Date of Award

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

Several genera of aminoglycoside resistant enteric bacteria were isolated from commercial turtle farms. Citrobacter freundii and Proteus vulgaris were the most prevalent organisms. Gentamicin resistance, measured by minimum inhibitory concentrations (MIC), ranged as high as 100 $\mu$g/ml. While resistance to other antibiotics was detected in gentamicin-resistant isolates, no correlation was observed between antibiotic resistance and plasmid content. Spontaneous mutation to gentamicin could not account for the high levels of gentamicin resistance found in the original isolates. Regions of chromosomal DNA encoding gentamicin resistance of 100 $\mu$g/ml were obtained from two Citrobacter freundii, two Proteus vulgaris, and two Salmonella enteritidis strains. These recombinant plasmids imparted a level of gentamicin resistance to Escherichia coli similar to that observed in the resistant isolates. The resistance gene was located on a 858 base pair DNA fragment. This fragment was common to both Citrobacter strains and hybridized with chromosomal DNA from several gentamicin-resistant enterics. The nucleotide sequences of the resistance genes obtained from Citrobacter freundii, Proteus vulgaris and Salmonella enteritidis shared complete homology. The region upstream of the C. freundii resistance gene shared only 49% homology with consensus promoter sequences in E. coli. The gentamicin resistance gene differed by only 3 bases from the aacC2 gene of plasmid pWP113a from Klebsiella. By substrate profiling, the 30,000 dalton protein encoded by the C. freundii resistance gene was identified as an aminoglycoside-(3)-N-acetyltransferase II. Gentamicin, kanamycin, and neomycin acetyltransferase activities of cell extracts correlated with the respective resistances against these antibiotics. Neither levels of gentamicin resistance nor acetyltransferase activities were affected by gene dosage or level of transcription. Consistent with the calculated codon usage index, the C. freundii acetyltransferase appears to be translated at low, constitutive levels. Based on aminoglycoside acetyltransferase assays and DNA hybridization there may be three different gentamicin resistance genes within the turtle population.

Pages

130

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