Rapid Screening of Fluoroquinolone Resistance Determinants in
            <i>Streptococcus pneumoniae</i>
            by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

Ip, Margaret; Chau, Shirley S.L.; Feng, Cheng; Qi, Amy; Lai, Robin Kit-Ho

doi:10.1128/jcm.44.3.970-975.2006

Cited by 13 publications

(9 citation statements)

References 32 publications

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“…Restriction fragment length polymorphism analysis is not a new technique for the typing of S. pneumoniae (1,3,6,12,18) but has been improved here by using an Agilent 2100 bioanalyzer for the accurate sizing of the DNA fragments. In addition, a PCR product of a noncoding region was used, which is likely to vary more between strains than a gene.…”

Section: Discussionmentioning

confidence: 99%

Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

et al. 2007

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Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.Streptococcus pneumoniae is an important human pathogen with a clonal population structure (4). Molecular typing has become increasingly important in order to understand the evolution of this pathogen and to trace clones with special traits such as antibiotic resistance. Several methods for genotypic analysis of S. pneumoniae are currently in use. The "gold standards" are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In PFGE, the whole genome is digested with a restriction endonuclease and run slowly through an agarose gel with an alternating current (10). The fragment banding pattern produced is used to characterize the strain. PFGE has the advantage of high discrimination between strains but is relatively laborious, time-consuming, and costly and tends to be used to compare strains within a laboratory rather than between laboratories because it involves the comparison of banding patterns rather than absolute values. MLST allows a direct comparison of genotypes by comparing sequences within several housekeeping genes (4). This technique has proven extremely useful in making comparisons between strains not only within a laboratory but also internationally by using a Web-based database. Sequencing, however, is currently expensive and time-consuming.Restriction fragment length polymorphism (RFLP) analysis is often used on a PCR amplification product that is subjected to endonuclease digestion followed by comparison of the resulting banding patterns produced on an agarose gel. This technique has been performed on coding regions such as the capsule genes (1) and penicillin-binding protein genes (12). It has some of the sho...

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Section: Discussionmentioning

confidence: 99%

Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

et al. 2007

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“…The mutations and the amino acid substitutions at the QRDRs in the gyrA, gyrB, parC, and parE genes were analyzed by PCR-RF-SSCP as previously described (17). Briefly, for each isolate, PCR amplicons of QRDRs of the gyrA and -B and parC and -E genes were digested with AluI, HinFI, Sau3AI, and MspI, respectively, and analyzed by SSCP.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we utilized a rapid screening method using PCR-restriction fragment (RF)-single-strand conformation polymorphism (SSCP) (17) to screen for variants/mutations at the quinolone resistance-determining regions (QRDRs) of the gyrase and topoisomerase genes in CIP-resistant S. pneumoniae (MIC Ն 4.0 g/ml). Using PCR-RF-SSCP, we identified a collection of atypical strains, presumed to be pneumococcus, with CIP MICs of Ն4.0 g/ml and unique patterns suggesting sequence variations within the QRDR in comparison with S. pneumoniae R6 and possible recombinational events with other streptococci.…”

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confidence: 99%

Fluoroquinolone Resistance in Atypical Pneumococci and Oral Streptococci: Evidence of Horizontal Gene Transfer of Fluoroquinolone Resistance Determinants from Streptococcus pneumoniae

Chau

Feng

et al. 2007

Antimicrob Agents Chemother

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Atypical strains, presumed to be pneumococcus, with ciprofloxacin MICs of >4.0 g/ml and unique sequence variations within the quinolone resistance-determining regions (QRDRs) of the gyrase and topoisomerase genes in comparison with the Streptococcus pneumoniae R6 strain, were examined. These strains were reidentified using phenotypic methods, including detection of optochin susceptibility, bile solubility, and agglutination by serotype-specific antisera, and genotypic methods, including detection of pneumolysin and autolysin genes by PCR, 16S rRNA sequencing, and multilocus sequence typing (MLST). The analysis based on concatenated sequences of the six MLST loci distinguished the "atypical" strains from pneumococci, and these strains clustered closely with S. mitis. However, all these strains and five of nine strains from the viridans streptococcal group possessed one to three gyrA, gyrB, parC, and parE genes whose QRDR sequences clustered with those of S. pneumoniae, providing evidence of horizontal transfer of the QRDRs of the gyrase and topoisomerase genes from pneumococci into viridans streptococci. These genes also conferred fluoroquinolone resistance to viridans streptococci. In addition, the fluoroquinolone resistance determinants of 32 well-characterized Streptococcus mitis and Streptococcus oralis strains from bacteremic patients were also compared. These strains have unique amino acid substitutions in GyrA and ParC that were distinguishable from those in fluoroquinolone-resistant pneumococci and the "atypical" isolates. Both recombinational events and de novo mutations play an important role in the development of fluoroquinolone resistance.It has been recognized that an overestimation of antimicrobial resistance may result from the misidentification of oral streptococci. Misleading macrolide and penicillin resistance rates in Streptococcus pneumoniae had resulted from the use of one or more phenotypic/genotypic characteristics in the identification of pneumococcus (28,38). In a study by Neeleman et al. (28), the identities of 141 macrolide-resistant presumptive pneumococcal isolates from 38 laboratories were reexamined. Only 65% (91/141) of the strains were confirmed as S. pneumoniae, while 23% (32/141) were Streptococcus mitis and the remaining streptococcal species. This led to significant overreporting of macrolide-resistant S. pneumoniae. Increasingly, "atypical" pneumococcal isolates that do not have the defining characteristics of S. pneumoniae, such as optochin susceptibility and deoxycholate (bile) solubility, have been identified, while other closely related oral streptococci, such as S. mitis and Streptococcus oralis, are found to harbor the pneumolysin (ply) and/or the autolysin (lytA) genes (13,14,39), causing confusion in the presumptive identification of S. pneumoniae in a diagnostic laboratory. Likewise, those strains which are discrepant in phenotype(s) and/or genotype(s) have also been identified from surveillance studies in Asian countries (22).The development of fluoroquinolone resi...

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“…Rapid detection of QRDR mutations is required and may constitute a more reliable approach than the current phenotypic method, which can represent susceptibility but cannot detect the potential of S. pneumoniae strains to harbor resistance. PCR-based techniques, such as PCR-restriction fragment length polymorphism analysis (1,15,20), single-stranded conformational polymorphism analysis (15), and PCR-oligonucleotide ligation (2), have been developed to detect QRDR mutations; however, these techniques are not appropriate for practical use, as they are complicated and time-consuming. More recently, real-time PCR methods combined with melting curve analysis (PCR-MCA) have been reported to be successful in the detection of key gene mutations associated with drug resistance in various microorganisms (12,25,26).…”

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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

Fukushima¹,

Hirakata

Sugahara³

et al. 2006

J Clin Microbiol

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Rapid Screening of Fluoroquinolone Resistance Determinants in Streptococcus pneumoniae by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

Cited by 13 publications

References 32 publications

Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

Fluoroquinolone Resistance in Atypical Pneumococci and Oral Streptococci: Evidence of Horizontal Gene Transfer of Fluoroquinolone Resistance Determinants from Streptococcus pneumoniae

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

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