Highly efficient in vitro photodynamic inactivation of Mycobacterium smegmatis

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Abstract
Efforts to control tuberculosis (TB) have been hampered by the emergence of multiple-drug resistant strains, necessitating pursuit of alternative approaches to the current antibiotic-based treatments. Herein, we explore the feasibility of photodynamic inactivation (PDI) of mycobacteria. In vitro PDI studies employing Mycobacterium smegmatis as a surrogate for Mycobacterium tuberculosis were performed examining photosensitizer (PS) type, concentration and light dose. M. smegmatis was grown to a concentration of 108 colony forming units (cfu) per mL, resuspended in PBS–0.5% Tween-80-containing buffer, incubated with the PS for 5 min and subsequently illuminated with white light (400–700 nm) at a fluence rate of 60 mW/cm2 for 1, 5, 15 or 30 min (equivalent to 3.4, 18, 54 or 108 J/cm2). The percentage survival was determined by the ratio of the colony count from illuminated and non-illuminated control cell suspensions. The PSs examined were 5,10,15,20-tetrakis(1-methyl-4-pyridinyl)porphyrin tetratosylate (TMPyP), 5,10,15,20-tetrakis(4-N,N,N-trimethylanilinium)porphyrin tetrachloride (TNMAP), methylene blue (MB), 5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin (TSPP), 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-Pd(II) (TCPP-Pd) and phthalocyanine tetrasulphonic acid (PhCS). Our best results demonstrate that PDI of M. smegmatis can achieve a noteworthy 5–6 log unit reduction in cfu (99.999% + viable cell eradication) when cationic PSs are employed in the nanomolar concentration range. Anionic PSs did not effectively mediate PDI of mycobacteria due to their inability to associate with the negatively charged mycobacterial cell membrane. PDI of M. smegmatis was found to be highly efficient in reducing the number of viable cells in vitro when cationic PSs were employed.

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