Correlation of Cefoxitin MICs with the Presence of mecA in Staphylococcus spp.

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of ≤4 μg/ml for mecA-negative and ≥6 or 8 μg/ml for mecA-positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of ≤2 μg/ml for mecA-negative and ≥4 μg/ml for mecA-positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.The use of a cefoxitin disk test to detect staphylococci that are likely to contain the mecA gene has been widely advocated since the test was first suggested (3, 6) and has been adopted by antimicrobial susceptibility testing organizations worldwide (http://www.bsac.org.uk/_db/_documents/version_6.1.pdf, http://www.srga.org/, http://www.clsi.org/). For laboratories that do not use disk diffusion as their primary testing method, performing disk diffusion requires additional expense and reagents. Consequently, we performed studies to determine if a cefoxitin broth microdilution MIC breakpoint would correlate with the presence of the mecA gene in staphylococci.Cation-adjusted Mueller-Hinton broths (CAMHB) from three manufacturers (BBL, BD Diagnostic Systems, Sparks, MD; Difco, BD Diagnostic Systems, Sparks, MD; and Oxoid, Basingstoke, Hampshire, England) (cation content adjusted if necessary), were used to prepare frozen microdilution panels containing cefoxitin concentrations of 0.5 to 32 μg/ml (including 6 μg/ml) and oxacillin concentrations of 0.06 to 16 μg/ml (in BBL CAMHB only). The frozen panels were shipped to all participants, along with 30-μg cefoxitin disks (BBL). Each laboratory used its current lot of Mueller-Hinton agar for disk diffusion testing. Participating laboratories included the Centers for Disease Control and Prevention (CDC), Atlanta, GA; BD Diagnostic Systems, Sparks, MD; bioMérieux, Inc., Hazelwood, MO; Duke University Hospital, Durham, NC; JMI Labs, North Liberty, IA; Massachusetts General Hospital, Boston, MA; Siemens Healthcare Diagnostics MicroScan, Inc., West Sacramento, CA; University of Rochester, Rochester, NY; Robert Wood Johnson Medical School, New Brunswick, NJ; Statens Serum Institut, Copenhagen, Denmark; and Trek Diagnostic Systems, Cleveland, OH.Each laboratory tested approximately 50 unique clinical isolates from their own collection (30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococci [CoNS]) and quality control strains for each test day (S. aureus ATCC 29213 [tested by MIC only] and S. aureus ATCC 25923 [tested by disk diffusion only]). Additional strains were tested at the CDC, totaling 167 S. aureus isolates and 22 CoNS. The total number of strains tested in all labs was 479 S. aureus isolates and 204 CoNS. All results were read after both 18-h and 24-h incubations. Endpoints were read as the lowest concentration at which no visible growth was observed. At 18 h of incubation, 97.3% of the MIC quality control results (for S. aureus ATCC 29213) and 97.9% of the disk diffusion quality control results (for S. aureus ATCC 25923) were within the range considered acceptable by the Clinical and Laboratory Standards Institute (CLSI).After testing, all strains were shipped to the CDC for further analysis. PCR testing for mecA was performed as previously described (8) on the following strains: (i) any S. aureus strain for which the cefoxitin MIC was <16 μg/ml in any of the three media tested (n = 180); (ii) any S. aureus strain for which the cefoxitin MIC was >8 μg/ml and the oxacillin MIC was <4 μg/ml in any of the three media tested (n = 5); and (iii) a sample of the 304 isolates (n = 16 [5%]) for which the cefoxitin MIC was >8 μg/ml and the oxacillin MIC was >2 μg/ml. All other S. aureus strains for which the cefoxitin MIC was >8 μg/ml and the oxacillin MIC was >2 μg/ml at 24 h of incubation were presumed to be mecA positive. All CoNS were tested by PCR for the presence of mecA and identified to species level by using previously described methods (1). For the purposes of data analysis, mecA or presumed mecA was used as the gold standard.Cefoxitin MIC results for the S. aureus group (which included eight isolates of Staphylococcus lugdunensis) showed a clear separation of mecA-positive and mecA-negative strains between 4 and 6 μg/ml; most isolates with cefoxitin MICs of 6 μg/ml contained the mecA gene (Table ​(Table1).1). Among 13 strains for which the cefoxitin MIC was 6 μg/ml in any medium at either incubation time period, 11 were mecA positive. All the S. lugdunensis strains tested were mecA negative and had cefoxitin MICs of ≤4 μg/ml. Using a cefoxitin breakpoint of ≤4 μg/ml for oxacillin-susceptible or mecA-negative isolates and that of ≥8 μg/ml (rounding 6 μg/ml up to 8 μg/ml) for oxacillin-resistant or mecA-positive isolates, sensitivity and specificity were excellent, 99.7 to 100%. If these breakpoints are applied to the isolate population in this study, there are some category discrepancies between cefoxitin susceptibility testing and oxacillin susceptibility testing. These were limited to isolates for which the cefoxitin MIC was ≥6 μg/ml and oxacillin MIC was <4 μg/ml. The number of isolates varied depending on the medium and time of incubation, with the lowest being three strains at 24 h in BBL CAMHB and the highest being seven strains at 18 h in Difco CAMHB. All were mecA positive except for two isolates. For the mecA-positive isolates, the oxacillin MICs were 1 or 2 μg/ml, and the cefoxitin MICs ranged from 6 to >32 μg/ml.

TABLE 1.

Cefoxitin MICs for 479 isolates of Staphylococcus aureus tested in three different CAMHB^a

Isolate	Incubation time (h)	CAMHB	No. of isolates with cefoxitin MIC (μg/ml) of:									No. (%) of correct resultsc
			≤0.5	1	2	4	6	8	16	32	>32
mecA positive (n = 312)b	18	BBL			1		2	8	38	146	117	311 (99.7)
		Difco					6	10	110	124	62	312 (100)
		Oxoid					9	8	101	127	67	312 (100)
	24	BBL			1		1	3	24	129	154	311 (99.7)
		Difco					3	10	70	142	87	312 (100)
		Oxoid					5	8	71	141	87	312 (100)
mecA negative (n = 167)	18	BBL	4	1	93	69						167 (100)
		Difco		4	93	69	1					166 (99.4)
		Oxoid		2	102	63						167 (100)
	24	BBL	3	1	80	83						167 (100)
		Difco		1	73	92	1					166 (99.4)
		Oxoid		1	84	81	1					166 (99.4)

Open in a separate window^aIncluded five strains of Staphylococcus lugdunensis.^bmecA determined by PCR if cefoxitin MIC is ≤8 μg/ml or if cefoxitin MIC is >8 μg/ml and oxacillin MIC is <4 μg/ml; others were presumed to be mecA positive.^cResults are considered correct for mecA-positive strains if MICs are ≥6 μg/ml; results are considered correct for mecA-negative strains if MICs are ≤4 μg/ml.The sensitivity and specificity of cefoxitin disk diffusion testing in this study were also excellent, 99.7 and 100%, respectively (data not shown). These results are similar to those from a previous study when cefoxitin MIC testing was done to determine cefoxitin disk diffusion breakpoints (9). In that 10-laboratory study using panels prepared with CAMHB of two manufacturers and the MIC breakpoints proposed here, the sensitivity was 98 to 99%, and specificity was 99 to 100% (9).Cefoxitin MIC results for CoNS showed no clear separation of mecA-positive and -negative strains (Table ​(Table2).2). However, test sensitivity using breakpoints of ≤2 μg/ml and ≥4 μg/ml differed substantially between the media used, as follows: ≥99% at both 18 h and 24 h for Difco and Oxoid CAMHB, but only 89% at 18 h and 94% at 24 h for BBL CAMHB. However, specificity for all media at both times was ≤80%.

TABLE 2.

Cefoxitin MICs for 203 CoNS tested in three different CAMHB

Isolate	Incubation time (h)	CAMHB	No. of isolates with cefoxitin MIC (μg/ml) of:									No. (%) of correct resultsb
			≤0.5	1	2	4	6	8	16	32	>32
mecA positive (n = 131)a	18	BBL	8	2	6	11	6	13	36	7	41	115 (88.5)
		Difco			2	14	16	3	39	23	34	129 (98.5)
		Oxoid			1	15	11	15	41	18	30	130 (99.2)
	24	BBL	2	3	4	13	5	13	33	13	44	122 (93.8)
		Difco			2	9	16	4	33	28	39	129 (98.5)
		Oxoid			1	11	8	17	31	28	35	130 (99.2)
mecA negative (n = 73)	18	BBL	10	21	27	6	7	2				58 (79.5)
		Difco	2	11	42	10	6	1	1			55 (75.3)
		Oxoid		7	47	11	7		1			54 (74.0)
	24	BBL	9	17	32	6	6	3				58 (79.5)
		Difco	2	8	45	10	5	2	1			55 (75.3)
		Oxoid		6	44	15	6	1	1			50 (68.5)

Open in a separate window^aThe number of isolates in BBL CAMHB was 130.^bResults are considered correct for mecA-positive strains if MICs are ≥4 μg/ml; results are considered correct for mecA-negative strains if MICs are ≤2 μg/ml.Comparing the performance for CoNS grouped by Staphylococcus epidermidis versus non-S. epidermidis isolates (Table ​(Table3),3), sensitivities were similar for both organisms groups (at 24 h, 94 to 99% for S. epidermidis and 91 to 100% for non-S. epidermidis). However, the specificity of the test was much better for S. epidermidis than that for non-S. epidermidis isolates (at 24 h, 85 to 91% for S. epidermidis versus 54 to 69% for non-S. epidermidis). Although the performance of the cefoxitin MIC test in this study for the prediction of mecA in CoNS is not ideal, its use might be considered if its performance were no worse than that of the oxacillin MIC test. Although our results and those from the previous CLSI study (9) showed that the two tests to have similar sensitivities (Table ​(Table4),4), the specificity of cefoxitin (69 to 90%) was not as good as that of oxacillin (89 to 91%).

TABLE 3.

Comparison of sensitivity and specificity of cefoxitin MICs of ≤2 μg/ml for mecA negativity and ≥4 μg/ml for mecA positivity for three groups

Groupc	CAMHB	Sensitivity (%) ata:		Specificity (%) atb:
		18 h	24 h	18 h	24 h
CoNS	BBL	89	94	80	80
	Difco	99	99	75	75
	Oxoid	99	99	74	69
S. epidermidis	BBL	88	94	91	91
	Difco	99	99	91	91
	Oxoid	99	99	88	85
Non-S. epidermidis	BBL	86	91	69	69
	Difco	97	97	62	62
	Oxoid	100	100	62	54

Open in a separate window^aSensitivity values are the percentage of mecA-positive results with cefoxitin MICs of ≥4 μg/ml. Values in boldface type are >90%.^bSpecificity values are the percentage of mecA-negative results with cefoxitin MICs of ≤2 μg/ml. Values in boldface type are >90%.^cmecA status for each group was as follows: 131 mecA-positive and 73 mecA-negative CoNS; 95 mecA-positive and 34 mecA-negative S. epidermidis isolates; and 36 mecA-positive and 39 mecA-negative non-S. epidermidis isolates. The number of mecA-positive CoNS in BBL CAMHB was 130.

TABLE 4.

Comparison of sensitivity and specificity of oxacillin and cefoxitin MICs for prediction of mecA presence in CoNS read at 24 h in two CLSI studies

Antimicrobial	CAMHB	Sensitivity (%) in thea:		Specificity (%) in theb:
		2004 study (n = 126)d	2007 study (n = 131)c	2004 study (n = 70)	2007 study (n = 72)
Oxacillin	BBL	95	95	89	89
	Difco	99		91
Cefoxitin	BBL	97	93	90	80
	Difco	100	99	84	75
	Oxoid		99		69

Open in a separate window^aSensitivity values are the percentage of mecA-positive results with cefoxitin MICs of ≥4 μg/ml.^bSpecificity values are the percentage of mecA-negative results with cefoxitin MICs of ≤2 μg/ml.^cCurrent study. The number of mecA-positive isolates in BBL CAMHB was 130.^dStudy by Swenson et al. (9).For many laboratories, the cefoxitin MIC test can be incorporated into routine testing of S. aureus when the drug is included in panels of automated antimicrobial susceptibility testing systems. Several automated susceptibility testing manufacturers have begun using cefoxitin MICs to predict mecA-mediated resistance in Staphylococcus spp. In a recent report, the cefoxitin MIC was a better predictor of mecA-mediated resistance in CoNS than is reported here (4). However, performance differences among commercial systems may reflect difference in the systems’ use of both oxacillin and cefoxitin data as well as the ability of device manufacturers to use a single-source optimized medium, whereas a reference method needs to work in media from multiple manufacturers.Recently, two groups of investigators demonstrated that moxalactam may be a better indicator of mecA in CoNS than is cefoxitin. One group recommended the use of a combination of oxacillin MIC and moxalactam MIC tests (7); the other recommended the use of a combination of cefoxitin disk and moxalactam disk tests (5). The use of moxalactam tests in combination with cefoxitin and/or oxacillin tests to predict the mecA status of CoNS may deserve further study, given that the use of cefoxitin MICs alone is not optimal for this purpose.In summary, in an 11-laboratory study, we found that cefoxitin broth microdilution breakpoints of ≤4 μg/ml for the prediction of mecA-negative results and those of ≥6 or 8 μg/ml for mecA-positive results in S. aureus isolates read at 18 h of incubation were highly sensitive and specific. These breakpoints for S. aureus were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing and were included in the M100-S18 tables published in January 2008 (2). For CoNS, we found that cefoxitin MIC levels were sensitive but not specific in identifying the presence of mecA and, thus, recommend that they not replace oxacillin MICs at this time.