Biofilm Degradation Research Articles

Sialidase of Streptococcus intermedius: a putative virulence factor modifying sugar chains

A sialidase gene of Streptococcus intermedius was cloned. It was most similar to nanA, a major sialidase gene in Streptococcus pneumoniae, and was expressed in Escherichia coli. Since the gene-knockout S. intermedius strain lost detectable sialidase activity, the gene might code, either solely or mainly, the glycosidase in the bacterial genome. Polymerase chain reaction using the primers for the nanA homologue in S. intermedius (described as nanA below) showed that this sialidase gene was commonly distributed within the isolates of S. intermedius, but not found in the strains of other species among the anginosus group. In biofilm formation assay under cultivation with mucin, the nanA-deleted S. intermedius maintained the amount of biofilm for 72 hr, while that of the parent strain decreased during incubation from 24 to 72 hr. Since sialidase activity in the parent strain increased during that time period, sialidase might contribute to the degradation of biofilm under sialic acid-rich conditions. When S. intermedius was added into the HepG2 hepatoma culture, the calculated disassociation constant (K(d)) of EDTA-releasable bacterial adhesion to the cells was higher in the nanA-deleted strain than in the parent. Furthermore, the rate constant, assuming endocytosis of the bacterium mediated by ASGP-R in HepG2 cells, seemed to be increased by sialidase pretreatment of the bacterial cells before addition to the cell culture. According to the results, modification of sugar chains by sialidase on the bacterial surface and in the surrounding environment might influence both bacterial interaction and host-bacterial interaction in S. intermedius.

Protease and amylase enzymes for biofilm removal and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas fluorescens bacteria

Removal of biofilms is difficult. In industrial settings, both the inactivation and removal of biofilms are of huge concern. If only disinfection without the removal of attached biofilms occurs, the inactivated biofilm cells may provide an ideal environment for further adhesion and growth, resulting in a complex matrix. Microbial resistance to biocides and their negative environmental impact are the main reasons for finding alternative biofilm control strategies. Enzymes may offer such an alternative. The objective of this study was to determine the effect of commercial proteases and amylases on biofilms formed by Pseudomonas fluorescens. Biofilms were grown in diluted medium containing glass wool used as the attachment surface. Extracellular polymeric substances (EPS) were extracted and EPS composition was determined. Protease (savinase, everlase and polarzyme) and amylase (Amyloglucosidase and Bacterial Amylase Novo) activity was tested on both biofilms and on extracted EPS. After testing enzymes, biofilm integrity was evaluated by scanning electron microscopy. EPS composition consisted predominantly of proteins. Everlase and Savinase were the most effective enzymatic treatments on removing biofilms and degrading the EPS. Key words: Biofilms, extracellular polymeric substances, Pseudomonas fluorescens, proteases, amylases.

Mutanase from Paenibacillus sp. MP-1 produced inductively by fungal α-1,3-glucan and its potential for the degradation of mutan and Streptococcus mutans biofilm

Laetiporus sulphureus is a source of α-1,3-glucan that can substitute for the commercially-unavailable streptococcal mutan used to induce microbial mutanases. The water-insoluble fraction of its fruiting bodies from 0.15 to 0.2% (w/v) induced mutanase activity in Paenibacillus sp. MP-1 at 0.35 μ ml−1. The mutanase extensively hydrolyzed streptococcal mutan, giving 23% of saccharification, and 83% of solubilization of glucan after 6 h. It also degraded α-1,3-polymers of biofilms, formed in vitro by Streptococcus mutans, even after only 3 min of contact.

Multifunctional nature of UV-irradiated nanocrystalline anatase thin films for biomedical applications

Anatase is known to decompose organic material by photocatalysis and to enhance surface wettability once irradiated by ultraviolet (UV) light. In this study, pulse magnetron-sputtered anatase thin films were investigated for their suitability with respect to specific biomedical applications, namely superhydrophilic and biofilm degrading implant surfaces. UV-induced hydrophilicity was quantified by static and dynamic contact angle analysis. Photocatalytic protein decomposition was analyzed by quartz crystal microbalance with dissipation. The surfaces were characterized by X-ray diffraction, atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. The radical formation on anatase, responsible for photocatalytic effects, was analyzed by electron spin resonance spectroscopy. Results have shown that the nanocrystalline anatase films, in contrast to reference titanium surfaces, were sensitive to UV irradiation and showed rapid switching towards superhydrophilicity. The observed decrease in carbon adsorbents and the increase in the fraction of surface hydroxyl groups upon UV irradiation might contribute to this hydrophilic behavior. UV irradiation of anatase pre-conditioned with albumin protein layers induces the photocatalytic decomposition of these model biofilms. The observed degradation is mainly caused by hydroxyl radicals. It is concluded that nanocrystalline anatase films offer different functions at implant interfaces, e.g. bedside hydrophilization of anatase-coated implants for improved osseointegration or the in situ decomposition of conditioning films forming the basal layer of biofilms in the oral cavity.

Kaempferia galanga L. Rhizome As a Potential Dental Plaque Preventive Agent

Dental plaque prevention can be achieved by inhibition of mouth cavity microbes to built biofilm. Kaempferia galanga rhizome has been known as a potential antibacterial agent. This research aimed to reveal the potency of Kaempferia galanga extract and essential oil as anti plaque active agents, based on their in vitro inhibitory activity against the planktonic growth and biofilm of Streptococcus mutans ATCC 21752. Kaempferia galanga extract was obtained by defatting dried-pulverized samples in petroleum ether prior to immersion in 70% ethanol. The fresh rhizome was steam-hydro distilled for 6 h to yield the essential oil. Antibacterial and anti biofilm assays were measured by micro dilution technique on polystyrene 96-wells micro titer plates at 37°C. The percentage of inhibition was calculated by comparing the absorbance of samples to the vehicle (control) measured by micro plate reader at 595 nm. Biofilms formed were first stained by 1% crystal violet. The above assays were performed in triplicates. This study revealed that both K. galanga rhizome essential oil and ethanolic extract showed antibacterial and antibiofilm activity towards S. mutans. The ethanol extract showed MIC90 value at 0.091% w/v and MBC at 2.724% w/v for antibacterial activity; IC50 at 0.048 % w/v for anti biofilm formation activity; and EC50 at 0.052%w/v for biofilm degradation activity. Until the highest concentration tested (0.6%w/v), the MIC90 and MBC values of the essential oil were not revealed, but higher biofilm inhibitory activity i.e. IC50 at 0.025 % w/v; and EC50 at 0.034 %w/v were observed. Key words: biofilm inhibitor, antibacterial, Kaempferia galanga

Production andIn VitroEvaluation of Soy Protein–Based Biofilms as a Support for Human Keratinocyte and Fibroblast Culture

This study presents results on soy protein isolate (SPI) biofilm production and the corresponding effect on the stability and toxicity of the derived films. SPI biofilms were prepared from SPI chemically treated with formaldehyde at various concentrations (0%, 1%, 2%, and 3%) as cross-linking agents. In vitro SPI biofilm degradation was evaluated as a function of water absorption leading to weight and size modifications. SPI biofilm toxicity was determined as a function of human keratinocyte and fibroblast adhesion, viability, and proliferation. Cytokine gene expression supported this using reverse transcriptase polymerase chain reaction techniques. Our results confirm that SPI can be used to produce biofilms. The resulting SPI biofilms without formaldehyde swell significantly, which leads to their physical instability. Formaldehyde treatment enhanced the mechanical properties of these biofilms by covalently cross-linking polypeptide chains. The decreased water absorption was dependent on the amount of formaldehyde present. SPI biofilms with 2% and 3% formaldehyde were highly stable and easier to manipulate than those with 0% and 1% formaldehyde. Tissue culture analyses revealed that the SPI biofilms without formaldehyde were non-toxic to human cells (keratinocytes and fibroblasts). The presence of formaldehyde in biofilms did not have any effects on cell viability, adhesion, or proliferation. This was supported by the high level of messenger RNA expression of interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha by the keratinocytes and of IL-6 and IL-8 by the fibroblasts. Overall, we produced a stable, non-toxic soy protein support, which may be of potential interest in medical applications such as cell culture matrices and damaged tissue replacement.

On the contribution of phytoplankton and benthic biofilms to the sediment record of marker pigments in high mountain lakes

This study compares the marker pigment composition in three different lake compartments: the water column; the surface sediment biofilm (0–0.5 cm), where production and main degradation processes take place; and the deep sediment (15–17 cm), where the signal is finally stored and marker pigments are used as a proxy in paleolimnological studies. The aim was to evaluate which marker pigments better record in the sediment the relative contributions of planktonic and benthic production in high mountain lakes, taking into account the differential preservation existing among pigments. A survey of 82 high mountain lakes distributed along the major environmental gradients was carried out in the Pyrenees. Comparison of pigments between the three compartments revealed that both water column and sediment biofilm signals could be distinguished in old sediment layers, despite the observation that some of the most characteristic carotenoids that appeared frequently and in high concentrations in the water column were already less common or even absent from the sediment biofilm. In the shallowest lakes, pigment composition in the sediment biofilm was typical of photosynthetically active communities and their deep sediment samples were characterised by a substitution of fucoxanthin by diatoxanthin as the dominant diatom marker pigment. However, in the deepest lakes the pigment composition of the sediment biofilm and deep sediment was similar, characterised by marker pigments of mainly planktonic algal groups and pheophytins, which are typical pigments of decaying communities. Results are discussed in terms of how pigment source (planktonic or benthic) and pigment type (lability of molecule) interact to shape pigment composition in the sediment record of high mountain lakes.

In vitro studies of the impact of the naked soil amoeba Thecamoeba similis Greef, feeding on phototrophic soil biofilms

The effect of grazing by the naked soil amoeba Thecamoeba similis feeding on algae of in vitro phototrophic soil biofilms was investigated in terms of carbon biomass of algae, bacteria and amoebae and compared to shifts in the microbial community determined by 16S rDNA t-RFLP patterns. In a first approach, grazing of T. similis on unialgal Xanthonema hormidioides biofilms growing on regosol was investigated. The growth rate of T. similis was 0.5 divisions per day and the production efficiency reached 25% (allocation of prey-carbon to predator-carbon). The algal biomass decreased from the original 100% down to 7% at the end of the experiment. The increase of egested residues enhanced bacterial growth; the biomass carbon ratio bacteria vs. algae increased from 0.25 to 4.5 indicating the degradation of the algal biofilm, while the 16S rDNA t-RFLP patterns indicated changes in the composition of the microbial community. A subsequent experiment was conducted by inoculating T. similis on a natural biofilm community consisting of trichomes of cyanobacteria, several species of Chlorophyceae and Xanthophyceae. The total algal biomass (including cyanobacteria) remained constant during the experiment. However, 73% of the algal biomass without cyanobacteria was grazed, while the amount of mucous Chlamydocapsa spec. doubled from 32% to 68%. The growth rate of T. similis was 0.4 divisions per day and the production efficiency was only 13%. The 16S rDNA t-RFLP patterns showed a shift of the microbial community. Our results give a first insight into the obviously high potential of naked soil amoebae feeding on algae to change the structures of both the algal and bacterial communities of phototrophic soil biofilms.

Saving a fragile legacy

Science and technology interact with art and culture in many ways. Both involve knowledge and investigation—some philosophers would maintain—and nature and its representation lie at their core (Frazzetto, 2004). But their interactions extend further: the arts draw both inspiration and new materials from science, while the scientific examination of art and artefacts has provided us with important insights into the progress of human civilizations. Now, science—in particular the biological sciences—might have an even more important role: to protect and conserve mankind's often fragile, cultural heritage for future generations. This task is particularly difficult given the magnitude and diversity of objects involved, and the enormous variety of different materials used—for example, stone, metal, ceramics, synthetic substances, and organic matter derived from plants and animals. A classic example of art under threat is found in the cave of Lascaux in southwest France. Discovered accidentally in 1940, the cave contains some of the finest paleolithic drawings and polychrome rock paintings in the world, dating back some 17,000 years. Herds of giant aurochs and other wild mammals—realistically depicted in vivid colours—still seem to roam the prairies while seeking salvation from hunting men (Fig 1). These strikingly beautiful images testify to the birth of humans using the abstract to represent reality, and the location was listed as a UNESCO World Heritage site in 1979. But an invading army of fungi, bacteria, algae and moss—their lives made easier by disputing conservators—threatens the artwork. Figure 1. White aurochs, Hall of the Bulls, Lascaux (Montignac, France). Image courtesy of Wikimedia Commons. The paintings remained intact and fresh until the cave was exposed to tourists after the Second World War, when the rise in temperature and humidity caused a burst of microbial growth, which was first noticed in 1955. The French authorities—worried about the future of the paintings—closed the cave to …

The Heat Shock Genes dnaK , dnaJ , and grpE Are Involved in Regulation of Putisolvin Biosynthesis in Pseudomonas putida PCL1445

Pseudomonas putida PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn5luxAB library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5luxAB flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a dnaK homologue which is located downstream of grpE and upstream of dnaJ. Analysis of putisolvin production and expression studies indicate that dnaK, together with the dnaJ and grpE heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires dnaK and dnaJ but not grpE. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that dnaK is positively regulated by GacA/GacS.

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 A resolution with a final R-factor of 16.8% for 25 to 1.8 A resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold. One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp(88) and Asp(149). This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149).

Water treatment using sequenced ozonation and SBR biofilm reactors

The degradation of 3-methylpyridine (3MEP), a model heterocyclic industrial molecule, was performed in a sequential batch ozonation–biofilm process. Four process steps (bubble-column ozonation, heterotrophic biofilm degradation, biofilm nitrification, and biofilm denitrification) were combined in different sequences. Three packed-bed biofilm reactors were started up so as to have separate, specific activities (heterotrophic, nitrification and denitrification). Batch experiments with acetate, ammonia, and nitrate proved that all reactors displayed degradation activity for all substances. Different batch sequences of these reactors were tested with the products of batch ozonation of 3MEP as the first step. The best results were obtained using a two-step process, in which the ozonation was followed by a single fluidized-bed, heterotrophic biofilm reactor. The high C/N ratio of 3MEP and the appreciable non-specific activity of this reactor made it possible to achieve all the biodegradation in the one reactor. Establishing the optimal batch ozonation time (80 min) was determined by an ozone electrode and by stopping the process when the dissolved ozone concentration rose above an initial low level. The identifiable products of 3MEP ozonation were nitrate, acetate, formate, pyruvate, oxalate and ammonium. A C-balance, compared with TOC measurement, indicated that about 50% of the carbon was in unidentified, but biogradable, ozonation products. Copyright © 2003 Society of Chemical Industry

26] Use of conductance measurements for determination of enzymatic degradation of microbial biofilm

Publisher Summary Using conductance measurements, the number of microorganisms left on substrata after enzyme treatment can be evaluated without removal of the cells from the substrata, simply by immersing the substrata in a conductance measuring cell. This chapter describes a method based on conductance measurements for evaluating the enzymatic degradation of microbial biofilm. The chapter describes Malthus method that is useful for the evaluation of enzyme activity against microbial biofilm as the number of microorganisms left on the substratum after enzyme treatment can be evaluated and enumerated without removal of the cells from the substratum. Using this method, bactericidal activity can be evaluated against biofilm cells without removal of the cells from the substratum by simply placing the substratum with biofilm in an conductance measuring cell and immersing the electrodes. The described method was particularly useful for the evaluation of enzymatic degradation of biofilm, as cells left on the substratum after enzyme treatment was more detachable than untreated biofilm cells.

義歯洗浄剤としての電解水の効果 ｉｎ ｖｉｔｒｏのＣａｎｄｉｄａ ａｌｂｉｃａｎｓバイオフィルムへの影響

Dentures may act as a reservoir for bacteria which cause opportunistic infection such as aspiration pneumonia, and denture cleaning could prevent such an infection. Recently, the bacteriocidal effect of electrolyzed water (EW) on bacteria has been reported.In this study, the influence of EWs on the Candida albicans biofilm in vitro was investigated. C. albicans adhered avidly to type IV collagen. The biofilm was established on the cover slip coated with type IV collagen using Luria broth at 37°C for 5 days. The biofilms were treated with several kinds of EWs, and the efficacy of EWs was investigated.C. albicans well adhered to collagen-coated celldesk, produced glycocalyx-like substance and formed a biofilm after five-day incubation. Scanning electron microscopy revealed degradation of biofilm treated with EW. Vital CFU in the biofilm decreased time-dependently and logarithmically. Five treatments for one minute was more effective than one treatment for five minutes. The effect of EW synthesized by a home care device was equal to or higher than that synthesized by a medical device. These results suggested that EW, even if synthesized by the home care device, is a potent denture-cleanser.

Predictive Modeling for Bioactive Fluidized Bed and Stationary Bed Reactors: Application to Dairy Wastewater

Bioadsorber models were formulated, and a systematic protocol was developed for adsorber modeling and design. These models were used for forecasting the performance of bioactive and non bioactive reactors in two configurations: fluidized bed reactor (with effluent recycle), and stationary bed reactor. The models incorporated important phenomena associated with adsorption and biofilm degradation, and used total organic carbon (TOC) as a lumped parameter for measuring the wastewater strength. The protocol could be easily applied to bioactive reactors with non-adsorbing media such as sand, polymers and glass. The protocol included the development of experimental techniques for evaluating model parameters. Laboratory-scale adsorption equilibrium and rate studies were conducted to evaluate the equilibrium and mass-transfer parameters, respectively. Biokinetic experiments were performed to determine the biodegradation parameters including kinetic coefficients for biological growth and decay, and the yield coefficient. Adsorption and biodegradation parameters were subsequently used as model inputs for predicting the dynamics of bioactive reactors. Laboratory-scale adsorber column studies for model verification indicated the good predictive capability of the models for bioactive and non bioactive reactors.

Modeling of bioactive carbon adsorbers: A hybrid weighted residual-finite difference numerical technique

Phenomenological mathematical models incorporating adsorption, mass transfer, and biofilm degradation were developed for performance prediction/simulation of bioactive carbon fixed-bed and fluidized-bed adsorbers in wastewater treatment. The model equations were solved by a numerical technique combining a weighted residual technique such as orthogonal collocation with finite difference method. This hybrid technique was numerically consistent and stable and provided accurate solutions at computing times lower than those corresponding to pure orthogonal collocation. The bioadsorber model parameters were independently determined from carefully designed laboratory-scale experiments and correlations. The model predictions of bioadsorber effluent concentration profiles were in strong agreement with the experimental data, illustrating the good predictive capability of the model. Sensitivity studies were performed to identify the influence of model parameters on the bioactive adsorber dynamics.