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PubMed2005

Use of microelectrodes to investigate the effects of 2-chlorophenol on microbial activities in biofilms.

In order to assess the applicability of using microelectrodes as a tool for inhibition tests, temporal and spatial inhibitory effects of 2-chlorophenol (2-CP) on O(2) respiration and nitrification activities in municipal wastewater biofilms were investigated using microelectrodes for O(2) and NH(4)(+). The time-course microelectrode measurements demonstrated that 2-CP inhibited O(2) respiration and nitrification activities within 6-18 min. The microbial activities were inhibited only in the upper 400 microm of the biofilms by 2-CP, and the bacteria present in the deeper parts of the biofilms were still active, probably due to limited penetration of 2-CP. These results could reasonably explain the difference in inhibitory ratios of the O(2) respiration and nitrification activities in the biofilms. O(2) respiration activity was incompletely inhibited, which was attributed to the presence of O(2) respiration activities in the deeper parts of the biofilm. In contrast, nitrification activity was significantly inhibited because ammonia-oxidizing bacteria were present in the upper parts of the biofilm. These results indicate that the microelectrodes with a very quick response time and a high spatial resolution are useful tools to study temporal and spatial inhibitory effects of inhibitors on in situ microbial activities in biofilms.

Bacterial Physiological Phenomena↗
PubMed2005

A general description of detachment for multidimensional modelling of biofilms.

A general method for describing biomass detachment in multidimensional biofilm modelling is introduced. Biomass losses from processes acting on the entire surface of the biofilm, such as erosion, are modelled using a continuous detachment speed function F(det). Discrete detachment events, i.e. sloughing, are implicitly derived from simulations. The method is flexible to allow F(det) to take several forms, including expressions dependent on any state variables such as the local biofilm density. This methodology for biomass detachment was integrated with multidimensional (2D and 3D) particle-based multispecies biofilm models by using a novel application of the level set method. Application of the method is illustrated by trends in the dynamics of biofilms structure and activity derived from simulations performed on a simple model considering uniform biomass (case study I) and a model discriminating biomass composition in heterotrophic active mass, extracellular polymeric substances (EPS) and inert mass (case study II). Results from case study I demonstrate the effect of applied detachment forces as a fundamental factor influencing steady-state biofilm activity and structure. Trends from experimental observations reported in literature were correctly described. For example, simulation results indicated that biomass sloughing is reduced when erosion forces are increased. Case study II illustrates the application of the detachment methodology to systems with non-uniform biomass composition. Simulations carried out at different bulk concentrations of substrate show changes in biofilm structure (in terms of shape, density and spatial distribution of biomass components) and activity (in terms of oxygen and substrate consumption) as a consequence of either oxygen-limited or substrate-limited growth.

Bacterial Adhesion↗
PubMed2006

Optical method for long-term and large-scale monitoring of spatial biofilm development.

A method was developed that allows biofilm monitoring on the square centimeter scale over extended periods of time. The method is based on image acquisition using a desktop scanner and subsequent image analysis. It was shown that results from grey level analysis are highly correlated with physical properties of the biofilm like average biomass and biofilm thickness. The scanner method was applied to monitor overall biofilm growth, detachment, and surface roughness during two 3 and 4 week long experiments. Two significantly different growth dynamics during the biofilm development could be identified, depending on the biofilm history. Surface roughness on transects in flow direction was always higher than on transects perpendicular to the flow, reflecting the anisotropic characteristics of biofilms growing in a flow field.

Bacteria↗
PubMed2006

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR.

Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results, the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities.

Bacteria↗
PubMed2001

Air-flow resistances of silicone rubber voice prostheses after formation of bacterial and fungal biofilms.

Laryngectomized patients use silicone rubber voice prostheses to rehabilitate their voice. However, biofilm formation limits the lifetime of voice prostheses by causing leakage or an increased air-flow resistance and the prosthesis has to be replaced. To determine which bacterial or yeast strains, isolated from explanted voice prostheses, contribute most to increases in air-flow resistance of silicone rubber voice prostheses, biofilms consisting of either a bacterial or a yeast strain were grown on voice prostheses in the artificial throat model. The effects of these biofilms on air-flow resistances were determined by calculating the difference in air-flow resistance of the individual voice prosthesis as covered with a 7-day-old biofilm with the situation prior to biofilm formation. Conspicuously, voice prosthetic biofilms formed by the bacterial strains Staphylococcus aureus GB 2/1 and Rothia dentocariosa GBJ 41/25B and their excreted organic matter showed larger increases in air-flow resistance (more then 30 cm H(2)O.s/L) than biofilms formed by Candida species. This is contrary to the literature, where there seems to be agreement that Candida species are mainly responsible for clinical failure of silicone rubber voice prostheses.

Bacteria↗
PubMed2005

Chlorhexidine-induced ultrastructural alterations in oral biofilm.

Chlorhexidine, the most used biocide in periodontology, alters the permeability of the bacterial cell membrane. However, the chlorhexidine-induced morphological alterations in the oral biofilm have not been studied. To examine the effects of chlorhexidine on oral biofilm on an electron microscopic level, gingival epithelial cells with attached biofilm were collected from 10 volunteers, subjected to 0.1% chlorhexidine for 1 or 5 min, stained with ruthenium red-tetroxide, and analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM visualized the bacterial glycocalyces and the biofilm matrix on the biofilm surface; however, no chlorhexidine-induced alterations were observed. TEM revealed loss of bacterial membrane integrity and fimbrial disintegration in a few bacteria. In the proximity of these alterations, a restricted matrix disintegration was also observed. However, the chlorhexidine-induced alterations only effected a minor part of the oral biofilm and did not cause its disintegration. These findings suggest the insufficient efficiency of chlorhexidine against oral biofilm.

Adolescent↗
PubMed2002

Oral bacteria in multi-species biofilms can be killed by red light in the presence of toluidine blue.

BACKGROUND AND OBJECTIVES: Oral bacteria can be killed by light in the presence of a suitable photosensitizer, and this could be used in the treatment of oral infections. In these diseases, however, bacteria are present as biofilms, which are refractive to antimicrobial agents. The purpose of this study was to determine whether oral bacterial biofilms were susceptible to lethal photosensitization. STUDY DESIGN/MATERIALS AND METHODS: Multi-species biofilms of oral bacteria were irradiated with light from a helium/neon laser in the presence of toluidine blue O (TBO) and the survivors enumerated. Controls examining the effects of light and TBO alone were also included. The biofilms were also examined by confocal scanning laser microscopy (CSLM). RESULTS: CSLM revealed that the biofilms had structures similar to those of dental plaque. Although, the biofilms consisted of extremely large numbers of bacteria ( approximately 9 x 10(9)), 97.4% were killed following irradiation with 31.5 J of laser light in the presence of 25 microg/ml TBO. CONCLUSIONS: Substantial numbers of oral bacteria in multi-species biofilms can be killed by light in the presence of TBO. This may be useful in the treatment of dental plaque-related diseases.

Adult↗
PubMed2006

Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA.

Bacterial colonisation and biofilm formation on the surface of urinary catheters is a common cause of nosocomial infection, and as such is a major impediment to their long-term use. Understanding the mechanisms of biofilm formation on urinary catheters is critical to their control and will aid the future development of materials used in their manufacture. In this report we have used proteomic analysis coupled with immunoassays to show that the major outer membrane protein (OmpA) of Escherichia coli is overexpressed during biofilm formation. A series of synthetic hydrogels being developed for potential use as catheter coatings were used as the substrata and OmpA expression was increased in biofilms on all these surfaces, as well as being a feature of both a laboratory and a clinical strain of E. coli. Up-regulation of OmpA may, therefore, be a common feature of E. coli biofilms. These findings present OmpA as a potential target for biofilm inhibition and may contribute to the rational design of biofilm inhibiting hydrogel coatings for urinary catheters.

Bacterial Outer Membrane Proteins↗
PubMed2006

Bacterial evasion of antimicrobial peptides by biofilm formation.

Biofilm formation is a main virulence determinant in many bacterial infections. It significantly increases bacterial resistance to antibiotics and innate host defense. In general, the specific physiology of biofilms and the barrier function of the extracellular biofilm matrix determine resistance to antibacterials. However, resistance to antimicrobial peptides appears to be mainly based on the interaction with biofilm and capsule exopolymers. These polymers may work by electrostatic repulsion and/or sequestration of antibacterial substances. As biofilm polymers play an eminent role in biofilm structuring and resistance, their destruction by dedicated enzymes is a promising attempt to prevent colonization and develop treatment for biofilm-associated infections.

Adhesins, Bacterial↗
PubMed1995

The effect of electrical currents and tobramycin on Pseudomonas aeruginosa biofilms.

The combined use of antibiotics with low levels of electrical current has been reported to be more effective in controlling biofilms (the bioelectric effect) than antibiotics alone. An electrical colonisation cell was designed to study the effect of antibiotics on biofilms formed on a dialysis membrane away from the electrode surface. To avoid the electrochemical generation of toxic products, Pseudomonas aeruginosa biofilms were formed in minimal salts medium that excluded chloride-containing compounds. Under these conditions, electrical currents of up to 20 mA cm-2 did not prevent biofilm formation or have any detrimental effect on an established biofilm. Tobramycin alone at concentrations of 10 micrograms ml-1 did not affect the biofilm, but were significantly enhanced by 9 mA cm-2. The effect of tobramycin concentrations of 25 micrograms ml-1 were enhanced by a 15 mA cm-2 electrical current. In both cases higher levels of electrical current, up to 20 mA cm-2, did not further enhance the effect of the antibiotic. The possible mechanisms of action of the bioelectric effect have been reported to involve electrophoresis, iontophoresis and electroporesis, thus overcoming the biofilm biomass and cell wall barriers. Our results suggest that other factors may also be important, such as the metabolic activity and growth rate of the bacteria. Such factors may be critical in maximising antibiotic efficacy.

Anti-Bacterial Agents↗
PubMed2001

Factors compromising antibiotic activity against biofilms of Staphylococcus epidermidis.

Several factors associated with bacterial biofilms were studied for their role in phenotypic resistance to antibiotics. These factors included bacterial slime extracted from biofilms, reduced growth rates of biofilm-embedded bacteria and high bacterial inocula. Antibiotic activity against suspended bacteria in the presence of these factors, either alone or combined, was compared with activity against adherent biofilms. All MICs, determined by standard susceptibility tests, were below the sensitivity breakpoints for Staphylococcus epidermidis strain V2. The addition of bacterial slime to suspended bacteria reduced the bactericidal activity of glycopeptides but had less or no effect on the activity of the other antibiotics tested. High bacterial inocula affected the activity of flucloxacillin and quinolones only moderately or not at all, though a more pronounced effect on glycopeptides was observed. In contrast, the bactericidal activity of most antibiotics was severely compromised when adherent bacterial biofilms were used as inocula. In conclusion, the presence of slime, slow growth rates and high bacterial counts may explain the poor activity of glycopeptides against biofilm-embedded organisms, but these factors, either alone or in combination, do not explain the lack of bactericidal activity of other drugs against biofilms. Thus, additional factors need to be identified.

Anti-Bacterial Agents↗
PubMed2007

Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform infrared spectroscopy and other corroborative techniques.

This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-microm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg(+)) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 microm in diameter and 24 microm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.

Biofilms↗
PubMed2003

An aufwuchs chamber slide for high-resolution confocal laser scanning microscopy and stereo imaging of microbial communities in natural biofilms.

Aufwuchs chamber slides were constructed by attaching a silicone rubber gasket to a glass slide with epoxy cement. For biofilm growth, the slides were suspended in Cayuga Lake near Ithaca, NY, for 27 days. Biofilms in the chamber were stained with 0.05% acridine orange. After rinsing, the chamber was filled with molten 1% agarose to stabilize filaments and delicate polymer structures at the biofilm surface. Areas of biofilm approximately 0.5 mm thick on the inner face of the wall of the chamber were selected for side-on optical sectioning in a confocal laser scanning microscope (CLSM). Stacks of high-resolution optical images captured by the CLSM z-sectioning software, were used to create left-right stereo image pairs. At low magnification the stereo pairs showed 3-D details of the microbial landscape in the mature biofilms. Channels, pores, and other structural features of the biofilm matrix were observed in peripheral regions. Higher magnification images revealed the 3-D distribution of specific biofilm components such as filaments of sheathed bacteria projecting outward into the liquid milieu, and organic coatings, including bacterial cells on the surfaces of mineral particles.

Acridine Orange↗
PubMed2002

Pitting corrosion inhibition of aluminum 2024 by Bacillus biofilms secreting polyaspartate or gamma-polyglutamate.

Pitting corrosion of aluminum 2024 in Luria Bertani medium was reduced by the secretion of anionic peptides by engineered and natural Bacillus biofilms and was studied in continuous reactors using electrochemical impedance spectroscopy. Compared to sterile controls, pitting was reduced dramatically by the presence of the biofilms. The secretion of a 20 amino acid polyaspartate peptide by an engineered Bacillus subtilis WB600/pBE92-Asp biofilm slightly reduced the corrosion rate of the passive aluminum alloy at pH 6.5; however, the secretion of gamma-polyglutamate by a Bacillus licheniformis biofilm reduced the corrosion rate by 90% (compared to the B. subtilis WB600/pBE92 biofilm which did not secrete polyaspartate or gamma-polyglutamate). The corrosion potential ( E(corr)) of aluminum 2024 was increased by about 0.15-0.44 V due to the formation of B. subtilis and B. licheniformis biofilms as compared to sterile controls. The increase of E(corr) and the observed prevention of pitting indicate that the pitting potential ( E(pit)) had increased. This result and the further decrease of corrosion rates for the passive aluminum alloy suggest that the rate of the anodic metal dissolution reaction was reduced by an inhibitor produced by the biofilms. Purified gamma-polyglutamate also decreased the corrosion rates of aluminum 2024.

Aluminum↗
PubMed2004

Gene expression in Escherichia coli biofilms.

DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p< or =0.05) compared to suspension cells, including several genes for the stress response ( hslS, hslT, hha, and soxS), type I fimbriae ( fimG), metabolism ( metK), and 11 genes of unknown function ( ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.

Biofilms↗
PubMed2006

Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber.

We have recently isolated a biofilm-producing strain (C208) of Rhodococcus ruber that degraded polyethylene at a rate of 0.86% per week (r2=0.98). Strain C208 adheres to polyethylene immediately upon exposure to the polyolefin. This initial biofilm differentiates (in a stepwise process that lasts about 20 h) into cell-aggregation-forming microcolonies. Further organization yields "mushroom-like" three-dimensional structures on the mature biofilm. The ratio between the population densities of the biofilm and the planktonic C208 cells after 10 days of incubation was about 60:1, indicating a high preference for the biofilm mode of growth. Analysis of extracellular polymeric substances (EPS) in the biofilm of C208 revealed that the polysaccharides level was up to 2.5 folds higher than that of the protein. The biofilm showed a high viability even after 60 days of incubation, apparently due to polyethylene biodegradation.

Biodegradation, Environmental↗
PubMed2006

Motility influences biofilm architecture in Escherichia coli.

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms ( approximately 43 microm thick, 21 to 34% surface coverage). JM109, DH5alpha, and MG1655 motA formed intermediate biofilms ( approximately 13 microm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5alpha) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).

Biofilms↗
PubMed2004

Impact of the intraoral location on the rate of biofilm growth.

The aims of the present study were: a) to assess the impact of the intraoral location on the rate of biofilm growth, and b) to establish an in vivo biofilm model to examine intraoral biofilm growth. Eight healthy volunteers wore acrylic splints with 15 glass slabs each in the upper and lower jaws to build up plaque. After 48 h, the specimens were removed and stained using the vital fluorescence technique. Biofilm thickness was evaluated by confocal laser scanning microscopy (CLSM). The mean plaque thickness amounted to 77.6 +/- 29.1 microm on the buccal sites of the upper jaw and 71.9 +/- 26.3 microm on the buccal sites of lower jaw. On the palatal site a biofilm of 52.1 +/- 26.2 microm thickness was grown, which was significantly less compared with the other locations evaluated (p < 0.001). The results demonstrate that the in situ biofilm thickness on the buccal sites was similar irrespective of the location in the oral cavity. The new splint system described may be a useful tool for further standardised experimental studies regarding influences on growth and structure of intraoral biofilms.

Adult↗