Identification of the novel spectinomycin resistance gene spw in methicillin-resistant and methicillin-susceptible Staphylococcus aureus of human and animal origin

Sir, Spectinomycin resistance in staphylococci is mostly mediated by spectinomycin 9-O-adenyltransferase encoded by the spc gene. This gene is located together with the macrolide–lincosamide– streptogramin B resistance gene erm(A) on transposon Tn554. The analysis of methicillin-resistant Staphylococcus aureus (MRSA) CC398 from cases of bovine mastitis showed that all spectinomycin-resistant isolates carried the gene spc along with erm(A), whereas MRSA CC398 isolates from turkey meat and turkey meat products occasionally exhibited spectinomycin resistance, but lacked the spc gene. This observation pointed towards the existence of other as yet unknown spectinomycin resistance genes in staphylococci. Most recently, the analysis of MRSA ST398 and methicillin-susceptible S. aureus (MSSA) ST9 isolates of human origin from Spain, but also MRSA ST9 isolates from swine in China, revealed the presence of multiresistance gene clusters that were most likely of enterococcal origin. – 7 So far, two different types of these multiresistance gene clusters have been identified, both of which carry the resistance genes aadE (streptomycin resistance), lnu(B) (lincosamide resistance) and lsa(E) (pleuromutilin– lincosamide–streptogramin A resistance). A closer look at these clusters revealed the presence of a reading frame for a putative spectinomycin resistance gene between aadE and lsa(E). This reading frame is 810 bp in size, has an unusual start codon (TTG) and codes for an adenyltransferase protein of 269 amino acids. Database searches identified identical or closely related proteins in Enterococcus faecium (e.g. GenBank accession number EFF20873), Enterococcus faecalis (e.g. GenBank accession number EEU82264) and Lactobacillus johnsonii (e.g. GenBank accession number EGP12870) (Figure 1). The corresponding proteins were referred to as streptomycin 3′′-adenylyltransferase, spectinomycin 9-O-adenylyltransferase, putative toxin-antitoxin system or a hypothetical protein in the respective database entries. Moreover, three database entries (GenBank accession numbers AAL05551, AFM38045 and AFU35063) reported a protein that was 27 amino acids shorter. This was due to the annotation of a wrong start codon (ATG) upstream of which no ribosome binding site was found. Analysis of the corresponding nucleotide sequences (GenBank accession numbers AF408195, JQ861959 and JX560992, respectively) confirmed the presence of the larger correct reading frame of 810 bp. To determine whether or not this protein confers resistance to streptomycin or spectinomycin, we decided to clone the gene and express it in an S. aureus host. For this, the recently developed PCR5 assay (forward: 5′-TTGGATTGCAGCATTATTGG-3′, reverse: 5′-ATTTGGTCGAAGCCTT GTTG-3′; annealing temperature 568C) was used to generate an amplicon of 1985 bp from the cloned 17.5 kb segment of plasmid pV7037 (GenBank accession number JX560992) from the porcine MRSA ST9 isolate SA7037. This amplicon included the entire reading frame for the putative resistance gene and 239 bp in the upstream and 936 bp in the downstream parts. The amplicon was initially cloned into pCR-Blunt II-TOPO (Life Technologies GmbH, Darmstadt, Germany), and the recombinant vector was transformed into Escherichia coli recipient strain TOP10. The amplicon was then cut off from this vector by EcoRI digestion and inserted into the single EcoRI site of the E. coli–S. aureus shuttle vector pLI50. This recombinant shuttle vector was first introduced by electrotransformation into E. coli TOP10 and subsequently into S. aureus RN4220. Antimicrobial susceptibility testing was performed by broth macrodilution for spectinomycin and streptomycin according to CLSI document M31-A3 with S. aureus ATCC 29213 as the quality control strain. The MICs of streptomycin were 4 mg/L for S. aureus RN4220, S. aureus RN4220 carrying pLI50 and S. aureus RN4220 carrying the recombinant pLI50, suggesting that the cloned gene does not confer streptomycin resistance. In contrast, the MICs of spectinomycin were 32 mg/L for S. aureus RN4220 and S. aureus RN4220 carrying pLI50, but were distinctly higher at 4096 mg/L for S. aureus RN4220 carrying the recombinant pLI50 with the cloned gene. This observation confirmed that the gene in question was a spectinomycin resistance gene, which was then designated spw. Besides the occurrence of spw in E. faecium, E. faecalis and L. johnsonii, a gene was detected in Streptococcus suis that encoded a protein of 262 amino acids (GenBank accession number AER19630) that showed 93.9% amino acid identity to Spw. In contrast, the Spw protein exhibited only 64.7% identity to the Tn554-associated Spc protein (Figure 1). To see whether spw was present in other staphylococci, a PCR assay (forward: 5′-CGGCAGTAATGGGTGGTTTA-3′, reverse: 5′-CAGCCACC TCAGATTCCATT-3′; annealing temperature 548C) was developed