The present study discusses a biofilm-positive P. aeruginosa isolate that survives at pH levels ranging from 4.0 to 9.0. The biofilm consortia were colonized with different phenotypes i.e., planktonic, slow-growing and metabolically inactive small colony variants (SCVs). The lower base of the consortia was occupied by SCVs. These cells were strongly attached to solid surfaces and interconnected through a network of nanotubes. Nanotubes were observed at the stationary phase of biofilm indwellers and were more prominent after applying weight to the consortia. The scanning electron micrographs indicated that the nanotubes are polar appendages with intraspecies connectivity. The micrographs indicated variations in physical dimensions (length, width, and height) and a considerable reduction in volume due to weight pressure. A total of 35 cells were randomly selected. The mean volume of cells before the application of weight was 0.288µm3, which was reduced to 0.144 µm3 after the application of weight. It was observed that a single cell may produce as many as six nanotubes, connected simultaneously to six neighbouring cells in different directions. The in-depth analysis confirmed that these structures were the intra-species connecting tools as no free nanotubes were found. Furthermore, after the application of weight, cells incapable of producing nanotubes were wiped out and the surface was covered by nanotube producers. This suggests that the nanotubes give a selective advantage to the cells to resist harsh environmental conditions and weight pressure. After the removal of weight and proper supply of nutrients, these phenotypes reverted to normal planktonic lifestyles. It is concluded that the nanotubes are not merely the phenomenon of dying cells; rather they are a connectivity tool which helps connected cells to tolerate and resist environmental stress.

Objectives: Objective: This study was designed to explore the role of different phenotypes of P. aeruginosa in the development, stability and persistence of biofilm. Methods: A total of seventeen (17) waterborne biofilm producing strains of P. aeruginosa were studied. These isolates were identified on the basis of typical phenotypic characters and the tube method was used for the study of biofilms. Population and phenotypic variance were studied by the drop plate method. The hydrophobicity of strains was evaluated by the bacterial adhesion to apolar solvent test. Results: Study showed that the subject isolates of P. aeruginosa adopted a biofilm life style after 36 h of incubation at 35 °C. After 24 h the adhesion started, but it was reversible and easily dispersed by simple washing. However, after 36 h the irreversible adhesion was noticed. The biofilm consortia harbor three different phenotypes: i. wild types, showed typical P. aeruginosa characters on Cetrimide agar; ii. Slow growers, showed poor pigmentation and take >36 h for colony development, and iii. Small colony variants (SCVs) are metabolically inactive and producing pinpointed non pigmented colonies. Comparative analysis showed that these phenotypes i.e. SCVs were highly hydrophobic and persistent in biofilm consortia due to the production of excessive amounts of exopolysaccharides. Conclusions: This study showed that phenotypic heterogeneity is a characteristic feature of P. aeruginosa biofilms and all of these phenotypes have a major role in stability and persistence of biofilm consortia.

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