Evaluation of different methods for detecting methicillin resistance in Staphylococcus aureus isolates in a university hospital located in the Northeast of Brazil

Brazilian Journal of Microbiology (2010) 41: 316-320 ISSN 1517-8382 EVALUATION OF DIFFERENT METHODS FOR DETECTING METHICILLIN RESISTANCE IN STAPHYLOCOCCUS AUREUS ISOLATES IN A UNIVERSITY HOSPITAL LOCATED IN THE NORTHEAST OF BRAZIL Francisco Canindé de Sousa Júnior1, Gildelane da Silva Néri1, Ana Karine Silva1, Bibiana Priscila Rodrigues Câmara de Araújo1, Myrian Júlia de Paiva Dourado Guerra1, Maria José de Britto Costa Fernandes1, Eveline Pipolo Milan2, Maria Celeste Nunes de Melo1* 1 Departamento de Microbiologia e Parasitologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil; 2Departamento de Infectologia, Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil. Submitted: May 22, 2009; Returned to authors for corrections: August 13, 2009; Approved: October 06, 2009. ABSTRACT Many methods have been described for the detection of methicillin-resistant Staphylococcus aureus (MRSA), but the heterogeneous expression of methicillin resistance affects the reliability of these methods. The aim of the present study was to evaluate some methods for detecting methicillin resistance in Staphylococcus aureus isolates in a university hospital located in the Northeast of Brazil. Among the isolates, 15 were methicillin-susceptible and 45 were methicillin-resistant, including low-level heterogeneous resistance strains. Both the 30 g-cefoxitin disk and PBP2a test had 100% sensibility/specificity and appear to be good options for the detection of MRSA in the clinical laboratory. Key words: MRSA, methicillin resistance, heterogeneous resistance, cefoxitin, PBP2a. INTRODUCTION recently recommended by the Clinical and Laboratory Standards Institute for prediction of mecA–mediated resistance Methicillin-resistant Staphylococcus aureus (MRSA) has (5). Finally, there are also mecA-specific tests such as mecA become the leading nosocomial pathogen worldwide and seems polymerase chain reaction and PBP2a latex agglutination test to have spread into the community (10). Methicillin resistance (21). However, detection of methicillin resistance in routine in staphylococci is caused by the expression of PBP2a encoded clinical laboratories has been problematic ever since the by the mecA gene that is located on a genetic element called emergence of MRSA during the 1960s (19). The difficulties are the staphylococcal cassette chromosome mec- SCCmec (9). associated mainly with heterogeneous expression of methicillin There are several methods available to laboratories for resistance in most MRSA strains currently prevalent (11, 24). detecting methicillin resistance. These include oxacillin disk Errors in the detection of methicillin resistance can have test, automated susceptibility testing systems, and oxacillin serious adverse clinical consequences. False-susceptibility agar screen plate. In addition, the cefoxitin disk test was results may result in treatment failure and the spread of MRSA *Corresponding Author. Mailing address: Departamento de Microbiologia e Parasitologia, Centro de Biociências, UFRN. Av. Senador Salgado Filho, S/N, 59072-970, Natal, RN, Brasil.; Fax: +55-84-3211-9210.; E-mail: 316 Sousa Júnior, F.C. et al. Methods for detecting methicilin resistance in S. aureus if appropriate infection control measures are not applied. were performed by observing the recommendation of the Conversely, false-resistance results may increase healthcare Clinical and Laboratory Standards Institute (5). The 25 g/mL costs following unnecessary isolation precautions and may lead methicillin test (25-Met) was carried using trypticase soy agar to overuse of glycopeptides (3, 23). Thus, as some controversy plates and heavy bacterial inoculums (109-1010 c.f.u.), as still exists over the inaccuracy of the recommended methods described previously (14). for identification of MRSA, we evaluated some of these methods for detecting methicillin resistance in Staphylococcus aureus isolates in a university hospital located in the Northeast of Brazil. Detection of penicillin-binding protein 2a (PBP2a test) The Slidex® MRSA Detection (Biomérieux, Paris, France), which is based on the agglutination of latex particles sensitized with monoclonal antibodies against PBP2a, was MATERIAL AND METHODS Bacterial strains A total of 60 strains of S. aureus isolated from clinical carried out and interpreted according to the manufacturer’s instructions. Population analysis profiling (PAP) samples in a university hospital in Natal city, RN, located in The expression of methicillin resistance was analyzed by the Northeast of Brazil were used in the study. The isolates PAP for strains that yielded isolated colonies on 25-Met. were identified as S. aureus using routine tests (Gram’s stain, Overnight cultures grown in TSB at 35ºC, containing 109–1010 catalase and free coagulase tests) and stored at -70 ºC in TSB c.f.u./mL, were plated at four dilutions (100, 10-1, 10-3 and 10-5) containing 10% (w/v) glycerol. The study was approved by the on to agar plates containing serial (two-fold) dilutions of Research Ethics Committee of the Universidade Federal do Rio methicillin at concentrations of 0 and 0.75 to 800 mg/mL. Grande do Norte (UFRN), according to protocol no. 109/2006. Colonies were counted after 48 h incubation at 35ºC. A graphic The methicillin-susceptible strain of S. aureus ATCC 25923 representation was constructed by plotting colony forming and the MRSA isolate BMB9393 were used to control. units per millilitre against the concentration of methicillin (22). Detection of the mecA gene We considered the presence of the mecA gene as the reference or “gold standard” method for establishing the sensitivity and specificity of each of the techniques studied. The DNA extraction was performed as described by Pacheco et al. (13) and the mecA gene was detected with the polymerase chain reaction (PCR) technique based on the procedure described by Oliveira and de Lencastre (12), with the following primers: mecA-F AAA ACT AGG TGT TGG TGA AGA TAT ACC and mecA-R GAA AGG ATC TGT ACT GGG TTA ATC AG, which amplify an internal region of 585bp of this gene. Amplicons were visualized following electrophoresis on agarose gels stained with ethidium bromide. Susceptibility tests RESULTS Among the 60 S. aureus isolates, 45 were MRSA (mecA positive) and 15 were MSSA (mecA negative; Fig. 1). Of the 45 MRSA isolates, 73% (33/45) belonged to the Brazilian Epidemic Clone (ST239- SCCmecIIIA), 7% (3/45) to the pediatric clone (ST5- SCCmecIV), and 20% (9/45) to the sporadic clones (20). Of all the mecA-positive isolates, one (NT42) displayed inhibition zone diameters of 13mm by a 1µg-oxacillin disk test (resistance breakpoint 10mm) and two (NT05 and NT80) showed small individual colonies or light growth within the inhibition zone >10mm and could have been erroneously classified as methicillin-susceptible isolates (sensi (...truncated)