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Updated: Jun 17, 2026
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I
M A Argudín1, A Fetsch, B-A Tenhagen
1Departmento de Biología Funcional (Area de Microbiología), University of Oviedo, Julían Clavería 6, E-33006 Oviedo, Spain,
Abstract:
During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.
Insights
Cfr9I pulsed-field gel electrophoresis (PFGE) effectively subtypes methicillin-resistant Staphylococcus aureus ST398, differentiating strains based on staphylococcal cassette chromosome mec (SCCmec) types. This method aids in tracking the successful ST398 clone in animal and food sources.
Area of Science:
- Microbiology
- Molecular Epidemiology
- Food Safety
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) ST398 is a significant zoonotic pathogen, frequently associated with livestock, particularly pigs.
- Standard subtyping methods like SmaI pulsed-field gel electrophoresis (PFGE) are ineffective for MRSA ST398 due to DNA restriction challenges.
- Cfr9I, a neoschizomer of SmaI, has recently been identified as a suitable enzyme for digesting ST398 DNA for PFGE analysis.
Purpose of the Study:
- To evaluate the utility of Cfr9I PFGE for subtyping a collection of German and Dutch MRSA ST398 isolates.
- To correlate Cfr9I PFGE profiles with staphylococcal cassette chromosome mec (SCCmec) types and protein A gene (spa) types.
- To investigate the genetic diversity and clonal structure within the MRSA ST398 population from various sources.
Main Methods:
- Analysis of 105 MRSA ST398 isolates from diverse sources including pigs (healthy carriers and clinical samples), farm dust, milk, and meat.
- Subtyping using Cfr9I PFGE, spa typing, and SCCmec typing.
- Comparative analysis of PFGE profiles, spa types, and SCCmec types, including dendrogram construction for Cfr9I patterns.
Main Results:
- Cfr9I PFGE generated 35 distinct profiles among the 105 ST398 isolates, showing a strong correlation with SCCmec types.
- Two major clusters were identified by Cfr9I PFGE patterns: Cluster A predominantly contained SCCmec type V isolates, while Cluster B included SCCmec types IVa and V*.
- The findings suggest the presence of a successful, diversifying subclone within the CC398 complex, predominantly associated with SCCmec type V.
Conclusions:
- Cfr9I PFGE is a valuable and effective tool for subtyping MRSA ST398, overcoming limitations of SmaI digestion.
- The combination of Cfr9I PFGE with spa and SCCmec typing reveals significant heterogeneity within the ST398 clone.
- This approach is highly applicable for epidemiological surveillance, outbreak investigations, and traceability studies of the emerging MRSA ST398 clone.
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