Ken Shimuta1, Gene Igawa2, Mitsuru Yasuda3, Takashi Deguchi4, Shu-Ichi Nakayama2, Makoto Ohnishi5. 1. Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan; Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address: shimuta@niid.go.jp. 2. Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan. 3. Center for Nutrition Support and Infection Control, Gifu University Hospital; Gifu University Center for Conservation of Microbial Genetic Resource, Organization for Research and Community Development. 4. Department of Urology, Graduate School of Medicine, Gifu University, Gifu, Japan. 5. Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address: ohnishi7@niid.go.jp.
Abstract
OBJECTIVES: Ceftriaxone (CRO) resistance is spreading worldwide, and hindering the effective treatment of gonococcal infections. This study developed a detection system for the genomic DNA of CRO-resistant Neisseria gonorrhoeae (N. gonorrhoeae) strains, in order to improve the surveillance of antimicrobial resistance. METHODS: A real-time PCR assay targeting the penA gene of recently isolated CRO-resistant N. gonorrhoeae strains was designed. Primer and probe sequence information was obtained from sequence comparisons between penA of Neisseria spp. and penA of CRO-resistant N. gonorrhoeae strains. RESULTS: Using this assay, a positive reaction was observed using the genomic DNA of three strains (GU140106, FC428, and A8806). The assay was evaluated using genomic DNA of 204 N. gonorrhoeae and 95 Neisseria spp. isolates with known minimum inhibitory concentrations of CRO. Following PCR assays for these strains, three FC428-related strains were positively identified, which possessed penA-60.001, whereas the remaining 201 N. gonorrhoeae strains and 95 Neisseria spp. strains were negative. CONCLUSIONS: A real-time PCR-based assay was designed to detect the genomic DNA of strains harbouring mosaic penA-59.001 (GU140106), penA-60.001 (FC428), and penA-64.001 (A8806) alleles and to discriminate them from N. gonorrhoeae and Neisseria spp. strains harbouring other genes.
OBJECTIVES:Ceftriaxone (CRO) resistance is spreading worldwide, and hindering the effective treatment of gonococcal infections. This study developed a detection system for the genomic DNA of CRO-resistant Neisseria gonorrhoeae (N. gonorrhoeae) strains, in order to improve the surveillance of antimicrobial resistance. METHODS: A real-time PCR assay targeting the penA gene of recently isolated CRO-resistant N. gonorrhoeae strains was designed. Primer and probe sequence information was obtained from sequence comparisons between penA of Neisseria spp. and penA of CRO-resistant N. gonorrhoeae strains. RESULTS: Using this assay, a positive reaction was observed using the genomic DNA of three strains (GU140106, FC428, and A8806). The assay was evaluated using genomic DNA of 204 N. gonorrhoeae and 95 Neisseria spp. isolates with known minimum inhibitory concentrations of CRO. Following PCR assays for these strains, three FC428-related strains were positively identified, which possessed penA-60.001, whereas the remaining 201 N. gonorrhoeae strains and 95 Neisseria spp. strains were negative. CONCLUSIONS: A real-time PCR-based assay was designed to detect the genomic DNA of strains harbouring mosaic penA-59.001 (GU140106), penA-60.001 (FC428), and penA-64.001 (A8806) alleles and to discriminate them from N. gonorrhoeae and Neisseria spp. strains harbouring other genes.