Rapid evaluation of the mycobactericidal efficacy of disinfectants in the quantitative carrier test EN 14563 by using fluorescent Mycobacterium terrae.

To prevent transmission of mycobacterial pathogens, medical devices must be disinfected by germicides with proven mycobactericidal activity. The quantitative carrier test EN 14563 provides an international standard for evaluation of the mycobactericidal activity of disinfectants under practical conditions. However, tests according to the EN 14563 standard are based on cultivation, and results are available only after 21 days. The aim of this study was to accelerate assessment of dosage and contact times of mycobactericidal preparations based on the EN 14563 standard. To this end, a gfp gene was constructed with a codon usage adapted for Mycobacterium tuberculosis. Expression of the gfp(m)(2+) gene in Mycobacterium terrae improved the detection sensitivity by 10-fold over that with a previously used reporter strain. Peracetic acid and a cation-active formulation were tested as commercially available disinfectants for medical devices. M. terrae expressing gfp(m)(2+) was used to determine dosage and contact times for the two test germicides. Fluorescence measurements correlated well with growth of the reporter strain, demonstrating that the fluorescence reliably indicated the number of viable cells. The fluorescence enabled us to determine the mycobactericidal efficacy of the test disinfectants according to the quantitative carrier test EN 14563 standard within at least 15 days. In conclusion, this study establishes gfp(m)(2+)-expressing M. terrae as a new reporter strain for reliable evaluation of mycobactericidal activities of disinfectants with a superior sensitivity and in a significantly shorter time than previously possible.