Oxacillin Susceptibility Testing of Staphylococci Directly from Bactec Plus Blood Cultures by the BBL Crystal MRSA ID System

Abstract

The BBL Crystal MRSA ID test (Becton Dickinson) was applied directly to blood culture vials containing clusters of gram-positive cocci. The sensitivity and specificity of the test were 84 and 100% and 54 and 100% for vials containing Staphylococcus aureus and coagulase-negative staphylococci, respectively. This test is a reliable method for direct detection of methicillin resistance in positive blood culture vials when S. aureus is identified in parallel by rapid identification procedures.

Detection of methicillin resistance of staphylococci is of importance in clinical practice, especially in blood culture isolates, since Staphylococcus aureus is a frequent cause of nosocomial bacteremia (1). Glycopeptides are still widely used in cases of severe disease while awaiting the results of antibiotic susceptibility testing due to the high prevalence of methicillin resistance in staphylococci (1). Standardized methods of oxacillin susceptibility testing have been developed for isolated colonies (2); therefore, the oxacillin susceptibility result is not usually available until at least 24 h after identification of a positive blood culture. The BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) applied to isolated colonies was recently reported to be an efficient, rapid, and specific alternative method allowing the detection of methicillin resistance in S. aureus within 4 to 6 h (4, 9, 11, 15, 16).

In the present study, our objective was to evaluate the reliability of this test, applied directly to blood culture vials positive for clustering gram-positive cocci, for the detection of methicillin resistance in coagulase-positive staphylococci and coagulase-negative staphylococci (CNS). The results of the BBL Crystal MRSA ID test were compared to those obtained by agar disk diffusion and mecA gene detection, the genotypic method being taken as the reference technique.

Over a 12-month period, all first blood vials (Bactec Plus aerobic/F and anaerobic/F; Becton Dickinson) containing staphylococcal clusters positive for Gram staining were retained for a given patient and tested by the BBL Crystal MRSA ID test, by agar disk diffusion, and by mecA gene detection. In order to complete rapid identification of S. aureus within the same time interval as oxacillin susceptibility testing, the clusters of gram-positive cocci were also identified by the coagulase tube test performed directly on positive blood vials. Briefly, a 50-μl aliquot was aseptically removed from each culture vial and diluted in 200 μl of brain heart broth medium (Sanofi Diagnostics Pasteur, Paris, France). After addition of 250 μl of citrated rabbit plasma, clotting was read after 4 and 24 h of incubation at 37°C. All CNS isolates for which the results of methicillin resistance detection by the BBL Crystal MRSA ID test were different from those of agar disk diffusion and/or mecA gene detection were identified by the API 32 Staph system (BioMérieux, Marcy-L’Etoile, France). The methicillin resistance detection test was performed by several methods. (i) Oxacillin disk diffusion was performed in accordance with the recommendations of the French Antibiogram Committee (3). Tests were carried out on Mueller-Hinton agar incubated for 48 h at 30°C and on Mueller-Hinton agar supplemented with 5% NaCl and incubated for 24 h at 37°C. (ii) The genotypic assay used the oligonucleotide mecA probe (5′-AGAGTAGCACTCGAATTAGGCAGT-3′) previously described by Shimaoka et al. (13). This probe was 5′ labeled with [γ-³²P]ATP as described by Sambrook et al. (12), and dot blot hybridization was performed as previously described (8). After hybridization, the filters were washed twice for 10 min (each time) at 35°C in 2× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA [pH 7.7]) buffer containing 0.1% (wt/vol) sodium dodecyl sulfate, air dried, and exposed overnight at −70°C to X-ray film (Fujifilm NIF) with two intensifying screens. (iii) The BBL Crystal MRSA ID test was prepared as follows. A 1-ml positive blood culture vial aliquot was put into a sterile Eppendorf microtube and centrifuged for 5 min at 10,000 × g. The supernatant was discarded and the pellet was resuspended in 1.5 ml of sterile bidistilled water for 30 s to lyse erythrocytes. Isotonicity was then restored with 90 μl of sterile 5 M NaCl solution and immediate mixing. These steps were repeated once if hemoglobin was still macroscopically present. The pellet was then adjusted to 0.5 McFarland unit in sterile saline buffer. Four drops of bacterial suspension were added to each of the three wells of the BBL Crystal MRSA ID test, incubated at 37°C, and observed after 4, 5, and 6 h of incubation. The test uses an oxygen-sensitive fluorescent indicator in the well test which detects oxygen consumption due to bacterial metabolism revealed with a longwave (365-nm) UV light source (9, 11). A test was considered to be interpretable if the well that contained no antibiotic was positive for fluorescence and the second well containing vancomycin at 16 μg/ml was negative and was otherwise defined as uninterpretable. In interpretable tests, the organism was considered to be methicillin resistant (MR) if the test well containing dried oxacillin at 4 μg/ml was positive or methicillin susceptible (MS) if the test well was negative. Throughout the study, S. aureus ATCC 25923 and ATCC 33592 were used as quality control organisms. Statistical calculations were carried out by using the formula of Ilstrup (7), taking the results of mecA detection as the reference.

Among 182 positive blood culture vials tested, 31 BBL Crystal MRSA ID tests were excluded due to the presence within the cultures of (i) a mixture of several bacterial species (more than one strain of Staphylococcus or strains belonging to Staphylococcus and to another bacterial genus) (27 tests) or (ii) gram-positive cocci other than Staphylococcus species (micrococci in 3 tests, enterococci in 1 test). Finally, 151 nonredundant blood culture vials were retained for further analysis.

The vials contained S. aureus in 39.1% of the cases (59 of 151) and CNS in 60.9% of the cases (92 of 151), with no discrepancies between results after 4 and 24 h of incubation in the coagulase tube test. Although some staphylococcal species other than S. aureus, such as, for instance, S. intermedius or S. schleiferi, produce a positive coagulase test result, their occurrence is extremely rare in clinical isolates (10). In previous work (10), among 3,397 consecutive isolates of coagulase-positive staphylococci examined in our laboratory, only 2, from a nasal swab and from pleural fluid, and none from blood cultures, contained S. intermedius.

Taking the mecA probe as a reference, methicillin resistance was present in 42.4% (25 of 59) of the S. aureus strains and in 58.7% (54 of 92) of the CNS strains. Among the 151 vials tested, no discrepancies were observed between the results of the two control methods, except in the case of a single S. epidermidis strain which appeared to be MS in two disk diffusion tests but gave a positive mecA signal (Table 1). Some BBL Crystal MRSA ID tests were uninterpretable. Their number decreased between 4 and 6 h of incubation (Table 1). After 6 h of incubation, all vials containing S. aureus were interpretable and only three containing CNS (one MR S. epidermidis strain, one MR S. haemolyticus strain, and one MS S. epidermidis strain) remained uninterpretable. This 3.3% uninterpretable tests could be due to slower growth, as reported by Ieven et al. (6), who tested isolated strains and proposed use of a 2 McFarland inoculum for testing of CNS vials rather than the 0.5 McFarland inoculum recommended by the manufacturer for S. aureus.

TABLE 1.

Oxacillin resistance determined by mecA gene detection, oxacillin disk diffusion, and the BBL Crystal MRSA ID test

Organism (no. of isolates)	mecA gene	No. of isolates	No. of strains positive by oxacillin disk diffusion after:		No. of isolates with following BBL Crystal MRSA ID test result after incubation for:
			48 h at 30°C	24 h at 37°C	4 h			5 h			6 h
					Sa	Rb	Uc	S	R	U	S	R	U
S. aureus (59)	Present	25	25	25	8	12	5	5	19	1	4	21	0
	Absent	34	34	34	34	0	0	34	0	0	34	0	0
CNS (92)	Present	54	53	53	23	14	17	25	23	6	24	28	2
	Absent	38	39	39	23	0	15	35	0	3	37	0	1

^aS, sensitive to oxacillin. 

^bR, resistant to oxacillin. 

^cU, uninterpretable test reaction. 

Among the 59 S. aureus strains, 25 were oxacillin resistant by mecA gene detection. The BBL Crystal MRSA ID test result was positive for 48% (12 of 25), 76% (19 of 25), and 84% (21 of 25) of these vials after 4, 5, and 6 h of incubation, respectively. Thus, four of the eight MR S. aureus strains falsely sensitive to oxacillin in a 4-h BBL test were found to be resistant after 6 h. All vials containing S. aureus strains negative by mecA detection were found to contain MS strains by the BBL Crystal MRSA ID test. Among the 92 CNS strains, 54 were MR by mecA gene detection. The BBL Crystal MRSA ID test result was positive for 25.9% (14 of 54), 42.6% (23 of 54), and 51.9% (28 of 54) of these vials after 4, 5, and 6 h of incubation, respectively. In the remaining 38 vials containing mecA-negative CNS, the BBL Crystal MRSA ID test revealed 60.5% (23 of 38), 92.1% (35 of 38), and 97.4% (37 of 38) of these strains as MS after 4, 5, and 6 h of incubation, respectively (Table 1). As no false methicillin resistance was detected in blood vials containing MS strains, the specificity and positive predictive value of the BBL Crystal MRSA ID assay were maximal (100%) for both S. aureus and CNS after only 4 h of incubation (Table 2).

TABLE 2.

Sensitivity, specificity, and positive and negative predictive values of interpretable BBL Crystal MRSA ID tests after 4, 5, and 6 h of incubation

Organism (no. of isolates)	Incubation time (h)	No. (%) of interpret-able tests	% Agree-ment	Sensi-tivity (%)	Speci-ficity (%)	PPVa (%)	NPVb (%)
S. aureus (59)	4	54 (91.5)	85.2	60.0	100	100	81.0
	5	58 (98.3)	91.4	79.2	100	100	87.2
	6	59 (100)	93.2	84.0	100	100	89.5
CNS (92)	4	60 (65.2)	61.7	37.8	100	100	50.0
	5	83 (90.2)	69.9	47.8	100	100	58.3
	6	89 (96.7)	73.0	53.8	100	100	60.7

^aPPV, positive predictive value. 

^bNPV, negative predictive value. 

The percentage of agreement among BBL Crystal MRSA ID tests was 93.2% for vials containing S. aureus and 73% for those containing CNS, while the sensitivity and negative predictive value reached 84 and 89%, respectively, after 6 h for S. aureus strains. These data are in accordance with the previous findings of Zambardi et al. (16) for S. aureus isolates. Those investigators observed sensitivities ranging from 71 to 100% and negative predictive values of 95 to 100% after 4 h of incubation. The sensitivity of the BBL Crystal MRSA ID assay to methicillin resistance was, however, less for CNS (53.8% after a 6-h incubation period), with only 70.6% of the MR CNS strains correctly identified after 6 h of incubation. Hence, the results of this test should be interpreted cautiously in the case of apparently MS CNS strains.

Automated systems such as the Vitek GPS-SA card (5) or the MicroScan apparatus (14) can produce results within 7 to 11 h, but this type of equipment is not available in the majority of hospitals. Molecular biology techniques are still at an experimental stage, and their use is restricted to a few medical centers. The BBL Crystal MRSA ID System could therefore represent a useful alternative for direct susceptibility testing. This assay does not require special equipment or expertise, is easy to perform in any laboratory, and can be quickly integrated into routine diagnostic procedures.