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Antimicrobial Agents and Chemotherapy, February 2008, p. 806-807, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00444-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Ciprofloxacin-Resistant Enterobacteria Harboring the aac(6')-Ib-cr Variant Isolated from Feces of Inpatients in an Intensive Care Unit in Uruguay{triangledown}


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The presence of aac(6')-Ib-cr is associated with decreased susceptibility to aminoglycosides (kanamycin, amikacin, and tobramycin) and to norfloxacin and ciprofloxacin (9). This allelic variant of aac(6')-Ib was found to be linked to the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-15 in isolates from many countries (4, 6, 7), while association of aac(6')-Ib with the blaCTX-M-2 ESBL gene has been widely reported in Uruguay and Argentina (3, 11).

In this work we looked for the presence of aac(6')-Ib and the aac(6')-Ib-cr variant and their putative ESBL coresistance markers in fecal isolates of enterobacteria resistant to ciprofloxacin and/or ceftazidime from inpatients in an intensive care unit (ICU) in Montevideo, Uruguay.

From 1 March to 31 October 2006, 106 patients were admitted to this ICU and followed daily until discharge. Rectal swabs obtained at 1, 4, 7, 10, 13, and 16 days after admission were plated on MacConkey agar plus ceftazidime (4 mg/liter) or ciprofloxacin (2 mg/liter). Enterobacterial isolates were identified by classical methods, including only the first isolate of each bacterial species per patient in this study.

Antibiotic resistance profiling, screening, and confirmatory testing for ESBL detection were performed by disk diffusion assay, and results were interpreted following the CLSI guidelines (2).

A total of 58/106 patients (55.2%) were colonized with ciprofloxacin- and/or ceftazidime-resistant enterobacteria, and 68 isolates were included in this study. Of these, 48 were resistant to gentamicin and 24 to amikacin (Table 1).


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TABLE 1. Main characteristics of the 68 studied isolatesa

 
All aminoglycoside-resistant isolates were screened for aac(6')-Ib by PCR; amplicons were analyzed by restriction with BstF5I, as described by Park et al. (8). PCR products that were not digested by the enzyme [tentatively assigned to aac(6')-Ib-cr] were confirmed to contain aac(6')-Ib-cr by double-strand sequencing. Only two Escherichia coli isolates were positive for aac(6')-Ib-cr detection.

Recalling the observed links between blaCTX-M-15 and aac(6')Ib-cr (4, 6, 7) and between aac(6')Ib and blaCTX-M-2, the two aac(6')-Ib-cr-positive isolates were further analyzed by PCR to detect CTX-M-1 and CTX-M-2 group ESBL genes using previously described primers (3, 5). Both isolates were positive only for CTX-M-1 group genes, identified as blaCTX-M-15 after sequencing.

Both isolates were obtained at the time of patient admission into the ICU and showed identical pulsed-field gel electrophoresis patterns (10). Both patients were previously hospitalized before ICU admission, suggesting that this strain could be endemic in the hospital, where it could be horizontally transferred. All the other E. coli isolates yielded different pulsotypes (data not shown) compared with these.

PCR assays for the detection of class 1 integrons and ISCR1 elements were performed according to the method of Di Conza et al. (3). Both isolates carried a class 1 integron containing the dfr17 and aadA5 gene cassettes, while ISCR1 elements were not detected.

So far we have not been able to transfer these resistance genes, either by transformation or by conjugation.

This is the first report of aac(6')-Ib-cr in Uruguay. In accordance with a previous report (6), blaCTX-M-15 and aac(6')-Ib-cr do not seem to be associated with class 1 integrons. Demonstration of a link to IS26 as previously reported (1) is pending.


    ACKNOWLEDGMENTS
 
This work was partially supported by grants from S/C/OP/76/30 PDT (Programa de Desarrollo Tecnológico, Ministerio de Educación y Cultura Uruguay) to R.V. and also by grants from CSIC (Comisión Sectorial de Investigación Científica Uruguay) to N.F.C. Part of this work was also supported by grants LSHM-CT-2003-503335 from the European Community and BFU2006-04574 from Ministerio de Educación y Ciencia, España, to J.A.A. and grants from UBACYT and ANPCYT and a Carrillo-Oñativia fellowship to G.G.


    FOOTNOTES
 
{triangledown} Published ahead of print on 12 November 2007. Back

{dagger} N.F.C. and L.R. contributed equally to the experimental work and to the elaboration of this report. Back


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  1. Boyd, D. A., S. Tyler, S. Christianson, A. McGeer, M. P. Muller, B. M. Willey, E. Bryce, M. Gardam, P. Nordmann, M. R. Mulvey, and the Canadian Nosocomial Infection Surveillance Program, Health Canada. 2004. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob. Agents Chemother. 48:3758-3764.[Abstract/Free Full Text]
  2. Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; 17th informational supplement. CLSI/NCCLS M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
  3. Di Conza, J., J. A. Ayala, P. Power, M. Mollerach, and G. Gutkind. 2002. Novel class 1 integron (InS21) carrying blaCTX-M-2 in Salmonella enterica serovar Infantis. Antimicrob. Agents Chemother. 46:2257-2261.[Abstract/Free Full Text]
  4. Karisik, E., M. J. Ellington, R. Pike, R. E. Warren, D. M. Livermore, and N. Woodford. 2006. Molecular characterization of plasmids encoding CTX-M-15 beta-lactamases from Escherichia coli strains in the United Kingdom. J. Antimicrob. Chemother. 58:665-668.[Abstract/Free Full Text]
  5. Kim, J., and Y. M. Lim. 2005. Prevalence of derepressed AmpC mutants and extended-spectrum β-lactamase producers among clinical isolates of Citrobacter freundii, Enterobacter spp., and Serratia marcescens in Korea: dissemination of CTX-M-3, TEM-52, and SHV-12. J. Clin. Microbiol. 43:2452-2455.[Abstract/Free Full Text]
  6. Machado, E., T. M. Coque, R. Cantón, F. Baquero, J. C. Sousa, L. Peixe, and the Portuguese Resistance Study Group. 2006. Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-producing Enterobacteriaceae strains containing the aac(6')-Ib-cr gene, which encodes an aminoglycoside- and fluoroquinolone-modifying enzyme. Antimicrob. Agents Chemother. 50:3220-3221.[Free Full Text]
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  8. Park, C. H., A. Robicsek, G. A. Jacoby, D. Sahm, and D. C. Hooper. 2006. Prevalence in the United States of aac(6')-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob. Agent Chemother. 50:3953-3955.[Abstract/Free Full Text]
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  11. Vignoli, R., N. Cordeiro, V. Seija, F. Schelotto, M. Radice, J. Ayala, P. Power, and G. Gudkind. 2006. Genetic environment of CTX-M-2 in Klebsiella pneumoniae isolates from hospitalized patients in Uruguay. Rev. Arg. Microbiol. 38:84-88.
Nicolás F. Cordeiro{dagger}
Luciana Robino{dagger}

Departamento de Bacteriología y Virología
Instituto de Higiene
Facultad de Medicina
Universidad de la República
11600 Montevideo, Uruguay

Julio Medina
Unidad Cuidados Intensivos del Hospital de Clínicas
Facultad de Medicina
Montevideo, Uruguay

Verónica Seija
Departamento de Laboratorio Clínico
Área Microbiología
Hospital de Clínicas Facultad de Medicina
Montevideo, Uruguay

Inés Bado
Virginia García

Departamento de Bacteriología y Virología
Instituto de Higiene
Facultad de Medicina
Universidad de la República
11600 Montevideo, Uruguay

Maximiliano Berro
Julio Pontet
Lucía López

Unidad Cuidados Intensivos del Hospital de Clínicas
Facultad de Medicina
Montevideo, Uruguay

Cristina Bazet
Departamento de Laboratorio Clínico
Área Microbiología
Hospital de Clínicas Facultad de Medicina
Montevideo, Uruguay

Gloria Rieppi
Unidad Cuidados Intensivos del Hospital de Clínicas
Facultad de Medicina
Montevideo, Uruguay

Gabriel Gutkind
Cátedra de Microbiología
Facultad de Farmacia y Bioquímica
Universidad de Buenos Aires
1113 Buenos Aires, Argentina

Juan A. Ayala
Centro de Biología Molecular Severo Ochoa
CSIC-UAM
Campus de Cantoblanco
28049 Madrid, Spain

Rafael Vignoli*
Departamento de Bacteriología y Virología
Instituto de Higiene
Facultad de Medicina
Universidad de la República
Alfredo Navarro 3051
11600 Montevideo, Uruguay

* Phone and fax: (598 2) 487 57 95
E-mail: rvignoli{at}higiene.edu.uy


Antimicrobial Agents and Chemotherapy, February 2008, p. 806-807, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00444-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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