Model system studies of the influence of bacterial biofilm formation on mineral surface reactivity

Biofilm development on mineral surfaces and related changes in surface reactivity were studied using batch and flow through experiments. An artificial groundwater was used as the primary nutrient medium, Pseudomonas aeruginosa (PAO1) was the model microbial organism and ‘mineral’ surfaces were kept as simple as possible by using glass or a polished quartz tile. Experiments were also completed with very low concentrations (100 mg l^?1) of iron, Fe²⁺ , in the solution. In situ confocal laser scanning microscopy of developing colonies during the live growth phase, and of thick, mature biofilms, revealed only sporadic coverage of biofilm cells and associated polymers at the ‘mineral–microbe interface’. Imaging and analysis of biofilm-conditioned surfaces doped with Fe²⁺ -rich solutions allowed the locus and form of Fe-rich mineral precipitation to be determined and show that biological surface components can cause mineral precipitation from dilute dissolved species which might otherwise remain in solution.     

Booster biocides and microfouling

Antifouling (AF) paints are used to prevent the attachment of living organisms to the submerged surfaces of ships, boats and aquatic structures, usually by the release of biocides. Apart from copper, organic booster biocides are the main active components in AF paints, but their use can have a negative impact on the marine environment. The direct effects of biocides on marine bacteria are poorly known. This work investigates the impact of two biocides, viz. diuron and tolylfluanid, on the growth and the viability of marine microorganisms and on their ability to form biofilms. The biocides in solution were found to inhibit growth of two strains of marine bacteria, viz. Pseudoalteromonas and Vibrio vulnificus, at a high concentration (1000 μg ml^?1), but only a small effect on viability was observed. Confocal laser scanning microscopy (CLSM) showed that the booster biocides decreased biofilm formation by both bacteria. At a concentration of 10 μg ml^?1, the biocides inhibited cell attachment and reduced biofilm thickness on glass surfaces. The percentage of live cells in the biofilms was also reduced. The effect of the biocides on two diatoms, Fragilaria pinnata and Cylindrotheca closterium, was also evaluated in terms of growth rate, biomass, chlorophyll a content and attachment to glass. The results demonstrate that diuron and tolylfluanid are more active against diatoms than bacteria.     

Characterization of planktonic and biofilm cells from two filamentous cyanobacteria using a shotgun proteomic approach

Abstract

Cyanobacteria promote marine biofouling with significant impacts. A qualitative proteomic analysis, by LC-MS/MS, of planktonic and biofilm cells from two cyanobacteria was performed. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4?s^?1 and 40?s^?1). For both strains and surfaces, biofilm development was higher at 4?s^?1. Biofilm development of Nodosilinea sp. LEGE 06145 was substantially higher than Nodosilinea sp. LEGE 06119, but no significant differences were found between surfaces. Overall, 377 and 301 different proteins were identified for Nodosilinea sp. LEGE 06145 and Nodosilinea sp. LEGE 06119. Differences in protein composition were more noticeable in biofilms formed under different hydrodynamic conditions than in those formed on different surfaces. Ribosomal and photosynthetic proteins were identified in most conditions. The characterization performed gives new insights into how shear rate and surface affect the planktonic to biofilm transition, from a structural and proteomics perspective.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

The effects of biofilms on chemical processes in surficial sediments

1. The objectives of the present work were: (a) to evaluate the effects of the development and the presence of a photosynthetically active algal biofilm on chemical fluxes and processes at the sediment–water interface; (b) to measure the effects of the biofilm on chemical concentration gradients in the bulk sediment; and (c) to monitor pH and dissolved oxygen concentration in the biofilm, and through the sediment–water interface using microelectrodes. 2. Two experiments were performed over a period of 8 weeks using a recirculating fluvarium channel containing river sediments with an exposed surface of 0.2 m² and 20 dm³ of overlying solution. The first experiment was in darkness with minimal effects of a photosynthetically active biofilm. The second experiment in natural light produced a complex photosynthetic biofilm involving a succession of diatoms, green algae and cyanobacteria. 3. The solution overlying the biofilm was monitored continuously for dissolved calcium, silicon, phosphorus, alkalinity and oxygen, as well as conductivity, temperature and pH. The surface of the sediment was also monitored for biological and physical changes as the biofilm developed. The overlying solution was analysed over a period of 48 h at 2-h intervals to examine the effects of a well-developed algal biofilm. At the end of the 48 h, pH and oxygen microelectrodes were used to measure gradients above and through the biofilm, and porewaters were analysed from sediments which had been longitudinally sectioned at a maximum depth resolution of 0.5 mm. 4. Biofilm development had a large influence on the composition of the overlying solution and the development of vertical concentration gradients of solutes in the porewater. Once a diatom community was established, the concentration of dissolved silicon was low (< 40 μm ), with all the silicon diffusing from the underlying sediment being consumed in the biofilm (≈ 0.026 μmol m^?2 s^?1 at the end of the experiment). 5. The concentrations of calcium, alkalinity and phosphorus in digested sediment increased near the sediment–water interface. X-ray diffraction analysis showed that calcite was formed at the surface. Estimation of the fluxes of alkalinity and calcium in the overlying solution were consistent with calcite formation during daylight and possible dissolution in darkness. The maximum precipitation flux of calcium was 0.87 μmol m^?2 s^?1 and the maximum net release flux was 0.89 μmol m^?2 s^?1. 6. The net loss of soluble reactive phosphorus from the overlying solution measured over the intensive sampling period of 48 h is consistent with a coprecipitation mechanism and a surface density of phosphorus included in the lattice of calcite of 0.097 μmol m^?2. Processes in the biofilm rather than diffusion from the sediment porewater control chemical fluxes of calcium, alkalinity and phosphorus from the sediment to the overlying water.     

Biofilm formation by the yeast Rhodotorula mucilaginosa: process,repeatability and cell attachment in a continuous biofilm reactor

Yeast biofilms contribute to quality impairment of industrial processes and also play an important role in clinical infections. Little is known about biofilm formation and their treatment. The aim of this study was to establish a multi-layer yeast biofilm model using a modified 3.7 l bench-top bioreactor operated in continuous mode (D = 0.12 h^?1). The repeatability of biofilm formation was tested by comparing five bioprocesses with Rhodotorula mucilaginosa, a strain isolated from washing machines. The amount of biofilm formed after 6 days post inoculation was 83 μg cm^?2 protein, 197 μg cm^?2 polysaccharide and 6.9 × 10⁶ CFU cm^?2 on smooth polypropylene surfaces. Roughening the surface doubled the amount of biofilm but also increased its spatial variability. Plasma modification of polypropylene significantly reduced the hydrophobicity but did not enhance cell attachment. The biofilm formed on polypropylene coupons could be used for sanitation studies.     

Towards a hybrid anaerobic digester-microbial fuel cell integrated energy recovery system: An overview of the development of an electrogenic biofilm

An electrogenic biofilm was developed on a macroporous chitosan-carbon nanotube (CHIT-CNT) electrode under constant poised potential (?0.25 V versus Ag/AgCl reference electrode) and flow through conditions utilizing the effluent of an anaerobic digester as both the inoculant and substrate for the electrogenic biofilm. After 125 days of inoculation the bioelectrode demonstrated an open circuit potential of ?0.62 V and a current density of 9.43 μA cm^?3 (at ?0.25 V). Scanning electron microscopy images indicate thorough surface coverage of the biofilm with a high density of bacterial nanowires physically connecting bacteria to bacteria and bacteria to carbon nanotube (electrode surface) suggesting the nanowires are electrically conductive. DGGE was used to identify the major bacterial and archaeal populations.     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

Increases in benthic community production and metabolism in response to marine‐derived nutrients from spawning Atlantic salmon (Salmo salar)

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Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC₅₀ and MBRC₅₀) of dextranase were 2 U ml^?1 and 5 U ml^?1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.     

In vitro actinomycete biofilm development and inhibition by the polyene antibiotic,nystatin, on IUD copper surfaces

The presence of intrauterine contraceptive devices (IUDs) gives a solid surface for attachment and an ideal niche for biofilm to form and flourish. Pelvic actinomycosis is often associated with the use of IUDs. Treatment of IUD-associated pelvic actinomycosis requires the immediate removal of the IUD. Therefore, this article presents in vitro evidence to support the use of novel antibiotics in the treatment of actinomycete biofilms. Twenty one clinical actinomycetes isolates from endocervical swabs of IUD wearers were assessed for their biofilm forming ability. An in vitro biofilm model with three isolates, Streptomyces strain A4, Nocardia strain C15 and Nocardia strain C17 was subjected to treatment with nystatin. Inhibition of biofilm formation by nystatin was found to be concentration dependent, with MBIC₅₀ values in the range 0.08–0.16 mg ml^?1. Furthermore, at a concentration of 0.16 mg ml^?1, nystatin inhibited the twitching motility of the isolates, providing evidence for a possible mechanism of biofilm inhibition.     

Conditioning film and initial biofilm formation on ceramics tiles in the marine environment

The formation of biofilm on surfaces in the marine environment is believed to be an important factor driving colonization and recruitment of some sessile invertebrate communities. The present study follows the process of biofilm buildup on unglazed ceramic tiles deployed into the marine environment in the northern Gulf of Eilat. PCR-DGGE of film eluted from the tile surface indicated the presence of bacteria as early as 2 h after deployment. The makeup of the biofilm bacterial community was dynamic. Bacterial presence was apparent microscopically 6 h after deployment, though a developed biofilm was not observed until 24 h following deployment. Total organic carbon (TOC) data suggest that a conditioning film was built within the first four hours following deployment. During this time period TOC reached the highest level possibly due to adhesion of organics (e.g., sugars, proteins and humic substances) from the water column. We suggest that the primary adhering bacteria, whilst still in the reversible stage of adhesion, utilize the conditioning film as food causing the decrease in TOC. Understanding the dynamics between these primary bacterial settlers is of importance, since they may play a role on the succession of invertebrate species settlement onto artificial surfaces.     

Antimicrobial and antifouling polymeric coating mitigates persistence of Pseudomonas aeruginosa biofilm

Abstract

Food wasted due to food spoilage remains a global challenge to the environmental sustainability and security of food supply. In food manufacturing, post-processing contamination of food can occur due to persistent bacterial biofilms, which can be resistant to conventional cleaning and sanitization. The objective was to characterize the efficacy of a polymeric coating in reducing Pseudomonas aeruginosa biofilm establishment and facilitating its removal. Viable cell density of a 48?h biofilm was reduced by 2.10 log cfu cm^?2 on the coated surface, compared to native polypropylene. Confocal laser scanning and electron microscopy indicated reductions in mature biofilm viability and thickness on the coated material. The antifouling coating improved cleanability, with ～2.5 log cfu cm^?2 of viable cells remaining after 105?min cleaning by water at 65?°C, compared to 4.5 log cfu cm^?2 remaining on native polypropylene. Such coatings may reduce the persistence of biofilms in food processing environments, in support of reducing food spoilage and waste     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

Effect of 2-hydroxyethyl methacrylate on Streptococcus spp. biofilms

Aims: The effect of different concentrations of 2‐hydroxyethyl methacrylate (HEMA) was evaluated on biofilm formation and preformed biofilm of Streptococcus mitis, Streptococcus mutans and Streptococcus oralis, alone or combined to each other. Methods and Results: Twofold serial dilution of HEMA ranged from 12 to 0·75 mmol l^?1 was added to Streptococcal broth cultures and mature biofilms in 96‐well‐microtitre plates to evaluate bacterial biomass and cell viability. HEMA affected the Streptococcal population in a strain‐specific way producing few significant effects. A reduction on biofilm formation and a detachment of preformed biofilm was recorded in Strep. mitis ATCC 6249, whereas in mixed cultures, the monomer expressed a general aggregative effect on mature biofilms. A reduction in cell viability was also recorded in an HEMA‐concentration‐dependent way in each experimental condition studied. Conclusions: These results suggest that the HEMA prevalent effects are both the reduction of bacterial adhesion to a polystyrene surface and the increase in dead cells also characterized by an aggregative status. Significance and Impact of the Study: Understanding the potential effect of HEMA, released from resin‐based materials, on oral bacteria may furnish information for surveillance of the risk reduction in secondary caries via hindering biofilm generation.     

Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity

Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 μg ml^?1) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections.     

Conditioning film formation and its influence on the initial adhesion and biofilm formation by a cyanobacterium on photobioreactor materials

Abstract

Although cyanobacteria are a common group of microorganisms well-suited to utilization in photobioreactors (PBRs), studies of cyanobacteria fouling and its prevention are scarce. Using a cyanobacterium, Anabaena sp. PCC 7120, which had been genetically modified to enhance linalool production, the formation of conditioning films and the effects of these on the physico-chemical surface properties of various PBR materials during initial adhesion and biofilm formation were investigated. The adhesion assay revealed that the overall attachment of Anabaena was substratum dependent and no correlation between the hydrophobicity/roughness of clean material and cell attachment was found. Surface hydrophilicity/hydrophobicity of all the materials changed within 12?h due to formation of conditioning films. ATR-FTIR spectroscopy revealed that the fractional change in protein deposition between 12 to 96?h was consistent with Anabaena cell attachment but polysaccharide deposition was material specific and did not correlate with cell attachment on the PBR materials. Also, the delay in conditioning film proteins on PVC and PTFE indicated that components other than proteins may be responsible for the decrease in contact angles on these surfaces within 12?h. This indicates the important role of the chemical nature of adsorbed conditioning films in determining the initial attachment of Anabaena to PBR materials. The lower rate of attachment of Anabaena on the hydrophilic surfaces (glass and PMMA) between 72?h to 96?h (regime 3) showed that these surfaces could potentially have low fouling characteristics at extended time scales and should be considered for further research.     

Dependence of the initial adhesion of biofilm forming Pseudomonas putida mt2 on physico-chemical material properties

Bacterial adhesion is strongly dependent on the physico-chemical properties of materials and plays a fundamental role in the development of a growing biofilm. Selected materials were characterized with respect to their physico-chemical surface properties. The different materials, glass and several polymer foils, showed a stepwise range of surface tensions (γ_s) between 10.3 and 44.7 mN m^?1. Measured zeta potential values were in the range between ?74.8 and ?28.3 mV. The initial bacterial adhesion parameter q _max was found to vary between 6.6 × 10⁶ and 28.1 × 10⁶ cm^?2. By correlation of the initial adhesions kinetic parameters with the surface tension data, the optimal conditions for the immobilization of Pseudomonas putida mt2 were found to be at a surface tension of 24.7 mN m^?1. Both higher and lower surface tensions lead to a smaller number of adherent cells per unit surface area. Higher energy surfaces, commonly termed hydrophilic, could constrain bacterial adhesion because of their more highly ordered water structure (exclusion zone) close to the surface. At low energy surfaces, commonly referred to as hydrophobic, cell adhesion is inhibited due to a thin, less dense zone (depletion layer or clathrate structure) close to the surface. Correlation of q _max with zeta potential results in a linear relationship. Since P. putida carries weak negative charges, a measurable repulsive effect can be assumed on negative surfaces.     

Anoxybacillus and Geobacillus biofilms in the dairy industry: effects of surface material,incubation temperature and milk type

Abstract

Anoxybacillus (A. flavithermus, A. kamchatkensis subsp. asachharedens, A. caldiproteolyticus and A. tepidamans) and Geobacillus (two strains of G. thermodenitrificans, G. thermoglucosidans and G. vulcanii) isolates and reference strains in whole milk were evaluated for their biofilm production on six different abiotic surfaces. G. thermodenitrificans DSM 465^T had the highest cell counts (>4 log₁₀ CFU cm^?2) on glass and stainless steel (SS) at 55 and 65?°C, respectively. G. thermodenitrificans D195 had the highest counts on SS at 55?°C (>5 log₁₀ CFU cm^?2) and polyvinyl chloride (PVC) at 65?°C (>4 log₁₀ CFU cm^?2), indicating the existence of strain variation. The ideal surfaces for all strains were SS and glass at 55?°C, but their preferences were polystyrene and SS at 65?°C. Moreover, Anoxybacillus members were more prone to form biofilms in skim milk than in semi-skim and whole milk, whereas the results were the opposite for Geobacillus. Both the attachment and sporulation of Geobacillus in whole milk was higher than in semi-skim or skim milk. This study proposes that the surface material, temperature and milk type had a cumulative effect on biofilm formation.     

An extraction free modified o-phthalaldehyde assay for quantifying residual protein and microbial biofilms on surfaces

Abstract

Biological contamination of surfaces in industry and healthcare is an important vector of disease transmission. Current assays for detecting surface-adherent contamination require extraction of biological soil. However, physical inaccessibility or poor solubility may limit recovery. Here, how the o-phthalaldehyde (OPA) protein assay can be modified to measure residual protein (modeled with bovine serum albumin) or biofilm on a surface without extraction is described. The assay limit of detection (LOD) for protein was 1.6 µg cm^?2. The detection threshold for Staphylococcus epidermis biofilm was 117 µg cm^?2. The clinical utility of the method was demonstrated for measurements taken from clinically used endoscopes. Since this method is more sensitive than extraction-based testing, clinical results should not be compared with conventional benchmarks. By enabling direct detection and quantification of soils in complex or hard-to-reach areas, this method has potential to improve the margin of safety in medical and industrial cleaning processes.