Dataset Information

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM.

ABSTRACT: Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m?C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m?C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m?C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.

SUBMITTER: Galimand M

PROVIDER: S-EPMC3022275 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM.

Galimand Marc M Schmitt Emmanuelle E Panvert Michel M Desmolaize Benoît B Douthwaite Stephen S Mechulam Yves Y Courvalin Patrice P

RNA (New York, N.Y.) 20101215 2

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides k ...[more]

PMID: 21159796

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Project description:Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like (rep17) plasmid backbone. Relative to a previously reported typing scheme for the vanA-carrying Tn1546, new variants of the Tn1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (ISEfa16, ISEfa17, and ISEfa18). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium. Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Dataset Information

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM.

Publications

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM.

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