococcus epidermidis) restores the fitness cost
associated with G2576T. Other authors suggested that L3 and 23S
rRNA alterations may act synergistically, requiring fewer mutated
copies of 23S rRNA alleles; therefore minimizing the fitness costs
associated with 23S rRNA mutations (Mendes et al., 2012).
It is important to mention that some alterations in L3 and
L4 have been observed among linezolid-susceptible isolates, such
as N158S in L4 (Wong et al., 2010). In addition, our group has
previously observed several amino acid alterations in L3 and L4 pro-
tein among linezolid-susceptible Gram-positive clinical organisms,
when studying resistance mechanisms other than those associated
with linezolid. Among these amino acid modifications, the follow-
ing were observed: A82, A83, G152, V188 and E191 in L3 protein
among staphylococci; A50 and V142 in S. aureus L4; and N130 and
G166 in E. faecium L4 protein (unpublished data).
4.2. Acquired resistance mechanism
Linezolid binding site alterations remain the main mechanism
of resistance detected among Gram-positive clinical organisms.
Although theoretically possible, transfer of ribosomal mutations
between pathogenic species has not been reported. However, a
more recent plasmid encoded resistance mechanism (transfer-
able), namely Cfr, has been observed. Cfr is closely related to
RlmN methyltransferase, which belongs to the radical S-adenosyl-
l-methionine (SAM) enzyme superfamily (Yan et al., 2010). RlmN
is an indigenous cellular enzyme that mono-methylates the C2
atom of A2503 at 23S rRNA. On the other hand, Cfr catalyzes the
post-transcriptional methylation of the C8 atom at the same posi-
tion (Toh et al., 2008; Atkinson et al., 2013). This modification
confers a MDR phenotype, affecting the susceptibility to pheni-
col, lincosamide, oxazolidinone, pleuromutilin, and streptogramin
A (PhLOPSA ) compounds (Long et al., 2006).
The Cfr-encoding gene was initially described in 2000 in a
17,108 bp 16.5-kb transferable plasmid (pSCFS1) from an animal
isolate of Staphylococcus sciuri (Schwarz et al., 2000; Kehrenberg
et al., 2004), and was detected in several other staphylococcal
organisms also from animal origin in Europe during investigations
associated with resistance to phenicols (florphenicol and chloram-
phenicol) (Kehrenberg and Schwarz, 2006; Kehrenberg et al., 2009).
In 2005, the first cfr-carrying S. aureus was recovered from a respi-
ratory tract specimen in a patient from Colombia (Toh et al., 2007).
This finding was followed by several other reports describing the
detection of cfr among staphylococci recovered from human spec-
imens (Mendes et al., 2008, 2009, 2010a, 2010b; Cui et al., 2013;
Chen et al., 2013; Febler et al., 2013; Gopegui et al., 2012; Quiles-
Melero et al., 2013; Shore et al., 2010).
All clinical isolates submitted as part of the SENTRY Antimi-
crobial Surveillance Programme, which includes the LEADER and
ZAAPS studies, displaying a linezolid MIC result at ≥4 mg/L have
been screened for cfr, and the presence of mutations in 23s rRNA
and L3 and L4 ribosomal mutations since 2002 (Table 4). The first
detection of cfr occurred in a MRSA recovered from blood cultures
in a hospitalized patient in Belgium in August 2006 (unpublished
data). This case was followed by the detection of cfr in staphylococci
from the USA in 2007 (Mendes et al., 2008).
The cfr gene has been, with rare exception (Toh et al., 2007),
plasmid-located and associated with a range of mobile genetic ele-
ments, such as transposons and insertion sequences. These mobile
structures have been detected among several Gram-positive iso-
lates other than staphylococci, such as E. faecalis (Diaz et al., 2012;
Liu et al., 2012), Macrococcus caseolyticus and Jeotgalicoccus pinni-
pedialis (Wang et al., 2012a), Bacillus spp. (Zhang et al., 2011; Wang
et al., 2012b; Dai et al., 2010), Streptococcus suis (Wang et al., 2013),
as well as in Gram-negative isolates of E. coli (Zhang et al., 2014)
and Proteus vulgaris (Wang et al., 2011) recovered from several dif-
ferent specimen sources collected from animals. However, recent
data has demonstrated the Bacillales order as the natural reservoirs
for the cfr genes (Hansen et al., 2012).
The diversity of organisms where cfr has been detected and the
association with mobile genetic structures indicates its propensity
for genetic mobilization. A complete review on the genetic envi-
ronment of cfr has been recently published (Shen et al., 2013); and
therefore will not be represented in this review. Interspecies dis-
semination of cfr has been previously documented (Mendes et al.,
2010b, 2013b), as well as outbreaks caused by either Cfr-producing
S. epidermidis or S. aureus (Bonilla et al., 2010; Cai et al., 2012;
Morales et al., 2010). Following the first outbreak caused by cfr-
carrying S. aureus in the ICU of Hospital Clínico San Carlos in 2008
(Morales et al., 2010), Baos et al. (2013) monitored the presence
of linezolid-resistant staphylococci from 2008 through 2011. The
authors did not recover linezolid-resistant S. aureus isolates dur-
ing the study period. However, the number of linezolid-resistant S.
epidermidis (58% cfr-positive) increase to 25% by 2010, when rates
decreased slightly to 20% in the next year. These increased rates of
linezolid-resistant S. epidermidis were observed despite implemen-
tation of infection control interventions.
Through the LEADER and ZAAPS Programme experiences, the
detection of cfr among Gram-positive clinical isolates remains spo-
radic with no apparent increase across the years. In addition, most
isolates seem to be endemic in specific hospitals in Arizona (Mendes
et al., 2008), Ohio (Mendes et al., 2008) Italy (Flamm et al., 2013b;
Mendes et al., 2010a) and Mexico (Flamm et al., 2013b; Mendes
et al., 2010b). Other cfr-positive strains have originated from ran-
dom sites in the USA (10 S. aureus, 2009–2012; two S. epidermidis,
2010; one Staphylococcus capitis, 2009; and one E. faecium, 2012),
Brazil (one S. aureus, 2012), France (one S. aureus, 2010; two S. epi-
dermidis, 2011), Spain (two S. epidermidis, 2010 and 2011), Panama
(two E. faecalis, 2011), Romania (one Staphylococcus cohnii, 2010)
and Thailand (one E. faecalis, 2010) (Jones et al., 2006, 2007a, 2008,
2009, 2010; Farrell et al., 2009, 2011; Flamm et al., 2012a, 2012b,
2013a, 2013b; Mendes et al., 2014b; Ross et al., 2007a, 2007b, 2009;
Diaz et al., 2012; Deshpande et al., 2013).
In a more recent investigation, Quiles-Melero et al. (2013) fur-
ther investigated linezolid-resistant staphylococci clinical isolates
recovered between 2005 and 2009 in a tertiary hospital in Madrid,
Spain. The authors included 256 isolates (2.5% of total) and all
seven linezolid-resistant S. aureus (1.0% of total) included were cfr-
positive. Six isolates had spa t067 and were recovered during 2008.
In the same study, cfr was detected among eight (8/125; 6.4%) of
125 (125/2183; 4.0%) linezolid-resistant S. epidermidis. Sierra et al.
 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
(2013), who evaluate the prevalence of cfr among 2215 bacteremic
MRSA clinical isolates in Hospital Universitari de Bellvitge (HUB)
from 1999 to 2010, observed only a single Cfr-producing MRSA
(0.05% of total) (Sierra et al., 2013).
The cfr gene has a great potential for dissemination due to its
association with mobile elements (Shen et al., 2013) and low fit-
ness cost (LaMarre et al., 2011). In addition, the MDR phenotype
conferred by cfr provides multiple possibilities for in vivo selec-
tion during antimicrobial therapy and several studies have reported
the detection of cfr-carrying isolates even without prior exposure
to linezolid (Baos et al., 2013; Deshpande et al., 2013; Gales et al.,
2014). Moreover, cfr is often associated with other resistance genes,
such as erm, fexA, lsa(B) and tet(L), which can assist co-selecting
the cfr gene and its spread (Shen et al., 2013; Gales et al., 2014;
Smith and Mankin, 2008). Also, several cfr-carrying isolates sub-
mitted though the SENTRY Programme, mainly S. aureus, exhibit
a linezolid MIC value at the extreme of the wildtype distribution
(i.e. 4 mg/L) and would be considered susceptible when applying
the current breakpoints (≤4 mg/L for susceptible). These charac-
teristics warrant active surveillance at local and national levels for
early detection and implementation of infection control policies.
4.3. Resistance mechanisms other than target site modifications
We have observed that some enterococci displaying elevated
MIC results for linezolid (MIC, 4–8 mg/L) included in the LEADER
and ZAAPS Programmes did not show any target site modifica-
tions associated with linezolid resistance (i.e. wildtype nucleotide
sequences for 23S rRNA and amino acid sequences for L3, L4 and
L22) or presence cfr. Isolates demonstrating such phenotypic and
genotypic characteristics have been collected from China, Panama,
Sweden and Taiwan (Table 4). Recent studies published in the lit-
erature, which evaluated the linezolid resistance mechanisms in
Gram-positive isolates from Canada and China, have also reported
similar results (Chen et al., 2013; Patel et al., 2013). Previous studies
have demonstrated that linezolid can be recognized as a substrate
of efflux-pump systems, which can extrude a wide range of struc-
turally dissimilar substrates (Sierra et al., 2009; Floyd et al., 2010).
Although the isolates above described were not evaluated for the
presence of efflux-pump or their expression levels, it is tempt-
ing to associate these low-level resistance phenotypes with the
over-expression of such MDR pumps. Further investigations should
determine the role of efflux-pump systems in these very rare but
geographically diverse cases of linezolid decreased susceptibility.
5. Conclusions
Linezolid has become and remains an important agent for
treating serious infections caused by Gram-positive organisms,
including MDR isolates. This oxazolidinone has been well studied
and much is currently known about its clinical applications, adverse
effects, mode of action, spectrum of activity, risks for resistance
development and resistance mechanisms. The knowledge acquired
during the past thirteen years has been applied for developing new
oxazolidinone agents, which have completed Phase 2 and 3 clinical
trials (Shaw and Barbachyn, 2011; Prokocimer et al., 2013). In addi-
tion, the linezolid patents in the USA and abroad will soon expire
and generic brands will certainly become available. These events
will likely establish a new era for this class of antimicrobial agents.
The occurrences of linezolid non-susceptible or resistant iso-
lates have remained relatively low since the drug introduction into
clinical use. However, sporadic cases of linezolid-resistant isolates
do occur, as well as epidemic outbreaks, emphasizing the impor-
tance for antimicrobial stewardship and surveillance for resistance.
As this review has described, it has certainly been interesting to
9
observe the changes associated with linezolid resistance mecha-
nisms. In the early 2000s, non-susceptible clinical isolates initially
harboured (almost exclusively) alterations in the 23S rRNA target,
which is still observed, and has remained the dominant mechanism
detected among enterococci. However, a limited number of clinical
isolates also carrying cfr.
In contrast, the scenario has changed significantly among S.
epidermidis and other species of CoNS, which were initially less
prevalent, and when detected presented the usual alterations in
23S rRNA. These isolates currently exhibit more diverse and com-
plex mechanisms of resistance, which consist of a greater number
of mutations in ribosomal proteins in conjunction with modifica-
tions in 23S rRNA and/or presence of cfr. Further investigations
will be required to understand whether these isolates now demon-
strating these complexes DNA alterations and presence of foreign
DNA belong to certain lineages that possess greater potential for
adapting to the nosocomial environment and antimicrobial selec-
tive pressure. The presence of linezolid resistance mechanisms in
CoNS may become a concern, since these organisms have gained
clinical relevance. Moreover, these CoNS can serve as a reservoir
for cfr.
S. aureus fortunately does not seem to behave like S. epidermidis
and other species of CoNS with regard to resistance mechanisms.
Among linezolid non-susceptible or resistant clinical isolates, S.
aureus remains rare in the original surveillance programmes. In
addition, and different from CoNS, most isolates demonstrate a sin-
gle linezolid resistance mechanism (cfr). However, this feature has
often produced isolates with linezolid MIC results at the end of
the wildtype distribution and borderline for susceptibility (i.e. MIC,
4 mg/L), which may complicate detection.
Acknowledgements
The authors wish to thank the following staff members at JMI
Laboratories (North Liberty, Iowa, USA): S. Benning, M. Castanheira,
P. Clark, A. Costello, D. Farrell, S. Farrell, R. Flamm, M. Konrardy, P.
Romberg, J. Ross, H. Sader, M. Stilwell, J. Streit and L. Woosley for
technical support and manuscript assistance.
References
Anderegg, T.R., Sader, H.S., Fritsche, T.R., Ross, J.E., Jones, R.N., 2005. Trends in
linezolid susceptibility patterns: report from the 2002–2003 worldwide Zyvox
Annual Appraisal of Potency and Spectrum (ZAAPS) Program. Int. J. Antimicrob.
Agents 26, 13–21.
Atkinson, G.C., Hansen, L.H., Tenson, T., Rasmussen, A., Kirpekar, F., Vester, B., 2013.
Distinction between the Cfr methyltransferase conferring antibiotic resistance
and the housekeeping RlmN methyltransferase. Antimicrob. Agents Chemother.
57, 4019–4026.
Auckland, C., Teare, L., Cooke, F., Kaufmann, M.E., Warner, M., Jones, G., Bamford, K.,
Ayles, H., Johnson, A.P., 2002. Linezolid-resistant enterococci: report of the first
isolates in the United Kingdom. J. Antimicrob. Chemother. 50, 743–746.
Ballow, C.H., Biedenbach, D.J., Rossi, F., Jones, R.N., 2002a. Multicenter assessment of
the linezolid spectrum and activity using the disk diffusion and Etest methods:
report of the Zyvox(R) Antimicrobial Potency Study in Latin America (LA-ZAPS).
Braz. J. Infect. Dis. 6, 100–109.
Ballow, C.H., Jones, R.N., Biedenbach, D.J., 2002b. A multicenter evaluation of line-
zolid antimicrobial activity in North America. Diagn. Microbiol. Infect. Dis. 43,
75–83.
Baos, E., Candel, F.J., Merino, P., Pena, I., Picazo, J.J., 2013. Characterization and
monitoring of linezolid-resistant clinical isolates of Staphylococcus epider-
midis in an intensive care unit 4 years after an outbreak of infection by
cfr-mediated linezolid-resistant Staphylococcus aureus. Diagn. Microbiol. Infect.
Dis. 76, 325–329.
Beckert, P., Hillemann, D., Kohl, T.A., Kalinowski, J., Richter, E., Niemann, S.,
Feuerriegel, S., 2012. rplC T460C identified as a dominant mutation in linezolid-
resistant Mycobacterium tuberculosis strains. Antimicrob. Agents Chemother. 56,
2743–2745.
Bell, J.M., Turnidge, J.D., Ballow, C.H., Jones, R.N., 2003. Multicentre evaluation of the
in vitro activity of linezolid in the Western Pacific. J. Antimicrob. Chemother. 51,
339–345.
10 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
Biedenbach, D.J., Jones, R.N., 1997. Disk diffusion test interpretive criteria and quality
control recommendations for testing linezolid (U-100766) and eperezolid (U-
100592) with commercially prepared reagents. J. Clin. Microbiol. 35, 3198–3202.
Biedenbach, D.J., Jones, R.N., 2001. In vitro activity of linezolid (U-100766) against
Haemophilus influenzae measured by three different susceptibility testing meth-
ods. Diagn. Microbiol. Infect. Dis. 39, 49–53.
Biedenbach, D.J., Jones, R.N., 2003. Revision of linezolid disk diffusion quality con-
trol guidelines for testing Staphylococcus aureus ATCC, 25923. An independent
seven-laboratory trial. Clin. Microbiol. Infect. 9, 1035–1037.
Billal, D.S., Feng, J., Leprohon, P., Legare, D., Ouellette, M., 2011. Whole genome anal-
ysis of linezolid resistance in Streptococcus pneumoniae reveals resistance and
compensatory mutations. BMC Genomics 12, 512.
Bock, A., Turnowsky, F., Hogenauer, G., 1982. Tiamulin resistance mutations in
Escherichia coli. J. Bacteriol. 151, 1253–1260.
Bolmstrom, A., Ballow, C.H., Qwarnstrom, A., Biedenbach, D.J., Jones, R.N., 2002. Mul-
ticentre assessment of linezolid antimicrobial activity and spectrum in Europe:
report from the Zyvox antimicrobial potency study (ZAPS-Europe). Clin. Micro-
biol. Infect. 8, 791–800.
Bonilla, H., Huband, M.D., Seidel, J., Schmidt, H., Lescoe, M., McCurdy, S.P., Lemmon,
M.M., Brennan, L.A., Tait-Kamradt, A., Puzniak, L., Quinn, J.P., 2010. Multicity
outbreak of linezolid-resistant Staphylococcus epidermidis associated with clonal
spread of a cfr-containing strain. Clin. Infect. Dis. 51, 796–800.
Bosling, J., Poulsen, S.M., Vester, B., Long, K.S., 2003. Resistance to the peptidyl
transferase inhibitor tiamulin caused by mutation of ribosomal protein L3.
Antimicrob. Agents Chemother. 47, 2892–2896.
Bozdogan, B., Appelbaum, P.C., 2004. Oxazolidinones: activity, mode of action, and
mechanism of resistance. Int. J. Antimicrob. Agents 23, 113–119.
Cai, J.C., Hu, Y.Y., Zhang, R., Zhou, H.W., Chen, G.X., 2012. Linezolid-resistant clinical
isolates of meticillin-resistant coagulase-negative staphylococci and Enterococ-
cus faecium from China. J. Med. Microbiol. 61, 1568–1573.
Chen, H., Wu, W., Ni, M., Liu, Y., Zhang, J., Xia, F., He, W., Wang, Q., Wang, Z., Cao, B.,
Wang, H., 2013. Linezolid-resistant clinical isolates of enterococci and Staphy-
lococcus cohnii from a multicentre study in China: molecular epidemiology and
resistance mechanisms. Int. J. Antimicrob. Agents 42, 317–321.
Clinical and Laboratory Standards Institute, 2006. CLSI Subcommittee on Antimicro-
bial Testing Meeting Minutes, January 2006. Clinical and Laboratory Standards
Institute, Miami, FL, USA.
Clinical and Laboratory Standards Institute, 2012a. CLSI Subcommittee on Antimi-
crobial Susceptibility Testing Meeting Minutes, January 2012. Clinical and
Laboratory Standards Institute, Tempe, AZ, USA.
Clinical and Laboratory Standards Institute, 2012b. M07-A9. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved
Standard, 9th edition. Clinical and Laboratory Standards Institute, Wayne, PA,
USA.
Clinical and Laboratory Standards Institute, 2013. M100-S23. Performance Stan-
dards for Antimicrobial Susceptibility Testing: 23rd Informational Supplement.
Clinical and Laboratory Standards Institute, Wayne, PA, USA.
Clinical and Laboratory Standards Institute, 2014. M07-A10. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved
Standard, 10th ed. Clinical and Laboratory Standards Institute, Wayne, PA, USA.
Colca, J.R., McDonald, W.G., Waldon, D.J., Thomasco, L.M., Gadwood, R.C., Lund, E.T.,
Cavey, G.S., Mathews, W.R., Adams, L.D., Cecil, E.T., Pearson, J.D., Bock, J.H., Mott,
J.E., Shinabarger, D.L., Xiong, L., Mankin, A.S., 2003. Cross-linking in the living
cell locates the site of action of oxazolidinone antibiotics. J. Biol. Chem. 278,
21972–21979.
Cui, L., Wang, Y., Li, Y., He, T., Schwarz, S., Ding, Y., Shen, J., Lv, Y., 2013. Cfr-mediated
linezolid-resistance among methicillin-resistant coagulase-negative Staphylo-
cocci from infections of humans. PLoS ONE 8, e57096.
Dai, L., Wu, C.M., Wang, M.G., Wang, Y., Wang, Y., Huang, S.Y., Xia, L.N., Li, B.B., Shen,
J.Z., 2010. First report of the multidrug resistance gene cfr and the phenicol
resistance gene fexA in a Bacillus strain from swine feces. Antimicrob. Agents
Chemother. 54, 3953–3955.
de Almeida, L.M., de Araujo, M.R., Sacramento, A.G., Pavez, M., de Souza, A.G.,
Rodrigues, F., Gales, A.C., Lincopan, N., Sampaio, J.L., Mamizuka, E.M., 2013.
Linezolid resistance in Brazilian Staphylococcus hominis strains is associated
with L3 and 23S rRNA ribosomal mutations. Antimicrob. Agents Chemother.
57, 4082–4083.
Deshpande, L.M., Vega, S., Ross, J.E., Jones, R.N., Mendes, R.N., 2013. Detection of
plasmid-encoded Cfr in clinical isolates of Enterococcus faecalis from Panama
City: report from the SENTRY Antimicrobial Surveillance Programme. In:
Abstract P1405. 23rd ECCMID, April 27–30, 2013, Berlin, Germany.
Diaz, L., Kiratisin, P., Mendes, R., Panesso, D., Singh, K.V., Arias, C.A., 2012. Trans-
ferable plasmid-mediated resistance to linezolid due to cfr in a human clinical
isolate of Enterococcus faecalis. Antimicrob. Agents Chemother. 56, 3917–3922.
Draghi, D.C., Sheehan, D.J., Hogan, P., Sahm, D.F., 2005. In vitro activity of linezolid
against key Gram-positive organisms isolated in the United States: results of
the LEADER 2004 surveillance program. Antimicrob. Agents Chemother. 49,
5024–5032.
Endimiani, A., Blackford, M., Dasenbrook, E.C., Reed, M.D., Bajaksouszian, S., Hujer,
A.M., Rudin, S.D., Hujer, K.M., Perreten, V., Rice, L.B., Jacobs, M.R., Konstan, M.W.,
Bonomo, R.A., 2011. Emergence of linezolid-resistant Staphylococcus aureus after
prolonged treatment of cystic fibrosis patients in Cleveland, Ohio. Antimicrob.
Agents Chemother. 55, 1684–1692.
EUCAST, 2013. Breakpoint tables for interpretation of MICs and zone diam-
eters. Version 3.1, February 2013., available at http://www.eucast.
org/clinical breakpoints/ (accessed August 2013).
Farrell, D.J., Mendes, R.E., Ross, J.E., Jones, R.N., 2009. Linezolid surveillance program
results for 2008 (LEADER Program for 2008). Diagn. Microbiol. Infect. Dis. 65,
392–403.
Farrell, D.J., Mendes, R.E., Ross, J.E., Sader, H.S., Jones, R.N., 2011. LEADER Program
results for 2009: an activity and spectrum analysis of linezolid using 6,414 clin-
ical isolates from the United States (56 Medical Centers). Antimicrob. Agents
Chemother. 55, 3684–3690.
Febler, A.T., Calvo, N., Gutierrez, N., Munoz Bellido, J.L., Fajardo, M., Garduno, E.,
Monecke, S., Ehricht, R., Kadlec, K., Schwarz, S., 2013. Cfr-mediated linezolid
resistance in methicillin-resistant Staphylococcus aureus and Staphylococcus
haemolyticus associated with clinical infections in humans: two case reports.
J. Antimicrob. Chemother. 69, 268–270.
Fines, M., Leclercq, R., 2000. Activity of linezolid against Gram-positive cocci pos-
sessing genes conferring resistance to protein synthesis inhibitors. J. Antimicrob.
Chemother. 45, 797–802.
Flamm, R.K., Farrell, D.J., Mendes, R.E., Ross, J.E., Sader, H.S., Jones, R.N., 2012a.
LEADER surveillance program results for 2010: an activity and spectrum anal-
ysis of linezolid using 6801 clinical isolates from the United States (61 medical
centers). Diagn. Microbiol. Infect. Dis. 74, 54–61.
Flamm, R.K., Farrell, D.J., Mendes, R.E., Ross, J.E., Sader, H.S., Jones, R.N., 2012b.
ZAAPS program results for 2010: an activity and spectrum analysis of linezolid
using clinical isolates from 75 medical centers in 24 countries. J. Chemother. 24,
328–337.
Flamm, R.K., Mendes, R.E., Ross, J.E., Sader, H.S., Jones, R.N., 2013a. Linezolid
surveillance results for the United States: LEADER Surveillance Program 2011.
Antimicrob. Agents Chemother. 57, 1077–1081.
Flamm, R.K., Mendes, R.E., Ross, J.E., Sader, H.S., Jones, R.N., 2013b. An international
activity and spectrum analysis of linezolid: ZAAPS program results for 2011.
Diagn. Microbiol. Infect. Dis. 76, 206–213.
Floyd, J.L., Smith, K.P., Kumar, S.H., Floyd, J.T., Varela, M.F., 2010. LmrS is a multidrug
efflux pump of the major facilitator superfamily from Staphylococcus aureus.
Antimicrob. Agents Chemother. 54, 5406–5412.
Gales, A.C., Sader, H.S., Andrade, S.S., Lutz, L., Machado, A., Barth, A.L., 2006.
Emergence of linezolid-resistant Staphylococcus aureus during treatment of pul-
monary infection in a patient with cystic fibrosis. Int. J. Antimicrob. Agents 27,
300–302.
Gales, A.C., Deshpande, L.M., Jones, R.N., De Souza, A.G., Pignatari, A.C., Mendes,
R.E., 2014. Fatal pneumonia due to methicillin-susceptible Staphylococcus aureus
ST398/t034 carrying cfr and erm(B) genes: first report in Brazil. J. Antimicrob.
Chemother. (in press).
Gentry, D.R., Rittenhouse, S.F., McCloskey, L., Holmes, D.J., 2007. Stepwise exposure
of Staphylococcus aureus to pleuromutilins is associated with stepwise acqui-
sition of mutations in rplC and minimally affects susceptibility to retapamulin.
Antimicrob. Agents Chemother. 51, 2048–2052.
Gopegui, E.R., Juan, C., Zamorano, L., Perez, J.L., Oliver, A., 2012. Transferable mul-
tidrug resistance plasmid carrying cfr associated with tet(L), ant(4 )-Ia, and dfrK
genes from a clinical methicillin-resistant Staphylococcus aureus ST125 strain.
Antimicrob. Agents Chemother. 56, 2139–2142.
Gould, I.M., Bal, A.M., 2013. New antibiotic agents in the pipeline and
how they can help overcome microbial resistance. Virulence 4, 185–
191.
Hansen, L.H., Planellas, M.H., Long, K.S., Vester, B., 2012. The order Bacillales hosts
functional homologs of the worrisome cfr antibiotic resistance gene. Antimicrob.
Agents Chemother. 56, 3563–3567.
Johnson, A.P., Warner, M., Livermore, D.M., 2000. Activity of linezolid against multi-
resistant Gram-positive bacteria from diverse hospitals in the United Kingdom.
J. Antimicrob. Chemother. 45, 225–230.
Johnson, A.P., Tysall, L., Stockdale, M.V., Woodford, N., Kaufmann, M.E., Warner, M.,
Livermore, D.M., Asboth, F., Allerberger, F.J., 2002. Emerging linezolid-resistant
Enterococcus faecalis and Enterococcus faecium isolated from two Austrian
patients in the same intensive care unit. Eur. J. Clin. Microbiol. Infect. Dis. 21,
751–754.
Jones, R.N., Johnson, D.M., Erwin, M.E., 1996. In vitro antimicrobial activities and
spectra of U-100592 and U-100766, two novel fluorinated oxazolidinones.
Antimicrob. Agents Chemother. 40, 720–726.
Jones, R.N., Ballow, C.H., Biedenbach, D.J., 2001. Multi-laboratory assessment of the
linezolid spectrum of activity using the Kirby-Bauer disk diffusion method:
report of the Zyvox Antimicrobial Potency Study (ZAPS) in the United States.
Diagn. Microbiol. Infect. Dis. 40, 59–66.
Jones, R.N., Ross, J.E., Fritsche, T.R., Sader, H.S., 2006. Oxazolidinone susceptibility
patterns in 2004: report from the Zyvox® Annual Appraisal of Potency and
Spectrum (ZAAPS) Program assessing isolates from 16 nations. J. Antimicrob.
Chemother. 57, 279–287.
Jones, R.N., Fritsche, T.R., Sader, H.S., Ross, J.E., 2007a. LEADER surveillance program
results for 2006: an activity and spectrum analysis of linezolid using clinical
isolates from the United States (50 medical centers). Diagn. Microbiol. Infect.
Dis. 59, 309–317.
Jones, R.N., Fritsche, T.R., Sader, H.S., Ross, J.E., 2007b. Zyvox® Annual Appraisal
of Potency and Spectrum Program results for 2006: an activity and spectrum
analysis of linezolid using clinical isolates from 16 countries. Diagn. Microbiol.
Infect. Dis. 59, 199–209.
Jones, R.N., Ross, J.E., Castanheira, M., Mendes, R.E., 2008. United States resistance
surveillance results for linezolid (LEADER Program for 2007). Diagn. Microbiol.
Infect. Dis. 62, 416–426.
Jones, R.N., Kohno, S., Ono, Y., Ross, J.E., Yanagihara, K., 2009. ZAAPS Interna-
tional Surveillance Program (2007) for linezolid resistance: results from 5591
 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
Gram-positive clinical isolates in 23 countries. Diagn. Microbiol. Infect. Dis. 64,
191–201.
Jones, R.N., Mendes, R.E., Farrell, D.J., Sader, H.S., Ross, J.E., 2010. Summary of
2009 oxazolidinone resistance (R) surveillance studies worldwide (LEADER and
ZAAPS Programs). In: Abstract 263. 48th IDSA, October 21–24, 2010, Vancouver,
B.C., Canada.
Kehrenberg, C., Schwarz, S., 2006. Distribution of florfenicol resistance genes fexA
and cfr among chloramphenicol-resistant Staphylococcus spp. isolates. Antimi-
crob. Agents Chemother. 50, 1156–1163.
Kehrenberg, C., Ojo, K.K., Schwarz, S., 2004. Nucleotide sequence and organization
of the multiresistance plasmid pSCFS1 from Staphylococcus sciuri. J. Antimicrob.
Chemother. 54, 936–939.
Kehrenberg, C., Cuny, C., Strommenger, B., Schwarz, S., Witte, W., 2009. Methicillin-
resistant and -susceptible Staphylococcus aureus strains of clonal lineages ST398
and ST9 from swine carry the multidrug resistance gene cfr. Antimicrob. Agents
Chemother. 53, 779–781.
Kosowska-Shick, K., Clark, C., Credito, K., McGhee, P., Dewasse, B., Bogdanovich, T.,
Appelbaum, P.C., 2006. Single- and multistep resistance selection studies on the
activity of retapamulin compared to other agents against Staphylococcus aureus
and Streptococcus pyogenes. Antimicrob. Agents Chemother. 50, 765–769.
Kosowska-Shick, K., Julian, K.G., McGhee, P.L., Appelbaum, P.C., Whitener, C.J., 2010.
Molecular and epidemiologic characteristics of linezolid-resistant coagulase-
negative staphylococci at a tertiary care hospital. Diagn. Microbiol. Infect. Dis.
68, 34–39.
LaMarre, J.M., Locke, J.B., Shaw, K.J., Mankin, A.S., 2011. Low fitness cost of the mul-
tidrug resistance gene cfr. Antimicrob. Agents Chemother. 55, 3714–3719.
LaMarre, J., Mendes, R.E., Szal, T., Schwarz, S., Jones, R.N., Mankin, A.S., 2013. The
genetic environment of the cfr gene and the presence of other mechanisms
account for the very high linezolid resistance of the Staphylococcus epidermidis
isolate 426-3147L. Antimicrob. Agents Chemother. 57, 1173–1179.
Leach, K.L., Swaney, S.M., Colca, J.R., McDonald, W.G., Blinn, J.R., Thomasco, L.M.,
Gadwood, R.C., Shinabarger, D., Xiong, L., Mankin, A.S., 2007. The site of action
of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol.
Cell 26, 393–402.
Lin, A.H., Murray, R.W., Vidmar, T.J., Marotti, K.R., 1997. The oxazolidinone eperezolid
binds to the 50S ribosomal subunit and competes with binding of chlorampheni-
col and lincomycin. Antimicrob. Agents Chemother. 41, 2127–2131.
Liu, Y., Wang, Y., Wu, C., Shen, Z., Schwarz, S., Du, X.D., Dai, L., Zhang, W., Zhang, Q.,
Shen, J., 2012. First report of the multidrug resistance gene cfr in Enterococcus
faecalis of animal origin. Antimicrob. Agents Chemother. 56, 1650–1654.
Locke, J.B., Hilgers, M., Shaw, K.J., 2009a. Mutations in ribosomal protein L3 are
associated with oxazolidinone resistance in staphylococci of clinical origin.
Antimicrob. Agents Chemother. 53, 5275–5278.
Locke, J.B., Hilgers, M., Shaw, K.J., 2009b. Novel ribosomal mutations in Staphylococ-
cus aureus strains identified through selection with the oxazolidinones linezolid
and torezolid (TR-700). Antimicrob. Agents Chemother. 53, 5265–5274.
Locke, J.B., Morales, G., Hilgers, M.G., Rahawi, C.K., Jose Picazo, S., Shaw, J., Stein,
K.J.J.L., 2010. Elevated linezolid resistance in clinical cfr-positive Staphylococcus
aureus isolates is associated with co-occurring mutations in ribosomal protein
L3. Antimicrob. Agents Chemother. 54, 5352–5355.
Long, K.S., Vester, B., 2012. Resistance to linezolid caused by modifications at its
binding site on the ribosome. Antimicrob. Agents Chemother. 56, 603–612.
Long, K.S., Poehlsgaard, J., Kehrenberg, C., Schwarz, S., Vester, B., 2006. The cfr
rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazo-
lidinones, pleuromutilins, and streptogramin A antibiotics. Antimicrob. Agents
Chemother. 50, 2500–2505.
Long, K.S., Munck, C., Andersen, T.M., Schaub, M.A., Hobbie, S.N., Bottger, E.C.,
Vester, B., 2010. Mutations in 23S rRNA at the peptidyl transferase center and
their relationship to linezolid binding and cross-resistance. Antimicrob. Agents
Chemother. 54, 4705–4713.
Marshall, S.H., Donskey, C.J., Hutton-Thomas, R., Salata, R.A., Rice, L.B., 2002. Gene
dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis.
Antimicrob. Agents Chemother. 46, 3334–3336.
Meka, V.G., Gold, H.S., 2004. Antimicrobial resistance to linezolid. Clin. Infect. Dis.
39, 1010–1015.
Meka, V.G., Pillai, S.K., Sakoulas, G., Wennersten, C., Venkataraman, L., DeGirolami,
P.C., Eliopoulos, G.M., Moellering Jr., R.C., Gold, H.S., 2004. Linezolid resistance in
sequential Staphylococcus aureus isolates associated with a T2500A mutation in
the 23S rRNA gene and loss of a single copy of rRNA. J. Infect. Dis. 190, 311–317.
Mendes, R.E., Deshpande, L.M., Castanheira, M., DiPersio, J., Saubolle, M.A., Jones,
R.N., 2008. First report of cfr-mediated resistance to linezolid in human staphy-
lococcal clinical isolates recovered in the United States. Antimicrob. Agents
Chemother. 52, 2244–2246.
Mendes, R.E., Sanchez, M., Deshpande, L.M., De la Torre, M.A., Jones, R.N., 2009. Detec-
tion of cfr-carrying Staphylococcus spp. isolates recovered from blood cultures
in a Spanish hospital during a Phase III clinical trial of tropical omiganan 1% gel
versus 10% povidone iodine. In: Abstract 928. 19th ECCMID, May 16–19, 2009,
Helsinki, Finland.
Mendes, R.E., Deshpande, L.M., Farrell, D.J., Spanu, T., Fadda, G., Jones, R.N., 2010a.
Assessment of linezolid resistance mechanisms among Staphylococcus epi-
dermidis causing bacteraemia in Rome, Italy. J. Antimicrob. Chemother. 65,
2329–2335.
Mendes, R.E., Deshpande, L., Rodriguez-Noriega, E., Ross, J.E., Jones, R.N., Morfin-
Otero, R., 2010b. First report of staphylococcal clinical isolates in Mexico with
linezolid resistance caused by cfr: evidence of in vivo cfr mobilization. J. Clin.
Microbiol. 48, 3041–3043.
11
Mendes, R.E., Deshpande, L.M., Costello, A., Farrell, D.J., 2012. Molecular epi-
demiology of Staphylococcus epidermidis clinical isolates from USA hospitals.
Antimicrob. Agents Chemother. 56, 4656–4661.
Mendes, R.E., Deshpande, L.M., Kim, J., Myers, D.S., Ross, J.E., Jones, R.N., 2013a. Strep-
tococcus sanguinis displaying a cross resistance phenotype to several ribosomal
RNA targeting agents. J. Clin. Microbiol. 51, 2728–2731.
Mendes, R.E., Deshpande, L.M., Bonilla, H.F., Schwarz, S., Huband, M.D., Jones, R.N.,
Quinn, J.P., 2013b. Dissemination of a pSCFS3-like cfr-carrying plasmid in Staphy-
lococcus aureus and Staphylococcus epidermidis clinical isolates recovered from
hospitals in Ohio. Antimicrob. Agents Chemother. 57, 2923–2928.
Mendes, R.E., Flamm, R.K., Hogan, P.A., Ross, J.E., Jones, R.N., 2014a. Summary of
linezolid activity and resistance mechanisms detected during the 2012 LEADER
surveillance program for the United States. Antimicrob. Agents Chemother. 58,
1243–1247.
Mendes, R.E., Hogan, P.A., Streit, J.M., Jones, R.N., Flamm, R.K., 2014b. Zyvox® Annual
Appraisal of Potency and Spectrum (ZAAPS) Program: report of linezolid activity
over nine years (2004–2012). J. Antimicrob. Chemother. (in press).
Michalska, K., Karpiuk, I., Krol, M., Tyski, S., 2013. Recent development of potent
analogues of oxazolidinone antibacterial agents. Bioorg. Med. Chem. Lett. 21,
577–591.
Miller, K., Dunsmore, C.J., Fishwick, C.W., Chopra, I., 2008. Linezolid and tia-
mulin cross-resistance in Staphylococcus aureus mediated by point mutations
in the peptidyl transferase center. Antimicrob. Agents Chemother. 52, 1737–
1742.
Morales, G., Picazo, J.J., Baos, E., Candel, F.J., Arribi, A., Pelaez, B., Andrade, R., de
la Torre, M.A., Fereres, J., Sanchez-Garcia, M., 2010. Resistance to linezolid is
mediated by the cfr gene in the first report of an outbreak of linezolid-resistant
Staphylococcus aureus. Clin. Infect. Dis. 50, 821–825.
Mutnick, A.H., Biedenbach, D.J., Turnidge, J.D., Jones, R.N., 2002. Spectrum and
potency evaluation of a new oxazolidinone, linezolid: report from the SENTRY
Antimicrobial Surveillance Program, 1998–2000. Diagn. Microbiol. Infect. Dis.
43, 65–73.
Mutnick, A.H., Enne, V., Jones, R.N., 2003. Linezolid resistance since 2001: SENTRY
Antimicrobial Surveillance Program. Ann. Pharmacother. 37, 769–774.
Noskin, G.A., Siddiqui, F., Stosor, V., Hacek, D., Peterson, L.R., 1999. In vitro
activities of linezolid against important Gram-positive bacterial pathogens
including vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 43,
2059–2062.
Patel, S.N., Memari, N., Shahinas, D., Toye, B., Jamieson, F.B., Farrell, D.J., 2013.
Linezolid resistance in Enterococcus faecium isolated in Ontario, Canada. Diagn.
Microbiol. Infect. Dis. 77, 350–353.
Peeters, M.J., Sarria, J.C., 2005. Clinical characteristics of linezolid-resistant Staphy-
lococcus aureus infections. Am. J. Med. Sci. 330, 102–104.
Poppe, S., Schaadt, R., Sheehan, D., Sahm, D., Zurenko, G., Shinabarger, D., 2006. Muta-
tion analysis of Staphylococcus aureus isolates demonstrating trailing linezolid
(LZD) endpoints in microdilution MIC assays. In: Abstr. A-088. 106th American
Society for Microbiology, Orlando, FL, USA.
Pournaras, S., Ntokou, E., Zarkotou, O., Ranellou, K., Themeli-Digalaki, K., Stathopou-
los, C., Tsakris, A., 2013. Linezolid dependence in Staphylococcus epidermidis
bloodstream isolates. Emerg. Infect. Dis. 19, 129–132.
Pringle, M., Poehlsgaard, J., Vester, B., Long, K.S., 2004. Mutations in ribosomal pro-
tein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated
with reduced susceptibility to tiamulin in Brachyspira spp. isolates. Mol. Micro-
biol. 54, 1295–1306.
Prokocimer, P., De Anda, C., Fang, E., Mehra, P., Das, A., 2013. Tedizolid phosphate vs
linezolid for treatment of acute bacterial skin and skin structure infections: the
ESTABLISH-1 randomized trial. J. Am. Med. Assoc. 309, 559–569.
Quiles-Melero, I., Gomez-Gil, R., Romero-Gomez, M.P., Sanchez-Diaz, A.M., de Pab-
los, M., Garcia-Rodriguez, J., Gutierrez, A., Mingorance, J., 2013. Mechanisms of
linezolid resistance among staphylococci in a tertiary hospital. J. Clin. Microbiol.
51, 998–1001.
Ross, J.E., Anderegg, T.R., Sader, H.S., Fritsche, T.R., Jones, R.N., 2005. Trends in line-
zolid susceptibility patterns in 2002: report from the worldwide Zyvox Annual
Appraisal of Potency and Spectrum Program. Diagn. Microbiol. Infect. Dis. 52,
53–58.
Ross, J.E., Fritsche, T.R., Sader, H.S., Jones, R.N., 2007a. Oxazolidinone susceptibility
patterns for 2005: international report from the Zyvox® Annual Appraisal of
Potency and Spectrum Study. Int. J. Antimicrob. Agents 29, 295–301.
Ross, J.E., Jones, R.N., Hogan, P.A., Sheehan, D.J., 2007b. Initial occurrences of linezolid
(LZD) resistance (R) from the Zyvox Annual Appraisal of Potency and Spectrum
(ZAAPS) Program (Europe, Latin, America, Asia Pacific). In: Abstract C2-865. 47th
ICAAC, September 17–20, 2007, Chicago, IL, USA.
Ross, J.E., Utsuki, U., Fumiaki, I., Kobayashi, I., Jones, R.N., Hogan, P.A., 2009. Zyvox®
Annual Appraisal of Potency and Spectrum (ZAAPS) Program 2008 (Europe, Latin
America, Canada, Asia Pacific): linezolid global in vitro susceptibility analyses.
In: Abstracts A-078. 109th ASM, May 17–21, 2009, Philadelphia, PA, USA.
Rybak, M.J., Cappelletty, D.M., Moldovan, T., Aeschlimann, J.R., Kaatz, G.W., 1998.
Comparative in vitro activities and postantibiotic effects of the oxazolidi-
none compounds eperezolid (PNU-100592) and linezolid (PNU-100766) versus
vancomycin against Staphylococcus aureus, coagulase-negative staphylococci,
Enterococcus faecalis, and Enterococcus faecium. Antimicrob. Agents Chemother.
42, 721–724.
Rybak, M.J., Hershberger, E., Moldovan, T., Grucz, R.G., 2000. In vitro activities
of daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin against
Staphylococci and Enterococci, including vancomycin-intermediate and -
resistant strains. Antimicrob. Agents Chemother. 44, 1062–1066.
12 R.E. Mendes et al. / Drug Resistance Updates 17 (2014) 1–12
Schwarz, S., Werckenthin, C., Kehrenberg, C., 2000. Identification of a plasmid-borne
chloramphenicol-florfenicol resistance gene in Staphylococcus sciuri. Antimi-
crob. Agents Chemother. 44, 2530–2533.
Shaw, K.J., Barbachyn, M.R., 2011. The oxazolidinones: past, present, and future. Ann.
N.Y. Acad. Sci. 1241, 48–70.
Shen, J., Wang, Y., Schwarz, S., 2013. Presence and dissemination of the multire-
sistance gene cfr in Gram-positive and Gram-negative bacteria. J. Antimicrob.
Chemother. 68, 1697–1706.
Shinabarger, D.L., Marotti, K.R., Murray, R.W., Lin, A.H., Melchior, E.P., Swaney,
S.M., Dunyak, D.S., Demyan, W.F., Buysse, J.M., 1997. Mechanism of action of
oxazolidinones: effects of linezolid and eperezolid on translation reactions.
Antimicrob. Agents Chemother. 41, 2132–2136.
Shore, A.C., Brennan, O.M., Ehricht, R., Monecke, S., Schwarz, S., Slickers, P.,
Coleman, D.C., 2010. Identification and characterization of the multidrug resis-
tance gene cfr in a Panton-Valentine leukocidin-positive sequence type 8
methicillin-resistant Staphylococcus aureus IVa (USA300) isolate. Antimicrob.
Agents Chemother. 54, 4978–4984.
Sierra, J.M., Ortega, M., Tarrago, C., Albet, C., Vila, J., Terencio, J., Guglietta, A., 2009.
Decreased linezolid uptake in an in vitro-selected linezolid-resistant Staphylo-
coccus epidermidis mutant. J. Antimicrob. Chemother. 64, 990–992.
Sierra, J.M., Camoez, M., Tubau, F., Gasch, O., Pujol, M., Martin, R., Dominguez, M.A.,
2013. Low prevalence of Cfr-mediated linezolid resistance among methicillin-
resistant Staphylococcus aureus in a Spanish hospital: case report on linezolid
resistance acquired during linezolid therapy. PLoS ONE 8, e59215.
Smith, L.K., Mankin, A.S., 2008. Transcriptional and translational control of the mlr
operon, which confers resistance to seven classes of protein synthesis inhibitors.
Antimicrob. Agents Chemother. 52, 1703–1712.
Stefani, S., Bongiorno, D., Mongelli, G., Campanile, F., 2010. Linezolid resistance in
staphylococci. Pharmaceuticals 3, 1–18.
Tenover, F.C., Williams, P.P., Stocker, S., Thompson, A., Clark, L.A., Limbago, B., Carey,
R.B., Poppe, S.M., Shinabarger, D., McGowan Jr., J.E., 2007. Accuracy of six antimi-
crobial susceptibility methods for testing linezolid against staphylococci and
enterococci. J. Clin. Microbiol. 45, 2917–2922.
Toh, S.M., Xiong, L., Arias, C.A., Villegas, M.V., Lolans, K., Quinn, J., Mankin, A.S., 2007.
Acquisition of a natural resistance gene renders a clinical strain of methicillin-
resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.
Mol. Microbiol. 64, 1506–1514.
Toh, S.M., Xiong, L., Bae, T., Mankin, A.S., 2008. The methyltransferase YfgB/RlmN is
responsible for modification of adenosine 2503 in 23S rRNA. RNA 14, 98–106.
Tsakris, A., Pillai, S.K., Gold, H.S., Thauvin-Eliopoulos, C., Venkataraman, L., Wenner-
sten, C., Moellering Jr., R.C., Eliopoulos, G.M., 2007. Persistence of rRNA operon
mutated copies and rapid re-emergence of linezolid resistance in Staphylococcus
aureus. J. Antimicrob. Chemother. 60, 649–651.
Tsiodras, S., Gold, H.S., Sakoulas, G., Eliopoulos, G.M., Wennersten, C., Venkataraman,
L., Moellering, R.C., Ferraro, M.J., 2001. Linezolid resistance in a clinical isolate
of Staphylococcus aureus. Lancet 358, 207–208.
von Eiff, C., Peters, G., 1999. Comparative in-vitro activities of moxifloxacin,
trovafloxacin, quinupristin/dalfopristin and linezolid against staphylococci. J.
Antimicrob. Chemother. 43, 569–573.
Wang, Y., Wang, Y., Wu, C.M., Schwarz, S., Shen, Z., Zhang, W., Zhang, Q., Shen, J.Z.,
2011. Detection of the staphylococcal multiresistance gene cfr in Proteus vulgaris
of food animal origin. J. Antimicrob. Chemother. 66, 2521–2526.
Wang, Y., Wang, Y., Schwarz, S., Shen, Z., Zhou, N., Lin, J., Wu, C., Shen, J., 2012a.
Detection of the staphylococcal multiresistance gene cfr in Macrococcus case-
olyticus and Jeotgalicoccus pinnipedialis. J. Antimicrob. Chemother. 67, 1824–
1827.
Wang, Y., Schwarz, S., Shen, Z., Zhang, W., Qi, J., Liu, Y., He, T., Shen, J., Wu, C., 2012b.
Co-location of the multiresistance gene cfr and the novel streptomycin resis-
tance gene aadY on a small plasmid in a porcine Bacillus strain. J. Antimicrob.
Chemother. 67, 1547–1549.
Wang, Y., Li, D., Song, L., Liu, Y., He, T., Liu, H., Wu, C., Schwarz, S., Shen, J., 2013. First
report of the multiresistance gene cfr in Streptococcus suis. Antimicrob. Agents
Chemother. 57, 4061–4063.
Wilson, P., Andrews, J.A., Charlesworth, R., Walesby, R., Singer, M., Farrell, D.J., Rob-
bins, M., 2003. Linezolid resistance in clinical isolates of Staphylococcus aureus.
J. Antimicrob. Chemother. 51, 186–188.
Wise, R., Andrews, J.M., Boswell, F.J., Ashby, J.P., 1998. The in vitro activity of line-
zolid (U-100766) and tentative breakpoints. J. Antimicrob. Chemother. 42, 721–
728.
Wolter, N., Smith, A.M., Farrell, D.J., Schaffner, W., Moore, M., Whitney, C.G.,
Jorgensen, J.H., Klugman, K.P., 2005. Novel mechanism of resistance to oxa-
zolidinones, macrolides, and chloramphenicol in ribosomal protein L4 of the
pneumococcus. Antimicrob. Agents Chemother. 49, 3554–3557.
Wong, A., Reddy, S.P., Smyth, D.S., Aguero-Rosenfeld, M.E., Sakoulas, G., Robinson,
D.A., 2010. Polyphyletic emergence of linezolid-resistant staphylococci in the
United States. Antimicrob. Agents Chemother. 54, 742–748.
Worth, S., Erwin, M.E., Jones, R.N., 1996. Quality control guidelines for amoxicillin,
amoxicillin-clavulanate, azithromycin, piperacillin-tazobactam, roxithromycin,
ticarcillin, ticarcillin-clavulanate, trovafloxacin (CP 99,219), U-100592, and
U-100766 for various National Committee for Clinical Laboratory Standards sus-
ceptibility testing methods. Results from multicenter trials. Diagn. Microbiol.
Infect. Dis. 24, 87–91.
Yan, F., LaMarre, J.M., Rohrich, R., Wiesner, J., Jomaa, H., Mankin, A.S., Fujimori,
D.G., 2010. RlmN and Cfr are radical SAM enzymes involved in methylation of
ribosomal RNA. J. Am. Chem. Soc. 132, 3953–3964.
Zhang, W.J., Wu, C.M., Wang, Y., Shen, Z.Q., Dai, L., Han, J., Foley, S.L., Shen, J.Z., Zhang,
Q., 2011. The new genetic environment of cfr on plasmid pBS-02 in a Bacillus
strain. J. Antimicrob. Chemother. 66, 1174–1175.
Zhang, W.J., Xu, X.R., Schwarz, S., Wang, X.M., Dai, L., Zheng, H.J., Liu, S., 2014.
Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine
origin that harbours the multiresistance gene cfr. J. Antimicrob. Chemother. 69,
385–389.
Zurenko, G.E., Yagi, B.H., Schaadt, R.D., Allison, J.W., Kilburn, J.O., Glickman, S.E.,
Hutchinson, D.K., Barbachyn, M.R., Brickner, S.J., 1996. In vitro activities of U-
100592 and U-100766, novel oxazolidinone antibacterial agents. Antimicrob.
Agents Chemother. 40, 839–845.
Zyvox, 2010. Zyvox Package Insert, available at http://www.pfizer.com/products/
rx/rx product zyvox.jsp (accessed January 2013).