Detection of two different kanamycin resistance genes in naturally occurring isolates of Campylobacter jejuni and Campylobacter coli

Abstract

A total of 225 isolates of Campylobacter jejuni and 54 isolates of Campylobacter coli were screened for resistance to kanamycin. Among these, five resistant isolates of C. jejuni and six resistant isolates of C. coli, all with different plasmid patterns, were identified. Each contained at least one plasmid greater than or equal to 41 kilobases in size. The MIC of kanamycin for all 11 strains was determined to be greater than or equal to 256 micrograms/ml by an agar dilution method. In addition, all of the strains exhibited resistance to tetracycline (greater than or equal to 16 micrograms/ml). Eight of the 11 strains transferred the kanamycin resistance phenotype to other Campylobacter strains by conjugation. DNA from 9 of the 11 strains hybridized to a DNA probe specific for the 3'-O-aminoglycoside phosphotransferase type III gene. The remaining two strains also failed to show homology with DNA probes specific for the genes encoding 3'-O-aminoglycoside phosphotransferase types I, II, and III. The novel kanamycin resistance gene was cloned into the vector pBR322 and was expressed in Escherichia coli. Phosphocellulose paper binding assays on sonicates of the E. coli strain carrying the cloned kanamycin determinant demonstrated significant activity against kanamycin, neomycin, and amikacin but not against butirosin, gentamicin, tobramycin, or lividomycin, suggesting that the enzyme is the product of a 3'-O-aminoglycoside phosphotransferase type of aminoglycoside resistance gene.