The smr gene resides on a novel plasmid pSP187 identified in a Staphylococcus pasteuri isolate recovered from unpasteurized milk.

UNLABELLED: This work describes a novel plasmid pSP187 (5550 bp) carrying the small multidrug resistance determinant smr encoding resistance to quaternary ammonium compounds (QACs). pSP187 was identified in a Staphylococcus pasteuri isolate recovered from bulk milk in a dairy cattle herd in Norway. Sequence analysis revealed 6 putative ORFs in addition to the smr gene within a cassette with identical genetic organization to that found in the pSK41-like Staphylococcus aureus plasmid pTZ22. A protein homology search suggested the gene product of ORF7 to be a putative replication initiation protein, while ORF2 was predicted to encode a protein homologous to members of FtsK/SpoIIIE cell division-DNA segregation protein families. Sequence similarities to some initiator proteins of rolling circle replicons (RCR) indicated that pSP187 uses a RCR mode of replication, supported by the detection of intermediate ssDNA using S1 nuclease treatment and hybridization analysis. Interestingly, a 30-bp sequence found upstream from ORF7 showed high similarity to other dyad symmetry motifs proposed as putative double-strand origins of replication in the plasmids pGI3 (Bacillus thuringiensis), pSTK1 (Bacillus stearothermophilus), and pER1-2 (Streptococcus thermophilus). IN CONCLUSION: The novel smr-containing plasmid pSP187 is the first member of RCR group VI to be identified in a Staphylococcus sp.

Deletion of pT181-like sequence in an smr-encoding mosaic plasmid harboured by a persistent bovine Staphylococcus warneri strain.

OBJECTIVES: The aim was to study the persistence and characteristics of Staphylococcus warneri strains resistant to quaternary ammonium compounds (QACs), including sequencing and analysis of two plasmids proved to carry the smr gene. METHODS: During a 3.5 year period quarter milk samples were collected on three occasions from all lactating cows in a dairy herd. The samples were screened with regard to QAC-resistant bacteria using a selective medium. Thirty randomly selected QAC-resistant S. warneri were typed by PFGE and subjected to plasmid isolation and analysis followed by gene detection using PCR. Two smr-containing plasmids in S. warneri isolates were sequenced. RESULTS: All isolates from the initial collection of quarter milk contained smr residing on a 5.8 kb plasmid (pSW174), which contained regions with high similarities to various plasmids, including pT181, pSK108 and pPI-2. The pT181-like sequence was flanked by 148 bp direct repeats, denoted ISLE49, with high similarity to previously reported sequences of approximately 148 bp, including ISLE39 flanking the insertion sequence IS257 in methicillin-resistant Staphylococcus aureus. All isolates from subsequent collections of quarter milk harboured a smaller smr-containing plasmid (pSW49). Sequence analyses revealed pSW49 (3552 bp) to be an in-part deleted version of pSW174 (5767 bp). CONCLUSIONS: The IS-associated elements found in this study may have a wider role in the integration and excision of DNA sequences in staphylococci than previously reported. The mosaic plasmid structure based on genetic elements of various origins contributes to further knowledge on the flexibility of smr-encoding plasmids.

Widespread distribution of disinfectant resistance genes among staphylococci of bovine and caprine origin in Norway.

We demonstrate here a widespread distribution of genes mediating efflux-based resistance to quaternary ammonium compounds (QACs) in staphylococci from unpasteurized milk from 127 dairy cattle herds and 70 dairy goat herds. QAC resistance genes were identified in 21% of the cattle herds (qacA/B, smr, qacG, and qacJ) and in 10% of the goat herds (qacA/B and smr). Further examination of 42 QAC-resistant bovine and caprine isolates revealed the following genes: qacA/B (12 isolates) was present in four different species of coagulase-negative staphylococci (CoNS), smr (27 isolates) was detected in eight different CoNS species and in Staphylococcus aureus on a previously reported plasmid (pNVH99), qacG (two isolates) was detected on two plasmids (pST94-like) in Staphylococcus cohnii and Staphylococcus warneri, and qacJ (two isolates) was found in Staphylococcus hominis and Staphylococcus delphini on a plasmid (pNVH01) previously found in equine staphylococci. Isolation of indistinguishable pulsed-field gel electrophoresis (PFGE) CoNS types from tank milk and mammary quarter milk samples in a dairy cattle herd suggested that these QAC-resistant staphylococci were of intramammary origin. Indistinguishable or closely related PFGE types of bovine QAC-resistant CoNS were observed in different herds. One particular bovine S. warneri PFGE type was isolated repeatedly from samples collected during a 30-month period in a herd, showing long-term persistence. In conclusion, it seems that the widespread distribution of staphylococci carrying QAC resistance genes in Norwegian dairy cattle and goat herds is the result of both the intra- and interspecies spread of QAC resistance plasmids and the clonal spread of QAC-resistant strains.

Novel plasmid-borne gene qacJ mediates resistance to quaternary ammonium compounds in equine Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus intermedius.

We identified a novel plasmid-borne gene (designated qacJ) encoding resistance to quaternary ammonium compounds (QACs) in three staphylococcal species associated with chronic infections in four horses. qacJ was located on a 2,650-bp plasmid (designated pNVH01), a new member of the pC194 family of rolling-circle replication plasmids. The 107-amino-acid protein, QacJ, showed similarities to known proteins of the small multidrug resistance family: Smr/QacC (72.5%), QacG (82.6%), and QacH (73.4%). The benzalkonium chloride MIC for a qacJ-containing recombinant was higher than those for otherwise isogenic recombinants expressing Smr, QacG, or QacH. Molecular epidemiological analyses by pulsed-field gel electrophoresis suggested both the clonal spread of a qacJ-harboring Staphylococcus aureus strain and the horizontal transfer of pNVH01 within and between different equine staphylococcal species. The presence of pNVH01 of identical nucleotide sequence in different staphylococcal species suggests that recent transfer has occurred. In three of the horses, a skin preparation containing cetyltrimethylammonium bromide had been used extensively for several years; this might explain the selection of staphylococci harboring the novel QAC resistance gene.