A high proportion of red colonies (smooth-domed-red, smooth-flat-red, red-rough) was generated by mutant MAV_2599 (Figure  3 I) additionally to smooth-opaque and smooth-transparent colonies. This mutant produced only few rough (rough-transparent, rough-red) colonies. Altogether, we observed a high frequency and intensity of morphological changes in the mutants pointing to involvement of the mutated genes in the composition GSK2399872A cost of cell wall structure. Since studies by different authors have related colony morphotype to virulence it would be of interest to investigate in further experiments if and to which degree

the different colony types are stable and differ in their virulence. Figure 3 Colony morphology upon plating on Congo Red agar plates. Well-grown

broth cultures of all strains were diluted 1:106 and 100 μl plated in triplicate onto Middlebrook agar with OADC containing 100 μg ml-1 Congo Red. Plates were incubated on average for three weeks. The arrows point to smooth-domed-opaque Pexidartinib (sdo), smooth-flat-red (sfr), smooth transparent (st), rough red (rr) and rough transparent (rt) colonies. A: WT; B: mutant MAV_2555; C: mutant MAV_1888; D: mutant MAV_4334; E: mutant MAV_5106; F: mutant MAV_1778; G: mutant MAV_3128; H: mutant MAV_3625; I: mutant MAV_2599. FK228 manufacturer pH-resistance The intraphagosomal pH of M. avium-containing phagosomes decreases to pH 5.2 in activated macrophages [59]. We therefore investigated the pH-resistance of the mutants compared to the WT by inoculating them into MB broth at pH 5 and pH 7 and measuring the growth during 11 days at 37°C by means of OD measurement and ATP quantification. ATP measurement represents a much more sensitive method than the OD measurement. Additionally, the OD of a culture not only

depends on cell number but also on the size of the cells, their morphology and the degree of clumping of the cells. For these reasons, ATP measurement was reported to be a more reliable method for quantification of mycobacteria in broth culture [41]. As shown in Figure  4, the WT grew better at neutral pH than at low pH. After 11 days of growth in neutral medium, it generated 722,491 RLU (relative light units), Idoxuridine while in medium with acidic pH only 143,082 RLU were achieved. The mutants MAV_2555, MAV_1888, MAV_4334 and MAV_5106 showed a similar growth pattern as the WT, both in neutral and acidic pH (data not shown). The mutants MAV_1778 and MAV_3128 grew similar as the WT at neutral pH; however, at low pH these strains enhanced their growth rate even above the level reached at neutral pH (Figure  4 A and B). While the mutant MAV_3128 showed enhanced growth in comparison to the WT at low pH already at day 1, the mutant MAV_1778 showed an identical growth rate as the WT at low pH until day 5 and then showed strongly enhanced growth.