Homozygous triplicate mutations in three 16S rRNA genes responsible for high-level aminoglycoside resistance in Nocardia farcinica clinical isolates from a Canada-wide bovine mastitis epizootic

Abstract

Nocardia farcinica strains showing high-level resistance to amikacin were isolated from clinical cases in a Canada-wide bovine mastitis epizootic. Shotgun cloning of the resistance genes in the amikacin-resistant mastitis isolate N. farcinica IFM 10580 (W6220 [Centers for Disease Control and Prevention]) using a multicopy vector system revealed that the 16S rRNA gene with an A-to-G single-point mutation at position 1408 (in Escherichia coli numbering) conferred "moderate" cross-resistance to amikacin and other aminoglycosides to an originally susceptible N. farcinica strain IFM 10152. Subsequent DNA sequence analyses revealed that, in contrast to the susceptible strain, all three chromosomal 16S rRNA genes of IFM 10580, the epizootic clinical strain, contained the same A1408G point mutations. Mutant colonies showing high-level aminoglycoside resistance were obtained when the susceptible strain N. farcinica IFM 10152 was transformed with a multicopy plasmid carrying the A1408G mutant 16S rRNA gene and was cultured in the presence of aminoglycosides for 3 to 5 days. Of these transformants, at least two of the three chromosomal 16S rRNA genes contained A1408G mutations. A triple mutant was easily obtained from a strain carrying the two chromosomal A1408G mutant genes and one wild-type gene, even in the absence of the plasmid. The triple mutant showed the highest level of resistance to aminoglycosides, even in the absence of the plasmid carrying the mutant 16S rRNA gene. These results suggest that the homozygous mutations in the three 16S rRNA genes are responsible for the high-level aminoglycoside resistance found in N. farcinica isolates of the bovine mastitis epizootic.

FIG. 1.

N. farcinica rRNA operons, rrnA, rrnB, rrnC, and upstream genes. Positions of forward and reverse PCR primers used in the present study are indicated by arrows. Partial sequences of the A-sites for wild-type (IFM 10152) and the epizootic aminoglycoside resistant (IFM 10580) strains are also shown.

FIG. 2.

Growth curves of N. farcinica strains: IFM 10152 (wild type) (•), the clinical isolate IFM 10580 (⧫), IFM 10152-M2 (▴), and IFM 10152-M3 (▪) in the presence of 1,024 μg of amikacin/ml.