16S Ribosomal RNA Methylation: Emerging Resistance Mechanism against Aminoglycosides

88 • CID 2007:45 (1 July) • ANTIMICROBIAL RESISTANCE

A N T I M I C R O B I A L R E S I S T A N C E I N V I T E D A R T I C L EGeorge M. Eliopoulos, Section Editor

16S Ribosomal RNA Methylation: Emerging ResistanceMechanism against Aminoglycosides

Yohei Doi1 and Yoshichika Arakawa2

1Division of Infectious Diseases, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; and 2Department of Bacterial Pathogenesis and Infection Control,National Institute of Infectious Diseases, Tokyo, Japan

Methylation of 16S ribosomal RNA (rRNA) has recently emerged as a new mechanism of resistance against aminoglycosides

among gram-negative pathogens belonging to the family Enterobacteriaceae and glucose-nonfermentative microbes, including

Pseudomonas aeruginosa and Acinetobacter species. This event is mediated by a newly recognized group of 16S rRNA

methylases, which share modest similarity to those produced by aminoglycoside-producing actinomycetes. Their presence

confers a high level of resistance to all parenterally administered aminoglycosides that are currently in clinical use. The

responsible genes are mostly located on transposons within transferable plasmids, which provides them with the potential

to spread horizontally and may in part explain the already worldwide distribution of this novel resistance mechanism. Some

of these organisms have been found to coproduce extended-spectrum b-lactamases or metallo-b-lactamases, contributing to

their multidrug-resistant phenotypes. A 2-tiered approach, consisting of disk diffusion tests followed by confirmation with

polymerase chain reaction, is recommended for detection of 16S rRNA methylase–mediated resistance.

Aminoglycosides continue to play an important role in the

management of serious infections caused by gram-negative

pathogens, often in combination with broad-spectrum b-lac-

tams. They bind specifically to the aminoacyl site (A-site) of

16S rRNA within the prokaryotic 30S ribosomal subunits and

interfere with protein synthesis [1]. The most commonly en-

countered mechanism of resistance to aminoglycosides is en-

zymatic inactivation, which is mediated by 3 classes of en-

zymes: acetyltransferases, nucleotidyltransferases, and

phosphotransferases [2]. They are further divided into sub-

classes that are based on the site of modification and the spec-

trum of resistance within the class of antimicrobials. Other

known mechanisms of aminoglycoside resistance include defect

of cellular permeability, active efflux, and, rarely, nucleotide

substitution of the target molecule [1].

Received 30 November 2006; accepted 24 March 2007; electronically published 21 May2007.

Reprints or correspondence: Dr. Yohei Doi, Div. of Infectious Diseases, University ofPittsburgh Medical Center, Ste. 3A, Falk Medical Bldg., 3601 Fifth Ave., Pittsburgh, PA 15213([email protected]).

Clinical Infectious Diseases 2007; 45:88–94� 2007 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2007/4501-0018$15.00DOI: 10.1086/518605

Aminoglycosides are produced by species of actinomycetes,

such as Streptomyces species and Micromonospora species. These

actinomycetes are intrinsically resistant to the aminoglycosides

that they produce [3]. In many cases, this resistance is caused

by ribosomal protection through methylation of specific nu-

cleotides within the A-site of 16S rRNA, which hampers binding

of aminoglycosides to the 30S ribosomal subunits and serves

as a means of self-protection. Until recently, this resistance

mechanism was believed to be absent in clinically relevant

species.

However, clinical strains of Pseudomonas aeruginosa and

Klebsiella pneumoniae that produced 16S rRNA methylases were

reported in 2003 [4, 5]. These enzymes were found to confer

extraordinarily high levels of resistance to clinically useful ami-

noglycosides, such as amikacin, tobramycin, and gentamicin.

Since 2003, the literature on this newly recognized resistance

mechanism has grown rapidly, documenting identification of

new enzymes and their spread to different species in various

parts of the world. In the present article, we will first review

aminoglycoside resistance caused by 16S rRNA methylation in

aminoglycoside-producing actinomycetes. We will then discuss

the current knowledge of this emerging, plasmid-mediated re-

sistance mechanism that is found among gram-negative path-

at California Institute of T

echnology on August 27, 2014

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ANTIMICROBIAL RESISTANCE • CID 2007:45 (1 July) • 89

Figure 1. Dendrogram of 16S rRNA methylases of gram-negative bacteria and of representative actinomycetes. KsgA is a nonresistance 16S rRNAmethylase intrinsic in Escherichia coli. The protein sequences were obtained from the databases of GenBank, European Molecular Biology Laboratory,and DNA Data Bank of Japan and were aligned using ClustalW [11]. M. echinospora, Micromonospora echinospora; M. inyonensis, Micromonosporainyonensis; M. olivasterospora, Micromonospora olivasterospora; M. purpurea, Micromonospora purpurea; M. rosea, Methylocystis rosea; M. zionensis,Micromonospora zionensis; S. hindustanus, Streptoalloteichus hindustanus; S. hirsuta, Saccharopolyspora hirsuta; S. kanamyceticus, Streptomyceskanamyceticus; S. tenebrarius, Streptomyces tenebrarius.

ogens, with an emphasis on the diagnostic and therapeutic

implications.

MODIFICATION OF RRNA IN BACTERIA

The ribosome is a large enzyme consisting of multiple proteins

and RNA components [6]. In bacteria, it comprises 30S and

50S subunits, the former containing 16S rRNA and the latter

containing 23S and 5S rRNAs. Posttranscriptional modification

events of RNAs, such as methylation of nucleosides, take place

following the generation of initial RNA transcripts. They are

predominantly reported in tRNA, but they are also reported

in rRNA. Escherichia coli, for example, is known to contain 10

methylated nucleosides in 16S rRNA and 14 methylated nu-

cleotides in 23S rRNA [7]. The primary roles of rRNA meth-

ylation likely include modulation of rRNA maturation, stabi-

lization of rRNA structures, and alteration of translation rates.

For instance, mutants of E. coli that were deficient in the pro-

duction of KsgA (RsmA) showed an increased leakiness of non-

sense and frameshift mutants and alteration in decoding fidelity

at both the A-site and peptidyl-tRNA site of 16S rRNA [8, 9].

In addition, some of the posttranscriptional methylation events

are known to confer resistance to antimicrobials that target

rRNA.

RESISTANCE IN ACTINOMYCETES MEDIATEDBY 16S RRNA METHYLATION

A number of actinomycetes are known to be intrinsically re-

sistant to aminoglycosides that they produce themselves. The

mechanisms of resistance include inactivation of aminoglyco-

sides by production of aminoglycoside-modifying enzymes and

protection of 16S rRNA within the 30S ribosome subunit by

production of 16S rRNA methylase. The latter mechanism re-

sults in high-level resistance to multiple aminoglycosides. It

represents an efficient means to avoid inhibition of their own

protein synthesis and is prevalent among aminoglycoside-pro-

ducing actinomycetes (figure 1) [3].

Two sites of methylation within 16S rRNA that lead to dif-

ferent aminoglycoside-resistance phenotypes have been iden-

tified [10]. One group of 16S rRNA methylases, such as that

produced by the istamycin producer Streptomyces tenjimarien-

sis, methylates residue A1408 (figure 2). Another group of 16S

rRNA methylases, exemplified by those produced by genta-

micin-producer Micromonospora purpurea, methylates residue

G1405. The former confers resistance to kanamycin and apra-

mycin but not gentamicin, whereas the latter confers resistance

to kanamycin and gentamicin but not apramycin. Both of these

residues are located within the A-site–decoding region of 16S




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Figure 2. The positions of modifications in the aminoacyl site (A-site)decoding region in 16S rRNA (modified from Beauclerk et al. [10] withpermission). ArmA is known to methylate G1405 [19]. Based on thecommon KanR-GenR phenotype, the other methylases in gram-negativeorganisms likely methylate the same residue. Methylases that modifyA1408 have only been reported in actinomycetes. Apr, apramycin; Gen,gentamicin; Kan, kanamycin.

rRNA, where aminoglycosides are known to bind and interfere

with accurate translation through blocking translocation of

peptidyl-tRNA from the A-site to the peptidyl-tRNA site [12].

RESISTANCE IN GRAM-NEGATIVE PATHOGENSMEDIATED BY 16S RRNA METHYLATION

Because it became evident that many aminoglycoside-produc-

ing actinomycetes used ribosomal resistance afforded by meth-

ylation of 16S rRNA, the question was raised as to why the

same resistance mechanism was not identified in clinically rel-

evant species. It was speculated that such resistance mechanisms

could exist but were possibly missed because of limited screen-

ing methods, because the resistance pattern could mimic that

of organisms producing multiple aminoglycoside modifying en-

zymes. In 2002, a gene encoding 16S rRNA methylase, later

designated ArmA, was deposited to the European Molecular

Biology Laboratory and GenBank as part of a plasmid sequence

from Citrobacter freundii in Poland (accession number

AF550415). No additional findings have been published to date.

Then, in 2003, an aminoglycoside-resistant P. aeruginosa clin-

ical isolate from Japan was reported to produce 16S rRNA

methylase [5]. The deduced amino acid sequence of this new

enzyme, designated RmtA, shared modest (up to 35%) identity

with 16S rRNA methylases of various actinomycetes. RmtA

displayed methylation activity against 16S rRNA of 30S ribo-

somal subunits derived from a susceptible strain of P. aerugi-

nosa. When rmtA was cloned and expressed in E. coli and P.

aeruginosa, it was found to confer a high degree of resistance

to all 4,6-disubstituted deoxystreptamines, which include gen-

tamicin, tobramycin, and amikacin. As described above, a pu-

tative 16S rRNA methylase gene was initially found in a C.

freundii clinical isolate from Poland. This gene was also iden-

tified in K. pneumoniae from France [4]. The gene product

ArmA was also shown to confer high-level resistance to 4,6-

disubstituted deoxystreptamines. The identity of the amino acid

sequence of ArmA with those of RmtA and other 16S rRNA

methylases from actinomycetes was only modest, ranging be-

tween 30% and 35%. The structural gene of RmtA was asso-

ciated with a genetic element that resembled a mercury-resis-

tance transposon Tn5041 on a transferable plasmid [13]. The

guanine cytosine content of rmtA was 55%, suggesting its origin

from some guanine cytosine–rich microbe, including actino-

mycetes. The structural gene for ArmA was reported to be

located on functional composite transposon Tn1548 [14]. The

guanine cytosine content of armA was 30%, suggesting that it

was derived from some microbe with lower guanine cytosine

content. These findings point to the possibility that these genes

were acquired horizontally from diverse nonpathogenic envi-

ronmental microbes, but their exact origins remain unknown.

Several other 16S rRNA methylases were subsequently dis-

covered among gram-negative bacteria, and a total of 5 are

known to date (figure 1). RmtB was identified in Serratia mar-

cescens from Japan [15]. RmtB is closest to RmtA, sharing 82%

identity at the amino acid level. The structural gene for RmtB

was located adjacent to a Tn3–like transposon on a large trans-

ferable plasmid. RmtC was found in a Proteus mirabilis clinical

strain from Japan that was rather distant in phylogeny from

the 3 enzymes already reported [16]. The structural gene for

RmtC is also located next to a transposon-mediated recom-

bination system termed ISEcp1, and the methylase gene was

shown to be mobilizable from plasmid to plasmid [17]. The

most recently identified 16S rRNA methylase is RmtD, which

shares moderate identity (40%–42%) with RmtA and RmtB

[18]. RmtD was found to be produced by a P. aeruginosa clinical

strain from Brazil, which also produced metallo-b-lactamase

SPM-1. This particular strain was, therefore, highly resistant to

carbapenems as well as to aminoglycosides.

These newly identified 16S rRNA methylases in gram-neg-

ative bacilli all confer resistance to 4,6-disubstituted deoxys-

treptamines, including gentamicin, tobramycin, and amikacin,

but not including 4,5-disubstituted deoxystreptamines, such as

neomycin, 4-monosubstituted deoxystreptamines, such as pa-

romomycin, or streptomycin, which lacks a deoxystreptamine

ring. The level of resistance to tobramycin appears to be slightly

lower than the level of resistance to other 4,6-disubstituted

deoxystreptamines when the responsible genes are cloned and

expressed in experimental strains of E. coli, such as XL1-Blue,

DH5a, or INVaF′ (MIC, 64–256 mg/mL) (table 1). However,




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Table 1. Aminoglycoside resistance pattern conferred by G140516S rRNA methylase.

Drug Susceptibility

4,6-disubstituted DOS Highly resistantGentamicin Highly resistantTobramycin Resistant, highly resistantAmikacin Highly resistant

4,5-disubstituted DOS; neomycin SusceptibleMonosubstituted DOS; apramycin SusceptibleNo DOS ring; streptomycin Susceptible

NOTE. DOS, deoxystreptamine.

Table 2. Genetic association and geographic distribution of 16S rRNA methylase genes.

16S rRNAmethylase gene

Guaninecytosine

content, %

Molecularweight of

product, kDaIS or

transposon

Associatedb-lactamase

genes Bacterial species (country or countries)Country,

reference(s)

rmtA 55.4 27.4 IS6100, kg

element,Tn4051

Pseudomonas aeruginosa (J) J [5, 21]

rmtB 55.6 27.4 Tn3 blaTEM-1, blaCTX-M-14 Serratia marcescens (J), Eschericia coli (J, T, C, Be),Klebsiella pneumoniae (J, T, K), Klebsiella oxytoca (J),Citrobacter freundii (K)

J [15], T [22],K [20, 23], C(DQ345788),Be [24]

rmtC 41.1 32.1 ISEcp1 Proteus mirabilis (J) J [16]

rmtD 59.3 27.7 ISCR blaSPM-1 P. aeruginosa (Br) Br [18]

armA 30.4 30.2 IS26,Tn1548

blaCTX-M-3 S. marcescens (J, F, K), C. freundii (P, K, F, Bu), Citrobac-ter amalonaticus (Be), K. pneumoniae (F, S,T, K, Bu,Be), K. oxytoca (Bu), E. coli (J, T, S, F, Bu, Be), Entero-bacter cloacae (F, K, Be), Enterobacter aerogenes (Bu,Be), P. mirabilis (F), Salmonella enterica Enteritidis (Bu),S. enterica Oranienburg (P), Shigella flexneri (Bu), Aci-netobacter species (J, K)

P (AY522431)[14], Bu [14],F [14], S[26], Be [24],J [21], T[22], K [20,23]

NOTE. DQ345788 and AY522431 are European Molecular Biology Laboratory and GenBank accession numbers. Be, Belgium; Br, Brazil; Bu, Bulgaria; C,China; F, France; I, India; IS, insertion sequence; J, Japan; K, South Korea; P, Poland; S, Spain; T, Taiwan.

the MICs of tobramycin for the clinical strains that produce

16S rRNA methylase usually range between 512 and 1024 mg/

mL or greater. This pattern of resistance resembles that of the

methylase of M. purpurea, which is known to methylate residue

G1405, as discussed above. Recently, residue G1405 of 16S

rRNA in the 30S ribosomal subunit was confirmed as the site

of methylation by ArmA using primer extension method (figure

2) [19]. No methylase that modifies residue A1408 has been

reported in gram-negative pathogens at this time.

PREVALENCE OF 16S RRNA METHYLASE–MEDIATED RESISTANCE

Data on the prevalence of aminoglycoside resistance mediated

by 16S rRNA methylation among gram-negative bacilli is still

scarce. The prevalence of RmtA among clinical isolates of P.

aeruginosa in Japan was estimated to be at least 0.4% during

a national surveillance when screened by high-level multiple-

aminoglycoside resistance followed by PCR confirmation [5].

Subsequently, RmtB has been detected in various species be-

longing to the family Enterobacteriaceae, including K. pneu-

moniae, Klebsiella oxytoca, E. coli, and C. freundii in Japan,

Taiwan, South Korea, China, and Belgium [20––25]. ArmA,

which was initially found in C. freundii and later characterized

in K. pneumoniae, has also been identified in clinical isolates

of E. coli, S. marcescens, Enterobacter cloacae, Salmonella enter-

ica, Shigella flexneri, and Acinetobacter species from various

countries in East Asia and Eastern and Western Europe (table

2) [14, 20–23]. A recent report from a university hospital in

Taiwan estimated the prevalence of ArmA and RmtB to be 0.9%

and 0.3%, respectively, among K. pneumoniae and E. coli when

screened by resistance to amikacin and confirmed by PCR [22].

RmtA and RmtD have only been reported from P. aeruginosa

in Japan and Brazil, respectively [5, 18, 21]. These findings

indicate that 16S rRNA methylase genes are already dissemi-

nated globally among pathogenic gram-negative bacilli, al-

though the overall prevalence appears to remain low.

Strains producing 16S rRNA methylase have been reported

from livestock, as well. Plasmid-mediated armA and rmtB genes

have been identified from E. coli in swine from Spain and China,

respectively [26, 25]. A large amount of aminoglycosides, in-

cluding kanamycin, gentamicin, apramycin, and streptomycin,

has been consumed in veterinary medicine. This may have

served as a selective pressure for enteric gram-negative organ-

isms to acquire 16S rRNA methylase genes, possibly from non-

pathogenic environmental actinomycetes that intrinsically pro-

duced aminoglycosides or similar 16S rRNA inhibitors, and

then maintain and spread them to humans through the food

supply chains. Monitoring for high-level aminoglycoside resis-

tance among gram-negative pathogens mediated by this new

resistance mechanism would, therefore, be important in live-

stock breeding environments, as well.




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Figure 3. Suggested approach in identifying organisms producing 16S rRNA methylase. aAlternatively, MICs of 256 mg/mL to all aminoglycosidestested may be used.

Figure 4. Electrophoresis profile of the PCR products. Lane M, 100–base pair (bp) marker; lane 1, rmtB; lane 2, rmtC; lane 3, armA; lane 4,rmtA; lane 5, rmtD.

CLINICAL IMPLICATION OF AMINOGLYCOSIDERESISTANCE DUE TO 16S RRNA METHYLATION

Despite its currently low prevalence, the global spread of gram-

negative bacilli producing 16S rRNA methylase is concerning

for several reasons. First, these gram-negative bacilli show a

very high level of resistance to most clinically useful aminog-

lycosides, including gentamicin, tobramycin, and amikacin,

which cannot be overcome by dose adjustments. Second, all of

the structural genes of known 16S rRNA methylases are as-

sociated with mobile genetic elements, such as transposon,

some of which have been proven functional, providing them

with the means to spread horizontally to other strains and

species. Third, these organisms appear to possess a high po-

tential for developing multidrug resistance, especially via ac-

quisition of various b-lactamase genes. Of 35 ArmA- and

RmtB-positive clinical isolates, 33 produced CTX-M- or SHV-

type extended-spectrum b-lactamases (ESBLs) in a Taiwanese

university hospital [22]. Similar observations have also been

made in South Korea [20]. It has been reported that the struc-

tural gene for ArmA, the most prevalent methylase thus far, is

located on a composite transposon Tn1548 on a transferable

plasmid and is frequently associated with CTX-M-3–type ESBL

genes [14]. Production of CTX-M-9–type ESBLs is seen in

many strains with RmtB [22] (K. Yamane, unpublished data).

RmtD was initially reported in a P. aeruginosa strain that co-

produced SPM-1 metallo-b-lactamase, which is endemic in

Brazil. The latter combination would render ineffective a potent

double-coverage regimen of carbapenem plus aminoglycoside.

Currently, there are no data regarding clinical outcome in pa-

tients infected with these organisms. Nonetheless, it would be

prudent to pay careful attention to the antibiogram and to

maintain a low threshold to screen for ESBL production when

these 16S rRNA methylase–producing gram-negative bacteria

are encountered in clinical situations. Contact precautions

should be used for patients when coproduction of 16S rRNA

methylase and ESBL or metallo-b-lactamase is highly suspected

or confirmed.

DETECTION OF AMINOGLYCOSIDERESISTANCE DUE TO 16S RRNA METHYLATION

Screening for 16S rRNA methylase–producing organisms may

be considered for epidemiologic purposes when nosocomial or

foodborne spread of such bacteria is suspected. Detection of

this resistance mechanism may pose a challenge in clinical lab-

oratories. Gram-negative bacilli commonly produce aminogly-

coside-modifying enzymes, such as acetyltransferases, nucleo-

tidyltransferases, and phosphotransferases. When 11 of these

enzymes are produced in single organisms, they could readily

become resistant to multiple aminoglycosides. The hallmark of

resistance mediated by 16S rRNA methylase that methylates

residue G1405 is the very high level of resistance to all par-

enterally formulated aminoglycosides (MIC is typically �256

mg/mL), except streptomycin. This, however, may not be dis-

cernible in routine susceptibility testing conducted in the clin-

ical laboratory, especially when automated susceptibility testing

systems are used that only measure MICs close to the break-

points of each aminoglycoside. One unique characteristic of

these methylases is the high-level resistance to arbekacin that

they confer. Arbekacin is a semisynthetic aminoglycoside de-

rived from dibekacin [27]. It has activity against staphylococci,




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Table 3. Primers for detection of 16S rRNA methylase genes.

Species, gene, primer Sequence (5′r3′)Amplicon size,

base pairs

Enterobacteriaceae and Acinetobacter speciesrmtB

rmtB-F GCT TTC TGC GGG CGA TGT AA 173rmtB-R ATG CAA TGC CGC GCT CGT AT

rmtCrmtC-F CGA AGA AGT AAC AGC CAA AG 711rmtC-R ATC CCA ACA TCT CTC CCA CT

armAarmA-F ATT CTG CCT ATC CTA ATT GG 315armA-R ACC TAT ACT TTA TCG TCG TC

Pseudomonas aeruginosarmtA

rmtA-F CTA GCG TCC ATC CTT TCC TC 635rmtA-R TTG CTT CCA TGC CCT TGC C

rmtDrmtD-F CGG CAC GCG ATT GGG AAG C 401rmtD-R CGG AAA CGA TGC GAC GAT

NOTE. Both PCR for rmtB, rmtC, and armA and PCR for rmtA and rmtD may be performed in a mutiplex protocol.Thermal cycling condition is as follows: initial denaturation at 96�C for 5 min, followed by 30 cycles at 96�C for 30 s,at 55�C for 30 s, at 72�C for 1 min, with a final extension at 72�C for 5 min.

as well as against gram-negative bacteria, and it is currently

approved only for treatment of multidrug-resistant Staphylo-

coccus aureus infections in Japan. It is generally stable against

the actions of aminoglycoside-modifying enzymes, with the ex-

ception of the bifunctional enzyme AAC(6′)/APH(2′′), which is

known to be produced by some multidrug-resistant S. aureus

and enterococcal strains, and it may result in low-level arbek-

acin resistance. However, arbekacin is not readily available in

many instances. Therefore, we propose the following approach

in screening for 16S rRNA methylase production (figure 3).

When a strain belonging to the family Enterobacteriaceae or

glucose nonfermentative species, such as P. aeruginosa or Aci-

netobacter species, meets the criteria set forth by the Clinical

and Laboratory Standards Institute for resistance to multiple

aminoglycosides, disk diffusion test using gentamicin, amika-

cin, and arbekacin (if available) may be performed. Production

of 16S rRNA methylase is suspected when no or little inhibitory

zone is observed with any of the aminoglycoside disks. The

addition of an arbekacin disk is desirable, because it raises the

positive predictive value of this method to �90%, compared

with ∼60% when performed only with amikacin [20]. Alter-

natively, if the MICs of these aminoglycosides are to be used,

a cutoff value of 256 mg/mL appears to provide excellent pos-

itive predictive value. All of the RmtA-, RmtB-, RmtC-, RmtD-

and ArmA-producing isolates that we have tested to date have

had MICs of these aminoglycosides �256 mg/mL—an obser-

vation confirmed elsewhere [20]. Currently, PCR is the only

confirmatory method available. Multiplex PCR may be per-

formed for armA, rmtB, and rmtC in strains belonging to the

family Enterobacteriaceae and Acinetobacter species and for

rmtA and rmtD in P. aeruginosa (figure 4). Recommended

primers and thermal cycling conditions are listed in table 3. Of

note, some other glucose nonfermentative organisms, such as

Stenotrophomonas maltophilia and Burkholderia cepacia, may

also present with panaminoglycoside-resistant phenotype, but

the mechanism of this resistance has not been elucidated.

CONCLUSIONS

Methylation of the 16S rRNA in the 30S ribosomal subunit

confers high-level resistance to most clinically useful aminog-

lycosides by inhibiting their access to the site of action. Gram-

negative pathogens possessing this mechanism were first re-

ported in 2003 and are increasingly reported worldwide. The

organisms that produce 16S rRNA methylase are often mul-

tidrug resistant, especially against broad-spectrum b-lactams via

production of ESBLs or metallo-b-lactamases, a process that is

likely to be facilitated by the association of 16S rRNA methylase

genes with genetic recombination systems. Although the clinical

outcome for patients infected with these organisms is still un-

known, early identification of the resistance mechanisms will

be helpful in optimizing antimicrobial therapy and infection-

control measures. A 2-tiered approach, consisting of disk dif-

fusion tests followed by PCR confirmation, is recommended

for detection of 16S rRNA methylase-mediated resistance.




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Acknowledgments

We thank Dr. Kunikazu Yamane and Dr. Jun-ichi Wachino for theircontribution to this article.

Financial support. Principal data on plasmid-mediated 16S rRNAmethylases demonstrated in this article were obtained by a series of in-vestigations supported by the Ministry of Health, Labor and Welfare, Japan.

Potential conflicts of interest. All authors: no conflicts.

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16S Ribosomal RNA Methylation: Emerging Resistance Mechanism against Aminoglycosides