Plasmid-Mediated 16S rRNA Methylases among Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Isolates

High levels of resistance to aminoglycosides conferred by plasmid-mediated mechanisms of resistance to aminoglycosides corresponding to 16S rRNA methylases have been reported since 2003 (3, 11). To date, six enzymes (ArmA, RmtA, RmtB, RmtC, RmtD, and NpmA) have been described, with ArmA being the most frequently identified methylase in Enterobacteriaceae (2). Associations between 16S rRNA methylase- and extended-spectrum β-lactamase (ESBL)-encoding genes such as the bla_CTX-M genes have been reported (1, 7, 8). The aim of this study was to evaluate the prevalence of 16S rRNA methylase genes among ESBL-producing Enterobacteriaceae isolates recovered in a French university hospital.

A total of 373 nonduplicate ESBL-producing enterobacterial clinical isolates, collected at the Bicêtre hospital between January 2005 and December 2007, have been studied. Among them, 19 isolates (5.1%) were resistant to all clinically used aminoglycosides. That collection was screened by PCR for detection of 16S rRNA methylase-encoding genes (armA, rmtA, rmtB, rmtC, rmtD, and npmA), as previously described (2). Four armA-positive isolates were detected in 2005 (n = 2) and in 2007 (n = 2), in addition to a single rmtB-positive isolate from 2006. No isolate was positive for the rmtA, rmtC, rmtD, and npmA genes. The armA-positive isolates produced the ESBL CTX-M-3 (n = 3) or CTX-M-14 (n = 1), whereas the single rmtB-positive isolate was a CTX-M-14 producer. (CTX-M enzymes were searched for as described previously [6].) The low prevalence rate of 16S rRNA methylase determinants among ESBL producers (1.3%) observed here is similar to that reported from Taiwan (1.1%) (8) and higher than those reported from Belgium (0.12%) and Japan (0.03%) (1, 10), but lower than that from Korea (3%) (7).

All 16S rRNA methylase and ESBL determinants were transferred by conjugation and transformation using Escherichia coli J53 and TOP10 recipient strains after selection on Trypticase soy agar containing amikacin (50 μg/ml) and/or cefotaxime (8 μg/ml) (6). The 16S rRNA methylase-positive transformants expressed a high level of resistance to aminoglycosides (Table 1). In three isolates, the armA and bla_CTX-M-3 genes were located on a same plasmid belonging to the IncL/M incompatibility group, as observed on the plasmid pCTX-M-3 (5), whereas the 16S rRNA methylase determinants were not physically linked to the ESBL genes for the two bla_CTX-M-14-positive remaining isolates. Sequencing of the 16S rRNA methylase-encoding genes showed a perfect identity with previously reported genes. The complete PCR-mapped region of 11.6 kb containing the armA gene was entirely sequenced for two isolates, showing its association with ISCR1 inside a sul1-type integron structure, the same configuration observed in Tn1548 (4). The rmtB gene was located on an 11.4-kb region bracketed by two IS26 elements containing tnpA, bla_TEM-1, intI1/groEL, and qepA and followed by an ISCR3 element, as observed on pHPA (9). Analysis of sequences flanking more closely the armA gene identified two novel insertion sequence elements, ISEc28 and ISEc29, belonging to the IS5 and IS4 families, respectively (http://www-is.biotoul.fr). No direct repeat sequence bracketing each of those IS elements and the overall ISEc28-armA-ISEc29 structure was identified.

TABLE 1.

MICs of antibiotics for clinical isolates producing 16S rRNA methylases, their corresponding armA- and rmtB-positive transformants, and E. coli TOP10^a

Clinical isolate or transformantb	16S rRNA methylase or/and ESBLs	MIC (μg/ml) ofc:									Plasmid size (kb)	Incompatibility group plasmid type
		AMK	NET	GEN	TOB	CTX	CAZ	IMP	NOR	CIP
E coli VOG	rmtB and blaCTX-M-14	>256	>256	>256	>256	>32	6	0.19	>256	>32	80, 50
E. coli TOP10(pVOG1)	rmtBd	>256	>256	>256	>256	0.047	0.38	0.19	0.032	0.003	80	F
E. coli TOP10(pVOG2)	blaCTX-M-14e	1.5	1.5	0.25	3	>32	2	0.19	<0.016	<0.002	50	NDf
E coli COP	armA and blaCTX-M-14	>256	>256	>256	>256	>32	1	0.19	>256	>32	60, 38
E. coli TOP10(pCOP1)	armA	>256	>256	>256	>256	0.047	0.38	0.19	<0.016	<0.002	60	F
E. coli TOP10(pCOP2)	blaCTX-M-14	1	0.125	0.125	0.19	8	1	0.19	<0.016	<0.002	38	I1
E coli MEZ	armA and blaCTX-M-3	>256	>256	>256	>256	>32	3	0.19	>256	>32	120
E. coli TOP10(pMEZ)	armA and blaCTX-M-3	>256	>256	>256	>256	>32	3	0.19	0.016	<0.002	120	L/M
Enterobacter cloacae BEH	armA and blaCTX-M-3	>256	>256	>256	>256	>32	16	0.19	1	0.19	130
E. coli TOP10(pBEH)	armA and blaCTX-M-3	>256	>256	>256	>256	>32	8	0.19	0.016	<0.002	130	L/M
Klebsiella pneumoniae DU	armA and blaCTX-M-3	>256	>256	>256	>256	>32	2	0.19	0.75	0.125	120
E. coli TOP10(pDU)	armA and blaCTX-M-3	>256	>256	>256	>256	>32	2	0.19	0.016	<0.002	120	L/M
E. coli TOP10		1	0.25	0.25	0.38	0.047	0.38	0.19	<0.016	<0.002

^aMICs of antibiotics were determined by using the Etest technique according to the manufacturer's recommendations. Analysis of plasmid content in the armA- and rmtB-positive isolates and their transformants was performed by the Kieser technique (7). The incompatibility group of plasmids was determined as previously described (2).

^bAll clinical isolates were also resistant to sulfonamides, trimethoprim, chloramphenicol, tetracycline, and rifampin.

^cAntibiotic-resistant marker abbreviations: AMK, amikacin; NET, netilmicin; GEN, gentamicin; TOB, tobramycin; CTX, cefotaxime; CAZ, ceftazidime; IMP, imipenem; NOR, norfloxacin; and CIP, ciprofloxacin.

^dMICs for E. coli TOP10 transformants harboring pVOG1 expressing RmtB, TEM-1, and QepA.

^eMICs for E. coli TOP10 transformants harboring pVOG2 expressing CTX-M-14 and AAC(6′)-Ib.

^fND, not determined.

This study underlines that the association on the same plasmid of 16S rRNA methylase- and ESBL-encoding genes remains rare. However, their localization on conjugative plasmids, and in some cases in association on the same plasmid, as observed here with CTX-M determinants, has the potential to further expand the threat of multidrug-resistant Enterobacteriaceae.

Nucleotide sequence accession numbers.

The nucleotide sequences of the regions surrounding qepA1 and rmtB have been submitted to the GenBank nucleotide sequence database under accession no. FJ183463 and FJ187822, respectively.