Ann Lab Med.  2020 Jan;40(1):27-32. 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

• Kakuta N
• Nakano R
• Nakano A
• Suzuki Y
• Tanouchi A
• Masui T
• Horiuchi S
• Endo S
• Kakuta R
• Ono Y
• Yano H

• KMID: 2457490
• DOI: http://doi.org/10.3343/alm.2020.40.1.27

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.

Keyword

Quinolone resistance-determining regions; PCR-restriction fragment length polymorphism; Acinetobacter baumannii; Fluoroquinolone resistance

MeSH Terms

Acinetobacter baumannii*
Acinetobacter*
Conserved Sequence
DNA Gyrase
DNA Topoisomerase IV
DNA-Directed RNA Polymerases
Polymerase Chain Reaction
Sequence Analysis
DNA Gyrase
DNA Topoisomerase IV
DNA-Directed RNA Polymerases

Figure

• 

  Fig. 1 Strategy used for mismatched PCR-RFLP of gyrA and parC QRDRs in A. baumannii. The reverse primers for gyrA and parC are located immediately downstream of the nucleotide sequences corresponding to GyrA87 and ParC84, respectively. The reverse primer for gyrA was designed with one mismatched nucleotide to create an XmnI recognition site (GAANNNNTTC) in the gyrA region containing the codon for Glu-87 (GAA). The reverse primer for parC was designed with two mismatched nucleotides to create an XmnI recognition site in the parC region containing the codon for Glu-84 (GAA). Boldface represents codons 83 and 87 of gyrA and codons 80 and 84 of parC. Underlined DNA sequences indicate restriction sites present in the QRDRs of FQ-susceptible strains.Abbreviations: FQ, fluoroquinolone; QRDR, quinolone resistance-determining region; RFLP, restriction fragment length polymorphism.

• 

  Fig. 2 PCR-RFLP patterns obtained following digestion with HinfI or XmnI for gyrA and parC. Lanes 1 to 3 and 4 to 6 show PCR-RFLP results for gyrA and parC, respectively. Lane: M, 20 bp DNA ladder marker. (A) PCR-RFLP results for A. baumannii ATCC19606. Lanes: 1, undigested (143 bp); 2, HinfI-digestion (103 bp and 40 bp); 3, XmnI-digestion (122 bp and 21 bp); 4, undigested (120 bp); 5, HinfI-digestion (85 bp and 35 bp); and 6, XmnI-digestion (104 bp and 16 bp). (B) PCR-RFLP results for the representative FQ-resistant A. baumannii strain possessing mutations in gyrA (83) and parC (80). Lanes: 1, undigested (143 bp); 2, HinfI-digestion (143 bp); 3, XmnI-digestion (122 bp and 21 bp); 4, undigested (120 bp); 5, HinfI-digestion (120 bp); and 6, XmnI-digestion (104 bp and 16 bp).Abbreviations: FQ, fluoroquinolone; RFLP, restriction fragment length polymorphism.

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