Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of
16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated
16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp
DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of
Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and
DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to
aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce
16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing
16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to
aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of
16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.