Production of Fructooligosaccharides by Aspergillus phoenicis Biofilm on Polyethylene as Inert Support

Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25% (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68% of 1-kestose and 32% of nystose, was obtained in Khanna medium maintained at 30 degrees C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30%, m/V), the recovery of FOS was higher than that observed at a low concentration (5%, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50%, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7% of total FOS after 72 h of cultivation at 30 C, and 10% corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.

In vitro enterococcus faecalis biofilm formation on five adhesive systems

Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness. Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness. Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond.

Effectiveness of Tooth Wipes in Removing Babies' Dental Biofilm

Purpose: To assess the effectiveness of tooth wipes in removing dental biofilm from babies' anterior teeth, as well as to evaluate the babies' behaviour and the guardians' preference concerning hygiene methods. Materials and Methods: In this random blind cross-over study, 50 high caries risk babies, from 8 to 15 months old, were divided into two groups: babies with oral hygiene performed by caregivers (n = 25) or by their mothers (n = 25). The caregivers and mothers removed biofilm using three methods of oral hygiene (tooth wipes, toothbrushes and gauze), one in each experimental phase. Professional cleaning was done before each phase, which had 2 days of biofilm accumulation and 1 experimental day, when caregivers and mothers used one method to remove biofilnn. Examiners blinded to the study design assessed the biofilm index at baseline, prior to and following biofilm removal using each method. The babies' behaviour and the mothers'/caregivers' preference were assessed. Results: The tooth wipes, toothbrushes and gauze significantly reduced the amount of biofilm (P < 0.001). The mothers' group removed more biofilm than the caregivers' group, using toothbrushes or tooth wipes (P < 0.05). Babies in the mothers' group had better behaviour using tooth wipes than toothbrushes (P < 0.05). Mothers and caregivers preferred to use tooth wipes. Conclusions: Tooth wipes are effective in removing biofilrn from babies' anterior teeth and are the method best accepted by mothers, caregivers and babies.

Antimicrobial effect of endodontic solutions used as final irrigants on a dentine biofilm model

Aim To evaluate the residual biovolume of live bacterial cells, the mean biofilm thickness and the substratum coverage found in mixed biofilms treated with different endodontic irrigant solutions. Methodology Twenty-five bovine dentine specimens were infected intraorally using a removable orthodontic device. Five samples were used for each irrigant solution: 2% chlorhexidine, 1% sodium hypochlorite (NaOCl), 10% citric acid, 17% EDTA and distilled water. The solutions were used for 5 min. The samples were stained using the Live/Dead technique and evaluated using a confocal microscope. Differences in the amount of total biovolume (mu m3), number of surviving cells (mu m3), mean biofilm thickness (mu m) and substratum coverage (%) of the treated biofilms were determined using nonparametric statistical tests (P < 0.05). Results Similar values of biovolume total, biovolume of live subpopulations and substratum coverage were found in 2% chlorhexidine, 10% citric acid, 17% EDTA and distilled water-treated biofilms (P > 0.05). The lower values of the studied parameters were found in 1% NaOCl-treated dentine (P < 0.05) with the exception of the mean biofilm height criteria that did not reveal significant differences amongst the irrigant solutions (P > 0.05). Conclusions One per cent sodium hypochlorite was the only irrigant that had a significant effect on biofilm viability and architecture.

Effect of Chlorhexidine on Denture Biofilm Accumulation

Purpose: Adequate denture hygiene can prevent and treat infection in edentulous patients, who are frequently elderly and have difficulty brushing their teeth. This study evaluated the efficacy of complete denture biofilm removal using a chlorhexidine solution in two concentrations: 0.12% and 2.0%. Materials and Methods: Sixty complete denture wearers participated in a trial for 21 days after receiving brushing instructions. They were distributed into three groups, according to the tested solution and regimen (n = 20): (G1) Control (daily overnight soaking in water); (G2) daily immersion at home in 0.12% chlorhexidine for 20 minutes after dinner; and (G3) a single immersion in 2.0% chlorhexidine for 5 minutes at the end of the experimental period, performed by a professional. Biofilm coverage area (%) was quantified on the internal surface of maxillary dentures at baseline and after 21 days. Afterward, the differences between initial and posttreatment results were compared by means of the Kruskal-Wallis test (a = 0.05). Results: Median values for biofilm coverage area after treatment were: (G1) 36.0%; (G2) 5.3%; and (G3) 1.4%. Differences were significant (KW = 35.25; p < 0.001), although G2 and G3 presented similar efficacy in terms of biofilm removal. Conclusions: Both chlorhexidine-based treatments had a similar ability to remove denture biofilm. Immersion in 0.12% or 2.0% chlorhexidine solutions can be used as an auxiliary method for cleaning complete dentures.

Abundance of biofilm on intertidal rocky shores: Can trampling by humans be a negative influence?

Trampling by human visitors to rocky shores is a known stressor on macroorganisms. However, the effects of trampling on rocky intertidal biofilm, a complex association of microorganisms of ecological importance in coastal communities, have not been quantified. We evaluated the impact of trampling frequency and intensity on total biomass of epilithic microalgae on intertidal rocky shores in the southeast of Brazil. There was a trend of increase in the variability of biomass of biofilm in function of intensity of trampling, but no significant effects emerged among trampling treatments. The low influence of trampling on biofilm might be a result of the small dimensions of the organisms coupled with their natural resilience and roughness of the substrate; the former preventing the removal of biofilm layers by shoes and facilitating their quick recovery. Our results provide insights for management and conservation of coastal ecosystems revealing a weaker impact of trampling on biofilm than that reported on macroorganisms. (C) 2012 Elsevier Ltd. All rights reserved.

Comparative analysis of Enterococcus Faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37 C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (mm3), live bacteria biovolume (mm3), and substrate coverage (%) were quantified by using the BioImage_L software. Results obtained were analyzed by nonparametric tests (P = .05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P < .05) and hydroxyapatite the highest at 21 days (P < .05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P < .05). The percentages of coverage were similar among all substrates at 21 days (P > .05), but at 14 days, bovine bone presented the highest coverage (P < .05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated

Antimicrobial activity of triantibiotic paste, 2% chlorhexidine gel, and calcium hydroxide on an intraoral-infected dentin biofilm model

Introduction: The purpose of this study was to evaluate the antimicrobial activity of calcium hydroxide, 2% chlorhexidine gel, and triantibiotic paste (ie, metronidazole, minocycline, and ciprofloxacin) by using an intraorally infected dentin biofilm model. Methods: Forty bovine dentin specimens were infected intraorally using a removable orthodontic device in order to induce the biofilm colonization of the dentin. Then, the samples were treated with the medications for 7 days. Saline solution was used as the control. Two evaluations were performed: immediately after the elimination of the medication and after incubation in brain-heart infusion medium for 24 hours. The Live/Dead technique (Invitrogen, Eugene, OR) and a confocal microscope were used to obtain the percentage of live cells. Nonparametric statistical tests were performed to show differences in the percentage of live cells among the groups (P < .05). Results: Calcium hydroxide and 2% chlorhexidine gel did not show statistical differences in the immediate evaluation. However, after application of the brain-heart infusion medium for 24 hours, 2% gel chlorhexidine showed a statistically lesser percentage of live cells in comparison with calcium hydroxide. The triantibiotic paste significantly showed a lower percentage of live cells in comparison with the 2% chlorhexidine gel and calcium hydroxide groups in the immediate and secondary (after 24 hours) evaluations. Conclusions: The triantibiotic paste was most effective at killing the bacteria in the biofilms on the intraorally infected dentin model in comparison with 2% chlorhexidine gel and calcium hydroxide

Analysis of the biofilm proteome of Xylella fastidiosa

Abstract Background Xylella fastidiosa is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of X. fastidiosa strain 9a5c, in comparison to planktonic growth condition. Results We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of X. fastidiosa. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments. Conclusions We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of X. fastidiosa, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.

Characterization of immobilized biomass by amplified rDNA restriction analysis (ARDRA) in an anaerobic sequencing-batch biofilm reactor (ASBBR) for the treatment of industrial wastewater

The performance of an anaerobic sequencing-batch biofilm reactor (ASBBR- laboratory scale- 14L )containing biomass immobilized on coal was evaluated for the removal of elevated concentrations of sulfate (between 200 and 3,000 mg SO4-2·L-1) from industrial wastewater effluents. The ASBBR was shown to be efficient for removal of organic material (between 90% and 45%) and sulfate (between 95% and 85%). The microbiota adhering to the support medium was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). The ARDRA profiles for the Bacteria and Archaea domains proved to be sensitive for the determination of microbial diversity and were consistent with the physical-chemical monitoring analysis of the reactor. At 3,000 mg SO4-2·L-1, there was a reduction in the microbial diversity of both domains and also in the removal efficiencies of organic material and sulfate.

Biofilm formation by the anaerobic pathogen Clostridium difficile

Clostridium difficile is an obligate anaerobic, Gram-positive, endospore-forming bacterium. Although an opportunistic pathogen, it is one of the important causes of healthcare-associated infections. While toxins TcdA and TcdB are the main virulence factors of C. difficile, the factors or processes involved in gut colonization during infection remain unclear. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Little is known about biofilm formation by anaerobic gut species. Biofilm formation by C. difficile could play a role in virulence and persistence of C. difficile, as seen for other intestinal pathogens. We demonstrate that C. difficile clinical strains, 630, and the strain isolated in the outbreak, R20291, form structured biofilms in vitro. Biofilm matrix is made of proteins, DNA and polysaccharide. Strain R20291 accumulates substantially more biofilm. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella and a putative quorum sensing regulator, LuxS, Spo0A, are required for maximal biofilm formation by C. difficile. Moreover we demonstrate that bacteria in C. difficile biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI, and that inhibitory and sub-inhibitory concentrations of the same antibiotic induce biofilm formation. Surprisingly, clinical C. difficile strains from the same out-break, but from different origin, show differences in biofilm formation. Genome sequence analysis of these strains showed presence of a single nucleoide polymorphism (SNP) in the anti-σ factor RsbW, which regulates the stress-induced alternative sigma factor B (σB). We further demonstrate that RsbW, a negative regulator of alternative sigma factor B, has a role in biofilm formation and sporulation of C. difficile. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Exploring the biofilm of Streptococcus agalactiae to identify virulence factors

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

Antibacterial effect of taurolidine (2%) on established dental plaque biofilm

Preliminary data have suggested that taurolidine may bear promising disinfectant properties for the therapy of bacterial infections. However, at present, the potential antibacterial effect of taurolidine on the supragingival plaque biofilm is unknown. To evaluate the antibacterial effect of taurolidine on the supragingival plaque biofilm using the vital fluorescence technique and to compare it with the effect of NaCl and chlorhexidine (CHX), 18 subjects had to refrain from all mechanical and chemical hygiene measures for 24 h. A voluminous supragingival plaque sample was taken from the buccal surfaces of the lower molars and wiped on an objective slide. The sample was then divided into three equal parts and mounted with one of the three test or control preparations (a) NaCl, (b) taurolidine 2% and (c) CHX 0.2%. After a reaction time of 2 min, the test solutions were sucked of. Subsequently, the plaque biofilm was stained with fluorescence dye and vitality of the plaque flora was evaluated under the fluorescence microscope (VF%). Plaque samples treated with NaCl showed a mean VF of 82.42 ± 6.04%. Taurolidine affected mean VF with 47.57 ± 16.60% significantly (p < 0.001, paired t test). The positive control CHX showed the lowest mean VF values (34.41 ± 14.79%; p < 0.001 compared to NaCl, p = 0.017 compared to taurolidine). Taurolidine possesses a significant antibacterial effect on the supragingival plaque biofilm which was, however, not as pronounced as that of CHX.

Microbiota in the oral subgingival biofilm is associated with obesity in adolescence

To test the hypothesis whether microbiota in oral biofilm is linked with obesity in adolescents we designed this cross-sectional study. Obese adolescents (n = 29) with a mean age of 14.7 years and normal weight subjects (n = 58) matched by age and gender were examined with respect to visible plaque index (VPI%) and gingival inflammation (bleeding on probing (BOP%)). Stimulated saliva was collected. They answered a questionnaire concerning medical history, medication, oral hygiene habits, smoking habits, and sociodemographic background. Microbiological samples taken from the gingival crevice was analyzed by checkerboard DNA-DNA hybridization technique. The sum of bacterial cells in subgingival biofilm was significantly associated with obesity (P < 0.001). The link between sum of bacterial cells and obesity was not confounded by any of the studied variables (chronic disease, medication, VPI%, BOP%, flow rate of whole saliva, or meal frequency). Totally 23 bacterial species were present in approximately threefold higher amounts, on average, in obese subjects compared with normal weight controls. Of the Proteobacteria phylum, Campylobacter rectus and Neisseria mucosa were present in sixfold higher amounts among obese subjects. The association between obesity and sum of bacterial cells in oral subgingival biofilm indicates a possible link between oral microbiota and obesity in adolescents.