Development and validation of a resazurin assay for in vitro susceptibility testing of Actinomadura madurae: a common causative agent of actinomycetoma

Objectives Actinomycetoma is a chronic granulomatous disease affecting skin, subcutaneous tissue, fascia, muscle and bones. With increasing resistance against commonly used treatment regimens, susceptibility testing is urgently needed. Methods We developed an in vitro susceptibility assay for Actinomadura madurae, one of the common causative agents of actinomycetoma, employing resazurin for endpoint reading. Using this assay, reproducible MICs were determined for the most commonly used antibacterial agents for actinomycetoma treatment. The tested antibacterial agents included trimethoprim/sulfamethoxazole, amikacin, streptomycin, amoxicillin, ceftriaxone, gentamicin, ciprofloxacin, doxycycline, imipenem, linezolid, penicillin G and rifampicin. Results Following the clinical breakpoints as stated by CLSI, 100% of the tested strains were susceptible to trimethoprim/sulfamethoxazole (MIC 0.03/0.59-1/19 mg/L), amikacin (MIC 0.0078-0.25 mg/L), doxycycline (MIC <0.25-1 mg/L) and linezolid (MIC <0.25-2 mg/L), 90% to ciprofloxacin (MIC <0.25-2 mg/L), 80% to ceftriaxone (MIC 64 mg/L) and imipenem (MIC <0.25-32 mg/L) and only 20% to amoxicillin (MIC 64 mg/L) and rifampicin (MIC 0.5 to >32 mg/L). Conclusions Determinations of MICs by visual readings of colour changes versus spectrophotometric readings were comparable. This convenient visual reading has the advantage of feasible implementation in endemic settings.

Automated summary

An in vitro susceptibility assay for Actinomadura madurae was developed, and the minimum inhibitory concentrations (MICs) of commonly used antibacterial agents were determined. The results showed that trimethoprim/sulfamethoxazole, amikacin, doxycycline, and linezolid had high susceptibility against Actinomadura madurae, while amoxicillin and rifampicin had low susceptibility.