Species specific susceptibility testing for ß-lactam antibiotics. With special reference to staphylococci

Abstract: The main objective of this thesis was to identify methods for the detection of ß-lactam resistance in staphylococci, bacteria often causing nosocomial infections. Detection of ß-lactam resistance in these species is difficult due to strong regulation of genes encoding for the two main resistance mechanisms, ß-lactamase production and the penicillin-binding protein (PBP) 2?. ß-lactamase has high affinity to penicillins and hydrolyse these drugs. In total ten applications were evaluated with respect to ability to identify ß-lactamase producing strains of staphylococci. Three applications, the starch-iodine plate test using induced and uninduced strains, and the nitrocefin spot test using induced strains grown on unsupplemented agar, efficiently identified ß-lactamase producing strains. The nitrocefin spot test proved to be the most appropriate test for the routine laboratory. Novobiocin-resistant coagulase-negative species, showed a high rate of false positive reactions in several tests and should not be tested for ß-lactamase production. PBP2? is encoded by the mecA gene and is only found in methicillin-resistant staphylococci, where it supersedes the regular PBPs in the presence of ß-lactam antibiotics. An appli- cation for detection of the mecA gene with polymerase chain reaction (PCR) was developed and used as reference method. The effect of the inoculum density, the incubation time and temperature, supplementation of the growth medium, and the disk content or drug, on the expression of methicillin-resistance with the disk diffusion method and MIC determination was evaluated. The disk diffusion method proved to be by far the most reliable method for the identification of methicillin-resistant strains if an inoculum of 108 cfu/ml, incubation at 30°C for 24 h, agar with no more than 0.5% NaCl, and disks with 1 and 5 µg oxacillin were applied. MIC determination as a tool for identification of methicillin- resistant strains is unsuitable, due to a poor discrimination between mecA+ and mecA- strains or a poor correlation to MIC limits.