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The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3-10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.

This study was performed in order to characterize the relationship between adhesion and biofilm formation abilities of drinking water-isolated bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.). Adhesion was assessed by two distinct methods: thermodynamic prediction of adhesion potential by quantifying hydrophobicity and the free energy of adhesion; and by microtiter plate assays. Biofilms were developed in microtiter plates for 24, 48 and 72 h. Polystyrene (PS) was used as adhesion substratum. The tested bacteria had negative surface charge and were hydrophilic. PS had negative surface charge and was hydrophobic. The free energy of adhesion between the bacteria and PS was > 0 mJ/m(2) (thermodynamic unfavorable adhesion). The thermodynamic approach was inappropriate for modelling adhesion of the tested drinking water bacteria, underestimating adhesion to PS. Only three (B. cepacia, Sph. capsulata and Staphylococcus sp.) of the six bacteria were non-adherent to PS. A. calcoaceticus, Methylobacterium sp. and M. mucogenicum were weakly adherent. This adhesion ability was correlated with the biofilm formation ability when comparing with the results of 24 h aged biofilms. Methylobacterium sp. and M. mucogenicum formed large biofilm amounts, regardless the biofilm age. Given time, all the bacteria formed biofilms; even those non-adherents produced large amounts of matured (72 h aged) biofilms. The overall results indicate that initial adhesion did not predict the ability of the tested drinking water-isolated bacteria to form a mature biofilm, suggesting that other events such as phenotypic and genetic switching during biofilm development and the production of extracellular polymeric substances (EPS), may play a significant role on biofilm formation and differentiation. This understanding of the relationship between adhesion and biofilm formation is important for the development of control strategies efficient in the early stages of biofilm development.

Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.

Sphingomonas species were commonly isolated from biofilms in drinking water distribution systems in Finland (three water meters) and Sweden (five water taps in different buildings). The Sphingomonas isolates (n = 38) were characterized by chemotaxonomic, physiological and phylogenetic methods. Fifteen isolates were designated to species Sphingomonas aromaticivorans, seven isolates to S. subterranea, two isolates to S. xenophaga and one isolate to S. stygia. Thirteen isolates represented one or more new species of Sphingomonas. Thirty-three isolates out of 38 grew at 5 degrees C on trypticase soy broth agar (TSBA) and may therefore proliferate in the Nordic drinking water pipeline where the temperature typically ranges from 2 to 12 degrees C. Thirty-three isolates out of 38 grew at 37 degrees C on TSBA and 15 isolates also grew on blood agar at 37 degrees C. Considering the potentially pathogenic features of sphingomonas, their presence in drinking water distribution systems may not be desirable.