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On-line version ISSN 1678-4405
Braz. J. Microbiol. vol.34 suppl.1 São Paulo Nov. 2003
Testes convencionais versus moleculares (Multiplex PCR e PCR gene mecA) na detecção de Staphylococcus aureus resistente a meticilina
Rosineide Marques RibasI; Paulo Pinto Gontijo-FilhoI; Ana Lúcia da Costa DariniII
IUniversidade Federal de Uberlândia, Uberlândia, MG, Brasil
IIFaculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, SP, Brasil
In this study, for detection of methicillin-resistant S. aureus (MRSA), a mecA multiplex PCR-based amplification was compared with the 1 µg oxacillin disk diffusion test, detection of minimal inhibitory concentration (MIC), and screening in agar with 4% NaCl and 6 µg/mL oxacillin. Among 24 isolates obtained from blood, mecA gene was detected in only 16 (66.7%) isolates by multiplex PCR. The MIC test showed a range of resistance to oxacillin from 0.19 to 512 µg/mL, among these isolates. Data obtained by screening and dilution tests showed that sensitivity to methicillin was 80.0% and 72.8%, respectively, when compared with the presence of mecA gene (multiplex). All isolates, including the negatives, when revaluated for mecA gene by PCR were positive. b-lactamase production was positive for 20/25 isolates (80.0%). About ¼ of patients died dispite most of them (83.3%) were adequately treated. The simultaneous identification of the bacteria and determination of this susceptibility to antibiotics are necessary for the choice of empiric antibiotic therapy in suspected staphylococcal sepse, but is important to considering the sensibility, specificity and validation of the available kits.
Key words: multiplex PCR; MRSA; b-lactamase; mecA gene.
Nesse estudo, para detecção de S. aureus resistente à meticilina (MRSA), a amplificação do gene mecA baseada no multiplex PCR foi comparada com os testes de difusão com disco para 1 µg/mL de oxacilina, detecção da concentração inibitória mínima (CIM), meio de triagem com 4% de NaCl e 6 µg/mL de oxacilina. Na investigação de 24 isolados obtidos de sangue, o genótipo mecA foi detectado em apenas 16 (66,7%) dos isolados pelo multiplex PCR. A CIM apresentou valores variando de 0,19 a 512 µg/mL entre os isolados de MRSA. Os dados obtidos pelos testes de triagem e diluição em ágar apresentaram sensibilidades de 80,0% e 72,8% respectivamente, quando comparados com a presença do gene mecA (multiplex). Todos os isolados, incluindo os negativos, quando reavaliados com a técnica de PCR exclusivo para este gene, o resultado foi positivo. A produção de b-lactamase foi positiva em 20/25 (80,0%) dos isolados. Cerca de ¼ dos pacientes evoluiu para óbito apesar da maioria (83,3%) ter sido tratada adequadamente. A identificação simultânea da bactéria e sua susceptibilidade aos antibióticos é necessária para a escolha de uma terapia adequada para casos de sepse estafilocócica, mas é importante considerar a sensibilidade, especificidade e validação dos kits disponíveis.
Palavras-chave: multiplex PCR; MRSA; b-lactamase; gene mecA.
Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide (1) and several classical methods have been used to detect the MRSA isolates including the 1 µg/mL oxacillin disk diffusion test, agar plate screen, agar dilution and the E'test (NCCLS) (2,3). Alternatively, molecular techniques mostly based on PCR amplification of mecA gene were used for rapid detection of MRSA (1). In the present study we compared the mecA multiplex PCR-based amplification and agar dilution, E'test, oxacillin agar screen results with the 1 µg disk diffusion test results that initially classified 24 isolates of MRSA.
MATERIALS AND METHODS
Hospital and design of the study
The 24 MRSA strains used in the present study were isolated by the microbiology laboratory of the Clinic Hospital of Uberlandia Federal University.
Oxacillin disk diffusion method and agar screening method for detection of MRSA
The 1 µg oxacillin disk diffusion method was performed on Mueller-Hinton agar. All isolates were also plated on Mueller-Hinton agar containing 4% NaCl and 6 µg/mL (4) oxacillin.
E'test for determining the MICs of oxacillin against S. aureus
For confirmation of mecA negative isolates, the E-test was used to determine the MICs of oxacillin.
Detection of b-lactamase production
b-lactamase production was tested using nitrocefin (Oxoid-Hampshire, UK).
PCR was performed with six complementary primers: mecA1 (5'- CTC Agg TAC TgC TAT CCA CC-3'); mecA2 (5' - CAC TTg gTA TAT CTT CAC C-3'); mupA1 (5' - TgA CAA TAg AAA Agg ACA gg-3'); mupA2 (5' - CTC TAA TTC AAC Tgg TAA Gcc-3'); 16S rRNA1 (5' - ggA ATT CAA A (T/g)g AAT TgA Cgg ggg C-3') and 16S rRNA2 (5' - Cgg gAT CCC Agg CCC ggg AAC gTA TTC AC- 3').
The results for the 25 MRSA isolates characterised by disk diffusion test are presented in Table 1. They are listed according to their level of MICs. Correlations between susceptibility testing and oxacillin disk test, oxacillin agar screening and presence of mecA gene are showed in Table 2. All isolates showed resistance to 1 µg/mL oxacillin disk diffusion test and 91.7% of isolates were considered resistant by the agar dilution test. Using the multiplex PCR-based amplification, mecA gene was detected in 16 of the 24 (66.7%) isolates. The negative isolates, revaluated for the mecA gene by PCR, were all mecA positive. b-lactamase production was detected in 20 of the 24 (83.3%) isolates. Among the four b-lactamase negative strains, two were positive for mecA.
We have compared multiplex PCR assays for the detection of antibiotic resistance genes with classical methods for the determination of susceptibility to antibiotics. In this study the 24 isolates initially classified as methicillin resistant based on disk diffusion showed discrepant results when tested with the multiplex PCR. The mecA was positive in 16 of 24 (66.7%) isolates and oxacillin MIC of > 128 µg/mL was found in 22 (91.7%) isolates. The discrepant findings in our study can be attributed to techinical problems. Investigators reported that the conventional susceptibility testing methods, including disk diffusion and agar dilution tests, are not always reliable in detecting methicillin resistant staphylococci (4). The staphylococcal strains showing discrepancies between the genotype ant the phenotype were revaluated by PCR of the mecA gene only, and all were positive. Some studies reported S. aureus strains carrying the mecA gene but susceptible to oxacillin. York et al. (5) observed that staphylococci strains with MIC less than 2 µg/mL may be methicillin resistant. In this work, two isolates with MIC < 0.25 µg/mL carried mecA gene. Although the expression of the mecA gene is considered an important mechanisms, of resistance, other mechanism alone or in combination have been detected in Staphylococcus strains. In our study, 83.3% of the S. aureus isolates were producers of b-lactamase.
1. Araj, G.F.; Talhouk, R.S; Simaan, C.J; Maasad, M.J. Discrepancies between mecA PCR and conventional tests used for detection of methicillin resistant Staphylococcus aureus. Intern. J. Antimicrob. Agents, 11:47-52. 1999. [ Links ]
2. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility testing. Sixth edition, Approved Standard, NCCLS, M2 A6, 1997a. [ Links ]
3. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Fourth edition, Approved Standard, NCCLS, M7 A4, 1997b. [ Links ]
4. Martineau, F.; Picard, F.J.; Lansac, N.; Nenard, C.; Roy, P.H.; Ovellehe, M.; Bergeron, M. Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and S. epidermidis. Antimicrobial Agents Chemother., 44:231-238, 2000. [ Links ]
5. York, M.K.; Gibbs L.; Chehab R.; Brooks, G.F. Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci. J. Clin. Microbiol., 34:249-253, 1996. [ Links ]
Rosineide Marques Ribas / Paulo Pinto Gontijo-Filho
Área de Imunologia, Microbiologia e Parasitologia
38400-902, Uberlândia, MG, Brasil
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This paper corresponds to an "extended abstract" selected for oral presentation in the 22nd Brazilian Congress of Microbiology, held in Florianópolis, SC, Brazil, in November 17-20, 2003