Biofilm formation in the life cycle of the cellulolytic actinomycete Thermobifida fusca

ABSTRACTActinomycetes have been used with enormous success in industrial processes; however, little is known about biofilm formation by these filamentous microbes, or community development on insoluble substrates such as cellulose. We hypothesized that biofilm formation is a general strategy used by actinomycetes in the degradation of cellulose, and that it may serve as a means for these microbes to secure nutrients and persist in their environments. The objective of this study was to examine biofilm production byThermobifida fusca, an actinomycete that rapidly degrades cellulose by means of a well-characterized extracellular cellulase system.Thermobifida fuscacells grew as biofilms attached to both nutritive (e.g. dialysis tubing membrane) and non-nutritive surfaces. Dialysis tubing was colonized byT. fuscaaleuriospores but not by mycelial pellets, except when mycelial pellets were disrupted by sonication. Microscopic examination of surface-attached growth revealed structures characteristic of biofilms, with cells embedded in fibrous material suggestive of an extracellular polymeric matrix. Concanavalin A bound to the extracellular polymeric substance of biofilms and mycelial pellets, indicating alpha-linkedd-mannosyl and/or alpha-linkedd-glucosyl residues. The carbohydrate content of both biofilms and mycelial pellets increased during growth. Also, DNase I inhibited biofilm production, suggesting a role for extracellular DNA inT. fuscabiofilm development. Cellulose degradation and expression ofcelE(encoding endoglucanase E5) was similar forT. fuscabiofilms and mycelial pellets. Results of this study indicate that, in the life cycle of this actinomycete, cellulose is specifically colonized by aleuriospores, which germinate, grow and degrade cellulose, ultimately developing into biofilms encased in a carbohydrate-containing exopolymeric matrix, a hallmark of biofilm production.

EDTA treatment diminishes the antibacterial and anti-adherence effect of calcium hydroxide on Enterococcus faecalis: an in vitro study

ABSTRACTThis study sought to understand the cell surface characteristics, viability and biofilm-forming potential ofEnterococcus faecaliscells sequentially exposed to EDTA and calcium hydroxide, as in endodontic treatment. Bacterial cells exposed to EDTA and calcium hydroxide were assayed for cell viability, membrane integrity, cell surface hydrophobicity and surface charge, while alteration in the surface topography ofE. faecaliscells was examined using atomic force microscopy (AFM). The bacterial adherence potential to type I collagen was also examined to assess the biofilm-forming capacity ofE. faecaliscells exposed to EDTA and calcium hydroxide. It was found that calcium hydroxide treatment reduced the viability ofE. faecalis. However, prior exposure to EDTA significantly reduced the antibacterial effect of calcium hydroxide (P< 0.05). Calcium hydroxide treatment resulted in impaired cell wall morphology, observed as increased surface roughness and pore formation under AFM. However, these topographical changes induced by calcium hydroxide were significantly reduced in EDTA pretreated cells (P< 0.05). Calcium hydroxide treatment caused reduction in hydrophobicity and adherence ofE. faecalisto type I collagen. These effects due to calcium hydroxide were also significantly altered in EDTA-pretreated cells (P< 0.001). The findings from this study showed that the antibacterial and anti-adherence effect of calcium hydroxide was diminished by prior exposure ofE. faecaliscells to EDTA.

Studies on enhancement of biofilm formation and adherence due to mechanical treatment of titanium surfaces in cooling-water systems

ABSTRACTTitanium has proven to be the heat exchanger material of choice for sea-water-cooled power plants owing to its outstanding resistance to pitting and crevice corrosion in a wide range of aggressive media. However, the inertness of the titanium surface makes it highly susceptible to biofilm formation and subsequent biofouling. This can hinder the heat transfer properties and flow of water. Fouling control strategies in condensers include a combination of mechanical, chemical and thermal treatments. However, reports from various industrial situations suggest that mechanical treatment may not have long-term effects. This study aimed to find out whether mechanical cleaning eventually enhances biofilm formation and increases the adherence of biofilm. In our studies epifluorescence micrographs of biofilms on control and mechanically treated titanium surfaces clearly showed accelerated biofilm formation as well as increased adherence on the mechanically cleaned surface. Total counts of viable bacteria acquired by culturing technique, and biofilm thickness measurements made using microscopic techniques, confirmed this observation. Surface profilometry showed increased roughness of the titanium surface, facilitating adherence of biofilm. The number of microbial species was higher on mechanically cleaned and re-exposed surfaces than on fresh titanium. Thus we concluded that mechanical cleaning can increase biofilm formation and adherence of biofilm, thereby increasing the potential of biofouling in the long term.

Effect of gravitational deposition on biofilm formation and development

ABSTRACTAlthough gravitational deposition is generally regarded to be important during biofilm development because it provides a mechanism by which bacteria can come into contact with a surface, this process is usually neglected in most biofilm studies. The purpose of this study was to develop a better understanding of the effect of gravitational deposition by comparing the development of biofilms on the upper and lower surfaces of a capillary glass tube biofilm reactor under various hydrodynamic conditions. Pure cultures ofPseudomonas fluorescensandShewanella oneidensisMR-1 were used for the test. Results demonstrated that gravitational deposition significantly influences biofilm development under slow laminar flow conditions, which may be attributable to the effect of gravity on both attachment and detachment during the initial reversible attachment phase and the later development phase. Additionally, it was shown that hydrodynamic conditions have the potential to reduce the impact of gravitational deposition on biofilm development, and that this became less significant with an increase in flow rate. These results will be useful for comparing biofilm development in different biofilm systems.

Conferences of interest to biofilms researchers

This list includes conferences that had been announced at the time of going to press. We are happy to include details of other biofilm-related conferences – please send details of these to the editor-in-chief.

Effects of smectite clay on biofilm formation by microorganisms

ABSTRACTWe have investigated the role of smectite clay particles in biofilm formation by several different species of bacteria (Pseudomonas syringae, Escherichia coli,Staphylococcus aureusandBacillus subtilis). We observed that the presence of clay particles enhances the formation of biofilms and, after 24 h, the bacterial populations in the clay mixtures were greater than the respective populations in media without clay. Smectite-bearing clay slurries uniformly develop bacteria–clay aggregates with a substantial biofilm component within 24 h, while the exclusively bacterial suspensions do not develop any observable biofilm component. The biofilm–clay aggregates vary in size from tens of micrometers to several millimeters. Biofilm formation was evaluated by phase contrast microscopy and fluorescence staining. Biofilm promotion by smectite clays may indicate the importance of transport of bacteria by aerosol dust particles.

Bacterial adhesion to stainless steel is reduced by aqueous fish extract coatings

ABSTRACTMicrobial adhesion and biofilm formation on surfaces pose major problems and risks to human health. One way to circumvent this problem is to coat surfaces (in this report stainless steel) with a non-toxic fish extract that generates an abiotic surface with less bacterial attachment than uncoated surfaces or surfaces coated with, for example, tryptone soy broth. The bacteria grow well in the fish extract; hence a general bacteriocidal effect is not the reason for the antifouling effect. Bacterial attachment was quantified by different methods including (a) direct fluorescence microscopy, (b) removal by ultrasound and subsequent quantification of the adhered bacteria, and (c) regrowth of the adhered bacteria measured by indirect conductometry. Surprisingly, the bacterial counts on surfaces coated with aqueous fish extract were 10–100 times lower than on surfaces coated with laboratory broths when surfaces were submerged in bacterial suspensions. The effect was seen forPseudomonas fluorescensAH2,Pseudomonas aeruginosaPAO1,Escherichia coliMG1655,Vibrio anguillarum90-11-287 andAeromonas salmonicidaJno 3175/88. It lasted for at least 7 days. Atomic force microscopy showed that steel surfaces conditioned with fish extract were covered by a thin layer of spherical, nanosized particles. Chemical analysis of the surfaces coated with adsorbed fish extract using X-ray photoelectron spectroscopy revealed that the layer was proteinaceous and had a thickness less than 2 nm. Numerous protein bands/peaks were also detected by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry techniques. We conclude that coating the stainless steel surface with fish extract results in a thin protein layer that reduces bacterial adhesion significantly.

Adaptation of microbial communities in soil contaminated with polychlorinated biphenyls, leading to the transformation of more highly chlorinated congeners in biofilm communities

ABSTRACTA site polluted for many years with polychlorinated biphenyls (PCB) was used to elucidate the metabolic adaptation of microbial communities to these xenobiotics. Soil samples taken along a gradient of PCB-pollution at this site were used to grow biofilm communities on PCB oil. The biofilm communities originating from the non-polluted soil formed rather uniform and thin bacterial layers on PCB oil, while the biofilms originating from contaminated soil samples formed agglomerated structures on the PCB droplets. Biofilm communities were very diverse but those from highly polluted soil were dominated byBurkholderiaspecies, a genus known for degrading several PCBs. All biofilm communities could transform low to medium chlorinated PCB congeners but a strong increase in the rate and degree of PCB transformation in communities from heavily polluted soil was observed. Notably, pentachlorinated congeners were transformed only by biofilms derived from the highly polluted soil but at the same time the content of trichlorinated congeners did not decrease. It is assumed that biofilms from the highly contaminated soil reductively dechlorinated PCB, converting pentachlorinated congeners to trichlorinated congeners in the spherical biofilm aggregates by diffusing to the surface of the aggregates, where aerobic transformation took place.

Effect of oxygen and growth medium onin vitrobiofilm formation byEscherichia coli

ABSTRACTThe effects of oxygen availability onin vitrobiofilm formation by anEscherichia coliK-12 strain and 13 clinicalE. colistrains were compared. AllE. colistrains were capable of forming monospecies biofilm on polystyrene in aerobic media. The K-12 strain produced biofilm in both aerobic glucose minimal medium (ABTG), and aerobic trypticase soy broth (TSB) whereas the majority of the clinical strains produced significant biofilm only in aerobic TSB (9 of 13). In anaerobic media,E. coliK-12 and 9 of the 13 clinical strains were capable of forming biofilmin vitro. Only three clinical strains formed biofilm in anaerobic TSB whereas six clinical strains produced detectable biofilm in anaerobic ABTG. None of the strains tested were capable of forming biofilm in both anaerobic ABTG and anaerobic TSB. Strains that were good biofilm formers in aerobic ABTG also produced the highest amount of biofilm in anaerobic ABTG (R2= 0.90). Image analysis revealed notable differences in architecture for biofilms grown in the presence and in the absence of oxygen. In aerobic ABTG, the biofilm was dominated by tall, mushroom-shaped microcolonies with pores and channels whereas biofilm in anaerobic ABTG was thinner and less heterogeneous, resulting in reduced maximum thickness and biovolume. Analysis of phospholipid fatty acid (PLFA) profiles fromE. coliK-12 and three clinical strains did not reveal a specific pattern associated with the biofilm phenotypes. Interestingly, the clinicalE. colistrains adjusted their PLFA composition much more than didE. coliK-12 in response to changes in growth regimens. Collectively, the results indicate that oxygen availability may affectE. colibiofilm formation in minimal and complex media. The results confirm thatE. coliK-12 and some clinicalE. colistrains are capable of formingin vitrobiofilm under anaerobic conditions. However, the data also suggest that this attribute is highly strain dependent and may vary significantly among clinical isolates.