New erm Gene in Staphylococcus aureus Clinical Isolates

Abstract

We have identified erm(Y), a novel gene class that was originally designated ermGM, from a Staphylococcus aureus strain that has a plasmid that also harbors msr(A) and mph(C), genes that encode an efflux mechanism and a putative phosphorylase, respectively. The nucleotide and deduced amino acid sequences of erm(Y) were 81 and 76% identical to those of erm(T), respectively.

Three mechanisms for resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics in staphylococci have been reported: modification of the target sites, active efflux, and inactivation (11, 18). Almost all strains of Staphylococcus aureus that are resistant to erythromycin (ERY) are resistant to MLS antibiotics, usually due to ribosomal modification by 23S rRNA methylase mediated by the erm(A) (10), erm(B) (20), erm(C) (13), and erm(F) (4) genes. These strains have been divided into two major phenotypic classes: (i) constitutively resistant strains that can grow in the presence of a high concentration (more than 100 μg/ml) of MLS antibiotics without prior induction and (ii) inducibly resistant strains whose high-level resistance to MLS antibiotics can be induced by subinhibitory concentrations (0.01 to 0.1 μg/ml) of ERY. We have previously reported that S. aureus MS8968 contains plasmid pMS97 (8), which carries three genes that appear to code for macrolide resistance determinants. MsrSA′ is ≈98% identical to Msr(A) from Staphylococcus epidermidis (15) and S. aureus (19) (see GenBank accession no. AF167161). Therefore, MsrSA′ has been reclassified as Msr(A) (14). Similarly, a putative inactivating gene also present on the same plasmid has been reclassified from mphBM to mph(C) (14). Here, we report on the sequence of erm(Y) and the detailed arrangement of this gene on pMS97.

Sequence analysis of erm(Y) gene.

Isolation of covalently closed circular plasmid DNA from S. aureus was performed by a previously described method (7). Universal primers (1) that could amplify a 530-bp product including the region of the methylase gene from plasmid pMS97 were used. The nucleotide sequence was determined and analyzed as described previously (8). The sequence of the 530-bp fragment had 76% nucleotide identity with erm(C) of pE194 (13) and 73% nucleotide identity with erm(G) of Bacillus sphaericus (9).

As a result of nucleotide sequence determination, two potential open reading frames (ORFs) were detected downstream from mph(C) (Fig. 1). The longer ORF, the structural gene for an erm methylase designated erm(Y) (M. Matsuoka, M. Inoue, and Y. Nakajima, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-35, p. 78, 1998), consists of 735 nucleotides and is predicted to encode a polypeptide of 244 amino acids. Moreover, erm(Y), previously named ermGM, met the criteria for a new gene class (14) (see GenBank accession no. AB014481). There are two potential ribosome binding sites (Shine-Dalgarno sequence), one for the leader peptide (MGNCSLFVINTVHYQPNEK) and another, 9 nucleotides upstream from the ATG codon, for erm(Y) (Fig. 2). The inducible regulation of erm(C) depends on the specific amino acid sequence of the leader peptide, including a crucial region defined by IFVI (18). Since the two leader peptides of erm(Y) and erm(C) are similar in length and IFVI is conserved in erm(Y) and erm(C), it is likely that induction of erm(Y) occurs as has been proposed for erm(C) (18). The predicted amino acid sequences of erm(Y) from S. aureus and erm(T) from Lactobacillus reuteri show that 187 (76%) of the 244 amino acids are identical. A dendrogram showing the possible evolutionary relationship between the methylase encoded by erm(Y) and other rRNA-methylating enzymes is shown in Fig. 3. Comparison of the nucleotide sequence of erm(Y) with those of other erm gene classes indicated that it is 63% identical to those of erm(A) and erm(B), 77% identical to that of erm(C), and 74% identical to that of erm(G). The erm(F), erm(D), erm(Q), and erm(X) genes have low levels of identity to erm(Y) since they consist of different numbers of nucleotides; erm(Y) is encoded by 735 nucleotides, whereas the other erm genes are encoded by 774 to 864 nucleotides. erm(Y) has only 26% identity to the sequence of erm(E), which consists of 1,146 nucleotides and which is from the ERY-producing strain Saccharopolyspora erythraea.

FIG. 1.

Schematic representation of plasmids pMS97 and pND503 including genes coding for resistance to macrolides. (a) pMS97 contains the entire msr-mph-erm three-gene cluster. The antibiogram shows the induction of resistance to rokitamycin (R), mycinamicin (M), clindamycin (C), and MKM-B (K) by ERY (E). OL (O) induces resistance to mycinamicin and mikamycin B. (b) pND503 contains erm(Y) and the C-terminal half of mph(C). The antibiogram shows the induction of resistance to rokitamycin (R), clindamycin (C), mycinamicin (M), and MKM-B (K) by ERY (E). S. aureus RN4220 harboring plasmid pMS97 and S. aureus RN4220 harboring plasmid pND503 are shown in insets a and b, respectively. Plasmid pND503 includes an XbaI-BamHI fragment present in fragment A from plasmid pMS97. Each disk contains one of the following drugs: ERY, 10 μg/disk; OL, 10 μg/disk; rokitamycin, 10 μg/disk; mycinamicin, 10 μg/disk; clindamycin, 2 μg/disk; MKM-B, 25 μg/disk. Two spacer sequences lie between the sequences of msr(A) and mph(C) (342 bp) and those of mph(C) and erm(Y) (536 bp), including a leader peptide sequence. The amino acid sequence deduced from the DNA sequence gave three large putative proteins, Msr(A), Mph(C), and Erm(Y), which contain 488, 299, and 244 amino acid residues, respectively. Bold lines represent the DNAs of pMS97 and of the cloned insert. The direction of transcription of the three genes is indicated by arrows. LP, leader peptides (indicated by open boxes); P, PstI; X, XbaI; B, BamHI. Fragment B containing msr(A) and mph(C) and fragment C containing erm(Y) were registered in GenBank and given accession nos. AB013298 and AB014481, respectively.

FIG. 2.

Comparison of the nucleotide sequences of erm(Y) from pMS97 and erm(T) from pGT633. The 26 bp of the duplicated sequence present in erm(T) (nucleotides 83 to 108) but absent from erm(Y) is indicated by a dashed line. SD1 and SD2 indicate the putative ribosome binding sites. A leader sequence is boxed. Identical nucleotides are shown by asterisks. The ATG and TAA codons marked with symbols (> and <) represent the initiation and termination codons for the erm coding region, respectively. The numbers refer to the deposited sequences [GenBank accession no. AB014481 for erm(Y) and GenBank accession no. M64090 for erm(T)].

FIG. 3.

Dendrogram of erm methylases. The source of the amino acid sequence is indicated by the GenBank accession number, the name of the gene, and the bacterial species. The predicted amino acid sequences of the polypeptides encoded by the erm(Y) gene in plasmid pMS97 show marked differences from those of other erm alleles. The sequences with accession nos. M14730 (4), M77505 (6), M64090 (17), AB014481 (this paper), V01278 (13), M15332 (9), X03216 (10), Y13600 (20), L22689 (3), and M36726 (16) have been described previously.

Susceptibilities of S. aureus RN4220(pND503) carrying the erm(Y) gene.

S. aureus RN4220(pMS97), which contains plasmid pMS97 from S. aureus MS8968 transformed into S. aureus RN4220, exhibited an inducible phenotype of resistance to MLS antibiotics with ERY as an inducer. However, only oleandomycin (OL) induced partial macrolide and streptogramin B (PMS) resistance. Therefore, there was a D-shaped zone of inhibition for all MLS antibiotics proximal to the ERY disks, while only mycinamicin and mikamycin B (MKM-B; synonymous with streptogramin B) resistance was inducible with OL (Fig. 1, inset a).

S. aureus RN4220 harboring pND503 on which erm(Y) and the C-terminal half of mph(C) are located exhibits inducible MLS resistance in the presence of a low concentration of ERY or azithromycin (AZM) but not in the presence of a low concentration of OL (Fig. 1, inset b). A zone of inhibition was not formed around ERY and OL disks for S. aureus RN4220(pMS97) (Fig. 1, inset a), whereas zones of inhibition were created around the disks for S. aureus RN4220(pND503) (Fig. 1, inset b). Such a disappearance of the zone of inhibition may result from the expression of the msr(A) gene coding for resistance (due to the efflux of a 14-membered-ring macrolide including mycinamicin) and/or the mph(C) gene coding for resistance (due to putative inactivation by a phosphotransferase). The profile of the antibiogram in inset a of Fig. 1 shows an apparently inducible MLS-type resistance in the presence of the ERY disk, although the small zone of inhibition around the ERY disk disappeared, unlike that in inset b of Fig. 1. That is the reason why the natural plasmid includes drug resistance genes [msr(A) and mph(C)], in addition to the erm(Y) gene. The profile of the antibiogram in inset b of Fig. 1 shows that expression of the erm(Y) gene alone was switched on with a better inducer (ERY) than OL because the C-terminal half of mph(C) is inactive. In short, the expression of three genes present in full-length plasmid pMS97 is likely to occur together in the presence of ERY. In contrast, S. aureus RN4220 bearing plasmid pND503 carrying erm(Y) and the C-terminal half of mph(C) appears to show MLS-type resistance but neither macrolide-lincosamide-type resistance nor resistance to drug inactivation. Further detailed research on these points remains to be done.

In S. aureus strains with inducible MLS resistance, it usually takes a long incubation time to express resistance in the presence of a high concentration of ERY or AZM, but the bacteria do not grow in the presence of antibiotics such as rokitamycin, clindamycin, or streptogramin B but occasionally grow in the presence of OL. This is confirmed by the fact that a resistant S. aureus strain, strain RN4220(pND503), showed inducible resistance to ERY or AZM after 36 to 38 h of incubation at 37°C (Table 1). The MICs are consistent with the fact that ERY and AZM act as better inducers than OL when the full-length plasmid (pMS97) is present. However, when erm(Y) is separated from its upstream genes, all three drugs are poor inducers, as noted by the delayed increase in the MICs of ERY or AZM and no changes in the MICs of OL. The 16-membered-ring MLS antibiotics are not inducers of resistance, even for the native plasmid.

TABLE 1.

MICs of MLS antibiotics for S. aureus strains

S. aureus strain	MIC (μg/ml)a
	ERY	OL	AZM	RKM	CLI	MKM-B
RN4220	0.20	0.78	0.78	0.39	0.05	6.25
4220(pMS97)	800	50	>100	0.39	0.05	6.25
4220(pND503)	1.56 (>800)	1.56	3.13 (>100)	0.39	0.05	6.25

^aAbbreviations: ERY, erythromycin; OL, oleandomycin; AZM, azithromycin; MCM, rokitamycin; CLI, clindamycin; MKM-B, mikamycin B. MICs in parentheses are those after 36 to 38 h of incubation.

In order to analyze the expression of resistance in erm(Y), we tried to construct a clone that contains the erm methylase gene and its putative promoter region. A plasmid harboring the erm gene was constructed as follows: the PCR product (1,829 bp) was amplified with a forward primer (5′-GATTTACACAACATACCAGA-3′) and a reverse primer (5′-GGACTGCCTAGATGGCAATC-3′) (GenBank accession nos. AB013298 and AB014481, respectively). The conditions for PCR with Taq DNA polymerase were 94°C for 135 s; then 30 cycles of 94°C for 45 s, 55°C for 30 s, and 72°C for 45 s; and finally, 72°C for 10 min. The amplified DNA fragment was cloned into the pT7Blue T vector. This recombinant plasmid was digested with XbaI and BamHI and was ligated into the same site of pND50 (5). The resultant plasmid was transformed into S. aureus RN4220 by electroporation (2, 12), and strains that showed inducible resistance to ERY were selected. The constructed plasmid (5.5 kb) was referred to as pND503. S. aureus RN4220(pND503) showed inducible resistance to rokitamycin, mycinamicin, clindamycin, and MKM-B in the presence of ERY but not in the presence of OL (Fig. 1, inset b). As shown in Table 1, the MICs of ERY and AZM, which act as inducers of resistance for S. aureus RN4220(pND503), were 1.56 and 3.13 μg/ml, respectively, after 20 h of incubation at 37°C; but these increased to >800 and >100 μg/ml, respectively, after an additional 16 to 18 h of incubation. Thus, it takes a longer incubation time for the expression of inducible resistance. The MICs of ERY and AZM for S. aureus RN4220(pMS97) seem to be reflected by the erm(Y) gene. It is unclear what gene(s) other than msr(A) and mph(C) on plasmid pMS97 is crucial in relation to the rate of induction, although it is an important and interesting issue.

Nucleotide sequence accession number.

The nucleotide sequence data for erm(Y) will appear in the GenBank nucleotide sequence database under accession no. AB014481.