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Nomenclature of Plasmid-Mediated 16S rRNA Methylases Responsible for Panaminoglycoside Resistance

American Society for Microbiology
Antimicrobial Agents and Chemotherapy
Authors:
Yohei Doi
Yohei Doi
Yohei Doi
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Jun-ichi Wachino
Jun-ichi Wachino
Jun-ichi Wachino
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Yoshichika Arakawa at Nagoya University
Yoshichika Arakawa
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2008, p. 2287–2288 Vol. 52, No. 6
0066-4804/08/$08.00⫹0 doi:10.1128/AAC.00022-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Nomenclature of Plasmid-Mediated 16S rRNA Methylases Responsible for
Panaminoglycoside Resistance
䌤
Production of 16S rRNA methylase has recently drawn at-
tention as a novel aminoglycoside resistance mechanism in
pathogenic gram-negative bacteria (1). It confers very-high-
level resistance to all aminoglycosides that are currently avail-
able for parenteral formulation. Six distinct genes, rmtA,rmtB,
rmtC,rmtD,armA, and npmA, encoding their respective en-
zymes have been identified in clinical and veterinary strains
from various geographic areas, including East Asia, Europe,
and the Americas, since 2003 (1, 10). NpmA is the only enzyme
among them that methylates residue A1408, whereas the oth-
ers methylate residue G1405, both within the aminoacyl site (A
site) of the 16S rRNA (7, 10). All six genes are confirmed to be
or are likely to locate on plasmids (3, 4, 10, 11, 12, 14). Recent
findings also indicate that some of these genes are capable of
crossing the barrier between glucose-fermenting and nonfer-
menting species. For instance, armA has been identified in
both members of the family Enterobacteriaceae and in Acineto-
bacter baumannii (5, 13), and rmtD has been identified in
Klebsiella pneumoniae and Pseudomonas aeruginosa (unpub-
lished data). We will likely see an increasing number of reports
about this resistance mechanism, including identification of
genes encoding new 16S rRNA methylases.
Historically, the nomenclature of genes and enzymes for
many resistance mechanisms has become complicated and
nonsystematic (6). An extreme example is that of aminoglyco-
side acetyltransferases, where new gene names are arbitrarily
assigned from one of the two coexisting nomenclature systems
(9). The situation is somewhat better with ␤-lactamases and
macrolide resistance genes, due to a registry and guidelines,
respectively (http://www.lahey.org/Studies/) (8). To prevent
confusion over the nomenclature of 16S rRNA methylases, we
would like to propose practical rules for the nomenclature of
these enzymes, which shall apply to any relevant enzymes to be
identified in the future.
Currently, the highest and lowest identities of amino acid
sequences among the G1405 16S rRNA methylases are 81.7%
between RmtA and RmtB and 25.8% between ArmA and
RmtD, respectively (2, 3). On the other hand, identities lower
than 10% are observed between the G1405 16S rRNA meth-
ylases and the NpmA that methylates A1408 (10) (Table).
Thus, we propose the following rules. A gene that has an
amino acid identity greater than 95% with the closest known
16S rRNA methylase gene will be assigned a variant number
starting from two in the order of the dates on which the se-
quences are deposited in the GenBank/EBML/DDBJ, e.g.,
rmtA2 and then rmtA3, analogous to the nomenclature of the
qnr genes. A gene that has between 50 and 95% amino acid
identity with the closest known 16S rRNA methylase gene will
be assigned a new alphabet letter according to the closest
existing gene name, e.g., rmtE,rmtF,armB,or armC, provided
that the gene is shown to confer a consistent aminoglycoside
resistance profile. A gene that has either an amino acid identity
of less than 50% with the closest known 16S rRNA methylase
gene or that is proven to methylate a new residue of 16S rRNA
may be assigned a brand new gene name, like npmA, contin-
gent upon demonstration of 16S rRNA methylation activity of
the gene product and attributable resistant phenotype. Data
regarding 16S rRNA methylase genes in pathogenic bacteria
will be accumulated and provided at the following website
http://www.nih.go.jp/niid/16s_database/index.html.
Studies of plasmid-mediated 16S rRNA methylases identified
among pathogenic microbes were supported by a grant (H18-Shinkou-
011) from the Ministry of Health, Labor and Welfare, Japan.
REFERENCES
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resistance mechanism against aminoglycosides. Clin. Infect. Dis. 45:88–94.
2. Doi, Y., D. de Oliveira Garcia, J. Adams, and D. L. Paterson. 2007. Copro-
duction of novel 16S rRNA methylase RmtD and metallo-␤-lactamase
SPM-1 in a panresistant Pseudomonas aeruginosa isolate from Brazil. Anti-
microb. Agents Chemother. 51:852–856.
3. Doi, Y., K. Yokoyama, K. Yamane, J. Wachino, N. Shibata, T. Yagi, K.
Shibayama, H. Kato, and Y. Arakawa. 2004. Plasmid-mediated 16S rRNA
methylase in Serratia marcescens conferring high-level resistance to amino-
glycosides. Antimicrob. Agents Chemother. 48:491–496.
4. Galimand, M., P. Courvalin, and T. Lambert. 2003. Plasmid-mediated high-
level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA
methylation. Antimicrob. Agents Chemother. 47:2565–2571.
5. Galimand, M., S. Sabtcheva, P. Courvalin, and T. Lambert. 2005. Worldwide
disseminated armA aminoglycoside resistance methylase gene is borne by com-
posite transposon Tn1548. Antimicrob. Agents Chemother. 49:2949–2953.
6. Hall, R., and S. Partridge. 2003. Unambiguous numbering of antibiotic resis-
tance genes. Antimicrob. Agents Chemother. 47:3998–3999.
7. Liou, G. F., S. Yoshizawa, P. Courvalin, and M. Galimand. 2006. Aminogly-
coside resistance by ArmA-mediated ribosomal 16S methylation in human
bacterial pathogens. J. Mol. Biol. 359:358–364.
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Seppala. 1999. Nomenclature for macrolide and macrolide-lincosamide-
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10. Wachino, J., K. Shibayama, H. Kurokawa, K. Kimura, K. Yamane, S. Su-
zuki, N. Shibata, Y. Ike, and Y. Arakawa. 2007. Novel plasmid-mediated 16S
rRNA m
1
A1408 methyltransferase, NpmA, found in a clinically isolated
Escherichia coli strain resistant to structurally diverse aminoglycosides. An-
timicrob. Agents Chemother. 51:4401–4409.
11. Wachino, J., K. Yamane, K. Shibayama, H. Kurokawa, N. Shibata, S. Su-
zuki, Y. Doi, K. Kimura, Y. Ike, and Y. Arakawa. 2006. Novel plasmid-
mediated 16S rRNA methylase, RmtC, found in a Proteus mirabilis isolate
demonstrating extraordinary high-level resistance against various aminogly-
cosides. Antimicrob. Agents Chemother. 50:178–184.
12. Yamane, K., F. Rossi, M. G. Barberino, J. M. Adams-Haduch, Y. Doi, and
D. L. Paterson. 2008. 16S ribosomal RNA methylase RmtD produced by
Klebsiella pneumoniae in Brazil. J. Antimicrob. Chemother. 61:746–747.
TABLE 1. Identity of amino acid residues among the sequences of
plasmid-mediated 16S rRNA methylases
Methylase Amino acid sequence identity (%)
a
RmtA RmtB RmtC RmtD ArmA NpmA
RmtA 100 81.7 27.7 41.2 29.2 ⬍10
RmtB 100 29.5 41.3 28.9 ⬍10
RmtC 100 26.0 27.8 ⬍10
RmtD 100 25.8 ⬍10
ArmA 100 ⬍10
NpmA 100
a
Identities were calculated by GENETYX, Macintosh version 14.0.1 (SDC
Co., Ltd., Tokyo, Japan).
2287
13. Yamane, K., J. Wachino, Y. Doi, H. Kurokawa, and Y. Arakawa. 2005.
Global spread of multiple aminoglycoside resistance genes. Emerg. Infect.
Dis. 11:951–953.
14. Yokoyama, K., Y. Doi, K. Yamane, H. Kurokawa, N. Shibata, K. Shibayama,
T. Yagi, H. Kato, and Y. Arakawa. 2003. Acquisition of 16S rRNA methylase
gene in Pseudomonas aeruginosa. Lancet 362:1888–1893.
Yohei Doi
Division of Infectious Diseases
University of Pittsburgh Medical Center
Pittsburgh, Pennsylvania
Jun-ichi Wachino
Yoshichika Arakawa*
Department of Bacterial Pathogenesis and Infection Control
National Institute of Infectious Diseases
4-7-1 Gakuen
Musashi-Murayama, Tokyo 208-0011
Japan
*Phone: 81-42-561-0771, ext. 500
Fax: 81-42-561-7173
E-mail: yarakawa@nih.go.jp
䌤
Published ahead of print on 31 March 2008.
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... The corresponding genes are associated with transposon structures located on transferable plasmids, enhancing their horizontal spread. Isolates producing 16S rRNA methylases are multidrug-resistant, particularly to broad spectrum beta lactams through the production of ESBLs or MBLs [21,22] . ...
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  • Jan 2004
  • LANCET
Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.
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Aminoglycoside Resistance by ArmA-mediated Ribosomal 16 S Methylation in Human Bacterial Pathogens
Article
  • Jul 2006
Aminoglycosides are a medically important class of antibiotics used to treat serious infections. Methylation of the ribosomal target is an emerging mechanism that produces a high level of resistance to all clinically available aminoglycosides for systemic therapy except streptomycin. ArmA was the first methyltransferase using this mechanism to be discovered in a clinical isolate. We demonstrate that ArmA methylates the N7 position of nucleotide G1405 in 16S rRNA. Methylation at this position is presumed to mediate cellular resistance by blocking aminoglycoside binding by ribosomes. To test this hypothesis, we measured the binding of gentamicin by 30S subunits. Under our conditions, we did not observe binding by ribosomes methylated by ArmA. Furthermore, the ArmA methylation reaction is specific for the 30S ribosomal subunit; neither 16S rRNA alone nor the 70S ribosome is a substrate for this reaction under our experimental conditions, implicating ribosomal proteins in substrate recognition. The biochemical characteristics of ArmA place it in the Agr family of methyltransferases, whose members are predominantly anti-suicide genes from Actinomycetes aminoglycoside producers. The discrepancy between the 30% GC content of armA and the >60% GC content of Actinomycetes, however, calls into question the origin of armA. We demonstrate that surprisingly, the natural promoter of armA from gram-negative Klebsiella pneumoniae was active in gram-positive Bacillus subtilis, suggesting that armA originated from a low-GC, gram-positive aminoglycoside-producing organism.
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