However, this approach was insufficient for identifying significant mutations linked to high-level FQ resistance because it determined only the presence or absence of gyrA mutations at codon 83 or parC mutations at codon 80. As an alternative, PCR-restriction fragment length polymorphism (RFLP) has been used to detect mutations associated with FQ resistance in A. baumannii and epidemiological studies of resistant strains.Īlthough DNA sequencing is a reliable technique for detecting mutations, it is costly, time-consuming, and laborious when analyzing numerous clinical strains. Detection of these mutations is therefore important for assessing FQ resistance in A. The single mutation affecting Glu-87 of GyrA, an important mutation associated with FQ resistance in other gram-negative microorganisms, has rarely been found in A. In particular, a double mutation, affecting the Ser-83 of GyrA and Ser-80 or Glu-84 of ParC, renders A. baumannii are within the QRDRs at Ser-83 in GyrA and at Ser-80 and Glu-84 in ParC. The most frequently described mutations in A. baumannii, FQ resistance occurs mainly through mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase ( gyrA) and topoisomerase IV ( parC), although overexpression of efflux pumps can contribute to FQ resistance. FQ resistance has increased globally in Acinetobacter species, which are clinically important pathogens that frequently cause infections among intensive care unit patients. Keywords: Quinolone resistance-determining regions, PCR-restriction fragment length polymorphism, Acinetobacter baumannii, Fluoroquinolone resistanceįluoroquinolones (FQs) are widely used to treat various bacterial infections. baumannii gyrA and parC mutations with FQ resistance. This assay specifically amplified gyrA and parC from A. 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. 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. 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 amplification of gyrA and parC was successful for all A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis. 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. Methodsīased on the conserved sequences of A. 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. Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase ( gyrA) and topoisomerase IV ( parC) are linked to fluoroquinolone (FQ) resistance. 1ġDepartments 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, Japanĭepartment of Microbiology and Infectious Diseases, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii
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