Cefoxitin Disk Diffusion Test Research Articles

Detection of methicillin resistance in Staphylococcus aureus isolated from pediatric patients: is the cefoxitin disk diffusion test accurate enough?

We evaluated the performance of several methods for the detection of methicillin resistance in Staphylococcus aureus using 101 clinical S. aureus isolates from pediatric patients in a tertiary hospital in Brazil; 50 isolates were mecA-positive and 51 were mecA-negative. The Etest and oxacillin agar screening plates were 100% sensitive and specific for mecA presence. Oxacillin and cefoxitin disks gave sensitivities of 96 and 92%, respectively, and 98% specificity. Alterations of CLSI cefoxitin breakpoints increased sensitivity to 98%, without decreasing specificity. Our results highlight the importance of a continuing evaluation of the recommended microbiological methods by different laboratories and in different settings. If necessary, laboratories should use a second test before reporting a strain as susceptible, especially when testing strains isolated from invasive or serious infections. With the new (2007) CLSI breakpoints, the cefoxitin-disk test appears to be a good option for the detection of methicillin resistance in S. aureus.

Evaluation of methicillin resistance by cefoxitin disk diffusion and PBP2a latex agglutination test in mecA-positive Staphylococcus aureus, and comparison of mecA with femA, femB, femX positivities

Methicillin resistance in staphylococci is primarily due to the presence of a mecA gene which encodes the novel penicillin binding protein2a. Some chromosomal factors such as femA and femB also participate in the expression of methicillin resistance. This study was designed to detect methicillin resistance by cefoxitin disk diffusion and penicillin binding protein2a latex agglutination methods, and to compare mecA, femA, femB and femX gene positivities. A total of 60 MRSA isolates were included in the evaluation. PCR analysis showed that all isolates were positive for mecA and femA genes. Seven of these isolates tested negative by the latex agglutination test. Fifteen isolates were positive for femB and 28 isolates for femX gene. This study implicated that for the determination of methicillin resistance, latex agglutination test is the least reliable method when compared to PCR and cefoxitin disk diffusion test. femA gene shows more correlation than femB and femX with methicillin resistance.

P1607 Evaluation of a direct cefoxitin disc diffusion test for the presumptive identification of MRSA from clinical specimens

P1607 Evaluation of a direct cefoxitin disc diffusion test for the presumptive identification of MRSA from clinical specimens

The cefoxitin disk test – what a clinical microbiologist needs to know

Accurate prediction of mecA- mediated resistance in Staphylococcus spp. has been a challenge for the clinical microbiologist ever since the discovery of methicillin resistance. In 2004, a cefoxitin disk diffusion test was recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) for prediction of mecA- mediated resistance in both Staphylococcus aureus and coagulase-negative staphylococci because of its high sensitivity and specificity compared to PCR tests for mecA. It is also much easier to read that the oxacillin disk diffusion test, not requiring close scrutiny of the zone of inhibition or 24 h of incubation. The test is used as a surrogate for oxacillin testing. In other words, when cefoxitin is tested, the results are applied to oxacillin and oxacillin is reported. For resistant strains, the results are applied not only to oxacillin, but also to all other β-lactam agents, which must also be reported as resistant if they are reported. There has been some confusion among clinical microbiologists about when and how the test should be used. We answer some of the questions that have been raised about the test.

Evaluation of different methods to detect oxacillin resistance in Staphylococcus aureus and their clinical laboratory utility

Methicillin-resistant Staphylococcus aureus strains (MRSA) are major human pathogens responsible for nosocomial and serious community-acquired infections worldwide. Methicillin resistance in S. aureus is primarily mediated by a modified penicillin-binding protein (PBP2a) encoded by the mecA gene, which is not present in susceptible strains [1–3]. Several additional gene products, such as those encoded by fem (factor essential for methicillin resistance) genes and auxiliary genes, can also contribute to MRSA resistance [1, 2, 4]. Rapid and accurate identification of MRSA is necessary for therapeutic and epidemiological purposes. Currently, several methods are available for detecting oxacillin resistance including phenotypic methods, such as disk diffusion, E-test and broth dilution tests, and genotypic methods that detect the mecA gene [1, 5, 6]. PCR amplification of the mecA gene has been used successfully and is presently considered the gold standard for detecting methicillin resistance in S. aureus [1]. Detection of oxacillin resistance using phenotypic methods is problematic because of variation in the degree of mecA gene expression in heterogeneous populations of MRSA; thus, routine oxacillin disk diffusion tests often fail to detect heterogeneous MRSA populations. The Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) has consequently recommended use of the cefoxitin disk diffusion test rather than the oxacillin disk diffusion test [7]. In the study presented here, the performance and utility of several different methods for detecting MRSA was evaluated. PCR for detecting the mecA gene was used as the gold standard assay against which we compared oxacillin and cefoxitin disk diffusion tests and the GenoType MRSA molecular genetic assay (Hain Lifescience, Nehren, Germany) for detecting MRSA. The objective was to determine whether these disk diffusion tests and GenoType MRSA could detect all MRSA as accurately as mecA PCR and to evaluate their suitability as routine methods for detecting oxacillin resistance in S. aureus in clinical microbiology laboratories. GenoType MRSA is a new DNA-based strip assay kit designed to detect oxacillin resistance in S. aureus. Its performance is based on amplification of a 71 bp fragment of the mecA gene and an 85 bp sequence highly specific for S. aureus and subsequent hybridization of the denatured amplicons to their complementary sequences coated on DNA strips. A total of 224 isolates of S. aureus collected from patients of Suleyman Demirel University Hospital were included in this study. The isolates were collected from different clinical specimens and at different times. All strains were identified using biochemical procedures. Reference strains included ATCC33591 (MRSA) and ATCC29213 (MSSA). Disk diffusion tests were performed for each of the 224 isolates using the following method recommended by the National Committee for Clinical Laboratory Standards [7]: 1 μg oxacillin disks (Oxoid, Basingstoke, UK) were incubated at 35°C and at 30°C on Mueller-Hinton agar (Difco, Detroit, MI, USA) with and without 2% NaCl, and 30 μg cefoxitin disks (Oxoid) were incubated at 35°C on Mueller-Hinton agar. After incubation, inhibition zone sizes for oxacillin and cefoxitin were noted and compared. For cefoxitin, isolates showing a zone of inhibition of ≤19 mm were considered Eur J Clin Microbiol Infect Dis (2006) 25:410–412 DOI 10.1007/s10096-006-0153-8

Results of Disk Diffusion Testing with Cefoxitin Correlate with Presence of mecA in Staphylococcus spp

The cefoxitin disk diffusion (DD) test for predicting mecA-mediated oxacillin resistance in staphylococci was assessed during a three-phase study. In phase 1, one laboratory tested 62 and 53 strains of Staphylococcus aureus and coagulase-negative staphylococci (CoNS), respectively. These data were used to choose the provisional cefoxitin DD breakpoints (resistant/susceptible) of < or =19 mm/> or =20 mm for S. aureus and < or =24 mm/> or =25 mm for CoNS for the next phase of testing. In phase 2, 10 laboratories each tested approximately 40 in-house strains of staphylococci (half of which were S. aureus) using Mueller-Hinton agar from different manufacturers. In this phase, the sensitivity and specificity, respectively, of the cefoxitin disk test were 98 and 100% for S. aureus and 99 and 96% for CoNS. The cefoxitin DD test performed equivalently to oxacillin broth microdilution (BMD) and to oxacillin DD tests among S. aureus and mecA-positive CoNS strains but gave better results than oxacillin BMD or oxacillin DD for mecA-negative strains of CoNS. The cefoxitin DD test also was much easier to read and did not require the use of transmitted light for detection of resistance. Based on data from the first two phases, the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) adopted the use of the cefoxitin DD test for predicting mecA-mediated oxacillin resistance in staphylococci and revised Table 2C in CLSI document M100-S14 to reflect the change. In the third phase, an additional 61 challenge strains of CoNS for which the oxacillin MICs were 0.5 to 2 microg/ml were tested in a single laboratory to determine the effectiveness of the cefoxitin DD test for this group of borderline-resistant isolates. These data were used to refine the description of the test in CLSI document M100-S15. The cefoxitin DD test is preferred over the oxacillin DD test for predicting mecA-mediated oxacillin resistance in S. aureus and CoNS.

Evaluation of a cefoxitin disk diffusion test for the routine detection of methicillin-resistant Staphylococcus aureus

Two oxacillin disk methods were compared with a cefoxitin disk diffusion test for detection of methicillin-resistant Staphylococcus aureus (MRSA), with PCR for mecA as the reference method. When tested with 115 MRSA and 350 methicillin-susceptible S. aureus isolates, the cefoxitin disk test (specificity 100%, sensitivity 96.5%) was superior to the oxacillin disk methods (specificity 99.1%, sensitivity 90.4%). Testing with both oxacillin and cefoxitin disks would give better sensitivity (100%) than the cefoxitin test alone, but at the expense of specificity (99.1%). The cefoxitin disk test required no special test conditions and would improve the reliability of routine tests for detection of MRSA.

Détection simple des staphylocoques résistants à la méticilline grâce à un disque de céfoxitine ou de latamoxef

Very-low level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We aimed at detecting the low-level MRSA by the disk diffusion method on a NaCl-free Mueller-Hinton agar plate flooded with 10 6 UFC/ml bacterial inoculum using the cephamycin antibiotics cefoxitin and latamoxef for 18 hours' incubation at 37°C. Detection of the mecA gene by PCR was the ⪡gold standard⪢. The cefoxitin and latamoxef disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm and latamoxef inhibition zone diameters <24 mm. Consequently, we evaluated the cefoxitin and latamoxef disk diffusion method to detect very low-level coagulase-negative staphylococci (MRCNS) or class 1 MRCNS. The cefoxitin and latamoxef disk diffusion detected 94% and 97% of all MRCNS respectively. Cefoxitin inhibition diameters were <27 mm and latamoxef diameters <22 mm. We studied the Slidex MRSA detection® (bioMérieux) to test the PLP2a in all S. aureus and coagulase negative staphylococci included in our trials with cephamycins. This test detected 98% MRSA, and 98% MRCNS so long as the CNS sample was grown on the edge of an oxacillin or a cephamycin disk. Therefore, we recommend the use of latamoxef disk diffusion method for routine detection of all classes of MRSA and MRCNS, and the subsequent PLP2a detection in cases of inhibition zone diameters in the range of 22–24 mm. Taken together, both tests allow one to detect all class 1 methicillin-resistant staphylococci within 24 hours.