Diversity of heterotrophic bacteria isolated from biofilm samples and cell surface hydrophobicity.

Biofi lm formation is a well known phenomenon in the medical and health care fi elds. Biofi lm infection has recently attained a greater importance as an intractable infectious disease since the number of persons easily infected is rising as the percentage of the elderly population increases (Costerton et al., 1999). The environmental hygienic equipment and paper manufacturing industries call this phenomenon slime, and reportedly have considerable diffi culty in preventing it (Blanco et al., 1997; Väisänen et al., 1994). Biofi lms exist in our life environment such as the kitchen and bathroom, and are typically described as ‘slimy’ or ‘clammy.’ We analyzed heterotrophic bacteria constituting the biofi lm produced in cooling towers for air-conditioning and houses (bathroom drain) from a new viewpoint in which the internal environment of the biofi lm is relatively eutrophic, and measured the hydrophobicity of the cell surface layer to investigate the adhesiveness of isolates. The test samples were as follows; A: This biofi lm was collected from the heat exchanger of a cooling tower installed to cool the production line in a food manufacturing plant in Ibaraki Prefecture. The cooling tower was installed in 1998, and 5 tons of water was retained. For supply water, industrial and well water was mixed and used (pH: 8.1, electric conductivity: 48 mS/m, all hardness: 137 mg/L, chloride ion: 39 mg/L, sulfuric acid ion: 83 mg/L, silica: 64 mg/L). The biofi lm sample was black and muddy. This biofi lm (350 g) was combined with 350 ml of sterile saline, vigorously shaken for 30 min, and kept at 4°C overnight. The supernatant was centrifuged at 1,000 rpm for 5 min, and the resulting supernatant was used as the sample. B: A biofi lm which had formed in a bathroom drain (material: polypropylene, thickness: about 1 mm) in a house in the Tokyo metropolitan area was scraped with sterile swabs and kept in sterile saline during transport. After macroscopic observation, the sample biofi lm was collected by centrifugation at 1,000 rpm for 1 min. This procedure was repeated twice to wash the biofi lm with sterile saline, followed by vigorous mixing using a vortex for 15 min, and the sample was fi nally adjusted to 20 ml (TOC: 100.6 mg/L, ammonium ion: 0.25 mg/L, phosphoric acid ion, nitric acid ion and nitrous acid ion: not detected). The number of constituent bacteria was measured in consideration of the nutrients in the medium and duration of culture. Eutrophic BHI agar medium (BD, USA) and oligotrophic R2A agar medium (0.5 g pepton, 0.5 g yeast extract, 0.5 g casamino acid, 0.5 g J. Gen. Appl. Microbiol., 55, 69‒74 (2009)