accepted - aac.asm.orgaac.asm.org/content/early/2008/03/24/aac.01150-07.full.pdfthe amplicon between...

1

Methicillin-resistant Staphylococcus saprophyticus Carrying Staphylococcal Cassette 1

Chromosome mec (SCCmec) Have Emerged in Urogenital Tract Infections 2

3

Masato Higashide1, 2

, Makoto Kuroda1, 3

*, Carlos Takashi Neves Omura1, Miyuki Kumano

1, 4

Saburo Ohkawa4, Sadahiro Ichimura

5, and Toshiko Ohta

1 5

6

1Department of Microbiology, Graduate School of Comprehensive Human Sciences, 7

University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; 2Kotobiken 8

Medical Laboratories Inc., 445-1 Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan; 9

3Laboratory of Bacterial Genomics, Center for Pathogen Genomics, National Institute of 10

Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; 4Department of 11

Microbiology, Kohjin Bio Co., Ltd., 5-1-3 Chiyoda, Sakado, Saitama 350-0214, Japan; 12

5Department of Bacteriology, BML, Inc., 1361-1 Matoba, Kawagoe-shi, Saitama 350-1101, 13

Japan. 14

15

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01150-07 AAC Accepts, published online ahead of print on 24 March 2008

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*Corresponding author 16

Mailing address: Laboratory of Bacterial Genomics, Center for Pathogen Genomics, 17

National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, 18

Japan. 19

Phone: +[81]-3-5285-1111 20

Fax: +[81]-3-5285-1166 21

Email: [email protected] 22

23

Running title: Characterization of SCCmec in S. saprophyticus 24 ACCEPTED


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Abstract 25

Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated 26

urinary tract infections (UTIs), particularly in female outpatients. We investigated the 27

dissemination and antimicrobial susceptibility of 101 S. saprophyticus isolates from the 28

genitourinary tracts of patients in Japan. Eight of these isolates were mecA-positive and 29

showed β-lactam resistance. Pulsed field gel electrophoresis (PFGE) showed that only some 30

isolates were isogenic, indicating that the mecA gene was apparently acquired independently 31

by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). 32

Type determination of SCCmec by multiplex PCR showed non-typeable element in the 8 33

mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. 34

saprophyticus strain revealed that it is non-typeable with the current SCCmec classification 35

due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI 36

genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are 37

similar to those of type I SCCmec in S. aureus NCTC 10442. Further, the genes around the 38

mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around 39

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the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 40

15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a 41

novel type. 42

43

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Introduction 44

Staphylococcus saprophyticus is a member of the coagulase-negative staphylococci (CoNS), 45

which frequently cause uncomplicated urinary tract infections (UTI) in young and 46

middle-aged female outpatients (8, 12, 15, 18, 21, 22, 23). Unlike most other CoNS, S. 47

saprophyticus is rarely resistant to most antibiotics active against gram-positive organisms 48

(10, 17). 49

Although UTI caused by S. saprophyticus have been well documented, the antimicrobial 50

resistance and dissemination of this species are not well studied. This study investigated the 51

current dissemination and antimicrobial resistance of S. saprophyticus isolated from the 52

urogenital tract of Japanese patients. In addition, characterization of a new type of SCCmec 53

element from a mecA-positive S. saprophyticus was carried out. 54

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Materials and Methods 56

Bacterial isolates 57

From April to December 2003, a total of 101 S. saprophyticus were isolated from urine (94 58

isolates) specimens of patients suffering from acute cystitis with bacterial counts of ≥104 59

CFU/ml or vaginal (7 isolates) specimens of patients suffering from bacterial vaginosis or 60

candidiasis related symptoms at the Clinical Microbiology laboratories of 65 different 61

Japanese hospitals. All isolates were from different patients, and duplicate isolates from the 62

same patient were excluded. In addition, specimens also yielding gram-negative bacteria, 63

which are often isolated in either uncomplicated or complicated urinary tract infections, were 64

not considered for further isolation of CoNS including S. saprophyticus. These isolates were 65

identified as CoNS by means of multiple assays, including Gram staining, catalase 66

production, coagulase production (Eiken, Japan), DNase production (Eiken, Japan), growth 67

on egg yolk mannitol salt agar (Becton Dickinson, NJ, USA), and the 68

novobiocin-susceptibility test (Showa, Japan). The final identification was carried out using 69

ID 32 APIStaph in the mini API system (bioMérieux, France). 70

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71

Antimicrobial susceptibility tests 72

The minimum inhibitory concentrations (MICs) were determined by the agar dilution 73

method, as described in CLSI and M7-A6 (3). The testing range of antimicrobial 74

concentrations was from 0.06 to 256 µg/ml. Ampicillin, cefoxitin, kanamycin, ofloxacin, and 75

oxacillin were purchased from Sigma (MO, USA). Cefazolin, erythromycin, and 76

vancomycin were purchased from Wako (Japan). The following antibiotics were obtained 77

from their respective manufacturers: arbekacin (Meiji, Japan), clarithromycin (Taisho, 78

Japan), fosfomycin (Meiji, Japan), imipenem (Banyu, Japan), teicoplanin (Fujisawa, Japan), 79

and trimethoprim-sulfamethoxazole (Shionogi, Japan). 80

In the case of oxacillin, Mueller-Hinton (MH) agar was supplemented with 2% (wt./vol.) 81

sodium chloride. The MIC of fosfomycin was determined on MH-agar supplemented with 25 82

µg/ml of glucose-6-phosphate. The agar plate was incubated at 35°C for 24 h. 83

84

Detection of mecA-positive S. saprophyticus 85

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The mecA-positive isolates were detected by dot-blot hybridization according to previously 86

described method (14). Briefly, S. saprophyticus cell lysates were denatured by alkaline 87

buffer, followed by spotting onto a GeneScreen Plus hybridization membrane (NEN, MA, 88

USA). The membrane was subjected to hybridization using a mecA gene-specific probe 89

labeled by the AlkPhos Direct Labelling kit (GE Healthcare, Buckinghamshire, England), 90

followed by the detection with CDP-Star™ along with the mecA PCR product and MRSA 91

N315 cell lysate as the positive control. 92

93

Pulsed-field gel electrophoresis (PFGE) analysis 94

The PFGE plug mold was prepared using the GenePath Group 1 reagent kit for 95

coagulase-negative staphylococci (Bio-Rad, CA, USA), according to the manufacturer’s 96

instruction. Chromosome DNA in the plug mold was digested with the SmaI restriction 97

enzyme. PFGE was performed under the following conditions: 6 V/cm, ±60° angle, 5.3 s 98

initial time, 34.9 s final time, and running for 20 h at 14°C with 0.5× Tris-borate-EDTA 99

buffer (TBE; Bio-Rad CHEF DRIII). 100

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A dendrogram showing similarity of the PFGE profiles was obtained by the unweighted 101

pair-group method with arithmetic mean (UPGMA) using the GelCompar software (Applied 102

Maths, Kortrijk, Belgium). 103

The mecA-positive SmaI-digested DNA fragment was detected by Southern hybridization 104

method using a mecA gene-specific probe labeled by the AlkPhos Direct Labelling kit (GE 105

Healthcare, Buckinghamshire, England). 106

107

Multiplex PCR for type assignment of the SCCmec element and mec/ccr gene 108

complexes 109

The multiplex PCR for the type assignment of SCCmec was performed according to the 110

reports of Oliveira et al. (19), but the thermostable DNA polymerase was exchanged with the 111

Phusion high-fidelity DNA polymerase (Finzyme, Espoo, Finland). Multiplex PCR for type 112

assignment of the mec or ccr gene complexes was performed according to the procedure of 113

Kondo et al. (11). 114

115

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Sequencing of the SCCmec element in the S. saprophyticus TSU33 strain 116

The SCCmec element from the TSU33 strain was partially amplified by PCR using the 117

ExTaq DNA polymerase (Takara, Japan) with oligonucleotide primers (Table 1) 118

corresponding to the consensus nucleotide sequence with the orfX, mecA, and ccrB genes 119

among the SCCmec elements. The PCR products for DNA sequencing were obtained as 120

follows. The amplicon between orfX and mecA was amplified using the orfX-201F and 121

mecA-33F primers. The amplicon between mecA and ccrB was amplified using the 122

mecA-381R and ccrB-F primers. To obtain an adjacent DNA fragment downstream ccr locus, 123

inversed-PCR was performed using HindIII digested/self-ligated chromosome DNA from 124

the TSU33 strain and the ccr-InvF and mecAccr-R4-2 primers under the following reaction 125

conditions: predenaturation at 94°C for 30 s; 30 cycles at 94°C for 30 s, 55°C for 1 min, and 126

68°C for 12 min; postextension at 72°C for 4 min; and soaking at 4°C. To obtain the adjacent 127

downstream DNA region, further inversed-PCR walking was performed using the same PCR 128

parameters as those described above and with the following primers and template DNA; 129

2nd

-InvF and 2nd

-InvR primers with EcoRI digested/self-ligated chromosome DNA as the 130

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template; 3rd

-InvF and 3rd

-InvR primers with PstI digested/self-ligated chromosome DNA as 131

the template. The DNA sequence was determined on both strands by the primer walking 132

method using the BigDye Terminator Cycle sequencing kit v.1.1 in an ABI 310 DNA 133

sequencer (Applied Biosystems, CA, USA). 134

135

Bioinformatic analysis of SCCmec elements 136

Putative open reading frames (ORFs) were predicted by the GLIMMER 2.0 program (5) with 137

manual determination of potential ribosome binding sequences. Functional assignments of 138

the predicted ORFs were based on a blastp homology search against the nonredundant 139

protein database (nr) (1). Pairwise alignment of SCCmec elements was performed by a blastn 140

homology search (1) between the elements, followed by visualization of the aligned images 141

with the ACT program (2). The phylogenetic tree of the amino acid sequences of CcrA and 142

CcrB recombinases was obtained by analysis with the clustalX program with 1000 times 143

bootstrapping (20). 144

145

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Nucleotide sequence accession number. 146

The complete sequence and annotation of the SCCmec element of the S. saprophyticus 147

TSU33 strain have been deposited in DDBJ (accession number: AB353724). 148

149

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Results and Discussion 150

Isolation and identification of S. saprophyticus from urine and vaginal specimens 151

S. saprophyticus is a novobiocin-resistant coagulase-negative staphylococcus (NRCoNS). 152

Overall, 96 NRCoNS isolates were obtained from 6,743 urine specimens of patients suffering 153

from acute cystitis with bacterial counts of ≥104 CFU/ml. Vaginal flora could be one of 154

potential reservoirs for S. saprophyticus urinary tract infections, 9 NRCoNS were isolated 155

from 12,153 vaginal specimens. Of the 105 NRCoNS isolates, 101 were identified as S. 156

saprophyticus, 3 as S. cohnii, and 1 as S. sciuri. Other NRCoNS species such as S. ariettae, S. 157

equorum, S. gallinarum, S. kloosii, and S. xylosus were not detected in this study. 158

159

Antimicrobial susceptibility of the S. saprophyticus isolates 160

We investigated the antimicrobial susceptibility of the 101 S. saprophyticus isolates (Table 161

2). All isolates showed oxacillin MICs in the resistance range (≥0.5 µg/ml) in accordance 162

with the CLSI M100-S18 guidelines for CoNS (4). However, the oxacillin susceptibility 163

pattern exhibited a clear bimodal distribution, as we previously reported (7). Screening for 164

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the presence of the mecA gene by dot-blot hybridization and PCR showed that the 93 isolates 165

with oxacillin MICs in the range 0.5 - 4 mg/l were all mecA-negative, while the eight isolates 166

with oxacillin MICs higher than 64 mg/l were mecA-positive (data not shown). These 167

mecA-positive isolates showed relatively high MICs with respect to β-lactams, macrolides, 168

and fosfomycin but not to other tested antibiotics (Table 3). 169

One mecA-positive isolate was from a vaginal specimen, and the other 7 mecA-positive 170

isolates were from urine specimens. Two of the 8 isolates were from the urine specimens of 171

males, and 1 of them was from an inpatient. No mecA-negative isolate was found in males, 172

suggesting that the UTIs caused by S. saprophyticus in males could be associated with 173

hospitalization, as reported previously (18). 174

Intriguingly, in comparison with other staphylococci, fosfomycin resistance was observed in 175

all S. saprophyticus isolates (Table 2), suggesting that this resistance is an intrinsic phenotype, 176

as reported previously (10). 177

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Distribution of multiple clones of mecA-positive S. saprophyticus 179

The PFGE analysis showed different SmaI-digestion profiles of the chromosomal DNA of 180

the mecA-positive S. saprophyticus isolates. Three of the 8 isolates showed identical or 181

similar PFGE profiles (Fig. 1), suggesting clonal relatedness. These isolates were from 182

different patients and hospitals, suggesting the potential for clonal spread. Southern 183

hybridization with the mecA gene-specific probe showed a uniform pattern for these three 184

isolates (Fig. 1). On the other hand, the results of PFGE profiling and Southern hybridization 185

for the other 5 isolates revealed different patterns, suggesting that multiple mecA-positive S. 186

saprophyticus strains might circulate in Japanese communities. Similar to our observation, 187

Widerstrom et al. also reported that multiple clones of S. saprophyticus were associated with 188

lower UTIs in women (24). 189

190

Type assignment of the SCCmec element in S. saprophyticus 191

The SCCmec type of the 8 mecA-positive isolates could not be assigned by the multiplex 192

PCR approach described by Oliveira et al. (19) (Fig. 2A), because these yielded a 193

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combination of DCS and mecI amplifcation products that was not consistent with those of 194

known types of SCCmec elements. Further classification of a mec and a ccr gene complex by 195

multiplex PCR methods (11) confirmed that the 8 isolates were non-typeable according to 196

current schemes, because all 8 isolates possessed a class A mec gene complex (Fig. 2B) but 197

yielded no amplification product for known ccr genes (Fig. 2C). 198

To characterize a novel SCCmec of S. saprophyticus, we selected vaginal isolate TSU33 199

strain, because vagina might be potential reservoir for UTI, in addition, TSU33 could be the 200

prototype of the clonally spreading strain among three isogenic isolates (TSU18, TSU33 and 201

TSU57 strain) described above. 202

The sequence of SCCmec from TSU33 was 23,743 bp long and composed of 26 ORFs (Table 203

4 and Fig. 3). Possible attachment sequences of attC (on the chromosome DNA) and attSCC 204

(on the SCC element) for SCCmec integration were investigated by direct repeat search, 205

which revealed that the attC/attSCC of this SCCmec element was most similar to that of type 206

I SCCmec of S. aureus NCTC 10442 (Fig. 4) (9). In addition, pairwise alignment of the 207

sequence around attC and attSCC showed that possible inverted-repeat sequences (IR-L and 208

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IR-R), which are predicted to be the cis-elements for CcrAB recombinases, were also similar 209

to those of type I SCCmec of S. aureus NCTC 10442 rather than to those of other types (Fig. 210

4). SCCmec in TSU33 carries the class A mec gene complex (IS431-mecA-mecR1-mecI 211

genes) that is found in type II SCCmec of S. aureus N315 or type III SCCmec of S. aureus 212

85/2082 (Table 4 and Fig. 3) (9). Around the mec gene complex, orf2 located in the junkyard 213

region J3 between the orfX and the mec gene complex was very similar to that of type I 214

SCCmec (S. aureus NCTC 10442) and type II SCCmec (S. aureus N315) (Table 4 and Fig. 3), 215

while it did not show any similarity to SCC15305RM in S. saprophyticus ATCC 15305 (Fig. 3) 216

(13). 217

The ccr genes and their composition are one of the key features used for categorizing 218

SCCmec types. CcrA and CcrB of SCCmec in TSU33 could be categorized as CcrA1 and 219

CcrB3, respectively, and had amino acid similarity (Fig. 5) but were not perfectly identical to 220

the known subclasses of CcrA or CcrB recombinases (Table 4). Apart from amino acid 221

similarity, this combination of ccr genes such as CcrA1 and CcrB3 has not yet been reported. 222

In fact, non-typeable ccr gene complexes seem to be widely distributed among CoNS (6). 223

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The ORFs around the ccr genes showed high similarity to SCC15305RM in S. saprophyticus 224

ATCC 15305 but were different from those found in other SCCmec elements, including type 225

I SCCmec, as shown in Fig. 3. 226

The ORFs of the junkyard J2 region between mecI and ccrB showed some similarity to those 227

of type III SCCmec in S. aureus 85/2082, while the ORFs of the junkyard J1 region between 228

ccrA and orf28, adjacent to the attC site, did not show significant similarity to those present 229

in other types SCCmec (Table 4). 230

231

232

233

Concluding remarks 234

Regarding the antimicrobial susceptibility of S. saprophyticus, we previously reported the 235

prevalence of β-lactam resistant S. saprophyticus isolates in Japan (7). In fact, there are few 236

reports on mecA-mediated resistance in S. saprophyticus. One of the reasons could be that S. 237

saprophyticus is not a commensal bacterium that colonizes human skin; thus, it is less likely 238

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to have become antimicrobial resistant by horizontal gene transfer from MRSA or MRSE. 239

Indeed, the SCCmec element of TSU33 shows unique gene organization in the ccr gene 240

complex. In addition, recent reports (6, 16) and our results suggest that CoNS are more likely 241

to contain several representatives of different ccr complexes. 242

243

244

Acknowledgements 245

This work was supported by grant from Kotobiken Medical Laboratories Inc. 246

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References 248

1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local 249

alignment search tool. J. Mol. Biol. 215:403–410. 250

2. Carver, T. J., K. M. Rutherford, M. Berriman, M. A. Rajandream, B. G. Barrell, 251

and J. Parkhill. 2005. ACT: the Artemis Comparison Tool. Bioinformatics 252

21:3422–3423. 253

3. Clinical and Laboratory Standards Institute. 2006. Methods for dilution 254

antimicrobial susceptibility tests for bacteria that grow aerobically, M7-A7. Approved 255

standard, 7th

ed. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania. 256

4. Clinical and Laboratory Standards Institute. 2008. Performance standards for 257

antimicrobial susceptibility testing, 18th information supplement. M100-S18. Clinical 258

and Laboratory Standards Institute, Wayne, Pennsylvania. 259

5. Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg. 1999. Improved 260

microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636–4641. 261

ACCEPTED


.org/D

ownloaded from

http://aac.asm.org/

21

6. Hanssen, A. M., and J. U. Sollid. 2007. Multiple staphylococcal cassette 262

chromosomes and allelic variants of cassette chromosome recombinases in 263

Staphylococcus aureus and coagulase-negative staphylococci from Norway. Antimicrob. 264

Agents Chemother. 51:1671–1677. 265

7. Higashide, M., M. Kuroda, S. Ohkawa, and T. Ohta. 2006. Evaluation of a cefoxitin 266

disk diffusion test for the detection of mecA-positive methicillin-resistant 267

Staphylococcus saprophyticus. Int. J. Antimicrob. Agents 27:500–504. 268

8. Ishihara, S., S. Yokoi, M. Ito, S. Kobayashi, and T. Deguchi. 2001. Pathologic 269

significance of Staphylococcus saprophyticus in complicated urinary tract infections. 270

Urology 57:17–20. 271

9. Ito, T., Y. Katayama, K. Asada, N. Mori, K. Tsutsumimoto, C. Tiensasitorn, and K. 272

Hiramatsu. 2001. Structural comparison of three types of staphylococcal cassette 273

chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus 274

aureus. Antimicrob. Agents Chemother. 45:1323–1336. 275

ACCEPTED


.org/D

ownloaded from

http://aac.asm.org/

22

10. Kahlmeter, G. 2003. An international survey of the antimicrobial susceptibility of 276

pathogens from uncomplicated urinary tract infections: the ECO.SENS Project. J. 277

Antimicrob. Chemother. 51:69–76. 278

11. Kondo, Y., T. Ito, X. X. Ma, S. Watanabe, B. N. Kreiswirth, J. Etienne, and K. 279

Hiramatsu. 2007. Combination of multiplex PCRs for staphylococcal cassette 280

chromosome mec type assignment: rapid identification system for mec, ccr, and major 281

differences in junkyard regions. Antimicrob. Agents Chemother. 51:264–274. 282

12. Kumari, N., A. Rai, C. P. Jaiswal, A. Xess, and S. K. Shahi. 2001. Coagulase 283

negative Staphylococci as causative agents of urinary tract infections-prevalence and 284

resistance status in IGIMS, Patna. Indian J. Pathol. Microbiol. 44:415–419. 285

13. Kuroda, M., A. Yamashita, H. Hirakawa, M. Kumano, K. Morikawa, M. 286

Higashide, A. Maruyama, Y. Inose, K. Matoba, H. Toh, S. Kuhara, M. Hattori, and 287

T. Ohta. 2005. Whole genome sequence of Staphylococcus saprophyticus reveals the 288

pathogenesis of uncomplicated urinary tract infection. Proc. Natl. Acad. Sci. U. S. A. 289

102:13272–13277. 290

ACCEPTED


.org/D

ownloaded from

http://aac.asm.org/

23

14. Kuroda, M., K. Kuwahara-Arai, and K. Hiramatsu. 2000. Identification of the up- 291

and down-regulated genes in vancomycin-resistant Staphylococcus aureus strains Mu3 292

and Mu50 by cDNA differential hybridization method. Biochem Biophys Res Commun 293

269:485-490. 294

15. Meers, P. D., W. Whyte, and G. Sandys. 1975. Coagulase-negative staphylococci and 295

micrococci in urinary tract infections. J. Clin. Pathol. 28:270–273. 296

16. Miragaia, M., J. C. Thomas, I. Couto, M. C. Enright, and H. de Lencastre. 2007. 297

Inferring a population structure for Staphylococcus epidermidis from multilocus 298

sequence typing data. J Bacteriol 189:2540-2552. 299

17. Nicolle, L. E., and G. K. Harding. 1982. Susceptibility of clinical isolates of 300

Staphylococcus saprophyticus to fifteen commonly used antimicrobial agents. 301

Antimicrob. Agents Chemother. 22:895–896. 302

18. Nicolle, L. E., S. A. Hoban, and G. K. Harding. 1983. Characterization of 303

coagulase-negative staphylococci from urinary tract specimens. J. Clin. Microbiol. 304

17:267–271. 305

ACCEPTED


.org/D

ownloaded from

http://aac.asm.org/

24

19. Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid 306

identification of structural types and variants of the mec element in methicillin-resistant 307

Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155–2161. 308

20. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 309

1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence 310

alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882. 311

21. Torres Pereira, A. 1962. Coagulase-negative strains of staphylococcus possessing 312

antigen 51 as agents of urinary infection. J. Clin. Pathol. 15:252–253. 313

22. von Eiff, C., G. Peters, and C. Heilmann. 2002. Pathogenesis of infections due to 314

coagulase-negative staphylococci. Lancet Infect. Dis. 2:677–685. 315

23. Wallmark, G., I. Arremark, and B. Telander. 1978. Staphylococcus saprophyticus: a 316

frequent cause of acute urinary tract infection among female outpatients. J. Infect. Dis. 317

138:791–797. 318

24. Widerstrom, M., J. Wistrom, S. Ferry, C. Karlsson, and T. Monsen. 2007. 319

Molecular epidemiology of Staphylococcus saprophyticus isolated from women with 320

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uncomplicated community-acquired urinary tract infection. J. Clin. Microbiol. 321

45:1561–1564. 322

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Figure Legends 323

Fig. 1. Pulsed-field gel electrophoresis (PFGE) analysis of 8 mecA-positive S. saprophyticus 324

strains. S. saprophyticus ATCC 15305 and S. aureus N315 were also included in this analysis 325

as reference strains. Composite clustering of the PFGE profiles is shown along with the 326

dendrogram with percentage of tree branch reliability obtained by the UPGMA method with 327

bootstrapping analysis. Arrows indicate the positive band detected by Southern hybridization 328

using the mecA-specific probe. S. saprophyticus type strain ATCC 15305 is mecA negative. 329

The profiles of 3 isolates are enclosed by a broken line; these are probably isogenic strains. 330

331

Fig. 2. Multiplex PCRs for the type assignment of SCCmec element of mecA-positive S. 332

saprophyticus. (A) for SCCmec type assignment (19). (B) for the determination of a mec 333

class (11). (C) for the determination of a ccr class (11). 334

335

Fig. 3. Schematic representation of SCCmec elements and pairwise alignment of their 336

nucleotide sequences. The arrows on the upper, middle, and lower lines show the 337

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organization of ORFs in the SCC15305RM of S. saprophyticus ATCC 15305, which is a 338

mecA-negative clinical isolate; SCCmec in S. saprophyticus TSU33 strain; and type I 339

SCCmec of S. aureus NCTC 10442 strain, respectively. Pairwise alignment was analyzed by 340

the ACT software using the results from the blastn homology search between nucleotide 341

sequences of the SCC elements. Matched sequences are shown as pink bars (same 342

orientation) and dark blue bars (inverted orientation) and those with a blastn score of less 343

than 200 were excluded. 344

345

Fig. 4. Pairwise alignment of the attachment site of SCCmec elements. Possible attachment 346

sequences of attC (on the chromosome DNA) and attSCC (on the SCC element) for SCCmec 347

integration are boxed. As an alignment control, S. aureus NCTC 8325 was used as the 348

SCCmec-negative strain. The same nucleotide sequence compared with SCCmec in TSU33 349

is shown in bold face. Possible inverted-repeat sequences (IR-L and IR-R) adjacent to attC 350

and attSCC, which are predicted to be the cis-elements for CcrAB recombinases, are shown 351

as arrows under the nucleotide sequences. The nucleotide sequence of SCCmec elements and 352

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the genome sequence have the following accession numbers: NCTC 10442, AB033763; 353

N315, D86934; 85/2082, AB037671; WIS, AB121219; and NCTC 8325, NC_007795. 354

355

Fig. 5. The phylogenetic tree obtained by the alignment of amino acid sequences among Ccr 356

recombinases. Basically, Ccr recombinases are classified as CcrA, CcrB, and CcrC and 357

categorized into subtypes, for example, CcrA1, as shown in the background of the circles. 358

The staphylococcus strains possessing Ccr recombinases are as follows: S. saprophyticus 359

ATCC 15305 and TSU33; S. aureus NCTC 10442, N315, MSSA476, MRSA252, 85/2082, 360

MW2, WIS, and HDE288; S. epidermidis ATCC 12228; and S. hominis ATCC 27844. The 361

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363

Table 1. Oligonucleotide primers used in this study Name Seq. (5′-3′) Reference

Amplification of the mecA gene for the detection by dot-blot hybridization

mecA-316F GGTAACATTGATCGCAACG This study

mecA-1642R GAGGTGCGTTAATATTGCC This study

Long PCR between the orfX and mecA genes

orfX.201F AAAACAGCAGTCCACGGTCATCAC This study

mecA-33F AATAGTTGTAGTTGTCGGGTTTGG This study

Long PCR between the mecA and ccrB genes

mecA-381R CCAATCTAACTTCCACATACCATC This study

ccrB-F CAGAGAACAAACGCAATCATTACG This study

1st inversed-PCR for the adjacent region of ccr genes

mecAccr-R4-2 ACCTATCTAACAAATATCTT This study

ccr-InvF TCATCCTTTCTGATTAAGCA This study

2nd inversed-PCR for the adjacent region of ccr genes

2nd-Inv-F ATAATGTCATCAACAGTTATTGTT This study

2nd-Inv-R CAGTCAAAAGAAAAAATTGATGAA This study

3rd inversed-PCR for the adjacent region of ccr genes

3rd-InvF TCCACCCATTACAGTGCCTT This study

3rd-InvR GAAATCAAGCACCGCAACTA This study

Multiplex PCR for type assignmnet of SCCmec

CIF2 F2 TTCGAGTTGCTGATGAAGAAGG 19

CIF2 R2 ATTTACCACAAGGACTACCAGC 19

KDP F1 AATCATCTGCCATTGGTGATGC 19

KDP R1 CGAATGAAGTGAAAGAAAGTGG 19

MECI P2 ATCAAGACTTGCATTCAGGC 19

MECI P3 GCGGTTTCAATTCACTTGTC 19

DCS F2 CATCCTATGATAGCTTGGTC 19

DCS R1 CTAAATCATAGCCATGACCG 19

RIF4 F3 GTGATTGTTCGAGATATGTGG 19

RIF4 R9 CGCTTTATCTGTATCTATCGC 19

RIF5 F10 TTCTTAAGTACACGCTGAATCG 19

RIF5 R13 GTCACAGTAATTCCATCAATGC 19

IS431 P4 CAGGTCTCTTCAGATCTACG 19

pUB110 R1 GAGCCATAAACACCAATAGCC 19

IS431 P4 CAGGTCTCTTCAGATCTACG 19

pT181 R1 GAAGAATGGGGAAAGCTTCAC 19

MECA P4 TCCAGATTACAACTTCACCAGG 19

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MECA P7 CCACTTCATATCTTGTAACG 19

Multiplex PCR for identification of mec classes, A, B, and C

mI6 CATAACTTCCCATTCTGCAGATG 11

IS7 ATGCTTAATGATAGCATCCGAATG 11

IS2(iS-2) TGAGGTTATTCAGATATTTCGATGT 11

mA7 ATATACCAAACCCGACAACTACA 11

Multiplex PCR for identification of ccr1, ccr2, ccr3, ccr4, and ccr5 (ccrC)

mA1 TGCTATCCACCCTCAAACAGG 11

mA2 AACGTTGTAACCACCCCAAGA 11

α1 AACCTATATCATCAATCAGTACGT 11

α2 TAAAGGCATCAATGCACAAACACT 11

α3 AGCTCAAAAGCAAGCAATAGAAT 11

βc ATTGCCTTGATAATAGCCITCT 11

α4.2 GTATCAATGCACCAGAACTT 11

β4.2 TTGCGACTCTCTTGGCGTTT 11

γR CCTTTATAGACTGGATTATTCAAAATAT 11 γF CGTCTATTACAAGATGTTAAGGATAAT 11

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Table 2. Distribution of MIC to tested antibiotics for 101 Staphylococcus saprophyticus isolates

No. of isolates for each tested antibiotic Antibiotics*

≤0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 ≥512 MIC50 (µg/ml) MIC90 (µg/ml)

OXA 54 37 1 1 1 5 2 0.5 1

AMP 74 16 2 1 8 0.25 1

CFZ 6 83 4 1 3 1 1 2 1 2

FOX 6 87 2 4 2 2 2

IPM 95 6 ≤0.06 ≤0.06

VAN 52 49 0.5 1

TEC 17 65 13 6 2 4

FOF 17 55 8 21 128 ≥512

OFX 79 22 0.5 1

ERY 4 79 5 1 2 9 1 0.125 2

CLR 73 14 2 2 2 7 1 ≤0.06 1

KAN 80 21 0.25 0.5

ABK 101 ≤0.06 ≤0.06

SXT 2 25 73 1 1 1

*OXA, Oxacillin; AMP, Ampicillin; CFZ, Cefazolin; FOX, Cefoxitin; IPM, Imipenem; VAN, Vancomycin; TEC, Teicoplanin; FOF, Fosfomycin;

OFX, Ofloxacin;

ERY, Erythromycin; CLR, Clarithromycin; KAN, Kanamycin; ABK, Arbekacin; SXT, Trimethoprim-Sulfamethoxazole (1:20)

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Table 3. Characteristics of 8 mecA-positive S. saprophyticus

MIC (µg/ml)c

Strains Gender

a

In/Outb

Source OX

A AMP CFZ

FO

X IPM VAN TEC FOF

OF

X ERY CLR KAN ABK

SX

T

TSU18 M Out Urine 128 8 16 16 ≤0.06 0.5 2 64 0.5 64 16 0.25 ≤0.06 1

TSU28 M In Urine 256 8 32 16 0.125 1 2 ≥512 0.5 128 32 0.25 ≤0.06 1

TSU33 F Out Vagina 128 8 1 8 ≤0.06 0.5 2 128 0.5 64 16 0.25 ≤0.06 1

TSU47 F Out Urine 64 4 4 8 0.125 1 4 128 0.5 64 16 0.25 ≤0.06 1

TSU57 F Out Urine 128 8 16 16 0.125 0.5 2 128 0.5 64 16 0.25 ≤0.06 1

TSU67 F Out Urine 256 8 128 32 0.125 1 2 256 1 0.25 0.125 0.25 ≤0.06 1

TSU69 F Out Urine 128 8 128 32 0.125 1 2 ≥512 1 0.25 0.125 0.5 ≤0.06 1

TSU90 F Out Urine 128 8 64 16 0.125 1 2 ≥512 0.5 64 16 0.5 ≤0.06 0.5

a M, male; F, female

b in, inpatient; out, outpatient

c OXA, Oxacillin; AMP, Ampicillin; CFZ, Cefazolin; FOX, Cefoxitin; IPM, Imipenem; VAN, Vancomycin; TEC, Teicoplanin; FOF,

Fosfomycin; OFX, Ofloxacin; ERY, Erythromycin; CLR, Clarithromycin; KAN, Kanamycin; ABK, Arbekacin; SXT,

Trimethoprim-Sulfamethoxazole (1:20)

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Table 4. Similarities of ORFs in the SCCmec element and in the region adjacent to the chromosome in the S. saprophyticus TSU33 strain

Best-matched gene by blastp search against nr database

ORFa Locationb

Directionc Gene Description Species and strainsd

SCCmece ORF ID Identitiesf GenBank ID

orf1 1 –

251 orfX hypothetical protein orfX,

partial

S. saprophyticus ATCC

15305 – SSP0026

76/77

(98%) YP_300116.1

orf2 432 –

1727 hypothetical protein S. aureus N315 II.1.1.1 SA0024 429/431

(99%) NP_373263.1

orf3 2926 –

2252 c tnp transposase IS431mec S. saprophyticus ATCC

15305 – SSP1641

223/224

(99%) YP_302591.1

orf4 2962 –

3351 putative HMG-CoA synthase S. aureus M03-68 IV.5.1.1 SA0035 129/129

(100%) AAZ76241.1

orf5 4148 –

4891 glycerophosphoryl diester

phosphodiesterase S. aureus N315 II.1.1.1 SA0036

246/247

(99%) NP_370563.1

orf6 4987 –

5415 hypothetical protein S. aureus N315 II.1.1.1 SA0037 141/142

(99%) NP_373277.1

orf7 7467 –

5461 c mecA penicillin binding protein 2' S. aureus COL I.1.1.1 SA0038 668/668

(100%) YP_184944.1

orf8 7567 –

9324 mecR1 signal transducer protein S. aureus N315 II.1.1.1 SA0039 584/585

(99%) NP_373279.1

orf9 9324 –

9695 mecI methicillin resistance

regulatory protein S. aureus N315 II.1.1.1 SA0040

123/123

(100%) NP_373280.1

orf10 9995 –

10384 putative phosphoesterase S. haemolyticus

JCSC1435 – –

49/124

(39%) YP_252305.1

orf11 10406 –

11170 putative glycosyltransferase E. coli O55:H7 – –

82/256

(32%) AAL67560.1

orf12 11751 –

11485 c hypothetical protein S. aureus 85/2082 III.1.1.1 CZ058 52/58

(89%) BAB47659.1

orf13 12591 –

12334 c hypothetical protein,

truncated S. aureus 85/2082 III.1.1.1 CZ059

74/87

(85%) BAB47660.1

orf14 12692 –

12549 c hypothetical protein,

truncated S. aureus 85/2082 III.1.1.1 CZ059

31/36

(86%) BAB47660.1

orf15 13292 –

12873 c hypothetical protein S. aureus N315 II.1.1.1 SA0053 95/101

(94%) NP_373293.1

orf16 13829 –

13296 c hypothetical protein S. saprophyticus ATCC

15305 – SSP0031

158/167

(94%) YP_300121.1

orf17 14154 –


15305 – SSP0032

102/103

(99%) YP_300122.1


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orf18 14588 –


15305 – SSP0034

103/112

(91%) YP_300137.1

orf19 15145 –

15432 hypothetical protein S. saprophyticus ATCC

15305 – SSP0051

26/53

(49%) YP_300141.1

orf20 17096 –

15468 c ccrB cassette chromosome

recombinase B S. aureus 85/2082 III.1.1.1 Z010

481/542

(88%) BAB47597.1

orf21 18466 –

17117 c ccrA cassette chromosome

recombinase A S. aureus MSSA476

– SAS0033

413/448

(92%) YP_042166.1

orf22 18901 –


15305 – SSP0037

39/45

(86%) YP_300127.1

orf23 20756 –


15305 – SSP0038

567/596

(95%) YP_300128.1

orf24 21049 –

20756 c hypothetical protein S. aureus RN7170 II.4.1.1 CRN05 61/95

(64%) BAF42865.1

orf25 22732 –

21221 c hypothetical protein S. aureus M03-68 IV.5.1.1 PK02 265/509

(52%) AAZ76227.1

orf26 23458

–

22892 c hypothetical protein

Candidatus

Protochlamydia

amoebophila UWE25 – –

21/49

(42%) YP_007751.1

orf27 24240 –

24452 hypothetical protein S. aureus 85/2082 III.1.1.1 CZ078 58/70

(82%) BAB47680.1

orf28 26687 –

24684 c γ-glutamyltranspeptidase,

putative S. aureus MRSA252

– SA0202

347/657

(52%) YP_039667.1

orf29 28176 –

26970 c RGD-containing lipoprotein,

partial S. aureus NCTC 8325

– SA0201

264/413

(63%) ABD29348.1

a ORFs in the region adjacent to the SCCmec element are underlined.

b Positions are based on the nucleotide sequence deposited in DDBJ under accession no. AB353724.

c "c" indicates the ORF located in the complementary strand. d Newly proposed SCCmec type by Kondo et al. and the SCCmec net web site (www.staphylococcus.net) is shown, instead of the earlier

classification. e Alignment lengths of the homologous regions; numbers in parentheses indicate percent identities of amino

acid sequences.

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