Search

Pseudomonas extremaustralis is a versatile Antarctic bacterium, able to grow under microaerobic and anaerobic conditions and is related to several non-pathogenic Pseudomonads. Here we report on the role of the global anaerobic regulator Anr, in the early steps of P. extremaustralis biofilm development. We found that the anr mutant was reduced in its ability to attach, to form aggregates and to display twitching motility but presented higher swimming motility than the wild type. In addition, microscopy revealed that the wild type biofilm contained more biomass and was thicker, but were less rough than that of the anr mutant. In silico analysis of the P. extremaustralis genome for Anr-like binding sites led to the identification of two biofilm-related genes as potential targets of this regulator. When measured using Quantitative Real Time PCR, we found that the anr mutant expressed lower levels of pilG, which encodes a component of Type IV pili and has been previously implicated in cellular adhesion. Levels of morA, involved in signal transduction and flagella development, were also lower in the mutant. Our data suggest that under low oxygen conditions, such as those encountered in biofilms, Anr differentially regulates aggregation and motility thus affecting the first stages of biofilm formation.

Removal of detrimental biofilms from surfaces exposed in the marine environment remains a challenge. A strain of Bacillus pumilus was isolated from the surface of titanium coupons immersed in seawater in the vicinity of Madras Atomic Power Station (MAPS) on the East coast of India. The bacterium formed extensive biofilms when compared to species such as Bacillus licheniformis, Pseudomonas aeruginosa PAO1 and Pseudomonas aureofaciens. A commercially available rhamnolipid was assessed for its ability to inhibit adhesion and disrupt pre-formed B. pumilus biofilms. The planktonic growth of B. pumilus cells was inhibited by concentrations >1.6mM. We studied the effect of various concentrations (0.05-100mM) of the rhamnolipid on adhesion of B. pumilus cells to polystyrene microtitre plates, wherein the effectiveness varied from 46 to 99%. Biofilms of B. pumilus were dislodged efficiently at sub-MIC concentrations, suggesting the role of surfactant activity in removing pre-formed biofilms. Scanning electron microscopy (SEM) confirmed the removal of biofilm-matrix components and disruption of biofilms by treatment with the rhamnolipid. The results suggest the possible use of rhamnolipids as efficient anti-adhesive and biofilm-disrupting agents with potential applications in controlling biofilms on surfaces., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The adhesion of microbial cells to metal surfaces in aqueous media is an important phenomenon in both the natural environment and engineering systems. The adhesion of two anaerobic sulfate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.) to four polished metal surfaces (i.e., stainless steel 316, mild steel, aluminum, and copper) was examined using a force spectroscopy technique with an atomic force microscope (AFM). Using a modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range) between the bacterial cell and the metal surface in aqueous media were quantified. Results show that the bacterial adhesion force to aluminum is the highest among the metals investigated, whereas the one to copper is the lowest. The bacterial adhesion forces to metals are influenced by both the electrostatic force and metal surface hydrophobicity. It is also found that the physiological properties of the bacterium, namely the bacterial surface charges and hydrophobicity, also have influence on the bacteria-metal interaction. The adhesion to the metals by Pseudomonas sp. and D. desulfuricans was greater than by the marine SRB isolate. The cell-cell interactions show that there are strong electrostatic repulsion forces between bacterial cells. Cell probe atomic force microscopy has provided some useful insight into the interactions of bacterial cells with the metal surfaces.

The adhesion of bacteria on metal surfaces in aqueous media and the development of biofilm and resultant biofouling are important phenomena in both the natural environment and engineering systems. This work reports on the use of a force microscopy technique to measure bacterial metal adhesion by two anaerobic sulphate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.). Using a modified bacteria tip, the atomic force microscope was able to quantify the attraction and repulsion force in the nano-Newton range between the bacteria cell and metal surface in aqueous media. Results show that increasing surface hydrophobicity of the metal, and increasing the ionic strength of the aqueous medium both enhance the adhesion force. The adhesion forces were also influenced by the physiological properties of the bacterium, such as the bacterial surface charges and hydrophobicity.

The effect of phenotypic variation on attachment of Pseudomonas tolaasii and P. putida to Agaricus bisporus mycelium was investigated. Quantitative studies demonstrated the ability of each isolate to attach rapidly and firmly to A. bisporus mycelium and significant differences in attachment of wild-type and phenotypic variant strains were observed. This was most pronounced in P. tolaasii, where the percentage attachment of the wild-type form was always greater than that of the phenotypic variant. The medium upon which the bacteria were cultured, prior to conducting an attachment assay, had a significant effect on their ability to attach. Attachment of the wild-type form of P. putida was enhanced when the assay was performed in the presence of CaCl2, suggesting the involvement of electrostatic forces. No correlation was observed between bacterial hydrophobicity and ability to attach to A. bisporus mycelium. Scanning electron microscopy confirmed the results obtained from the quantitative studies and provided further evidence for marked differences in the ability of the pseudomonads to attach to mycelium. Fibrillar structures and amorphous material were frequently associated with attached cells and appeared to anchor bacteria to each other and to the hyphal surface. A time-course study of attachment using transmission electron microscopy revealed the presence of uneven fibrillar material on the surface of cells. This material stained positive for polysaccharide and may be involved in ensuring rapid, firm attachment of the cells.

Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

A marine Pseudomonas sp. S9 produced and released an extracellular polysaccharide during complete energy and nutrient starvation in static conditions. The presence of the polysaccharide on the cell surface, demonstrable by immune transmission electron microscopy, correlated with changes in the degree of adhesion to hydrophobic surfaces. Polysaccharide coated cells showed a lower degree of adhesion than did cells devoid of the polymer. After 10 h of starvation, no ruthenium red stained antibody stabilized polysaccharides could be observed on the cell surface. The polysaccharide was not produced during growth since lysates of mid-log phase cells did not precipitate the antiserum. The relative proportions of sugars in the polysaccharide were 28% glucose, 35% N-acetyl-glucosamine and 37% N-acetylgalactosamine. The released polysaccharide did not significantly alter the physical parameters of surface tension and viscosity of the starvation regime. Cells starved in agitated conditions did not produce any extracellular polysaccharides and exhibited a different adhesion pattern to hydrophobic surfaces.

Small bowel biopsy specimens were taken from 21 patients undergoing gastrointestinal endoscopy to detect a possible adhesion of bacteria to the mucous layer of the upper gastrointestinal tract. In 30 control biopsy specimens taken from 10 patients free from gastrointestinal pathology, no associated bacteria were found, whereas in 23 biopsy specimens taken from eight gastrectomized patients an associated bacterial flora including E. coli or Pseudomonas was grown. Bacterial adhesion was confirmed by means of transmission electron microscopy of the eight patients yielding positive cultures. Bacterial adhesion induced local alterations of the brush border membrane. These results suggest that adherent bacteria may be present in hypochlorhydric patients. Pathophysiologic consequences require further studies.