Linezolid-resistant Staphylococcus aureus strain 1128105, the first

Linezolid-resistant Staphylococcus aureus strain 1128105, the first known clinical isolate 1

possessing the cfr multidrug resistance gene 2

3

Running title: cfr-positive S. aureus isolate 1128105 4

5

Jeffrey B. Locke1#, Douglas E. Zuill1, Caitlyn R. Scharn3, Jennifer Deane2, Daniel F. Sahm2, 6

Gerald A. Denys4, Richard V. Goering3, and Karen J. Shaw1 7

8

1Trius Therapeutics, Inc., 6310 Nancy Ridge Drive, Suite 105, San Diego, CA 92121 9 10 2Eurofins Global Central Laboratory, 14100 Park Meadow Drive, Suite 110, Chantilly, VA 11 20151 12 13 3Department of Medical Microbiology and Immunology, Creighton University School of 14 Medicine, 2500 California Plaza, Omaha, NE 68178 15 16 4Indiana University Health Pathology Laboratory, 350 West 11th Street, Room 6027B 17 Indianapolis, IN 46202 18 19

#Corresponding author: 20

Jeffrey B. Locke, Ph.D. 21

Cubist Pharmaceuticals, Inc. 22

6310 Nancy Ridge Drive, Suite 105 23

San Diego, CA 92121 24

Phone: (858) 452-0370 x226 25

Fax: (858) 452-0412 26

E-mail: [email protected] 27

Key words: cfr, linezolid, tedizolid, oxazolidinone, Staphylococcus aureus 28

AAC Accepts, published online ahead of print on 25 August 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.03493-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 29

The Cfr methyltransferase confers resistance to six classes of drugs which target the 30

peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones such as 31

linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the 32

late 1990s, although the earliest report of a clinical cfr-positive strain was 2005 Colombian 33

MRSA isolate CM05. Here, through retrospective analysis of LZDr clinical strains from a US 34

surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, 35

predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique PFGE profile 36

most similar to USA100, spa type t002 and multilocus sequence type 5 (ST5). In addition to cfr, 37

LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S 38

rRNA gene mutation T2500A. Transformation of the ~37 kb conjugative p1128105 cfr-bearing 39

plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in 40

recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 41

7 kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the 42

Chinese veterinary Proteus vulgaris isolate PV-01 and in US clinical S. aureus isolate 1900, 43

although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment 44

in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-45

negative strains of clinical and veterinary origin and has been associated with multiple mobile 46

elements, highlighting the versatility of this multidrug resistance gene and its potential for further 47

dissemination. 48

49

50

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52

INTRODUCTION 53

Linezolid (LZD) resistance predominantly occurs through structural alterations to the 54

oxazolidinone binding site in the 50S peptidyl transferase center (PTC) (1). These 55

conformational changes are the result of mutations in genes encoding 23S rRNA (2, 3) or 56

ribosomal proteins L3 and L4 (4, 5) or via posttranscriptional modification of 23S rRNA base 57

A2503 by the Cfr methyltransferase (6). Highly LZDr isolates have been identified that possess 58

both the cfr-gene and chromosomally-encoded mutations (7-10). 59

The horizontally-transferrable, plasmid-borne nature of cfr makes this resistance 60

determinant inherently more worrisome than chromosomally-encoded resistance mechanisms 61

that must arise independently and/or disseminate clonally (11, 12). Adding to the potential for 62

spread is the low fitness cost of this gene (13) and the broad spectrum of resistance conferred by 63

Cfr to drugs included in the PhLOPSA phenotype (phenicol, lincosamide, oxazolidinone, 64

pleuromutilin, and streptogramin A class antibiotics) (6, 12, 14) as well as 16-member-ring 65

macrolides (15). Within the oxazolidinone class there are differences in susceptibility to strains 66

possessing cfr depending on structural features at both ends of the molecule. The addition of D-67

ring systems that pick up additional binding interactions in the PTC and the substitution of an A-68

ring C-5 hydroxymethyl groups (in place of the bulkier acetamide-containing substituent found 69

on LZD and other oxazolidinones) allow oxazolidinones such as tedizolid (16) to retain greater 70

potency than LZD in the presence of Cfr methylation (17). 71

The cfr gene was first identified on the pSCFS1 plasmid in a Staphylococcus sciuri 72

isolate recovered from a florfenicol-treated calf in Bavaria in 1997 (18). Since then, a variety of 73

other veterinary staphylococci have been identified possessing cfr-bearing plasmids sharing 74


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either similarity to pSCFS1 or to one of two other groups possessing regions containing the cfr 75

gene flanked by either IS256 or IS21-558 mobile elements (19). The first clinical cfr-positive 76

strains reported were Staphylococcus aureus isolates recovered in 2005 from Ireland and 77

Colombia. Irish MRSA isolate M05/0060 (no specific collection date given) (20) possessed the 78

cfr-bearing pSCFS7 plasmid which has similarity to IS21-558-carrying veterinary plasmid 79

pSCFS3 (21) although only a truncated portion of the IS21-558 element remained (22). 80

Colombian MRSA isolate CM05 was recovered in May of 2005 from a patient in Medellin who 81

had briefly undergone LZD therapy (2 doses) (6, 23). The cfr gene in CM05 was chromosomally 82

located and shared flanking sequence with high similarity to the pSM19035 Gram-positive 83

multidrug resistance plasmid, including an IS21-558 element (24, 25). Subsequently, additional 84

cfr-positive clinical staphylococci and enterococci have been recovered from diverse 85

geographical origins: United States (26), Belgium (27), Germany (28), France (29), Italy (30), 86

Spain (31), Mexico (32), Panama (33), Brazil (34), Thailand (35), Canada (36), India (37), and 87

China (38). The cfr gene has also been found in Gram-negative species including Escherichia 88

coli (39) and Proteus vulgaris (40), although in these species the Gram-negative IS26 element 89

appears to have been involved in cfr mobility (19). 90

To investigate the potential existence of clinical cfr-positive strains predating CM05 we 91

screened historical LZDr Gram-positive isolates that were collected as part of an ongoing 92

surveillance initiative conducted by Eurofins Medinet to monitor antimicrobial resistance trends 93

among key Gram-positive pathogens encountered across the USA. Among these was a 2005 94

LZDr MRSA isolate (strain # 1128105) that was selected for further phenotypic and genotypic 95

analysis. Here we characterize the genetic determinants conferring LZD resistance in 1128105, 96

describe the genetic environment of the cfr gene and assess its transmissibility. 97


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98

CASE REPORT 99

In January 2005, a 36-year-old female patient with end-stage cystic fibrosis presented to 100

the emergency room (Indianapolis, IN) with increased cough, low grade fever (100.8 ºF), 101

decreased appetite, and left sided chest pain. Chest X-ray on admission showed acute left mid 102

lung airspace disease with left pleural effusion. The S. aureus respiratory isolate resistant to 103

linezolid (strain 1128105) recovered during the course of her hospitalization is described below. 104

The patient had numerous prior hospitalizations with a complicated history of end stage 105

cystic fibrosis, cystic fibrosis related diabetes, pulmonary hypertension, hemoptysis, hypoxemia, 106

and chronic renal failure. The patient was admitted to the ICU on 1-2-05 requiring oxygen and 107

Albuterol and Pulmozyme nebulizer treatments. She was also started on IV ceftazidime and 108

tobramycin because previous hospitalization cultures grew multi-drug resistant Pseudomonas 109

aeruginosa. The patient’s lung disease showed improvement on X-ray and she was transferred 110

to the floor on 1-4-05. Over a course of several days the patient’s pulmonary functions 111

worsened. Repeat X-rays showed chronic lung changes, and sputum cultures from 1-12-05 grew 112

P. aeruginosa and MRSA susceptible to linezolid (MIC 4 μg/ml). Oral linezolid was started on 113

1-15-05. The patient completed 13 days of oral linezolid and was continued on IV ceftazidime 114

and tobramycin throughout hospitalization. Repeat sputum cultures from 1-20-05 showed no 115

changes with both P. aeruginosa and MRSA present. The MIC of linezolid, however, was 116

elevated (16 μg/ml) from the previous culture on 1-12-05. The patient was stable on 1-28-05 and 117

discharged home on IV ceftazidime and tobramycin and Albuterol, colistin, and Pulmozyme 118

nebulizer treatments. 119

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MATERIALS AND METHODS 121

Bacterial strains and culture conditions. S. aureus strains 1128105 (initially 122

designated as 3133832) (41), ATCC 29213, ATCC 29213 T2500A, RN4220 and transformants 123

thereof were cultured aerobically at 37°C on cation-adjusted Mueller-Hinton II agar (MHA, 124

Becton Dickinson, Franklin Lakes, NJ) or in MH broth (MHB). The ATCC 29213 T2500A 125

strain possesses a single copy (allele 4) of the 23S rRNA gene mutation T2500A (E. coli 126

numbering) and was selected as a spontaneous mutant with reduced LZD susceptibility in a 127

previous study (42). For conjugation experiments, RN4220 was transduced to novobiocin 128

resistance from S. aureus strain U9NO (43) as an additional strain selection marker. 129

Antimicrobial agents. Vancomycin (VAN), florfenicol (FFC), erythromycin (ERY), 130

clindamycin (CLI), tobramycin (TOB), trimethoprim (TMP), ciprofloxacin (CIP), oxacillin 131

(OXA), ceftazidime (CAZ), and novobiocin (NOV) were purchased from Sigma-Aldrich (St. 132

Louis, MO). Other compounds were obtained as follows: TZD (Trius Therapeutics, San Diego, 133

CA), LZD (ChemPacific Corp., Baltimore, MD), tiamulin (TIA, Wako Pure Chemical Industries, 134

Ltd., Richmond, VA), daptomycin (DAP, TSZ CHEM, Framingham, MA). All compounds were 135

prepared fresh in DMSO, ethanol or deionized water prior to use in MIC assays or selective 136

media. 137

MIC assays. MIC assays were performed via broth microdilution in accordance with 138

CLSI guidelines (44) with the exception that test compounds were made up at 50X concentration 139

(2 µl added to 98 µl of broth + cells) and that MIC values were determined visually through 140

detection with alamarBlue® (Invitrogen Corp., Carlsbad, CA) as previously described (45). 141

Enumeration of colony-forming units was performed by serially diluting bacteria in PBS and 142

plating on MHA. MIC assays containing DAP used MHB supplemented with 50 mg/l Ca2+. 143


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Plasmid isolation and transformation. Plasmid DNA was isolated from 1128105 via 144

lysostaphin digestion and subsequent processing using a miniprep kit (Qiagen, Venlo, 145

Netherlands). Competent cell preparation and transformation of ATCC 29213 and RN4220 146

background strains was performed as previously described (46). Transformant cells were plated 147

on MHA containing 5 µg/ml of TIA. 148

PCR and plasmid sequencing. De novo plasmid sequencing of p1128105 was 149

performed using Illumina HiSeq (Genewiz, South Plainfield, NJ). 23S rRNA alleles from rrn 150

operons 1, 3, 4, and 5 were amplified by PCR using primers previously described (47). Allele 2 151

was amplified with primers rrn6_RF (reverse complement to rrn6_R (48, 49)) and rrn2_R (47) 152

and allele 6 was amplified using 16S_F and rrn6_R (48, 49). Genes encoding ribosomal proteins 153

L3 (rplC) and L4 (rplD) were amplified as previously described (42). Sequence data were 154

analyzed using Vector NTI Advance 11TM software (Invitrogen) and annotated based on 155

homology to sequence data from NCBI BLAST analyses (50). 156

Molecular typing. For pulsed-field gel electrophoresis (PFGE), chromosomal DNA was 157

prepared in agarose plugs, digested with SmaI restriction endonuclease, and analyzed as 158

previously described (51). Staphylococcal protein A (spa) typing and multi-locus sequence 159

typing (MLST) were also performed using published protocols (52, 53). 160

Filter mating. Conjugation experiments were performed by filter mating as described 161

previously (54). Briefly, overnight donor and recipients cultures were resuspended in 5 ml 0.5% 162

NaCl to an optical density (OD540) of 1.0. The suspensions were combined and collected on a 163

0.45 μm nitrocellulose filter which was incubated upright on a brain heart infusion (BHI) 164

(Becton, Dickinson and Company, Sparks, MD) agar plate overnight at 37°C. The filter was 165

resuspended in 10 ml BHI broth, vigorously vortexed, and the cells recovered by centrifugation 166


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for 10 minutes at 2,700 × g. The resulting cell pellet was resuspended in 1.0 ml BHI broth and 167

plated on BHI agar containing 5 μg/ml TIA and 10 μg/ml NOV for transconjugant selection after 168

overnight incubation at 37°C. Resulting colonies were further subcultured twice on BHI agar 169

with TIA and NOV followed by PFGE analysis to confirm the transconjugant lineage. 170

Plasmid analysis and Southern hybridization. Plasmid DNA was isolated by the 171

method of Holmes and Quigley (55) and visualized on 0.8% SEAKems LE agarose gels 172

(LONZA, Rockland, ME) with electrophoresis for 12 hours at 6V/cm. A digoxigenin-labeled 173

full length cfr-specific 1,050 bp probe was synthesized using cfr_F/cfr_R primers (25) and the 174

Roche PCR DIG Probe Synthesis Kit (Roche Diagnostics, Mannheim, Germany). Southern 175

hybridization was performed essentially by the method of Sambrook and Russell (56) using the 176

Roche DIG Nucleic Acid Detection Kit following the manufacturer’s instructions. 177

Nucleotide sequence accession number. A 7,020 bp region of the p1128105 plasmid 178

containing the cfr gene has been deposited in the NCBI database under the GenBank accession 179

number KJ866414. 180

181

RESULTS 182

1128105 has a molecular profile most similar to USA100. Molecular analysis of S. aureus 183

strain 1128105 revealed a PFGE banding pattern most similar to that of healthcare-associated 184

MRSA USA100 (88% related to USA100 by UPGMA/Dice Coefficient) (Fig. 1). Results of spa 185

typing (i.e., t002) and MLST (clonal complex 5, sequence type 5) were also consistent with this 186

relationship (57). 187

The 1128105 antibiogram is consistent with the presence of cfr. Sequence analysis of LZD 188

resistance determinants in 1128105 (cfr, 23S rRNA, rplC, rplD) identified the presence of the cfr 189


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gene and a single copy of the T2500A 23S rRNA gene mutation (allele 4). T2500A has been 190

previously associated with reduced susceptibility to oxazolidinones (42, 48) and a single copy of 191

this mutation is consistent with a TZD MIC value of 1 μg/ml in S. aureus (42). The 1128105 192

antibiogram coincides with the PhLOPSA phenotype conferred by Cfr, including resistance to 193

LZD, FFC, TIA, and CLI (Table 1) (14, 17). Additionally, 1128105 demonstrated resistance to 194

TOB, TMP, CIP, ERY, OXA, and CAZ. Of the drugs tested, 1128105 was only susceptible to 195

VAN and DAP. 196

The 1128105 cfr gene is plasmid-borne. To determine whether the cfr gene was located on a 197

plasmid or the chromosome, total 1128105 plasmid DNA was transformed into the S. aureus 198

ATCC 29213 wild-type and 23S rRNA T2500A mutant backgrounds and plated on selective 199

media containing TIA. The presence of TIAr colonies that were PCR-positive for cfr suggested 200

that the cfr gene was plasmid-borne. The transformant strains recapitulated the MIC profile of 201

1128105 for drugs falling within the Cfr resistance spectrum (i.e. LZD, CLI, TIA, and FFC) 202

(Table 1). In addition, no change in MIC was observed for TZD in either the S. aureus ATCC 203

29213 wild-type (TZD MIC 0.5 µg/ml) or ATCC 29213 23S rRNA T2500A (MIC 1 µg/ml) 204

strains transformed with cfr, consistent with previous findings (17). The resulting MIC values 205

for TZD and LZD (1 and 16 µg/ml, respectively) in the ATCC 29213 T2500A background were 206

identical to the combination of the cfr and T2500A resistance determinants in the 1128105 207

isolate. Southern hybridization confirmed the location of the cfr gene on a plasmid ca. 37 kb in 208

size in S. aureus strain 1128105 and RN4220 + p1128105 transconjugants (Fig. 2, lanes 2-3 and 209

6-7, respectively). Transformation of p1128105 in the ATCC 29213 wild-type and T2500A 210

backgrounds also conferred resistance to TMP and TOB (Table 1), likely accounted for by the 211

dfrA and aacA-aphD genes identified in other de novo plasmid sequencing contigs. 212


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The p1128105 cfr plasmid is conjugative. Filter mating experiments demonstrated conjugal 213

transfer of the cfr plasmid (p1128105) from S. aureus strain 1128105 to recipient RN4220 at a 214

frequency ranging from 1.0 × 10-8 to 4.0 × 10-9 transconjugates per recipient cell. Agarose gel 215

electrophoresis of plasmid preparations from transconjugants confirmed the presence of 216

p1128105 (data not shown). 217

The p1128105 cfr gene environment has high similarity to those found in P. vulgaris PV-01 218

and S. aureus 1900 isolates. A 7,020 bp contig containing the cfr gene was identified from de 219

novo sequencing of 1128105 total plasmid DNA. This 7 kb region of p1128105 was nearly 220

identical to that found chromosomally in the Chinese PV-01 P. vulgaris veterinary isolate 221

(GenBank accession no. JF969273) (40) and in the pSA1900 plasmid of the recently described 222

clinical S. aureus isolate 1900 recovered from an Ohio hospital in during a 2006-2007 cfr 223

outbreak (GenBank accession no. KC561137; deposited as “pSA8589”) (58) (Fig. 3). In both 224

1128105 and 1900, the cfr gene is plasmid-borne whereas it is chromosomally located in PV-01, 225

although it is thought to have arisen through integration of a cfr-bearing plasmid via 226

recombination between an IS26 element present in a plasmid and one inserted into the 227

chromosomal fimD gene (40). 228

The p1128105 sequence contains two internal sequence discrepancies with PV-01 and 229

pSA1900/pSA8589: 1) 5’-AGCGTACAAC-3’ insertion in p1128105 311 bp upstream from cfr 230

not present in pSA1900/pSA8589 or PV-01 (Fig. 3B), and 2) an “A” present in p1128105 and 231

PV-01 58 bp downstream of cfr but not found in pSA1900/pSA8589 (Fig. 3C). The 5’-232

AGCGTACAAC-3’ insertion within the 300-bp “∆tnpB” ORF of PV-01 and pSA1900/pSA8589 233

results in the generation of a 513 bp ORF in p1128105. This ORF, as well as the “∆tnpB” ORFs 234

of PV-01 and pSA1900/pSA8589, however lack any significant sequence similarity with the 235


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tnpB gene of Tn554 (GenBank accession no. X03216) (59). The p1128105 ORF shares 97% 236

sequence identity with the 226 bp 5’ portion of the 357 bp “∆tnpB” ORF of the pSCFS1 cfr 237

plasmid (GenBank accession no. AJ579365) (60), although this similarity ends around the 3’ 140 238

bp of pSCFS1 ∆tnpB sequence which has 100% nucleotide identity with the 3’ end of the Tn554 239

tnpB gene, hence this ORF has been designated simply as a hypothetical protein (“ORF1”) in 240

p1128105. The p1128105 ORF1 sequence, including the 5’-AGCGTACAAC-3’ insertion, also 241

has high similarity with sequences found in multidrug resistance plasmids pKKS825 from S. 242

aureus (GenBank accession no. FN377602) (61) and pDB2011 from Listeria innocua (GenBank 243

accession no. KC456362) (62). 244

Both PV-01 and p1128105 cfr environments contain the ORF1-cfr-rec-hp-pre/mob gene 245

insertion within identical rep gene sequences; however the rep gene is disrupted in different 246

locations. In p1128105 insertion of a mobile element resulted in the direct duplication of the 5’-247

ATATTAAA-3’ insertion site sequence within rep which is flanked by the 16-bp perfect 248

terminal inverted repeats (TIRs) (5’-ACTTTGCAACAGAACC-3’ and 5’-249

GGTTCTGTTGCAAAGT-3’), while PV-01 contains a direct duplication of the 5’-CTTTAGAT-250

3’ rep insertion site sequence flanked by IS26 14-bp TIRs (5’-TTTGCAACAGTGCC-3’ and 5’-251

GGCACTGTTGCAAA-3’) (Fig. 3A, D) (40, 63). The p1128105 TIRs as well as flanking 252

sequence of the contig both upstream (5’-TTTT-3’) and downstream (5’-TGA-3’) of the 5’ and 253

3’ TIRs are identical to sequences of IS431 (64, 65). The pSA1900/pSA8589 plasmid also 254

shares the conserved ORF1-cfr-rec-hp-pre/mob genetic composition, however this plasmid 255

contains an uninterrupted rep gene. Two additional, larger cfr-bearing plasmids are present in S. 256

aureus 1900 and it is unknown how this 7 kb plasmid may integrate into those plasmids and 257

whether such integrations may involve mobile elements inserting within rep (58). 258


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259

DISCUSSION 260

Linezolid-resistant S. aureus 1128105 represents the earliest documented cfr-positive 261

clinical isolate, predating CM05 by 5 months. Of the three 2005 cfr clinical isolates described to 262

date (CM05, M05/0060, and 1128105,) all have originated from geographically-distinct regions 263

and possess unique cfr gene environments. The cfr gene in CM05 is found in a pSM19035-like 264

chromosomally-integrated region (25); the cfr environment of M05/0060 is a pSCFS3-like 265

background (22); while here, we show that the p1128105 cfr-flanking sequence is highly similar 266

to the cfr gene chromosomal insertion in P. vulgaris (40) and the pSA1900/pSA8589 plasmid of 267

S. aureus 1900 (58), although unique in its association with IS431-like sequences. The IS431 268

element has only been found in one other cfr-bearing plasmid, pERGB (66), although it is an 269

element commonly found in SCCmec cassettes and Gram-positive plasmids (67). The TIRs of 270

IS431 are identical to those found in Gram-positive insertion sequences ISSau10 (68), IS257 (69) 271

and are highly similar to those of the Gram-negative IS26 element (63, 64). These associations 272

further support the emerging picture of cfr as a widely disseminated multidrug resistance drug 273

gene found in a diverse set of genetic environments in a wide variety of clinical and veterinary 274

pathogens (19). The ongoing use of drug classes such as phenicols and pleuromutilins in 275

veterinary medicine (i.e. florfenicol, tiamulin, valnemulin) will provide continued positive 276

selective pressure for cfr and increases the likelihood of transfer of this gene into clinical strains 277

where other members of these drug classes (i.e. chloramphenicol, retapamulin) are used in 278

human medicine. 279

Analysis of the remainder of p1128105 sequence could provide further clues about the 280

origins of early clinical cfr plasmids and the likely transfer from genetic environments found in 281


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veterinary pathogens to those found in clinical pathogens and/or transfer between Gram-positive 282

and Gram-negative species. Although the first documented occurrence of cfr was in a veterinary 283

isolate of S. sciuri, there have not been worldwide longitudinal surveys of veterinary or 284

environmental isolates to identify the origin or comprehensive species distribution of the gene in 285

non-clinical settings. Therefore, the abundance of cfr in P. vulgaris, and whether the gene 286

originated from or was transferred to staphylococci, is still unknown. More conclusive 287

documentation of the early dissemination of cfr however will perhaps require a different strategic 288

approach than was taken here (analyzing linezolid-resistant strains) since: 1) a recent report that 289

describes an E. faecalis veterinary isolate that failed to confer the typical Cfr antibiogram profile 290

likely due to unknown strain factors, as the gene itself was still transcribed and translated (70), 2) 291

LZD surveillance studies in recent years that have identified LZDs cfr-positive S. aureus clinical 292

isolates (MIC=4 µg/ml) (71, 72), and 3) the identification of cfr in Gram-negative species (39, 293

40) which are intrinsically resistant to some drugs within the Cfr resistance spectrum, such as 294

oxazolidinones and pleuromutilins. Identifying potential pre-2005 clinical cfr isolates would 295

therefore benefit from the use of MIC-independent genotypic screening strategies. 296

Although the strain was not retained, the S. aureus isolate collected from the patient on 1-297

12-05, 3 days prior to starting LZD therapy, had a LZD MIC value of 4 µg/mL, which is 298

consistent with the MIC of the ATCC 29213 background single-copy 23S rRNA T2500A mutant 299

strain used in this study and could represent a cfr-negative background of 1128105 or one in 300

which the cfr gene was not expressed. The subsequent therapeutic regiment containing LZD and 301

TOB would have provided dual selective pressure for the acquisition and/or expression of the 302

cfr-containing multidrug resistance plasmid p1128105. The conjugative potential of p1128105 303

adds credence to the horizontal transmissibility of this particular cfr plasmid. It is interesting to 304


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note that the 1900 S. aureus isolate, which also possesses a plasmid-borne P. vulgaris-like cfr 305

gene environment, was isolated as part of an outbreak of cfr strains from 2006-2007 in a 306

bordering state (Ohio). 1900, like 1128105, was also multilocus sequence type 5, spa type t002 307

and has a PFGE profile most similar to USA100 (58). 308

Similarly to 1128105, the cfr gene has been found in a large number of strains with co-309

occurring chromosomal mutations in 23S rRNA and/or ribosomal protein L3 and L4 mutations 310

that selectively confer reduced susceptibility to oxazolidinones (8, 10, 73). The presence of cfr 311

in strains with such background mutations may favor the propagation of these strains even in the 312

absence of selective pressure from LZD, since any member of the 6 classes of agents falling 313

within the Cfr resistance spectrum could select for its retention. As demonstrated with previous 314

structure-activity relationship studies, MIC values of TZD were maintained in the presence of cfr 315

likely due to the presence of a potency-adding D-ring system and reduced steric interaction of 316

the methylated A2503 23S rRNA with the hydroxymethyl A-ring C-5 substituent of TZD, 317

relative to the more bulky C-5 acetamide-containing group of linezolid (17). 318

The expansive drug resistance profile of 1128105 highlights the importance of continuing 319

to develop Gram-positive agents which maintain activity against a wide variety of resistance 320

mechanisms. VAN and DAP were the only two anti-staphylococcal agents tested in this study to 321

which 1128105 remained susceptible. The β-lactam and aminoglycoside resistance mechanisms 322

in this strain additionally conferred resistance to broad spectrum agents such as ceftazidime and 323

tobramycin used to target the patient’s co-occurring P. aeruginosa infection. Although it is the 324

earliest clinical cfr-positive isolate described to date, 1128105 possesses a diverse set of 325

mechanisms capable of evading many contemporary antimicrobial therapeutics. 326

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ACKNOWLEDGEMENTS 328

Portions of this work were presented at the 23rd European Congress of Clinical Microbiology 329

and Infectious Diseases (41). This study was funded in part by Trius Therapeutics. Jeffrey B. 330

Locke is a current employee of Cubist Pharmaceuticals. Douglas E. Zuill and Karen J. Shaw 331

were employees of Trius Therapeutics at the time this study was conducted. All authors had full 332

access to the data. The authors had final responsibility for the decision to submit for publication. 333

334

TABLES AND FIGURES 335

Table 1. MIC profiling of S. aureus 1128105 and ATCC 29213 transformants possessing the 336

p1128105 cfr-bearing plasmid 337

338

TZD, tedizolid; LZD, linezolid; FFC, florfenicol; TIA, tiamulin; CLI, clindamycin; TOB, 339

tobramycin; TMP, trimethoprim; CIP, ciprofloxacin; ERY, erythromycin; OXA, oxacillin; CAZ, 340

ceftazidime; VAN, vancomycin; DAP, daptomycin. 341

342

343

FIG 1 PFGE profile comparison of 1128105 with USA100 and USA800 reference strains. 344

345


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346

FIG 2 Southern blot analysis of cfr in 1128105 and the RN4220 p1128105 transformant. 347

Agarose gel electrophoresis of plasmid preparations (A) and Southern hybridization analysis 348

using a cfr-specific probe (B) with S. aureus strains 1128105 (lanes 2 and 3), RN4220 recipients 349

(lanes 4 and 5) and TIAr/NOVr RN4220 p1128105 transconjugants (lanes 6 and 7) (see text). S. 350

aureus USA300 strain FPR3757 (lane 1) (74) was included as a plasmid size reference. To 351

demonstrate reproducibility, donor and recipient isolates were analyzed in duplicate lanes. 352

353

354


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355

FIG 3 Comparative analysis of the p1128105 cfr locus. Alignment of cfr gene environments 356

from S. aureus 1128105 plasmid p1128105 (7,020 bp), S. aureus 1900 plasmid 357

pSA1900/pSA8589 (6,962 bp) (58), and P. vulgaris PV-01 chromosomal sequence (11,228 bp) 358

(40) reveal almost 7 kb of near 100% identity (gray shading). Direct repeats (DRs) and adjacent 359

terminal inverted repeats (TIRs) are shown in boxes. Key differentiating sequence features 360

among the three cfr loci include: (A) rep gene 8-bp direct repeats in p1128105 at the site of 361

insertion of a IS431-like element flanked by 16-bp TIRs, (B) 10-bp insertion in p1128105 but not 362

present in pSA1900/pSA8589 or PV-01, (C) a single adenine base present in p1128105 and PV-363

01 but not in pSA1900/pSA8589, (D) 8-bp DRs in the PV-01 rep gene and 8-bp fimD DRs at the 364

site of insertion of IS26 and its 14-bp TIRs. The ORFs located upstream of cfr in 365

pSA1900/pSA8589 and PV-01 are labeled here as “ΔtnpB*” rather than their “∆tnpB” 366

designation in GenBank due to a lack of sequence similarity with the tnpB gene of Tn554 367

(GenBank accession no. X03216) (59). In p1128105, the 5’-AGCGTACAAC-3’ sequence 368

present upstream of cfr extends this putative ORF (“ORF1”), which has been designated as a 369

hypothetical protein, beyond the length of the ΔtnpB* ORFs of pSA1900/pSA8589 and PV-01. 370


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The pSA1900/pSA8589 sequence is a complete circular plasmid that was linearized here for 371

illustrative purposes by splitting the rep gene to optimize alignment with the linear p1128105 372

and PV-01 sequences. 373

374

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strain cfr MIC (µg/ml)

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


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Documents

Linezolid-resistant Staphylococcus aureus strain 1128105, the first