Title page for ETD etd-11182005-035932

Type of Document Dissertation
Author Diaz, Maria Alejandra
Author's Email Address mdiaz3@lsu.edu
URN etd-11182005-035932
Title Plasmid-Mediated High-Level Gentamicin Resistance in Bacteria from the Turtle Farm Environment in Louisiana
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
Richard K. Cooper Committee Chair
Anne Grove Committee Member
Huangen Ding Committee Member
Patrick J. DiMario Committee Member
Albert R. Cunningham Dean's Representative
  • integrons
  • high-level gentamicin resistance
  • salmonella
Date of Defense 2005-11-11
Availability unrestricted
The sale of small turtles is banned from the US 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 1000 μg/ml gentamicin sulfate baths 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 Salmonella enterica subsp. enterica, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2000 μg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) contributing to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate their incidence in these bacteria, as well as to explore the molecular elements that contribute to the dissemination of gentamicin resistance in this context. R-plasmids from gentamicin resistant strains were transferred by conjugation and transformation to naīve Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of aminoglycoside acetyltransferase genes aac(3)IIa and aac(3)VIa. Multiplex PCR assays showed that every gentamicin resistant isolate carried either one of these acetyltransferase genes. Pulsed-Field Gel Electrophoresis and restriction enzyme digestion analysis of R-plasmids carrying these genes revealed plasmids with sizes ranging from ca. 45 kb to 145 kb, with different restriction profiles, supporting their presence on mobile molecular elements. In effect, gene aac(3)VIa was present as a gene cassette of a class 1 integron. Furthermore, evidence suggests that gene aac(3)IIa may be encoded as part of a complex class 1 integron. The presence of aac(3)IIa and aac(3)VIa in mobile molecular elements explains the distribution of these genes in the different plasmids and organisms described in this study. Further studies are required to characterize these molecular elements, and to detect genes which may be co-selected in the presence of gentamicin. This work is one of the first to report dissemination of high-level gentamicin resistance in Gram-negative bacteria, particularly in environmental isolates of Salmonella enterica subsp. arizonae.
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