Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides

Abstract

Serratia marcescens S-95, which displayed an unusually high degree of resistance to aminoglycosides, including kanamycins and gentamicins, was isolated in 2002 from a patient in Japan. The resistance was mediated by a large plasmid which was nonconjugative but transferable to an Escherichia coli recipient by transformation. The gene responsible for the aminoglycoside resistance was cloned and sequenced. The deduced amino acid sequence of the resistance gene shared 82% identity with RmtA, which was recently identified as 16S rRNA methylase conferring high-level aminoglycoside resistance in Pseudomonas aeruginosa. Histidine-tagged recombinant protein showed methylation activity against E. coli 16S rRNA. The novel aminoglycoside resistance gene was therefore designated rmtB. The genetic environment of rmtB was further investigated. The sequence immediately upstream of rmtB contained the right end of transposon Tn3, including bla(TEM), while an open reading frame possibly encoding a transposase was identified downstream of the gene. This is the first report describing 16S rRNA methylase production in S. marcescens. The aminoglycoside resistance mechanism mediated by production of 16S rRNA methylase and subsequent ribosomal protection used to be confined to aminoglycoside-producing actinomycetes. However, it is now identified among pathogenic bacteria, including Enterobacteriaceae and P. aeruginosa in Japan. This is a cause for concern since other treatment options are often limited in patients requiring highly potent aminoglycosides such as amikacin and tobramycin.

FIG. 1.

(a) Schematic presentation of the 4.6-kb BamHI fragment of pS95B2 and 1.2-kb Sau3AI fragment of pS95S8. Shaded boxes indicate terminal inverted repeats (IR) of Tn3. The 1.2-kb insert of pS95S8 carries only IR and the rmtB gene as well as its possible promoter. (b) Region upstream of the rmtB gene. The nucleotide sequence containing the inverted repeat (IR) of the transposon 3 (Tn3) and region upstream of the rmtB gene are shown. The open reading frame of bla_TEM is terminated at ^1,238TAA. Several dyad symmetries are indicated with horizontal arrows. Possible −35 and −10 regions are boxed. IR sequence of Tn3 is enclosed with an open oblong box. A Shine-Dalgarno-like sequence (S.D.) (^1,399AGGAG) is located just upstream of the initiation codon (¹⁴¹⁰ATG) of the rmtB gene.

FIG. 2.

Alignment of the deduced amino acid sequence of RmtB with those of RmtA and ArmA. Asterisks indicate amino acid identities, and dots denote conserved replacements. Residues conserved among representative 16S rRNA methylases (GenBank accession no. AB083212, AB103506, AY220558, M55520, M55521, M87057, S60108, and Y15838) are underlined.

FIG. 3.

Methylation of 16S rRNA. The 16S rRNA from E. coli XL1-Blue was incubated with purified RmtB using [methyl-³H]SAM as a cofactor. The value of each point was calculated with three data points. Error bars, standard deviations.