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.Prevention of infections by mycobacteria is an important goal due to their paramount clinical relevance. This task presents a significant challenge, since mycobacteria are intrinsically resistant to many chemical agents (2). In order to avoid device-related transmission of mycobacterial pathogens, germicides developed for disinfection of medical devices must be proven to possess mycobactericidal activity. Conventional testing of disinfectants for their tuberculocidal activity is based on cultivation techniques. Mycobacterium terrae, which has been demonstrated to possess resistance to chemical disinfectants similar to that of Mycobacterium tuberculosis (5), is used as a surrogate for establishing tuberculocidal activity of germicides. However, such an assay takes at least 3 weeks because of the slow growth of M. terrae. In order to accelerate the process of screening chemicals for mycobactericidal activity, Zafer et al. has described the use of gfp-expressing M. terrae in quantitative suspension tests (18). Generally, suspension tests are used to determine the overall efficacy of disinfectants against mycobacteria. However, mycobacteria are used in cell suspensions in these experiments and thus are likely to be more accessible to disinfectants. Therefore, it is essential to demonstrate the mycobactericidal efficacy of disinfectants for medical devices under practical conditions. For these reaso...
MspA is the major porin of Mycobacterium smegmatis and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the mspA gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of mspA was identified by single point mutations in lacZ fusions and resembled A promoters of M. smegmatis. However, a 500-bp upstream fragment including P mspA in a transcriptional fusion with lacZ yielded only low -galactosidase activity, whereas activity increased 12-fold with a 700-bp fragment. Activation of P mspA by the 200-bp element was almost eliminated by increasing the distance by 14 bp, indicating binding of an activator protein. The chromosomal mspA transcript had a size of 900 bases and was very stable with a half-life of 6 minutes, whereas the stabilities of episomal mspA transcripts with three other 5 untranslated region (UTRs) were three-to sixfold reduced, indicating a stabilizing role of the native 5 UTR of mspA. Northern blot experiments revealed that the amount of mspA mRNA was increased under nitrogen limitation but reduced under carbon and phosphate limitation at 42°C in stationary phase in the presence of 0.5 M sodium chloride, 18 mM hydrogen peroxide, and 10% ethanol and at acidic pH. These results show for the first time that M. smegmatis regulates porin gene expression to optimize uptake of certain nutrients and to protect itself from toxic solutes.
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