Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.     

Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecA(LGA251)

The recent finding of a new mecA homologue, mecA(LGA251) , with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA(LGA251) from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n=185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA(LGA251) . The mecA(LGA251) gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA(LGA251) in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MRSA isolates.     

实时荧光PCR在检测MRSA引起血流感染中的研究

目的建立检测引起血流感染中耐甲氧西林金黄色葡萄球菌（MRSA）mecA基因的实时荧光PCR法。方法设计mecA引物,优化荧光实时PCR实验条件,并用该法检测临床分离的78株金黄色葡萄球菌引起血流感染标本,其结果与头孢西丁纸片扩散法的结果进行比较。结果 78株金黄色葡萄球菌中,荧光实时PCR法检测出mecA基因阳性38株、阴性40株;头孢西丁纸片扩散法检测出MRSA 27株、MSSA 51株,经配对卡方检验,荧光实时PCR法检出率显著高于头孢西丁纸片扩散法（P〈0.01）。结论荧光实时PCR法能敏感、特异检测MRSA引起的血流感染,比一般MRSA表型检测更加简便、快速。     

Local variants of Staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci: evidence of horizontal gene transfer?

The mecA gene in Staphylococcus aureus is located on the genetic element staphylococcal cassette chromosome (SCC). Different SCCmecs have been classified according to their putative recombinase genes (ccrA and ccrB) and overall genetic composition. Clinical isolates of coagulase-negative staphylococci (CoNS; n = 39) and S. aureus (n = 20) from Norway, India, Italy, Finland, the United States, and the United Kingdom were analyzed by pulsed-field gel electrophoresis, which showed that most isolates were genetically unrelated. Cluster analyses of 16S rRNA gene and pta sequences confirmed the traditional biochemical species identification. The mecI, mecR1, mecA, and ccrAB genes were detected by PCRs, identifying 19 out of 20 S. aureus and 17 out of 39 CoNS isolates as carriers of one of the three published ccrAB pairs. New variants of SCCmec were identified, as well as CoNS isolates containing ccrAB genes without the mec locus. ccrAB and mec PCRs were verified by hybridization. Sequence alignments of ccrAB genes showed a high level of diversity between the ccrAB alleles from different isolates, i.e., 94 to 100% and 95 to 100% homology for ccrAB1 and ccrAB2, respectively. All of the ccrAB3 genes identified were identical. Genetically unique and sporadic methicillin-resistant S. aureus (MRSA) contained local variants of ccrAB gene pairs identical to those found in MR-CoNS but different from those in MRSA from other regions. Allelic variants of ccrAB in isolates from the same geographic region showed sequence conservation independent of species. The species-independent sequence conservation found suggests that there is a closer genetic relationship between ccrAB2 in Norwegian staphylococci than between ccrAB2 sequences in international MRSA and Norwegian MRSA. This might indicate that different staphylococcal species acquire these genes locally by horizontal gene transfer.     

Rapid MRSA detection by a latex kit.

Methicillin resistant strains of Staphylococcus aureus (MRSA) are implicated in serious infections and nosocomial outbreaks, and show resistance to a wide range of antibiotics, thus limiting the treatment options. Therefore, rapid detection is clinically crucial for both treatment and infection control measures. This study assessed the performance of a rapid latex agglutination kit marketed to detect MRSA clinical isolates (MRSA-Screen test Denka Seiken Co Ltd, Tokyo, Japan) based on detecting a specific penicillin binding protein 2a (PBP2a) in comparison to the NCCLS oxacillin salt agar screen plate, the 1 microg oxacillin disk diffusion test, and the oxacillin MIC by E-test. Testing was carried out on 133 isolates consisting of 99 MRSA and 34 methicillin sensitive strains of S. aureus (MSSA). Concordant results were observed between the latex kit and all the other tests for the 99 MRSA isolates. Only 1 of the 34 MSSA isolates gave a positive agglutination reaction in the latex kit. The kit sensitivity and specificity were determined to be 100% and 97%, respectively. This reliable performance indicates that the MRSA-Screen latex test is very useful test for the rapid detection of MRSA isolates in the clinical microbiology laboratory.     

耐甲氧西林金黄色葡萄球菌产色培养基临床应用评估

目的通过头孢西丁纸片法、耐甲氧西林金黄色葡萄球菌（MRSA）产色筛选平板法和聚合酶链反应（PCR）检测MRSA,评估MRSA产色筛选平板的敏感性和特异性。方法收集仁济医院2008年8月至9月临床分离的金黄色葡萄球菌68株,分别采用头孢西丁纸片法、MRSA产色筛选平板法和PCR检测MRSA,以PCR检测femA基因和mecA基因结果为金标准,比较MRSA产色筛选平板的敏感性和特异性。结果甲氧西林敏感金黄色葡萄球菌（MSSA）除万古霉素、替考拉宁和利奈唑胺外,对其他11种抗菌药物的敏感率明显高于MRSA。68株金黄色葡萄球菌中,头孢西丁纸片法筛选出50株MRSA,产色平板法筛选出51株MRSA,PCR检测到51株mecA基因阳性,MRSA产色筛选平板结果和mecA基因检测结果符合率为100%。结论MRSA产色筛选平板可用于临床快速检测MRSA。     

多重PCR检测MRSA的SCCmec基因分型

目的了解我院MRSA的流行状况。方法收集2005年1—6月65株社区感染MRSA及60株医院感染MRSA，应用多重PCR对MRSA染色体Dlec基因盒（staphylococcal cassette chromosomeSCCmec）分型及杀白细胞毒素（PVI。）基因检测，应用K—B纸片法进行药敏分析。结果125株MRSA的mecA基因阳性，其中SCCmecII型1株，SCCmecⅢ型120株，SCCmecⅣ型3株，未分型1株；未发现携带PVL基因的MRSA。携带SCCmecII型、SCCmecⅢ型的菌株均为多重耐药株，而携带SCCmecⅣ型的菌株除对8内酰胺类药物耐药外，对其他类别的抗菌药敏感。结论本院分离的MRSA以SCCmecⅢ型为主，发现SCCmecIV型CA-MRSA，但不携带PVL基因；携带SCCmecⅡ、SCCmecⅢ的临床分离株耐药严重。     

In vivo transfer of high-level mupirocin resistance from Staphylococcus epidermidis to methicillin-resistant Staphylococcus aureus associated with failure of mupirocin prophylaxis

OBJECTIVES: We examined the molecular basis of the emergence of mupirocin resistance in a methicillin-resistant Staphylococcus aureus (MRSA) strain colonizing a nursing home resident undergoing mupirocin prophylaxis. Patient and methods: A persistent carrier of mupirocin-susceptible MRSA participated in a trial of mupirocin for nasal decolonization among nursing home residents. During prophylaxis a high-level mupirocin-resistant MRSA emerged in the nasal isolates from this patient. S. aureus and coagulase-negative staphylococci were isolated prior to, during and after 14 days of mupirocin treatment. The staphylococcal isolates and their plasmids were examined by molecular genetic methods. RESULTS: All mupirocin-susceptible and -resistant MRSA isolates possessed the same genotype. The patient was also colonized by a single mupirocin-resistant Staphylococcus epidermidis strain. The mupirocin-resistant MRSA and S. epidermidis strains harboured identical plasmids that carried the mupA determinant and genes for conjugative DNA transfer in staphylococci. These plasmids could be transferred in vitro from both clinical isolates to S. aureus RN2677. CONCLUSIONS: The MRSA strain contained a conjugative plasmid expressing mupA that was identical with that found in the S. epidermidis strain which colonized the patient. These findings suggest that transfer of mupA from S. epidermidis to MRSA probably occurred during mupirocin prophylaxis.     

复合PCR鉴别葡萄球菌及其多重耐药基因

目的　建立既可鉴别金黄色葡萄球菌又可同时检测其耐药基因的分子诊断方法。方法　对应于femB、mecA、ileS基因的 3对引物与快速提取的单菌落模板DNA进行单管同步扩增 ,电泳观察PCR片段 ;mecA、ileS耐药基因扩增结果分别与苯唑西林、莫匹罗星药敏试验对比 ,分析菌株的耐药性。结果　检测femB基因可快速特异性地筛选出金黄色葡萄球菌 ,mecA基因的检出与常规药敏试验鉴定耐甲氧西林葡萄球菌 (MRS)的结果基本一致 ,而拥有ileS基因的全部葡萄球菌分离株对莫匹罗星耐药。结论　复合PCR可快速敏感地从葡萄球菌中区分金黄色葡萄菌 ,并同时检出MRSA和耐莫匹罗星的多重耐药菌株。     

In vivo deletion of the methicillin resistance mec region from the chromosome of Staphylococcus aureus strains

Two sets of Staphylococcus aureus isolates recovered from two patients exhibited similar susceptibility profiles except for oxacillin susceptibility (MSSA) or resistance (MRSA). SMA:I macrorestriction and inter-IS256 PCR analysis showed patterns closely related to the Belgian epidemic MRSA clone 1 in each pair of MSSA/MRSA strains. Loss of one large SMA:I DNA fragment and concurrent gain of a smaller fragment in the MSSA isolates was observed. The mecA sequence present in the MRSA was absent in the MSSA variant. Therefore, in vivo deletion of the mec region may occur in some lineages of S. aureus more frequently than previously thought.     

Methicillin-resistant Staphylococcus aureus (MRSA) isolated in hospitals in Fukuoka City area]

Bacteriological and clinical studies were carried out on 280 strains of Staphylococcus aureus isolated in hospitals in Fukuoka city area from September 1990 to March 1991. Of all S. aureus strains studied 116 (41.4%) were methicillin-resistant. The proportion of MRSA in S. aureus isolates from outpatients was 10% (11/109), 69.2% (90/130) in those from inpatients. The average age of patients with isolated MRSA was 70.5 +/- 16.9 years and that of patients with isolated MSSA, 44.2 +/- 29.3. MRSA strains were recovered mainly from sputum and pus. The isolation rate did not vary significantly with hospitals of different size (number of beds). Of all MRSA strains 48 (41.3%) produced coagulase type VII. As for drug susceptibility, MRSA strains with coagulase type VII were more sensitive to clindamycin and more resistant to minocyclin compared to MRSA with other coagulase types.     

National survey of molecular epidemiology of Staphylococcus aureus colonization in Belgian cystic fibrosis patients

OBJECTIVES: Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is poorly defined in cystic fibrosis (CF) patients, and S. aureus detection may be hampered by the presence of small colony variants (SCVs). We conducted a multicentre survey to determine the prevalence of S. aureus and MRSA colonization in Belgian CF patients and characterize the phenotype and clonal distribution of their staphylococcal strains. METHODS: S. aureus isolated from CF patients attending nine CF centres were collected. Oxacillin resistance was detected by oxacillin agar screen and mecA PCR. Antibiotic susceptibility was tested by microdilution. MRSA strains were genotyped by PFGE and SCCmec typing and compared with hospital-associated MRSA strains. RESULTS: Laboratories used a diversity of sputum culture procedures, many of which appeared substandard. S. aureus was isolated from 275/627 (44%) CF patients (20% to 72% by centre). The prevalence of SCV colonization was 4%, but SCVs were almost exclusively recovered from patients in two centres performing an SCV search. Phenotypically, 14% of S. aureus isolates were oxacillin-resistant: 79% carried mecA and 19% were SCVs lacking mecA. The mean prevalence of 'true' MRSA colonization was 5% (0% to 17% by centre). By PFGE typing, 67% of CF-associated MRSA were related to five epidemic clones widespread in Belgian hospitals. CONCLUSIONS: This first survey of S. aureus colonization in the Belgian CF population indicated a diversity in local prevalence rates and in proportion of oxacillin-resistant and SCV phenotypes, probably related to variation in bacteriological methods. These findings underscore the need for standard S. aureus detection methods and MRSA control policies in Belgian CF centres.     

Diagnostic PCR analysis of the occurrence of methicillin and tetracycline resistance genes among Staphylococcus aureus isolates from phase 3 clinical trials of tigecycline for complicated skin and skin structure infections

Diagnostic PCR assays were developed to track common genetic determinants of oxacillin resistance as well as resistance to classical tetracyclines in Staphylococcus aureus isolates from the recently completed worldwide phase 3 clinical trials of tigecycline. A total of 503 unique S. aureus strains isolated from complicated skin and skin structure infections were analyzed. The mecA gene was amplified from 120 strains (23.9%) determined to be resistant to oxacillin (MICs > or = 4 microg/ml). The prevalence of the mecA gene was found to vary regionally from 6.5% to 50.9% among isolates originating in Eastern Europe and North America, respectively. The presence of a tetracycline resistance determinant, tet(M) or tet(K), among methicillin-resistant S. aureus (MRSA) isolates also varied regionally, with a range of 11.9% to 46.2% among isolates tested from North America and Eastern Europe, respectively. The occurrence of a tetracycline resistance marker in methicillin-susceptible S. aureus (MSSA) strains varied from 2.5 to 16.1% among the isolates tested across the regions of study. The presence of tet(M) or tet(K) had no discernible effect on the tigecycline MICs for either MRSA or MSSA strains, which is consistent with the ability of the glycylcyclines to retain activity in the presence of both the ribosomal protection and efflux mechanisms of resistance to the tetracyclines.     

Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in Central Sydney,Australia

Ninety clinical Staphylococcus aureus isolates from separate patients were examined phenotypically and genotypically for susceptibility to methicillin/oxacillin. Thirty were methicillin/oxacillin susceptible and 60 were methicillin and oxacillin resistant (MRSA). The 60 MRSA isolates examined were subdivided into two groups according to their antibiotic profiles and comprised 30 non-multidrug-resistant (NMDR) isolates, resistant to less than two non-beta-lactam antibiotics, and 30 multidrug-resistant (MDR) isolates, resistant to three or more non-beta-lactam antibiotics. Phenotypic and genotypic analysis of methicillin/oxacillin showed that despite use of the guidelines published by the NCCLS for the testing of S. aureus susceptibility to methicillin/oxacillin, MIC values of some NMDR MRSA isolates fell below the NCCLS-recommended breakpoints. Etest strips failed to detect two NMDR MRSA isolates tested with oxacillin and four tested with methicillin. Lowering the NCCLS-recommended oxacillin screen agar concentration from 6 to 2 mg/L and temperature of incubation to 30 degrees C, improved the specificity and sensitivity of NMDR MRSA detection from 87% to 100%. On PFGE analysis these NMDR MRSA strains were genotypically different. Genotypic tests, such as multiplex PCR for the mecA/nuc genes and DNA hybridization for the mecA gene, or phenotypic monoclonal antibody-based tests to detect penicillin-binding protein 2a (PBP2a) offer advantages for problematic isolates in detecting or confirming low-level phenotypic heterogeneous mecA expression of oxacillin and methicillin resistance in NMDR MRSA.     

The distribution of mecA, mecR1 and mecI and sequence analysis of mecI and the mec promoter region in staphylococci expressing resistance to methicillin.

The presence and sequences of genes that regulate the expression of methicillin resistance was investigated in 42 isolates of Staphylococcus aureus and 102 isolates of coagulase-negative staphylococci (CNS). PCR was used to detect mecA and the regulatory genes mecR1 and mecI. In a selected group of isolates, the sequences of mecI and the mec promoter region were also determined and compared with the sequences obtained from pre-MRSA strain N315. The genetic diversity of the collection was assessed by pulsed-field gel electrophoresis (PFGE). mecA was present in 21 S. aureus and 44 CNS. mecR1 was associated with mecA in all S. aureus and in all CNS, except two isolates of Staphylococcus haemolyticus. mecI was present in 48% of mecA-positive S. aureus and 50% of mecA-positive CNS. In six S. aureus isolates, mecI contained a termination codon at nucleotide 202 which would truncate the MecI protein. No mutation was found in the mecI gene of the four other S. aureus and 15 CNS sequenced. Seven isolates of Staphylococcus simulans had a single nucleotide substitution in the mec promoter region. Expression of methicillin resistance could be explained for all mecA-positive staphylococci with mutations within mecI or in the mec promoter region or in which mecI was deleted. However, the 'wild type' sequences observed in four S. aureus and eight CNS suggest that there is another mechanism for overcoming the repression of resistance caused by mecI.     

Comparison of the Velogene Rapid MRSA Identification Assay, Denka MRSA-Screen Assay, and BBL Crystal MRSA ID System for rapid identification of methicillin-resistant Staphylococcus aureus.

Early detection of methicillin-resistant S.aureus (MRSA) is critical for both the management of infected patients, and the timely institution of appropriate infection control measures. Although detection of the mecA gene by PCR remains the gold standard, this technology is inaccessible for many laboratories. Therefore, we sought to evaluate several new rapid identification systems and compare them to PCR. A total of 71 methicillin-susceptible S. aureus (MSSA), 25 borderline oxacillin-resistant S. aureus (BORSA), and 213 MRSA were selected for study. S.aureus was identified using standard methods. Initial screening was performed on a Mueller-Hinton agar plate with 6 mg/L of oxacillin. MRSA strains were identified using PCR with primers specific for the mecA gene. PCR was used as the reference method. All isolates were tested using the BBL Crystal MRSA ID System (Becton Dickinson Microbiology Systems, Maryland, USA), the MRSA-Screen Assay (Denka Seiken Co., Ltd., Tokyo, Japan), and the Velogene Rapid MRSA Identification Assay (ID Biomedical Corp, Vancouver, BC). With minor modifications, all assays were performed according to manufacturers' instructions. Overall, the 3 commercial assays performed well. The sensitivity and specificity of the BBL, Denka, and Velogene systems were 99%/100%, 99%/100%, and 96%/100% respectively. The advantages of the phenotypic tests-BBL Crystal Kit and Denka MRSA-Screen Assay include lower cost per test, shelf-life, ease of use, and rapid turn-around times. Advantages of the Velogene Rapid MRSA include ability to perform genotypic high-volume testing without the equipment requirements and technical complexity involved with PCR. Turn-around times ranged from 15 min for the Denka MRSA-Screen Assay, 2 h for the Velogene Rapid MRSA, and 4 h for the BBL Crystal. The BBL Crystal, Denka MRSA-Screen, and Velogene Rapid MRSA identification systems are rapid, easy to perform, and provide accurate identification of MRSA. These rapid kits offer an acceptable alternative for smaller, non-reference, laboratories and reduce the dependency on PCR in larger laboratories for routine confirmation.     

Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus

Methicillin resistance in staphylococci is mediated by penicillin binding protein 2a (PBP 2a), encoded by mecA on mobile staphylococcal cassette chromosome mec (SCCmec) elements. In this study, two clonal complex 130 (CC130) methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients in Irish hospitals were identified that were phenotypically PBP 2a positive but lacked mecA by conventional PCR and by DNA microarray screening. The isolates were identified as methicillin-susceptible S. aureus using the GeneXpert real-time PCR assay. Whole-genome sequencing of one isolate (M10/0061) revealed a 30-kb SCCmec element encoding a class E mec complex with highly divergent blaZ-mecA-mecR1-mecI, a type 8 cassette chromosome recombinase (ccr) complex consisting of ccrA1-ccrB3, an arsenic resistance operon, and flanking direct repeats (DRs). The SCCmec element was almost identical to that of SCCmec type XI (SCCmec XI) identified by the Sanger Institute in sequence type 425 bovine MRSA strain LGA251 listed on the website of the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements. The open reading frames (ORFs) identified within SCCmec XI of M10/0061 exhibited 21 to 93% amino acid identity to ORFs in GenBank. A third DR was identified ca. 3 kb downstream of SCCmec XI, indicating the presence of a possible SCC remnant. SCCmec XI was also identified in the second CC130 MRSA isolate by PCR and sequencing. The CC130 MRSA isolates may be of animal origin as previously reported CC130 S. aureus strains were predominantly from bovine sources. The highly divergent nature of SCCmec XI relative to other SCCmec elements indicates that it may have originated in another taxon.     

Characterization of oxacillin-susceptible <Emphasis Type="Italic">mecA</Emphasis>-positive <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>: a new type of MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) has been defined as S. aureus having the mecA gene or showing a minimum inhibitory concentration (MIC) of oxacillin higher than 4 mg/l. However, some clinical isolates are mecA-positive and oxacillin-susceptible. Therefore, we surveyed the occurrence of S. aureus having the mecA gene and an MIC of oxacillin of less than 2 mg/l (oxacillin-susceptible MRSA; OS-MRSA) in a total of 480 strains of S. aureus collected from 11 hospitals in different location in Japan isolated from 2003 through 2005. We found 6 strains matching the criteria for OS-MRSA. All 6 strains were staphylococcal cassette chromosome (SCC) mec-positive, without exception, and 4 strains showed the SCCmec type III-variant, which is unique in Japan. These OS-MRSAs were least resistant to oxacillin among the MRSAs tested and they were within the susceptible range to seven other beta-lactam antibiotics tested. Thus, OS-MRSA may become a high-resistant MRSA upon the treatment of patients with beta-lactam antibiotics. To characterize whether these OS-MRSAs were hospital-acquired or community-acquired MRSAs, we tested for the presence of the genes encoding toxins. Genes encoding hemolysin, exfoliative toxin, enterotoxin, toxic shock syndrome toxin-1, and Panton-Valentine leukocidin were found in 6, 4, 0, 0, and 0 strains, respectively. These results revealed that OS-MRSAs could be classified as a new type of MRSA that exhibits properties distinguishable from either hospital- or community-acquired MRSA. Coagulase typing of the OS-MRSAs supported the above conclusion. In this study, the occurrence of OS-MRSA at a certain frequency was noted; precautions are called for in the classification of oxacillin-resistant S. aureus and in the treatment of OS-MRSA infection.     

Rapid detection of mecA in methicillin resistant Staphylococcus aureus using cycling probe technology.

A novel method has been developed for the detection of the mecA gene, that confers the principle mechanism of methicillin resistance in staphylococci. Cycling Probe Technology (CPT) is a rapid, simple, isothermal method for the detection of specific target sequences. CPT utilizes a unique chimeric DNA-RNA-DNA probe sequence that provides an RNase H sensitive scissile link when hybridized to a complementary target DNA sequence. In the presence of target DNA, the cycling reaction converts full-length chimeric probe into cleaved probe fragments, which accumulate and are quantified. A cycling probe designed for detection of a specific sequence within the mecA gene was used to develop a culture confirmation assay for methicillin resistant Staphylococcus aureus. The CPT assay was used to screen 238 S. aureus isolates and the results were in complete agreement with detection of the mecA gene by polymerase chain reaction (PCR). Detection of mecA should be considered the gold standard for determining methicillin resistance in S. aureus. This study demonstrates the feasibility of using CPT to meet this need.