Abstract
A number of real-time PCR assays for direct detection of methicillinresistant (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic T(m) value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.
Highlights
Rates of methicillin-resistant Staphylococcus aureus (MRSA) infections have steadily increased during the past two decades and the occurrence and spread of MRSA strains in healthcare facilities as well as in the community is a growing problem worldwide [1,2,3]
Molecular characteristics of MRSA isolates derived from the Bavarian livestock-associated MRSA (LA-MRSA) survey By sampling the nares of 634 swine and 116 farmers on 60 geographically distinct farms in Bavaria during the course of an ongoing study, a total number of 245 MRSA strains from pigs and 34 MRSA strains from farmers were grown from the collected swabs
Novel single nucleotide polymorphisms in the staphylococcal cassette chromosome mec (SCCmec)-orfX integration site of LA-MRSA isolates By systematic sequencing of the SCCmec-orfX integration sites of MRSA isolates of animal origin, all of the 44 sequences obtained from Bavarian porcine isolates (Table, rows 1 to 6) were found to be identical in a multiple alignment (using pileup from the HUSAR sequence analysis package from the German Cancer Research Center (DKFZ), http://genius.embnet.dkfz-heidelberg.de, data not shown)
Summary
Rates of methicillin-resistant Staphylococcus aureus (MRSA) infections have steadily increased during the past two decades and the occurrence and spread of MRSA strains in healthcare facilities as well as in the community is a growing problem worldwide [1,2,3]. Classically considered as a nosocomial pathogen, reports of MRSA carriage or its acquisition in the community have become increasingly common during the past decade [2,4]. Molecular characterisation revealed that a distinct clone of MRSA is predominant within this collective: LA-MRSA strains are grouping within the new clonal complex 398 (CC398) with sequence type 398 (ST398) as the most prevalent type. Animals carrying MRSA represent a reservoir for transmission to humans [13,14,15]. The MRSA strains from animal origin have been shown to be pathogenic for humans and can cause severe infections such as endocarditis and ventilatorassociated pneumonia [16]. A number of diagnostic strategies have been published on the molecular characterisation of the MRSA CC398 clonal lineage, using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) or other laborious techniques based on genome sequencing [1,19,20]
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