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16S rRNA methyltransferase RmtC in Salmonella enterica serovar Virchow.

Aminoglycosides are used in treating a wide range of infections caused by both gram-negative and gram-positive bacteria and have been classified by the World Health Organization as critically important antimicrobial drugs in human medicine (1). They inhibit bacterial protein synthesis by binding irreversibly to the bacterial 16S ribosomal subunit, which thereby leads to cell death. Resistance to these antimicrobial agents usually results from production of aminoglycoside-modifying enzymes (such as acetyltransferases, phosphorylases, and adenyltransferases), reduced intracellular antimicrobial drug accumulation, or mutation of ribosomal proteins or rRNA. An additional mechanism, methylation of the aminoacyl site of 16S rRNA, confers high-level resistance to clinically important aminoglycosides such as amikacin, tobramycin, and gentamicin. Six types of 16S rRNA methyltransferase genes conferring resistance to these antimicrobial agents, armA, rmtA, rmtB, rmtC, rmtD, and npmA, have been identified (2,3). armA and rmtB are spread in enterobacteria worldwide, and the presence of other methyltransferase genes have not previously been reported in Europe (3). With the exceptions of armA and rmtB in porcine Escherichia coli from Spain and the People's Republic of China, respectively (4,5), all methyltransferase genes described have been identified in human clinical samples, for which a possible role for food in transmission of these determinants remains largely unknown. Despite large surveys performed to identify 16S rRNA methyltransferases, the rmtC gene has been detected in only 2 Proteus mirabilis clinical isolates from Japan and Australia in 2006 and 2008, respectively (3,6,7). In this study, 81,632 Salmonella and 10,700 Escherichia coli isolates obtained from the Health Protection Agency (HPA) Centre for Infections culture collection (isolated from January 2004 through December 2008) were screened for the presence of 16S rRNA methyltransferases.

The Study

Salmonella enterica (56 isolates) and Escherichia coli (24 isolates) were selected from the HPA collection based on their resistance to amikacin (breakpoint concentration routinely used in HPA Salmonella Reference Unit = 4 [micro]g/mL). Because 16S rRNA methyltransferases confer high-level resistance to amikacin, 13 S. enterica isolates were selected on the basis of ability to grow on Isosensitest agar containing 500 L g/mL amikacin, whereas none of the E. coli isolates grew under these conditions. All isolates belonged to serotype Virchow. Further antimicrobial susceptibility testing by microdilution by using dehydrated Sensititer plates following the CLSI guidelines confirmed high-level resistance to 4,6-disubstituted 2-deoxystreptamines (Table 1). PCR screening of the 13 isolates for armA, rmtA, rmtB, rmtC, and rmtD (8) identified rmtC. Nucleotide sequencing of the amplicons confirmed an rmtC gene with 100% identity with those originally identified in Proteus mirabilis strain ARS68 isolated from an inpatient in Japan (6) and P. mirabilis strain JIE273 from Australia (7). To our knowledge, this is the third report of rmtC-bearing bacteria. Class one integrons were amplified (9), and sequenced. Isolates resistant to neomycin bore the aac(6')-Ib gene cassette, whereas the dfrA1 gene was responsible for resistance to trimethoprim.

Twelve of the 13 S. enterica strains were originally isolated over a 4-year period from patients with clinical infection; 1 strain was obtained from frozen produce. Seven of 12 strains were obtained from patients with histories of foreign travel; 4 of the 7 patients had reported recent travel to India (Table 2). P. mirabilis strain JIE273 was also isolated from a patient who had recently returned from India (7). Investigations to ascertain the presence of rmtC genes in India are under way. To identify a possible link between the isolates, chromosomal DNA was embedded in agarose plugs prepared according to the pulsed-field gel electrophoresis (PFGE) protocol of PulseNet Europe (10). PFGE patterns showed only 1-2-band differences (Figure 1) and correlated with phage typing data (Table 1). All clinical isolates were recovered from feces, except a blood isolate recovered from a patient with invasive salmonellosis (Table 2). The temporal and geographic distribution of the isolates suggested independent acquisition of infections in most cases and possibly epidemiologically linked cases, e.g., strains 9 and 10 (Table 2; Figure 2).

PCR with primers ISEcp1R-F and rmtC-down (7) showed that the rmtC gene and immediate upstream sequences (GenBank accession nos. FJ984623-FJ984634 for human isolates and GQ131574 for the food isolate) were identical to those previously identified in P. mirabilis (6,7), in which ISEcp1 has been shown to play a role in the expression and transposition of the rmtC gene (11). However, the complete ISEcp1 element could not be amplified by using primers ISEcp1 5' and ISEcp1 reverse, which suggests either partial deletion of this element or involvement of a different ISEcp1-like element in spread of rmtC in Salmonella (6,12). Attempts to isolate rmtC by conjugal transfer to rifampin-resistant E. coli 20R764 were unsuccessful, as was electroporation into E. coli LMG194 and ElectroMAX DH10B cells (both Invitrogen, Paisley, UK) by using plasmid preparations. An [approximately equal to] 100-kb rmtC-bearing plasmid was previously transferred from P. mirabilis ARS68 by electroporation but could not be mobilized by conjugation (6), and attempts to transfer the rmtC plasmid from P. mirabilis JIE273 by electroporation and conjugation failed (7). This finding contrasts with some qualities of the other methyltransferases, such as armA and rmtB, which are mostly located on conjugative plasmids (8,13).

The location of the rmtC gene was determined with PFGE by using I-CeuI nuclease treatment. Agarose plugs were digested with 9.5 U I-CeuI nuclease (New England Biolabs, Beverly, MA, USA). Separated DNA fragments were transferred onto a nylon membrane (GE Healthcare, Madrid, Spain) and hybridized with 16S rDNA and rmtC probes labeled with DIG-11-dUTP. Hybridization, labeling, and detection were performed according to the manufacturer's recommendations (Roche Applied Science, Mannheim, Germany). A DNA band hybridized with both probes, showing that the rmtC gene was located on the chromosome. Results of hybridization of plasmid extractions (Plasmid Midi kit; QIAGEN, Inc., Chatworth, CA, USA) with the rmtC probe were negative (data not shown).


We describe the occurrence of 16S rRNA methyltransferase rmtC in Salmonella isolates and the rmtC gene in Europe. We also report that a producer of 16S rRNA methyltransferase was isolated from food.


The overall isolation frequency of 16S rRNA methyltransferase-producing S. enterica is low (13/81,632 strains) in the United Kingdom, and these genes were absent in E. coli. However, spread of multidrug-resistant isolates that express 16S rRNA methyltransferases, amplified by the association of these genes with the ISEcp1 element, raises clinical concern that further spread is likely. Ongoing surveillance of 16S rRNA methyltransferases in isolates found in food products and in humans and animals is crucial to delay the spread of resistance to these classes of antimicrobial agents.


While this manuscript was under revision, an S. enterica ser. Virchow isolate bearing the rmtC gene isolated from a child with a history of travel to India was reported in the United States (14).


DOI: 10.3201/eid1604.090736


We thank Michel Doumith of the HPA Antimicrobial Resistance Monitoring and Reference Laboratory for technical advice and assistance with the electroporation experiments.

This study was supported by work package 29 of the MedVet-Net Network of Excellence (FOOD-CT-2004-506122). Strain requests should be addressed to the Health Protection Agency Centre for Infections (


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(2.) Yamane K, Wachino J, Doi Y, Kurokawa H, Arakawa Y. Global spread of multiple aminoglycoside resistance genes. Emerg Infect Dis. 2005;11:951-3.

(3.) Fritsche TR, Castanheira M, Miller G, Jones R, Armstrong E. Detection of methyltransferases conferring high-level resistance to aminoglycosides in enterobacteriaceae from Europe, North America, and Latin America. Antimicrob Agents Chemother. 2008;52:1843-5. DOI: 10.1128/AAC.01477-07

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(6.) Wachino J, Yamane K, Shibayama K, Kurokawa H, Shibata N, Suzuki S, et al. Novel plasmid-mediated 16S rRNA methylase, RmtC, found in a Proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides. Antimicrob Agents Chemother. 2006;50:178-84. DOI: 10.1128/AAC.50.1.178184.2006

(7.) Zong Z, Partridge S, Iredell J. RmtC 16S rRNA methyltransferase in Australia. Antimicrob Agents Chemother. 2008;52:794-5. DOI: 10.1128/AAC.01399-07

(8.) Doi Y, Arakawa Y. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin Infect Dis. 2007;45:88-94. DOI: 10.1086/518605

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Author affiliations: Health Protection Agency Centre for Infections, London, UK (K.L. Hopkins); and Universidad Complutense de Madrid, Madrid, Spain (J.A. Escudero, L. Hidalgo, B. Gonzalez-Zorn)

Dr Hopkins is a researcher in the Gastrointestinal, Emerging and Zoonotic Infections Department at the Health Protection Agency Centre for Infections (London, UK). Her research interests focus on mechanisms of antimicrobial resistance in enteric pathogens and development and application of DNA-based typing methods for epidemiologic investigations.

Address for correspondence: Bruno Gonzalez-Zorn, Departamento de Sanidad Animal, Facultad de Veterinaria and VISAVET, Universidad Complutense de Madrid, 28040, Spain; email:
Table 1. Phage types for Salmonella enterica serovar Virchow
isolates bearing rmtC and MICs of selected antimicrobial agents *

Isolate        type    GEN    KAN    AMK    TOB    ARB    NEO

HO 5164 0340    ND     >512   >512   >512   >512   >512   64

HO 5366 0426    30     >512   >512   >512   >512   >512    2

HO 6018 0151    30     >512   >512   >512   >512   >512    2
HO 6316 0322    30     >512   >512   >512   >512   >512   32

HO 6398 0463    30     >512   >512   >512   >512   >512   32

HO 7078 0136    30     >512   >512   >512   >512   >512    2

HO 7310 0210    31     >512   >512   >512   >512   >512    4
HO 7468 0335    25     >512   >512   >512   >512   >512    2
HO 7474 0467    25     >512   >512   >512   >512   >512    4

HO 7496 0137    25     >512   >512   >512   >512   >512    2
HO 7512 0259    25     >512   >512   >512   >512   >512    4
HO 8354 0857    25     >512   >512   >512   >512   >512    4

HO 8512 0713    25     >512   >512   >512   >512   >512    4

Isolate             TMP        CPX         AMP

HO 5164 0340   [less than or   0.5          1
               equal to] 0.5
HO 5366 0426        >32        0.25   [less than or
                                      equal to] 0.05
HO 6018 0151        >32        0.25         1
HO 6316 0322   [less than or   0.25         1
               equal to] 0.5
HO 6398 0463   [less than or   0.25         1
               equal to] 0.5
HO 7078 0136        >32        0.25   [less than or
                                      equal to] 0.05
HO 7310 0210        >32        0.25         1
HO 7468 0335        >32        0.5          1
HO 7474 0467        >32        0.25   [less than or
                                      equal to] 0.05
HO 7496 0137        >32        0.25         1
HO 7512 0259        >32        0.25         1
HO 8354 0857        >32        0.25   [less than or
                                      equal to] 0.05
HO 8512 0713        >32        0.25         1

* MICs are given in [micro]g/mL. GEN, gentamicin; KAN, kanamycin;
AMK, amikacin; TOB, tobramycin; ARB, arbekacin; NEO, neomycin;
TMP, trimethoprim; CPX, ciprofloxacin; AMP, ampicillin; ND, not

Table 2. Epidemiologic information from rmtC-positive Salmonella
enterica serovar Virchow isolates, United Kingdom, 2004-2008 *

Isolate        Date received   Map no.      Location       Sample type

HO 5164 0340    2005 Apr 20       1          Reading          Feces
HO 5366 0426    2005 Sept 8       2          London           Feces
HO 6018 0151    2006 Jan 6        3          Wexham           Feces
HO 6316 0322    2006 Aug 3        4      Nottinghamshire      Feces
HO 6398 0463   2006 Sept 29       5          London           Blood
HO 7078 0136    2007 Feb 16       6         Orpington         Feces
HO 7310 0210   2007 July 30       7          Wrexham          Feces
HO 7468 0335    2007 Nov 16       8          Bedford          Feces
HO 7474 0467    2007 Nov 21       9        West Sussex        Feces
HO 7496 0137    2007 Dec 6       10        West Sussex        Feces
HO 7512 0259    2007 Dec 18      11          Surrey           Feces
HO 8354 0857    2008 Aug 27      12           Kent             ND
HO 8512 0713    2008 Dec 16      13         Spalding          Food

Isolate             Symptoms          Travel history

HO 5164 0340           ND                   ND
HO 5366 0426        Diarrhea        Unknown destination
HO 6018 0151           ND                   ND
HO 6316 0322        Diarrhea         No recent travel
HO 6398 0463   Fever and diarrhea          India
HO 7078 0136        Diarrhea               India
HO 7310 0210        Diarrhea        Unknown destination
HO 7468 0335       Enteritis               India
HO 7474 0467        Diarrhea                ND
HO 7496 0137        Diarrhea                ND
HO 7512 0259        Diarrhea               India
HO 8354 0857        Diarrhea        Unknown destination
HO 8512 0713           NA                   NA

* ND, not determined; NA, not applicable.
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Article Details
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Title Annotation:DISPATCHES
Author:Hopkins, Katie L.; Escudero, Jose A.; Hidalgo, Laura; Gonzalez-Zorn, Bruno
Publication:Emerging Infectious Diseases
Article Type:Clinical report
Geographic Code:4EUUK
Date:Apr 1, 2010
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