Klebsiella pneumoniae Sequence Type 11 Isolate Producing RmtG 16S rRNA Methyltransferase from a Patient in Miami, Florida

LETTER

High-level resistance to aminoglycosides mediated by production of 16S rRNA methyltransferase (16S-RMTase) is increasingly reported among various Gram-negative pathogens (1). The 16S-RMTases most commonly encountered in Enterobacteriaceae worldwide include ArmA and RmtB. RmtG was first reported in Klebsiella pneumoniae producing KPC-2 in Brazil (2) and then also in K. pneumoniae producing CTX-M-2 in Chile (3). Here, we report the identification of K. pneumoniae producing RmtG coupled with both CTX-M-2 and CTX-M-15 at a hospital in Miami, FL.

The patient was a 64-year-old Hispanic male with a recent diagnosis of bladder cancer after an initial episode of hematuria while visiting Cuba in 2012. Throughout 2012, he had seven acute hospitalizations and underwent four rectal cultures to rule out carriage of carbapenem-resistant Enterobacteriaceae (part of standard infection control practices at the hospital). The last rectal screening culture grew K. pneumoniae isolate K-109-R; the previous three cultures obtained within a span of 3 months were negative.

K. pneumoniae K-109-R was resistant to all aminoglycosides tested as well as ertapenem and meropenem, as determined by the broth microdilution method (Sensititre GNX2F; TREK Diagnostics, Oakwood Village, OH) and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints (Table 1) (4). Multilocus sequence typing was performed as described previously (5), and the sequence type was determined as ST11, an ST with global distribution among KPC- and CTX-M-producing K. pneumoniae isolates as well as 16S-RMTase-producing K. pneumoniae isolates (6–9). PCR for bla_KPC was negative, and a modified Hodge test was also negative for carbapenemase production. PCR and sequencing confirmed the presence of bla_CTX-M-2, bla_CTX-M-15, and rmtG for the clinical isolate. The Escherichia coli TOP10 transformant (selected with gentamicin at 25 μg/ml) and J53AziR transconjugant (selected with gentamicin at 25 μg/ml and sodium azide at 150 μg/ml) strains containing plasmid pK190R1, which was transferred from the clinical isolate by electroporation and conjugation, respectively, harbored bla_CTX-M-2 and rmtG but not bla_CTX-M-15. The conjugation frequency was approximately 1 × 10⁻⁵ plasmids/donor. They were accordingly highly resistant to aminoglycosides and cephalosporins (Table 1). Resistance to trimethoprim-sulfamethoxazole was also cotransferred. pK190R1 was estimated to be approximately 150 kb in size by S1 nuclease pulsed-field gel electrophoresis (PFGE) (2) and belonged to IncA/C, as determined by the standard replicon typing method (10). rmtG was located on an IncN plasmid harboring bla_CTX-M-59 in Brazil (2) and on an IncA/C plasmid lacking bla_CTX-M in Chile (3). Thus, pK190R1 appears to differ from these previously reported plasmids harboring rmtG.

TABLE 1.

MICs of Klebsiella pneumoniae K-190-R and its rmtG-positive Escherichia coli transformant and transconjugant

Antimicrobial agent	MIC (μg/ml) for:
	K. pneumoniae K-190-R	E. coli TOP10(pK190R1)a	E. coli J53AziR(pK190R1)b	E. coli TOP10	E. coli J53AziR
Amikacin	>32	>32	>32	≤4	≤4
Gentamicin	>8	>8	>8	≤1	≤1
Tobramycin	>8	>8	>8	≤1	≤1
Ticarcillin-clavulanate	>128/2	>128/2	>128/2	≤16/2	≤16/2
Piperacillin-tazobactam	>64/4	16/4	16/4	≤8/4	≤8/4
Cefotaxime	>32	>32	>32	≤1	≤1
Ceftazidime	>16	8	8	≤1	≤1
Cefepime	>16	>16	>16	≤2	≤2
Aztreonam	>16	>16	>16	≤2	≤2
Ertapenem	>4	≤0.25	≤0.25	≤0.25	≤0.25
Meropenem	4	≤1	≤1	≤1	≤1
Imipenem	≤1	≤1	≤1	≤1	≤1
Ciprofloxacin	>2	≤0.25	≤0.25	≤0.25	≤0.25
Doxycycline	16	≤2	4	1	2
Trimethoprim-sulfamethoxazole	>4/76	>4/76	>4/76	≤0.5/9.5	≤0.5/9.5
Tigecycline	≤0.25	≤0.25	≤0.25	≤0.25	≤0.25
Colistin	0.5	≤0.25	≤0.25	≤0.25	≤0.25

^aTransformant.

^bTransconjugant.

A cloning experiment was then performed. pK190R1 was extracted, digested with HindIII, and ligated with the vector pBC-SK−. E. coli TOP10 was transformed with these plasmids, which yielded a recombinant plasmid with a 1,628-bp insert containing rmtG. This fragment was sequenced in full using custom sequencing primers. The genetic environment of rmtG was identical to that reported to occur in K. pneumoniae isolate 350/10 from Brazil, where a putative tRNA ribosyltransferase gene precedes rmtG, with an overlap of 8 nucleotides (2).

RmtG, along with RmtD1/D2, has been considered a 16S-RMTase unique to South America, with no cases or isolates reported outside this region thus far. In addition, Enterobacteriaceae isolates producing aminoglycoside-resistant 16S-RMTases have remained extremely rare in the United States. However, the recent spread of Enterobacteriaceae producing NDM-1 metallo-β-lactamase warrants increased awareness of this resistance mechanism, since 16S-RMTase genes are often borne by the plasmid containing bla_NDM-1 (11). No further isolates with 16S-RMTase have been identified since at this hospital.

Nucleotide sequence accession number.

The nucleotide sequence reported here was deposited into the GenBank database under accession no. KJ004567.