Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus

Abstract

Resistance to quaternary ammonium compounds (QAC) in staphylococci is common in hospital environments and has been described in the food industry. Little is known about staphylococcal QAC resistance associated with animal disease, although such disinfectants are widely used in veterinary medicine. In order to investigate the occurrence of QAC resistance in staphylococci isolated from QAC-exposed animals, 32 penicillin- and tetracycline-resistant and 23 penicillin- and tetracycline-susceptible Staphylococcus aureus isolates collected from milk from cows with mastitis during a 4-year period were selected for QAC susceptibility studies and genetic characterization. The isolates originated from four different herds that used a common pasture with a joint milking parlor in the summer. During the pasture season, a teat cream containing the QAC cetyltrimethylammonium bromide had been used daily for more than 10 years for mastitis control. Three of the penicillin- and tetracycline-resistant isolates, which were recovered from three different cows during a 20-month period, were resistant to QAC. Plasmid analysis, PCR, and DNA sequencing revealed a novel plasmid of 2,239 bp containing the smr gene. The plasmid, designated pNVH99, has similarities to small, smr-containing staphylococcal plasmids previously found in human and food isolates. pNVH99 is a new member of the pC194 family of rolling-circle replication plasmids. The three QAC-resistant isolates, as well as 28 of the 29 remaining penicillin- and tetracycline-resistant isolates, were indistinguishable by pulsed-field gel electrophoresis. The study indicates that the occurrence and spread of QAC-resistant S. aureus among dairy cows may be a problem that needs further investigation.

FIG. 1

Nucleotide sequence of pNVH99 (GenBank accession no. AJ296103). Putative promoter sequences (−10 and −35) and ribosome binding sites (RBS) are indicated (underlined) for both smr and repNVH99. The DNA sequence resembling the pC194 plasmid family replication nick site is indicated by a vertical arrow and underlined. Direct repeat sequences previously described (14), viz., DR1_C and DR2_C, and the single-strand origin sequence SSO_A (formerly designated palA) (1) are boxed. Upstream from the smr gene is a DNA-sequence of 18 bp (nt 1290 to 1308, italic letters) directly repeated downstream (DR₁₈₊₁₈ in Fig. 2) and overlapping DR1_C by 10 bp (DR_10, bold letters). DR2_C is truncated by a 96-bp sequence (italicized), followed by a 299-bp sequence showing a close relationship to pSK89 and other small staphylococcal plasmids (4, 15).

FIG. 2

Genetic organization of pNVH99 in relation to other staphylococcal plasmids that contain a proposed smr gene cassette, extending from the putative replication nick site (indicated by a vertical arrow) to the end of the direct repeat region downstream from the smr gene. The directions of the marked ORFs are denoted by horizontal arrows within the boxes. Direct repeat sequences previously described, DR1_C, ′DR1_C, DR2_C, DR1_D, DR2_D (14), and the 10-bp direct repeat sequence within DR1_C in pNVH99, DR₁₀, are denoted by solid boxes. The 18-bp direct repeat sequence DR₁₈₊₁₈ is indicated by a bracket. The single-strand origin sequence SSO_A (formerly designated palA) (1) within some of the DR sequences is shown as a white box.