Reliability of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay in detecting MRSA isolates with a variety of genotypes from the United States and Taiwan.
ABSTRACT: The BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay is a molecular screening test for detection of MRSA in nasal colonization. This assay coamplifies the extremity of staphylococcal chromosome cassette mec (SCCmec) and adjacent chromosomal DNA at the SCCmec insertion site. Increasing reports of novel SCCmec types and the diverse genetic backgrounds of MRSA strains prompted us to test the accuracy of the BD GeneOhm MRSA kit with 914 MRSA isolates with a variety of SCCmec types harbored in 21 genetic backgrounds, as determined by the multilocus sequence type (ST). The BD GeneOhm MRSA assay was performed on colony lysates; purified genomic DNA (0.2 pg/?l and 0.2 ng/?l) was tested to confirm negative results from lysates. Of 914 MRSA isolates tested, 911 tested positive (detection rate, 99.7%). The SCCmec types carried by assay-positive isolates were I, II, III, IV, V, V(5C2&5), VI, and VIII and SCCmec composite islands with mec class A and ccr complexes 2 and 4. One of the assay-negative isolates had a community-associated genotype: ST8, SCCmec type IV. However, this was an outlier among the 99.8% (434/435) ST8, SCCmec type IV-containing isolates that tested positive. The two other assay-negative isolates had a health care-associated genotype (ST5); both carried a distinct, uncommon, composite SCCmec type. In summary, the BD GeneOhm MRSA assay had a high rate of detection of MRSA isolates harboring common and uncommon SCCmec types from the United States and Taiwan.
Project description:Methicillin-susceptible Staphylococcus aureus (MSSA) can arise from methicillin-resistant S. aureus (MRSA) following partial or complete excision of staphylococcal cassette chromosome mec (SCCmec). This study investigated whether multiresistant MSSA isolates from Irish hospitals, where MRSA has been endemic for decades, harbor SCCmec DNA. Twenty-five multiresistant MSSA isolates recovered between 2002 and 2006 were tested for SCCmec DNA by PCR and were genotyped by multilocus sequence typing and spa typing. All isolates lacked mecA. Three isolates (12%) harbored SCCmec DNA; two of these (genotype ST8/t190) harbored a 26-kb SCCmec IID (II.3.1.2) remnant that lacked part of mecI and all of mecR1, mecA, and IS431; the third isolate (ST8/t3209) harbored the SCCmec region from dcs to orfX. All three isolates were detected as MRSA using the BD GeneOhm and Cepheid's Xpert MRSA real-time PCR assays. Six isolates (ST8/t190, n = 4; ST5/t088, n = 2), including both isolates with the SCCmec IID remnant, harbored ccrAB4 with 100% identity to ccrAB4 from the Staphylococcus epidermidis composite island SCC-CI. This ccrAB4 gene was also identified in 23 MRSA isolates representative of ST8/t190-MRSA with variant SCCmec II subtypes IIA to IIE, which predominated previously in Irish hospitals. ccrAB4 was located 5,549 bp upstream of the left SCCmec junction in both the MRSA and MSSA isolates with SCCmec elements and remnants and 5,549 bp upstream of orfX in the four MSSA isolates with ccrAB4 only on an SCC-CI homologous region. This is the first description of a large SCCmec remnant with ccr and partial mec genes in MSSA and of the S. epidermidis SCC-CI and ccrAB4 genes in S. aureus.
Project description:The arginine catabolic mobile element (ACME) is prevalent among methicillin-resistant Staphylococcus aureus (MRSA) isolates of sequence type 8 (ST8) and staphylococcal chromosomal cassette mec (SCCmec) type IVa (USA300) (ST8-MRSA-IVa isolates), and evidence suggests that ACME enhances the ability of ST8-MRSA-IVa to grow and survive on its host. ACME has been identified in a small number of isolates belonging to other MRSA clones but is widespread among coagulase-negative staphylococci (CoNS). This study reports the first description of ACME in two distinct strains of the pandemic ST22-MRSA-IV clone. A total of 238 MRSA isolates recovered in Ireland between 1971 and 2008 were investigated for ACME using a DNA microarray. Twenty-three isolates (9.7%) were ACME positive, and all were either MRSA genotype ST8-MRSA-IVa (7/23, 30%) or MRSA genotype ST22-MRSA-IV (16/23, 70%). Whole-genome sequencing and comprehensive molecular characterization revealed the presence of a novel 46-kb ACME and staphylococcal chromosomal cassette mec (SCCmec) composite island (ACME/SCCmec-CI) in ST22-MRSA-IVh isolates (n=15). This ACME/SCCmec-CI consists of a 12-kb DNA region previously identified in ACME type II in S. epidermidis ATCC 12228, a truncated copy of the J1 region of SCCmec type I, and a complete SCCmec type IVh element. The composite island has a novel genetic organization, with ACME located within orfX and SCCmec located downstream of ACME. One PVL locus-positive ST22-MRSA-IVa isolate carried ACME located downstream of SCCmec type IVa, as previously described in ST8-MRSA-IVa. These results suggest that ACME has been acquired by ST22-MRSA-IV on two independent occasions. At least one of these instances may have involved horizontal transfer and recombination events between MRSA and CoNS. The presence of ACME may enhance dissemination of ST22-MRSA-IV, an already successful MRSA clone.
Project description:The BD GeneOhm MRSA assay could identify methicillin-resistant Staphylococcus aureus (MRSA) strains at a high ratio (97.8%). Analysis of 11 assay-negative MRSA strains suggested that insertion of non-mec staphylococcal cassette chromosome elements (SCCs) downstream of orfX, and carriage of SCCmecs with a left extremity that cannot be detected by the kit, might lead to their being given an incorrect negative status.
Project description:In methicillin resistant Staphylococcus aureus (MRSA), the arginine catabolic mobile element (ACME) was initially described in USA300 (t008-ST8) where it is located downstream of the staphylococcal cassette chromosome mec (SCCmec). A common health-care associated MRSA in Copenhagen, Denmark (t024-ST8) is clonally related to USA300 and is frequently PCR positive for the ACME specific arcA-gene. This study is the first to describe an ACME element upstream of the SCCmec in MRSA. By traditional SCCmec typing schemes, the SCCmec of t024-ST8 strain M1 carries SCCmec IVa, but full sequencing of the cassette revealed that the entire J3 region had no homology to published SCCmec IVa. Within the J3 region of M1 was a 1705 bp sequence only similar to a sequence in S. haemolyticus strain JCSC1435 and 2941 bps with no homology found in GenBank. In addition to the usual direct repeats (DR) at each extremity of SCCmec, M1 had two new DR between the orfX gene and the J3 region of the SCCmec. The region between the orfX DR (DR1) and DR2 contained the ccrAB4 genes. An ACME II-like element was located between DR2 and DR3. The entire 26,468 bp sequence between DR1 and DR3 was highly similar to parts of the ACME composite island of S. epidermidis strain ATCC12228. Sequencing of an ACME negative t024-ST8 strain (M299) showed that DR1 and the sequence between DR1 and DR3 was missing. The finding of a mobile ACME II-like element inserted downstream of orfX and upstream of SCCmec indicates a novel recombination between staphylococcal species.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in Irish hospitals between 1971 and 2002 were characterized using multilocus sequence typing (MLST) (n = 130) and SCCmec typing (n = 172). Where atypical SCCmec typing results were obtained, PCR amplification of entire SCCmec elements, analysis of amplimer mobility, and nucleotide sequencing were undertaken. MLST revealed that 129/130 isolates had the same genotypes as internationally spread MRSA clones, including ST239, ST247, ST250, ST5, ST22, ST36, and ST8. A novel genotype, ST496, was identified in one isolate. Half of the isolates (86/172) had SCCmec type I, IA, II, III, or IV. The remaining 86 isolates harbored novel SCCmec variants in three distinct genetic backgrounds: (i) 74/86 had genotype ST8 and either one of five novel SCCmec II (IIA, IIB, IIC, IID, and IIE) or one of two novel SCCmec IV (IVE and IVF) variants; (ii) 3/86 had genotype ST239 and a novel SCCmec III variant; (iii) 9/86 had a novel SCCmec I variant associated with ST250. SCCmec IVE and IVF were similar to SCCmec IVc and IVb, respectively, but differed in the region downstream of mecA. The five SCCmec II variants were similar to SCCmec IVb in the region upstream of the ccr complex but otherwise were similar to SCCmec II, except for the following regions: SCCmec IIA and IID had a novel mec complex, A.4 (Delta mecI-IS1182-Delta mecI-mecR1-mecA-IS431mec); SCCmec IIC and IIE had a novel mec complex, A.3 (IS1182-Delta mecI-mecR1-mecA-IS431mec); SCCmec IID and IIE lacked pUB110; SCCmec IIC and IIE lacked a region of DNA between Tn554 and the mec complex; and SCCmec IIB lacked Tn554. This study has demonstrated a hitherto-undescribed degree of diversity within SCCmec.
Project description:In this study, 425 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in the Dutch-German Euregio were investigated for the presence of the arginine catabolic mobile element (ACME). Sequence analysis by whole-genome sequencing revealed an entirely new organization of the ACME-staphylococcal cassette chromosome mec composite island (SCCmec-CI), with truncated ACME type II located downstream of SCCmec. An identical nucleotide sequence of ACME-SCCmec-CI was found in two distinct MRSA lineages (t064-ST8 and t002-ST5), which has not been reported previously in S. aureus.
Project description:This study determined the performance of BD Max StaphSR and the rate of methicillin-resistant Staphylococcus aureus (MRSA) with an unrecognized staphylococcal cassette chromosome mec (SCCmec) right-extremity junction (MREJ) region among 907 methicillin-resistant S. aureus (MRSA) and 900 methicillin-susceptible S. aureus (MSSA) isolates. The rate of mecA/mecC dropout mutants was also evaluated. Only three MRSA isolates (99.7% sensitivity; 904/907) were classified as MSSA by the BD Max StaphSR assay, due to negative results for MREJ. Eight MSSA isolates (99.1% sensitivity; 892/900) were assigned as MRSA. However, six of these MSSA isolates had the mecA gene confirmed by PCR and sequencing (99.8% sensitivity; 898/900). Overall, 7.1% (64/900) of MSSA isolates showed results compatible with a mecA dropout genotype.
Project description:Increasing frequencies of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain isolation have been reported from many countries. The overall prevalence of MRSA in Norway is still very low. MRSA isolates (n = 67) detected between 1995 and 2003 in northern Norway were analyzed by pulsed-field gel electrophoresis, multilocus sequence typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Sixty-seven isolates were associated with 13 different sequence types. Two successful MRSA clones predominated. Sequence type 8 (ST8) (40%) and ST80 (19%) containing SCCmec type IV were detected in hospitals and communities in different geographic regions during a 7-year period. In general, there was a low level of antimicrobial resistance. Only 26% of the isolates were multiresistant. International epidemic clones were detected. The frequent findings of SCCmec type IV (91%) along with heterogeneous genetic backgrounds suggest a horizontal spread of SCCmec type IV among staphylococcal strains in parallel with the clonal spread of successful MRSA strains.
Project description:Forty-one methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates that clearly differed from the six major pandemic clones of MRSA in pulsed-field gel electrophoresis type, mecA and Tn554 polymorphism, and epidemic behavior were selected from an international strain collection for more detailed characterization. SpaA typing, multilocus sequence typing, and SCCmec (staphylococcal cassette chromosome mec) typing demonstrated extensive diversity among these sporadic isolates both in genetic background and also in the structure of the associated SCCmec elements. Nevertheless, the isolates could be grouped into restricted clonal complexes by using the BURST (i.e., based upon related sequence types) program algorithm, which predicted that most sporadic MRSA isolates evolved from pandemic MRSA clones. Several of the sporadic MRSA resembled community-acquired MRSA isolates in properties that included a relatively limited multiresistance pattern, faster growth rates, diversity of genetic backgrounds, and a frequent association with SCCmec type IV.
Project description:Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.