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Susceptibility

Correlation between the Resistance Genotype Determined by Multiplex PCR Assays and the Antibiotic Susceptibility Patterns of Staphylococcus aureus andStaphylococcus epidermidis

Francis Martineau, François J. Picard, Nicolas Lansac, Christian Ménard, Paul H. Roy, Marc Ouellette, Michel G. Bergeron
Francis Martineau
Centre de Recherche en Infectiologie de l'Université Laval,
Division de Microbiologie, Faculté de Médecine, Université Laval, Québec G1V 4G2, and
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François J. Picard
Centre de Recherche en Infectiologie de l'Université Laval,
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Nicolas Lansac
Centre de Recherche en Infectiologie de l'Université Laval,
Division de Microbiologie, Faculté de Médecine, Université Laval, Québec G1V 4G2, and
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Christian Ménard
Centre de Recherche en Infectiologie de l'Université Laval,
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Paul H. Roy
Centre de Recherche en Infectiologie de l'Université Laval,
Département de Biochimie, Université Laval, Québec G1K 7P4, Canada
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Marc Ouellette
Centre de Recherche en Infectiologie de l'Université Laval,
Division de Microbiologie, Faculté de Médecine, Université Laval, Québec G1V 4G2, and
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Michel G. Bergeron
Centre de Recherche en Infectiologie de l'Université Laval,
Division de Microbiologie, Faculté de Médecine, Université Laval, Québec G1V 4G2, and
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DOI: 10.1128/AAC.44.2.231-238.2000
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    Fig. 1.

    Example of multiplex PCR amplifications with themecA-, S. aureus-, S. epidermidis-, and internal control-specific primer pairs. PCR assays were performed either from 10 pg of purified genomic DNA (lanes 2 to 14) or from 1 μl of a bacterial suspension whose turbidity was adjusted to that of a 0.5 McFarland standard (lanes 15 to 18). The content of each lane is as follows: 2, S. aureus ATCC 43300 (Oxar); 3,S. aureus ATCC 25923 (Oxas); 4, S. epidermidis ATCC 35983 (Oxar); 5, S. epidermidis ATCC 14990 (Oxas); 6, S. capitis ATCC 49326 (Oxar); 7, S. haemolyticus ATCC 29970 (Oxas); 8, S. hominis ATCC 27844 (Oxas); 9, S. saprophyticus ATCC 15305 (Oxas); 10, S. simulans ATCC 27848 (Oxas); 11, S. warneriATCC 27836 (Oxas); 12, Enterococcus faecalisATCC 29212; 13, Enterococcus faecium ATCC 51559; 14,Streptococcus pneumoniae ATCC 27336; 15, S. aureus ATCC 43300 (Oxar); 16, S. aureusATCC 25923 (Oxas); 17, S. epidermidis ATCC 35983 (Oxar); and 18, S. epidermidis ATCC 14990 (Oxas). Lanes 1 and 19, controls to which no DNA was added; lane M, 50-bp ladder molecular size standard.

Tables

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  • Table 1.

    Antibiotic resistance gene-specific and species-specific primers used in this study

    Target geneAccession number (GenBank)Primer sequenceAnnealing position
    aac(6′)-aph(2")M137715′-TTG GGA AGA TGA AGT TTT TAG A-3′159–180
    5′-CCT TTA CTC CAA TAA TTT GGC T-3′311–332
    blaZM602535′-ACT TCA ACA CCT GCT GCT TTC-3′511–531
    5′-TGA CCA CTT TTA TCA GCA ACC-3′663–683
    ermAK029875′-TAT CTT ATC GTT GAG AAG GGA TT-3′370–392
    5′-CTA CAC TTG GCT TAG GAT GAA A-3′487–508
    ermBU352285′-CTA TCT GAT TGT TGA AGA AGG ATT-3′366–389
    5′-GTT TAC TCT TGG TTT AGG ATG AAA-3′484–507
    ermCM179905′-CTT GTT GAT CAC GAT AAT TTC C-3′214–235
    5′-ATC TTT TAG CAA ACC CGT ATT C-3′382–403
    Internal control (16S rRNA gene)J018595′-GGA GGA AGG TGG GGA TGA CG-3′1244–1263
    5′-ATG GTG TGA CGG GCG GTG TG-3′1469–1489
    mecAX525945′-AAC AGG TGA ATT ATT AGC ACT TGT AAG-3′1059–1085
    X525935′-ATT GCT GTT AAT ATT TTT TGA GTT GAA-3′1206–1232
    msrAX520855′-TCC AAT CAT TGC ACA AAA TC-3′891–910
    5′-AAT TCC CTC TAT TTG GTG GT-3′1034–1053
    S. aureusaAF0331915′-AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG-3′5–34
    5′-CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA-3′83–112
    S. epidermidisaNAb5′-ATC AAA AAG TTG GCG AAC CTT TTC A-3′21–45
    5′-CAA AAG AGC GTG GAG AAA AGT ATC A-3′121–145
    • ↵a The S. aureus- and S. epidermidis-specific primer pairs were derived from genomic DNA fragments of unknown coding potential (34, 35).

    • ↵b NA, not applicable.

  • Table 2.

    Multiplex PCR assays

    Multiplex no.aPCR primer pairs combined (amplicon sizes)
    1S. aureus (108 bp) + S. epidermidis (125 bp) +mecA (174 bp)
    2S. aureus (108 bp) +S. epidermidis (125 bp) +aac(6′)-aph(2") (174 bp)
    3S. aureus (108 bp) + S. epidermidis (125 bp) +ermA (139 bp)
    4S. aureus (108 bp) +S. epidermidis (125 bp) + ermB (142 bp)
    5S. aureus (108 bp) + S. epidermidis (125 bp) + ermC (190 bp)
    6S. aureus (108 bp) +S. epidermidis (125 bp) + msrA (163 bp)
    7S. aureus (108 bp) + S. epidermidis (125 bp) + blaZ (173 bp)
    • ↵a All multiplex PCR assays included an internal control (241 bp).

  • Table 3.

    Correlation between gentamicin resistance and the presence of the resistance gene aac(6′)-aph(2")

    SpeciesNo. of strains (type) tested by disk diffusion with gentamicinPCR results foraac(6′)-aph(2")
    No. positiveNo. negative
    S. aureus41 (resistant)410
    S. aureus165 (susceptible)0165
    S. epidermidis131 (resistant)1310
    S. epidermidis57 (susceptible)057
  • Table 4.

    Correlation between oxacillin resistance and the presence of the resistance gene mecA

    SpeciesNo. of strains (type) tested by disk diffusion with oxacillinPCR results for mecA
    No. positiveNo. negative
    S. aureus66 (resistant)624
    S. aureus140 (susceptible)1139
    S. epidermidis146 (resistant)1460
    S. epidermidis42 (susceptible)339
  • Table 5.

    Correlation between erythromycin resistance and the presence of the resistance gene ermA, ermB,ermC, or msrA

    SpeciesNo. of strains (type) tested by disk diffusion with erythromycinNo. of strains positive by PCR for:No. of PCR-negative strains
    ermAermBermCmsrA
    S. aureus73 (resistant)4352122
    S. aureus133 (susceptible)0040129
    S. epidermidis142 (resistant)9112480
    S. epidermidis46 (susceptible)000046
  • Table 6.

    Characteristics of the four strains resistant to oxacillin but negative for mecA by PCR

    StrainsOxacillin MIC (μg/ml)Nitrocefin test resultDisk diffusion test result (mm) amoxicillin with clavulanic acidablaZ (PCR detection)
    S. aureusR6204+24.0+
    S. aureus R6268+22.0+
    S. aureus R6274+24.5+
    S. aureusSTA5728+23.3+
    S. aureus ATCC 43300b64+15.2+
    S. aureus ATCC 25923b1−28.3−
    • ↵a Strains were considered β-lactamase hyperproducers when (i) the diameter of the inhibition zone with the amoxicillin-clavulanic acid (20 and 10 μg, respectively) disk exceeded 20 mm and (ii) they were resistant to oxacillin (2).

    • ↵b S. aureus ATCC 43300 is a nonhyperproducing strain carrying mecA, while S. aureus ATCC 25923 is a β-lactamase-negative strain not carrying mecA.

  • Table 7.

    S. aureus (n = 11) andS. epidermidis (n = 3) strains for which the resistance genotype was discordant with the resistance phenotype

    Group and strainaProfileMIC (μg/ml)MIC after selectionb (μg/ml)
    Group 1
     S. aureusR632mecA, Oxas2>64
     S. epidermidisR588mecA, Oxas0.125>64
     S. epidermidis 184mecA, Oxas1>64
     S. epidermidis3348mecA, Oxas0.25>64
     S. aureus ATCC 29213mecA negative, Oxas0.50.5
     S. aureus18ermC, Erys1>64
     S. aureus 75ermC, Erys1>64
     S. aureus 93ermC, Erys1>64
     S. aureus5919ermC, Erys1>64
     S. aureus ATCC 29213ermC negative, Erys0.50.5
    Group 2
     S. aureus R572mecA negative, Oxar8
     S. aureus R620mecA negative, Oxar4
     S. aureusR626mecA negative, Oxar8
     S. aureus R627mecA negative, Oxar4
     S. aureus MA-5091ermABC negative,msrA negative, Eryr>32
     S. aureus R566ermABC negative, msrAnegative, Eryr>32
    • ↵a Group 1, resistance gene present, susceptible; group 2, resistance gene absent, resistant. MIC column data for group 1 strains are the initial MICs.

    • ↵b The selection of resistant cells was done by plating on media with increasing gradients of the target antibiotic.

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Correlation between the Resistance Genotype Determined by Multiplex PCR Assays and the Antibiotic Susceptibility Patterns of Staphylococcus aureus andStaphylococcus epidermidis
Francis Martineau, François J. Picard, Nicolas Lansac, Christian Ménard, Paul H. Roy, Marc Ouellette, Michel G. Bergeron
Antimicrobial Agents and Chemotherapy Feb 2000, 44 (2) 231-238; DOI: 10.1128/AAC.44.2.231-238.2000

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Correlation between the Resistance Genotype Determined by Multiplex PCR Assays and the Antibiotic Susceptibility Patterns of Staphylococcus aureus andStaphylococcus epidermidis
Francis Martineau, François J. Picard, Nicolas Lansac, Christian Ménard, Paul H. Roy, Marc Ouellette, Michel G. Bergeron
Antimicrobial Agents and Chemotherapy Feb 2000, 44 (2) 231-238; DOI: 10.1128/AAC.44.2.231-238.2000
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KEYWORDS

Drug Resistance, Microbial
Genes, Bacterial
Polymerase Chain Reaction
Staphylococcus aureus
Staphylococcus epidermidis

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