Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Influence of substratum hydrophobicity on salivary pellicles: organization or composition?

Different physico-chemical properties (eg adsorption kinetics, thickness, viscoelasticity, and mechanical stability) of adsorbed salivary pellicles depend on different factors, including the properties (eg charge, roughness, wettability, and surface chemistry) of the substratum. Whether these differences in the physico-chemical properties are a result of differences in the composition or in the organization of the pellicles is not known. In this work, the influence of substratum wettability on the composition of the pellicle was studied. For this purpose, pellicles eluted from substrata of different but well-characterized wettabilities were examined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that substratum hydrophobicity did not have a major impact on pellicle composition. In all substrata, the major pellicle components were found to be cystatins, amylases and large glycoproteins, presumably mucins. In turn, interpretation of previously reported data based on the present results suggests that variations in substratum wettability mostly affect the organization of the pellicle components.     

Effect of cadmium and zinc on attachment and detachment interactions of Pseudomonas fluorescens H2 with glass.

The physiological and physicochemical bases for the effect of 5, 10, 50, or 100 micrograms of Cd and Zn ml-1 on the attachment and detachment interactions of Pseudomonas fluorescens H2 with glass substrata were determined. Attachment and detachment varied with the type and concentration of metal and the time at which cells were exposed to the metal. The largely inhibitory effect of the metals on bacterial motility and physiological activity did not directly influence attachment. The amount of Cd or Zn accumulated by the cells increased with metal concentration and was greater for free than for attached cells. The hydrophobicity and negative and positive charges of the bacterial surfaces (measured by hydrophobic and electrostatic interaction chromatography) were increased by cell exposure to the metals, particularly after Cd treatment. Cells exposed to Cd prior to attachment showed increased adhesion. Zinc-treated cells did not. There was a positive correlation between adhesion and Cd concentration in the attachment solution. No such relationship existed for Zn. P. fluorescens H2 exposed to Cd prior to attachment desorbed similarly to untreated controls. Zinc pretreatment resulted in decreased desorption. Cells attached in 5 or 10 micrograms of Cd or Zn ml-1 detached less than those attached in 50 or 100 micrograms of Cd or Zn ml-1. The presence of Cd or Zn during detachment had little effect on desorption. The dominant influence of Cd and Zn on attachment and detachment appears to be through modification of the bacterial surface. In natural ecosystems, heavy metals may influence the distribution of bacteria between the solid and liquid phases.     

Involvement of a rhamnolipid-producing strain of Pseudomonas aeruginosa in the degradation of polycyclic aromatic hydrocarbons by a bacterial community

A rhamnolipid-producing strain of Pseudomonas aeruginosa GL1 was isolated from a bacterial community growing on a mixture of polycyclic aromatic hydrocarbons (PAH) as sole carbon source. Strain GL1 did not grow on PAH but grew on known degradation metabolites of phenanthrene ( o -phthalic acid) and of naphthalene (salicylic acid). In co-culture with a phenanthrene-degrading strain, Ps. aeruginosa GL1 accelerated the degradation of phenanthrene. Strain GL1 was resistant to toxic amphiphilic compounds such as cationic and anionic detergents. Rhamnolipid production took place in a late stage growth in cultures of strain GL1 on glycerol or n -hexadecane. It coincided with a substantial decrease in cell hydrophobicity and with morphological changes of the outer membrane as observed by transmission electronic microscopy. The rhamnolipids produced inhibited the growth of bacteria such as Rhodococcus erythropolis , Bacillus cereus and Ps. fluorescens . The overall results suggested an outer membrane origin for the rhamnolipids. They also indicate that the utilization of PAH metabolites by strain GL1 is important for the stability of the PAH-degrading community.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Intrinsic and acquired resistance to quaternary ammonium compounds in food-related Pseudomonas spp

AIMS: To determine the sensitivity of a strain used for disinfectants testing (Pseudomonas aeruginosa ATCC 15442) and food-associated isolates to benzalkonium chloride and didecyl dimethylammonium chloride (DDAC). To determine whether the increase in bacterial resistance after adaptation to DDAC can be associated with phenotypic changes. To test the activity of alternative disinfectants to eliminate resistant Pseudomonas spp. METHODS AND RESULTS: Pseudomonas aeruginosa ATCC 15442 was among the most resistant strains tested using a bactericidal suspension test. Growth of a sensitive Ps. fluorescens in gradually higher concentrations of DDAC resulted in stable higher resistance and to some cross-resistance to several antibacterial agents, with the exception of disinfectants containing chloramine T, glutaraldehyde or peracetic acid. It was shown by microscopy that adaptation was followed by loss of flagella, and slime formation. Removal of the slime by sodium dodecyl sulphate resulted in partial loss of the acquired resistance. CONCLUSIONS: Pseudomonas spp. may adapt to survive against higher concentrations of quaternary ammonium compounds (QACs), but resistant strains can be eliminated with chemically unrelated disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: The work supports the rotation of disinfectants in food processing environments for avoiding the development of bacterial resistance to QACs. The alternating disinfectants should be chosen carefully, because of possible cross-resistance.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water

M. GENNARI AND F. DRAGOTTO. 1992. Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water.

Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

Enzymatic removal and disinfection of bacterial biofilms.

Model biofilms of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and Pseudomonas aeruginosa were made on steel and polypropylene substrata. Plaque-resembling biofilms of Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum were made on saliva-coated hydroxyapatite. The activity of enzymes against bacterial cells in biofilm was measured by fluorescence microscopy and an indirect conductance test in which evolution of carbon dioxide was measured. Glucose oxidase combined with lactoperoxidase was bactericidal against biofilm bacteria but did not remove the biofilm from the substrata. A complex mixture of polysaccharide-hydrolyzing enzymes was able to remove bacterial biofilm from steel and polypropylene substrata but did not have a significant bactericidal activity. Combining oxidoreductases with polysaccharide-hydrolyzing enzymes resulted in bactericidal activity as well as removal of the biofilm.     

ppGpp accumulation in Pseudomonas aeruginosa and Pseudomonas fluorescens subjected to nutrient limitation and biocide exposure

The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps. fluorescens, was examined. It is concluded that BIT is able to induce a stringent response in Ps. aeruginosa and Ps. fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue. This is the first report of a RelA-like protein in pseudomonads.     

The diversity of lipases from psychrotrophic strains of Pseudomonas : a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene ( lipA ) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens , the gene encodes a lipase of M_r 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M_r approximately 30 kDa comprising sequences from Ps. fragi , Ps. aeruginosa , Ps. fluorescens C9 and Burkholderia , and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities

Aims:  To investigate the mechanism of insoluble phosphate (P) solubilization and plant growth-promoting activity by Pseudomonas fluorescens RAF15.
Methods and Results:  We investigated the ability of Ps. fluorescens RAF15 to solubilize insoluble P via two possible mechanisms: proton excretion by ammonium assimilation and organic acid production. There were no clear differences in pH and P solubilization between glucose-ammonium and glucose-nitrate media. P solubilization was significantly promoted with glucose compared to fructose. Regardless of nitrogen sources used, Ps. fluorescens RAF15 solubilized little insoluble P with fructose. High performance liquid chromatography analysis showed that Ps. fluorescens RAF15 produced mainly gluconic and tartaric acids with small amounts of 2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with soluble P concentration. Ps. fluorescens RAF1 showed the properties related to plant growth promotion: pectinase, protease, lipase, siderophore, hydrogen cyanide, and indoleacetic acid.
Conclusion:  This study indicated that the P solubility was directly correlated with the organic acids produced.
Significance and Impact of the Study:  Pseudomonas fluorescens RAF15 possessed different traits related to plant growth promotion. Therefore, Ps. fluorescens RAF15 could be a potential candidate for the development of biofertilizer or biocontrol agent.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

Siderophore production by using free and immobilized cells of two pseudomonads cultivated in a medium enriched with Fe and/or toxic metals (Cr, Hg, Pb)

Pseudomonads are serious candidates for siderophore production applied to toxic metal (TM) solubilization. The bioaugmentation of contaminated soils by these TM-solubilizing bacteria combined with phytoextraction is an emerging clean-up technology. Unfortunately, siderophore synthesis may be drastically reduced by soluble iron in soils and bacteria can suffer from TM toxicity. In this study, we compared siderophore production by Pseudomonas aeruginosa and Pseudomonas fluorescens by using free and immobilized cells in Ca-alginate beads incubated in a medium containing Fe and/or TM (mixture of Cr, Hg, and Pb in concentrations which represented the soluble fraction of a contaminated agricultural soil). Free cell growth was stimulated by Fe, whatever the microorganism, the inoculum size and the presence or not of TM might have been. P. aeruginosa was less sensitive to TM than P. fluorescens. By comparison with free cells, immobilization with the high inoculum size showed less sensitivity to TM most probably because of lower metal diffusion in beads. Indeed, a maximum of 99.1% of Cr, 57.4% of Hg, and 99.6% of Pb were adsorbed onto beads. The addition of iron in the culture medium reduced significantly siderophore production of free cells while it led only to a low decrease with their immobilized counterparts, in particular with P. aeruginosa. In culture medium enriched with Fe and/or TM, siderophore-specific production of immobilized cells was higher than for free cells.     

Adsorption of flavobacterium breve and pseudomonas fluorescens p17 on different metals: Electrochemical polarization effect

The electrochemical polarization effect on early adsorption of Flavobacterium breve and Pseudomonas fluorescens P17 to platinum, titanium, stainless steel, copper, aluminum alloy and mild steel was studied. A well‐defined peak characterized the bacterial adsorption dependence on externally applied potential. Maximal adsorption occurred in the potential range of ‐0.5 to 0.5 V (SCE) for all tested metals. A shift of applied potential towards both a positive and a negative direction from the maximal adsorption potential (Emax,ad) was accompanied by a gradual decrease in bacterial adsorption. The extent of bacterial adsorption strongly depended on the nature of the metallic substratum and decreased accordingly as follows: platinum > titanium > stainless steel > aluminum alloy > carbon steel > copper. Adsorption on all tested metals was approximately two orders of magnitude higher with the relatively more hydrophobic F. breve compared to the less hydrophobic P. fluorescens P17. The effect of electrochemical polarization on the initial stages of bacterial adsorption onto metallic substrata is further discussed.     

Effects of Temperature and Nutrients on Proteinase Production by Pseudomonas fluorescens and Ps. aeruginosa in Broth and Milk

Temperature and the composition of the medium influenced the production of proteinase by Pseudomonas fluorescens and Pseudomonas aeruginosa isolated from raw milk. Many isolates of Ps. fluorescens digested litmus milk at 10° but not at 5° or 2°. With Ps. fluorescens proteinase production per unit of growth in a Peptone–Yeast Extract broth declined progressively as the incubation temperature was reduced from 20° to 5°. At 30° there was heavy growth in the same medium but only slight proteinase production whereas enzyme production by Ps. aeruginosa was maximal at this temperature. Proteinase production by both species in semi-defined media was essentially a function of the organic nitrogen content of the medium; there was evidence of catabolite repression by glucose and, to a lesser extent, lactate. In milks seeded with these pseudomonads, the extent of proteolysis was either increased markedly or slightly decreased when glucose was included.     

Zinc and glycerol enhance the production of nematicidal compounds in vitro and improve the biocontrol of Meloidogyne javanica in tomato by fluorescent pseudomonads

AIMS: To assess the effects of various carbon and mineral sources on the nematicidal potential of biocontrol inoculants of Pseudomonas aeruginosa IE-6S+ and Ps. fluorescens CHA0 under laboratory and glasshouse conditions. METHODS AND RESULTS: Culture filtrates of strains IE-6S+ and CHA0, cultured in nutrient yeast extract broth, caused substantial mortality of the juveniles of Meloidogyne javanica. The nematicidal activities of the culture filtrates were altered after amendment with various carbon and mineral sources. Soil amendment with zinc alone or in combination with glycerol improved the biocontrol efficacy against root-knot nematode, promoted tomato plant growth and enhanced bacterial rhizosphere and endophytic colonization. CONCLUSIONS: Appropriate quantities of glycerol and zinc alone or in combination enhance the nematicidal activity of Ps. aeruginosa and Ps. fluorescens. Glucose reduces the activity of these bacteria against nematodes. SIGNIFICANCE AND IMPACT OF THE STUDY: Minerals and carbon sources are appealing because they are easy and economical to provide during liquid fermentation of inoculants or as fertilizer amendments to improve the biocontrol activity of indigenous and introduced bacteria.     

Kinetics of adhesion of the oral bacterium Streptococcus sanguis CH3 to polymers with different surface free energies.

The kinetics of adhesion of Streptococcus sanguis CH3 from suspension to polymers with different surface free energies were studied by using three bacterial concentrations (2.5 X 10(7), 2.5 X 10(8), and 2.5 X 10(9) cells per ml-1). Substratum surface free energies (gamma s) ranged from 18 to 120 erg cm-2. The kinetics of bacterial adhesion to these surfaces showed a typical two-step adhesion process, indicating an equilibrium in both steps. In the initial adhesion step (step 1), low equilibrium numbers of adhering bacteria were counted on substrata with surface free energies lower than 55 erg cm-2. A maximal number adhered on substrata with higher surface free energies. At the lowest bacterial concentration tested, the highest number of bacteria were found on substrata with a surface free energy around 55 erg cm-2. For each substratum, step 2 started after a characteristic time interval tau, being short (30 min) for gamma s less than 50 and long (120 min) for gamma s greater than 50 erg cm-2. The relationship between the substratum surface free energy and the number of bacteria adhering at equilibrium after step 2 was similar to, although less distinct than, that during step 1 with a slight indication of a bioadhesive minimum around gamma s = 35 erg cm-2. The results are indicative of a two-step adhesion model, in which step 1 is controlled by macroscopic substratum properties.     

Kinetics of adhesion of the oral bacterium Streptococcus sanguis CH3 to polymers with different surface free energies

The kinetics of adhesion of Streptococcus sanguis CH3 from suspension to polymers with different surface free energies were studied by using three bacterial concentrations (2.5 X 10(7), 2.5 X 10(8), and 2.5 X 10(9) cells per ml-1). Substratum surface free energies (gamma s) ranged from 18 to 120 erg cm-2. The kinetics of bacterial adhesion to these surfaces showed a typical two-step adhesion process, indicating an equilibrium in both steps. In the initial adhesion step (step 1), low equilibrium numbers of adhering bacteria were counted on substrata with surface free energies lower than 55 erg cm-2. A maximal number adhered on substrata with higher surface free energies. At the lowest bacterial concentration tested, the highest number of bacteria were found on substrata with a surface free energy around 55 erg cm-2. For each substratum, step 2 started after a characteristic time interval tau, being short (30 min) for gamma s less than 50 and long (120 min) for gamma s greater than 50 erg cm-2. The relationship between the substratum surface free energy and the number of bacteria adhering at equilibrium after step 2 was similar to, although less distinct than, that during step 1 with a slight indication of a bioadhesive minimum around gamma s = 35 erg cm-2. The results are indicative of a two-step adhesion model, in which step 1 is controlled by macroscopic substratum properties.     

Surface hydrophobicity and dispersal of Pseudomonas aeruginosa from biofilms

A novel method of cell culture was employed to control the growth-rate of bacterial biofilms [1]. Cell-surface hydrophobicity increased progressively with growth rate for planktonic, chemostatgrown Pseudomonas aeruginosa and also for cells, resuspended from the biofilms. Dependence of surface hydrophobicity upon growth rate was greater for the planktonic cells. Newly-formed daughter cells, shed from the biofilms, were in all cases more hydrophilic than their adherent counterparts and demonstrated only slight growth rate dependency for this property.