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Bacteriology

Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus

Pascal Lapierre, Ann Huletsky, Véronique Fortin, François J. Picard, Paul H. Roy, Marc Ouellette, Michel G. Bergeron
Pascal Lapierre
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
2Division de Microbiologie, Faculté de Médecine
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Ann Huletsky
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
2Division de Microbiologie, Faculté de Médecine
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Véronique Fortin
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
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François J. Picard
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
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Paul H. Roy
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
3Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Sainte-Foy, Québec, Canada
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Marc Ouellette
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
2Division de Microbiologie, Faculté de Médecine
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Michel G. Bergeron
1Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL)
2Division de Microbiologie, Faculté de Médecine
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  • For correspondence: michel.g.bergeron@crchul.ulaval.ca
DOI: 10.1128/JCM.41.7.3246-3251.2003
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    FIG. 1.

    Example showing the real-time detection on a Smart Cycler of a fluoroquinolone-sensitive S. aureus strain (ATCC 25923) containing no mutation in the grlA gene (A) and a fluoroquinolone-resistant S. aureus strain (CCRI-12876) containing mutations at both codons 80 and 84 (B). For every sample tested, two separate PCRs, one containing probes S80-FAM and Sau-TET and one containing probes E84-FAM and Sau-TET, were performed. The left graphs show the fluorescence curves for the Sau-TET probe (hatched curve) and the S80-FAM probe (plain curve). The right graphs show the fluorescence curves for the Sau-TET probe (hatched curve) and the E84-FAM probe (plain curve). PCR assays were performed with crude DNA extracts prepared from bacterial suspensions whose turbidities were adjusted to that of a 0.5 McFarland standard.

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  • TABLE 1.

    Amplification primers and molecular beacon probes used in this study

    OligonucleotideSequencePositiona
    Primers
        grlA205 (Forward)5′-GAT GTT ATT GGT CAA TAT CAT CCA-3′205-228
        grlA396 (Reverse)5′-AAG AAA CTG TCT CTT TAT TAA TAT CAC GT-3′396-424
    Probesb
        S80-FAM5′ (FAM)-CAG CGT ACA CTG AGG AGT CTC CGC TG-3′ (DABCYL)233-248
        E84-FAM5′ (FAM)-CGC GAA GTG TAC GAA GCA ATG GTC GCG-3′ (DABCYL)243-259
        Sau-TET5′ (TET)-CCG GGT ATC GAT AAT GAT CCG CCA GCC CGG-3′ (DABCYL)324-343
    • ↵ a Nucleotide positions in the grlA gene (start codon, 1 to 3) from GenBank accession no. D67075 .

    • ↵ b The underlined sequences constitute the stem of each molecular beacon. FAM, 6-carboxylfluorescein; TET, tetrachloro-6-carboxylfluorescein.

  • TABLE 2.

    Evaluation of the real-time PCR assay for the detection of fluoroquinolone-resistant Staphylococcus aureus using a variety of bacteria

    Bacterial species (strain number)Fluorescent signal with the indicated test and probea
    PCR no. 1PCR no. 2
    Sau-TETS80-FAMSau-TETE84-FAM
    Abiotrophia defectiva (ATCC 49176)−−−−
    Bacillus cereus (ATCC 13472)−−−−
    Bacillus mycoides (ATCC 10206)−−−−
    Enterococcus faecalis (ATCC 29212)−−−−
    Enterococcus faecalis (ATCC 19433)−−−−
    Enterococcus flavescens (ATCC 49996)−−−−
    Granulicatella adiacens (ATCC 49175)−−−−
    Lactococcus lactis (ATCC 11454)−−−−
    Listeria innocua (ATCC 33090)−−−−
    Listeria monocytogenes (ATCC 15313)−−−−
    Staphylococcus auricularis (ATCC 33753)−−−−
    Staphylococcus capitis subsp. urealyticus (ATCC 49326)−−−−
    Staphylococcus carnosus (ATCC 51365)−−−−
    Staphylococcus chromogenes (ATCC 43764)−−−−
    Staphylococcus epidermidis (ATCC 35983)−−−−
    Staphylococcus epidermidis (ATCC 35984)−−−−
    Staphylococcus gallinarum (ATCC 35539)−−−−
    Staphylococcus haemolyticus (ATCC 29970)−−−−
    Staphylococcus haemolyticus (LSPQ 2514)−−−−
    Staphylococcus hominis subsp. hominis (ATCC 27844)−−−−
    Staphylococcus lentus (ATCC 29070)−−−−
    Staphylococcus lugdunensis (ATCC 43809)−−−−
    Staphylococcus saccharolyticus (ATCC 14953)−−−−
    Staphylococcus saprophyticus (ATCC 15305)−−−−
    Staphylococcus saprophyticus (ATCC 35552)−−−−
    Staphylococcus saprophyticus (ATCC 43867)−−−−
    Staphylococcus simulans (ATCC 27848)−−−−
    Staphylococcus warneri (ATCC 27836)−−−−
    Staphylococcus xylosus (ATCC 29971)−−−−
    Streptococcus agalactiae (ATCC 12400)−−−−
    Streptococcus pneumoniae (ATCC 49619)−−−−
    S. aureus (ATCC 25923)b++++
    S. aureus (ATCC 29213)b++++
    S. aureus (CCRI-2024)b+−++
    S. aureus (9191)b+−++
    S. aureus (9299)b+−++
    S. aureus (9015)b+++−
    S. aureus (9283)b+++−
    S. aureus (CCRI-12876)b+−+−
    • ↵ a All PCR tests were performed using 0.1 ng of purified genomic DNA.

    • ↵ b These strains were chosen to represent different mutations at codons 80 and 84 of grlA. The quinolone resistance-determining regions of grlA from these strains were sequenced in this study. S. aureus strains ATCC 25923 and ATCC 29213 have no mutation at codons 80 and 84; strains CCRI-2024, 9191, and 9299 have a Ser-80-to-Phe (TCC→TTC) mutation; strain 9015 has a Glu-84-to-Gly (GAA→GGA) mutation; strain 9283 has a Glu-84-to-Lys (GAA→AAA) mutation; and strain CCRI-12876 has both Ser-80-to-Tyr (TCC→TAC) and Glu-84 to Gly (GAA→GGA) mutations.

  • TABLE 3.

    Correlation between fluoroquinolone susceptibility and the real-time PCR assay

    FluoroquinoloneEtest result (no.)PCR result
    Susceptible (no.)Resistant (no.)
    CiprofloxacinSusceptible (35)350
    Resistant (50)149
    LevofloxacinSusceptible (37)361
    Resistant (48)048
    GatifloxacinSusceptible (37)361
    Resistant (48)048
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Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus
Pascal Lapierre, Ann Huletsky, Véronique Fortin, François J. Picard, Paul H. Roy, Marc Ouellette, Michel G. Bergeron
Journal of Clinical Microbiology Jul 2003, 41 (7) 3246-3251; DOI: 10.1128/JCM.41.7.3246-3251.2003

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Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus
Pascal Lapierre, Ann Huletsky, Véronique Fortin, François J. Picard, Paul H. Roy, Marc Ouellette, Michel G. Bergeron
Journal of Clinical Microbiology Jul 2003, 41 (7) 3246-3251; DOI: 10.1128/JCM.41.7.3246-3251.2003
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KEYWORDS

Anti-Infective Agents
DNA Topoisomerase IV
Drug Resistance, Bacterial
mutation
polymerase chain reaction
Staphylococcus aureus

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