Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa

Abstract

Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing™ for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing™ as a substitute for traditional methods as it provides a rapid and reliable technique for determinating the antibiotic resistance pattern of a given bacterial strain in <1 h.

Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance

“…The isolates were recovered from various clinical sites, and most of them exhibited resistance not only to fluoroquinolones but also to various other antimicrobial compounds (see Table S1 in the supplemental material). Pyrosequencing technology has been proven to be time and cost competitive and to allow efficient detection of SNPs in localized regions where the nucleotide variants are known (24). We have designed two different pyrosequencing assays for sequencing analysis of the most prominent mutation hot spots in the QRDR of the A subunit of the DNA gyrase, encoded by gyrA, which spans amino acid positions 83 to 87, and in the QRDR of the A subunit of topoisomerase IV, encoded by parC, which spans amino acid positions 82 to 84.…”

Molecular Characterization of Extraintestinal Escherichia coli Isolates in Japan: Relationship between Sequence Types and Mutation Patterns of Quinolone Resistance-Determining Regions Analyzed by Pyrosequencing

“…Because pyrosequencing is less labor-and time-intensive than the conventional Sanger method for nucleotide sequence analysis, this method has already been used successfully to identify the resistance-conferring genes of several bacterial species (14)(15)(16)(17). This method appears to be especially suitable as a tool for identifying "hot spot" mutations of QRDRs, namely, at amino acid positions 83 and 87 in GyrA and at positions 80 and 84 in ParC of E. coli.…”

Assesment of the Resistance of Clinical Isolates Pseudomonas Aeruginosa to Quinolonones

“…But the problem, which is very important for the treatment of infections, caused by these microorganisms, is the resistance development to quinolones in the last years. The main mechanisms of resistance are mutations in the target genes, those encoding DNA gyrase (gyrA) and topoisomerase IV (parC) (2,5,6) and in regulatory genes for drug efflux pumps (7).…”