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Antimicrobial Agents and Chemotherapy, December 2003, p. 3840-3845, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3840-3845.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

GeneHunter, a Transposon Tool for Identification and Isolation of Cryptic Antibiotic Resistance Genes

Stephen J. Salipante, Miriam Barlow, and Barry G. Hall^*

Biology Department, University of Rochester, Rochester, New York

Received 29 July 2003/ Returned for modification 19 August 2003/ Accepted 29 August 2003

GeneHunter is a transposon tool designed for the experimental activation and identification of silent antibiotic resistance genes. The method permits the identification of novel resistance genes that lack previously identified homologues. Using Salmonella enterica serovar Typhimurium strain LT2 as a test organism for the in vivo version of the GeneHunter method, we were able to activate, clone, and identify two cryptic antibiotic resistance genes, the aminoglycoside acetyltransferase aac(6')-Iaa and the probable Mar-A regulon activator rma. Because the method requires being able to electroporate the host with an efficiency of at least 10¹⁰ transformants per microgram, the in vivo method is not applicable to most microorganisms. We therefore developed an in vitro transposition method, showed that it can also recover the cryptic rma gene from S. enterica serovar Typhimurium strain LT2, and showed that it is generally applicable to a variety of microorganisms by using it to recover a cryptic metallo-ß-lactamase gene from the gram-positive organism Bacillus cereus. It is anticipated that the GeneHunter method will be used to identify potential resistance genes during the development and testing of novel antibiotics, new variants of existing antibiotics, and drug inhibitor combinations.

Present address: University of Washington, Seattle, WA 98195-7470.

Present address: Department of Epidemiology, Rollins School of Public Health of Emory University, Atlanta, GA 30322.

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