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.

INTRODUCTION

Staphylococcus aureus is an important pathogen in both community and hospital settings. Although methicillin-resistant S. aureus (MRSA) has accounted for the majority of recent nosocomial S. aureus infections in our hospital (National Taiwan University Hospital, Taipei, Taiwan [NTUH]) (71 to 78% in 2000 to 2006 and 55 to 68% in 2007 to 2009), methicillin-susceptible S. aureus (MSSA) accounted for nearly 40% of all nosocomial S. aureus infections from 2007 to 2009 (1), indicating that MSSA infections also pose a serious threat to human health.

Erythromycin, a macrolide antibiotic, acts by binding to the peptidyl transferase active site of the 50S ribosomal subunit, thereby inhibiting protein synthesis (2). Erythromycin was approved in Taiwan in 1968 (3). During the long period in which erythromycin has been used in Taiwan, S. aureus strains have become highly resistant to the drug (4).

Two major mechanisms have been identified that lead to macrolide-lincosamide-streptogramin (MLS) resistance or macrolide-streptogramin (MS) resistance in S. aureus. The MLS resistance phenotype is due to a methylase (encoded by erm) that methylates 23S rRNA, expressing it either constitutively (cMLS) or inducibly (iMLS) (5). The MS resistance phenotype functions via an efflux pump encoded by msrA or msrB (5). In general, the iMLS phenotype is predominant in MSSA, and cMLS is predominant in MRSA. The most prevalent erythromycin resistance genes in S. aureus were the ermA, ermB, ermC, ermT, and msrA and msrB genes. ermA is the most common determinant in cMLS and iMLS MRSA (4, 6, 7). ermC is the most common in iMLS MSSA from Taiwan (4), China, and Europe (8, 9). In Japan, both ermA and ermC are common in iMLS MSSA (10). In China, both ermB and ermC are common in cMLS MSSA (8).

A 29-kb plasmid, pI258, carrying ermB was first recovered from S. aureus in Japan in 1963 and is harbored on the transposon Tn551 (11). However, ermB was rarely found in S. aureus until recently. A study in China showed that among cMLS MSSA, 43% carried ermB and 42% carried ermC (8). Another report showed that in sequence type 59 (ST59) MRSA, ermB is harbored on mobile element structures (MES) on the chromosome flanked by IS1216V. A MES is a composite transposon consisting of Tn551—a Tn5405-like transposon carrying aph(3′)-IIIa, aadE, and cat (12)—carrying ermB. The ermB gene is commonly found in many species (13). ermA is often harbored on the transposon Tn554 and has been described as being able to insert itself into different regions of the S. aureus chromosome (14), including staphylococcal cassette chromosome mec (SCCmec) type II and SCCmec type III (15). The ermC gene in staphylococci is typically found on small 2.5- to 5-kb plasmids (14, 16). The ermT gene, flanked by insertion sequence 1216V (IS1216V), is present in Streptococcus gallolyticus subsp. pasteurianus (17). Recently, the ermT gene was found in ST398 MRSA and MSSA (18, 19). The msrA and msrB genes are present in clinical S. aureus isolates (20) and Enterococcus faecalis isolates (21).

In this study, we found that the ermB gene was the most prevalent (97/274) determinant encoding erythromycin resistance in MSSA. Of 97 ermB-positive isolates, 89 carried the MES element, which has been previously detected in MRSA. The remaining 8 isolates harbored a Tn1546-like element and contained Tn551 and IS1216V. This is the first report that MSSA carries a Tn1546-like structure.

MATERIALS AND METHODS

Bacterial isolates.

A total of 2,240 nonrepetitive MSSA isolates were recovered from blood cultures during the period from 2000 to 2012 at the Bacteriology Laboratory, NTUH, a 2,500-bed teaching hospital in northern Taiwan. Among the 2,240 MSSA isolates, 274 (12.2%) were resistant to erythromycin.

Determination of MIC by agar dilution.

The MICs of erythromycin, clindamycin, gentamicin, kanamycin, chloramphenicol, and streptomycin (Sigma-Aldrich) were determined using the agar dilution method with Mueller-Hinton agar (MHA) as described by the CLSI (22). S. aureus ATCC 29213 was used as the reference strain.

Identification of the iMLS phenotype using the D test.

All of the erythromycin-resistant strains were further tested for clindamycin susceptibility to determine whether they have the iMLS phenotype using the double-disc diffusion assay (D test) according to CLSI guidelines. The presence of a D-shaped zone was reported as positive for the iMLS phenotype.

Detection of resistance genes in erythromycin-resistant isolates by PCR.

Detection of erythromycin resistance genes was performed by PCR using primer pairs specific for ermA, ermB, ermC, ermT, msrA, and msrB (23).

PFGE.

The genetic relatedness of the 97 ermB-carrying MSSA isolates was determined by pulsed-field gel electrophoresis (PFGE). The DNA in gel plugs was digested with SmaI (New England BioLabs, Ipswich, MA) and then separated in a CHEF-DR III apparatus (Bio-Rad Laboratories). The plugs were applied to wells of 0.8% (wt/vol) agarose gels (Bio-Rad). PFGE was carried out at 200 V and 12°C for 20 h, with a pulse angle of 120° and pulse times ranging from 5 to 60 s. The pulsotypes were analyzed using BioNumerics software version 4.0 (Applied Maths, Sint-Martens-Latem, Belgium).

S1 nuclease PFGE.

S1 nuclease PFGE was used to detect the possible presence of plasmids (24). Agarose gel plugs of total DNA were prepared for PFGE as described previously (25). Gel slices were incubated at 37°C for 45 min with 1 to 10 units of Aspergillus oryzae S1 nuclease (Invitrogen). The reaction was stopped by transferring the slices to ES buffer (0.5 mM EDTA, 1% [wt/vol] Sarkosyl, pH 9.0) at 4°C for 10 min. The plugs were applied to wells of 1.2% (wt/vol) agarose gels (Bio-Rad) and run in a CHEF-DR III apparatus (Bio-Rad Laboratories) with a pulse angle of 120° and pulse times of 45 s for 14 h and 25 s for 6 h at 200 V in 0.5× Tris-borate-EDTA (TBE) (24). Each band was considered a unit length linear plasmid.

Southern blot hybridization.

DNA was digested with restriction enzymes (New England BioLabs, Ipswich, MA) and then electrophoresed, depurinated, denatured, neutralized, and transferred to a Hybond-N⁺ nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) using a vacuum blotting system (VacuGene XL; Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Hybridization with the PCR DIG (digoxigenin) probe synthesis kit (Roche Diagnostics GmbH, Penzberg, Germany) was performed using the Hybridization Incubator model 1000 (Robbins Scientific). Detection with anti-digoxigenin-allophycocyanin (AP) (Roche Diagnostics GmbH, Penzberg, Germany) and the DIG luminescent detection kit (Roche Diagnostics GmbH, Penzberg, Germany) was performed using the LAS-4000 imaging system (Fuji Film Life Science, Japan).

Sequencing of ermB and flanking regions.

To determine the sequence of the entire ermB gene and its flanking regions in 8 sasK-intact isolates, a long and accurate PCR (LA-PCR) in vitro cloning kit (TaKaRa Shuzo Co. Ltd., Japan) and inverse PCR were used, with subsequent sequencing using the Applied Biosystems 3730xl DNA sequencer.

spa typing and MLST.

The spa repeat region was amplified, purified, and sequenced using primers Sa0122-F and Sa0122-R (26). Multilocus sequence typing (MLST) was carried out to determine the ST of each ermB-carrying MSSA isolate. STs were assigned using the S. aureus MLST database (http://www.mlst.net) (27).

Conjugation test.

Strain RN2677 was used as the recipient in the conjugation test, and mating was carried out on LB agar (28). After 24 h of incubation, the mixed cultures were taken from the plates, suspended in Mueller-Hinton broth, and incubated at 37°C with shaking at 240 rpm. After 4 h of incubation, the cells were collected by centrifugation and plated on MHA containing erythromycin (0.5 μg/ml) and rifampin (80 μg/ml) and then incubated at 37°C for 24 h. Confirmation that conjugation had taken place in RN2677 was determined by testing for the presence of the ermB gene by PCR. The transconjugants were also checked by spa typing (spa type t211).

Accession number.

The nucleotide sequences of the 14,566-bp element containing the ermB gene characterized in this study were submitted to GenBank and assigned accession number LC102479.

RESULTS

Resistance phenotypes and erythromycin resistance determinants.

Of 274 erythromycin-resistant MSSA isolates recovered from 2000 to 2012 (Fig. 1), 130 (47%) displayed the cMLS phenotype, 98 (36%) exhibited the iMLS phenotype, and 46 (17%) exhibited the MS resistance phenotype (Table 1). Of the 130 cMLS isolates, 97 (75%) contained ermB, 26 (20%) contained ermC, and 10 (8%) contained ermA. Of 98 iMLS isolates, 49 (50%) contained ermC and 48 (49%) contained ermA. Of the 46 isolates with the MS resistance phenotype, 36 (78%) contained msrA and msrB (Table 1). The MICs for MSSA containing erm genes were higher (MIC ≥ 256 μg/ml) than those for isolates with other erythromycin resistance determinants (Table 1). Of 97 ermB-carrying MSSA isolates, seven distinct resistance patterns were found by MIC analysis. Half of the isolates were also resistant to other classes of antibiotics, including kanamycin, streptomycin, and chloramphenicol, showing marked multidrug resistance.

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.

TABLE 1.

Distribution of resistance determinants in strains with different phenotypes of erythromycin-resistant MSSA and correlation with MIC

Resistance gene(s)	No. of strains	No. of strains with phenotype:			No. of strains with erythromycin MIC (μg/ml) of:
		iMLS	cMLS	MS	>256	256	128	64	32	16	8
ermA	56	46	10	0	55	1	0	0	0	0	0
ermB	94	0	94	0	79	11	3	1	0	0	0
ermC	71	49	22	0	68	0	1	2	0	0	0
msrA + msrB	36	0	0	36	1	0	3	17	15	0	0
ermB + ermC	3	0	3	0	3	0	0	0	0	0	0
ermA + msrA + msrB	2	2	0	0	2	0	0	0	0	0	0
ermC + msrA + msrB	1	0	1	0	1	0	0	0	0	0	0
ermT	1	1	0	0	1	0	0	0	0	0	0
Unknowna	10	0	0	10	0	0	0	1	1	4	4
Total	274	98	130	46	210	12	7	21	16	4	4

^aUnknown, none of the listed genes was detected.

Genotyping of 97 ermB-carrying MSSA isolates.

ermB was the most frequent erythromycin resistance determinant in MSSA isolates in the present study, which is different from other reports. One of the possible causes may be clonal spreading. Thus, PFGE, spa typing, and MLST were performed to determine the genetic relatedness of ermB-carrying MSSA isolates. Of the 97 isolates (94 carried ermB alone and 3 carried both ermB and ermC), 28 pulsotypes were identified (using an 80% similarity cutoff). There was no predominant pulsotype. A total of 16 spa types were found. The most frequent spa type was t437 (71%; 69/97), followed by t441 (9%; 9/97). Seven spa types (t437, t441, t3385, t1151, t216, t3592, and t7496, all belonging to ST59) were found in 84 isolates. Selected isolates of each spa type were examined for MLST, and the spa types matched their corresponding STs.

Genetic contents of ermB-carrying elements.

Half of the 97 ermB-carrying isolates were also resistant to other class of antibiotics, and we detected genes encoding resistance to kanamycin, streptomycin, chloramphenicol, and gentamicin and the gene content of MES_PM (which includes three variants, MES_PM1, MES_PM9, and MESP_M18) or MES_6272-2-like. There were seven distinct patterns (Table 2). All ermB-carrying MSSA isolates contained IS1216V. In order to compare their differences in gene organization, we also used PCR mapping based on previously reported MES_PM sequences (12). The results indicate that 47 isolates containing ermB, aph(3′)-IIIa, aadE, and cat were MES_PM1 and 33 isolates containing ermB, aph(3′)-IIIa, and aadE were MES_PM9 (Table 2 and Fig. 2). Nine carried other MES (Table 2). In particular, 8 isolates were sasK intact (Table 2). Unlike isolates with sasK insertions, which were mostly ST59, the spa types of 8 sasK-intact isolates were very diverse (t796 [n = 4], t002 [n = 2], t242 [n = 1], and t216 [n = 1]), corresponding to ST7 (n = 4), ST5 (n = 3), and ST59 (n = 1) (Table 2). The 8 sasK-intact MSSA isolates were isolated in different years (2002 [n = 1], 2004 [n = 1], 2009 [n = 1], 2010 [n = 2], and 2012 [n = 3]).

TABLE 2.

Distribution of spa types, resistance genes, and MES in 97 ermB-carrying MSSA isolates

No. of strains	spa type(s) (no. of isolates)	Drug resistance genea					MES structure
		ermB	aph(3′)-IIIa	aadE	cat	aacA-aphD	sasKb	IS1216V	MESc
47	t437 (36), t441 (5), t3385 (2), t1380 (1), t3592 (1), t7496 (1), t8886 (1)	+	+	+	+	−	−	+	MESPM1
33	t437 (28), t441 (2), t1151 (1), t3401 (1), NDd (1)	+	+	+	−	−	−	+	MESPM9
1	t437 (1)	+	−	−	+	−	−	+	MES6272-2-like
1	t437 (1)	+	−	−	−	−	−	+	Partial MESPM18
2	t437 (1), t3517 (1)	+	+	+	+	+	−	+	MES6272-2-like
5	t437 (2), t441 (2), t3515 (1)	+	+	+	−	+	−	+	MES6272-2-like
8	t796 (4), t002 (2), t242 (1), t216 (1)	+	−	−	−	+	Intact	+	Absent

^aermB encodes erythromycin/clindamycin resistance, aph(3′)-IIIa encodes kanamycin resistance, aadE encodes streptomycin resistance, cat encodes chloramphenicol resistance, and aacA-aphD encode kanamycin/gentamicin resistance. +, present; −, absent.

^b−, split and negative.

^cThe structures of MES_PM1, MES_PM9, and MES_PM18 found in ST59/SCCmec type V MRSA were reported in 2012 (12); MES_6272-2 was found in ST59/SCCmec type IV MRSA (unpublished results).

^dND, not determined (unknown).

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).

Localization of the ermB element in sasK-intact MSSA.

To determine the location of the novel ermB genetic element in sasK-intact MSSA isolates, PFGE separation of S1 nuclease-digested DNAs followed by Southern blotting hybridization with a DIG-labeled ermB-specific probe was performed for three isolates (ST5 NTUH_9448, ST7 NTUH_1027, and ST59 NTUH_3874) and their transconjugants (RN-NTUH_9448, RN-NTUH_1027, and RN-NTUH_3874) (Fig. 3). The results indicated that the ermB element was located on a plasmid with an estimated size ranging from 23.1 to 48.5 kb (Fig. 3). The PFGE bands of the three transconjugants displayed patterns identical to those of the respective parental strains (Fig. 3). In addition, the MICs of erythromycin and gentamicin for the transconjugants increased >512-fold and 64-fold compared to those for the parental recipient strain, RN2677.

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.

Tn1546-ermB element in NTUH 3874.

The nucleotide sequence of a 14,566-bp element containing the ermB gene in ST59 NTUH_3874 was determined by LA-PCR and inverse PCR (GenBank accession number LC102479). The 14,566-bp element was composed of 11 putative open reading frames (ORFs), including three transposase genes (tnp) for Tn1546, Tn551, and IS1216V; three resolvase genes (tnpR); two drug resistance genes (ermB and aacA-aphD, conferring resistance to erythromycin and kanamycin/gentamicin, respectively); one leader peptide for the ermB gene; one thymidylate synthase gene (thyX); and one dihydrofolate reductase gene (drf-like) (Table 3 and Fig. 4). There are two 38-bp transposase distal terminal inverted repeats (att sites; the att-1 sequence is 5′-GGGGTAGCGTCAGGAAAATGCGGATTTACAACGTTAAG-3′, and the att-2 sequence is 5′-CTTAGCGTTGTAAATCCGCATTTTCCTGACGGTACCCC-3′) belonging to transposon Tn1546, which was originally described in Enterococcus (GenBank accession number M97297) (Fig. 4). Hence, the entire 14,566-bp element has the structure of a Tn1546-like transposon with the ermB gene located within Tn551 (Fig. 4). By comparing the sequences with others in the NCBI database, the best match for the ermB-carrying regions is the M. caseolyticus plasmid pMCCL2 DNA (GenBank accession number AP009486) (Fig. 4). However, the upstream region containing tnp (IS1216V) in NTUH_3874 (positions 1 to 3596) is 99% identical in sequence to the downstream region of tnp (IS1216V) (positions 56061 to 59659) in pMCCL2, but with the opposite orientation (Fig. 4).

TABLE 3.

ORFs of partial plasmid elements in MSSA NTUH_3874^a

No.	ORF	Location (bp)	Size of encoded product (aa)	Homologous protein
1	thyX	103–810	235 (complement)	Thymidylate synthase
2	drf-like	807–1193	128 (complement)	Dihydrofolate reductase
3	tnpR	1382–1876	164 (complement)	Resolvase
4	tnpR	2281–2823	180	Resolvase
5	tnp	3597–4283	228	Transposase for IS1216V
6	aacA-aphD	4596–6071	491	Bifunctional aminoglycoside N-acetyltransferase and aminoglycoside phosphotransferase
7	ORF1	6444–6527	27	Leader peptide
8	ermB	6652–7389	245	rRNA adenine N-6-methyltransferase
9	tnpR	7744–8298	184	Resolvase
10	tnp	8302–10,875	857	Transposase for Tn551
11	tnp	11,529–14,492	987	Transposase for Tn1546

^aSource, M. caseolyticus pMCCL2, accession no. AP009486.

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.

The ermB element in other sasK-intact isolates.

PCR mapping using four sets of primers covering 8 kb was employed to determine whether the above-described structure was present in the remaining seven ermB-carrying and sasK-intact isolates. Seven isolates (four ST7 MSSA [NTUH_9033, NTUH_1027, NTUH_433, and NTUH_1023570] and three ST5 MSSA [NTUH_My675, NTUH_798, and NTUH_9448]) were positive in all PCRs generating predicted product sizes, indicating that their ermB-containing structures were similar to that of NTUH_3874. Furthermore, we used an additional four sets of primers to sequence the border regions of Tn1546, which include transposase and resolvase genes and att. The sequences of the transposase and resolvase genes and att for Tn1546 of ST5 NTUH_9448 and ST7 NTUH_1027 were 100% identical to those of NTUH_3874.

DISCUSSION

Erythromycin resistance rates in MSSA in NTUH from 2000 to 2012 ranged from 8% to 16% per year, which were much lower than in MRSA (29). In addition, the erythromycin resistance rate in the present study is slightly lower than that in a previous report (21%) that investigated 729 MSSA isolates from 1995 to 2006 in southern Taiwan (4).

The percentage of cMLS resistance (47%; 130/274) in the present study was similar to that found in an earlier study in southern Taiwan from 1995 to 2006 (52%; 77/149) (4) but much higher than in several other countries, such as Japan (10%; 48/478) (30), southeastern Turkey (8%; 1/13) (31), and Korea (20%; 40/205) (32). The iMLS phenotype was detected in 36% (98/274) of erythromycin-resistant MSSA isolates, which equals 68% (98/144) of erythromycin-resistant, clindamycin-susceptible strains, slightly lower than in a previous report from southern Taiwan (76%; 55/72) (4) or in Japan (93%; 397/428) (30) but higher than in Korea (35%; 58/165) (32) or in the United States (60%; 73/122) (33). The distribution of resistance determinants is related to phenotypes. In particular, a high prevalence of ermB was found in our erythromycin-resistant MSSA isolates. So far, the ermB gene has not been commonly found in S. aureus but is very common in several Gram-positive bacteria, such as Streptococcus and Enterococcus.

The majority of erythromycin-resistant MSSA isolates harboring the ermB gene (80/97; 82%) belonged to ST59 (spa types t437, t441, and t3385), and many carried MES_PM1 or MES_PM9, suggesting that the high prevalence of ermB in MSSA isolates in Taiwan may be partly due to ST59 spreading. ST59 is the most common community-associated (CA)-MRSA ST in Taiwan (34). It has been reported that a single endemic MRSA ST59 strain with high macrolide resistance was predominant in previously healthy children in northern Taiwan (35). ST59 S. aureus is the major genotype present in both MSSA and MRSA in Taiwan (36). Since ST59 MSSA and MRSA have already spread in many countries, for example, Singapore in 2001 (37), Western Australia in 2003 (38), the United States in 2004 and 2005 (39), and Japan in 2009 (40), the presence of ermB in ST59 MSSA needs more attention.

For the 8 sasK-intact ermB-carrying MSSA, similar to MES, the ermB gene was harbored on the transposon Tn551 but was flanked by one copy of IS1216V, linked with aacA-aphD, not aph(3′)-IIIa and aadE, and a transposase (Tn1546), which means this is a new structure in S. aureus. The nucleotide sequence of the Tn1546-ermB element in strain NTUH_3874 was 99.9% identical to that on plasmid pMCCL2 of M. caseolyticus. ermB in pMCCL2 is flanked by two inverse copies of a transposase (IS1216V), indicating that it is a mobile element. The 38-bp att sequence in strain NTUH_3874 was 100% identical to that on pMCCL2 of M. caseolyticus and nearly identical to that of Tn1546 of Enterococcus faecium (with a 1-nucleotide difference). Moreover, the Tn1546-like structure of the 14,566-bp element was present in at least three distinct sequence types (ST5, ST7, and ST59) of MSSA. IS1216V is recognized as an enterococcal insertion sequence but is rarely found in S. aureus (41). In E. faecium, a 92-kb plasmid, pHKK701, has been reported to contain a 39-kb large mobile element, Tn5506, which carries ermB and IS1216V, and also contains Tn1546 with vanA (encoding vancomycin resistance) (12, 42). The unusual presence of both IS1216V and ermB in S. aureus implies that the ermB gene in S. aureus may have originated from Enterococcus. Tn1546 is recognized as an enterococcal transposon and has been reported in clonal complex 5 (CC5) MRSA/vancomycin-resistant S. aureus (VRSA) in the United States (43). Previous reports have indicated that the horizontal transfer of Tn1546 elements plays an important role in the spread of VanA-type vancomycin-resistant Enterococcus (VRE) (44) and VRSA (45). However, so far, no MSSA isolate has been reported to carry Tn1546. Although non-MES_PM1 or non-MES_PM9 ermB-carrying MSSA isolates are only a minority in the present study, the presence of resistance genes in the mobile element Tn1546 on plasmids is alarming and merits continued attention. In conclusion, this is the first report that a Tn1546-ermB-like element has been found in S. aureus.