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Antimicrobial Agents and Chemotherapy, March 2009, p. 1177-1184, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.00485-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genetic Variability among ampC Genes from Acinetobacter Genomic Species 3{triangledown}

Alejandro Beceiro,1 Astrid Pérez,1 Felipe Fernández-Cuenca,2 Luis Martínez-Martínez,3 Alvaro Pascual,2 Jordi Vila,4 Jesús Rodríguez-Baño,5 Jose Miguel Cisneros,6 Jerónimo Pachón,6 Germán Bou,1* and the Spanish Group for Nosocomial Infection (GEIH)

Servicio de Microbiología, Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Coruña,1 Servicios de Microbiología y,2 Enfermedades Infecciosas, Hospital Virgen Macarena, 41071 Sevilla,5 Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Santander,3 Servei de Microbiología, Hospital Clinic, 08036 Barcelona,4 Servicio de Enfermedades Infecciosas, Hospital Universitario Virgen del Rocío, 41013 Sevilla, Spain6

Received 14 April 2008/ Returned for modification 24 June 2008/ Accepted 13 November 2008

As a part of a nationwide study in Spain, 15 clinical isolates of Acinetobacter genomic species 3 (AG3) were analyzed. The main objective of the study was to characterize the ampC genes from these isolates and to determine their involvement in β-lactam resistance in AG3. The 15 AG3 isolates showed different profiles of resistance to ampicillin (range of MICs, 12 to >256 µg/ml). Nucleotide sequencing of the 15 ampC genes yielded 12 new AmpC enzymes (ADC-12 to ADC-23). The 12 AG3 enzymes showed 93.7 to 96.1% amino acid identity with respect to the AmpC enzyme from Acinetobacter baumannii (ADC-1 enzyme). Eight out of fifteen ampC genes were expressed in Escherichia coli cells under the control of a common promoter, and with the exception of one isolate (isolate 65, which showed lower β-lactam MICs), significant differences in overall β-lactam MICs for E. coli cells expressing AG3 ampC genes were not revealed. No significant differences in ampC gene expression in AG3 clinical isolates were revealed by reverse transcription-PCR analysis. A detailed analysis of the 12 AmpC protein sequences revealed that amino acid replacements (in comparison with those of ADC-1) occurred mainly in the same positions, although none were located in important functional domains such as the {Omega}- loop or conserved β-lactamase motifs. Kinetic experiments performed with three representative AmpC enzymes (ADC-14, -16, and -18) in some cases revealed dramatic changes in Km and kcat values for β-lactams. No ISAba1 was detected upstream of the ampC genes. Our results reveal 12 new ampC genes in AG3. The enzymes showed a moderate degree of variability, and they are tentatively named ADC-12 to ADC-23.

* Corresponding author. Mailing address: Servicio de Microbiología, Complejo Hospitalario Universitario Juan Canalejo-INIBIC, C/Xubias de Arriba 84, 15006 La Coruña, Spain. Phone: 34-981-176087. Fax: 34-981-17697. E-mail: germanbou{at}canalejo.org

{triangledown} Published ahead of print on 24 November 2008.

Antimicrobial Agents and Chemotherapy, March 2009, p. 1177-1184, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.00485-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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