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A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii.
Ann Lab Med. 2020 Jan; 40(1):27-32.AL

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.

Authors+Show Affiliations

Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan. rnakano@naramed-u.ac.jp.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan. Department of Otolaryngology-Head and Neck Surgery, Nara Medical University, Nara, Japan.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan.International University of Health and Welfare, Shioya Hospital, Tochigi, Japan.Department of Otolaryngology-Head and Neck Surgery, Tohoku University Graduate School of Medicine, Miyagi, Japan.Department of Microbiology and Immunology, Teikyo University School of Medicine, Tokyo, Japan.Department of Microbiology and Infectious Diseases, Nara Medical University, Nara, Japan.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31432636

Citation

Kakuta, Naoki, et al. "A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter Baumannii." Annals of Laboratory Medicine, vol. 40, no. 1, 2020, pp. 27-32.
Kakuta N, Nakano R, Nakano A, et al. A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii. Ann Lab Med. 2020;40(1):27-32.
Kakuta, N., Nakano, R., Nakano, A., Suzuki, Y., Tanouchi, A., Masui, T., Horiuchi, S., Endo, S., Kakuta, R., Ono, Y., & Yano, H. (2020). A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii. Annals of Laboratory Medicine, 40(1), 27-32. https://doi.org/10.3343/alm.2020.40.1.27
Kakuta N, et al. A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter Baumannii. Ann Lab Med. 2020;40(1):27-32. PubMed PMID: 31432636.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii. AU - Kakuta,Naoki, AU - Nakano,Ryuichi, AU - Nakano,Akiyo, AU - Suzuki,Yuki, AU - Tanouchi,Ayako, AU - Masui,Takashi, AU - Horiuchi,Saori, AU - Endo,Shiro, AU - Kakuta,Risako, AU - Ono,Yasuo, AU - Yano,Hisakazu, PY - 2019/02/15/received PY - 2019/03/31/revised PY - 2019/07/26/accepted PY - 2019/8/22/entrez PY - 2019/8/23/pubmed PY - 2020/2/27/medline KW - Acinetobacter baumannii KW - Fluoroquinolone resistance KW - PCR-restriction fragment length polymorphism KW - Quinolone resistance-determining regions SP - 27 EP - 32 JF - Annals of laboratory medicine JO - Ann Lab Med VL - 40 IS - 1 N2 - 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. SN - 2234-3814 UR - https://www.unboundmedicine.com/medline/citation/31432636/A_Novel_Mismatched_PCR_Restriction_Fragment_Length_Polymorphism_Assay_for_Rapid_Detection_of_gyrA_and_parC_Mutations_Associated_With_Fluoroquinolone_Resistance_in_Acinetobacter_baumannii_ L2 - http://www.annlabmed.org/journal/viewJournal.html?year=2020&vol=40&page=27 DB - PRIME DP - Unbound Medicine ER -