High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types

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.

FIG. 1.

Cfr9I PFGE analysis of Staphylococcus aureus ST398 isolates. Lane λ, lambda ladder PFGE marker (New England Biolabs, Frankfurt am Main, Germany); lane A, Cfr9I profile of strain ATCC 29213; lane N, Cfr9I profile of strain NCTC 8325 included as quality control; lanes S1 to S4, SmaI profiles of out-group isolates; lanes C1 to C35, Cfr9I profiles generated from ST398 isolates.

FIG. 2.

Dendrogram showing the relatedness between Cfr9I macrorestriction fragment profiles generated from the S. aureus ST398 isolates, the out-group isolates, and the control (NCTC 8325 and ATCC 29213) strains. At a Jaccard's coefficient of similarity (J) of 0.63, two clusters (labeled A and B, the latter with three subclusters) were detected grouping the ST398 isolates. The SCCmec and spa types and origin associated with each Cfr9I profile are also indicated. −, no SCCmec (MSSA isolates); CP, carrier pig at abattoir; CS, clinical sample from pig; D, dust; FP, fecal sample from pig; M, milk; MC, meat from chicken; MP, meat from pig; MT, meat from turkey.