Electrofiltration improves dead-end filtration of hyaluronic acid and presents an alternative downstream processing step that overcomes technological challenges of conventional methods.

Hyaluronic acid (HA) dispersion obtained from the bacteria Streptococcus equi was concentrated by electrofiltration. In the conventional downstream processing of HA, extraction and precipitation lead to increase in environmental issues, structural changes, and time and energy related costs. Using electrofiltration as an alternative technology delivers solutions to these limitations. Experiments were conducted in order to test the applicability of electrofiltration to downstream processing of the negatively charged HA. The structural changes and molecular weight distributions, often a consequence of the employed separation method, were tested by analysis of the initial dispersions and final products. In comparison to the conventional filtration, concentration factors were increased up to almost four times without any detectable structural change in the final product.

Effect of dental cements on peri-implant microbial community: comparison of the microbial communities inhabiting the peri-implant tissue when using different luting cements.

BACKGROUND: Cementing dental restorations on implants poses the risk of undetected excess cement. Such cement remnants may favor the development of inflammation in the peri-implant tissue. The effect of excess cement on the bacterial community is not yet known. The aim of this study was to analyze the effect of two different dental cements on the composition of the microbial peri-implant community. METHODS: In a cohort of 38 patients, samples of the peri-implant tissue were taken with paper points from one implant per patient. In 15 patients, the suprastructure had been cemented with a zinc oxide-eugenol cement (Temp Bond, TB) and in 23 patients with a methacrylate cement (Premier Implant Cement, PIC). The excess cement found as well as suppuration was documented. Subgingival samples of all patients were analyzed for taxonomic composition by means of 16S amplicon sequencing. RESULTS: None of the TB-cemented implants had excess cement or suppuration. In 14 (61%) of the PIC, excess cement was found. Suppuration was detected in 33% of the PIC implants without excess cement and in 100% of the PIC implants with excess cement. The taxonomic analysis of the microbial samples revealed an accumulation of oral pathogens in the PIC patients independent of the presence of excess cement. Significantly fewer oral pathogens occurred in patients with TB compared to patients with PIC. CONCLUSION: Compared with TB, PIC favors the development of suppuration and the growth of periodontal pathogens.

Microbial analysis of biofilms on cement surfaces: An investigation in cement-associated peri-implantitis.

The cementation of implant-supported restorations always poses the risk of excess cement retained in the peri-implant sulcus despite careful clinical control. Excess cement can become the basis of colonization by oral microorganisms. As a result of the biofilm formation peri-mucositis or peri-implantitis may develop. Complications were observed in the routine prosthetic restoration of implants when a methacrylate-based cement was used. These developed a few weeks after cementation of the suprastructure and caused bleeding on probing as well as suppuration from the peri-implant tissue. In the revision therapy, excess cement in the peri-implant sulcus was found in many cases. This excess cement was sampled from ten patients and investigated for biofilm formation. For this purpose, the cement samples were collected and analyzed for bacterial in situ colonization by 16S rDNA-based methods. In laboratory experiments, the methacrylate-based cement and two other dental cements were then investigated for their proneness to form biofilm. The results of the in situ and in vitro investigations revealed a strong tendency towards bacterial invasion of the methacrylate-based cement by opportunistic species and pathogens.

The Pseudomonas quinolone signal (PQS) stimulates chemotaxis of polymorphonuclear neutrophils.

Cross-talk between bacteria and mammalian cells is increasingly recognized as an important factor, especially during chronic infections. In particular, the interaction of extracellular bacterial signaling molecules with cells of the innate immune response is of special interest. In this context, we investigated whether the Pseudomonas quinolone signal (PQS) which is a quorum sensing molecule produced by bacteria and participates in biofilm formation and virulence has any influence on polymorphonuclear neutrophils (PMN), the cells of the "first line defense" against bacterial infections. We found that PQS did not enhance the bactericidal activity of PMN and did not induce apoptosis at concentrations up to 100 µM. However, PQS stimulated chemotaxis of PMN in doses of 10-100 µM. This PQS-dependent chemotaxis could be inhibited with SB203580 which blocks MAPkinase p38, suggesting a signaling pathway similar to AHL-12 induction. Using bacterial cell culture supernantants of Pseudomonas aeruginosa wild-type cells and a PQS-deficient mutant strain support the in vivo relevance of these findings. Since PQS is produced in the early phase of biofilm formation, PMN infiltration could be timely enough to eradicate bacteria before biofilm formation is completed, which confers the bacteria with a relative resistance to host defense mechanisms.

Swimming behavior of Pseudomonas aeruginosa studied by holographic 3D tracking.

Holographic 3D tracking was applied to record and analyze the swimming behavior of Pseudomonas aeruginosa. The obtained trajectories allow to qualitatively and quantitatively analyze the free swimming behavior of the bacterium. This can be classified into five distinct swimming patterns. In addition to the previously reported smooth and oscillatory swimming motions, three additional patterns are distinguished. We show that Pseudomonas aeruginosa performs helical movements which were so far only described for larger microorganisms. Occurrence of the swimming patterns was determined and transitions between the patterns were analyzed.

Cement-associated peri-implantitis: a retrospective clinical observational study of fixed implant-supported restorations using a methacrylate cement.

BACKGROUND: Cement-retained fixed implant-supported restorations involve the risk of excess cement, which can associate peri-implantitis. In connection with routine therapy using a methacrylate cement (Premier Implant Cement, Premier(®) Dental Products Company, Plymouth Meeting, PA, USA) to retain fixed implant-supported restorations, complications, that is, inflammations, were developed in some cases. After removing the suprastructure and the abutment, residual excessive cement was found. For this reason, all implant-supported restorations that had been fixed with this type of methacrylate cement were reevaluated and retreated. METHODS: In a retrospective clinical observational study including 71 patients with 126 implants, the findings made during retreatment were documented. In all cases, the suprastructure and the abutment were removed. For recementation, Temp Bond (Kerr Sybron Dental Specialities, Washington, D.C., USA) was used. If an inflammation had developed, a follow-up appointment was scheduled 3-4 weeks later. RESULTS: In 59.5% of the implants, cement residues were identified. Bleeding on probing was diagnosed at 80% of the implants with excess cement and suppuration at 21.3% of the implants. After removal of the excess cement and recementation with Temp Bond, a 76.9% reduction in bleeding on probing was found at follow-up. Suppuration was not found around any of the implants at follow-up. CONCLUSION: Excess cement left in the implant-mucosal interface caused bleeding on probing in most cases and suppuration in some. The removal of excess cement after cementation should be given high priority. In this retrospective observational study, an unusually high number of implants with excess cement after cementation was found with the methacrylate cement applied in the study.

Quartz crystal microbalance with dissipation coupled to on-chip MALDI-ToF mass spectrometry as a tool for characterising proteinaceous conditioning films on functionalised surfaces.

Proteinaceous conditioning films (pCFs) are thought to play a key role in microbial adhesion, leading to the fouling of technical and biomedical devices and biofilm formation, which in turn causes material damage or persistent infections, respectively. However, little is definitively known about the process of surface conditioning via proteins. Herein, we demonstrate the potential of quartz crystal microbalance with dissipation coupled to MALDI-ToF mass spectrometry (QCM-D-MALDI) to investigate protein adsorption on different surfaces, enabling both the monitoring of CF formation and the determination of the molecular composition of CFs. After running QCM-D experiments, a subsequent tryptic on chip digestion step allows the identification of the proteins deposited on the sensor chip surface via MALDI-ToF mass spectrometry. Prominent blood plasma proteins, i.e., human serum albumin (HSA), fibrinogen (FG) and fibronectin (FN), were used. Chemically well defined sensor surfaces were prepared, among others, via self-assembled monolayer (SAM) technology. In cases where protein adsorption was observed by QCM-D, the adsorbed proteins were clearly detected and identified using MALDI-ToF/MS for both single-protein solutions of HSA, FG and FN as well as for protein mixtures. However, for equimolar protein mixtures on TiO2 surfaces, only signals attributed to FG and FN were observed in the mass spectra. No signals indicating the presence of HSA could be detected. This finding leads to the assumption that only FG and FN attach to the TiO2 sensor surface under the given experimental conditions.

DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.

Drinking water biofilms on copper and stainless steel exhibit specific molecular responses towards different disinfection regimes at waterworks.

Biofilms growing on copper and stainless steel substrata in natural drinking water were investigated. A modular pilot-scale distribution facility was installed at four waterworks using different raw waters and disinfection regimes. Three-month-old biofilms were analysed using molecular biology and microscopy methods. High total cell numbers, low counts of actively respiring cells and low numbers of cultivable bacteria indicated the high abundance of viable but not cultivable bacteria in the biofilms. The expression of the recA SOS responsive gene was detected and underlined the presence of transcriptionally active bacteria within the biofilms. This effect was most evident after UV disinfection, UV oxidation and UV disinfection with increased turbidity at waterworks compared to chemically treated and non-disinfected systems. Furthermore, live/dead staining techniques and environmental scanning electron microscopy imaging revealed the presence of living and intact bacteria in biofilms on copper substrata. Cluster analyses of DGGE profiles demonstrated differences in the composition of biofilms on copper and steel materials.

Hydrophobic liquid-infused porous polymer surfaces for antibacterial applications.

Biofilms represent a fundamental problem in environmental biology, water technology, food hygiene as well as in medical and technical systems. Recently introduced slippery liquid-infused porous surface (SLIPS) showed great promise for preventing biofilm formation owing to the low surface energy of such surface in combination with its self-cleaning properties. In this study we demonstrated a novel hydrophobic liquid-infused porous poly(butyl methacrylate-co-ethylene dimethacrylate) surface (slippery BMA-EDMA) with bacteria-resistance in BM2 mineral medium and long-term stability in aqueous environments. We showed that the slippery BMA-EDMA surface prevents biofilm formation of different strains of opportunistic pathogen Pseudomonas aeruginosa for at least up to 7 days in low nutrient medium. Only ∼1.8% of the slippery surface was covered by the environmental P. aeruginosa PA49 strain under investigation. In uncoated glass controls the coverage of surfaces reached ∼55% under the same conditions. However, in high nutrient medium, more relevant to physiological conditions, the biofilm formation on the slippery surface turned out to be highly dependent on the bacterial strain. Although the slippery surface could prevent biofilm formation of most of the P. aeruginosa strains tested (∼1% surface coverage), the multiresistant P. aeruginosa strain isolated from wastewater was able to cover up to 12% of the surface during 7 days of incubation. RAPD-PCR analysis of the used P. aeruginosa strains demonstrated their high genome variability, which might be responsible for their difference in biofilm formation on the slippery BMA-EDMA surface. The results show that although the slippery BMA-EDMA surface has a great potential against biofilm formation, the generality of its bacteria resistant properties is still to be improved.

Diversity of patients microflora on orthopaedic and dental implants.

The aim of this study was to compare the diversity of microbial colonization on implant material from different individuals. Eubacterial DNA was extracted, separated and sequenced from orthopaedic metallic implant material, tissues or body fluids, and skin of 4 patients as well as from identical dental cement material from 10 individuals after revision and routine removal. Additionally, the composition of the bacterial population of the dental cement and the oral swab sample from one individual after direct extraction of bacterial DNA was compared to extraction after conventional microbiological enrichment. The latter investigation proved that the commonly used cultivation technique gave different results than direct extraction of DNA, especially as regards the detection of anaerobes. Comparing the bacterial colonization of implant materials from different patients showed significant individual diversity. The common focus on a constricted pathogen spectrum may have to be expanded toward a multispecies population. Moreover, the dependence of the bacterial population on the individual host has to be integrated in discussing implant colonization and infection.

Immune defense against S. epidermidis biofilms: components of the extracellular polymeric substance activate distinct bactericidal mechanisms of phagocytic cells.

Bacteria, organized in biofilms, are a common cause of relapsing or persistent infections and the ultimate cause of implant-associated osteomyelitis. Bacterial biofilms initiate a prominent local inflammatory response with infiltration of polymorphonuclear neutrophils (PMN), the main protagonists of the local innate host defense against bacteria. In our previous work we found that PMN recognize and adhere to biofilms, and that phagocytosis and degranulation of bactericidal substances, such as lactoferrin, were initiated. In contrast to the situation with planktonic bacteria, opsonization of biofilms with immunoglobulin and complement was not required for PMN activation, suggesting that biofilms contain signaling components for PMN. In the present study we identified in the bacteria-free extracellular substance of Staphylococcus epidermidis biofilms protein fractions that activated PMN in vitro.

Electrofiltration as a purification strategy for microbial poly-(3-hydroxybutyrate).

The biodegradable polyester poly-(3-hydroxybutyrate) (PHB), produced by Ralstonia eutropha in batch and fed-batch processes, was purified by electrofiltration. The protein film on PHB granules determines their high negative zeta potential, enabling the application of electrofiltration as an integrated technology in the downstream processing of PHB. In order to determine the optimal purification parameters, various pressure and electric field strength conditions were tested. Electrofiltration of PHB at 4bars and 4V/mm provided an up to four times higher concentration factor than conventional filtration. FT-Raman spectroscopy demonstrated that electrofiltration did not result in structural changes to the products. The study demonstrates the efficiency and practical advantages of electrofiltration as a promising downstream step in the PHB production technology.

Filtration kinetics of chitosan separation by electrofiltration.

Downstream processing of chitosan requires several technological steps that contribute to the total production costs. Precipitation and especially evaporation are energy-consuming processes, resulting in higher costs and limiting industrial scale production. This study investigated the filtration kinetics of chitosan derived from cell walls of fungi and from exoskeletons of arthropods by electrofiltration, an alternative method, thus reducing the downstream processing steps and costs. Experiments with different voltages and pressures were conducted in order to demonstrate the effect of both parameters on filtration kinetics. The concentration of the biopolymer was obtained by the average factor of 40 by applying an electric field of 4 V/mm and pressure of 4 bars. A series of analytical experiments demonstrated the lack of structural and functional changes in chitosan molecules after electrofiltration. These results, combined with the reduction of energy and processing time, define the investigated method as a promising downstream step in the chitosan production technology.

Effects of synbiotic fermentation products on primary chemoprevention in human colon cells.

The consumption of synbiotics, a mixture of probiotics and indigestible food constituents such as dietary fiber, has been reported to reduce colon cancer risk. We investigated the effects of fermented wheat aleurone enriched with the probiotics Lactobacillus rhamnosus GG/Bifidobacterium animalis supsp. lactis on the gene expression and functional end points related to cellular defence in HT29 and primary human colon cells. Aleurone was digested and fermented in vitro with/without probiotics. The resulting fermentation supernatants (fs) were analyzed for concentrations of deoxycholic acid and ammonia. The cells were treated with the fs, and effects on gene expression of catalase, GSTP1 and SULT2B1, enzyme activity of catalase and glutathione S-transferase as well as H2O2-induced DNA damage were examined. Fermentation of aleurone reduced deoxycholic acid concentration by 84%, while the probiotics enhanced this effect. Ammonia was increased by fs aleurone, whereas a reduction occurred by the addition of L. rhamnosus GG/B. animalis supsp. lactis 12. GSTP1 expression tended to result in an increase by the fs aleurone in both cell types, whereas the probiotics could not additionally increase the effect. Catalase was not modulated by fs aleurone enriched with probiotics. Only in HT29 cells, expression of SULT2B1 was enhanced by fs aleurone. Enzyme activity of catalase and glutathione S-transferase was induced (2-3.6 fold, 72 h) in HT29 cells only. Addition of probiotics had no influence on this effect. In HT29 cells, a reduced H2O2-induced DNA damage by the fs aleurone after 48 h, enhanced by the addition of probiotics, was detected. The observed effects could improve detoxification of xenobiotics and therefore may lower colon cancer risk.

The impact of recombinant fusion-hydrophobin coated surfaces on E. coli and natural mixed culture biofilm formation.

The impact of increased surface hydrophobicity on biofilms regarding retardation, repulsion, or attraction was studied with hydrophobin modified glass substrata. Recombinantly produced fungal hydrophobins forming self-assembled monolayers were used as the surface coating. The adsorption dynamics of hydrophobins were analysed with a quartz crystal microbalance which showed the surface coating to be rapid and stable. The change of surface wettability was determined by water contact angle measurements and demonstrated an increase in hydrophobicity in range of 60-62°. The homogeneity of the monolayers was demonstrated by immunofluorescence microscopy. Atomic force microscopy was applied to visualise the uniform texture of the coated materials. The hydrophobin coatings had no impact on different biofilms in terms of spatial distribution, cell numbers, and population composition. In consequence, hydrophobicity might not represent an important parameter for biofilm formation. Nevertheless, recombinant hydrophobins are suitable for large scale surface modification and functionalization with bioactive molecules.

Bacterial DNA from orthopedic implants after routine removal.

Bacterial 16S rDNA was monitored and identified from orthopedic metallic implants after routine or septic removal from patients in a German hospital. From March to June 2009, 28 metallic implants, 10 human biopsies, and 6 foam dressings from 28 patients were investigated. After analysis of this first collective, the methods were optimized to enhance sensitivity and to reduce interference with human DNA. Then a second collective consisting of 21 metallic implants from 21 patients was investigated from June 2009 to January 2010. In the first collective, 71% of the metallic implants were negative for eubacterial DNA. Pathogens such as Staphylococcus aureus and opportunists such as Lactobacillus rhamnosus were identified in 11% of the samples, whereas the residual 18% positive results were classified as from skin sources or could not be confirmed. Tissue, secretion, and bone samples as well as foam dressings from the same collective also contained pathogens and opportunists. After the optimization of the methods, a considerable increase of positive samples was seen: in the second collective 19 of the 21 metallic implants proved to be positive for eubacterial 16S rDNA. Bacterial DNA from environmental sources was detected in 13 samples, and in 20 specimens, predominantly mostly the skin. Opportunistic pathogens were detected in 19 samples. Interestingly, septic complications did not occur despite the presence of bacterial DNA. The results obtained up to now encourage us not only to continue a directed monitoring of bacterial DNA on orthopedic implants in practice but also to look intensely for possible sources of bacterial contamination during and after insertion or during removal of such implants.

MALDI-ToF mass spectrometry-multivariate data analysis as a tool for classification of reactivation and non-culturable states of bacteria.

Some bacterial life states are only difficult to describe and to detect because they are on the border of active metabolism. A prominent example is the so-called viable but non-culturable state, which is mainly characterized by the inability of bacteria to grow on synthetic media. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF/MS) in combination with multivariate data analysis represents a powerful tool for mass-spectrometric pattern recognition of biological samples. This method is already used for differentiation of bacterial strains. In this study we present a rapid readout method based on MALDI-ToF/MS in combination with principal component analysis to classify the bacterial non-culturable state using Enterococcus faecalis as a model organism. By applying this technique to samples of different physiological states, distinct clusters were calculated and all mass spectra were classified correctly into groups of similar type concerning their physiological state.

A lysozyme and magnetic bead based method of separating intact bacteria.

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Modification of an in vitro model simulating the whole digestive process to investigate cellular endpoints of chemoprevention.

In vitro gut fermentation systems are relevant tools to study health benefits of foodstuffs. Most of them are commonly used to investigate the degradation of nutrients or the development of gut flora. Using these models, strong cytotoxic effects of the resulting samples on cultured cells were observed. Hence, the aim of the present study was to develop a modified in vitro fermentation model that simulates the whole digestive tract and generates fermented samples that are suitable for testing in cell culture experiments. Wholemeal wheat flour (wwf) was digested and fermented in vitro with a fermentation model using different ox gall concentrations (41·6 and 0·6 g/l). The resulting fermentation supernatants (fs) were characterised for metabolites and biological effects in HT29 cells. The fermentation of wwf increased chemopreventive SCFA and decreased carcinogenic deoxycholic acid (DCA). The strong cytotoxic effects of the fs, which were partly due to cholic acid and DCA, were diminished by lowering the ox gall concentration, allowing the use of the samples in cell culture experiments. In conclusion, an in vitro digestion model, which can be used to study the effects of foodstuffs on chemoprevention and gut health in colon cells, is introduced and its physiological relevance is demonstrated.