High Heterogeneity within Methicillin-Resistant Staphylococcus aureus ST398 Isolates, Defined by Cfr9I Macrorestriction-Pulsed-Field Gel Electrophoresis Profiles and spa and SCC mec 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.

Livestock-Associated MRSA and Its Current Evolution

“…Although the use of Cfr9I allowed direct comparison with SmaI-PFGE profiles, differentiation of LA-MRSA CC398 was based mainly on SCCmec and spa typing. Initially, CC398 SCCmec was thought to be type III; however, later it was proven that it was mainly types IV and V, and the isolates typed as SCCmec III corresponded to type V [58,59]. To date, little variation has been seen in the prevalence of CC398 strains; however, with increased use of zinc oxide in some European countries, SCCmec type V (5C2&5) may increase.…”

“…The CC398 isolates firstly identified as carrying SCCmec type III, via the SCCmec PCR typing scheme developed by Zhang et al [57] corresponded in fact to isolates carrying SCCmec type V [58,59]. However, LA-MRSA CC398 harboring SCCmec type III was described in bovines [22] and pigs [2•] by researchers using a SCCmec PCR typing scheme developed by Kondo et al [60].…”

“…Although several enzymes were used as an alternative [117], their profiles were not comparable with those generated by SmaI in non-CC398 isolates. To overcome this problem, the use of Cfr9I, a neoschizomer (an enzyme that cuts within the same recognition sequence) of SmaI, was proposed [116], and this enzyme was used successfully for PFGE typing of CC398 isolates [59]. Although the use of Cfr9I allowed direct comparison with SmaI-PFGE profiles, differentiation of LA-MRSA CC398 was based mainly on SCCmec and spa typing.…”

Epidemiology and Genotypic Characteristics of Methicillin-Resistant Staphylococcus aureus Strains of Porcine Origin

“…Pulsed-field gel electrophoresis was conducted following a modified protocol as described previously (25). As ST398 strains are nontypeable by PFGE using the standard SmaI macrorestriction enzyme, an isoschizomer enzyme, Cfr9I, which has the same recognition site as SmaI but cuts a different location within the site, was used in this study (1,4). In brief, isolates were grown overnight at 37°C on MH agar plates (BD).…”

Livestock-associated strains of methicillin resistant Staphylococcus aureus (LA-MRSA) – the current state of knowledge

“…Szczepy należące do typu sekwencyjnego 398 określane są jako nietypowalne przy pomocy tej metody, ponieważ ich DNA genowego nie poddaje się trawieniu enzymem restrykcyjnym SmaI stosowanym w uniwersalnym protokole PFGE (44). Do trawienia DNA S. aureus ST398 należy zastosować enzym ApaI, BstZI, SacII lub Cfr9I (3,40).…”