First report of plasmid-mediated resistance to quinolones and cefotaxime in an Enterobacter cloacae strain isolated from an outpatient in Brazil

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ANTIMICROBIALAGENTS ANDCHEMOTHERAPY, Jan. 2007, p. 401–402 Vol. 51, No. 1 0066-4804/07/$08.00⫹0 doi:10.1128/AAC.00935-06

First Report of Plasmid-Mediated Resistance to Quinolones and Cefotaxime in

an

Enterobacter cloacae

_{Strain Isolated from an Outpatient in Brazil}

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Recently, low-level quinolone resistance has been associated with DNA acquired from transferable plasmids. Several stud-ies showed a worldwide dissemination of QnrA determinants among enterobacterial isolates (1, 5). However, the presence of the qnrgene in clinical isolates from outpatients has not been hitherto reported for Brazil. The aim of this study was to determine the occurrence of the qnrgene 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 theqnrgene.

A total of 257 unique nalidixic acid-resistant enterobacterial isolates were collected between January 2000 and May 2005. The presence of theqnrgene 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 usingEscherichia coliJ53 AzR as the recipient strain were performed. Transconjugants were selected on MacConkey agar plates containing sodium azide (100␮g/ml) and ceftazidime (2 ␮g/ml) to select for plasmid-encoded resistance. Analysis of plasmid content in donor cells and transconjugants performed by the Kieser method (4) iden-tified a 180-kb plasmid that hybridized with a qnrA-specific probe.

Genomic DNA ofE. cloacaeJF 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 digoxi-genin-labeled DNA probe specific for theqnrgene. The hy-bridization assay was performed according to the DIG System User Guide for filter hybridization (Boehringer, Mannheim, Germany). PFGE analysis showed that recipient and transcon-jugant strains exhibited similar patterns, except for one unique fragment which presented signal hybridization with the qnr

probe (Fig. 1).

Antimicrobial susceptibilities of the donor strain, the recip-ient 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 re-sistance was transferred by conjugation. Rere-sistance to other antimicrobial agents, excepting cefoxitin, was also transferred with the plasmid.␤-Lactamase extracts from cultures of both

E. cloacae JF 277 and the transconjugant were subjected to analytical isoelectric focusing, as previously described (7).

Iso-electric focusing analysis of the extracts showed two ␤ -lac-tamase bands with pIs of 5.4 and 8.2. Moreover, a band with a pI of 9.0 was observed, corresponding to chromosomal␤ -lac-tamase AmpC inE. cloacaeJF 277. According to sequencing results, the genes corresponding to␤-lactamases were identi-fied asblaTEM-1andblaSHV-5.

The chromosome-encoded quinolone resistance determi-nant mutations were assessed by sequencing ofgyrAandparC

ofE. cloacaeaccording to the method of Mammeri et al. (5). No amino acid change in the quinolone resistance determin-ing region was observed, although two nucleotide substitu-tions 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.

Theqnrgene was sequenced directly from the PCR-ampli-fied DNA, which showed that the isolate contained a nucleo-tide sequence identical to that of theqnrgene originally re-ported for aKlebsiella pneumoniaeisolate (6). Analysis of gene structure indicated the possibility that qnr was carried on a

FIG. 1. PFGE macrorestriction pattern of positive control, recipi-ent, donor, and transconjugant strains (a) and Southern hybridization with theqnrprobe (b). Lines 1 to 5: Lambda ladder PFG marker, positiveqnrcontrol,E. cloacaeJF 277,E. coliJ53, and the transcon-jugant.

TABLE 1. Resistance profiles of theE. coliJ53 recipient strain,E. cloacae, and transconjugant

Strain MIC of drug (␮g/ml)

a

CTX CTX/AC CAZ FEP FOX NAL CIP NOR OFL LVX GAT MOX IMP

E. coliJ53 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. cloacaeJF 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

a_{CTX, 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.

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class 1 integron and located between 3⬘ conserved sequence (CS) regions, downstream from orf513 and directly upstream fromampR, a regulator of the expression ofampC. 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 theqnrAgene was detected in a fluo-roquinolone-susceptible (MIC,ⱕ2␮g/ml) and cefotaxime-re-sistant 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 se-quences determined in this study have been deposited in the GenBank databases and assigned the following accession num-bers:qnrA, DQ983226; gyrA, DQ983227; andparC, DQ983228.

This work was supported by Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP) and Coordenaçaõ de Aperfeiçoa-mento de Pessoal de Nı´vel Superior (CAPES).

We are grateful to George A. Jacoby for kindly providingE. coliJ53 AzR, to Joseane C. Ferreira for performing the PFGE assay, and to Izabel C. V. Palazzo for help in the MIC determinations.

REFERENCES

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2005. Plasmid-mediated resistance to ciprofloxacin and cefotaxime in clinical isolates ofSalmonella entericaserotype Enteritidis in Hong Kong. J. Antimi-crob. Chemother.56:586–589.

2.Clinical and Laboratory Standards Institute.2005. Performance standards for antimicrobial susceptibility testing, 15th informational supplement M100-S15. Clinical and Laboratory Standards Institute, Wayne, Pa.

3.Jacoby, G. A., N. Chow, and K. B. Waites.2003. Prevalence of plasmid-mediated quinolone resistance. Antimicrob. Agents Chemother.47:559–562. 4.Kieser, T.1984. Factors affecting the isolation of CCC DNA from

Streptomy-ces lividansandEscherichia coli. Plasmid12:19–36.

5.Mammeri, H., M. Van De Loo, L. Poirel, L. Martinez-Martinez, and P. Nordmann.2005. Emergence of plasmid-mediated quinolone resistance in

Escherichia coliin Europe. Antimicrob. Agents. Chemother.49:71–76. 6.Martinez-Martinez, L., A. Pascual, and G. A. Jacoby.1998. Quinolone

resis-tance from a transferable plasmid. Lancet351:797–799.

7.Matthew, M., A. M. Harris, M. J. Marshall, and G. W. Ross.1975. The use of analytical isoelectric focusing for detection and identification of␤-lactamases. J. Gen. Microbiol.88:169–178.

Luciene A. R. Minarini

Departamento de Ana´lises Clıńicas, Toxicolo´gicas e Bromatolo´gicas Faculdade de Cieˆncias Farmaceûticas de Ribeiraõ Preto

Universidade de Sa˜o Paulo Av. do Cafe´, s/n

14040-903 Ribeira˜o Preto, Sa˜o Paulo, Brazil

Ana C. Gales Laborato´rio Alerta

Universidade Federal de Sa˜o Paulo Rua Pedro de Toledo, 781

Vila Clementino, 04039-032 Sa˜o Paulo, Brazil

Ana Lucia C. Darini*

Departamento de Ana´lises Clıńicas, Toxicolo´gicas e Bromatolo´gicas Faculdade de Cieˆncias Farmaceûticas de Ribeiraõ Preto

Universidade de Sa˜o Paulo Av. do Cafe´, s/n

14040-903 Ribeira˜o Preto, Sa˜o Paulo, Brazil

*Phone: 55 16 3602-4291 Fax: 55 16 3602-4878 E-mail: aldarini@fcfrp.usp.br

䌤_{Published ahead of print on 23 October 2006.}