Molecular analysis of high-level streptomycin resistance in Erwinia amylovora

Streptomycin-resistant and -sensitive strains of Erwinia amylovora from New Zealand and five states in the United States were compared on streptomycin- and myomycin-amended media. Strains with high resistance to streptomycin (HR strains) were insensitive to myomycin, an antibiotic resembling streptomycin in its mode of action, whereas strains with medium resistance (MR) or sensitivity (S) to streptomycin were sensitive to myomycin. No mutations were found in the 16S rRNA gene of nine HR strains of E. amylovora that could account for streptomycin resistance. Nucleotide sequence and allele-specific amplification-polymerase chain reaction analyses were used to examine S, MR, and HR strains for mutations in the rpsL gene. All 102 HR strains of E. amylovora contained a single base-pair mutation in codon 43 of their rpsL gene that resulted in an amino acid substitution in ribosomal protein S12. Codon 43, which encodes lysine in S and MR strains, was converted to a codon for arginin in 96 HR strains, a codon for asparagine in three HR strains, and a codon for threonine in three HR strains. In gene complementation studies sensitivity to streptomycin and myomycin was restored to E. amylovora and Escherichia coli HR strains with a plasmid carrying the wild-type E. amylovora rpsL gene. Conversely, resistance to streptomycin and insensitivity to myomycin in E. amylovora and E. coli S strains was restored only when the plasmid carried a mutant rpsL gene with the lysine-to-arginine substitution in codon 43. We conclude that mutation in a single codon of ribosomal protein S12 gene rpsL have resulted in high-level streptomycin resistance in E. amylovora. Two genetic base of streptomycin resistance have now been identified in E. amylovora. These results indicate that the genetic mechanism determines the level of streptomycin resistance and the expression of myomycin resistance and that the presence or relative importance of the two mechanisms differ among various geographic regions