Rapid microbiological methods (RMMs) for evaluating the activity of cephalosporin antibiotics employing triphenyltetrazolium chloride

Agar diffusion method has been used to evaluate antimicrobial activity since the discovery of penicillin. Nevertheless, little progress has occurred in reducing the time required to determine growth inhibition zones. The aim of this work was to develop, optimize and validate rapid microbiological methods (RMMs) for cephalosporin antibiotics using triphenyltetrazolium chloride (TTC) to reduce the incubation time of the assays. Through a factorial design in which the inoculum suspension, incubation time, and percentage of the TTC solution were varied, it was possible to validate the RMMs for cefazolin, cefuroxime, ceftriaxone, and cefepime. The validated conditions employed 5 mL of MHA medium inoculated with 2% of Staphylococcus aureus suspension, incubation time of 5 h and 30 min at 37 +/- 1 degrees C and the addition of 0.3% TTC solution in 1% agar, cefazolin and cefuroxime in concentrations from 15 to 60 mu g mL(-1) ceftriaxone and cefepime in concentrations from 20 to 80 mu g mL(-1). The methods were selective, linear (for standard and sample curves, respectively), accurate, precise, robust and rugged. The results found by the RMMs were statistically similar to those found by conventional microbiological methods, but the advantages of the former decreased the incubation time from 22 h to 5 h and 30 min. Therefore, RMMs can be used in the evaluation and quantification of cephalosporin antibiotics, ensuring their quality, safety, and therapeutic efficacy.