Four novel plasmids from Staphylococcus hyicus and CoNS that carry a variant of the spectinomycin resistance gene spd.

Sir, During recent years, Staphylococcus aureus isolates of animal origin have been identified that have exhibited high MICs of spectinomycin (≥512 mg/L). – 3 The molecular analysis for resistance genes in these isolates identified frequently the Tn554-borne spectinomycin resistance gene spc. For more than 25 years, spc was the only spectinomycin resistance gene known to occur in staphylococci. In 2013, a second spectinomycin resistance gene, spw, was identified in staphylococci. The gene spw was part of variants of a presumably enterococcal multiresistance gene cluster, which was identified on a plasmid or in the chromosomal DNA of MRSA and MSSA of human and animal origin in Spain, Germany, mainland China and Hong Kong. – 8 Most recently, a third spectinomycin resistance gene, spd, was detected first in MRSA ST398 from various human and animal sources in Belgium and soon thereafter in MRSA ST398 and MSSA ST433 from poultry and pig samples in the Netherlands, Germany and Austria. In contrast to the genes spc and spw, the gene spd was located exclusively on small plasmids of ,5 kb in size. So far, two different types of spd-carrying plasmids have been identified. The aim of this study was to investigate staphylococci other than S. aureus that exhibit high spectinomycin MICs for the presence of the spd gene and to investigate plasmids that carry the spd gene for their structure and organization. In the BfT-GermVet study, 179/248 staphylococcal isolates from diseased pigs, dogs and cats belonged to species other than S. aureus. Among them, seven isolates showed high spectinomycin MICs (≥512 mg/L). These comprised two Staphylococcus hyicus from urinary/genital tract infections in pigs, single S. hyicus, Staphylococcus chromogenes and Staphylococcus equorum from skin infections in pigs, and two Staphylococcus simulans from respiratory tract infections in cats (Table S1, available as Supplementary data at JAC Online). Species assignment was confirmed by biochemical profiling using ID32Staph (bioMérieux, Nürtingen, Germany) and 16S rDNA sequencing as previously described. The isolates were tested for the presence and the genetic environment of the three known staphylococcal spectinomycin resistance genes and other resistance genes by previously described PCRs. –10 PCR analyses identified the spc gene in the two S. simulans, in a S. hyicus and in a S. chromogenes isolate. In all cases, except the S. chromogenes, the spc gene was linked to the macrolide/lincosamide/streptogramin B resistance gene erm(A) in a chromosomally located Tn554. In the S. chromogenes, which only displayed spectinomycin resistance, the spc gene was not plasmid-borne based on the results of Southern blot analyses and repeatedly negative transformation assays performed as previously described. A single S. hyicus isolate was positive for the spw gene and harboured a part of the multiresistance gene cluster as confirmed by overlapping PCR assays. This part of the cluster also comprised the resistance genes erm(B), aadE, lsa(E) and lnu(B) (Table S1). The spd gene was found in two S. hyicus isolates and single S. equorum and S. chromogenes isolates, of which one S. hyicus and the S. chromogenes were also positive for the gene spc. Plasmid profiling followed by Southern blotting and electrotransformation revealed the presence of small spd-carrying plasmids in all four isolates. These spd-carrying plasmids, designated pSWS118, pSWS961, pSWS1211 and pSWS2482, were completely sequenced by primer walking on both strands starting with primers located in the spd gene. They had sizes between 3780 and 4229 bp (Figure 1) and showed differences in their pre/mob and rep genes. The spd genes of all four plasmids were identical but had only one of two 12 bp tandem repeats at the 3′-terminus in comparison with spd from the S. aureus plasmids pDJ91S and pSWS2889. This 12 bp deletion had no impact on the high spectinomycin MIC conferred by these plasmids. The Rep proteins of the four novel plasmids varied in size between 311 and 314 amino acids and the corresponding Pre/Mob proteins between 355 and 417 amino acids. A comparison between these four Rep and Pre/Mob proteins showed identities of 82.6%–99.7% and 70.4%–97.5%, respectively. A comparison with the Rep and Pre/Mob proteins of the previously described spd-carrying plasmids pDJ91S and pSWS2889 revealed identities of 79.1%–87.6% and 68.3%–87.6%, respectively (Figure S1a and b). Database comparisons of each of the four novel Rep proteins revealed highest identities of 89.5%–91.8% to the same or similarly sized Rep proteins of S. aureus, Staphylococcus lugdunensis or Staphylococcus epidermidis (GenBank accession numbers EZW76123, AAB18269 and ADA80324). The Pre/Mob proteins of pSWS118 (409 amino acids), pSWS961 (410 amino acids) and pSWS1211 (355 amino acids) showed 96.5% –100% identity to the corresponding parts of a 403 amino acid Pre/Mob protein