Assessment of a microplate method for determining the post-antibiotic effect in Staphylococcus aureus and Escherichia coli

Objectives: The post-antibiotic effect (PAE) is an important parameter of antibiotic action that is widely used as a predictor of pharmacodynamic activity. Traditionally, PAE has been determined by a labour-intensive method involving determination of viable cell numbers. New methods using spectrophotometric procedures could offer significant advantages for PAE determinations, particularly in terms of speed. A number of such methods have been described in the literature, but extensive comparison with the classical procedure for determining PAEs has not been carried out. We have now compared PAE values obtained using a rapid microplate method with those achieved by the classical viable count procedure. Methods: We determined PAE values for a variety of antibiotics against Staphylococcus aureus and Escherichia coli following exposure to 5 x MIC drug concentrations for 60 min in Mueller-Hinton Broth (MHB). The duration of the PAE was obtained by following the recovery of bacterial growth in antibiotic-free MHB measured either as colony forming units on Mueller-Hinton agar, or as culture absorbance (600 nm) in a microplate reader. Results: For bacteriolytic agents there was poor correlation between the two methods for both S. aureus (R-2=0.096) and E. coli (R-2=0.5456). However, when PAEs for bacteriostatic agents and non-lytic bactericidal agents were compared, correlation between the two methods was high for both S. aureus (R-2=0.7529) and E. coli (R-2=0.7687). Conclusions: The spectrophotometric microplate method for determining PAEs may be a suitable alternative to the classical method for those antibiotics that do not induce bacterial cell lysis.