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

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 (Oxa^r); 3,S. aureus ATCC 25923 (Oxa^s); 4, S. epidermidis ATCC 35983 (Oxa^r); 5, S. epidermidis ATCC 14990 (Oxa^s); 6, S. capitis ATCC 49326 (Oxa^r); 7, S. haemolyticus ATCC 29970 (Oxa^s); 8, S. hominis ATCC 27844 (Oxa^s); 9, S. saprophyticus ATCC 15305 (Oxa^s); 10, S. simulans ATCC 27848 (Oxa^s); 11, S. warneriATCC 27836 (Oxa^s); 12, Enterococcus faecalisATCC 29212; 13, Enterococcus faecium ATCC 51559; 14,Streptococcus pneumoniae ATCC 27336; 15, S. aureus ATCC 43300 (Oxa^r); 16, S. aureusATCC 25923 (Oxa^s); 17, S. epidermidis ATCC 35983 (Oxa^r); and 18, S. epidermidis ATCC 14990 (Oxa^s). Lanes 1 and 19, controls to which no DNA was added; lane M, 50-bp ladder molecular size standard.

Table 1.

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

Target gene	Accession number (GenBank)	Primer sequence	Annealing position
aac(6′)-aph(2")	M13771	5′-`TTG GGA AGA TGA AGT TTT TAG A`-3′	159–180
		5′-`CCT TTA CTC CAA TAA TTT GGC T`-3′	311–332
blaZ	M60253	5′-`ACT TCA ACA CCT GCT GCT TTC`-3′	511–531
		5′-`TGA CCA CTT TTA TCA GCA ACC`-3′	663–683
ermA	K02987	5′-`TAT CTT ATC GTT GAG AAG GGA TT`-3′	370–392
		5′-`CTA CAC TTG GCT TAG GAT GAA A`-3′	487–508
ermB	U35228	5′-`CTA TCT GAT TGT TGA AGA AGG ATT`-3′	366–389
		5′-`GTT TAC TCT TGG TTT AGG ATG AAA`-3′	484–507
ermC	M17990	5′-`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)	J01859	5′-`GGA GGA AGG TGG GGA TGA CG`-3′	1244–1263
		5′-`ATG GTG TGA CGG GCG GTG TG`-3′	1469–1489
mecA	X52594	5′-`AAC AGG TGA ATT ATT AGC ACT TGT AAG`-3′	1059–1085
	X52593	5′-`ATT GCT GTT AAT ATT TTT TGA GTT GAA`-3′	1206–1232
msrA	X52085	5′-`TCC AAT CAT TGC ACA AAA TC`-3′	891–910
		5′-`AAT TCC CTC TAT TTG GTG GT`-3′	1034–1053
S. aureus^a	AF033191	5′-`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. epidermidis^a	NA^b	5′-`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.^a	PCR primer pairs combined (amplicon sizes)
1	S. aureus (108 bp) + S. epidermidis (125 bp) +mecA (174 bp)
2	S. aureus (108 bp) +S. epidermidis (125 bp) +aac(6′)-aph(2") (174 bp)
3	S. aureus (108 bp) + S. epidermidis (125 bp) +ermA (139 bp)
4	S. aureus (108 bp) +S. epidermidis (125 bp) + ermB (142 bp)
5	S. aureus (108 bp) + S. epidermidis (125 bp) + ermC (190 bp)
6	S. aureus (108 bp) +S. epidermidis (125 bp) + msrA (163 bp)
7	S. aureus (108 bp) + S. epidermidis (125 bp) + blaZ (173 bp)

Table 3.

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

Species	No. of strains (type) tested by disk diffusion with gentamicin	PCR results foraac(6′)-aph(2")
Species		No. positive	No. negative
S. aureus	41 (resistant)	41	0
S. aureus	165 (susceptible)	0	165
S. epidermidis	131 (resistant)	131	0
S. epidermidis	57 (susceptible)	0	57

Table 4.

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

Species	No. of strains (type) tested by disk diffusion with oxacillin	PCR results for mecA
Species		No. positive	No. negative
S. aureus	66 (resistant)	62	4
S. aureus	140 (susceptible)	1	139
S. epidermidis	146 (resistant)	146	0
S. epidermidis	42 (susceptible)	3	39

Table 5.

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

Species	No. of strains (type) tested by disk diffusion with erythromycin	No. of strains positive by PCR for:				No. of PCR-negative strains
Species		ermA	ermB	ermC	msrA	No. of PCR-negative strains
S. aureus	73 (resistant)	43	5	21	2	2
S. aureus	133 (susceptible)	0	0	4	0	129
S. epidermidis	142 (resistant)	9	1	124	8	0
S. epidermidis	46 (susceptible)	0	0	0	0	46

Table 6.

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

Strains	Oxacillin MIC (μg/ml)	Nitrocefin test result	Disk diffusion test result (mm) amoxicillin with clavulanic acid^a	blaZ (PCR detection)
S. aureusR620	4	+	24.0	+
S. aureus R626	8	+	22.0	+
S. aureus R627	4	+	24.5	+
S. aureusSTA572	8	+	23.3	+
S. aureus ATCC 43300^b	64	+	15.2	+
S. aureus ATCC 25923^b	1	−	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 strain^a	Profile	MIC (μg/ml)	MIC after selection^b (μg/ml)
Group 1
S. aureusR632	mecA, Oxa^s	2	>64
S. epidermidisR588	mecA, Oxa^s	0.125	>64
S. epidermidis 184	mecA, Oxa^s	1	>64
S. epidermidis3348	mecA, Oxa^s	0.25	>64
S. aureus ATCC 29213	mecA negative, Oxa^s	0.5	0.5
S. aureus18	ermC, Ery^s	1	>64
S. aureus 75	ermC, Ery^s	1	>64
S. aureus 93	ermC, Ery^s	1	>64
S. aureus5919	ermC, Ery^s	1	>64
S. aureus ATCC 29213	ermC negative, Ery^s	0.5	0.5
Group 2
S. aureus R572	mecA negative, Oxa^r	8
S. aureus R620	mecA negative, Oxa^r	4
S. aureusR626	mecA negative, Oxa^r	8
S. aureus R627	mecA negative, Oxa^r	4
S. aureus MA-5091	ermABC negative,msrA negative, Ery^r	>32
S. aureus R566	ermABC negative, msrAnegative, Ery^r	>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.