The soil saprophyte Bacillus cereus forms biofilms at solid-liquid interfaces. The composition of the extracellular polymeric matrix is not known, but biofilms of other bacteria are encased in polysaccharides, protein, and also extracellular DNA (eDNA). A Tn917 screen for strains impaired in biofilm formation at a solid-liquid interface yielded several mutants. Three mutants deficient in the purine biosynthesis genes purA, purC, and purL were biofilm impaired, but they grew planktonically like the wild type in Luria-Bertani broth. Biofilm populations had higher purA, purC, and purL transcript ratios than planktonic cultures, as measured by real-time PCR. Laser scanning confocal microscopy (LSCM) of BacLight-stained samples indicated that there were nucleic acids in the cell-associated matrix. This eDNA could be mobilized off the biofilm into an agarose gel matrix through electrophoresis, and it was a substrate for DNase. Glass surfaces exposed to exponentially growing populations acquired a DNA-containing conditioning film, as indicated by LSCM. Planktonic exponential-phase cells released DNA into an agarose gel matrix through electrophoresis, while stationary-phase populations did not do this. DNase treatment of planktonic exponential-phase populations rendered cells more susceptible than control populations to the DNA-interacting antibiotic actinomycin D. Exponential-phase purA cells did not contain detectable eDNA, nor did they convey a DNA-containing conditioning film to the glass surface. These results indicate that exponential-phase cells of B. cereus ATCC 14579 are decorated with eDNA and that biofilm formation requires DNA as part of the extracellular polymeric matrix.

Bacillus is commonly isolated from soils, with organisms of Bacillus cereus sensu lato being prevalent. Knowledge of the ecology of B. cereus and other Bacillus species in soil is far from complete. While the older literature favors a model of growth on soil-associated organic matter, the current paradigm is that B. cereus sensu lato germinates and grows in association with animals or plants, resulting in either symbiotic or pathogenic interactions. An in terra approach to study soil-associated bacteria is described, using filtersterilized soil-extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM. B. cereus ATCC 14579 displayed a life cycle, with the ability to germinate, grow, and subsequently sporulate in both the liquid SESOM extract and in ASM inserted into wells in agar medium. Cells grew in liquid SESOM without separating, forming multicellular structures that coalesced to form clumps and encasing the ensuing spores in an extracellular matrix. Bacillus was able to translocate from the point of inoculation through soil microcosms as shown by the emergence of outgrowths on the surrounding agar surface. Microscopic inspection revealed bundles of parallel chains inside the soil. The motility inhibitor L-ethionine failed to suppress outgrowth, ruling out translocation by a flagellar-mediated mechanism such as swimming or swarming. Bacillus subtilis subsp. subtilis Marburg and four Bacillus isolates taken at random from soils also displayed a life cycle in SESOM and ASM and were all able to translocate through ASM, even in presence of L-ethionine. These data indicate that B. cereus is a saprophytic bacterium that is able to grow in soil and furthermore that it is adapted to translocate by employing a multicellular mode of growth.

Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods. B. cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2-and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up-and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and L-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both B and H , could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.

Nontuberculous mycobacteria (NTM) are ubiquitous and have been isolated from a variety of environmental sources, including water. Various NTM were isolated from biofilms in drinking water distribution systems in two urban and two semiurban areas in South Africa. Most of the isolates belonged to opportunistic pathogenic species of the NTM group, but none belonged to the Mycobacterium avium complex.The genus Mycobacterium comprises both the strictly pathogenic species that are transmitted by human or animal reservoirs only (M. tuberculosis, M. leprae) and the so-called nontuberculous mycobacteria (NTM) (3,13,14). The NTM have generally been associated with soil and water, and while many of them are considered to be nonpathogenic, an increasing number are being reported as opportunistic pathogens (3,5,16). This growing number of atypical pathogenic mycobacteria includes M. abscessus (8), M. chelonae (10), M. fortuitum (16,27), M. gordonae (19), M. mageritense (9), and M. xenopi (5). Several NTM constitute a risk not only to immunosuppressed persons but also to otherwise healthy persons (10). They can cause pulmonary and cutaneous diseases, lymphadenitis, and other infections (14).M. abscessus, M. gilvum, M. gordonae, and M. mageritense have been associated with municipal water supplies (7,9,12). In a recent report, furunculosis, caused by M. mageritense, was also linked to the water supply of a salon where two women received footbaths (9). In another report, cervical lymphadenitis in children below 2 years of age has been linked with mycobacteria in the United States, the United Kingdom, and Australia (21). These infections were linked to the prevalence of M. avium and M. scrofulaceum in water (21). There are also reports that NTM can be present in aerosols, such as at swimming pools and spas, of water that may contain mycobacteria and that individuals exposed to the aerosols for extended periods are more at risk of contracting an infection (6). In the United States alone, over a million workers are exposed to aerosols generated by metal grinding, and exposure to such aerosols can lead to hypersensitivity, pneumonitis, and chronic obstructive pulmonary disease (6, 21).NTM are tolerant to a much wider pH and temperature range than are most other bacterial pathogens detected in municipal water supplies. They are also generally tolerant to chlorine, making them potentially more difficult to eliminate (12, 13). Adding to this is their ability to form biofilms on surfaces in drinking water distribution systems (12, 24). The growth of NTM in biofilms may lead to dissemination into the bulk water, constituting a risk to consumers both by drinking and by inhalation of aerosols though showering and swimming.NTM can form biofilms under low-nutrient conditions, making surfaces of drinking water distribution systems an environment for their growth and possible dissemination (12). The aim of this study was to determine the presence and diversity of NTM in biofilms in drinking water distribution systems by analyzing samples from...

We investigated the composition of soil-extracted solubilized organic and inorganic matter (SESOM) prepared from three different soils. Growth of various bacterial strains in these soil extracts was evaluated to find appropriate conditions for ecophysiological approaches. Analysis of SESOM by (1)H-NMR and gas chromatography/mass spectrometry revealed a complex mixture of organic compounds. An oak forest SESOM supported the growth of several gram-positive and gram-negative soil-derived heterotrophic bacteria, whereas beech forest and grassland soil extracts did not. A metabolomic approach was performed by determining the extracellular metabolite profile of Bacillus licheniformis in SESOM. The results demonstrated that determination of the organic composition of SESOM during batch culturing is feasible. This makes SESOM amenable to studying the ecophysiology of a range of soil bacteria growing on soil-dissolved organic matter under more defined laboratory conditions. SESOM may also increase success in isolating previously uncultured or novel soil bacteria. Cell populations and the corresponding extracellular medium can be obtained readily and specific components extracted, paving the way for proteomic, transcriptomic, and metabolomic analyses. The synthetic carbon mixture based on SESOM, which mimics soil abilities, shows a positive impact on higher cell yields and longer cultivation time for biotechnological relevant bacteria.