Evaluation of methods for detecting oxacillin resistance in coagulase-negative staphylococci including cefoxitin disc di usion

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1 Evaluation of methods for detecting oxacillin resistance in coagulase-negative staphylococci including cefoxitin disc di usion Izabel Cristina V. Palazzo 1,2 & Ana Lúcia C. Darini 1 1 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil; and 2 Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP, Brazil Correspondence: Ana Lúcia C. Darini, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Via do Café, s/n1, Ribeirão Preto, SP, Brazil. Tel.: ; fax: ; aldarini@fcfrp.usp.br Received 26 August 2005, revised 31 January 2006, accepted 1 February First published online 7 March doi: /j x Editor: Stefan Schwarz Abstract The aim of this study was to evaluate the accuracy of cefoxitin disc diffusion as a prediction of oxacillin resistance in coagulase-negative staphylococci (CoNS), and also to compare genotypic and phenotypic methods for detecting this resistance property. A total of 151 clinical CoNS isolates were tested by PCR for the presence of the meca gene (gold standard method). The isolate susceptibilities were determined by the disc diffusion method with oxacillin (1 mg) and cefoxitin (30 mg) and by the agar dilution method for cefoxitin and oxacillin. Although none of the techniques showed 100% sensitivity and 100% specificity, the cefoxitin disc diffusion and oxacillin agar dilution were the best methods for detecting resistance to oxacillin among CoNS as these methods produced the best negative and positive predictive values. A combination of methods can be used routinely to identify resistance to oxacillin in CoNS. Keywords staphylococci; antibiotic resistance; meca gene; methicillin; cefoxitin. Introduction Coagulase-negative staphylococci (CoNS) are the major cause of bacteraemia in hospitalized patients and infections related to prostheses (Rupp & Archer, 1994). A substantial increase in the frequency of oxacillin resistance in CoNS isolates has occurred over the last few decades. Currently, more than 70% of the CoNS isolates worldwide are resistant to methicillin or oxacillin. In addition, those CoNS strains acquired in hospitals have become resistant to various other antimicrobial agents (Diekema et al., 2001). Few clinical isolates of methicillin-resistant CoNS (MRCoNS) express homogeneous methicillin resistance (i.e. 1in10 2 cells express high-level resistance): the majority of isolates have heterogeneous drug resistance (heteroresistance) under routine growth conditions (Sakoulas et al., 2001). Phenotypic expression of resistance can vary depending on the growth conditions (e.g. temperature, osmolarity, and culture medium supplements such as NaCl or sucrose) (Tenover et al., 1999; Sakoulas et al., 2001; Ferreira et al., 2003), making susceptibility testing of MRCoNS by standard microbiological methods potentially problematic, despite the guidelines published by the Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards). Methicillin-resistant CoNS become resistant by acquisition of the PBP2a-encoding gene, meca, which is a chromosomally derived gene, not found in susceptible strains of staphylococci. The PBP2a produced has a low affinity for b-lactam antibiotics, and is thought to function in their presence to confer resistance to the bacteria. Additionally, the meca gene is highly conserved in staphylococcal strains and thus it is a useful molecular marker of methicillin/ oxacillin resistance (Ferreira et al., 2003). Studies focusing on methods for phenotypic detection of oxacillin susceptibility in CoNS isolates are necessary, as few laboratories have the technical and/or economic capabilities to apply molecular methods to detect oxacillin resistance in staphylococci. Thus, the aim of this study was to evaluate the accuracy of phenotypic methods to detect resistance in CoNS clinical isolates including the cefoxitin disc diffusion (DD) method, and compare the results with the presence of the meca gene detected by PCR.

2 300 I.C.V. Palazzo & A.L.C. Darini Materials and methods Bacterial isolates and species identification The CoNS isolates (n = 151) analysed in our study were randomly collected from March 1995 to August 2004 from patients admitted to the University Hospital, Faculty of Medicine, Ribeirão Preto, University of Sao Paulo, Brazil. The hospital, also a district general hospital comprising 551 beds, offers medical assistance in accidents and emergencies, in maternity and high-risk baby care units and in acute medical and surgical cases. The bacterial isolates recovered from blood cultures, intravascular catheters, surgical wounds, and cerebrospinal and peritoneal fluids were clinically significant. Only one isolate per patient was analysed. Coagulase-positive and coagulase-negative staphylococci were differentiated by free coagulase tests and slide tests for bound coagulase (Difco Laboratories, Detroit, MI). CoNS were identified up to the species level by the API20 Staph and ID32 Staph ATB test (biomérieux, Marcy-l Étoile, France) and complemented by the following additional tests when necessary: [pyrrolidonyl arylamidase (PYR), Difco Laboratories] oxidase and resistance to polymyxin B and novobiocin (Difco Laboratories). In some cases, the strains were also identified through an automated Microscan System (Dade Behring, Deerfield, IL). Control strains The quality control for species identification was performed using the following standard CoNS strains (American Type Culture Collection, ATCC, Rockville, MD): Staphylococcus epidermidis ATCC 14990, Staphylococcus hominis ATCC 27844, Staphylococcus haemolyticus ATCC 29970, Staphylococcus caprae ATCC 35538, Staphylococcus chromogenes ATCC 43764, Staphylococcus warneri ATCC 27836, Staphylococcus sciuri ATCC 29062, Staphylococcus lugdunensis ATCC 43809, and Staphylococcus capitis ATCC Antimicrobial susceptibility testing The susceptibilities to oxacillin and cefoxitin were determined by the DD method on Mueller Hinton agar (Oxoid, Basingstoke, UK) plates using bacterial suspensions whose turbidity was adjusted to a 0.5 McFarland standard (Densimat, Rome, Italy), after incubation at 35 1C for 24 h and 18 h for oxacillin and cefoxitin, respectively. The results were interpreted according to CLSI (2005) guidelines. Cefoxitin disc (30 mg, Oxoid) diffusion was performed to evaluate its performance in predicting oxacillin resistance. CLSI (2005) criteria were used for cefoxitin susceptibility testing of CoNS isolates, that is, 24 mm resistant, and Z25 mm susceptible, except for S. lugdunensis, which was evaluated by the same criteria proposed by CLSI for Staphylococcus aureus (Z20 mm susceptible, and 19 mm resistant). Staphylococcus aureus ATCC was used as control. The minimum inhibitory concentrations (MICs) for oxacillin and cefoxitin were determined by the agar dilution (AD) method, according to CLSI guidelines. Briefly, for each strain, colonies isolated from an overnight growth were transferred to sterile saline; the suspensions were adjusted to a 0.5 McFarland standard (10 8 CFU ml 1 ), and inoculated on Mueller Hinton agar plates containing 0, , 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 or 256 mg of cefoxitin ml 1 using a replicator. The oxacillin Mueller Hinton agar plates were supplemented with 2% NaCl and incubated at 35 1C for 24 h. Detection of the meca gene The meca gene was detected by PCR with specific primers meca1 (5 0 -CTCAGGTACTGCTATCCACC-3 0 ) and meca2 (5 0 -CACTTGGTATATCTTCACC-3 0 ) (Invitrogen Life Technologies, Carlsbad, CA). Staphylococcal DNA was extracted after cell lysis in the presence of lysozyme (100 mg ml 1 ) and lysostaphin (1 mg ml 1 ), using standard protocols (Bignardi et al., 1996). Subsequently, 2 ml of the bacterial DNA was added to the PCR tube, followed by a 25 ml mixture containing 0.2 mm of each deoxynucleoside triphosphate (Eppendorf, Wetsbury, NY), U of Taq polymerase (Invitrogen Life Technologies), 1 PCR buffer (20 mm Tris-HCl, 50 mm KCl), 2 mm MgCl 2, and 50 ng ml 1 of each primer. PCR was performed under the following conditions: initial denaturation for 5 min at 94 1C, followed by 30 cycles of 60 s at 94 1C, 30 s at 52 1C, and 30 s at 72 1C, and a final extension step at 72 1C for 10 min. A positive result was indicated by the presence of a 490 bp amplified DNA fragment, which was revealed after electrophoresis on a 1% agarose gel by staining with ethidium bromide and UV light. The strain S. aureus N315 (meca positive) was used as control. Results and discussion The most frequent species among the 151 CoNS isolated samples were Staphylococcus epidermidis (99 isolates; 65.6%), followed by Staphylococcus haemolyticus (18 isolates; 11.9%), Staphylococcus hominis (17 isolates; 11.2%), Staphylococcus capitis (five isolates; 3.3%), Staphylococcus warneri (four isolates; 2.6%), Staphylococcus caprae (two isolates; 1.3%) and Staphylococcus lugdunensis (two isolates; 1.3%). There was only one isolate of Staphylococcus chromogenes, Staphylococcus sciuri, Staphylococcus gallinarum, and Staphylococcus auricularis. The majority of the CoNS samples were isolated from blood (108 isolates; 71.5%), followed by a catheter (23 isolates; 15.2%). The CoNS isolated from liquor, peritoneal fluid, and surgery wounds represented 13.3% of all isolates.

3 Detecting oxacillin resistance in coagulase negative staphylococci 301 Table 1. Genotypic and phenotypic tests for the characterisation of 151 clinical isolates of coagulase-negative staphylococci Susceptibility methods w Agar dilution Disk diffusion Species Number of strains meca gene OXA FOX OXA FOX Staphylococcus epidermidis 48 S S S S 35 1 R R R R 09 1 R S R R 04 1 S S S S 01 1 R S S S 01 S S R S 01 R S S S Staphylococcus haemolyticus 14 1 R R R R 03 1 R S R R 01 S S S S Staphylococcus hominis 09 1 R R R R 07 S S S S 01 1 S S S R Staphylococcus caprae 01 S S S S 01 1 R S R S Staphylococcus chromogenes 01 1 R R R R Staphylococcus warneri 03 S S S S 01 R S R R Staphylococcus capitis 05 S S S S Staphylococcus sciuri 01 S S R S Staphylococcus lugdunensis 01 1 R S R R 01 S S S S Staphylococcus gallinarum 01 1 R R R R Staphylococcus auricularis 01 S S S S meca gene was detected by PCR; 1, gene detected;, gene not detected. w The interpretative criteria used in susceptibility methods were defined in Materials and methods; R, resistant; S, susceptible; OXA, oxacillin; FOX, cefoxitin. Total number of isolates tested: 151. Table 1 shows the results for susceptibility to antimicrobial agents of the 151 CoNS isolates, as determined by the genotypic method (PCR) and phenotypic methods such as AD and DD for oxacillin, and cefoxitin. Fifty-three per cent of the isolates were considered oxacillin resistant based on the presence of the meca gene detected by PCR. Oxacillin resistance predicted by results obtained from phenotypic methods with cefoxitin were 50% for DD and 39.7% for AD. Table 1 also shows the discrepant results among phenotypic and genotypic methods. One S. hominis that harboured the meca gene showed susceptibility for the majority of phenotypic methods, except in the cefoxitin DD method, according to which the strain was resistant. Four strains identified as S. epidermidis harboured the meca gene, but they were classified as oxacillin susceptible based on all phenotypic methods. Furthermore, two CoNS strains (one S. epidermidis and one S. caprae) that harboured the meca gene showed cefoxitin susceptibility and oxacilin resistance by DD or AD methods. The third type of discrepancy was encountered in four meca-negative strains (two S. epidermidis, one S. warneri, and one S. sciuri), which were oxacillin resistant by the DD or AD methods. Other important discrepancies were obtained for 13 CoNS strains (nine S. epidermidis, three S. haemolyticus, and one S. lugdunensis) that harboured the meca gene, but were classified as cefoxitin susceptible by the AD method. As the majority of methicillin-resistant staphylococci harbour the meca gene, tests based on amplification, such as PCR or DNA hybridization, have been expected to identify even the most heterogeneous strains correctly (Jaffe et al., 2000). Although PCR is considered the best method for detecting oxacillin-resistant staphylococci, several studies have reported discrepancies showing that some strains do not harbour the meca gene, but show phenotypic resistance to oxacillin. This finding has already been reported and may be associated with the hyperproduction of b-lactamase in these isolates. However, other strains with the meca gene showed phenotypic susceptibility to oxacillin. It has been suggested that the conflicting findings could be because of a variable and complex expression of the resistance among the different staphylococci strains (Sakoulas et al., 2001; Ferreira et al., 2003; Velasco et al., 2005). Thus,

4 302 I.C.V. Palazzo & A.L.C. Darini studies based on classic methods provide information that molecular procedures alone are unable to generate and it is also necessary to assume that several factors can produce PCR-biased results. The mec operon of strains that harbour the meca gene, but are oxacillin susceptible, is being sequenced to determine the alterations responsible for this behaviour. As shown in Table 2, the majority of the CoNS (70 out of 80 CoNS strains) showed MICs ranging from 8 to 256 mg ml 1. Ten out of 80 CoNS strains that were meca positive showed MICs from 0.5 to 4 mgml 1 and from these seven CoNS strains appeared susceptible in cefoxitin DD and three CoNS strains were cefoxitin resistant by the DD method. Recommendations for using cefoxitin to define methicillin resistance using AD were not made by the CLSI, and our observations with this test were not very encouraging. Although the method has been more specific (100% of specificity) for predicting oxacillin resistance, its low sensitivity prevents its use. Therefore, if a breakpoint of cefoxitin could be changed to 4 mgml 1 as proposed by Fernandes et al. (2005), our sensitivity value would be 87.5%. However, this breakpoint alteration is not a sufficient sensitivity improvement justifying its use as a diagnostic test in CoNS. Some authors proposed the cefoxitin DD method as being helpful in predicting oxacillin resistance in S. aureus strains (Felten et al., 2002; Skov et al., 2003; Cauwelier et al., 2004; Fernandes et al., 2005; Skov et al., 2005; Velasco et al., 2005), and the utilization of the same test for CoNS was standardized by the CLSI, although the proposed critical diameters for CoNS and S. aureus are different. Most reports show the sensitivity and specificity of the cefoxitin DD for S. aureus, which we also compared with the data obtained in this study for CoNS, although variable conditions have been used in tests based on cefoxitin to predict oxacillin resistance in S. aureus. In some cases, the culture medium, incubation temperature, and inoculum size were different from those proposed by CLSI. According to Felten et al. (2002), the cefoxitin DD method was 100% sensitive and 100% specific for detecting MRSA strains by using a critical diameter for cefoxitin o 27 mm, low-density inoculums at 10 6 CFU per ml, and 37 1C as the incubation temperature. According to the results obtained by Skov et al. (2003), using a semiconfluent inoculum (equivalent to 10 6 CFU per ml), overnight incubation at C,and an interpretative zone diameter breakpoint of R o 29 mm, the sensitivity and specificity for cefoxitin 30 mg discs for S. aureus were 100% and 99%, respectively. Using the conditions proposed by CLSI, Velasco et al. (2005) obtained 100% sensitivity and 98% specificity in the cefoxitin DD method for the detection of MRSA. According to data obtained in this study, the specificity of the cefoxitin DD method for CoNS is similar to that obtained for S. aureus using the conditions proposed by CLSI. However, the low sensitivity is related to isolates that have problems in expressing methicillin resistance. This fact seems to be more common in CoNS than in S. aureus and could be responsible for the lower test sensitivities. According to Skov et al. (2005), in CoNS there are substantial overlaps between meca-positive and mecanegative isolates in all methodological variants. Thus, a false resistance of 1 9% is expected. Although the conditions in this study were different, we obtained a 1.3% value of false resistance for the cefoxitin DD method. According to the results obtained in this study, cefoxitin DD and oxacillin AD methods could be used as initial tests for determining methicillin susceptibility in CoNS isolates but combining the two methods could, in addition, minimize errors in the detection of the methicillin resistance in the isolates. However, the possibility of identification errors is not excluded and these results should be confirmed by meca PCR. We consider that utilization of the two methods in clinical laboratories is feasible because they are not expensive and may be performed in any laboratory without acquisition of extra equipment. Although the AD method is very laborious, it is still necessary as a quantitative method to improve detection of oxacillin resistance in CoNS. The cefoxitin DD improved the detection of oxacillin resistance in S. aureus (Swenson & Tenover, 2005); however, recent reports (Frigatto et al., 2005; Swenson & Tenover, 2005) Table 2. Comparison of cefoxitin minimum inhibitory concentrations by agar dilution method with cefoxitin diameter zones and meca polymerase chain reaction-based determinations in coagulase-negative staphylococci clinical isolates Number of strains with cefoxitin MIC (mgml 1 ) Number of strains Cefoxitin zones (mm) Total number of strains tested: 151. The shaded numbers of strains in the table harboured the meca gene. The current CLSI (2005) criteria for cefoxitin MIC: susceptible at 8 mgml 1 and resistant Z32 mgml 1.

5 Detecting oxacillin resistance in coagulase negative staphylococci 303 indicate that the high sensitivity and specificity of this method are not seen in CoNS. According to the results obtained in this study the breakpoint in the cefoxitin DD method for CoNS, as proposed by CLSI, is correct because CoNS strains that not harbour the meca gene showed diameter zones of mm in 53 strains (75.7%) and mm in 17 strains (24.3%). Thus, changes of the breakpoint do not improve the test sensitivity as false-negative results remain and keep an unchanged picture. Several authors (Udo et al., 2000; Petinaki et al., 2002; Swenson & Tenover, 2005) have proposed utilization of the PBP2a latex agglutination test as a possible tool for routine microbiological diagnostic in the detection of oxacillin resistance in CoNS. However, it is an expensive test and the reports show values of sensitivity and specificity varying according to the conditions used in this test (i.e. size of inoculums, induction of resistance mechanism, time of reading the results, and heterogeneity of the population studied). Published data regarding methicillin resistance in staphylococci have to be considered in order to understand the association between resistance to methicillin and cefoxitin. Cefoxitin induces PBP2a production (Okonogi et al., 1989; Dancer, 2001) and it probably has a high affinity for staphylococcal PBP2 because a spontaneous PBP2-deficient mutant of methicillin-resistant S. aureus showed increased susceptibility to cephamycin-type antibiotics (Murakami et al., 1987). This fact leads to the conclusion that cefoxitinresistant strains harbour PBP2 with a low-level affinity for cefoxitin. Furthemore, heterogeneous methicillin resistance may be related to PBP2 overproduction combined with other concomitant phenotypic changes (Yoshida et al., 2003). Thus, it can be hypothesized that there is a cooperative mechanism among oxacillin and cefoxitin-resistant strains when resident PBP2 aids PBP2a production in the expression of resistance to methicillin induced by cefoxitin. Table 3 summarizes the data showing the sensitivity, specificity, predictive values, efficiency, and concordance of the phenotypic methods considering PCR meca as the gold standard method. The ratio of the summed true positive and negative results to the total number in the population studied expresses the efficiency of the test. The k concordance index (nonparametric test) was used to evaluate the degree of concordance between phenotypic methods and the meca PCR. Although sensitivities to cefoxitin and oxacilin measured by the DD method have the same percentage (92.5%), the combination of high sensitivity and high specificity obtained in oxacillin resistance determination in CoNS by the cefoxitin DD method argues for the better performance of this test. Six false-negative results obtained for cefoxitin discs caused a sensitivity reduction in this test. Using the breakpoint proposed by CLSI for cefoxitin AD, a high number of false-negative results was obtained for predicting oxacillin resistance in CoNS by this method. Table 3 also shows the results obtained by a combination of DD methods for cefoxitin and oxacillin AD. The combination between these tests improved the sensitivity for detecting oxacillin resistance in CoNS as well as a negative predictive value. Table 3. Sensitivity, specificity, predictive values, efficiency and concordance of the methods used to detect oxacillin resistance in coagulase negative staphylococci isolates considering PCR meca gene as gold standard method Oxacillin resistance detection methods PCR meca Phenotypic methods Positive Negative Sens (%) Specif (%) PPV NPV Efficiency (%) Concordance (%) DD oxacillin R S 6 68 DD cefoxitin R S 6 70 AD oxacillin R S 5 69 AD cefoxitin R S DD fox1ad oxa R S 4 70 Sens, sensitivity; Specif, specificity; PPV, positive predictive values; NPV, negative predictive values; DD, disc diffusion method; AD, Agar dilution method; R, resistant; S, sensitive; FOX, cefoxitin; OXA, oxacillin.

6 304 I.C.V. Palazzo & A.L.C. Darini As expected, the sensitivity and specificity of the tests used for S. aureus are lower for CoNS strains because standard susceptibility testing is less accurate in determining oxacillin resistance in meca-positive CoNS than in MRSA (Hussain et al., 2000; Caieirão et al., 2004; Ghoshal et al., 2004). In summary, although none of the techniques compared here showed 100% sensitivity and specificity for detecting methicillin resistance in CoNS, the combination of the cefoxitin DD and oxacillin AD may reduce errors in the detection of this resistance property. Thus, a combination of methods should be routinely used to detect methicillin resistance in CoNS. Acknowledgements The authors thank technicians Sohaila Mohamed Kassin Hussain and Gustavo Rafael Nascimento Silva of the Hospital das Clínicas de Ribeirão Preto (USP) for providing the bacterial isolates, and Keiichi Hiramatsu (Juntendo University, Japan) for supplying S. aureus N315. 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