Rapid Screening of Topoisomerase Gene Mutations by a Novel Melting Curve Analysis Method for Early Warning of Fluoroquinolone-Resistant
            <i>Streptococcus pneumoniae</i>
            Emergence

Fukushima, Kazuko Y.; Hirakata, Yoichi; Sugahara, Kazuyuki; Yanagihara, Katsunori; Kondo, Akira; Kohno, Shigeru; Kamihira, Shimeru

doi:10.1128/jcm.01887-06

Cited by 11 publications

(8 citation statements)

References 27 publications

(30 reference statements)

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“…As shown in Table 4, the mutation profiles for the QRDRs in the gyrA and parC genes revealed a close relationship between the MIC level and the number of QRDR mutations. Previous studies have found that the conventional phenotypic method failed to detect strains that have a single QRDR mutation; these strains have the potential to develop into a highly resistant pathogen (8). Several reports have noted that a significant number of Streptococcus pneumoniae isolates already have a single-step mutation and are prone to acquiring a second-step mutation (19).…”

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confidence: 99%

Melting Curve Analysis for Rapid Detection of Topoisomerase Gene Mutations inHaemophilus influenzae

Nakamura¹,

Yanagihara²,

Morinaga³

et al. 2009

J Clin Microbiol

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We established a real-time PCR assay with melting curve analysis to rapidly genotype quinolone resistancedetermining regions (QRDRs) of gyrase A and topoisomerase IV genes in Haemophilus influenzae. This assay is a useful tool for the detection of fluoroquinolone resistance and for the early detection of preexisting QRDR mutations.Haemophilus influenzae is a major causative pathogen isolated from infections, including acute and chronic respiratory infections, acute otitis media, sinusitis, and meningitis in pediatric patients. Recent reports have noted the prevalence of fluoroquinolone (FQ)-resistant H. influenzae (1,3,4,9,11,14,20,22,26). FQ-resistant H. influenzae often carries mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and the parC genes, which encode subunits of DNA gyrase and topoisomerase IV, respectively (9,11,16,18,23). A combination of real-time PCR methods and melting curve analysis (PCR-MCA) is a useful tool for the rapid detection of key gene mutations associated with drug resistance in various microorganisms (13,24,25), but there are no reports about H. influenzae. The aim of this study was to develop a PCR-MCA method for detecting H. influenzae strains by targeting a total of four QRDR positions in the gyrA (codons 84 and 88) and the parC (codons 84 and 88) genes that are frequently associated with FQ resistance (9,11,16,23). This current method could simultaneously identify the gyrA and the parC mutations by using only one PCR performance.Seventeen H. influenzae clinical isolates were used. Ten of the strains were susceptible to FQ, and seven of the strains had low susceptibility or were resistant to FQ. The seven FQresistant/low-susceptibility strains consisted of one strain (NUH-1) from Nagasaki University Hospital, one strain (BY-1) from Bayer (Osaka, Japan), two strains (DR-1 and DS-2) from Daiichi-Sankyo (Tokyo, Japan), and three strains (MSC24060, MSC27995, and MSC11438) kindly provided by Meiji-Seika Kaisha (Tokyo, Japan) (21). The 10 FQ-susceptible strains were isolated from patients at Nagasaki University Hospital. Identification of H. influenzae was confirmed by colony morphology, Gram staining, growth on chocolate agar, and the X and V factor requirements. The MICs of ciprofloxacin (CPFX), sparfloxacin, levofloxacin (LVFX), gatifloxacin, moxifloxacin, garenoxacin, and sitafloxacin were determined by a broth dilution method using Haemophilus test medium according to the recommendations of the Clinical and Laboratory Standards Institute (5). H. influenzae ATCC 51907 was used for quality control.DNA was extracted from each strain by using a QIAamp DNA mini-kit (Qiagen, Hilden, Germany). Sequences of the oligonucleotides and probes are shown in Tables 1 and 2. The sequences are from the known sequences of the parC and gyrA genes, which were derived from GenBank accession no. NP439678 and NP439419, respectively. To identify mutations in the QRDRs of gyrA and parC in these strains, we performed PCR and direct DNA sequencing according to the method described ...

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confidence: 99%

Melting Curve Analysis for Rapid Detection of Topoisomerase Gene Mutations inHaemophilus influenzae

Nakamura¹,

Yanagihara²,

Morinaga³

et al. 2009

J Clin Microbiol

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“…Isolates D001 and D002 had both GyrA and ParC mutations (for D001, Ser79Phe in ParC and Gly85Lys in GyrA; for D002, Ser79Phe in ParC and Ser81Phe in GyrA). All the strains were exposed to 2,4,8,16,32, and 64ϫ the MICs for ciprofloxacin (CIP), LVX, GAT, MXF, and GAR for 48 to 72 h at 37°C in 5% CO 2 . The MPCs were measured using a procedure previously described (2).…”

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In Vitro Activity of Garenoxacin against Streptococcus pneumoniae Mutants with Characterized Resistance Mechanisms

Yamamoto

Yanagihara

Sugahara

et al. 2009

Antimicrob Agents Chemother

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“…However, this is highly improbable, since molecular typing of 9 of these Several real-time PCR methods based on the detection of point mutations have been developed to differentiate between drug-susceptible and -resistant bacteria (4,6,12,14,15,16). However, to the best of our knowledge, only two real-time PCR assays followed by high-resolution melt (HRM) curve analysis have been developed to track antibiotic resistance in Mycoplasma pneumoniae (12,15), both for macrolides.…”

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confidence: 99%

Development and Evaluation of a Novel Single-Nucleotide-Polymorphism Real-Time PCR Assay for Rapid Detection of Fluoroquinolone-Resistant Mycoplasma bovis

Shabat¹,

Mikula²,

Gerchman³

et al. 2010

J Clin Microbiol

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Monitoring of the susceptibility of Mycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However, in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains of M. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan singlenucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification of M. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of the parC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific for M. bovis, with a detection limit of 5 fg/l (about 5 M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility of M. bovis to fluoroquinolones.Mycoplasma bovis is an important and emerging cause of bovine respiratory disease (BRD), calf pneumonia, mastitis, arthritis, and otitis media, as well as various less common presentations (11). Clinical disease associated with M. bovis is often chronic, debilitating, and poorly responsive to antimicrobial therapy, resulting in significant economic loss, the full extent of which is sometimes difficult to estimate (5, 11). Moreover, since there is no effective vaccine for M. bovis, use of antibiotics is the main control method for this infection.Fluoroquinolones are broad-spectrum antimicrobials highly effective for the treatment of a variety of clinical and veterinary infections and frequently used for the treatment of M. bovis infection in cattle. Their antibacterial activity is due mainly to inhibition of DNA replication. A major mechanism of fluoroquinolone resistance in prokaryotes (including Mollicutes) involves alterations in the target enzymes DNA gyrase and DNA topoisomerase IV, thereby altering affinity for the drug (2, 8). The mutations associated with resistance have been localized within the quinolone resistance-determining regions (QRDRs) of DNA gyrase subunits GyrA and GyrB and/or topoisomerase IV subunits ParC and ParE.Recently, the QRDRs of these genes in 42 M. bovis clinical isolates exhibiting various levels of susceptibility to fluoroquinolones were characterized (10). The data showed that 10/...

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Rapid Screening of Topoisomerase Gene Mutations by a Novel Melting Curve Analysis Method for Early Warning of Fluoroquinolone-Resistant Streptococcus pneumoniae Emergence

Cited by 11 publications

References 27 publications

Melting Curve Analysis for Rapid Detection of Topoisomerase Gene Mutations inHaemophilus influenzae

Melting Curve Analysis for Rapid Detection of Topoisomerase Gene Mutations inHaemophilus influenzae

In Vitro Activity of Garenoxacin against Streptococcus pneumoniae Mutants with Characterized Resistance Mechanisms

Development and Evaluation of a Novel Single-Nucleotide-Polymorphism Real-Time PCR Assay for Rapid Detection of Fluoroquinolone-Resistant Mycoplasma bovis

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