However, viable wild-type M. smegmatis bacteria decreased

rapidly after lysozyme treatment for 4 h. A significant difference (P < 0.01) in viability was observed between M. smegmatis/Rv1096 and wild-type M. smegmatis after lysozyme treatment for 9 h. About 107 wild-type M. smegmatis cells survived, whereas only 1016 M. smegmatis/Rv1096 cells survived. Figure 4 Lysozyme susceptibility assay. A) Lysozyme treatment growth curves for M. smegmatis/Rv1096 and wild-type M. smegmatis. M. smegmatis/Rv1096 (square) and wild-type M. smegmatis (triangle) were grown in LBT medium at 37°C to an OD600 of 0.2; the cultures were then divided into two parts. One part (closed symbol) was treated with lysozyme, the other part was not. Three microliter samples from each culture were collected

at 1 h intervals for OD600 measurements. M. smegmatis/Rv1096 showed PI3K inhibitor LEE011 datasheet significantly SN-38 manufacturer greater resistance to lysozyme than did wild-type M. smegmatis (**P < 0.01). Values are means ± SD. B) Cell survival curves for M. smegmatis/Rv1096 and wild-type M. smegmatis under lysozyme treatment. M. smegmatis/Rv1096 (square) and wild-type M. smegmatis (triangle) were each grown in LBT medium at 37°C to an OD600 of 0.2, then the cultures were divided into two parts. One part (closed symbol) was treated with lysozyme, the other part was not. Three microliter culture samples were collected at 1 h intervals to measure CFU/ml. M. smegmatis/Rv1096 exhibited greater cell survival than that of the

wild-type bacterium (**P < 0.01). Values are means ± SD. The M. smegmatis/Rv1096cell wall was undamaged by 9 h of lysozyme treatment Because the most apparent differences in bacterial growth and viability were observed (Figures 4A and B) after treatment with lysozyme for 9 h, morphological observations were performed at this time point. The results of the Ziehl-Neelsen acid-fast staining showed that wild-type M. smegmatis lost its acid-fastness and became blue dyed, whereas M. smegmatis/Rv1096 retained its acid-fastness (Figure 5). Scanning electronic microscopy (SEM) showed that the wild-type M. smegmatis had an irregular appearance (enlarged shape, destructed cell wall and wrinkled surface) in the presence of lysozyme, Progesterone whereas M. smegmatis/Rv1096 had a regular shape, undamaged cell wall and smooth surface after 9 h lysozyme treatment (Figure 6). Figure 5 Acid-fast staining of M. smegmatis/Rv1096 and wild-type cells. A) Wild-type M. smegmatis without lysozyme treatment, B) wild-type M. smegmatis with lysozyme treatment, C) M. smegmatis/Rv1096 without lysozyme treatment and, D) M. smegmatis/Rv1096 with lysozyme treatment (×1000). Lysozyme treatment was for 9 h. Figure 6 Scanning electron micrographs of M. smegmatis/Rv1096 and wild-type M. smegmatis . A) Wild-type M. smegmatis without lysozyme treatment, B) wild-type M. smegmatis with lysozyme treatment, C) M. smegmatis/Rv1096 without lysozyme treatment and, D) M.