JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2007, p. 141–146 0095-1137/07/$08.00⫹0 doi:10.1128/JCM.01228-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 1
Rapid Multiplex PCR Assay for Identification of USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates䌤 Kristin K. Bonnstetter,1 Daniel J. Wolter,1 Fred C. Tenover,2 Linda K. McDougal,2 and Richard V. Goering1* Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska 68178,1 and Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia 303332 Received 15 June 2006/Returned for modification 5 August 2006/Accepted 27 October 2006
Recent reports have noted a discernible increase in the number of community-associated methicillinresistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature “AT repeat” sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3ⴕ end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of >6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had >6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for >6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates. leukocidin (PVL) genes lukS and lukF, which produce cytotoxins that cause leukocyte destruction and tissue necrosis (13). Strains producing PVL have been associated with skin abscess formation, furunculosis, and severe cases of necrotizing pneumonia (21). The presence of PVL genes may also be associated with increased disease severity (6, 10). Despite their community-associated designation, CA-MRSA strains are frequently isolated from and transmitted among patients within the hospital setting (30). CA-MRSA strains have also been associated with increased patient morbidity and mortality, costly treatment, and extensive eradication procedures, which underscores the value of active surveillance for the presence of these strains (29). Molecular typing methods used to characterize MRSA strains include pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and PCR amplification of target genes (32). By PFGE, CA-MRSA isolates in the United States have thus far been classified as pulsed-field types (PFTs) USA300 (sequence type 8 [ST8]), USA400 (ST1) (23), USA1000 (ST59), and USA1100 (ST30) by the Centers for Disease Control and Prevention (L. K. McDougal, W. Zhu, J. B. Patel, and F. C. Tenover, Abstr. 104th Annu. Meet. Am. Soc. Microbiol., abstr. C-220, 2004). S. aureus strain MW2, responsible for fatal infections in four children from North Dakota and Minnesota between 1997 and 1999, is considered the prototype community-associated MRSA strain belonging to the USA400 PFT (3). However, recent years have seen an alarming rise in the number of USA300 isolates identified in a variety of community populations, including children, sports participants, prisoners, military recruits, and men who have sex with men (1, 2,
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are now a major cause of clinical concern. Although first identified in the United States among intravenous drug users (31) followed by other high-risk populations (e.g., prison inmates, athletes, etc.), hospitals nationwide have noted an increasing trend in the number of CA-MRSA infections seen in young, healthy populations without predisposing risk factors (4, 5, 11, 20, 22, 26). A prospective cohort study published by Naimi et al. found the median age of patients with CA-MRSA infections to be significantly lower than those with healthcare-associated MRSA, at 30 years versus 70 years, respectively (26). Numerous studies have also indicated that CA-MRSA infections are frequently seen among infants and children, again suggesting that the likelihood of contracting such infections is no longer limited to traditional at-risk populations (2, 3, 12, 17, 25). CA-MRSA strains commonly harbor the staphylococcal cassette chromosome mec (SCCmec) type IV element and are susceptible to multiple non--lactam antibiotics. This is in contrast to healthcare-associated strains, such as USA100 isolates, which carry the SCCmec type II element and are resistant to a wide range of antibiotics due to the presence of multiple mobile and nonmobile genetic elements (23). However, community-associated strains typically carry the Panton-Valentine
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178. Phone: (402) 280-4098. Fax: (402) 280-1875. E-mail:
[email protected]. 䌤 Published ahead of print on 8 November 2006. 141
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BONNSTETTER ET AL. TABLE 1. S. aureus isolate characteristics
Isolate
956 CH17 CH18 CH33 CH62 CH48 947 948 1244 1245 1226 1112 1116 1117 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1233 1235 1335 1330 1118 1119 1234 1236 1336 1333 1334 1337 1237 1238 1331 1228 1230 1231 511 513 515 517 519 521 523 525
MLSTa
CC1:ST1 CC1:ST1f CC1:ST1f CC1:ST1f CC1:ST1f CC1:ST1f CC5:ST5 CC5:ST5 CC5:ST5 CC5:ST5 CC5:ST228 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8f CC8:ST8f CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST8f CC8:ST8f CC8:ST8f CC8:ST8j CC8:ST8 CC8:ST8 CC8:ST8f CC8:ST8 CC8:ST8 CC8:ST8 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239
PFTb or strain designation
PVL genec
No. of AT repeatsd
Sourcee
USA400 (MW2) USA400 USA400 USA400 USA400 USA400 USA100 (N315) USA100 (Mu50) USA800 USA800 Denmark 3727-03 NCTC8325g USA300-0114h USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0114 USA300-0045i USA300-0045i USA300-0068j USA300-0120k USA300-0120 USA300-0120 USA300-0120 USA300-0120 USA300-0120 USA300-0120 USA300-0120i USA300-0247h USA300-0251h USA300-0272l USA500 USA500 USA500 Brazilian Brazilian Brazilian Brazilian Brazilian Brazilian Brazilian Brazilian
⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
ND ND ND ND ND ND 5 5 ⱕ5 ⱕ5 ⱕ5 5 6 6 6 6 6 8 8 6 8 6 6 6 6 6 6 6 8 6 6 6 6 8 6 6 6 6 6 6 6 ⱖ6 ⱖ6 ⱖ6 ⱖ6 6 6 ⱖ6 6 ⱖ6 ⱖ6 ⱖ6 ⱖ6 6 ⱖ6 ⱖ6 ⱖ6 6 6 ND ND ND ND ND ND ND ND
CDC This study This study This study This study This study CDC CDC CDC CDC SSI CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC Continued on following page
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TABLE 1—Continued b
Isolate
MLSTa
PFT or strain designation
PVL genec
No. of AT repeatsd
Sourcee
1186 1187 1188 1189 1191 1192 1184 1190 1225 1185 510 512 514 516 518 520 522 524 1227 1229 1241 930 1246 1220 1221 1224 1232 1172 1248 958 1223 1222 1242 1247 1243 1197 1198 1212 1214
CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST239 CC8:ST240 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST247 CC8:ST250 CC15:ST15 CC22:ST22 CC22:ST22 CC22:ST22 CC22:ST22 CC30:ST30 CC30:ST30 CC30:ST30 CC30:ST36 CC30:ST579 CC45:ST45 CC59:ST59 CC72:ST72 CC80:ST80 CC80:ST80 CC80:ST80 CC80:ST80
Brazilian Brazilian Brazilian Brazilian Brazilian Brazilian Brazilian Brazilian Denmark 2731-03 Brazilian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian Iberian USA500 (COL) USA900 Denmark 40135 Denmark 22744-99 Denmark 848-03 EMRSA15 USA200/EMRSA16 USA1100 USA200 Denmark 2551-03 Denmark 1391-03 USA600 USA1000 USA700 Denmark 188851-95 Denmark 11819-97 France HT0401 Greece 14
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹
ND ND ND ND ND ND ND ND ND ND ⱕ5 ⱕ5 ⱕ5 ⱕ5 ⱕ5 ⱕ5 ⱕ5 ⱕ5 5 5 ⱕ5 5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
CDC CDC CDC CDC CDC CDC CDC CDC SSI CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC SSI SSI SSI CDC SMRSAL CDC SMRSAL SSI SSI CDC CDC CDC SSI SSI SSI SSI
a
Given in the format clonal complex:sequence type. Pulsed-field type as described by McDougal et al. (23). c ⫹, present; ⫺, absent. d ND, not detected. Numbers indicate the number of AT repeats confirmed with sequencing (numbers with ⱕ or ⱖ signs are the number of AT repeats detected via PCR assay). e CDC, Centers for Disease Control and Prevention, Atlanta, GA; SSI, Robert Skov, Statens Serum Institute, Copenhagen, Denmark; SMRSAL, Donald Morrison, Scottish MRSA Reference Laboratory, Glasgow, United Kingdom. f The MLST type has been assumed according to PFGE pattern. g This strain is a methicillin-susceptible Staphylococcus aureus strain. h SCCmec type IVa. USA300-0114 isolates were independent isolates from known geographic locations known to be epidemiologically unrelated. i SCCmec type IVb. j SCCmec type was nontypeable and was not IVa, IVb, or IVc. k USA300-0120 isolates (except isolate 1337) were SCCmec type IVb. l SCCmec type IVc. b
15, 18, 24, 27, 28). Detection of USA300 CA-MRSA strains has traditionally required the use of PFGE, MLST, and PCR (i.e., PVL), which, taken together, are time-consuming and require equipment that may not be readily available to the routine clinical laboratory. In addition, the newly described arginine catabolic mobile element recently described by Diep et al. (7) appears to be present only in USA300 strains carrying SCCmec type IVa (L. K. McDougal, G. E. Fosheim, K. K. Bonnstetter, F. C. Tenover, D. J. Wolter, and R. V. Goering, Abstr. 46th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-603, 2006). Thus, the goal of this study was to develop a more
unified molecular approach to the rapid identification of USA300 CA-MRSA isolates.
MATERIALS AND METHODS Bacterial strains. A total of 106 S. aureus strains (105 MRSA strains and 1 methicillin-susceptible S. aureus strain) were examined in this study (Table 1). Strains were chosen according to MLST and PFGE type, with 44 belonging to USA300. Of these, 30 isolates belonged to USA300-0114 clonal complex 8 (CC8: ST8) (9); all were independent isolates known to be epidemiologically unrelated. Other isolates were included on the basis of their genetic relatedness to USA300 strains as determined by MLST BURST analysis (http://www.mlst.net).
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FIG. 1. Diagram showing the location of the multiplex PCR primers with reference to S. aureus strain COL (GenBank accession number CP000046). The black bar is the SCCmec resistance element. DR is the 3⬘ direct repeat region flanking the SCCmec element. Gray indicates the 3.3-kb chromosomal sequence 3⬘ to SCCmec, including SACOL0058 and the AT repeat sequence. PCR amplification using primers lnaAT (nucleotides 69490 to 69511) and ATreg-2 (nucleotides 70831 to 70855) indicates the presence of ⱖ6 AT repeats. SACOL0058 is detected using ATreg-1 (nucleotides 69954 to 69977) and ATreg-2. Amplification of the PVL genes using primers designed by Lina et al. (21) occurs at a separate location within the chromosome. The lower black box demonstrates hybridization of the lnaAT primer with the AT repeat sequence as it would occur in a USA300 MRSA strain.
PFGE. All strains were analyzed by PFGE. Bacterial DNA was prepared according to the rapid protocol as previously described (14). Pulsed-field patterns were analyzed using BioNumerics software (version 4.6; Applied Maths, Kortrijk, Belgium) according to criteria published previously (23). PCR. Chromosomal DNA was isolated for PCR using the method described by Enright et al. on the MLST website (http://saureus.mlst.net/misc/info.asp) (8). Detection of PVL genes was performed using primers luk-PV-1, 5⬘-ATCATT AGGTAAAATGTCTGGACATGATCCA-3⬘, and luk-PV-2, 5⬘-GCATCAACT GTATTGGATAGCAAAAGC-3⬘, generating a 433-bp product as described by Lina et al. (21). The primers used to detect the conserved hypothetical gene SACOL0058 (S. aureus strain COL, GenBank accession number CP000046) were ATreg-1, 5⬘-GGAAATGGAATAGAGTTGGCAGAC-3⬘ (nucleotides 69954 to 69977), and ATreg-2, 5⬘-CAATTAACGATGATATTCCCGATAG-3⬘ (nucleotides 70831 to 70855), resulting in an amplification product of 902 bp. Reaction mixtures (100 l total volume) contained 1.5 mM MgCl2, 200 M deoxynucleoside triphosphate mix, primers at a final concentration of 0.5 mM, 2.5 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), and 1 l (ca. 1 g) of template DNA. Amplification was carried out for 34 cycles with denaturation at 94°C for 30 s, annealing at 66°C for 30 s, extension at 72°C for 1 min 30 s, and a final extension at 72°C for 5 min. The primer designed to discriminate the number of AT repeats present within SACOL0058 was lnaAT: 5⬘-TGLCTLCGALCGTCAALTALTATATATAT-3⬘ (nucleotides 69490 to 69511, designed with an additional AT at the 3⬘ end of the primer). Locked nucleic acid (LNA) oligonucleotides (Sigma-Proligo) within the primer are indicated by a superscript L (33). This primer was coupled with the ATreg-2 primer with PCR conditions as described above for detection of SACOL0058 but with an annealing temperature of 67°C for 30 s and 5 U of Taq polymerase, yielding a product 1,366 bp in size. Multiplex PCR, to simultaneously detect PVL, SACOL0058, and the number of AT repeats, was performed as for SACOL0058, but with primers at the following concentrations: luk-PV-1, luk-PV-2, and ATreg-1 at 0.05 M; ATreg-2 at 0.75 M; lnaAT at 0.5 M; and 5 U of Taq DNA polymerase per reaction. Amplification reactions were visualized by agarose gel electrophoresis (1.5% SeaKem LE [FMC BioProducts, Rockland, ME]) in 1⫻ Tris-borate-EDTA buffer. PCR products were sequenced at the Creighton University Molecular Biology Core Facility using an ABI Prism 3100 Avant genetic analyzer (Applied Biosystems, Foster City, CA).
RESULTS Identification of USA300 “signature” AT repeat sequence. The SCCmec chromosomal region in MRSA isolates is known
to be recombinogenic, resulting in a variety of SCCmec types (16). We sought potential USA300-specific sequences in genomic regions directly flanking SCCmec, hypothesizing that these areas might also be subjected to higher rates of recombination. Using a variety of primers, analysis of ca. 3 kb of genomic sequence upstream of the orfX side of SCCmec revealed 100% homology across all 30 USA300-0114 (CC8:ST8) strains studied. In addition, the sequence was similar to seven nonUSA300 Staphylococcus aureus genomes (MW2, COL, Mu50, N315, NCTC8325, MRSA252, and MSSA476) (http://www.ncbi .nlm.nih.gov and data not shown). However, analysis of 3.3 kb of
FIG. 2. (A) LNA primer identification of USA300 MRSA strains. Lane 1, 1-kb DNA ladder (Invitrogen, Carlsbad, CA); lanes 2 and 3, USA300:ST8 strains CRG-1130 and CRG-1128, containing eight and six AT repeats, respectively; lane 4, strain CRG-930 (USA500:ST250) containing five AT repeats; and lane 5, water control. (B) Multiplex PCR assay differentiates USA300 strains from other MRSA strains. Lane 1, 1-kb DNA ladder (Invitrogen); lanes 2 and 3, USA300:ST8 strains CRG-1130 and CRG-1128, containing eight and six AT repeats, respectively, as well as SACOL0058 and PVL; lane 4, strain CRG-1112 (mecA negative) containing five AT repeats; lane 5, strain CRG-1231 (USA500:ST8) containing six AT repeats and SACOL0058; lane 6, strain MW2 (CC1:ST1) (SACOL0058 negative); and lane 7, water control.
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genomic sequence extending downstream of the J1-SCCmec chromosomal junction (19) revealed a region containing either six or eight repetitions of an adenine-thymine base pair in all USA300 isolates examined. Sequence comparison with S. aureus strain COL (CC8:ST250) located the AT repeat region approximately 1.4 kb downstream from the J1-SCCmec chromosomal junction, within the conserved hypothetical gene SACOL0058. However, SACOL0058 contained only five AT repeats in COL. In addition, PCR analysis revealed the presence of SACOL0058 in PVL-negative USA100, USA500, and USA800 isolates. SACOL0058 was absent from the chromosome of the prototypical community-associated USA400 MRSA strain MW2 (CC1: ST1). LNA PCR to detect the presence and extent of the AT repeat sequence. Traditional oligonucleotide primers were not suitable for AT repeat detection due to the potential of the multiple 3⬘ repeats to facilitate in hairpin formation, primer dimers, etc. Therefore, LNA oligonucleotides were used to ensure correct hybridization and discrimination between 5 and ⱖ6 AT repeats. This specificity results from the fact that LNA oligonucleotides are modified, with a 2⬘-O, 4⬘-C methylene bridge forming a thermodynamically stable primer with improved target specificity under stringent annealing conditions (33). An LNA PCR primer was designed with 6 AT repeats at the 3⬘ end and LNA-modified bases near the 5⬘ end to strongly drive correct hybridization and PCR amplification when used with an appropriate reverse primer in isolates with ⱖ6 AT repeats (Fig. 1). As shown in Fig. 2A, amplification was observed only in strains containing ⱖ6 (i.e., 6 or 8) AT repeats. These results were 100% concordant with DNA sequence analysis (data not shown). LNA PCR was used to examine a variety of strains belonging to MLST clonal complexes CC5 and CC8, including single locus variants of both groups and double or triple locus variants of CC8. In addition to USA300:ST8 isolates, SACOL0058 was found in CC8:ST247 (Iberian clone), CC8:ST250 (USA500), CC5:ST5 (USA100 and USA800), and CC5:ST228 isolates but was absent from CC8:ST239 and CC8: ST240, isolates of the Brazilian clone. However, as noted previously, only USA300 isolates, which carry PVL genes, possessed either six or eight AT repeats. Results for all isolates examined are shown in Table 1. Multiplex PCR for rapid identification of USA300 CAMRSA isolates. Using minor modification of the PCR, primers were combined to create a multiplex PCR assay with the potential to differentiate USA300 isolates from other MRSA strains. As shown in Fig. 2B, lanes 2 and 3, USA300 strains were identified by the amplification of three PCR products: (i) SACOL0058, (ii) LNA-based amplification of ⱖ6 AT repeats, and (iii) PVL genes. A USA500:ST8 isolate, CRG-1231, containing six AT repeats, was distinguished from the USA300 isolates by the absence of the PVL band (Fig. 2B, lane 5). The mecA-negative isolate CRG-1112 (NCTC8325) was a positive control for SACOL0058 (Fig. 2B, lane 4), while strain CRG956 (USA400, MW2) served as a positive control for PVL (Fig. 2B, lane 6). Discrepant amplification products, when observed, were not problematic due to their minor intensity and size variation.
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DISCUSSION USA300 CA-MRSA isolates are a clear and emerging clinical concern. However, the definitive identification of these strains has traditionally involved a combination of tests and protocols (i.e., PFGE, MLST, SCCmec, and PVL) which require specialized expertise and several days to complete. In addition, the arginine catabolic mobile element recently described by Diep et al. (7) appears to be present only in USA300 strains carrying SCCmec type IVa (L. K. McDougal, G. E. Fosheim, K. K. Bonnstetter, F. C. Tenover, D. J. Wolter, and R. V. Goering, Abstr. 46th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-603, 2006). The multiplex assay described here differentiates USA300 CA-MRSA strains with a variety of SCCmec IV subtypes (Table 1) from other MRSA strains. In this study, only USA300 isolates contained either six or eight AT repeats as well as PVL genes. In some instances, isolates with related sequence types, such as USA500 (ST8), exhibited ⱖ6 AT repeats, but never in combination with PVL. Other isolates (e.g., ST80) encoded PVL but always contained ⬍6 AT repeats. Thus, the combined detection of these elements via multiplex PCR allowed USA300 isolates to be quickly and specifically identified without sequencing. As with any assay, variant strains that could be difficult to detect with this method may exist. Nevertheless, our results suggest that the LNA assay has potential to be used as a rapid, cost-effective approach for identifying USA300 CA-MRSA, a significant pathogen with increasing prevalence in many hospital and community settings. In the CC8 isolates examined, SACOL0058 was present in ST8, ST247, and ST250 but not in ST239 and ST240, consistent with MLST analysis as discussed by Enright et al. (9). Interestingly, SACOL0058 was also found in CC5. Protein sequence analysis of the conserved hypothetical gene SACOL0058 via the Accelrys GCG translation program (San Diego, CA) showed that MRSA strains containing five AT repeats may possess a fully functional protein. However, an additional AT repeat (i.e., six AT repeats) resulted in a reading frame shift producing a stop codon at amino acid 286 (data not shown). With eight AT repeats, the first 285 amino acids of the protein remain homologous to the original with divergence thereafter. While these data raise serious questions regarding a functional role for SACOL0058 in staphylococcal isolates, the region appears to remain conserved among strains, especially including the USA300 genotype. AT repeat PCR, in combination with PCR for the presence of PVL genes and SACOL0058, has the potential to identify USA300 CA-MRSA strains in a rapid, cost-efficient manner. Accurate results can be obtained by carefully following optimized PCR conditions, allowing valuable diagnostic and surveillance data to be collected quickly without the need for sequencing. ACKNOWLEDGMENT The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. REFERENCES 1. Begier, E. M., K. Frenette, N. L. Barrett, P. Mshar, S. Petit, D. J. Boxrud, K. Watkins-Colwell, S. Wheeler, E. A. Cebelinski, A. Glennen, D. Nguyen, and
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2.
3. 4.
5. 6. 7.
8.
9. 10. 11.
12.
13.
14.
15.
16. 17.
BONNSTETTER ET AL. J. L. Hadler. 2004. A high-morbidity outbreak of methicillin-resistant Staphylococcus aureus among players on a college football team, facilitated by cosmetic body shaving and turf burns. Clin. Infect. Dis. 39:1446–1453. Buckingham, S. C., L. K. McDougal, L. D. Cathey, K. Comeaux, A. S. Craig, S. K. Fridkin, and F. C. Tenover. 2004. Emergence of community-associated methicillin-resistant Staphylococcus aureus at a Memphis, Tennessee Children’s Hospital. Pediatr. Infect. Dis. J. 23:619–624. Centers for Disease Control and Prevention. 1999. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus—Minnesota and North Dakota, 1997-1999. Morb. Mortal. Wkly. Rep. 48:707–710. Centers for Disease Control and Prevention. 2003. Methicillin-resistant Staphylococcus aureus infections among competitive sports participants— Colorado, Indiana, Pennsylvania, and Los Angeles County, 2000-2003. Morb. Mortal. Wkly. Rep. 52:793–795. Centers for Disease Control and Prevention. 2003. Methicillin-resistant Staphylococcus aureus infections in correctional facilities—Georgia, California, and Texas, 2001-2003. Morb. Mortal. Wkly. Rep. 52:992–996. Chambers, H. F. 2005. Community-associated MRSA—resistance and virulence converge. N. Engl. J. Med. 352:1485–1487. Diep, B. A., S. R. Gill, R. F. Chang, T. H. Phan, J. H. Chen, M. G. Davidson, F. Lin, J. Lin, H. A. Carleton, E. F. Mongodin, G. F. Sensabaugh, and F. Perdreau-Remington. 2006. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant [sic] Staphylococcus aureus. Lancet 367:731–739. Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008–1015. Enright, M. C., D. A. Robinson, G. Randle, E. J. Feil, H. Grundmann, and B. G. Spratt. 2002. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc. Natl. Acad. Sci. USA 99:7687–7692. Etienne, J. 2005. Panton-Valentine leukocidin: a marker of severity for Staphylococcus aureus infection? Clin. Infect. Dis. 41:591–593. Francis, J. S., M. C. Doherty, U. Lopatin, C. P. Johnston, G. Sinha, T. Ross, M. Cai, N. N. Hansel, T. Perl, J. R. Ticehurst, K. Carroll, D. L. Thomas, E. Nuermberger, and J. G. Bartlett. 2005. Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes. Clin. Infect. Dis. 40:100–107. Fridkin, S. K., J. C. Hageman, M. Morrison, L. T. Sanza, K. Como-Sabetti, J. A. Jernigan, K. Harriman, L. H. Harrison, R. Lynfield, and M. M. Farley. 2005. Methicillin-resistant Staphylococcus aureus disease in three communities. N. Engl. J. Med. 352:1436–1444. Genestier, A. L., M. C. Michallet, G. Prevost, G. Bellot, L. Chalabreysse, S. Peyrol, F. Thivolet, J. Etienne, G. Lina, F. M. Vallette, F. Vandenesch, and L. Genestier. 2005. Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J. Clin. Investig. 115:3117–3127. Goering, R. V. 1993. Pulsed field gel electrophoresis, p. 185–196. In D. H. Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.), Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, DC. Gonzalez, B. E., G. Martinez-Aguilar, K. G. Hulten, W. A. Hammerman, J. Coss-Bu, A. Avalos-Mishaan, E. O. Mason, Jr., and S. L. Kaplan. 2005. Severe staphylococcal sepsis in adolescents in the era of community-acquired methicillin-resistant Staphylococcus aureus. Pediatrics 115:642–648. Hanssen, A. M., and J. U. Ericson Sollid. 2006. SCCmec in staphylococci: genes on the move. FEMS Immunol. Med. Microbiol. 46:8–20. Herold, B. C., L. C. Immergluck, M. C. Maranan, D. S. Lauderdale, R. E. Gaskin, S. Boyle-Vavra, C. D. Leitch, and R. S. Daum. 1998. Communityacquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 279:593–598.
J. CLIN. MICROBIOL. 18. Hidron, A. I., E. V. Kourbatova, J. S. Halvosa, B. J. Terrell, L. K. McDougal, F. C. Tenover, H. M. Blumberg, and M. D. King. 2005. Risk factors for colonization with methicillin-resistant Staphylococcus aureus (MRSA) in patients admitted to an urban hospital: emergence of community-associated MRSA nasal carriage. Clin. Infect. Dis. 41:159–166. 19. Ito, T., K. Okuma, X. X. Ma, H. Yuzawa, and K. Hiramatsu. 2003. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist. Updates 6:41–52. 20. Kazakova, S. V., J. C. Hageman, M. Matava, A. Srinivasan, L. Phelan, B. Garfinkel, T. Boo, S. McAllister, J. Anderson, B. Jensen, D. Dodson, D. Lonsway, L. K. McDougal, M. Arduino, V. J. Fraser, G. Killgore, F. C. Tenover, S. Cody, and D. B. Jernigan. 2005. A clone of methicillin-resistant Staphylococcus aureus among professional football players. N. Engl. J. Med. 352:468–475. 21. Lina, G., Y. Piemont, F. Godail-Gamot, M. Bes, M. O. Peter, V. Gauduchon, F. Vandenesch, and J. Etienne. 1999. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin. Infect. Dis. 29:1128–1132. 22. Lindenmayer, J. M., S. Schoenfeld, R. O’Grady, and J. K. Carney. 1998. Methicillin-resistant Staphylococcus aureus in a high school wrestling team and the surrounding community. Arch. Intern. Med. 158:895–899. 23. McDougal, L. K., C. D. Steward, G. E. Killgore, J. M. Chaitram, S. K. McAllister, and F. C. Tenover. 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol. 41:5113–5120. 24. Miller, L. G., F. Perdreau-Remington, G. Rieg, S. Mehdi, J. Perlroth, A. S. Bayer, A. W. Tang, T. O. Phung, and B. Spellberg. 2005. Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles. N. Engl. J. Med. 352:1445–1453. 25. Mishaan, A. M., E. O. Mason, Jr., G. Martinez-Aguilar, W. Hammerman, J. J. Propst, J. R. Lupski, P. Stankiewicz, S. L. Kaplan, and K. Hulten. 2005. Emergence of a predominant clone of community-acquired Staphylococcus aureus among children in Houston, Texas. Pediatr. Infect. Dis. J. 24:201–206. 26. Naimi, T. S., K. H. LeDell, K. Como-Sabetti, S. M. Borchardt, D. J. Boxrud, J. Etienne, S. K. Johnson, F. Vandenesch, S. Fridkin, C. O’Boyle, R. N. Danila, and R. Lynfield. 2003. Comparison of community- and health careassociated methicillin-resistant Staphylococcus aureus infection. JAMA 290: 2976–2984. 27. Nguyen, D. M., L. Mascola, and E. Brancoft. 2005. Recurring methicillinresistant Staphylococcus aureus infections in a football team. Emerg. Infect. Dis. 11:526–532. 28. Pan, E. S., B. A. Diep, E. D. Charlebois, C. Auerswald, H. A. Carleton, G. F. Sensabaugh, and F. Perdreau-Remington. 2005. Population dynamics of nasal strains of methicillin-resistant Staphylococcus aureus—and their relation to community-associated disease activity. J. Infect. Dis. 192:811–818. 29. Rubin, R. J., C. A. Harrington, A. Poon, K. Dietrich, J. A. Greene, and A. Moiduddin. 1999. The economic impact of Staphylococcus aureus infection in New York City hospitals. Emerg. Infect. Dis. 5:9–17. 30. Saiman, L., M. O’Keefe, P. L. Graham III, F. Wu, B. Said-Salim, B. Kreiswirth, A. LaSala, P. M. Schlievert, and P. Della-Latta. 2003. Hospital transmission of community-acquired methicillin-resistant Staphylococcus aureus among postpartum women. Clin. Infect. Dis. 37:1313–1319. 31. Saravolatz, L. D., D. J. Pohlod, and L. M. Arking. 1982. Community-acquired methicillin-resistant Staphylococcus aureus infections: a new source for nosocomial outbreaks. Ann. Intern. Med. 97:325–329. 32. Shopsin, B., and B. N. Kreiswirth. 2001. Molecular epidemiology of methicillin-resistant Staphylococcus aureus. Emerg. Infect. Dis. 7:323–326. 33. Vester, B., and J. Wengel. 2004. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43:13233–13241.
