First Report of Plasmid-Mediated Resistance to Quinolones and Cefotaxime in an Enterobacter cloacae Strain Isolated from an Outpatient in Brazil

Recently, low-level quinolone resistance has been associated with DNA acquired from transferable plasmids. Several studies showed a worldwide dissemination of QnrA determinants among enterobacterial isolates (1, 5). However, the presence of the qnr gene in clinical isolates from outpatients has not been hitherto reported for Brazil. The aim of this study was to determine the occurrence of the qnr gene in enterobacterial isolates from outpatients in a private laboratory located in Juiz de Fora, state of Minas Gerais, Brazil, and to analyze the transferability and the structure of plasmid DNA adjacent to the qnr gene.

A total of 257 unique nalidixic acid-resistant enterobacterial isolates were collected between January 2000 and May 2005. The presence of the qnr gene was investigated by the colony blotting method and PCR assays according to the method of Jacoby et al. (3, 6). One Enterobacter cloacae (0.39%) qnr-positive strain was isolated from a wound in a 62-year-old man in January 2005.

Conjugation experiments using Escherichia coli J53 AzR as the recipient strain were performed. Transconjugants were selected on MacConkey agar plates containing sodium azide (100 μg/ml) and ceftazidime (2 μg/ml) to select for plasmid-encoded resistance. Analysis of plasmid content in donor cells and transconjugants performed by the Kieser method (4) identified a 180-kb plasmid that hybridized with a qnrA-specific probe.

Genomic DNA of E. cloacae JF 277 and of the recipient and transconjugant strains was extracted and digested with XbaI. After pulsed-field gel electrophoresis (PFGE), DNA was transferred to a nylon membrane and hybridized with a digoxigenin-labeled DNA probe specific for the qnr gene. The hybridization assay was performed according to the DIG System User Guide for filter hybridization (Boehringer, Mannheim, Germany). PFGE analysis showed that recipient and transconjugant strains exhibited similar patterns, except for one unique fragment which presented signal hybridization with the qnr probe (Fig. 1).

FIG. 1.

PFGE macrorestriction pattern of positive control, recipient, donor, and transconjugant strains (a) and Southern hybridization with the qnr probe (b). Lines 1 to 5: Lambda ladder PFG marker, positive qnr control, E. cloacae JF 277, E. coli J53, and the transconjugant.

Antimicrobial susceptibilities of the donor strain, the recipient strain, and the transconjugant were determined by agar dilution in accordance with the guidelines of the CLSI (2). MICs are reported in Table 1. Quinolone and cefotaxime resistance was transferred by conjugation. Resistance to other antimicrobial agents, excepting cefoxitin, was also transferred with the plasmid. β-Lactamase extracts from cultures of both E. cloacae JF 277 and the transconjugant were subjected to analytical isoelectric focusing, as previously described (7). Isoelectric focusing analysis of the extracts showed two β-lactamase bands with pIs of 5.4 and 8.2. Moreover, a band with a pI of 9.0 was observed, corresponding to chromosomal β-lactamase AmpC in E. cloacae JF 277. According to sequencing results, the genes corresponding to β-lactamases were identified as bla_TEM-1 and bla_SHV-5.

TABLE 1.

Resistance profiles of the E. coli J53 recipient strain, E. cloacae, and transconjugant

Strain	MIC of drug (μg/ml)a
	CTX	CTX/AC	CAZ	FEP	FOX	NAL	CIP	NOR	OFL	LVX	GAT	MOX	IMP
E. coli J53 AzR	<0.125	0.125	0.062	0.031	2	4	<0.031	0.031	0.031	<0.031	<0.016	<0.016	0.06
E. cloacae JF 277	128	64	128	2	>256	128	2	2	2	1	0.25	1	0.25
Transconjugant	128	0.125	8	<0.5	4	128	0.125	0.5	0.5	0.25	0.125	0.25	0.25

^aCTX, cefotaxime; CTX/AC, cefotaxime plus clavulanic acid; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; NAL, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; OFL, ofloxacin; LVX, levofloxacin, GAT, gatifloxacin; MOX, moxifloxacin; IMP, imipenem.

The chromosome-encoded quinolone resistance determinant mutations were assessed by sequencing of gyrA and parC of E. cloacae according to the method of Mammeri et al. (5). No amino acid change in the quinolone resistance determining region was observed, although two nucleotide substitutions were identified in gyrA, codons encoding Ile-89 and Val-90, and four substitutions in parC, codons encoding Val-71, Gly-73, Tyr-75, and Gly-79.

The qnr gene was sequenced directly from the PCR-amplified DNA, which showed that the isolate contained a nucleotide sequence identical to that of the qnr gene originally reported for a Klebsiella pneumoniae isolate (6). Analysis of gene structure indicated the possibility that qnr was carried on a class 1 integron and located between 3′ conserved sequence (CS) regions, downstream from orf513 and directly upstream from ampR, a regulator of the expression of ampC. Between the 5′ CS and the first copy of the 3′ CS, only a gene cassette (aadA2) was inserted into the integron.

Our study shows that the qnrA gene was detected in a fluoroquinolone-susceptible (MIC, ≤2 μg/ml) and cefotaxime-resistant isolate of Enterobacter from one outpatient in Brazil and confirms previous findings that the qnrA gene may be detected in ciprofloxacin-susceptible isolates.

Nucleotide sequence accession numbers.

The nucleotide sequences determined in this study have been deposited in the GenBank databases and assigned the following accession numbers: qnrA, DQ983226; gyrA, DQ983227; and parC, DQ983228.