RmtC 16S rRNA Methyltransferase in Australia

Proteus mirabilis isolate JIE273 was recovered in March 2007 from the urine of an inpatient at Blacktown Hospital, Sydney. In vitro susceptibility tests (Phoenix NMIC/ID-101 panel; BD, Franklin Lakes, NJ) indicated that JIE273 was susceptible to imipenem (MIC, ≤2 μg/ml), cefoxitin (MIC, ≤4 μg/ml), amoxicillin-clavulanate (MIC, ≤4/2 μg/ml), and ticarcillin-clavulanate (MIC, ≤2/2 μg/ml). It had “intermediate” susceptibility to ticarcillin (MIC, 32 μg/ml) but was resistant to ampicillin (MIC, >16 μg/ml), cephalothin (MIC, >16 μg/ml), cefuroxime (MIC, >16 μg/ml), cefotaxime (MIC, >32 μg/ml), ceftriaxone (MIC, >32 μg/ml), ceftazidime (MIC, >16 μg/ml), aztreonam (MIC, >16 μg/ml), trimethoprim-sulfamethoxazole (MIC, >2/38 μg/ml), ciprofloxacin (MIC, >2 μg/ml), levofloxacin (MIC, >4 μg/ml), and nalidixic acid (MIC, >32 μg/ml).

JIE273 was also highly resistant to amikacin, gentamicin, and tobramycin, and this was confirmed by E-test (AB Biodisk, Solna, Sweden) at >256, >1,024, and >256 μg/ml, respectively, suggesting the presence of a 16S rRNA methyltransferase (3). PCR screening of JIE273 for armA (8), rmtA (17), rmtB (19), rmtC (18), and rmtD (5) revealed rmtC only. This gene was originally discovered in P. mirabilis ARS68 in Japan in 2003 (16) and, to our knowledge, has not been reported elsewhere. The patient harboring JIE273 was born in and had recently returned to India but had no history of travel to Japan.

The PCR product obtained with primers ISEcpIR-F (5′-CAATGTGTGAGAAGCAGTCTAAA-3′) and rmtC-down (5′-GCGAGGTGTGTTAGAATTTGC-3′) contained a gene and flanking sequences identical to those of the original rmtC sequence with GenBank accession no. AB194779 (15). ISEcp1 has been previously shown to promote both the expression and transposition of rmtC (15), and an additional PCR with ISEcp1-5′ (6) and RMTC-R rmtC (18) confirmed a complete, uninterrupted copy of ISEcp1 adjacent to rmtC, as in ARS68.

The rmtC gene was transferred from ARS68 to Escherichia coli by electroporation but not conjugation (16). Attempts to transfer rmtC from JIE273 to a rifampin-resistant variant of E. coli DH5α (DH5αRf) by a filter-based (14) or a broth-based (2) conjugation method with selection on amikacin (16 μg/ml) and rifampin (80 μg/ml) were unsuccessful. Repeated attempts to obtain amikacin-resistant (MIC, >16 μg/ml) transformants of E. coli DH5α by electroporation with DNA prepared from JIE273 by alkaline lysis (13) were also unsuccessful, despite successful transfer of control plasmids.

A “keyhole” effect in a double-disc synergy test (9) with 30-μg cefotaxime, ceftazidime, and aztreonam discs placed 15 mm from a 20/10-μg amoxicillin-clavulanate disc (Bio-Rad, Hercules, CA) suggested an extended-spectrum β-lactamase in JIE273. Known associations between 16S RNA methylases and extended-spectrum β-lactamase genes (3) include bla_CTX-M and/or bla_SHV with armA or rmtB (19). Neither the bla_CTX-M (10), nor the bla_SHV (12), nor the bla_TEM (1) gene was amplified from JIE273, but a bla_VEB (11) amplicon was obtained. A single nucleotide change from bla_VEB-4 (GenBank accession no. EF136375) predicts only a conservative substitution (Ile18Val) in the leader peptide. This β-lactamase was designated VEB-6 (http://www.lahey.org/Studies/), and we believe it is the first report of a bla_VEB-like gene in Australia.

The general problem of resistance to β-lactams, including carbapenems, is significantly exacerbated by aminoglycoside resistance, since they are often employed together in the treatment of the critically ill. In contrast to patterns observed elsewhere (4), amikacin susceptibility is currently preserved in gentamicin- and tobramycin-resistant isolates with transmissible metalloenzymes in Australia (7). However, the emergence of genes such as rmtC with a broad aminoglycoside resistance spectrum (including amikacin) will limit our future options for dealing with all β-lactamases.

Nucleotide sequence accession numbers.

The nucleotide sequence of the rmtC gene has been submitted to GenBank under accession no. EU144360, and the bla_VEB-6 gene has been assigned accession no. EU259884.