Ann Lab Med 2020; 40(1): 27-32  https://doi.org/10.3343/alm.2020.40.1.27
A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii
Naoki Kakuta, M.D.1,*, Ryuichi Nakano, Ph.D.1,*, Akiyo Nakano, M.S.1, Yuki Suzuki, M.S.1, Ayako Tanouchi, M.S.1, Takashi Masui, M.D.1,2, Saori Horiuchi, M.S.1, Shiro Endo, M.D.3, Risako Kakuta, M.D.4, Yasuo Ono, M.D.5, and Hisakazu Yano, M.D.1
1Departments of Microbiology and Infectious Diseases and 2Otolaryngology-Head and Neck Surgery, Nara Medical University, Nara, Japan; 3International University of Health and Welfare, Shioya Hospital, Tochigi, Japan; 4Department of Otolaryngology-Head and Neck Surgery, Tohoku University Graduate School of Medicine, Miyagi, Japan; 5Department of Microbiology and Immunology, Teikyo University School of Medicine, Tokyo, Japan
Corresponding author: Ryuichi Nakano, Ph.D.
Department of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
Tel: +81-744-29-8839 Fax: +81-744-29-8839 E-mail: rnakano@naramed-u.ac.jp

* These authors contributed equally to this work.
Received: February 15, 2019; Revised: March 31, 2019; Accepted: July 26, 2019; Published online: January 1, 2020.
© Korean Society for Laboratory Medicine. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrAse (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii.
Methods: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.
Results: PCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain.
Conclusions: This assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.
Keywords: Quinolone resistance-determining regions, PCR-restriction fragment length polymorphism, Acinetobacter baumannii, Fluoroquinolone resistance



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