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Journal of Clinical Microbiology, November 2008, p. 3836-3838, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.01276-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Differentiation of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible Staphylococcus aureus from Blood Cultures by Use of a Direct Cefoxitin Disk Diffusion Test {triangledown}

Kelly Bennett and Susan E. Sharp*

Kaiser Permanente, Airport Way Laboratory, 13705 N.E. Airport Way, Portland, Oregon 97230

Received 7 July 2008/ Returned for modification 18 August 2008/ Accepted 19 September 2008


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ABSTRACT
 
A total of 276 blood culture bottles with Staphylococcus aureus were tested by direct cefoxitin disk diffusion testing; 105 (38.1%) had zone sizes of ≤17 mm (all 105 had methicillin-resistant S. aureus [MRSA]), 18 (6.5%) had zone sizes that measured 18 mm (17 had MRSA and 1 had methicillin-susceptible S. aureus [MSSA]), 8 (2.9%) had zone sizes that measured 19 mm (6 had MRSA and 2 had MSSA), 8 (2.9%) had zone sizes that measured 20 mm (6 had MRSA and 2 had MSSA), and 137 (49.6%) had zone sizes of ≥21 mm (all 137 had MSSA). Detection of MRSA/MSSA in blood cultures could be reported 10 to 24 h earlier for 88% of cultures with total accuracy.


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TEXT
 
The incidence of health care- and community-acquired Staphylococcus aureus bloodstream infections has significantly increased over the past few decades (20). The National Nosocomial Infections Surveillance System report from January 1992 through June 2001 stated that more than 55% of the S. aureus isolates were resistant to methicillin, oxacillin, or nafcillin (16). Risk factors associated with methicillin-resistant S. aureus (MRSA) bacteremia include the following: residence in an extended-care facility, prior antibiotic exposure, insulin-dependent diabetes, prolonged hospitalization, urinary catheterization, nasogastric tube placement, prior surgery, and having an underlying disease (13, 18, 19). The elderly population (≥65 years old) is at a significantly higher risk of death due to MRSA bacteremia than are younger populations (18, 22).

Infection with MRSA places a patient at increased risk for further health problems. MRSA bacteremia has been associated with an increased risk of acute renal failure, longer hospital and intensive-care-unit stays, development of ventilator dependency, and increased hospital costs (1, 6, 15). Several studies have shown that patients with MRSA bloodstream infections are at a significantly higher risk for mortality than are patients with methicillin-susceptible S. aureus (MSSA) bloodstream infections (1, 5, 19). Fatality rates for patients that develop MRSA bacteremia are estimated to be between 23% and 54% (1, 8).

Patients with MRSA bacteremia have also been shown to be at increased risk for delayed treatment (15). Inappropriate antibiotic choices for patients with S. aureus bacteremia have also been associated with higher hospital mortality rates (10). Inappropriate therapies consisted of β-lactams for MRSA bacteremia and vancomycin for MSSA bacteremia. The initial choice of vancomycin for MSSA bacteremia has been associated with a higher incidence of delayed clearance of infection, and delayed appropriate therapy for MRSA patients is an independent predictor of mortality (10, 15).

The choice of therapy for bloodstream infections has become critical with the increasing antibiotic resistance seen with many organisms. The increase in gram-positive organisms that are resistant to vancomycin threatens many accepted treatment regimens and justifies the empirical use of glycopeptides for bloodstream infections (12). Thus, the more rapid characterization of S. aureus isolates from blood cultures as being oxacillin resistant or oxacillin susceptible is an important factor in the prompt and accurate treatment of bacteremic patients.

Blood culture samples were collected in aerobic and anaerobic bottles (BacT/Alert FA [aerobic] and BacT/Alert FN [anaerobic] bottles; bioMérieux, Durham, NC) and assayed in the BacT/Alert 3D (bioMérieux, Durham, NC). Samples of blood cultures that showed "gram-positive cocci in clusters" on Gram stains were soaked on a swab from an aliquot derived from the blood bottle by using the same technique one would use in the preparation of a disk diffusion susceptibility test (3). The swab was then used to streak the entire surface of a 100-mm tryptic soy blood agar plate (BD BBL, Franklin Lakes, NJ), as would also be done for a disk diffusion test. Subsequently, a cefoxitin disk (BBL Sensi-Disc, Franklin Lakes, NJ) was placed in the center of the plate, and the plates were incubated overnight (10 to 24 h) at 35 to 37°C in ambient air. The following morning, cefoxitin zone sizes were measured and recorded. The measured cefoxitin zone size results were compared with the results obtained with confirmatory testing. Confirmatory assays included testing the isolated organism for susceptibility to oxacillin by both the Vitek 2 GP-63 susceptibility card (bioMéreiux, Durham, NC) and the standardized cefoxitin disk diffusion testing (3, 4). The organisms were identified as S. aureus by a 4-h tube coagulase test performed directly from the blood culture broth; identifications were reported the same day the blood culture was determined to be positive. If the direct tube coagulase was negative, slide and/or tube coagulase testing was performed on isolated colonies the following morning when the cefoxitin zone sizes were read.

A total of 782 blood culture samples were tested, 276 of which grew a pure culture of S. aureus (from 103 patients). A total of 489 samples grew coagulase-negative staphylococci (CNS), 12 grew Micrococcus species, 4 grew both S. aureus and CNS, and 1 grew Micrococcus species and CNS. Of the 276 pure cultures of S. aureus, 105 (38.1%) had direct cefoxitin zone sizes that measured ≤17 mm, 18 (6.5%) had those that measured 18 mm, 8 (2.9%) had those that measured 19 mm, 8 (2.9%) had those that measured 20 mm, and 137 (49.6%) had those that measured ≥21 mm (Fig. 1). All samples that had zone sizes that measured ≤17 mm with the direct cefoxitin disk test were confirmed as oxacillin resistant by both of the confirmatory methods, and all samples with zone sizes that measured ≥21 mm with the direct cefoxitin disk test were confirmed to be oxacillin susceptible by both confirmatory methods. Thus, detection of MRSA or MSSA in blood cultures could be reported 10 to 24 h earlier for 88% of cultures with total accuracy.


Figure 1
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  		FIG. 1. This figure shows the number of samples seen at each zone diameter when tested with the direct cefoxitin disk test. Those cultures/organisms that were determined to be MRSA upon definitive identification testing are represented by the black bars, while those likewise determined to be MSSA are indicated by the white bars. The criteria for using the direct cefoxitin disk test with 100% accuracy were as follows: ≤17-mm zone diameter = MRSA; ≥21-mm zone diameter = MSSA; 18- to 20-mm zone diameter = indeterminate result.

Thirty-four samples with zone sizes that measured between 18 and 20 mm contained a combination of both MRSA and MSSA isolates. Of these 34 samples, 29 (85.3%) were confirmed as MRSA, and 5 (14.7%) were confirmed as MSSA. This zone-size range was then described as "indeterminate," as the oxacillin results for organisms falling into this range could not be ascertained by direct cefoxitin disk testing with 100% accuracy but must wait for confirmatory testing to be performed. One might even consider calling all organisms with zone diameters of between 18 and 20 mm as presumptive MRSA, as over 85% of these samples were determined to be MRSA upon confirmatory testing. These zone ranges (MRSA, ≤20 mm; MSSA, ≥21 mm) would make the accuracy of the direct cefoxitin disk test >96%.

Incubation times were assessed for all study isolates with zone diameters of 18 to 20 mm to determine if samples with shorter incubation times were likely to result in indeterminate zone sizes. Of the 34 samples which fell into the indeterminate zone range, 3 (9%) had incubation times of 10 to 15 h, 9 (26%) were incubated for between 15 and 20 h, and 22 (65%) were incubated for 20 to 24 h. There was no correlation between incubation times and indeterminate zone diameters; a majority of the indeterminate samples had full incubations of between 20 and 24 h, with <10% having <15 h of incubation.

Assessing samples per patient, 39 of the 103 patients had MRSA, and 64 had MSSA. Using the first blood culture positive for each patient, 8 of the 39 MRSA patients had indeterminate results (zone diameters of between 18 and 20 mm), as did 2 of the 64 patients with MSSA. Thus, assessing the patient's first positive blood culture with the direct cefoxitin disk test to determine the oxacillin result, 93 of the 103 (>90%) patients' organisms would have been called with 100% accuracy by using the parameters of ≤17-mm zone diameters for MRSA and ≥21-mm zone diameters for MSSA, which is very close to the 88% seen when testing each blood culture. Although we did not see any mixed infections of MRSA and MSSA (one followed by the other in subsequent blood culture draws), we recommend testing all "gram-positive cocci in clusters" from blood cultures with the direct cefoxitin disk test, as this scenario could occur.

The need to differentiate MRSA from MSSA from blood culture specimens in a timely manner is of importance in providing the best care to patients through the use of appropriate antibiotics as soon as possible. Several mechanisms for determining Staphylococcus species and/or oxacillin results directly from blood culture bottles include selective differentiation chromogenic agar (17), direct identification methods such as the BBL Crystal MRSA ID test (Becton Dickinson Microbiology Systems, Cockeysville, MD) (11), and molecular methods, including the BD GeneOhm StaphSR assay (Becton Dickinson, Sparks, MD), Xpert MRSA/SA blood culture test from Cepheid (Sunnyvale, CA), multiplex detection of mecA and the nuc genes (12), detection of mecA and orfX genes by real-time PCR (21), and the IDI real-time assay (Infectio Diagnostic, Sainte-Foy, Quebec, Canada) using the SmartCycler (Cepheid, Sunnyvale, CA) (7). In addition, the PBP2a MRSA latex agglutination test (Oxoid Ltd., Basingstoke, United Kingdom) could be used the morning following subculture of positive samples if sufficient growth occurs (incubation time dependent). This would give a similar turnaround time as our protocol; however, the PBP2a latex test requires well-isolated colonies, works most efficiently when batched, takes additional staff time for testing, and is more costly.

All of the techniques mentioned above utilize additional laboratory resources, such as kit ID systems and/or specialized media or molecular assays. Although rapid and accurate, these tests are often best utilized for batch testing, are all more costly and labor intensive for the laboratory, and, with molecular testing, can often require additional molecular expertise and instrumentation that may not be readily available. This study outlines an approach for determining the oxacillin result of S. aureus isolates directly from blood culture specimens that can be used cost-effectively and accurately and need not be batch tested, but can be read quickly and individually at each work station. Understandably, the laboratory would be testing all blood cultures showing gram-positive cocci in clusters, but as the materials and expertise needed to perform the direct cefoxitin disk test are already a part of the routine diagnostic microbiology laboratory, the added cost of a tryptic soy blood agar plate and a cefoxitin disk is well worth the modest expense (~$0.50/SA culture) in order to report MSSA or MRSA more rapidly from blood cultures. For direct cefoxitin disk results of ≤17-mm or ≥21-mm zone sizes, the accuracy of reporting the oxacillin results for S. aureus isolates was 100%. By utilizing this protocol the laboratory can give an accurate oxacillin result up to 24 h faster than waiting for the routine susceptibilities to be completed. It should be noted that these breakpoints were determined by the results of this study and do not exactly coincide with those published by the CLSI for assessment of oxacillin resistance by using cefoxitin disks from isolated colonies of staphylococci (4). In addition, as this study was not based on CLSI guidelines, blood agar was chosen for use over the classic Mueller-Hinton agar utilized in standardized testing protocols, as blood agar was less expensive (by approximately 40% in our institution) and would be more readily available to laboratories that primarily use automated instrumentation for susceptibility testing.

By more-rapidly reporting oxacillin results for S. aureus isolates from cases of bacteremia, patients could be treated earlier with the most effective antibiotic. Appropriately and rapidly treating a patient with the most effective antibiotic will decrease complications for the patient, reduce the length of hospital stay for the patient, and decrease patient morbidity and mortality, resulting in increased savings for medical institutions (10, 14, 15, 18). In addition, the more appropriate use of vancomycin will decrease the emergence of resistance to this drug in gram-positive organisms (2, 9).


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FOOTNOTES
 
* Corresponding author. Mailing address: Kaiser Permanente NW, 13705 N.E. Airport Way, Portland, OR 97230. Phone: (503) 258-6824. Fax: (503) 258-6864. E-mail: susan.e.sharp{at}kp.org Back

{triangledown} Published ahead of print on 1 October 2008. Back


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Journal of Clinical Microbiology, November 2008, p. 3836-3838, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.01276-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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