Aims: To study the effect of hypermutation on the architecture and phenotypic diversification of P. aeruginosa (PA) biofilms. Methods: PA reference strain PAO1, environmental strain Hex1T and their respective mutS mutants, PAOMS and Hex1TMS, were used. Strains were gfp-tagged using the mini-Tn7 transposon system. Biofilms were grown at 30°C in three-channel flow cells irrigated with FAB minimal medium plus 0.3 mM glucose. Channels were inoculated with 250 ìl of 0.001 OD600 bacterial cultures. Pictures were taken at 2, 4 and 6 days. To study the phenotypic diversification, 6days-biofilms were harvested, plated on PA isolation agar and the morphology of approx. 2.000 colonies per strain was examined. All microscopic observations were performed by a confocal laser scanning microscope (CLSM). For structural analysis and processing of images software packages COMSTAT and IMARIS were used. Structural data were compared using a Student t test. P value of <0.05 was considered statistically significant. Results: Initial inocula were approx. 1.5 x106 CFU increasing by 3 log after 6 days for all strains. Biofilms formed by PAOMS were thicker and showed more biomass than those formed by PAO1 (P <0.05 at 4d). However, although no significant differences were found at days 2 and 4, Hex1TMS biofilms were thinner and showed less biomass than Hex1T at 6d (P <0.05). CLSM images of biofilms formed by hypermutable strains showed irregular biofilms structures, randomly smooth, rough and filamentous. Empty mushroom-like structures were characteristically observed in Hex1TMS biofilms. Examination of colony morphology after 6d-biofilm revealed up to 6 different morphotypes in hypermutable and only 2 within wild-type strains. Normal, giant, mini, wrinkly, mucoid, brown and orange pigmented were between the different morphotypes found. Total percentages of colonies with different morphotypes were 8.3 and 0.05 % for PAOMS and PAO1, respectively, and 2.3 and 0.8% for Hex1TMS and Hex1T, respectively. Conclusions: Mismatch repair system deficiency increases the structural diversity of PA biofilms and this could probably be a microscopic visible evidence of the acceleration of phenotypic diversification.
