Difficult-To-Detect Staphylococcus aureus: mecA-Positive Isolates Associated with Oxacillin and Cefoxitin False-Susceptible Results

LETTER

In August of 2018, the United States Food and Drug Administration (FDA) announced a class I recall associated with a methicillin-resistant Staphylococcus aureus (MRSA) safety alert. This was due to failure to identify certain isolates as MRSA when using Gram-positive antimicrobial susceptibility testing (AST) cards with the Vitek 2 automated testing instrument (ATI) (1). To assist with the investigation, the U.S. Centers for Disease Control and Prevention (CDC) obtained 30 isolates identified during the initial recall from the National Reference Center for Staphylococci, Lyon, France. These isolates, reported as PCR-positive for mecA, produced susceptible results for oxacillin and cefoxitin (a surrogate for oxacillin) using AST cards on an ATI. Here, we describe the characterization of these isolates and announce their availability in the CDC and FDA Antibiotic Resistance Isolate Bank (AR Isolate Bank; https://wwwn.cdc.gov/ARIsolateBank/).

All isolates were tested using established Clinical and Laboratory Standards Institute AST methods and interpretive criteria, including disk diffusion (DD) for cefoxitin and in-house prepared reference broth microdilution (BMD) panels for oxacillin and cefoxitin (Table 1) (2–4). BMD was performed on each isolate at least three times by at least three different operators; the modal MIC was used for analysis. Twenty-eight isolates were categorized as cefoxitin resistant by DD and 23 by BMD. Two isolates, AR-998 and AR-1003, tested susceptible to cefoxitin by DD and by BMD. Five isolates displayed susceptibility to cefoxitin by BMD but resistance by DD. By BMD, six isolates were oxacillin resistant; the remaining 24 isolates were susceptible (MIC ≤ 2 μg/ml) (Table 1). Twelve isolates displayed MICs spanning multiple dilutions for either oxacillin or cefoxitin; eleven isolates displayed MIC values associated with different interpretive categories. In this limited collection of isolates, cefoxitin DD performed better than reference BMD; the PBP2a SA culture colony test (Table 1) also detected all isolates that were resistant by cefoxitin DD. Additional nonreplicate testing with two other ATIs (MicroScan WalkAway [Beckman Coulter, Brea, CA] and Phoenix [Becton, Dickinson and Company, Franklin Lakes, NJ]) also failed to uniformly detect these strains as MRSA or produce concordance across the ATIs (data not shown).

TABLE 1.

Phenotypic and genotypic characteristics of 30 difficult-to-detect mecA-positive Staphylococcus aureus isolates

Isolate	CDC reference laboratory resulte
	CMRSAII	mecAa	PBP2a	FOX DD (mm)	FOX DD INTPN	FOX BMD MIC (μg/ml)	FOX BMD INTPN	OXA BMD MIC (μg/ml)	OXA BMD INTPN	Clonal complex	SCCmec type
AR-0977	+	+	+	21	R	4	S	≤0.25	S	CC34	V
AR-0978	+	+	+	12	R	16	R	16	R	CC5	VI
AR-0979	+	+	+	14	R	8	R	8	R	CC5	VI
AR-0980	+	+	+	16	R	8	R	0.5	S	CC5	VI
AR-0981	+	+	+	19	R	4c,d	S	≤0.25	S	CC8	IV
AR-0982	+	+	+	16	R	8	R	0.5	S	CC5	VI
AR-0983	+	+	+	17	R	8	R	1	S	CC5	VI
AR-0984	+	+	+	16	R	8	R	0.5	S	CC5	VI
AR-0985	+	+	+	19	R	8	R	2c,d	S	CC5	IV
AR-0986	+	+	+	19	R	8	R	0.5	S	CC8	V
AR-0987	+	+	+	19	R	8	R	0.5	S	CC8	IV
AR-0988	+	+	+	17	R	8	R	1	S	CC8	IV
AR-0989	+	+	+	19	R	8	R	0.5	S	CC5	VI
AR-0990	+	+	+	17	R	8	R	0.5c,d	S	CC5	VI
AR-0991	−	+	+	18	R	4d	S	1	S	CC1	IV
AR-0992	−	+	+	18	R	4d	S	1	S	CC1	IV
AR-0993	+	+	+	13	R	16	R	4c,d	R	CC22	IV
AR-0994	−	+	+	18	R	4d	S	1	S	CC1	IV
AR-0995	+	+	+	15	R	16	R	0.5	S	CC8	IV
AR-0996	+	+	+	18	R	8	R	0.5	S	CC8	IV
AR-0997	+	+	+	12	R	16	R	16	R	CC5	IV
AR-0998b	−	+	+	27	S	4	S	≤0.25	S	CC8	IV
AR-0999	+	+	+	17	R	8	R	0.5	S	CC88	IV
AR-1000	+	+	+	14	R	16	R	2d	S	CC5	VI
AR-1001	+	+	+	17	R	8	R	4d	R	CC5	VI
AR-1002	+	+	+	15	R	8d	R	2d	S	CC5	VI
AR-1003b	−	+	−	26	S	4d	S	0.5	S	CC93	IV
AR-1004	+	+	+	18	R	8	R	2	S	CC30	IV
AR-1005	−	+	+	17	R	8	R	2	S	CC8	V
AR-1006	+	+	+	17	R	>16c	R	>16c	R	CC8	I
ATCC 29213	−	−	−	N/A	N/A	≤2	N/A	≤0.25	N/A	N/A	N/A
ATCC 43300	+	+	+	11	N/A	16	N/A	16	N/A	N/A	N/A

^aA CDC laboratory-developed test was used for mecA detection by PCR.

^bIsolate with a mutation in the mecA region.

^cUpon replicate testing, isolate displayed MIC values spanning more than 3 log₂ dilutions.

^dUpon replicate testing, isolate displayed MIC values associated with different interpretive categories; S, susceptible; R, resistant.

^eInterpretations are based on the individual drug MIC and not considering other drug results, supplemental phenotypic tests, or genomic data. MICs displayed are modal values. Interpretive criteria were applied according to CLSI performance standards for antimicrobial susceptibility testing M100 (2); oxacillin, R ≥ 4 μg/ml; cefoxitin, R ≥ 8 μg/ml; cefoxitin DD, R ≤ 21 mm. CMRSAII, CHROMagar BBL (Becton, Dickinson); PBP2a, Alere PBP2a SA culture colony test (Abbott); DD, disk diffusion (Becton, Dickinson); BMD, broth microdilution; FOX, cefoxitin; OXA, oxacillin; INTPN, interpretation; N/A, not applicable; +, positive; −, negative.

Whole-genome sequencing (WGS) was performed on all isolates (MiSeq platform; Illumina) and analyzed using the QuAISAR-H pipeline (https://github.com/nvlachos/QuAISAR-H). Sequence types were assigned using multilocus sequence typing (https://pubmlst.org/saureus/) and grouped into eight different clonal complexes (CCs) using the eBURST algorithm (5). Staphylococcal cassette chromosome mec (SCCmec) typing was performed as previously described (6), and all isolates were mecA-positive with four different SCCmec types identified (Table 1). The mecA full-length coding sequence was present in the genome assemblies of all isolates. AR-998 contained a nucleotide insertion (adenine at position 952 of mecA; see GenBank accession number CP026073.1) predicted to result in a frameshift mutation causing premature termination of MecA. AR-1003 is missing the full mecA promoter sequence (7), and work is ongoing to further characterize the SCCmec element. These mutations are likely the basis for the oxacillin and cefoxitin susceptible results consistently observed in AR-998 and AR-1003.

The data gathered to date indicate that these isolates represent a diverse strain collection with no single distinguishing feature and are seemingly limited in number. Work is ongoing to complete more comprehensive analyses of the observations reported herein, which are distinct from recently described mechanisms associated with “stealth MRSA” due to mecA sequence instability (8). In May of 2019, the FDA closed out the recall (9). With the addition of the “difficult-to-detect Staphylococcus aureus-harboring mecA” panel to the AR Isolate Bank, laboratories are encouraged to request these isolates to evaluate the performance of their local AST methods. Clinical laboratories should be aware that the S. aureus isolates described in this report exist.

Data availability.

All sequence data have been deposited with links to BioProject accession number PRJNA592083 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).