Linezolid-Resistant Staphylococcus aureus Strain 1128105, the First Known Clinical Isolate Possessing the cfr Multidrug Resistance Gene

Abstract

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZD^r clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination.

INTRODUCTION

Linezolid (LZD) resistance occurs predominantly through structural alterations to the oxazolidinone binding site in the 50S peptidyl transferase center (PTC) (1). These conformational changes are the result of mutations in genes encoding 23S rRNA (2, 3) or the ribosomal proteins L3 and L4 (4, 5) or via posttranscriptional modification of 23S rRNA base A2503 by the Cfr methyltransferase (6). Highly LZD^r isolates that possess both the cfr gene and chromosomally encoded mutations have been identified (7–10).

The horizontally transferrable, plasmid-borne nature of cfr makes this resistance determinant inherently more worrisome than chromosomally encoded resistance mechanisms that must arise independently and/or disseminate clonally (11, 12). Adding to the potential for spread is the low fitness cost of this gene (13) and the broad spectrum of resistance conferred by Cfr to drugs included in the PhLOPS_A phenotype (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A class antibiotics) (6, 12, 14), as well as 16-member-ring macrolides (15). Within the oxazolidinone class, there are differences in susceptibilities of strains possessing cfr depending on structural features at both ends of the molecule. The addition of D-ring systems that pick up additional binding interactions in the PTC and the substitution of an A-ring C-5 hydroxymethyl group (in place of the bulkier acetamide-containing substituent found on LZD and other oxazolidinones) allow oxazolidinones such as tedizolid (TZD) (16) to retain greater potency than LZD in the presence of Cfr methylation (17).

The cfr gene was first identified on the pSCFS1 plasmid in a Staphylococcus sciuri isolate recovered from a florfenicol-treated calf in Bavaria in 1997 (18). Since then, a variety of other veterinary staphylococci possessing cfr-bearing plasmids sharing similarity either to pSCFS1 or to one of two other groups possessing regions containing the cfr gene flanked by either IS256 or IS21-558 mobile elements have been identified (19). The first clinical cfr-positive strains reported were Staphylococcus aureus isolates recovered in 2005 from Ireland and Colombia. Irish methicillin-resistant S. aureus (MRSA) isolate M05/0060 (no specific collection date given) (20) possessed the cfr-bearing pSCFS7 plasmid, which has similarity to the IS21-558-carrying veterinary plasmid pSCFS3 (21), although only a truncated portion of the IS21-558 element remained (22). Colombian MRSA isolate CM05 was recovered in May 2005 from a patient in Medellin, Colombia, who had briefly undergone LZD therapy (2 doses) (6, 23). The cfr gene in CM05 was chromosomally located and shared flanking sequence with high similarity to the pSM19035 Gram-positive multidrug resistance plasmid, including an IS21-558 element (24, 25). Subsequently, additional cfr-positive clinical staphylococci and enterococci have been recovered from diverse geographical origins: the United States (26), Belgium (27), Germany (28), France (29), Italy (30), Spain (31), Mexico (32), Panama (33), Brazil (34), Thailand (35), Canada (36), India (37), and China (38). The cfr gene has also been found in Gram-negative species, including Escherichia coli (39) and Proteus vulgaris (40), although in these species the Gram-negative IS26 element appears to have been involved in cfr mobility (19).

To investigate the potential existence of clinical cfr-positive strains predating CM05, we screened historical LZD^r Gram-positive isolates that were collected as part of an ongoing surveillance initiative conducted by Eurofins Medinet to monitor antimicrobial resistance trends among key Gram-positive pathogens encountered across the United States. Among these was a 2005 LZD^r MRSA isolate (strain 1128105), which was selected for further phenotypic and genotypic analysis. Here we characterize the genetic determinants conferring LZD resistance in 1128105, describe the genetic environment of the cfr gene, and assess its transmissibility.

(Portions of this work were presented at the 23rd European Congress of Clinical Microbiology and Infectious Diseases, 2013 [41]).

CASE REPORT

In January 2005, a 36-year-old female patient with end-stage cystic fibrosis presented to the emergency room (Indianapolis, IN) with increased cough, low-grade fever (100.8°F), decreased appetite, and left-sided chest pain. Chest X-ray on admission showed acute left midlung airspace disease with left pleural effusion. The S. aureus respiratory isolate resistant to linezolid (strain 1128105) that was recovered during the course of her hospitalization is described below.

The patient had numerous prior hospitalizations, with a complicated history of end-stage cystic fibrosis, cystic fibrosis-related diabetes, pulmonary hypertension, hemoptysis, hypoxemia, and chronic renal failure. The patient was admitted to the intensive care unit (ICU) on 2 January 2005, requiring oxygen and Albuterol and Pulmozyme nebulizer treatments. She was also started on intravenous (i.v.) ceftazidime and tobramycin because previous hospitalization cultures grew multidrug-resistant Pseudomonas aeruginosa. The patient's lung disease showed improvement on X-ray analysis, and she was transferred to the pulmonary unit on 4 January 2005. Over a course of several days, the patient's pulmonary functions worsened. Repeat X-rays showed chronic lung changes, and sputum cultures from 12 January 2005 grew P. aeruginosa and MRSA susceptible to linezolid (MIC, 4 μg/ml). Oral linezolid was started on 15 January 2005. The patient completed 13 days of oral linezolid and was continued on i.v. ceftazidime and tobramycin throughout hospitalization. Repeat sputum cultures from 20 January 2005 showed no changes, with both P. aeruginosa and MRSA present. The MIC of linezolid, however, was elevated (16 μg/ml) from the previous culture on 12 January 2005. The patient was stable on 28 January 2005 and discharged home on i.v. ceftazidime and tobramycin and Albuterol, colistin, and Pulmozyme nebulizer treatments.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

S. aureus strains 1128105 (initially designated 3133832) (41), ATCC 29213, ATCC 29213 T2500A, RN4220, and transformants thereof were cultured aerobically at 37°C on cation-adjusted Mueller-Hinton II agar (MHA) (Becton Dickinson, Franklin Lakes, NJ) or in MH broth (MHB). The ATCC 29213 T2500A strain possesses a single copy (allele 4) of the 23S rRNA gene mutation T2500A (E. coli numbering) and was selected as a spontaneous mutant with reduced LZD susceptibility in a previous study (42). For conjugation experiments, RN4220 was transduced to novobiocin resistance from S. aureus strain U9NO (43) as an additional strain selection marker.

Antimicrobial agents.

Vancomycin (VAN), florfenicol (FFC), erythromycin (ERY), clindamycin (CLI), tobramycin (TOB), trimethoprim (TMP), ciprofloxacin (CIP), oxacillin (OXA), ceftazidime (CAZ), and novobiocin (NOV) were purchased from Sigma-Aldrich (St. Louis, MO). Other compounds were obtained as follows: TZD (Trius Therapeutics, San Diego, CA), LZD (ChemPacific Corp., Baltimore, MD), tiamulin (TIA) (Wako Pure Chemical Industries, Ltd., Richmond, VA), and daptomycin (DAP) (TSZ CHEM, Framingham, MA). All compounds were prepared fresh in dimethyl sulfoxide (DMSO), ethanol, or deionized water prior to use in MIC assays or selective media.

MIC assays.

MIC assays were performed via broth microdilution in accordance with CLSI guidelines (44), with the exception that test compounds were made up at a 50× concentration (2 μl added to 98 μl of broth plus cells) and that MIC values were determined visually through detection with alamarBlue (Invitrogen Corp., Carlsbad, CA) as previously described (45). Enumeration of CFU was performed by serially diluting bacteria in phosphate-buffered saline (PBS) and plating on MHA. MIC assays containing DAP used MHB supplemented with 50 mg/liter Ca²⁺.

Plasmid isolation and transformation.

Plasmid DNA was isolated from 1128105 via lysostaphin digestion and subsequent processing using a miniprep kit (Qiagen, Venlo, Netherlands). Competent cell preparation and transformation of ATCC 29213 and RN4220 background strains was performed as previously described (46). Transformant cells were plated on MHA containing 5 μg/ml of TIA.

PCR and plasmid sequencing.

De novo plasmid sequencing of p1128105 was performed using the Illumina HiSeq system (Genewiz, South Plainfield, NJ). 23S rRNA alleles from rrn operons 1, 3, 4, and 5 were amplified by PCR using primers previously described (47). Allele 2 was amplified with primers rrn6_RF (reverse complement to rrn6_R [48, 49]) and rrn2_R (47), and allele 6 was amplified using 16S_F and rrn6_R (48, 49). Genes encoding ribosomal proteins L3 (rplC) and L4 (rplD) were amplified as previously described (42). Sequence data were analyzed using the Vector NTI Advance 11 software program (Invitrogen) and annotated based on homology to sequence data from NCBI BLAST analyses (50).

Molecular typing.

For pulsed-field gel electrophoresis (PFGE), chromosomal DNA was prepared in agarose plugs, digested with SmaI restriction endonuclease, and analyzed as previously described (51). Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) were also performed using published protocols (52, 53).

Filter mating.

Conjugation experiments were performed by filter mating as described previously (54). Briefly, overnight donor and recipient cultures were resuspended in 5 ml 0.5% NaCl to an optical density at 540 nm (OD₅₄₀) of 1.0. The suspensions were combined and collected on a 0.45-μm nitrocellulose filter, which was incubated upright on a brain heart infusion (BHI) (Becton, Dickinson and Company, Sparks, MD) agar plate overnight at 37°C. The filter was resuspended in 10 ml BHI broth and vigorously vortexed, and the cells recovered by centrifugation for 10 min at 2,700 × g. The resulting cell pellet was resuspended in 1.0 ml BHI broth and plated on BHI agar containing 5 μg/ml TIA and 10 μg/ml NOV for transconjugant selection after overnight incubation at 37°C. Resulting colonies were further subcultured twice on BHI agar with TIA and NOV followed by PFGE analysis to confirm the transconjugant lineage.

Plasmid analysis and Southern hybridization.

Plasmid DNA was isolated by the method of Holmes and Quigley (55) and visualized on 0.8% SEAKems LE agarose gels (Lonza, Rockland, ME) with electrophoresis for 12 h at 6 V/cm. A digoxigenin-labeled full-length cfr-specific 1,050-bp probe was synthesized using the cfr_F/cfr_R primers (25) and the Roche PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany). Southern hybridization was performed essentially by the method of Sambrook and Russell (56) using the Roche DIG nucleic acid detection kit, following the manufacturer's instructions.

Nucleotide sequence accession number.

A 7,020-bp region of the p1128105 plasmid containing the cfr gene has been deposited in the NCBI database under the GenBank accession number KJ866414.

RESULTS

1128105 has a molecular profile most similar to that of USA100.

Molecular analysis of S. aureus strain 1128105 revealed a PFGE banding pattern most similar to that of health care-associated MRSA USA100 (88% related to USA100 by the unweighted-pair group method using average linkages (UPGMA)/Dice coefficient) (Fig. 1). Results of spa typing (i.e., t002) and MLST (clonal complex 5, sequence type 5) were also consistent with this relationship (57).

FIG 1.

PFGE profile comparison of 1128105 with USA100 and USA800 reference strains.

The 1128105 antibiogram is consistent with the presence of cfr.

Sequence analysis of LZD resistance determinants in 1128105 (cfr, 23S rRNA, rplC, and rplD) identified the presence of the cfr gene and a single copy of the T2500A 23S rRNA gene mutation (allele 4). T2500A has been previously associated with reduced susceptibility to oxazolidinones (42, 48), and a single copy of this mutation is consistent with a TZD MIC value of 1 μg/ml in S. aureus (42). The 1128105 antibiogram coincides with the PhLOPS_A phenotype conferred by Cfr, including resistance to LZD, FFC, TIA, and CLI (Table 1) (14, 17). Additionally, 1128105 demonstrated resistance to TOB, TMP, CIP, ERY, OXA, and CAZ. Of the drugs tested, 1128105 was susceptible only to VAN and DAP.

TABLE 1.

MIC profiling of S. aureus 1128105 and ATCC 29213 transformants possessing the p1128105 cfr-bearing plasmid

Strain	Presence of cfr	MIC of drug (μg/ml)a
		TZD	LZD	FFC	TIA	CLI	TOB	TMP	CIP	ERY	OXA	CAZ	VAN	DAP
1128105	+	1	16	>128	>128	>128	>128	>128	>128	>128	>128	>128	2	0.5
29213	−	0.5	2	8	0.5	0.062	0.5	2	0.5	0.25	0.5	16	1	0.25
29213 + p1128105	+	0.5	16	>128	>128	>128	64	>128	0.5	0.25	0.25	16	1	0.25
29213 T2500A	−	1	4	8	1	0.062	0.5	2	0.5	0.25	0.25	16	1	0.25
29213 T2500A + p1128105	+	1	16	>128	>128	>128	64	>128	0.5	0.25	0.25	16	1	0.25

^aTZD, tedizolid; LZD, linezolid; FFC, florfenicol; TIA, tiamulin; CLI, clindamycin; TOB, tobramycin; TMP, trimethoprim; CIP, ciprofloxacin; ERY, erythromycin; OXA, oxacillin; CAZ, ceftazidime; VAN, vancomycin; DAP, daptomycin.

The 1128105 cfr gene is plasmid borne.

To determine whether the cfr gene was located on a plasmid or the chromosome, total 1128105 plasmid DNA was transformed into the S. aureus ATCC 29213 wild-type and 23S rRNA T2500A mutant backgrounds and plated on selective medium containing TIA. The presence of TIA^r colonies that were PCR positive for cfr suggested that the cfr gene was plasmid borne. The transformant strains recapitulated the MIC profile of 1128105 for drugs falling within the Cfr resistance spectrum (i.e., LZD, CLI, TIA, and FFC) (Table 1). In addition, no change in MIC was observed for TZD in either the S. aureus ATCC 29213 wild-type (TZD MIC, 0.5 μg/ml) or ATCC 29213 23S rRNA T2500A (MIC, 1 μg/ml) strain transformed with cfr, consistent with previous findings (17). The resulting MIC values for TZD and LZD (1 and 16 μg/ml, respectively) in the ATCC 29213 T2500A background were identical to the combination of the cfr and T2500A resistance determinants in the 1128105 isolate. Southern hybridization confirmed the location of the cfr gene on a plasmid ca. 37 kb in size in S. aureus strain 1128105 and the RN4220 plus p1128105 transconjugants (Fig. 2, lanes 2 and 3 and 6 and 7, respectively). Transformation of p1128105 in the ATCC 29213 wild-type and T2500A backgrounds also conferred resistance to TMP and TOB (Table 1), likely accounted for by the dfrA and aacA-aphD genes identified in other de novo plasmid sequencing contigs.

FIG 2.

Southern blot analysis of cfr in 1128105 and the RN4220 p1128105 transformant. Agarose gel electrophoresis of plasmid preparations (A) and Southern hybridization analysis using a cfr-specific probe (B) with S. aureus strains 1128105 (lanes 2 and 3), RN4220 recipients (lanes 4 and 5), and TIA^r NOV^r RN4220 p1128105 transconjugants (lanes 6 and 7) (see the text) are shown. S. aureus USA300 strain FPR3757 (lane 1) (74) was included as a plasmid size reference. To demonstrate reproducibility, donor and recipient isolates were analyzed in duplicate lanes. chrom, chromosomal DNA.

The p1128105 cfr plasmid is conjugative.

Filter mating experiments demonstrated conjugal transfer of the cfr plasmid (p1128105) from S. aureus strain 1128105 to recipient RN4220 at a frequency ranging from 1.0 × 10⁻⁸ to 4.0 × 10⁻⁹ transconjugates per recipient cell. Agarose gel electrophoresis of plasmid preparations from transconjugants confirmed the presence of p1128105 (data not shown).

The p1128105 cfr gene environment has high similarity to those found in the P. vulgaris PV-01 and S. aureus 1900 isolates.

A 7,020-bp contig containing the cfr gene was identified from de novo sequencing of 1128105 total plasmid DNA. This 7-kb region of p1128105 was nearly identical to that found chromosomally in the Chinese PV-01 P. vulgaris veterinary isolate (GenBank accession no. JF969273) (40) and in the pSA1900 plasmid of the recently described clinical S. aureus isolate 1900, recovered from an Ohio hospital in during a 2006-2007 cfr outbreak (GenBank accession no. KC561137; deposited as “pSA8589”) (58) (Fig. 3). In both 1128105 and 1900, the cfr gene is plasmid borne, whereas it is chromosomally located in PV-01, although it is thought to have arisen through integration of a cfr-bearing plasmid via recombination between an IS26 element present in a plasmid and one inserted into the chromosomal fimD gene (40).

FIG 3.

Comparative analysis of the p1128105 cfr locus. Alignment of cfr gene environments from S. aureus 1128105 plasmid p1128105 (7,020 bp), S. aureus 1900 plasmid pSA1900/pSA8589 (6,962 bp) (58), and P. vulgaris PV-01 chromosomal sequence (11,228 bp) (40) reveal almost 7 kb of near-100% identity (gray shading). Direct repeats (DRs) and adjacent terminal inverted repeats (TIRs) are shown in boxes. Key differentiating sequence features among the three cfr loci include rep gene 8-bp direct repeats in p1128105 at the site of insertion of an IS431-like element flanked by 16-bp TIRs (A), a 10-bp insertion in p1128105 but not present in pSA1900/pSA8589 or PV-01 (B), a single adenine base present in p1128105 and PV-01 but not in pSA1900/pSA8589 (C), 8-bp DRs in the PV-01 rep gene and 8-bp fimD DRs at the site of insertion of IS26 and its 14-bp TIRs (D). The ORFs located upstream of cfr in pSA1900/pSA8589 and PV-01 are labeled here as “ΔtnpB*” rather than their “ΔtnpB” designation in GenBank due to a lack of sequence similarity with the tnpB gene of Tn554 (GenBank accession no. X03216) (59). In p1128105, the 5′-AGCGTACAAC-3′ sequence present upstream of cfr extends this putative ORF (“ORF1”), which has been designated a hypothetical protein, beyond the length of the ΔtnpB* ORFs of pSA1900/pSA8589 and PV-01. The pSA1900/pSA8589 sequence is a complete circular plasmid that was linearized here for illustrative purposes by splitting the rep gene to optimize alignment with the linear p1128105 and PV-01 sequences.

The p1128105 sequence contains two internal sequence discrepancies with PV-01 and pSA1900/pSA8589: (i) a 5′-AGCGTACAAC-3′ insertion in p1128105, 311 bp upstream from cfr, not present in pSA1900/pSA8589 or PV-01 (Fig. 3B) and (ii) an “A” present in p1128105 and PV-01, 58 bp downstream of cfr, but not found in pSA1900/pSA8589 (Fig. 3C). The 5′-AGCGTACAAC-3′ insertion within the 300-bp “ΔtnpB” open reading frame (ORF) of PV-01 and pSA1900/pSA8589 results in the generation of a 513-bp ORF in p1128105. This ORF, as well as the “ΔtnpB” ORFs of PV-01 and pSA1900/pSA8589, however, lack any significant sequence similarity with the tnpB gene of Tn554 (GenBank accession no. X03216) (59). The p1128105 ORF shares 97% sequence identity with the 226-bp 5′ portion of the 357-bp “ΔtnpB” ORF of the pSCFS1 cfr plasmid (GenBank accession no. AJ579365) (60), although this similarity ends around the 3′ 140 bp of the pSCFS1 ΔtnpB sequence, which has 100% nucleotide identity with the 3′ end of the Tn554 tnpB gene; hence, this ORF has been designated simply a hypothetical protein (“ORF1”) in p1128105. The p1128105 ORF1 sequence, including the 5′-AGCGTACAAC-3′ insertion, also has high similarity with sequences found in the multidrug resistance plasmids pKKS825 from S. aureus (GenBank accession no. FN377602) (61) and pDB2011 from Listeria innocua (GenBank accession no. KC456362) (62).

Both the PV-01 and p1128105 cfr environments contain the ORF1-cfr-rec-hp-pre/mob gene insertion within identical rep gene sequences; however, the rep gene is disrupted in different locations. In p1128105, insertion of a mobile element resulted in the direct duplication of the 5′-ATATTAAA-3′ insertion site sequence within rep, which is flanked by the 16-bp perfect terminal inverted repeats (TIRs) (5′-ACTTTGCAACAGAACC-3′ and 5′-GGTTCTGTTGCAAAGT-3′), while PV-01 contains a direct duplication of the 5′-CTTTAGAT-3′ rep insertion site sequence flanked by IS26 14-bp TIRs (5′-TTTGCAACAGTGCC-3′ and 5′-GGCACTGTTGCAAA-3′) (Fig. 3A and D) (40, 63). The p1128105 TIRs, as well as flanking sequence of the contig both upstream (5′-TTTT-3′) and downstream (5′-TGA-3′) of the 5′ and 3′ TIRs, are identical to sequences of IS431 (64, 65). The pSA1900/pSA8589 plasmid also shares the conserved ORF1-cfr-rec-hp-pre/mob genetic composition; however, this plasmid contains an uninterrupted rep gene. Two additional, larger cfr-bearing plasmids are present in S. aureus 1900, and it is unknown how this 7-kb plasmid may integrate into those plasmids and whether such integrations may involve mobile elements inserting within rep (58).

DISCUSSION

Linezolid-resistant S. aureus 1128105 represents the earliest documented cfr-positive clinical isolate, predating CM05 by 5 months. Of the three 2005 cfr clinical isolates described to date (CM05, M05/0060, and 1128105,) all have originated in geographically distinct regions and possess unique cfr gene environments. The cfr gene in CM05 is found in a pSM19035-like chromosomally integrated region (25), and the cfr environment of M05/0060 is a pSCFS3-like background (22), while here, we show that the p1128105 cfr-flanking sequence is highly similar to the cfr gene chromosomal insertion in P. vulgaris (40) and the pSA1900/pSA8589 plasmid of S. aureus 1900 (58), although unique in its association with IS431-like sequences. The IS431 element has been found in only one other cfr-bearing plasmid, pERGB (66), although it is an element commonly found in SCCmec cassettes and Gram-positive plasmids (67). The TIRs of IS431 are identical to those found in the Gram-positive insertion sequences ISSau10 (68) and IS257 (69) and are highly similar to those of the Gram-negative IS26 element (63, 64). These associations further support the emerging picture of cfr as a widely disseminated multidrug resistance drug gene found in a diverse set of genetic environments in a wide variety of clinical and veterinary pathogens (19). The ongoing use of drug classes such as phenicols and pleuromutilins in veterinary medicine (i.e., florfenicol, tiamulin, and valnemulin) will provide continued positive selective pressure for cfr and increases the likelihood of transfer of this gene into clinical strains, where other members of these drug classes (i.e., chloramphenicol and retapamulin) are used in human medicine.

Analysis of the remainder of p1128105 sequence could provide further clues about the origins of early clinical cfr plasmids and the likely transfer from genetic environments found in veterinary pathogens to those found in clinical pathogens and/or transfer between Gram-positive and Gram-negative species. Although the first documented occurrence of cfr was in a veterinary isolate of S. sciuri, there have not been worldwide longitudinal surveys of veterinary or environmental isolates to identify the origin or comprehensive species distribution of the gene in nonclinical settings. Therefore, the abundance of cfr in P. vulgaris and whether the gene originated with or was transferred to staphylococci are still unknown. More conclusive documentation of the early dissemination of cfr, however, will perhaps require a different strategic approach than was taken here (analyzing linezolid-resistant strains), for the following reasons: (i) a recent report describes an E. faecalis veterinary isolate that failed to confer the typical Cfr antibiogram profile, likely due to unknown strain factors, since the gene itself was still transcribed and translated (70), (ii) LZD surveillance studies in recent years have identified LZD^s cfr-positive S. aureus clinical isolates (MIC = 4 μg/ml) (71, 72), and (iii) cfr has been identified in Gram-negative species (39, 40), which are intrinsically resistant to some drugs within the Cfr resistance spectrum, such as oxazolidinones and pleuromutilins. Identifying potential pre-2005 clinical cfr isolates would therefore benefit from the use of MIC-independent genotypic screening strategies.

Although the strain was not retained, the S. aureus isolate collected from the patient on 12 January 2005, 3 days prior to starting LZD therapy, had an LZD MIC value of 4 μg/ml, which is consistent with the MIC of the ATCC 29213 background single-copy 23S rRNA T2500A mutant strain used in this study and could represent a cfr-negative background of 1128105 or one in which the cfr gene was not expressed. The subsequent therapeutic regimen containing LZD and TOB would have provided dual selective pressure for the acquisition and/or expression of the cfr-containing multidrug resistance plasmid p1128105. The conjugative potential of p1128105 adds credence to the horizontal transmissibility of this particular cfr plasmid. It is interesting to note that the 1900 S. aureus isolate, which also possesses a plasmid-borne P. vulgaris-like cfr gene environment, was isolated as part of an outbreak of cfr strains from 2006-2007 in a bordering state (Ohio). 1900, like 1128105, was also multilocus sequence type 5 and spa type t002 and has a PFGE profile most similar to that of USA100 (58).

Similarly to the case with 1128105, the cfr gene has been found in a large number of strains with co-occurring chromosomal mutations in 23S rRNA and/or ribosomal protein L3 and L4 mutations that selectively confer reduced susceptibility to oxazolidinones (8, 10, 73). The presence of cfr in strains with such background mutations may favor the propagation of these strains even in the absence of selective pressure from LZD, since any member of the 6 classes of agents falling within the Cfr resistance spectrum could select for its retention. As demonstrated with previous structure-activity relationship studies, MIC values of TZD were maintained in the presence of cfr, likely due to the presence of a potency-adding D-ring system and reduced steric interaction of the methylated A2503 23S rRNA with the hydroxymethyl A-ring C-5 substituent of TZD, relative to the more bulky C-5 acetamide-containing group of linezolid (17).

The expansive drug resistance profile of 1128105 highlights the importance of continuing to develop Gram-positive agents which maintain activity against a wide variety of resistance mechanisms. VAN and DAP were the only two antistaphylococcal agents tested in this study to which 1128105 remained susceptible. The β-lactam and aminoglycoside resistance mechanisms in this strain additionally conferred resistance to broad-spectrum agents such as ceftazidime and tobramycin, used to target the patient's co-occurring P. aeruginosa infection. Although it is the earliest clinical cfr-positive isolate described to date, 1128105 possesses a diverse set of mechanisms capable of evading many contemporary antimicrobial therapeutics.