title: Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.
abstract: 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.
authors.affiliation: Department of Microbiology and Infectious Disease, Toho University School of Medicine, Tokyo, Japan; Department of Laboratory Medicine, Tohoku University Hospital, Miyagi, Japan; Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address: nagasawa-m@umin.net.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Nagasawa
authors.affiliation: Department of Infection Control and Laboratory Diagnostics, Tohoku University Graduate School of Medicine, Miyagi, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Kaku
authors.affiliation: Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Kamachi
authors.affiliation: Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Shibayama
authors.affiliation: Department of Bacteriology/Drug Resistance and Pathogenesis, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Yoshichika
authors.initials: Y
authors.lastname: Arakawa
authors.affiliation: Department of Microbiology and Infectious Disease, Toho University School of Medicine, Tokyo, Japan.
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Department of Microbiology and Infectious Disease, Toho University School of Medicine, Tokyo, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
mesh_terms: D000150:Acinetobacter / Q000235:genetics; D000617:Aminoglycosides / Q000494:pharmacology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004755:Enterobacteriaceae / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000235:genetics*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000617:Aminoglycosides; D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008780:Methyltransferases
keywords: 16S rRNA methylase; Aminoglycoside resistance; Loop-mediated isothermal amplification; armA; rmtA; rmtB