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Applied and Environmental Microbiology, January 2006, p. 306-312, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.306-312.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Plasmid-Mediated High-Level Gentamicin Resistance among Enteric Bacteria Isolated from Pet Turtles in Louisiana

María Alejandra Díaz,1* Richard Kent Cooper,2 Axel Cloeckaert,3 and Ronald John Siebeling1,4,{dagger}

Department of Biological Sciences, 107 Life Sciences Building, Louisiana State University, Baton Rouge, Louisiana 70803,1 Department of Veterinary Science, 111 Dalrymple Building, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803,2 UR86 Bio-Agresseurs, Santé, Environnement, Institut National de la Recherche Agronomique, Centre de Recherche de Tours, 37380 Nouzilly, France,3 Department of Biological Sciences, 202 Life Sciences Building, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 708034

Received 23 June 2005/ Accepted 7 October 2005

The sale of small turtles is banned by the Food and Drug Administration from the U.S. market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses gentamicin sulfate baths (1,000 µg/ml) to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria, such as Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2,000 µg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) which contributes to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate the incidence of such genes in these bacteria. R plasmids from gentamicin-resistant strains were transferred by conjugation and transformation to naive Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of the aminoglycoside acetyltransferase genes aac(3)-IIa and aac(3)-VIa; the latter was present as a gene cassette of a class 1 integron. Multiplex PCR assays showed that every gentamicin-resistant isolate carried one of these acetyltransferase genes. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of R plasmids carrying these genes revealed different restriction profiles and sizes, indicating a dissemination of the gentamicin resistance genes through mobile molecular elements. The data presented highlight the need to develop an alternate method for the eradication of Salmonella spp. from turtle eggs.

* Corresponding author. Mailing address: Department of Biological Sciences, 107 Life Sciences Building, Louisiana State University, Baton Rouge, LA 70803. Phone: (225) 578-5421. Fax: (225) 578-4890. E-mail: mdiaz3{at}lsu.edu

{dagger} Deceased (14 September 2002).

Applied and Environmental Microbiology, January 2006, p. 306-312, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.306-312.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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