TABLE 4.

Summary of reported studies evaluating phenotypic methods for detection of oxacillin (methicillin) resistance in staphylococci^ab

Author(s) (reference)	Organism, no. of isolates	Reference standard	Test methodsc	% of isolates detected				% Overall agreement	Notes
				S. aureus		CoNS
				Resistant	Susceptible	Resistant	Susceptible
Thornsberry and McDougal (20)	S. aureus, 45; CoNS, 12	None (all isolates previously characterized as methicillin susceptible or resistant)	Broth dilution: CSMH with 2% NaCl; 24 h at 35°C	100		100			Increasing NaCl concentration to 5% resulted in better growth than that with 2% NaCl for resistant isolates; for susceptible isolates 5% NaCl resulted in higher MICs; 10% NaCl inhibited growth
Agar spot screen: MH with 4% NaCl; 24 h at 35°C	100		100
Hindler and Inderlied (8)	S. aureus, 10	None (all isolates previously characterized as methicillin resistant)	Disk diffusion: MH; 16 to 24 h at 35°C, reincubated additional 24 h at 30°C	100					Different commercial sources of MH were evaluated and differences in results were noted, especially for agar dilution
			Agar dilution: MH; 16 to 18 h at 35°C
Hansen and Pope (7)	S. aureus, 161	None (all isolates previously characterized as methicillin susceptible or resistant)	Broth screen: CSMH with 2% NaCl; ∼5 h at 35°C	96.9	100				Broth test vials were placed on a rotary shaker for 5 h at 35°C; tetrazolium salt respiratory indicator was added, and vials were observed for the development of a pink color after 30 min of additional incubation
Coudron et al. (4)	S. aureus, 95; CoNS, 175	Agar dilution (MH with 4% NaCl; 24 and 48 h incubation at 35°C) or broth dilution (CSMH with 2% NaCl; 24 and 48 h incubation at 35°C)	Disk diffusion: MH with  10-μg/ml methicillin and  0% NaCl						Comprehensive study which evaluated an array of different concentrations of NaCl and different incubation periods
			18 h	35	100	15	100
			48 h	61	96	45	100
			4% NaCl
			18 h	89	98	88	100
			48 h	100	76	95	100
			Agar swab screen: MH with  10-μg/ml methicillin and 0% NaCl
			24 h	87	100	46	100
			48 h	89	100	94	92
			4% NaCl
			24 h	91	100	95	97
			48 h	94	100	98	90
			Agar spot screen: MH with 10-μg/ml methicillin and  4% NaCl
			24 h	89	98	96	97
			48 h	89	96	99	95
Hindler and Warner (9)	S. aureus, 109	Broth microdilution: CSMH with 2% NaCl, 24 h at 35°C	Agar spot screen: MH with 4% NaCl, 24 h at 35°C	90–100e	100				MH media from five different commercial sources were evaluated. Results varied according to the source of MH medium.
Milne et al. (16)	S. aureus, 50; CoNS, 135	None (all isolates previously characterized as methicillin susceptible or resistant)	Disk Diffusion, 10-μg/ml methicillin:						Other media (Diagnostic Sensitivity Test agar, Sensitest agar, and Isosensitest agar) tested but results were worse than those for COL and MH agars.
			COL agar and 0% NaCl,  30°C
			18 h	90	95	66	93
			40 h	97	63	81	83
			COL agar and 5% NaCl,  35°C
			18 h	90	63	90	94
			40 h	97	63	92	93
			MH agar and 0% NaCl,  30°C
			18 h	77	100	50	93
			40 h	84	100	75	97
			MH agar and 5% NaCl,  35°C
			18 h	74	89	32	97
			40 h	87	63	50	96
Huang et al. (10)	S. aureus and CoNS, 223; no differentiation among species	mecA gene	Agar dilution: MH agar, 24 h at 35°C						The authors concluded that 2% NaCl is required for agar-based dilution methods; however, incubation time was limited to 24 h.
			0% NaCl					78
			2% NaCl					83
			4% NaCl					71
			5% NaCl					72
			Broth microdilution: CSMH, 24 h at 35°C
			0% NaCl					64
			2% NaCl					94
			5% NaCl					91
			E-test: MH, 24 h at 35°C
			0% NaCl					71
			2% NaCl					78
			4% NaCl					69
			5% NaCl					66
Baker et al. (1)	S. aureus and CoNS, 223 (no differentiation among species)	Broth microdilution: MH with 2% NaCl, 24 h at 35°C	Agar dilution: MH, 24 h at 35°C						The authors recommended 2% NaCl supplementation for both broth and agar dilution; the presence of mecA gene was also determined in isolates. The best correlation with mecA and results for broth microdilution was achieved using MH supplemented with 2% NaCl.
			0% NaCl					89
			2% NaCl					96
			4% NaCl					88
			5% NaCl					87
			E-test: MH, 24 h at 35°C
			0% NaCl					88
			2% NaCl					97
			4% NaCl					90
			5% NaCl					88
			Broth microdilution: MH,  24 h at 35°C
			0% NaCl					87
			4% NaCl					92
			5% NaCl					91
Knapp et al. (14)	S. aureus, 67; CoNS, 47 (all species were S. epidermidis and all were oxacillin resistant)	All isolates determined to be resistant by broth microdilution: CSMH with 2% NaCl, 24 h at 35°C; heterogeneous population analysis and oxacillin agar screen method: MH with 4% NaCl, 24 h	Disk diffusion, MH, 35°C
			Inoculation 107 CFU/plate
			24 h	82		87
			48 h	97		89
			Inoculation, 109 CFU/plate
			24 h	88		100
			48 h	97
			Vitek GPS-SA card software 6.1 and 7.1	98		100
Woods et al. (23)	S. aureus, 92; CoNS, 103	Microscan 24 h MIC panels. Discrepants resolved by oxacillin agar screening: MH with 4% NaCl, 24 h at 35°C	Microscan rapid panel	97		72			22 of 100 SCN isolates did not grow in the rapid panels.
Geha et al. (6)	S. aureus, 228; CoNS, 272	mecA gene	Agar dilution (MH with 0% NaCl, 48 h at 30°C) confirmed by disk diffusion (MH with 0% NaCl, 24 h at 35°C)	100		93			3 of 4 S. aureus isolates that were mecA-negative were resistant by agar dilution/disk diffusion method. These isolates were demonstrated to be hyperproducers of β-lactamase. No CoNS isolates were mecA negative and resistant by agar dilution/disk diffusion method.
York et al. (24)	CoNS, 140	mecA gene	Broth microdilution: CSMH  with 2% NaCl at 35°C						Authors concluded that a lower breakpoint for resistance (2 μg/ml) than recommended by the NCCLS (4 μg/ml) would improve results for the microdilution method.
			24 h			50
			48 h			77
			Disk diffusion: MH at 35°C
			24 h			84
			48 h			82
			Agar swab screen: MH with 4% NaCl at 35°C
			24 h			70
			48 h			100
Wallet et al. (21)	S. aureus, 57; CoNS, 100	mecA gene	Disk diffusion: MH, 24 h at 35°C	100		91 (91)d			S. epidermidis and SCN isolates were separately analyzed.
			Automated API-plus system (bioMerieux): MH with 5% NaCl, 24 h at 35°C	100		86 (85)d
			BBL Crystal MRSA ID system (Becton-Dickinson)
			4 h	92		79 (81)d
			6 h	96		86 (88)d
			Agar dilution: MH 2% of NaCl, 24 h at 35°C	96		88 (88)d
Mulder (17)	CoNS, 319	mecA gene	Disk diffusion: MH						Disk diffusion results best with MH agar supplemented with 2% NaCl and oxacillin (5 μg disks). The results for the E-test were interpreted by using 2 μg/ml as the breakpoint for oxacillin resistance.
Methicillin, 5 μg, 48 h at 30°C	84
Oxacillin, 1 μg, 24 h at 35°C	95
Oxacillin, 5 μg/ml, 24 h at 35°C	86
Disk diffusion: MH with 2% NaCl
Methicillin, 5 μg, 48 h at 30°C	96
Oxacillin, 1 μg, 24 h at 35°C	96
Oxacillin, 5 μg, 24 h at 35°C	99
			E-test: MH with 2% NaCl, 48 h at 35°C			99.6
Baker and Tenover (2)	S. aureus, 54; CoNS, 22	Broth microdilution: CSMH with 2% NaCl, 24 h at 35°C	Alamar colorimetric broth microdilution: MH with saline diluent, 24 h at 35°C					97.9	63% of isolates were resistant by reference methods.
Weller et al. (22)	S. aureus, 44; CoNS, 120	mecA gene	Agar dilution:
			Methicillin
			COL with 5% NaCl, 48 h at 35°C		100		83
			IS with 5% NaCl, 48 h at 35°C		67		77
			E-test:
			Methicillin
			COL with 5% NaCl, 48 h at 35°C		100		81
			IS with 5% NaCl, 48 h at 35°C		100		63
			Oxacillin
			COL with 5% NaCl, 48 h at 35°C		95		97
			IS with 5% NaCl, 48 h at 35°C	90		17
Jarløv et al. (11)	CoNS, 359	mecA gene/agar dilution: COL with 4.5% NaCl, 42 to 46 h at 35°C	Disk diffusion: 42 to 46 h at 35°C						Based on the best agreement shown for the mecA gene assay among four agar dilution assays for determining the MIC, a COL agar assay with a heavy inoculum (105 to 106 CFU (spot) was used as the reference MIC method. A total of 19 different methods using different concentrations of oxacillin, methicillin, and different media were used for disk diffusion, but only the methods with the best performance are shown. S. epidermidis and CoNS isolates were separately analyzed.
			Oxacillin, 1 μg
			MH			97 (84)d
			COL with 4.5% NaCl			96 (86)d
			COL			97 (86)d
			DBA			98 (93)d
			Beef agar with 7.5% NaCl			96 (82)d
			IS			99 (81)d
			IS with heavy inoculum			81 (99)d
			Oxacillin 5 μg/ml
			DBA			96 (84)d
			Beef agar with 7.5% NaCl			95 (68)d
Frebourg et al. (5)	S. aureus, 64; CoNS, 76 (All isolates carried mecA gene)	mecA gene and oxacillin agar screening: MH with 4% NaCl, 6-μg/ml oxacillin, 48 h at 35°C	E-test: MH with 2% NaCl, 24 h at 35°C	95		67			The authors note that the performance of the E-test and Vitek methods may be enhanced if the NCCLS oxacillin breakpoint for resistance is changed.
			ATB Staph (bioMerieux): MH with 5% NaCl, 24 h at 35°C	89		58
			Rapid ATB: MH with 5% NaCl, 24 h at 35°C	77
			Vitek GPS-503 card	95		80
Kampf et al. (12)	S. aureus, 136	mecA gene	Disk diffusion: MS, 24 h at 36°C	100					Evaluated only S. aureus isolates
			Agar spot screen: 2-μg/ml oxacillin, MS, 24 h at 36°C	98

^aSummary includes only recent reports for commercial (including automated) systems as many of early reports indicated inferior performance for these methods. 

^bAbbreviations: MH, Mueller-Hinton medium; MS, mannitol salt medium; CSMH, cation-supplemented MH medium; COL, Columbia medium; TS, tryptic soy medium; IS, isosensitest medium; DBA, Danish blood agar. 

^cAll incubations performed in ambient air; results for oxacillin unless otherwise indicated; 6 μg of oxacillin/ml was used for agar screen methods unless otherwise stated; 1-μg oxacillin disks were used for disk diffusion unless otherwise stated. 

^dThe first number refers to percentage for S. epidermidis isolates; the second number refers to percentage for all other CoNS isolates. 

^eThe percentages varied for different sources of MH medium.