A New Variant of 16S rRNA Methylase, RmtD3, in a Clinical Isolate of Pseudomonas aeruginosa in Myanmar

LETTER

Acquired 16S rRNA methylases are responsible for an extremely high level of resistance against various aminoglycosides in Gram-negative pathogens, including Enterobacteriaceae and glucose-nonfermentative bacteria (1). To date, 10 kinds of 16S rRNA methylases, including ArmA, RmtA, RmtB, RmtC, RmtD, RmtE, RmtF, RmtG, RmtH, and NpmA, have been found in clinical isolates (2–5). One of these, RmtD, was first detected in a clinical isolate of Pseudomonas aeruginosa from an inpatient in 2005 in Brazil (6). Subsequently, RmtD2, a variant of RmtD, was found in 7 clinical isolates of Enterobacteriaceae, including Citrobacter freundii, Enterobacter aerogenes, and Enterobacter cloacae, in Argentina in 2011 (7).

P. aeruginosa strain MyNCGM481 was isolated from a sputum sample from an inpatient in 2014 in Yangon, Myanmar. Bacterial identification was performed by the Vitek 2 system (bioMérieux, Marcy l'Etoile, France) and confirmed by 16S rRNA sequences (8). The O serotypes of isolates were determined with a slide agglutination test kit (Denka Seiken Co., Tokyo, Japan) and sequence analysis of serotype-specific genes (9).

MICs were determined using the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (10). The rmtD3 gene was cloned into the corresponding sites of the vector plasmid pSTV28 (TaKaRa, Shiga, Japan) using the primer set SalI-rmtD3-F (5′-atgtcgacatgagcgaactgaaggaaaaac-3′) and SphI-rmtD3-R (5′-atgcatgctcattttcgtttcagcacgtaa-3′). Escherichia coli DH5α was transformed with pSTV28-RmtD3, and the transformant was selected on chloramphenicol-containing plates (30 μg/ml). The DNA was extracted from the isolate using the DNeasy blood and tissue kit (Qiagen, Tokyo, Japan), and the entire genome was sequenced by MiSeq (Illumina, San Diego, CA) using the MiSeq reagent kit v.3 (Illumina). Raw reads were assembled using CLC genomics workbench version 8.0 (CLC bio, Tokyo, Japan). Drug-resistant genes encoding β-lactamases, aminoglycoside modification enzymes, and 16S rRNA methylases were detected using ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/). The sequences of the quinolone resistance genes (11) gyrA and parC were determined using the CLC genomics workbench (version 8.0). Multilocus sequence types (MLSTs) were deduced as described in the protocols of the PubMLST databases (https://pubmlst.org/paeruginosa/). DNA plugs of MyNCGM481, digested with S1 nuclease or I-CeuI, were prepared, separated by pulsed-field gel electrophoresis, and subjected to Southern hybridization using rmtD3 or 16S rRNA probes. Signals were detected using a digoxigenin (DIG) High Prime DNA labeling and detection starter kit II (Roche Applied Science, Indianapolis, IN).

As shown in Table 1, P. aeruginosa MyNCGM481 was highly resistant to the aminoglycosides tested, including amikacin, arbekacin, gentamicin, and tobramycin, with MICs of >1,024 μg/ml. It was also resistant to penicillins, cefotaxime, carbapenems, and fluoroquinolones, whereas it was susceptible to ceftazidime and colistin. The E. coli transformant expressing rmtD3 was highly resistant to aminoglycosides, including arbekacin, amikacin, gentamicin, kanamycin, and tobramycin (Table 1). The O serotype of the strain was O11.

TABLE 1.

MICs of various antibiotics for P. aeruginosa MyNCGM481 and the E. coli transformant expressing rmtD3

Antibiotics	MIC (μg/ml) for:
	P. aeruginosa MyNCGM481	E. coli (pSTV28-rmtD3)
Ampicillin	>1,024	NDb
Ampicillin-sulbactama	512	ND
Penicillin	>1,024	ND
Ceftazidime	8	ND
Cefotaxime	128	ND
Aztreonam	16	ND
Imipenem	16	ND
Meropenem	16	ND
Amikacin	>1,024	>1,024
Arbekacin	>1,024	>1,024
Gentamicin	>1,024	>1,024
Kanamycin	>1,024	>1,024
Tobramycin	>1,024	>1,024
Ciprofloxacin	32	ND
Levofloxacin	64	ND
Minocycline	256	ND
Tetracycline	>1,024	ND
Tigecycline	32	ND
Chloramphenicol	>1,024	ND
Fosfomycin	128	ND
Colistin	1	ND

^aThe ratio of ampicillin to sulbactam was 2:1.

^bND, not determined.

P. aeruginosa MyNCGM481 contained a new variant of rmtD, designated rmtD3.

Compared with RmtD, the amino acid sequence of RmtD3 had 9 amino acid substitutions: Trp26Cys, Val39Ala, Met66Leu, Ser102Ile, Thr130Ala, Asn165Asp, Leu169Met, Ala181Thr, and Gly236Ser. Compared with RmtD2, the amino acid sequence of RmtD3 had 4 amino acid substitutions: Cys26Trp, Ala69Thr, Asp186Glu, and Ser236Gly. The isolate harbored genes encoding β-lactamases (bla_OXA-50 and bla_PAO) and genes encoding aminoglycoside-modification enzymes [aph(3′)-IIb, aac(6′)-Ib-cr, and aadA10]. The isolate had a point mutation in the quinolone resistance-determining regions of gyrA with amino acid substitution Ser83Ile in GyrA. It also had fosA encoding a fosfomycin modification enzyme. The MLST of the isolate was ST316.

The sequence was tnp (IS91 family)-orfA-orfB-mraW-rmtD3-orfC-orfD-yraL-tnp (IS91 family) (with orfA, orfC, and orfD encoding hypothetical protein and orfB encoding a tRNA-guanine family transglycosylase). Compared with the genetic environments surrounding rmtD and rmtD2, the genomic environment surrounding rmtD3 had a unique genetic structure between two genes encoding the IS91 family transposase (Fig. 1). Pulsed-field gel electrophoresis (PFGE) and Southern hybridization revealed that the rmtD3 gene was located on the chromosome (data not shown).

FIG 1.

The genetic environments surrounding rmtD in P. aeruginosa PA0905 (GenBank accession no. DQ914960), rmtD2 in Enterobacter cloacae Q4010 (GenBank accession no. HQ401566), and rmtD3 in P. aeruginosa MyNCGM481 (GenBank accession no. LC229801). orfA, orfC, and orfD are hypothetical protein-encoding genes, and orfB is a tRNA-guanine family transglycosylase-encoding gene.

This is the first report of a P. aeruginosa clinical isolate producing a new RmtD variant, RmtD3. Strain MyNCGM481 seems to be different from the epidemic strain of multidrug-resistant P. aeruginosa, because of its different MLST (ST316 versus ST235). To date, 20 P. aeruginosa isolates belonging to ST316 have been registered on the PubMLST website (https://pubmlst.org/paeruginosa/). Of them, PubMLST identification no. 16 (Pseudomonas aeruginosa 8297), which was first registered in 2005 in the United Kingdom, had the same O11 serotype as MyNCGM481, although the serotypes of the others were not reported.

Accession number(s).

The nucleotide sequence obtained in this study was submitted to the GenBank database and assigned accession no. LC229801.