Identification and characterization of nine novel types of small staphylococcal plasmids carrying the lincosamide nucleotidyltransferase gene lnu(A)

Objectives: To date, very little is known about lincosamide resistance plasmids in staphylococci with only a single Inu(A)-carrying staphylococcal plasmid having been sequenced completely. The aim of this study was to characterize small Inu(A)-carrying plasmids isolated from bovine coagulase-negative staphylococci (CoNS). Methods: Nine CoNS isolates with MICs of the lincosamide pirlimycin of 1-4 mg/L were tested for the presence of the Inu(A) gene. Its location was determined by Southern-blot hybridization. The Inu(A)carrying plasmids were transformed into Staphylococcus aureus RN4220 and compared by restriction mapping and subsequent sequencing. Selected plasmids were investigated for their copy number and their lnu(A) gene expression via FIT real-time PCR. Results: The lnu(A) gene was detected on plasmids in all isolates. Sequence analysis revealed that these plasmids carried a rep gene, coding for the replication initiator protein, and the resistance gene Inu(A), coding for a lincosamide nucleotidyltransferase. While the Lnu(A) proteins were closely related (91.3-100% amino acid identity), the Rep proteins differed distinctly (27.4-100% amino acid identity), but showed similarity (81.4-98.5%) to Rep proteins of other small staphylococcal resistance plasmids. Sequence features of rolling-circle plasmids, such as the single-strand (ssoA) and double-strand (dso) origins of replication, were identified. For two plasmid types detected, the lincosamide resistance level varied with regard to the amounts of Inu(A) transcripts detected. Conclusions: Structurally different Inu(A)-carrying plasmids were detected in various CoNS species. The detection of the same lnu(A) gene in different plasmid backbones suggested the exchange of the gene via interplasmid recombinational events.