Novel Structure of Enterococcus faecium-Originated ermB-Positive Tn1546-Like Element in Staphylococcus aureus

Abstract

We determined the resistance determinants in 274 erythromycin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) isolates during a 13-year period, 2000 to 2012. The resistance phenotypes, inducible macrolide-lincosamide-streptogramin (iMLS), constitutive MLS (cMLS), and macrolide-streptogramin (MS) resistance phenotypes, were examined by a double-disk diffusion D test. The ermB gene was more frequent (35%; 97/274) than ermC (27%; 75/274) or ermA (21%; 58/274). All 97 ermB-positive isolates harbored Tn551 and IS1216V The majority (89/97) of ermB-positive isolates displayed the cMLS phenotype and carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA). The remaining 8 ermB-carrying isolates, belonging to ST7 (n = 4), ST5 (n = 3), and ST59 (n = 1), were sasK intact and did not carry MES-like structures. Unlike a MES-like structure that was located on the chromosome, the ermB elements on sasK-intact isolates were located on plasmids by S1 nuclease pulsed-field gel electrophoresis (PFGE) analysis and conjugation tests. Sequence data for the ermB-containing region (14,566 bp) from ST59 NTUH_3874 revealed that the best match was a Tn1546-like element in plasmid pMCCL2 DNA (GenBank accession number AP009486) of Macrococcus caseolyticus Tn1546 is recognized as an enterococcal transposon and was known from the vancomycin resistance gene cluster in vancomycin-resistant Enterococcus (VRE). So far, acquisitions of Tn1546 in S. aureus have occurred in clonal complex 5 (CC5) MRSA, but not in MSSA. This is the first report that MSSA harbors an Enterococcus faecium-originated ermB-positive Tn1546-like element located on a plasmid.

FIG 1

Distribution of erythromycin resistance genes among MSSA strains isolated during a 13-year period. Of the 2,240 MSSA strains isolated over a 13-year period, 2000 to 2012, 274 were resistant to erythromycin. “Others” includes one ermT strain and strains in which none of the genes shown were detected.

FIG 2

PCR mapping of the MES_PM1 and MES_PM9 structures in isolates carrying ermB, aph(3′)-IIIa, aadE, and cat and carrying ermB, aph(3′)-IIIa, and aadE (Table 2), respectively. MES_PM1 and MES_PM9 are shaded in gray. (A) There are six pairs of primers for the MES_PM1 structure: 11F and ermBR, ermB and aadE, 1F and 35R, 35F and LA-R2, LA-F1 and 23R, and 23F and 48R3. The PCR product sizes are approximately 6 kb, 4 kb, 1.2 kb, 1.5 kb, 1.5 kb, and 1.6 kb, respectively. (B) There are four pairs of primers for the MES_PM9 structure: 11F and ermBR, ermB and aadE, 1F and 23R, and 23F and 48R3. The PCR product sizes are approximately 6 kb, 4 kb, 1.1 kb, and 1.6 kb, respectively. The primers are the same as those in the report of Hung et al. (12).

FIG 3

S1 nuclease PFGE patterns of MSSA isolates and Southern blot hybridization with ermB. (A) The PM1 strain harboring a 26-kb plasmid was used as a positive control. Strain RN2677, lacking the plasmid, was used as a negative control. The plasmid DNAs of three isolates, ST5 NTUH_9448, ST7 NTUH_1027, and ST59 NTUH_3874, and three transconjugants from the above-mentioned isolates, named RN-NTUH_9448, RN-NTUH_1027, and RN-NTUH_3874, were digested with S1 nuclease and separated from chromosomal DNA by PFGE. (B) The PM1 strain carrying the ermB gene on the chromosome was used as a positive control. RN2677, containing no ermB, was used as a negative control. DNA was hybridized with the DIG-labeled ermB-specific probe and amplified by PCR using primers ermB-f and ermB-r. The sizes of the plasmids are between 23.1 kb and 48.5 kb.

FIG 4

Structure of the ermB resistance element in MSSA NTUH_3874. Shown is a comparison of the structures of the ermB resistance element and flanking regions in ST59 NTUH_3874 (accession number LC102479), plasmid pMCCL2 of M. caseolyticus (accession number AP009486), and Tn1546 of E. faecium (accession number M97297). The arrows indicate the lengths and transcriptional orientations of the tnp (transposase), tnpR (resolvase), ermB (combined resistance to MLS), aacA-aphD (resistance to kanamycin and gentamicin), leader peptide, thyX (thymidylate synthase), and drf-like (dihydrofolate reductase) genes. The 38-bp inverted repeats upstream and downstream of the NTUH_3874 14,566-bp element are also shown.