This study demonstrated that the homozygous triplicate point mutations that occurred at the position 1408 (in E. coli numbering) of each chromosomal 16S rRNA gene were responsible for the high-level 2-DOS aminoglycoside cross-resistance observed in the N. farcinica isolates as a causative pathogen of Canada-wide bovine mastitis epizootic.
Aminoglycosides have been divided into two major classes: those containing the streptamine moiety, such as streptomycin, and those containing the 2-DOS moiety, such as the neomycins. The 2-DOS-containing aminoglycosides bind to the end of helix 44 in 16S rRNA near the location of the A-site, which binds tRNA (20
). The nucleotide at position 1408 resides in the A-site and 2-DOS aminoglycosides block bacterial growth through interference with protein synthesis. High-level aminoglycoside resistance attributable to alterations of antibiotic-binding targets might involve methylation of conserved regions within 16S rRNA molecules, e.g., an adenine at either position 1405 or position 1408 of 16S rRNA. Modifications of these types have been found in aminoglycoside-producing organisms such as Streptomyces
) and recently in clinical strains of Gram-negative bacteria such as Pseudomonas aeruginosa
), Klebsiella pneumoniae
), and Serratia marcescens
). On the other hand, mutational alterations of 16S rRNA at position 1408 have been found to mediate acquired high-level cross-resistance to 2-DOS aminoglycosides in E. coli
and M. smegmatis in vivo
). However, clinical high-level resistance to aminoglycosides attributable to 1408G mutation in the 16S rRNA has been found only in Mycobacterium
species carrying a single chromosomal rRNA operon, such as M. tuberculosis
, M. abscessus
, and M. chelonae
); it has not been found in organisms that carry more than two rRNA operons, such as members of the enterobacteria family, staphylococci, and streptococci. It has been considered that acquisition of aminoglycoside resistance by rRNA mutation might not occur or that it might be rare because most bacteria harbor multiple rRNA operons and because the mutant allele is usually recessive in strains that are heterozygous for wild-type and mutated rRNAs, as shown in the M. smegmatis
strain, which carries two rRNA operons (24
), although partial dominance of aminoglycoside resistance attributable to 16S rRNA mutation has also been reported in E. coli
). The strains of N. farcinica
have three chromosomal rRNA operons. The bovine mastitis epizootic clinical strains of N. farcinica
, including IFM 10580, represent the first clinical case of high-level aminoglycoside resistance attributable to rRNA alteration in bacterial species carrying more than two copies of chromosomal rRNA operons.
The results of these analyses of transformants carrying 1408G mutated rRNA and spontaneous mutants that acquired the 1408G point mutation in the chromosomal 16S rRNA genes revealed that aminoglycoside resistance mediated by the rRNA mutation was recessive in N. farcinica
strains. Introduction of mutated 16S rRNA gene by extrachromosomal replication of the multicopy plasmid [pNV19-16S (1408G)] conferred moderate levels of cross-resistance to 2-DOS aminoglycosides (e.g., MIC ≈ 64 μg/ml for amikacin) to susceptible strain IFM 10152. On the other hand, highly resistant (MIC > 1,024 for amikacin) mutants were obtained spontaneously when moderately resistant cells were cultured in the presence of 1,024 μg of amikacin/ml for a few days (Table , last column). The results show that the strains which are highly resistant to aminoglycosides had two 1408G point-mutated copies (rrnA 16S
and rrnC 16S
) and one wild-type copy in chromosomal 16S rRNA genes [Table , analysis3, IFM 10152-M2/pNV19-16S(1408G)]. The heterozygous mutants were amikacin susceptible (MIC < 1 μg/ml) in the absence of the plasmid carrying mutated rRNA (Table , analysis 4, IFM 10152-M2) but were highly resistant to amikacin (MIC > 1,024 μg/ml) in the presence of the plasmid (Table , analysis 3). The cells became highly resistant when the third chromosomal 16S rRNA gene, rrnB 16S
, obtained A1408G mutation (Table , analysis 5, IFM 10152-M3). These results suggest rRNA mutation-mediated aminoglycoside resistance is recessive, supporting a previous report in M. smegmatis
, where A1408G point mutations were artificially introduced into one or two of 16S rRNA genes (24
At present, it remains unknown how the clinical amikacin-resistant strains acquired multiple homozygous A1408G point mutations in the three chromosomal 16S rRNA genes. We think it is unlikely that the highly amikacin-resistant clinical strains arose through recombination between chromosomal 16S rRNA genes and plasmids, as observed in experimental conditions (24, present work, Table ). We assume in the epizootic a single A1408G mutation occurred at either one of the three 16S rRNA genes, followed by successive homozygous mutations in the other two rrn 16S
genes in response to the continued used of aminoglycosides at a high concentration, resulting in the amikacin-resistant clinical strains. Indeed, the highly resistant chromosomal mutants (IFM 10152-M3) were easily obtained from the susceptible chromosomal mutant without carrying the plasmids (IFM 10152-M2) in the presence of amikacin (at a frequency of one mutant from 2.3 × 103
cells) (Table ). The underlying mechanisms for the homozygous mutation might be gene conversions among 16S rRNAs, as have been reported for E. coli
, Vibrio parahaemolyticus
, and other strains (11
), or other novel but unknown mechanisms. Similar homologous recombination has also been hypothesized in Staphylococcus aureus
23S rRNA genes, where mutant gene dose-dependent increase in MIC to linezolid was observed (3
). It is of great importance to reveal the mechanisms involved in the homozygous mutations for purposes of clinical treatment and antibiotic therapy, as well as for biological interests.
Although the transformants of the susceptible strain IFM 10152 carrying plasmid-encoded mutated rRNA gene [IFM 10152/pNV19-16S(1408G)] acquired high-level resistance to 2-DOS aminoglycosides upon prolonged incubation in the presence of amikacin (Table , day 5), the spectrum of the aminoglycoside sensitivities differed slightly from that of the clinical resistant strain IFM 10580 (Table , e.g., netilmicin). Such a difference might be explained by a ratio or amounts of the wild-type (1408A) and the mutant (1408G) 16S rRNA molecules in the respective strains (only the mutant 16S rRNA is present in the clinical resistant strains, but both wild-type and the mutant rRNA are present in the transformants), and also by a difference in affinities of aminoglycosides to the two different types of the rRNA molecular complexes. Alternatively, some aminoglycosides, e.g., netilmicin, might have some mode of action in addition to binding to the A-site of the 16S rRNA complex.
The results of the present work generalized the previous finding that the mutant allele of 16S rRNA, which confers aminoglycoside resistance, is recessive in microbes having more than two 16S rRNA operons. Nevertheless, the present evidence conflicts with the idea that aminoglycoside resistance might seldom be obtained by multiple mutations of 16S rRNA genes in microbes having multiple 16S rRNA operons. We have not encountered clinical cases of “human” infection with aminoglycoside-resistant microbe harboring the serial 16S rRNA mutations, but we must remain aware of such cases for the future.