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.

Spread of Novel Aminoglycoside Resistance Gene aac ( 6 ′)- Iad among Acinetobacter Clinical Isolates in Japan

“…PCR analysis was performed for the 16 non-amikacinsusceptible Acinetobacter strains with primers ABA-F (5Ј-TTT GGC TAT GAT CCT ATG-3Ј) and ABA-R (5Ј-CAT GTC GAA CAA GTA CGC-3Ј) to amplify an internal fragment of the aac(6Ј)-Iad gene. The conditions used have been described previously (7). When amplicons were obtained, they were directly sequenced with the same primers.…”

“…Conjugation experiments were conducted by using rifampin-resistant E. coli CSH2 and Acinetobacter calcoaceticus DU1, a rifampin-resistant derivative of A. calcoaceticus ATCC 33305, as the recipients by the broth mating method (7). Transconjugants were selected on LB agar supplemented with rifampin (50 g/ml) and kanamycin (10 g/ml).…”

Functional importance of individual rRNA 2′-O-ribose methylations revealed by high-resolution phenotyping

“…Since perturbing the topology of the binding pocket is enough to induce resistanceconferring phenotypes, nucleotide modifications, or lack thereof, it should also be expected to have the same effect. Indeed, it is known that many aminoglycoside-producing organisms express rRNA methylases which methylate specific rRNA residues, resulting in protection from their own antibiotic products, as in the case of paromomycin and anisomycin resistance of Serratia marcescens (Doi et al 2004). …”

Plasmid-borne armA methylase gene, together with bla CTX-M-15 and bla TEM-1, in a Klebsiella oxytoca isolate from China

“…16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD and npmA) were detected by PCR using primers and conditions described previously (Yokoyama et al, 2003;Doi et al, 2004;Yamane et al, 2005;Wachino et al, 2007;Yu et al, 2007b). For the 16S rRNA methylase-producing isolates, amplification and identification of ESBL-encoding genes were performed with previously described primers (Yu et al, 2007a) for bla TEM , bla SHV , bla CTX-M-1 -like, bla CTX-and gyrA genes were detected as reported previously (Maurin et al, 2001;Decré et al, 2004).…”