This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Felten, A.
Right arrow Articles by Casin, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Felten, A.
Right arrow Articles by Casin, I.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, August 2002, p. 2766-2771, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.2766-2771.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant Staphylococcus aureus (MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test

Annie Felten,* Bernadette Grandry, Philippe Henri Lagrange, and Isabelle Casin

Service de Bactériologie-Virologie-Hygiène, Hôpital Saint-Louis, 75475 Paris Cedex 10, France

Received 15 January 2002/ Returned for modification 7 March 2002/ Accepted 8 May 2002


arrow
ABSTRACT
 
Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-µg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter-1 for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-µg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-µg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.


arrow
INTRODUCTION
 
Staphylococcus aureus causes serious community-acquired and nosocomial infections. The introduction of benzylpenicillin had a dramatic effect on mortality rates due to invasive S. aureus. The increasing prevalence of benzylpenicillin-resistant S. aureus was initially overcome by the introduction of semisynthetic penicillins. Since that time, methicillin-resistant S. aureus (MRSA) has rapidly emerged and become a major clinical problem. In 1996, 57% of S. aureus isolates from French patients with nosocomial infections were methicillin resistant (1). Epidemiological studies on high-level MRSA, which is resistant to numerous antibiotics and antiseptics, revealed nosocomial outbreaks with clones disseminating nationally and internationally (2). Infections caused by very-low-level MRSA were first reported in Japan, where MRSA was more prevalent than elsewhere (14, 21). Subsequently, an increasing number of endemic and epidemic low-level MRSA strains that either were susceptible to most antistaphylococcal antibiotics, were susceptible to gentamicin, or did not produce ß-lactamase were detected (4, 9, 23). The prevalence of community-acquired skin, soft-tissue, and disseminated infections caused by very-low-level MRSA in people without known risk factors increased recently in the United States and Europe (6, 10, 12). Four phenotypic classes of MRSA were identified by Tomasz et al. on the basis of population analyses of methicillin MICs in vitro. Three of these classes were heterogeneous (classes 1 to 3), and one was homogeneous (class 4); the methicillin MIC for class 4 was >800 mg liter-1. For the major populations of class 1 to 3 isolates, methicillin MICs were 1.5 to 100 mg liter-1, respectively, and for the minor populations, 10-8 to 10-2, respectively, methicillin MICs were >100 mg liter-1 (27).

Nearly all MRSA isolates produce an additional penicillin-binding protein (PBP), named PBP2a. PBP2a binds ß-lactams with a lower affinity than PBP2, the major physiological methicillin target. PBP2a is encoded by the mecA gene, a component of a larger DNA fragment designated the mec region (7, 14). The standard test used to identify MRSA is amplification of the mecA gene (22). The mec elements upstream and downstream of mecA are polymorphic. Nonetheless, two upstream genes, mecR1 and mecI, are thought to regulate methicillin resistance (14, 15). Many other factors are involved in modulating the expression of methicillin resistance (7, 14).

Routine oxacillin tests often fail to detect very heterogeneous MRSA populations, which consequently are considered methicillin-susceptible S. aureus (MSSA) because of their usual susceptibility to most non-ß-lactam antistaphylococcal antibiotics. Therefore, a number of parameters have been recommended to improve results: increasing the inoculum, growth at a low temperature, an oxacillin screen test with NaCl, or protracted incubation (17). The tests currently recommended by the NCCLS (National Committee for Clinical Laboratory Standards) are the oxacillin disk method, using 1 µg of oxacillin on a swab-inoculated Mueller-Hinton agar (MHA) plate supplemented with 2% NaCl and incubated at 35°C, and the oxacillin agar screen test using MHA supplemented with 4% NaCl. The Comité de l'Antibiogramme de la Société Française de Microbiologie (CASFM) recommends use of the oxacillin disk method, with 5 µg of oxacillin on an MHA plate, either supplemented with NaCl at 37°C or without NaCl at 30°C, flooded with the bacterial inoculum (24). More recent methods for detection of MRSA include the oxacillin E-test (AB BIODISK, Solna, Sweden) for determination of MICs, the automated Vitek 2 system (bioMérieux, La Balme les Grottes, France), and the MRSA-screen latex agglutination test (Denka, Seiken Co. Ltd., Tokyo, Japan), which detects PBP2a (3, 16, 18, 26, 27). Cephamycins were used extensively in Japan in the early 1980s, and as a result some MRSA and MSSA isolates became resistant to cefoxitin. Surprisingly, cefoxitin induced production of PBP2a in vitro in MSSA isolates for which cefoxitin MICs were high, and the disk diffusion assay with ceftizoxime (a cephamycin) proved to be a good assay for detection of low-level MRSA in Japan (19, 21).

We compared three methods for detection of MRSA, particularly the low-level class 1 MRSA: the cefoxitin and moxalactam (cephamycins) disk diffusion assays, the Vitek 2 system, and the MRSA-screen test. We subsequently measured the prevalence of clinical class 1 MRSA isolates among hospitalized patients and studied the distribution of mecA-regulatory genes in MRSA isolates.


arrow
MATERIALS AND METHODS
 
Bacterial strains. We studied 152 clinical S. aureus isolates from the St. Louis Hospital, Paris, France. These isolates formed three groups. The first comprised low-level MRSA index cases. These included eight isolates taken from seven patients and from the nose of a surgeon in the plastic surgery department in November and December 1998; three were class 1 MRSA and five were class 2 MRSA. The second group was a series of 95 consecutive clinical isolates collected between January and March 1999 from patients hospitalized in all departments except the intensive care unit and used to study the current prevalence of very-low-level MRSA. These isolates were collected from 90 patients, since 3 patients were found to harbor several S. aureus isolates with different resistance phenotypes. The third group comprised a selection of 49 clinical MRSA and MSSA isolates with different antibiotypes. Eight of these isolates were MSSA and were resistant to various antistaphylococcal antibiotics, and 41 were MRSA, including 13 class 1 isolates. Isolates were stored at -80°C. They were grown in air at 37°C on Columbia agar supplemented with 5% sheep blood before testing. S. aureus was identified by the mannitol fermentation test on Chapman medium, the coagulase-binding latex slide agglutination test (Fumouze Diagnostics, Levallois Perret, France), the tube coagulase test with oxalated rabbit serum and 18-h Staphylocoagulase broth (1 ml, incubated 4 h at 37°C; Bio-Rad, Marnes la Coquette, France), thermonuclease production on plates seeded with DNA (Bio-Rad) (100 µl of the 18-h Staphylocoagulase broth was heated for 5 min at 95°C, and DNase excretion was detected with 1 M HCl), and the API 32 Staph strip (bioMerieux) when necessary. Control strains used for all assays included the MSSA ß-lactamase-negative strain ATCC 25923, the BORSA (ß-lactamase-positive, borderline methicillin-resistant, mecA-negative S. aureus) strain CCUG 35302, the heterogeneous class 1 MRSA strain ATCC 43300T, and the homogeneous MRSA strain CCUG 31966 from the Swedish Collection. Micrococcus luteus ATCC 4698 was used to test ß-lactamase production.

Analysis of mec gene complex by amplification. DNA was extracted from three or four colonies suspended in 1.5 ml of water. After centrifugation, the pellet was resuspended in 200 µl of InstaGene Matrix (Bio-Rad). After 20 min at 56°C, the lysate was stirred and boiled for 8 min and then centrifuged to collect the DNA in the supernatant. PCR amplification was performed by using 1 U of Taq DNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany). The reaction was carried out by using a Gene Amp PCR System 9600 (Perkin-Elmer). The mecA gene was amplified as described by Predari et al. (22). These conditions yielded a 528-bp PCR product corresponding to mecA. When mecA was present, the DNA was tested for the upstream regulatory genes mecR1 and mecI with the primers and PCR conditions described by Kobayashi et al. (15). The 5'-end mecRA and 3'-end mecRB regions of the regulatory gene mecR1 yielded PCR products of 310 and 236 bp, respectively. When mecRB was present, the DNA was tested for the presence of a mecI PCR product of 481 bp. For visualization of PCR products, 8-µl samples of the product were electrophoresed in 0.8% 1x TBE (8.9 M Tris, 8.9 M boric acid, 0.2 M EDTA) for 45 min at 100 V, stained with ethidium bromide, and photographed under UV illumination. The PCR products of the clinical isolates were visually compared with those of the reference strains.

Susceptibility testing methods. (i) Inocula for susceptibility testing. High-density inocula were made by diluting five colonies grown overnight on Columbia agar supplemented with 5% sheep blood (Bio-Rad) in 5 ml of Mueller-Hinton broth (Bio-Rad) or distilled water to prepare a suspension equivalent in density to 0.5 McFarland barium sulfate standard unit (average turbidity, 108 CFU ml-1). Low-density inocula were made by diluting 200 µl of the high-density suspension in 20 ml of distilled water to a final concentration of approximately 106 CFU ml-1.

(ii) Disk diffusion tests for resistance to oxacillin, cefoxitin, and moxalactam. The entire surface of the MHA plate (diameter, 90 mm) (Bio-Rad) was covered with the required inoculum, and the plate was air dried for 15 min before the disks were laid on the surface and incubation was performed for 18 h at the required temperature. Oxacillin resistance was determined with 1- and 5-µg disks according to the NCCLS and CASFM critical diameters, <13 and <20 mm, respectively (17, 24). In cases of heterogeneous growth, defined as the occurrence of small colonies in the circular growth inhibition area, the diameter of the inner limit of the small colonies' inhibition zone was taken into account.

(iii) Oxacillin agar screen. One hundred microliters of the high-density inocula was dropped onto MHA plates with 2% NaCl containing 6 µg of oxacillin ml-1. If any growth occurred within 48 h, the isolate was considered to be oxacillin resistant.

(iv) Determination of oxacillin and cefoxitin MICs by the E-test and of moxalactam MICs by dilutions in agar. E-tests were performed according to the manufacturer's instructions on 150-mm-diameter MHA plates inoculated by swabbing in three directions. If heterogeneous growth occurred, the highest MIC (inner limit of the inhibition zone) was read. Moxalactam in the form of a dry powder (Chiomarin, Shionogi Co., Osaka, Japan) was diluted in MHA plates from 0.5 and 32 mg liter-1. One microliter of the bacterial suspension (about 104 bacteria) was laid on the surface with an A400 multipoint inoculator (Dynex, Issy les Moulineaux, France). S. aureus ATCC 25923 was tested with each batch of medium. The MIC was defined as the lowest concentration that inhibited bacterial growth.

(v) Vitek 2. Susceptibility testing with the Vitek 2 system was performed according to the manufacturer's instructions. Readings were automatically taken every 15 min. The current NCCLS breakpoints for oxacillin susceptibility were used: MICs of <=2 mg liter-1 indicated susceptibility, and MICs of >=4 mg liter-1 indicated resistance.

(vi) Detection of PBP2a. The MRSA-screen test, which is based on the agglutination of latex particles sensitized with monoclonal antibodies against PBP2a, was used according to the manufacturer's instructions.

(vii) ß-Lactamase production. ß-Lactamase production was measured by two methods. (i) Colonies from the edge of the inhibition zone of a 10-IU benzylpenicillin disk were streaked onto a nitocefin disk (bioMérieux), and ß-lactamase production was characterized by the appearance of pink colonies within 15 min. (ii) If the first test was negative, Got's test was performed on an MHA plate containing approximately 108 CFU of M. luteus ml-1. A benzylpenicillin disk was placed in the center of the agar plate, and a loopful of each isolate was streaked radially. After 24 h at 37°C, ß-lactamase-producing S. aureus induced the growth of M. luteus in the vicinity of the streak.

Each set of tests was carried out by an investigator unaware of the individual oxacillin status of the isolates, and each was read by two independent observers.

(viii) Classification of MRSA isolates. We adopted the 72-h protracted incubation of the 5-µg oxacillin disk diffusion method to fit the classification system described by Tomasz et al. (25). Class 1 MRSA gave an inhibition zone larger than 20 mm within 18 h and a few colonies inside this zone within 36 to 72 h; classes 2 and 3 gave an inhibition zone larger than 20 mm within 18 h and a hazy growth of colonies inside this zone within 18 to 48 h; and class 4 showed either no inhibition zone or a zone of <20 mm within 18 h.


arrow
RESULTS
 
Performances of the different methods for the detection of MRSA. Among the 152 S. aureus isolates, 83 were MRSA, mecA positive, and 69 were MSSA, mecA negative.

Oxacillin 6-µg agar screen and oxacillin disk diffusion methods. The sensitivity of the agar screen test was 94% when the 31 MRSA isolates that gave hazy growth after 48 h were included (Table 1). The sensitivity of the 5-µg oxacillin disk method was 95.2% with the high-density inoculum at 37°C (Table 1), 41% with the low-density inoculum at 37°C, and 31.3% with the low-density inoculum at 30°C (data not shown). The 1-µg oxacillin disk diffusion test was 96.4% sensitive with the high-density inoculum at 37°C (Table 1). All of the methods except the 1-µg oxacillin disk test (specificity, 97.1%) were 100% specific.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Sensitivities of the recommended oxacillin methods and of the new methods for detection of 83 MRSA clinical isolates

Cefoxitin and moxalactam disk diffusion tests. The ranges of the inhibition zone diameters for the MRSA and the MSSA isolates were very distinct, except with the low-density inoculum at 30°C for one class 1 MRSA isolate (isolate 112) and one MSSA isolate (isolate 9), both of which gave a 26-mm cefoxitin inhibition zone diameter (Table 2). With the low-density inoculum at 37°C, all MRSA isolates showed cefoxitin inhibition zone diameters of <27 mm and moxalactam inhibition zone diameters of <24 mm, and all MSSA isolates showed larger diameters. With these critical diameters, cefoxitin and moxalactam disk diffusion tests were 100% sensitive and 100% specific (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Inhibition zone diameters of cefoxitin and moxalactam disk diffusion tests for 83 mecA-positive (MRSA) and 69 mecA-negative (MSSA) S. aureus isolates

Oxacillin, cefoxitin, and moxalactam MICs. In cases of heterogeneous growth with the E-test, the recorded MIC corresponded to the highest limit of the inhibition of growth of the most resistant population. The E-test oxacillin MICs for the MSSA isolates were all <2 mg liter-1, and those for the MRSA isolates were between 0.38 and 256 mg liter-1 (>2 mg liter-1 for 91.6% of MRSA isolates, and 0.38 to 1.5 mg liter-1 for 8.4%—all class 1 isolates) (Fig. 1). For all MSSA isolates, E-test cefoxitin MICs were <4 mg liter-1, whereas for all MRSA isolates, cefoxitin MICs were >=4 mg liter-1 (Fig. 1). The median cefoxitin MIC for MSSA isolates was 2 mg liter-1, and the median cefoxitin MICs for class 1, classes 2 and 3, and class 4 MRSA isolates were 32, 48, and 256 mg liter-1, respectively. For all MSSA isolates, moxalactam MICs were <=8 mg liter-1, and for all MRSA isolates except one (isolate 128, for which the moxalactam MIC was 4 mg liter-1), moxalactam MICs were >8 mg liter-1 (Fig. 1). The median moxalactam MIC for MSSA isolates was 8 mg liter-1, and the median MICs for heterogeneous and homogeneous MRSA isolates were 32 and >32 mg liter-1, respectively. The moxalactam MIC for S. aureus ATCC 25923 was 2 or 4 mg liter-1, depending on the batch of medium.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1. Distribution of oxacillin, cefoxitin, and moxalactam cumulative MICs in mecA-negative (MSSA) and mecA-positive (MRSA) S. aureus isolates.

Vitek 2. All of the MSSA isolates were Oxascreen negative, and oxacillin MICs for all MSSA isolates were <=1 mg liter-1. For 10 MRSA isolates, oxacillin MICs were <4 mg liter-1. Five of these isolates, all of which belonged to classes 1 to 3, did not grow on the Oxascreen either, and were misclassified as oxacillin susceptible (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Characteristics of the seven MRSA isolates that were misclassified by the Vitek 2 or the MRSA-screen test

MRSA-screen test. Two mecA-positive isolates gave false-negative results repeatedly on the MRSA-screen. Neither of these isolates belonged to class 1 (Table 3).

All the above assays gave satisfactory results with the reference strains.

Classification of isolates according to MRSA class, mecA status, and mecA-regulatory gene status. (i) mecA status and MRSA class. Sixty-nine of the 152 clinical S. aureus isolates studied were mecA negative and oxacillin susceptible. Of the 83 mecA-positive isolates, 26 belonged to class 1, 37 belonged to class 2 or 3, and 20 belonged to class 4.

(ii) mecA-regulatory genes. Seventy nine MRSA isolates harbored a mecR1 gene; of these, 2 harbored the complete mecRA-mecRB (pattern of isolate 88) and 77 harbored the truncated mecRA only (pattern of isolate 112) (Fig. 2). Four MRSA isolates harbored no mecR1 gene (pattern of isolate 105) (Fig. 2). mecI was found in the heterogeneous class 1 strain ATCC 43300T but in no clinical isolate, unlike most class 1 Japanese isolates (14, 15). Neither MRSA class nor test performance was related to mec-regulatory genes: all of the 26 class 1 MRSA isolates, and all of the 7 MRSA isolates that were misclassified either by the Vitek 2 or by the MRSA-screen test, were of the predominant genotype.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 2. Gel image of representative PCR mec gene products, mecA (528 bp), mecRA (310 bp), mecRB (236 bp), and mecI (481 bp), from different MRSA isolates, obtained with specific primers. Lane M, DNA molecular weight markers. Lanes 1, 7, 12, and 17, MRSA strain ATCC 44300; lanes 2, 8, and 13, MRSA strain CCUG 31966; lane 3, BORSA strain CCUG 35302; lanes 4, 9, 14, and 18, MRSA isolate 88; lanes 5, 10, and 15, MRSA isolate 112; lanes 6, 11, and 16, MRSA isolate 105.


arrow
DISCUSSION
 
The cefoxitin and moxalactam disk diffusion tests were found 100% sensitive and specific for MRSA under all conditions tested, except for one isolate under test conditions of a low-density inoculum and incubation at 30°C. This implies that the cephamycin disk test (cefoxitin or moxalactam) is an available alternative to the oxacillin disk method for routine antibiotic susceptibility testing at 37°C (24). According to our results, an S. aureus isolate that gives a cefoxitin diameter of <27 mm or a moxalactam diameter of <24 mm can be identified as MRSA. These diameters are higher than those corresponding to the respective MICs indicating resistance according to CASFM or NCCLS disk diffusion values (24). The strong correlation between cephamycin diameters and oxacillin resistance is mediated by still unknown mechanisms (14). This strong correlation between cephamycin MICs (cefoxitin MIC of >=4 mg liter-1, moxalactam MIC of >8 mg liter-1) and methicillin resistance may be due to the interaction between PBP2a and various PBPs by still unknown mechanisms (14). Compared to cephalosporins, cephamycins have a high affinity for S. aureus PBP4, a protein which is involved in cell wall cross-linking (13, 20). Previous experiments showed a relationship between PBP2, PBP4, and methicillin resistance. The cephamycin MIC for an MRSA strain was 100 mg liter-1, whereas that for its isogenic mutant, which was defective in PBP2, was 3 mg liter-1 (20). In vitro induction of overexpression of PBP4 in an MSSA strain resulted in methicillin resistance (11). Ceftriaxone, an expanded-spectrum cephalosporin, has also recently been advocated as a presumptive test for MRSA; its performance (95% sensitivity and 97% specificity) (5) was lower than those of cephamycins.

The Vitek 2 test was 94% sensitive and 100% specific. The isolates which were misclassified were heterogeneous MRSA isolates. The sensitivity of the 6-µg/ml oxacillin agar screen method was also 94%. Previous trials showed that detection sensitivities were 95.3% with Vitek on heterogeneous French MRSA isolates and 97% with Vitek GPS 106 on multinational bloodstream isolates (3, 27). These trials did not detail results for very-low-level MRSA isolates. The Vitek 2 test produces rapid results: oxacillin susceptibility results within 5 h, identification to the species level and complete antimicrobial susceptibility results within 8 h.

The MRSA-screen test was 97.6% sensitive and did not misclassify any class 1 MRSA isolate as MSSA; the two misclassified isolates belonged to different clones (Centre National de Référence pour les Staphylocoques, Institut Pasteur, Paris, France). In previous assays, the MRSA-screen test was found to be 97 to 100% sensitive and 100% specific (5, 16, 18, 27). The MRSA-screen test is easy to perform, and the results are available within 15 min. Thus, it was the best commercially available test for distinguishing very-low-level class 1 MRSA from MSSA.

The detection sensitivities of the reference methods, oxacillin MICs of >2 mg liter-1, the oxacillin agar screen, and disk diffusion tests with 5- or 1-µg oxacillin disks, were 91.6, 94, 95.2, and 96.4%, respectively. The oxacillin E-test failed to detect seven isolates correctly (MICs, 0.38 to 1.5 mg liter-1; median MIC, 0.5 mg liter-1), all of which were class 1 MRSA isolates. Surprisingly, the sensitivity of the oxacillin agar screen on MHA plates with 2% NaCl was lower than that of the 1-µg oxacillin disk diffusion method (with a high inoculum). In contrast, national quality control of class 1 MRSA testing performed by the Laboratory Proficiency Testing Program of Ontario showed that 21% of laboratories using the standard disk test and 1% of those using an oxacillin agar screen reported an incorrect MSSA result (17). In the present study, all of the tests were 100% specific, except for the disk diffusion tests with 1 µg of oxacillin (97.1%) and 30 µg of cefoxitin at 30°C (98.6%). Specificities averaging 80% have been reported with the 1-µg oxacillin disk (7).

In this assay, the prevalence of MRSA was approximately 36%, and more than 10% of all S. aureus isolates were class 1 MRSA, as in most French hospitals. Nevertheless, class 1 MRSA may be misdiagnosed as MSSA with the usual tests, and the prevalence of their susceptibility to other antibiotics (benzylpenicillin [20% in the consecutive isolates], fosfomycin, rifampin, gentamicin, and erythromycin) is misleading. Community-acquired MRSA isolates from skin and soft tissue infections are usually susceptible to various antibiotics (10, 12). Many antibiotics, and even penicillins with good affinity for PBP2a, such as amoxicillin and benzylpenicillin, may be effective against ß-lactamase-negative class 1 MRSA isolates (8). Regardless of the susceptibilities of these isolates to many classes of antibiotics, four recent cases of pediatric invasive community-acquired sepsis with very-low-level MRSA were fatal (6). In our institution, two-thirds of the S. aureus isolates originated from skin specimens, and 13% of these were class 1 MRSA. MSSA isolates from skin lesions probably acquired the mecA gene by horizontal transfer from other skin staphylococcal species before becoming class 1 to 4 in turn (14, 25). The index cases of heterogeneous MRSA formed a cluster of nosocomial skin infections in the plastic surgery department following reconstructive surgery after carcinoma removal. They belonged to three clones, which differed according to their MRSA classes (1, 2, and 3, respectively), their phage types, their antibiotypes, and their DNA profiles as determined by pulsed-field gel electrophoresis (data not shown).

In conclusion, the cefoxitin and moxalactam disk diffusion method was very suitable for detection of MRSA, particularly class 1 isolates. The MRSA-screen test identified all class 1 isolates. The Vitek 2 test (Oxascreen and oxacillin MIC) presented no benefit over the oxacillin MIC test alone. With some adaptation, these tests would also improve the detection of methicillin-resistant, coagulase-negative staphylococci [A. Felten, B. Grandry, P. H. Lagrange, and I. Casin, abstract from the 11th European Congress of Clinical Microbiology and Infectious Diseases 2001, Clin. Microbiol. Infect. 7(Suppl. 1):13, abstr. O-100, 2001].


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Service de Microbiologie, Hôpital Saint-Louis, 1 avenue Vellefaux, 75475 Paris Cedex 10, France. Phone: 33 1 42 49 93 48. Fax: 33 1 42 49 92 00. E-mail: annie.felten{at}sls.ap-hop-paris.fr. Back


arrow
REFERENCES
 
    1
  1. Astagneau, P., and The French Prevalence Survey Study Group. 2000. Prevalence of nosocomial infections in France: results of the nationwide survey in 1996. J. Hosp. Infect. 46:186-193.[CrossRef][Medline]
    2. 2
  2. Ayliffe, G. A. J. 1997. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clin. Infect. Dis. 24(Suppl. 1):S74-S79.
    3. 3
  3. Barbier Frebourg, N., D. Nouet, L. Lemée, E. Martin, and J. F. Lemeland. 1998. Comparison of ATB staph, rapid ATB staph, Vitek and E-test for detection of oxacillin heteroresistance in staphylococci possessing mecA. J. Clin. Microbiol. 36:52-57.[Abstract/Free Full Text]
    4. 4
  4. Blanc, D. S., C. Petignat, P. Moreillon, J. M. Entenza, M.-C. Eisenring, H. Kleiber, A. Wenger, N. Troillet, C.-H. Blanc, and R. Francioli. 1999. Unusual spread of a penicillin-susceptible methicillin-resistant Staphylococcus aureus clone in a geographic area of low incidence. Clin. Infect. Dis. 29:1512-1518.[CrossRef][Medline]
    5. 5
  5. Cavassini, M., A. Wenger, K. Jaton, D. S. Blanc, and J. Bille. 1999. Evaluation of MRSA-screen, a simple anti-PBP2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus. J. Clin. Microbiol. 37:1591-1594.[Abstract/Free Full Text]
    6. 6
  6. Centers for Disease Control and Prevention. 1999. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus—Minnesota and North Dakota, 1997-1999. JAMA 282:1123-1125.[Free Full Text]
    7. 7
  7. Chambers, H. F. 1997. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin. Microbiol. Rev. 10:781-791.[Abstract]
    8. 8
  8. Franciolli, M., J. Bille, M. P. Glauser, and P. Moreillon. 1991. ß-Lactam resistance mechanisms of methicillin resistant Staphylococcus aureus. J. Infect. Dis. 163:514-523.[Medline]
    9. 9
  9. Galbart, J. O., A. Morvan, and N. el Sohl. 2000. Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in France from 1995 to 1997. J. Clin. Microbiol. 38:185-190.[Abstract/Free Full Text]
    10. 10
  10. Gorak, E. J., S. M. Yamada, and J. D. Brown. 1999. Community-acquired methicillin-resistant Staphylococcus aureus in hospitalized adults and children without known risk factors. Clin. Infect. Dis. 29:797-800.[Medline]
    11. 11
  11. Henze, U. U., and B. Berger-Bächi. 1996. Penicillin-binding protein 4 overproduction increases ß-lactam resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 40:2121-2125.[Abstract]
    12. 12
  12. Herold, B. C., L. C. Immergluck, M. C. Maranan, D. S. Lauderdale, R. E. Gaskin, S. Boyle-Vavra, C. D. Leitch, and R. S. Daum. 1998. Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 279:593-598.[Abstract/Free Full Text]
    13. 13
  13. Higashi, Y., A. Wakabayashi, Y. Matsumoto, Y. Watanabe, and A. Ohno. 1999. Role of inhibition of penicillin binding proteins and cell wall cross-linking by beta-lactam antibiotics in low- and high-level methicillin resistance of Staphylococcus aureus. Chemotherapy 45:37-47.[CrossRef][Medline]
    14. 14
  14. Hiramatsu, K. 1995. Molecular evolution of MRSA. Microbiol. Immunol. 39:531-543.[Medline]
    15. 15
  15. Kobayashi, N., K. Taniguchi, K. Kojima, S. Urusawa, N. Uehara, Y. Omizu, Y. Kishi, A. Yagihashi, I. Kurokawa, and N. Watanabe. 1996. Genomic diversity of mec regulator genes in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Epidemiol. Infect. 117:289-295.[Medline]
    16. 16
  16. Louie, L., S. O. Matsumura, E. Choi, M. Louie, and A. E. Simor. 2000. Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus. J. Clin. Microbiol. 38:2170-2173.[Abstract/Free Full Text]
    17. 17
  17. Mackenzie, A. M. R., H. Richardson, R. Lannigan, and D. Wood. 1995. Evidence that the National Committee for Clinical Laboratory Standards disk test is less sensitive than the screen plate for detection of low-expression-class methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 33:1909-1911.[Abstract]
    18. 18
  18. Marriott, D. J. E., T. Karagiannis, J. H. Harkness, and P. Kearney. 1999. Further evaluation of the MRSA-screen kit for rapid detection of methicillin resistance. J. Clin. Microbiol. 37:3783-3784.[Free Full Text]
    19. 19
  19. Moriyasu, I., J. Igari, N. Yamane, T. Oguri, A. Takahashi, M. Tosaka, K. Takemori, S. Toyoshima, and W. Minamide. 1994. Multicenter evaluation of Showa ceftizoxime disk susceptibility test to discriminate between the strains of methicillin-resistant Staphylococcus aureus (MRSA) and those susceptible (MSSA). Rinsho Byori 42:271-277.[Medline]
    20. 20
  20. Murakami, K., K. Nomura, M. Doi, and T. Yoshida. 1987. Increased susceptibility to cephamycin-type antibiotics of methicillin-resistant Staphylococcus aureus defective in penicillin-binding protein 2. Antimicrob. Agents Chemother. 31:1423-1425.[Abstract/Free Full Text]
    21. 21
  21. Okonogi, K., Y. Nogi, M. Kondo, A. Imada, and T. Yokota. 1989. Emergence of methicillin-resistant clones from cephamycin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 24:637-645.[Abstract/Free Full Text]
    22. 22
  22. Predari, S. C., M. Ligozzi, and R. Fontana. 1991. Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction. Antimicrob. Agents Chemother. 35:2568-2573.[Abstract/Free Full Text]
    23. 23
  23. Sa-Leao, R., I. Santos Sanches, D. Dias, I. Peres, R. M. Barros, and H. de Lencastre. 1999. Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain? J. Clin. Microbiol. 37:1913-1920.[Abstract/Free Full Text]
    24. 24
  24. Soussy, C. J., G. Carret, J. D. Cavallo, H. Chardon, C. Chidiac, P. Choutet, P. Courvalin, H. Dabernat, H. Drugeon, L. Dubreuil, F. Goldstein, V. Jarlier, R. Leclercq, M. H. Nicolas-Chanoine, A. Philippon, C. Quentin, B. Rouveix, and J. Sirot. 2000. Comité de l'Antibiogramme de la Société Française de Microbiologie. Communiqué 2000-2001. Pathol. Biol. 48:832-871.
    25. 25
  25. Tomasz, A., S. Nachman, and H. Leaf. 1991. Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci. Antimicrob. Agents Chemother. 35:124-129.[Abstract/Free Full Text]
    26. 26
  26. van Griethuysen, A., M. Pouw, N. van Leeuwen, M. Heck, P. Willemse, A. Buiting, and J. Kluytmans. 1999. Rapid slide latex agglutination test for detection of methicillin resistance in Staphylococcus aureus. J. Clin. Microbiol. 37:2789-2792.[Abstract/Free Full Text]
    27. 27
  27. Yamazumi, T., S. A. Marshall, W. W. Wilke, D. J. Diekema, M. A. Pfaller, and R. N. Jones. 2001. Comparison of Vitek gram-positive susceptibility 106 card and the MRSA-screen latex agglutination test for determining oxacillin resistance in clinical bloodstream isolates of Staphylococcus aureus. J. Clin. Microbiol. 39:53-56.[Abstract/Free Full Text]

Journal of Clinical Microbiology, August 2002, p. 2766-2771, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.2766-2771.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Swenson, J. M., Brasso, W. B., Ferraro, M. J., Hardy, D. J., Knapp, C. C., Lonsway, D., McAllister, S., Reller, L. B., Sader, H. S., Shortridge, D., Skov, R., Weinstein, M. P., Zimmer, B. L., Patel, J. B. (2009). Correlation of Cefoxitin MICs with the Presence of mecA in Staphylococcus spp.. J. Clin. Microbiol. 47: 1902-1905 [Abstract] [Full Text]  
  • Skov, R., Smyth, R., Yusof, A., Karlsson, A., Mills, K., Frimodt-Moller, N., Kahlmeter, G. (2009). Effects of temperature on the detection of methicillin resistance in Staphylococcus aureus using cefoxitin disc diffusion testing with Iso-Sensitest agar. J Antimicrob Chemother 63: 699-703 [Abstract] [Full Text]  
  • John, M. A., Burden, J., Stuart, J. I., Reyes, R. C., Lannigan, R., Milburn, S., Diagre, D., Wilson, B., Hussain, Z. (2009). Comparison of three phenotypic techniques for detection of methicillin resistance in Staphylococcus spp. reveals a species-dependent performance. J Antimicrob Chemother 63: 493-496 [Abstract] [Full Text]  
  • Baldoni, D., Hermann, H., Frei, R., Trampuz, A., Steinhuber, A. (2009). Performance of Microcalorimetry for Early Detection of Methicillin Resistance in Clinical Isolates of Staphylococcus aureus. J. Clin. Microbiol. 47: 774-776 [Abstract] [Full Text]  
  • Broekema, N. M., Van, T. T., Monson, T. A., Marshall, S. A., Warshauer, D. M. (2009). Comparison of Cefoxitin and Oxacillin Disk Diffusion Methods for Detection of mecA-Mediated Resistance in Staphylococcus aureus in a Large-Scale Study. J. Clin. Microbiol. 47: 217-219 [Abstract] [Full Text]  
  • Roisin, S., Nonhoff, C., Denis, O., Struelens, M. J. (2008). Evaluation of New Vitek 2 Card and Disk Diffusion Method for Determining Susceptibility of Staphylococcus aureus to Oxacillin. J. Clin. Microbiol. 46: 2525-2528 [Abstract] [Full Text]  
  • Jain, A., Agarwal, A., Verma, R. K. (2008). Cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. J Med Microbiol 57: 957-961 [Abstract] [Full Text]  
  • von Ah, U., Wirz, D., Daniels, A. U. (2008). Rapid Differentiation of Methicillin-Susceptible Staphylococcus aureus from Methicillin-Resistant S. aureus and MIC Determinations by Isothermal Microcalorimetry. J. Clin. Microbiol. 46: 2083-2087 [Abstract] [Full Text]  
  • Sauer, P., Sila, J., Stosova, T., Vecerova, R., Hejnar, P., Vagnerova, I., Kolar, M., Raclavsky, V., Petrzelova, J., Loveckova, Y., Koukalova, D. (2008). Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic. J Med Microbiol 57: 403-410 [Abstract] [Full Text]  
  • Cherkaoui, A., Renzi, G., Francois, P., Schrenzel, J. (2007). Comparison of four chromogenic media for culture-based screening of meticillin-resistant Staphylococcus aureus. J Med Microbiol 56: 500-503 [Abstract] [Full Text]  
  • Skov, R., Smyth, R., Larsen, A. R., Bolmstrom, A., Karlsson, A., Mills, K., Frimodt-Moller, N., Kahlmeter, G. (2006). Phenotypic Detection of Methicillin Resistance in Staphylococcus aureus by Disk Diffusion Testing and Etest on Mueller-Hinton Agar. J. Clin. Microbiol. 44: 4395-4399 [Abstract] [Full Text]  
  • Perazzi, B., Fermepin, M. R., Malimovka, A., Garcia, S. D., Orgambide, M., Vay, C. A., de Torres, R., Famiglietti, A. M. R. (2006). Accuracy of cefoxitin disk testing for characterization of oxacillin resistance mediated by penicillin-binding protein 2a in coagulase-negative staphylococci.. J. Clin. Microbiol. 44: 3634-3639 [Abstract] [Full Text]  
  • Stepanovic, S., Hauschild, T., Dakic, I., Al-Doori, Z., Svabic-Vlahovic, M., Ranin, L., Morrison, D. (2006). Evaluation of Phenotypic and Molecular Methods for Detection of Oxacillin Resistance in Members of the Staphylococcus sciuri Group.. J. Clin. Microbiol. 44: 934-937 [Abstract] [Full Text]  
  • Fang, H., Hedin, G. (2006). Use of Cefoxitin-Based Selective Broth for Improved Detection of Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 44: 592-594 [Abstract] [Full Text]  
  • Stoakes, L., Reyes, R., Daniel, J., Lennox, G., John, M. A., Lannigan, R., Hussain, Z. (2006). Prospective Comparison of a New Chromogenic Medium, MRSASelect, to CHROMagar MRSA and Mannitol-Salt Medium Supplemented with Oxacillin or Cefoxitin for Detection of Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 44: 637-639 [Abstract] [Full Text]  
  • Brown, D. F. J., Edwards, D. I., Hawkey, P. M., Morrison, D., Ridgway, G. L., Towner, K. J., Wren, M. W. D., on behalf of the Joint Working Party of the Britis, (2005). Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrob Chemother 56: 1000-1018 [Abstract] [Full Text]  
  • Smyth, R. W., Kahlmeter, G. (2005). Mannitol Salt Agar-Cefoxitin Combination as a Screening Medium for Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 43: 3797-3799 [Abstract] [Full Text]  
  • Swenson, J. M., Tenover, F. C., the Cefoxitin Disk Study Group, (2005). Results of Disk Diffusion Testing with Cefoxitin Correlate with Presence of mecA in Staphylococcus spp.. J. Clin. Microbiol. 43: 3818-3823 [Abstract] [Full Text]  
  • Miller, M. B., Meyer, H., Rogers, E., Gilligan, P. H. (2005). Comparison of Conventional Susceptibility Testing, Penicillin-Binding Protein 2a Latex Agglutination Testing, and mecA Real-Time PCR for Detection of Oxacillin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococcus. J. Clin. Microbiol. 43: 3450-3452 [Abstract] [Full Text]  
  • Frigatto, E. A. M., Machado, A. M. O., Pignatari, A. C. C., Gales, A. C. (2005). Is the Cefoxitin Disk Test Reliable Enough To Detect Oxacillin Resistance in Coagulase-Negative Staphylococci?. J. Clin. Microbiol. 43: 2028-2029 [Full Text]  
  • Fernandes, C. J., Fernandes, L. A., Collignon, P., on behalf of the Australian Group on Antimicrobial, (2005). Cefoxitin resistance as a surrogate marker for the detection of methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 55: 506-510 [Abstract] [Full Text]  
  • Velasco, D., del Mar Tomas, M., Cartelle, M., Beceiro, A., Perez, A., Molina, F., Moure, R., Villanueva, R., Bou, G. (2005). Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus. J Antimicrob Chemother 55: 379-382 [Abstract] [Full Text]  
  • Skov, R., Smyth, R., Larsen, A. R., Frimodt-Moller, N., Kahlmeter, G. (2005). Evaluation of cefoxitin 5 and 10 {micro}g discs for the detection of methicillin resistance in staphylococci. J Antimicrob Chemother 55: 157-161 [Abstract] [Full Text]  
  • Perry, J. D., Davies, A., Butterworth, L. A., Hopley, A. L. J., Nicholson, A., Gould, F. K. (2004). Development and Evaluation of a Chromogenic Agar Medium for Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 42: 4519-4523 [Abstract] [Full Text]  
  • Darini, A. L. d. C., Palazzo, I. C. V., Felten, A. (2004). Cefoxitin Does Not Induce Production of Penicillin Binding Protein 2a in Methicillin-Susceptible Staphylococcus aureus Strains. J. Clin. Microbiol. 42: 4412-4413 [Full Text]  
  • Donay, J.-L., Mathieu, D., Fernandes, P., Pregermain, C., Bruel, P., Wargnier, A., Casin, I., Weill, F. X., Lagrange, P. H., Herrmann, J. L. (2004). Evaluation of the Automated Phoenix System for Potential Routine Use in the Clinical Microbiology Laboratory. J. Clin. Microbiol. 42: 1542-1546 [Abstract] [Full Text]  
  • Skov, R., Smyth, R., Clausen, M., Larsen, A. R., Frimodt-Moller, N., Olsson-Liljequist, B., Kahlmeter, G. (2003). Evaluation of a cefoxitin 30 {micro}g disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 52: 204-207 [Abstract] [Full Text]  
  • Poulsen, A. B., Skov, R., Pallesen, L. (2003). Detection of Low-Level Methicillin-Resistant Staphylococcus aureus with Commercially Available Tests. J. Clin. Microbiol. 41: 3458-3458 [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Felten, A.
Right arrow Articles by Casin, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Felten, A.
Right arrow Articles by Casin, I.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»