ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2008, p. 2244–2246
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 6
First Report of cfr-Mediated Resistance to Linezolid in Human
Staphylococcal Clinical Isolates Recovered in the United States?
Rodrigo E. Mendes,1Lalitagauri M. Deshpande,1Mariana Castanheira,1Joseph DiPersio,2
Michael A. Saubolle,3and Ronald N. Jones1*
JMI Laboratories, North Liberty, Iowa1; Summa Health System, Akron, Ohio2; and Laboratory Sciences of
Arizona, Banner Health, Phoenix, Arizona3
Received 19 February 2008/Returned for modification 5 March 2008/Accepted 30 March 2008
Linezolid resistance has dominantly been mediated by mutations in 23S rRNA or ribosomal protein L4
genes. Recently, cfr has demonstrated the ability to produce a phenotype of resistance to not only oxazolidi-
nones, but also other antimicrobial classes (phenicols, lincosamides, pleuromutilins, and streptogramin A). We
describe the first detection of cfr-mediated linezolid resistance in Staphylococcus aureus and Staphylococcus
epidermidis recovered from human infection cases monitored during the 2007 LEADER Program.
Linezolid, the first oxazolidinone class agent used in clinical
practice, has demonstrated potent antimicrobial activity
against gram-positive pathogens, including methicillin-resis-
tant Staphylococcus aureus, vancomycin-resistant enterococci,
and Streptococcus spp. (3). According to the LEADER Pro-
gram (5), nearly all S. aureus strains (?99.9%) and coagulase-
negative staphylococci (98.4%) isolated in the United States
were susceptible to linezolid. Furthermore, similar results
(99.8% susceptibility) were observed when testing a global
collection of gram-positive isolates evaluated by the ZAAPS
Program in the same year (5, 6). Linezolid resistance has ap-
peared only sporadically since its introduction in 2000, and it is
usually mediated by the presence of mutations in one or more
alleles of the target 23S rRNA gene (4, 11). However, some
linezolid-resistant isolates fail to display these mutations, indi-
cating the presence of other resistance mechanisms.
Previously, the cfr gene was described as a chloramphenicol
resistance mechanism in Staphylococcus sciuri (14). The cfr-
encoded product, a methyltransferase, provides posttranscrip-
tional methylation of the 23S rRNA at position A2503. This
methylation affects the binding of at least four antimicrobial
classes (phenicols, lincosamides, pleuromutilins, and strepto-
gramin A), leading to a multidrug-resistant phenotype (10).
This gene has been detected in Staphylococcus spp. of animal
origin in Europe (7, 8, 10, 14). One recent report described the
detection of cfr in a Staphylococcus aureus isolate recovered
from the respiratory tract of an infected patient in Colombia
The LEADER Program evaluates the activity of linezolid
and numerous comparator agents against gram-positive clini-
cal isolates recovered from more than 50 medical centers
within the United States. During the 2007 LEADER Program,
linezolid-resistant S. aureus (004-737X) and Staphylococcus
epidermidis (426-3147L) were forwarded to JMI Laboratories
(North Liberty, IA) and tested for susceptibility by the Clinical
and Laboratory Standards Institute (CLSI) reference broth
microdilution method (1). The S. aureus strain was isolated
from a 45-year-old paraplegic female patient residing in a
nursing home. She was admitted to a hospital in Ohio showing
symptoms of urinary tract infection, pneumonia, and sepsis,
which required mechanical ventilation. Candida spp. were re-
covered from a blood culture, and the patient received an
antifungal agent plus ciprofloxacin therapy. Pseudomonas spp.
were also recovered from a urine culture. S. aureus (004-737X),
Klebsiella pneumoniae, and Pseudomonas alcaligenes were re-
covered from a bronchio-alveolar lavage specimen. The patient
subsequently received azithromycin, vancomycin, linezolid,
and piperacillin-tazobactam and remained hospitalized for 1
month secondary to respiratory failure. S. epidermidis (426-
3147L) was isolated from a 79-year-old female living in a long-
term care facility. Between July and September 2007, she was
admitted to a hospital in Arizona and returned to the long-
term care facility multiple times before being placed in hospital
care. S. epidermidis was recovered from a blood culture 3 days
after the second hospital admission. She received vancomycin,
cefepime, and ampicillin/sulbactam. No linezolid use could be
Both isolates (004-737X and 426-3147L) displayed linezolid-
nonsusceptible phenotypes (MICs of 8 and ?256 ?g/ml, re-
spectively), which were confirmed by the Etest (AB Biodisk,
Solna, Sweden) and the disk diffusion methods (2), with results
at 8 and ?256 ?g/ml or 19 and 6 mm, respectively. Addition-
ally, the isolates showed resistance to chloramphenicol, clin-
damycin, quinupristin-dalfopristin, retapamulin, oxacillin, cip-
rofloxacin, erythromycin (S. aureus only), tetracycline, and
trimethoprim-sulfamethoxazole but remained susceptible to
vancomycin (Table 1). These results led to screenings for the
G2576T mutation in the 23S rRNA genes (11) and the previ-
ously described cfr gene (8). The G2576T mutation was not
present, but a positive PCR result was obtained using cfr-
specific primers, which was confirmed in both isolates by se-
quencing. S. aureus 004-737X was further analyzed for the
characterization of SCCmec types and the presence of PVL
genes (lukF-PV and lukS-PV) (12) and was subjected to pulsed-
field gel electrophoresis (PFGE). The PFGE pattern was com-
pared to those of contemporary community-acquired and hos-
* Corresponding author. Mailing address: JMI Laboratories, 345
Beaver Kreek Centre, Suite A, North Liberty, IA 52317. Phone: (319)
665-3370. Fax: (319) 655-3371. E-mail: email@example.com.
?Published ahead of print on 7 April 2008.
pital-associated methicillin-resistant S. aureus clones prevalent
in the United States (15). Additionally, both isolates were
screened for erythromycin resistance determinants, as previ-
ously described (9). Characterization of SCCmec types (I
through VI) of S. aureus isolate 004-737X was unsuccessful,
and the reaction for the presence of PVL genes was negative.
The isolate showed a unique PFGE pattern compared to those
of the predominant U.S. clones, and erythromycin resistance in
the isolate 004-737X was mediated by ermA, while the isolate
426-3147L showed negative results for the most common
ermA, ermB, ermC, and mefA resistance genes. This latter re-
sult does not exclude the possibility that the isolate 426-3147L
harbored other ribosomal methylation or efflux pump genes,
which could explain the decreased susceptibility (4 ?g/ml) to
Plasmid DNA was extracted using the plasmid DNA midi kit
(Qiagen GmbH, Hilden, Germany), separated on 1% agarose
gel in Tris-acetate-EDTA buffer on a Criterion sub-cell GT
system (Bio-Rad, Hercules, CA), and transferred onto a nylon
membrane by Southern blotting (13). A labeled cfr probe was
used for hybridization, which was revealed with a nonradioac-
tive DIG-High Prime DNA labeling and detection kit (Roche
Diagnostics GmbH, Mannheim, Germany). Plasmid sizes were
determined by comparison with standard plasmid DNAs ex-
tracted from Escherichia coli NCTC 50192 and NCTC 50193.
Analysis of the plasmid content of isolates 004-737X and 426-
3147L revealed the presence of two plasmids in each isolate
(550 and 55 kb, and 175 and 75 kb, respectively). Experiments
showed that the 55- and 175-kb plasmid DNAs from isolates
004-737X and 426-3147L, respectively, hybridized with the cfr-
specific probe (data not shown).
Surrounding cfr DNA sequences were accessed by primer
walking. Downstream of the cfr gene, the presence of ?tnpB
was noted in the S. aureus isolate, which was identical to the
structure described for the pSCFS3 plasmid found in an S.
aureus isolate collected from the respiratory tract of a swine (7)
(AM086211) (Fig. 1). The DNA sequence upstream of the cfr
gene in the S. aureus isolate showed the presence of istAS and
istBS genes, which were also identical to those of the pSCFS3
plasmid, suggesting that these insertion sequences may be in-
volved in the mobilization of the cfr gene (7). However, a PCR
using a primer targeting ?tnpA (tnpA-F), which was located
further upstream of the cfr gene on the pSCFS3 plasmid,
yielded a negative result, suggesting that the upstream region
of cfr on this isolate significantly differed from that of the
pSCFS3 plasmid. PCRs performed with the primers cfr-F and
cfr-R2 or cfr-F2 and tnpB-R for the 426-3147L isolate yielded
negative results, also indicating additional distinct DNA se-
quences upstream and downstream of the cfr gene on this
isolate. Further analysis of the cfr genetic context in the 426-
3147L isolate is ongoing.
Linezolid resistance, as described in numerous earlier re-
ports, has been mediated by mutations in 23S rRNA or other
TABLE 1. Antimicrobial susceptibility profiles of cfr-harboring
S. aureus (004-737X) and S. epidermidis (426-3147L) isolates
aErythromycin resistance is mediated by ermA.
bNo macrolide resistance mechanism was detected.
FIG. 1. Schematic representation of cfr surrounding DNA sequences in S. aureus (004-737X) and S. epidermidis (426-3147L) isolates. The
genetic context of pSCFS3 is also shown for comparison purposes (AM086211). Genes are indicated by boxes, and the arrows indicate their
transcriptional orientations. Small arrows indicate primers targeting regions and their respective orientations. Dashes indicate unknown DNA
sequences. The background shading indicates identity to the pSCFS3 DNA sequence.
VOL. 52, 2008NOTES2245
ribosomal protein genes, implying the slow dissemination of
resistance by these mechanisms (10). However, the detection
of a plasmid-borne cfr-mediated linezolid resistance gene in
staphylococci recovered from human specimens in the United
States adds a new dimension to the threat against the clinical
utility of several antimicrobial classes, including the oxazolidi-
Although S. aureus 004-737X did not belong to one of the
prevalent clones in the United States (15) and cfr-carrying
Staphylococcus sp. isolates appear rare (10), these data require
continued active resistance surveillance programs (such as
LEADER and ZAAPS). This must be combined with effective
infection control strategies in case further spread of this resis-
tance mechanism is observed by those programs. The dissem-
ination of the cfr-mediated resistance genes among staphylo-
coccal clinical isolates is especially worrisome given the
potential for rapid simultaneous increases in resistance rates
for several antimicrobial classes.
Nucleotide sequence accession number. The nucleotide se-
quences of the cfr gene from S. aureus 004-737X have been
deposited in the GenBank database under the accession num-
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2246 NOTESANTIMICROB. AGENTS CHEMOTHER.