Tn560, a Novel Tn554 Family Transposon from Porcine Methicillin-Resistant Staphylococcus aureus ST398, Carries a Multiresistance Gene Cluster Comprising a Novel spc Gene Variant and the Genes lsa(E) and lnu(B)

LETTER

Combined resistance to pleuromutilins, lincosamides, and streptogramin A antibiotics (PLS_A) in staphylococci is often conferred by ABC-F genes, including lsa(E) (1, 2). The lsa(E) gene is commonly part of multiresistance gene clusters, in which the region comprising the streptomycin resistance gene aadE, the spectinomycin resistance gene spw, the lincosamide resistance gene lnu(B), and the PLS_A resistance gene lsa(E) is conserved (3–7). During the analysis of the whole-genome sequence of the porcine methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) strain GDC6P096P from China, obtained by hybrid assembly of Illumina short-read and MinION long-read data, we identified a novel 12,518-bp Tn554-like transposon harboring part of the lsa(E) gene cluster. This transposon, designated Tn560, differed from all previously reported transposons of the Tn554 family in its structure and organization (8–10).

Tn560 was inserted into the chromosomal radC gene. It displayed two 6-bp sequences, 5′-GATGTA-3′ at the left-end junction and 5′-TGATAA-3′ at the right-end junction. While the transposase genes tnpA, tnpB, and tnpC of Tn560 were identical to those of Tn554 and the lnu(B) and lsa(E) genes of Tn560 shared 99.88% and 99.93% nucleotide sequence identities with the previously reported prototype genes (11, 12), the Tn560-borne “spw” gene showed only 78.38% nucleotide sequence identity with the original spw gene (13). However, this gene showed 93.74% nucleotide sequence identity with the Tn554-borne spc gene. The deduced amino acid sequence revealed that the N-terminal 200 amino acids (aa) were identical to those of Spc, whereas the C-terminal 68 aa were identical to those of Spw, suggesting that the novel spc gene variant, designated spc_V, resulted from the in-frame in vivo recombination of the two related genes spc and spw. A similar finding has been reported for the macrolide-lincosamide-streptogramin B resistance gene erm(33), which resulted from the in vivo recombination of the genes erm(A) and erm(C) (14).

Tn560 most likely originated from recombination of a Tn554 transposon with the spw-lsa(E)-lnu(B)-carrying, 74,612-bp multiresistance plasmid pE508-2 from porcine Enterococcus faecalis (GenBank accession number MK784778.1). A 7,588-bp segment of Tn560, which comprised part of the spw gene and reached until the region downstream of lnu(B), exhibited 99.95% nucleotide sequence identity with the corresponding region in plasmid pE508-2 (Fig. 1a). Two potential recombination sites were identified (Fig. 1b). Recombination site A is located within the spc gene of Tn554 and includes a stretch of 14 bp of 100% identity between the spc and spw genes. Upstream of recombination site A, the sequence of Tn560 corresponds to spc of Tn554, downstream of it to spw of pE508-2. Recombination site B was located downstream of the gene lnu(B) within an open reading frame (ORF) for a hypothetical protein and comprised a stretch of 35 bp, with 100% and 85.71% identities to the corresponding regions of pE508-2 and Tn554, respectively. Upstream of recombination site B, the sequence of Tn560 corresponded to the sequence of pE508-2, downstream of it to Tn554.

FIG 1.

(a) Comparison of Tn560, Tn554, and a part of plasmid pE508-2. The areas of >99% identity are shaded in gray. Antimicrobial resistance genes are shown in red, transposase genes are in blue, and hypothetical proteins are in gray. The positions of the two recombination sites A and B are indicated by circles. (b) Detailed structures of the two recombination sites A and B. Vertical bars indicate identical bases with respect to the Tn560 sequence. The positions refer to the corresponding database entries (GenBank accession numbers are in parentheses). The boxed areas indicate the areas where the crossover most likely took place.

The detection of the circular Tn560 by PCR (primers Tn560-fw [5′-CGGCTTATTCTCCACTTCT-3′] and Tn560-rev [5′-CCTGGATGCCAATTCCATA-3′], with an annealing temperature of 50°C and an amplicon size of 460 bp) indicated that this transposon is functionally active in this strain (Fig. 2). Sequence analysis of the amplicon showed that it comprised 175 bp from the left terminus and 285 bp from the right terminus of Tn560.

FIG 2.

Detection of the 460-bp amplicon obtained from the circular intermediate of Tn560 that has been formed after the excision of Tn560 from the chromosomal DNA. Lanes M contain the GeneRuler 100-bp Plus DNA ladder (Thermo Fisher Scientific, Dreieich, Germany), lane + contains the PCR amplicon obtained from S. aureus GDC6P096P, and lane − is the negative control, i.e., demineralized water. The sizes of selected fragments of the DNA ladder are indicated in base pairs.

Data availability.

The data have been deposited in GenBank under accession number MW832219.