Novel Plasmid-Mediated 16S rRNA Methylase, RmtC, Found in a Proteus mirabilis Isolate Demonstrating Extraordinary High-Level Resistance against Various Aminoglycosides

Abstract

Proteus mirabilis ARS68, which demonstrated a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. The aminoglycoside resistance of this strain could not be transferred to recipient strains Escherichia coli CSH-2 and E. coli HB101 by a general conjugation experiment, but E. coli DH5α was successfully transformed by electroporation with the plasmid of the parent strain, ARS68, and acquired an unusually high degree of resistance against aminoglycosides. Cloning and sequencing analyses revealed that the presence of a novel 16S rRNA methylase gene, designated rmtC, was responsible for resistance in strain ARS68 and its transformant. The G+C content of rmtC was 41.1%, and the deduced amino acid sequences of the newly identified 16S rRNA methylase, RmtC, shared a relatively low level of identity (≤29%) to other plasmid-mediated 16S rRNA methylases, RmtA, RmtB, and ArmA, which have also been identified in pathogenic gram-negative bacilli. Also, RmtC shared a low level of identity (≤28%) with the other 16S rRNA methylases found in aminoglycoside-producing actinomycetes. The purified histidine-tagged RmtC clearly showed methyltransferase activity against E. coli 16S rRNA in vitro. rmtC was located downstream of an ISEcp1-like element containing tnpA. Several plasmid-mediated 16S rRNA methylases have been identified in pathogenic gram-negative bacilli belonging to the family Enterobacteriaceae, and some of them are dispersing worldwide. The acceleration of aminoglycoside resistance among gram-negative bacilli by producing plasmid-mediated 16S rRNA methylases, such as RmtC, RmtB, and RmtA, may indeed become an actual clinical hazard in the near future.

Aminoglycosides have been widely used for the treatment of a variety of bacterial infections (9). These agents bind to the A site of the 16S rRNA of prokaryotic 30S ribosomal subunits and subsequently block bacterial growth through interference with protein synthesis (17). On the other hand, bacteria have acquired resistance to aminoglycosides by producing aminoglycoside-modifying enzymes, such as aminoglycoside acetyltransferases, aminoglycoside nucleotidyltransferases, and aminoglycoside phosphotransferases (17, 24). Moreover, reduction of affinity for the target site within 16S rRNA by nucleic acid point mutations, the excretion of aminoglycosides by the augmented function of efflux systems, and altered membrane permeability, which leads to the reduced penetration of these agents, also contribute to the intrinsic clinical resistance of bacteria (3, 17).

Recently, as a new mechanism of resistance against aminoglycosides among clinically important pathogenic bacteria, plasmid-mediated 16S rRNA methylase (RmtA) was first characterized in a clinically isolated Pseudomonas aeruginosa strain, strain AR-2. This strain was isolated in 1997 in a Japanese hospital and demonstrated consistent resistance to various clinically important aminoglycosides (29). The total sequence of a large plasmid carrying genes for both CTX-M-3 and 16S rRNA methylase was then submitted to the EMBL/GenBank database (accession no. AF550415) on 18 October 2002 by M. Golebiewski et al., although they did not seem to be aware of the presence of the armA gene in the sequence deposited in the database. In 2003, the armA gene, found in a clinically isolated Klebsiella pneumoniae strain, was reported from France (7). RmtB, which was encoded on a nonconjugative plasmid of a clinically isolated Serratia marcescens strain, was also reported from Japan in 2004 (6). At present, the three types of plasmid-mediated 16S rRNA methylases described above have been found in pathogenic gram-negative rods. More recently, nosocomial outbreaks caused by 16S rRNA methylase-producing gram-negative bacteria was reported from Taiwan (28). The further global dissemination of 16S rRNA methylase genes among pathogenic bacilli will be a cause of great concern in the near future, because these genes were mediated by some bacterial site-specific recombination and translocation systems such as a transposon (6, 7, 26).

A Proteus mirabilis strain, strain ARS68, which displayed a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. A preliminary PCR analysis, however, failed to detect any of the known three plasmid-mediated 16S rRNA methylase genes, rmtA, rmtB, and armA, in this strain. Therefore, it was strongly suspected that the P. mirabilis ARS68 strain would have a novel 16S rRNA methylase gene. In the present study, the molecular mechanism underlying a very high level of resistance against various aminoglycosides found in strain ARS68 was elucidated.

MATERIALS AND METHODS

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are listed in Table 1. P. mirabilis strain ARS68 was isolated in August 2003 from a throat swab of an inpatient admitted to a general hospital in Japan. Biochemical phenotypic identification of this strain was performed with a commercially supplied API 20E system (bioMerieux, Marcy l'Etoile, France).

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Characteristicsa	Source
Strains
P. mirabilis ARS68	Clinical isolate resistant to various aminoglycosides	This study
E. coli DH5α	supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 acrAB+	TOYOBO
E. coli CSH-2	metB F− nalidixic acidr rifampinr	T. Sawai, Chiba University
E. coli HB101	thi-1 hsdS20(rB− mB+) supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 rps20 (Strr) xyl-5 mtl-1	A. Ohta, Tokyo University
E. coli BL21(DE3)pLysS	F−ompT hsdSB (rB− mB−) gal dcm (DE3) pLysS	Novagen
Plasmids
pARS68	A natural plasmid carrying rmtC of P. mirabilis ARS68	This study
pBC-E1	A recombinant plasmid carrying a 7.7-kb EcoRI fragment containing rmtC	This study
pBC-KB1	A recombinant plasmid carrying a PCR-amplified fragment containing rmtC and its promoter	This study
pBC-Sa1	A recombinant plasmid carrying aph(3′)	This study
pGEM-rmtC	A recombinant plasmid carrying PCR-amplified rmtC ligated to the pGEM-T vector	This study
pET-His-rmtC	A recombinant plasmid carrying rmtC ligated to pET29a(+)	This study
pBCSK+	A cloning vector, chloramphenicolr	Stratagene
pGEM-T	A cloning vector, ampicillinr	Promega
pET29a(+)	An expression vector, kanamycinr	Novagen

^ar, resistant to the indicated antimicrobial agent.

Antibiotic susceptibility testing.

The MICs of antimicrobial agents were determined by the agar dilution method with Mueller-Hinton agar plates, according to the protocol recommended by CLSI (formerly the National Committee for Clinical Laboratory Standards) (18). The following antibiotics were obtained from the indicated sources: amikacin, Bristol Pharmaceuticals K. K., Tokyo, Japan; arbekacin, kanamycin, and streptomycin, Meiji Seika Kaisha, Ltd., Tokyo, Japan; gentamicin and sisomicin, Schering-Plough K. K., Osaka, Japan; isepamicin, Asahi Kasei Corporation, Tokyo, Japan; neomycin, Nippon Kayaku Co., Ltd., Tokyo, Japan; and tobramycin, Shionogi & Co. Ltd., Osaka, Japan.

PCR amplification.

The sets of PCR primers and amplification conditions used to detect the three 16S rRNA methylase genes, rmtA, rmtB, and armA, are referred to in our recent study (27).

Transfer of aminoglycoside resistance.

Conjugal transfer was performed by using E. coli CSH-2 (F⁻ metB, resistant to both nalidixic acid and rifampin) or E. coli HB101 (resistant to streptomycin) as a recipient by a filter-mating method. Transconjugants were selected on Luria-Bertani (LB) agar plates containing rifampin (100 μg/ml) and kanamycin (30 μg/ml) or arbekacin (10 μg/ml) when E. coli CSH-2 was used as the recipient. Two kinds of streptomycin-containing (50 μg/ml) LB agar plates supplemented with kanamycin (30 μg/ml) or arbekacin (10 μg/ml) were also prepared when E. coli HB101 was used as the recipient. The plasmid DNA of P. mirabilis ARS68 was prepared by the method of Kado and Liu (14). E. coli DH5α was transformed with the plasmids of P. mirabilis ARS68 by electroporation techniques. Transformants were selected on LB agar plates supplemented with arbekacin (4 μg/ml) or kanamycin (10 μg/ml).

Cloning and sequencing of aminoglycoside resistance determinants.

Both total DNA and plasmid DNA were prepared from the bacterial strains as described previously (23) and restricted with endonucleases according to the recommendations of the supplier. The digested fragments were ligated to restriction enzyme-cleaved pBCSK+ (Stratagene, La Jolla, Calif.), and E. coli competent cells were transformed by electroporation with the mixture of recombinant plasmids. Transformants were selected on LB agar plates containing chloramphenicol (30 μg/ml) and arbekacin (4 μg/ml) or kanamycin (10 μg/ml). Both strands of the nucleotide sequences of the cloned fragment encoding the gene responsible for aminoglycoside resistance were determined with BigDye Terminator cycle sequencing ready reaction kits and an ABI 3100 DNA analyzer (Applied Biosystems, Foster City, Calif.) by using several custom sequencing primers.

PCR cloning of aminoglycoside resistance gene.

The DNA fragment carrying the aminoglycoside resistance gene and its promoter region was amplified by PCR with the primers rmtC-F (5′-CGC GGA TCC AGT GTA TGA AAA ATG TCT GG-3′) and rmtC-R (5′-CGG GGT ACC GGT GTG TTA GAA TTT GCC TT-3′) (where the underlining indicates the restriction site of BamHI or KpnI). The resultant fragments were digested with BamHI and KpnI and ligated to pBCSK+ (Stratagene).

Expression and purification of histidine-tagged enzyme.

The gene responsible for aminoglycoside resistance was amplified from plasmid pBC-E1 by using primers that introduced NdeI and XhoI sites at the ends of the amplified fragments. This fragment was ligated to the pGEM-T vector (Promega, Madison, Wis.), and one plasmid with no amplification error (pGEM-rmtC) was selected. A single nucleotide mutation which leads to the silent mutation (T to C) at position 171 was introduced to destroy the NdeI site within the fragment inserted on pGEM-rmtC by using an LA PCR in vitro mutagenesis kit (Takara Bio Inc., Ohtsu, Japan). A resultant plasmid was digested with NdeI and XhoI and ligated into the pET-29a(+) vector (Novagen, Madison, Wis.) restricted with the same enzymes. The newly constructed expression vector, pET-His-rmtC, was introduced into E. coli (DE3)pLysS (Novagen) and cultured in 1 liter of LB broth containing both kanamycin (50 μg/ml) and chloramphenicol (30 μg/ml). Isopropyl-β-d-thiogalactopyranoside (0.5 mM) was added when the culture reached an A₆₀₀ of 0.55, and the culture was incubated for an additional 3 h. The bacterial pellet harvested by centrifugation was washed with 50 mM phosphate buffer (pH 7.0) and suspended in 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl and 10 mM imidazole. The suspension was passed through a French pressure cell (Ohtake Works Co., Ltd., Tokyo, Japan) at 120 MPa and then centrifuged at 100,000 × g for 1 h. The supernatant containing the fusion protein was loaded onto a HisTrap HP column and purified according to the manufacturer's instructions (Amersham Biosciences, K. K., Tokyo, Japan). The eluted fusion protein was dialyzed against 20 mM Tris-HCl buffer (pH 7.5), applied to an anion-exchange HiTrap Q HP column (Amersham Biosciences), and eluted with a linear gradient of NaCl. Finally, size-exclusion chromatography was performed with a Superdex 200 HR10/30 column (Amersham Biosciences). The purified protein was dialyzed against HRS buffer (10 mM HEPES-KOH, pH 7.5; 10 mM MgCl₂; 50 mM NH₄Cl; 3 mM 2-mercaptoethanol). The purity was checked by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The protein concentration was estimated by use of the Coomassie Plus protein assay reagent and bovine serum albumin as a standard (Pierce Biotechnology, Rockford, Ill.). The N-terminal sequence of the purified protein was obtained by Edman degradation in a Shimadzu model PPSQ-23 automated protein sequencer.

Preparation of 30S ribosomal subunits.

The 30S ribosomal subunits of E. coli DH5α were prepared as described by Skeggs et al. (25). After ultracentrifugation with sucrose density gradients, fractions of the 30S ribosomal subunits were collected and concentrated by centrifugation with an Ultrafree-15 centrifugal filter device (Millipore Corporation, Bedford, Mass.). The purity of the 30S ribosomal subunit was checked by denatured agarose gel electrophoresis of the 16S rRNA derived from the material, and the 30S ribosomal subunit was stored at −80°C in aliquots until use.

Methylation assay of 30S ribosomal subunits.

The methylation assay of the 30S ribosomal subunits was carried out as described by Doi et al. (6), with some modifications, as follows. The reaction mixture contained 20 pmol 30S ribosomal subunits from E. coli DH5α, 20 pmol histidine-tagged RmtC, and 5 μCi S-adenosyl-l-[methyl-³H]methionine ([methyl-³H]SAM); and this mixture was adjusted to 200 μl with methylation buffer (50 mM HEPES-KOH, pH 7.5; 7.5 mM MgCl₂; 37.5 mM NH₄Cl; 3 mM 2-mercaptoethanol). In control experiments, histidine-tagged RmtC was replaced by an equal volume of heat-inactivated histidine-tagged RmtC, bovine serum albumin, and HRS buffer. Samples (35 μl) were taken at 0, 5, 15, 30, and 60 min and purified with an RNeasy Mini kit (QIAGEN K. K., Tokyo, Japan), according to the instructions provided by the manufacturer. Two micrograms of eluted 16S rRNA was spotted onto a DEAE filter mat for MicroBeta (Perkin-Elmer Life Sciences Japan Co., Ltd., Tokyo, Japan). The filter mat was then covered with MeltiLex for MicroBeta filters (Perkin-Elmer) on a hot plate. Finally, it was applied to a 1450 MicroBeta TRILUX (Perkin-Elmer), and the radioactivity of each spot was counted.

Nucleotide sequence accession number.

The open reading frame of rmtC was deposited in the EMBL and GenBank databases through the DDBJ database and has been assigned accession number AB194779.

RESULTS

Characteristics of P. mirabilis strain ARS68.

Clinically isolated P. mirabilis strain ARS68 showed an extraordinary high level of resistance (MIC, ≥1,024 μg/ml) to the various clinically important aminoglycosides except streptomycin and neomycin, as shown in Table 2. PCR analyses were performed preliminarily to detect three 16S rRNA methylase genes, rmtA, rmtB, and armA, which were previously found in pathogenic gram-negative bacilli; but none of them was detected in this strain.

TABLE 2.

Results of antibiotic susceptibility testing

Aminoglycoside	MIC (μg/ml)
	P. mirabilis ARS68(pARS68)	E. coli DH5α(pARS68)	E. coli DH5α(pBC-E1)	E. coli DH5α(pBC-KB1)	E. coli DH5α(pBCSK+)
4,6-Substituted deoxystreptamine antimicrobials
Kanamycin group
Arbekacin	>1,024	512	512	>1,024	0.25
Amikacin	>1,024	1,024	512	>1,024	0.5
Kanamycin	>1,024	>1,024	>1,024	>1,024	1
Tobramycin	1,024	256	128	512	0.25
Gentamicin group
Gentamicin	>1,024	256	512	>1,024	0.13
Sisomicin	>1,024	512	256	>1,024	0.13
Isepamicin	>1,024	>1,024	1,024	>1,024	0.13
4,5-Substituted deoxystreptamine antimicrobials
Neomycin	512	16	0.5	1	0.5
Another aminoglycoside
Streptomycin	4	2	2	2	2

Transfer of aminoglycoside resistance.

The aminoglycoside resistance of P. mirabilis strain ARS68 could not be transferred to the recipients E. coli CSH-2 and E. coli HB101 by conjugation under the experimental conditions used in this study. However, E. coli DH5α was successfully transformed by electroporation with the plasmid, pARS68, prepared from P. mirabilis ARS68. The size of plasmid pARS68 was estimated to be ca. >100 kb by summation of the SacI-digested DNA fragment sizes observed by agarose gel electrophoresis (data not shown). E. coli DH5α(pARS68) demonstrated a very high degree of resistance to various aminoglycosides, as was observed in the parent strain (Table 2).

Cloning of aminoglycoside resistance determinant.

A cloning experiment was performed to confirm the genetic aminoglycoside resistance determinant of P. mirabilis ARS68 and its transformant, E. coli DH5α(pARS68). As a result, one recombinant plasmid (pBC-E1) with a 7.7-kb EcoRI insert derived from pARS68 was obtained by selection with arbekacin and chloramphenicol, and the insert was then sequenced. A part of the cloned fragment sequenced is shown in Fig. 1A. The first 0.5 kb of the insert contained the 3′ end of the tnpA gene with a terminal inverted repeat (IR). This region containing the IR had a high degree of similarity at the nucleotide level with the ISEcpI element, which was often identified upstream of several genes encoding CTX-M-type β-lactamases and CMY-type cephalosporinases (2, 4, 10, 20, 21). One open reading frame, which encoded 281 amino acids, was located downstream of tnpA. A BLAST analysis of the deduced amino acid sequence revealed that the gene product exhibited low-level identities to the 16S rRNA methylases, RmtA, RmtB, and ArmA (28%, 29%, and 28%, respectively), found in pathogenic gram-negative bacilli. The predicted enzyme was designated RmtC, and a comparison of the deduced amino acid sequences of RmtA, RmtB, and ArmA is shown in Fig. 2. RmtC also has a low degree of similarity (≤28%) to other 16S rRNA methylases found in aminoglycoside-producing Streptomyces and Micromonospora species. The amino acid similarities among 16S rRNA methylases are summarized in Table 3. The putative promoter region of rmtC appeared to be located within the ISEcpI-like element, just upstream of the IR generally found among several CTX-M-type and CMY-type β-lactamase genes (Fig. 1B) (4, 10, 20, 22). One Sau3AI fragment carrying the aminoglycoside phosphotransferase gene, aph(3′), was also cloned from P. mirabilis strain ARS68 when kanamycin was used as a selection marker.

FIG. 1.

(A) Schematic presentation of the 7.7-kb EcoRI fragment on pBC-E1 and the 1.2-kb PCR fragment on pBC-KB1. (B) Part of the nucleotide sequences encoding the 3′ end of an ISEcp1-like element and the start region of rmtC. The predicted −35 and −10 promoter sequences and the +1 position of the putative transcriptional start of rmtC are boxed. These positions were cited elsewhere (4). Arrows indicate the transcription orientation. The deduced amino acid sequences are designated in single-letter code. The right inverted repeat (IR) of an ISEcp1-like element is underlined.

FIG. 2.

Alignment of the deduced amino acid sequence of RmtC with those of RmtA, RmtB, and ArmA. Asterisks indicate the conserved residues among the above four 16S rRNA methylases.

TABLE 3.

Amino acid identities among various 16S rRNA methylases

16S rRNA methylase	G+C content (%)	Identity (%) of amino acid residues
		Plasmid-mediated 16S rRNA methylases among pathogenic gram-negative bacilli			Chromosomally encoded 16S rRNA methylases among aminoglycoside-producing actinomycetes
		RmtA	RmtB	ArmA	GrmA	KgmB	GrmO	FmrO	Kmr
RmtC	41.1	27.7	29.5	27.8	26.5	23.1	25.4	23.0	22.0
RmtA	55.4		82.0	29.2	31.7	29.5	28.1	27.3	28.7
RmtB	55.6			28.9	31.7	26.4	28.9	28.5	26.3
ArmA	30.4				26.3	26.6	20.6	28.0	24.4

Antibiotic susceptibilities.

The MICs of the aminoglycosides for parental strain P. mirabilis ARS68, E. coli DH5α(pARS68), E. coli DH5α(pBC-E1), and E. coli DH5α(pBC-KB1) are shown in Table 2. E. coli DH5α(pARS68) demonstrated resistance to all the various aminoglycosides except streptomycin and neomycin. RmtC-producing strains E. coli DH5α(pBC-E1) and E. coli(pBC-KB1) showed high levels of resistance to 4,6-disubstituted deoxystreptamine antimicrobials belonging to the kanamycin and gentamicin groups but were susceptible to the 4,5-disubstituted deoxystreptamine antimicrobial neomycin and another aminoglycoside, streptomycin. E. coli DH5α(pBC-Sa1), which carried the aminoglycoside phosphotransferase gene, aph(3′), showed resistance to both neomycin (MIC, 1,024 μg/ml) and kanamycin (MIC, >1,024 μg/ml). The resistance to neomycin found in strain ARS68 seemed to be attributable to the presence of aph(3′).

Identification of RmtC as a 16S rRNA methyltransferase.

Histidine-tagged RmtC-producing E. coli BL21(DE3)pLysS showed resistance to arbekacin, while E. coli BL21(DE3)pLysS and E. coli BL21(DE3)pLysS, which carried the pET29a(+) vector, were susceptible to arbekacin. This finding indicated that the production of histidine-tagged RmtC was responsible for the aminoglycoside resistance in E. coli BL21(DE3)pLysS. The N-terminal sequence of the purified protein was determined to be MKTND. The result of the methylation assay is shown in Fig. 3. Purified histidine-tagged RmtC readily methylated 30S ribosomal subunits prepared from E. coli DH5α in the presence of the methyl group donor [methyl-³H]SAM as a cosubstrate in a time-dependent manner. On the other hand, incubation with heat-inactivated histidine-tagged RmtC did not increase the counts of radioactivity. When an equal volume of bovine serum albumin or HRS buffer was used in place of purified histidine-tagged RmtC, no increase in the radioactivity counts was observed (data not shown).

FIG. 3.

Methylation of 16S rRNA. The 16S rRNA from E. coli DΗ5α was incubated with purified histidine-tagged RmtC (His-RmtC) by using [methyl-³H]SAM as a cofactor. The value of each point was calculated with three datum points. Error bars indicate standard deviations.

DISCUSSION

In the present study, we found a new 16S rRNA methylase gene, rmtC, in a clinical P. mirabilis isolate and characterized it precisely. The production of RmtC conferred a high degree of resistance mainly against 4,6-disubstituted deoxystreptamines but not against non-4,6-disubstituted deoxystreptamines, such as streptomycin and neomycin, as did RmtA, RmtB, and ArmA. Although the methylation site in the 16S rRNA has not been clarified yet, it was speculated that G1405 within the A site of 16SrRNA would be methylated by these plasmid-mediated 16S rRNA methylases, since the methylation of G1405 by some 16S rRNA methylases produced by actinomycetes was reported to confer resistance against 4,6-disubstituted deoxystreptamines but not against 4,5-disubstituted deoxystreptamines, such as neomycin (1). RmtC as well as RmtA, RmtB, and ArmA might well confer resistance against 4,6-disubstituted deoxystreptamines through a manner similar to that in aminolycoside-producing actinomycetes. The methylation site in the 16S rRNA introduced by these enzymes will be elucidated in a forthcoming study.

Interestingly, all the plasmid-mediated 16S rRNA methylase genes found so far were associated with some genes implicated in gene recombination systems. For example, the rmtA gene was flanked by a 262-bp sequence called the κγ element that was initially found in Tn5041 and that was predicted to be a relic of mobile genetic elements (26). The rmtB gene was located just downstream of the 3′ end of the insertion sequence of Tn3 (6). As for the two genes described above, the mode of actual translocation of the fragments containing the 16S rRNA methylase genes has not been elucidated in detail. On the other hand, it was reported that the armA gene was mediated by a composite transposon Tn1548 and was successfully transposed in vitro (8). Although the rmtC gene was also associated with a tnpA gene encoding a probable transposase, the actual mode of translocation of the regions carrying the rmtC gene is unclear. However, it is speculated that the presence of an ISEcpI-like element located upstream of rmtC would be responsible for the actual translocation process, because several CTX-M-type β-lactamase genes located downstream of tnpA within the ISEcpI element were able to be transposed in vitro (4, 21). Characterization of the genetic environment mediating the rmtC gene and the mode of translocation will be undertaken in another study.

As was observed in the phylogenic tree (Fig. 4), a cluster of plasmid-mediated 16S rRNA methylases is antithetical to that of the 16S rRNA methylases from actinomycetes. Although no progenitor of plasmid-mediated 16S rRNA methylases, including RmtA, RmtB, RmtC, and ArmA, has been found to date, these genes might have been derived from unknown environmental aminoglycoside-producing bacteria.

FIG. 4.

Dendrogram of 16S rRNA methylases. Sequences are from P. mirabilis (RmtC; GenBank accession number AB194779), P. aeruginosa (RmtA; GenBank accession number AB083212) (29), S. marcescens (RmtB; GenBank accession number AB103506) (6), K. pneumoniae (ArmA; GenBank accession number AY220558) (7), Micromonospora zionensis (Sgm; GenBank accession number A45282) (16), Micromonospora rosea (Grm; GenBank accession number M55521) (15), Micromonospora inyoensis (Srm1; GenBank accession number AY661430), Micromonospora echinospora (GrmA; GenBank accession number AY524043), Streptomyces sp. (Kan; GenBank accession number AJ414669), Streptomyces tenebrarius (NebM; GenBank accession number AJ550991), S. tenebrarius (KgmB; GenBank accession number S60108) (13), Streptoalloteichus hindustanus (NbrB; GenBank accession number AF038408), Streptoalloteichus kanamyceticus (Kmr; GenBank accession number AJ582817) (5), Micromonospora olivasterospora (FmrO; GenBank accession number D13171) (19), M. echinospora (GrmO; GenBank accession number AY524043), and Chlorobium tepidum TLS (putative methytransferase; GenBank accession number AAM72273). The “0.1” scale represents a genetic unit reflecting 10% of the amino acid substitutions calculated with the ClustalW program (http://www.ddbj.nig.ac.jp/search/Welcome-e.html) provided by the DDBJ (http://www.ddbj.nig.ac.jp/Welcome-e.html).

In conclusion, we identified a novel plasmid-mediated 16S rRNA methylase, RmtC, in a clinical P. mirabilis isolate that demonstrated an extraordinarily high level of aminoglycoside resistance like actinomycetes. The nosocomial transmission of pathogens that produce plasmid-dependent 16S rRNA methylases has recently been reported from Taiwan (28), and an ArmA-producing E. coli isolate was isolated from the feces of a diarrheic pig in Spain in 2002 (11, 12). Special caution should be taken because of the emergence and spread of pathogenic bacteria that have acquired various new antimicrobial resistance genes, including rmtC, rmtB, and rmtA, especially in both clinical and livestock farming environments, where large amounts of antimicrobial agents have routinely been used.