Identification of the novel spectinomycin resistance gene spd in a different plasmid background among methicillin-resistant Staphylococcus aureus CC398 and methicillin-susceptible S. aureus ST433.

Sir, In a recent study, a novel spectinomycin resistance gene, designated spd, was identified in eight methicillin-resistant Staphylococcus aureus (MRSA) isolates of sequence type (ST) 398 from chicken, horse, rat, cattle, pig and human origin in Belgium. Little is currently known about how widespread this gene is among MRSA and methicillin-susceptible S. aureus (MSSA) from different human and animal sources outside Belgium. During recent years, several survey studies have been conducted in Germany, Austria and the Netherlands in which livestock-associated MRSA were investigated for their resistance phenotypes and genotypes. These studies included: the analysis of MRSA CC398 and CC9 from broiler chickens at slaughter and abattoir workers in the Netherlands, and from broiler farms, humans and their households in the Netherlands; the analysis of MRSA isolates from food and food products of poultry origin in Germany and Austria; the analysis of MRSA isolates from diseased pigs in the GERM-Vet program; and the analysis of coagulase-positive and coagulase-variable staphylococci from diseased pigs, dogs and cats in the BfT-GermVet study. In all these studies, 31 isolates were identified that showed high MICs (≥512 mg/L) of spectinomycin, but were negative for the Tn554-associated spectinomycin resistance gene spc (Table S1, available as Supplementary data at JAC Online). Subsequent analysis of these isolates also revealed that all but one were negative for the spectinomycin resistance gene spw (Table S1), which has recently been described to be part of a multiresistance gene cluster present on plasmids or in the chromosomal DNA of MRSA ST398 and MRSA/MSSA ST9. The origin and characteristics of the 31 isolates, which comprise 30 MRSA CC398 and a single MSSA ST433, are shown in Table 1. Spectinomycin MIC testing of the 31 isolates was repeated by broth macrodilution according to CLSI document VET01-A4, and all isolates showed MICs of ≥8192 mg/L. The spd-specific PCR assay described by Jamrozy et al. was applied, and all 31 isolates yielded a PCR product of the expected size. Two randomly selected PCR amplicons were sequenced for confirmatory reasons and their sequence corresponded exactly to that of spd. To determine whether spd was located on plasmids, plasmid profiles were prepared by alkaline lysis following lysostaphin treatment, subjected to gel electrophoresis and transferred to a nylon membrane by Southern blotting. Hybridization of the membranes was carried out with the aforementioned 317 bp internal spd PCR amplicon as an spd-specific gene probe. This probe was labelled using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics GmbH, Mannheim, Germany). Detection followed the recommendations of the manufacturer. Again, all 31 isolates revealed the presence of a small 3.9 kb plasmid that hybridized with the spd probe (data not shown). Electrotransformation into S. aureus RN4220, with subsequent selection of the transformants on media containing 512 mg/L spectinomycin, was conducted. For this, the plasmid present in MRSA isolate 2889 obtained from the nasal swab of an abattoir worker collected in 2009 in the Netherlands was used. The electrotransformants harboured the expected small plasmid and susceptibility testing confirmed that this plasmid confers only spectinomycin resistance. The plasmid, designated pSWS2889, was completely sequenced on both strands by primer walking, starting with the previously described spd-specific primers: spd_F, 5′-CATGAAAATGAAAATTGGTCTTATCC-3′; and spd_R, 5′-CCT GTTTCATAAGTTACGATC-3′. Sequence analysis of pSWS2889 revealed a total size of 3898 bp (European Nucleotide Archive accession no. HG803547), which is 30 bp smaller than plasmid pDJ91S. Similarly to pDJ91S, pSWS2889 also harboured three reading frames: for a plasmid replication protein of 314 amino acids; the spectinomycin resistance protein Spd of 257 amino acids; and a plasmid recombination/mobilization protein of 405 amino acids. While the spd gene and the pre/mob gene were indistinguishable from the respective genes of pDJ91S, considerable differences were noticed between the two rep genes. A comparison based on sequence alignments (DNAMAN software, Lynnon Corporation, Quebec, Canada) between the 969 bp rep gene of pDJ91S and the 945 bp rep gene of pSWS2889 showed an overall identity of 82.6%. The corresponding proteins exhibited an overall identity of 81.1%. The smaller sizes of the pSWS2889associated rep gene and Rep protein were due to the loss of one