Dissemination of a pSCFS3-like cfr-carrying plasmid in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates recovered from hospitals in Ohio

Abstract

Nineteen linezolid-resistant Staphylococcus epidermidis and two Staphylococcus aureus isolates recovered from two medical institutions in northeast Ohio and an S. aureus cfr index strain previously collected in the same facilities during the 2007 SENTRY Antimicrobial Surveillance Program were investigated for the genetic basis of oxazolidinone resistance and the location of cfr. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST). The location of cfr was determined by Southern blotting and hybridization. Plasmid sequencing was performed using the 454 Life Sciences (Roche) GS-FLX DNA platform. The two S. aureus isolates showed unique PFGE patterns but were multilocus sequence type 5 (ST5) and spa type t002, whereas the S. aureus index strain was ST239 and t037. Southern blot and hybridization experiments showed that cfr was plasmid located and that the S. epidermidis isolates, one of the S. aureus isolates, and the S. aureus index strain shared an identical cfr-carrying plasmid (39.3 kb). Sequencing results confirmed these findings. A 10-kb fragment containing cfr showed the highest identity (99.9%) to a 9.5-kb fragment of plasmid pSCFS3 from a bovine Staphylococcus lentus isolate from Germany. In addition, these 39.3-kb plasmids from human S. epidermidis and S. aureus exhibited BglII restriction profiles very similar to that observed for plasmid pSCFS3. The cfr-carrying plasmid detected in the remaining S. aureus isolate (7.9 kb) was distinct and showed the highest identity to the chromosomal cfr integrate found in the chromosomal DNA of a Proteus vulgaris isolate from a pig in China.

Fig 1

PFGE profiles of cfr-carrying S. aureus 1900 and 1609 (hospital B) compared with 737 (hospital B complex facilities) and a representative strain of the USA100 clone (NRS382). NA, not applicable.

Fig 2

(A) S1 partially digested genomic DNA of representative S. epidermidis isolates from each hospital, A and B (1243 and 1519, respectively), and S. aureus isolates 1609 and 1900 (from hospital B). “737” represents the cfr-carrying S. aureus index strain 737 (from hospital B complex facilities) (23). “λ” and “+C” represent lambda ladder PFGE marker and positive control (diluted PCR product), respectively. (B) Hybridization signals (arrows) obtained with a cfr-specific probe. Hybridization signals for one of the S. epidermidis (1243) are not visible in this particular membrane.

Fig 3

(A) Schematic representation of DNA sequences surrounding cfr detected in S. aureus 737 and 1609 and S. epidermidis 1243, as well as those from pSCFS3 (9), pJP1 (14), and pERGB (35). (B) Representation of DNA sequence surrounding cfr detected in S. aureus 1900, as well as the P. vulgaris chromosomal DNA fragment containing cfr (19). The 6- and 8-bp repeats are boxed.