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Sensitive EDTA-Based Microbiological Assays for Detection of Metallo-ß-Lactamases in Nonfermentative Gram-Negative Bacteria

Departamento de Microbiología,¹ Departamento de Química Biológica, Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,² Centro de Especialidades Médicas Ambulatorias de Rosario, Departamento Bioquímico Municipal, Secretaría de Salud Pública, Municipalidad de Rosario 2000 Rosario,³ Servicio Antimicrobianos, Departamento Bacteriología, Instituto Nacional de Enfermedades Infecciosas—ANLIS "Dr. Carlos G. Malbrán," Ciudad Autónoma de Buenos Aires, Argentina⁴

Received 26 July 2005/ Returned for modification 15 August 2005/ Accepted 31 August 2005

The worldwide spread of metallo-ß-lactamase (MBL)-producing gram-negative bacilli represents a great concern nowadays. Sensitive assays for their specific detection are increasingly demanded to aid infection control and to prevent their dissemination. We have developed a novel microbiological assay employing crude bacterial extracts, designated EDTA-imipenem microbiological assay (EIM), to identify MBLs in nonfermentative gram-negative clinical strains. We also evaluated the ability of EIM to detect MBLs in comparison to those of other currently employed screening methods, such as the EDTA disk synergy test (EDS) with imipenem as a substrate and the Etest method. The sensitivities of EIM and Etest were similar (1 versus 0.92, respectively) and much higher than that of EDS (0.67). Moreover, both EIM and Etest displayed the maximum specificity. Modifications were introduced to EDS, including the simultaneous testing of three different ß-lactams (imipenem, meropenem, and ceftazidime) and two different EDTA concentrations. This resulted in a sensitivity improvement (0.92), albeit at a cost to its specificity. A simple strategy to accurately detect MBL producers is proposed; this strategy combines (i) an initial screening of the isolates by the extended EDS assay to select the potential candidates and (ii) confirmation of the true presence of MBL activity by EIM.

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