Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin

Abstract

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6?h day^?1) to 1% sucrose for 4?days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student’s-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48?h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.     

On the release of fluoride from biofilm reservoirs during a cariogenic challenge: an in situ study

Abstract

Biofilm fluoride reservoirs may be a source of fluoride to the fluid phase during a sugar challenge reducing tooth mineral loss. However, the evidence for that is conflicting and has not been studied in biofilms containing different fluoride levels. In order to test fluoride release from biofilms with distinct fluoride concentrations, biofilms were grown in situ exposed to a combination of placebo, calcium and fluoride rinses forming biofilms with no (fluoride-free rinses), low (fluoride-only rinses) or high (calcium followed by fluoride rinses) fluoride concentrations, and collected before and 5?min after a sucrose challenge. Rinsing with fluoride increased fluoride concentration in the biofilm (p?<?0.05), mainly when a calcium pre-rinse was used before the fluoride (p?<?0.05). However, after a sugar challenge, no significant increase in the biofilm fluid fluoride concentration was observed, even in the fluoride-rich biofilms (p?>?0.05). Fluoride-rich biofilms do not release fluoride to the fluid phase during a sugar challenge.     

Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

An in vitro dynamic microcosm biofilm model for caries lesion development and antimicrobial dose-response studies

Some dynamic biofilm models for dental caries development are limited as they require multiple experiments and do not allow independent biofilm growth units, making them expensive and time-consuming. This study aimed to develop and test an in vitro dynamic microcosm biofilm model for caries lesion development and for dose-response to chlorhexidine. Microcosm biofilms were grown under two different protocols from saliva on bovine enamel discs for up to 21 days. The study outcomes were as follows: the percentage of enamel surface hardness change, integrated hardness loss, and the CFU counts from the biofilms formed. The measured outcomes, mineral loss and CFU counts showed dose-response effects as a result of the treatment with chlorhexidine. Overall, the findings suggest that biofilm growth for seven days with 0.06 ml min^?1 salivary flow under exposure to 5% sucrose (3 × daily, 0.25 ml min^?1, 6 min) was suitable as a pre-clinical model for enamel demineralization and antimicrobial studies.     

Use of dissolved ozone for controlling planktonic and sessile bacteria in industrial cooling systems

Cooling water treatment requires effective, environmentally-safe biocides compatible with system operation. The unique combination of high biocidal activity during use with no toxic discharge, could render dissolved ozone a safe biocide for cooling water treatment. Planktonic and sessile cells of Pseudomonas fluorescens (a frequent microbial contaminant of industrial systems) were used in this work to assess the biocidal effectiveness of ozone. Dissolved ozone showed to be effective at concentrations between 0.1 and 0.3 ppm, to eliminate completely the levels of planktonic cells used in this paper (10⁷–10⁸ cell/ml) within a range of contact times between 10 and 30 min. However, ozone at 0.15 ppm was only able to diminish sessile cell population by two or three orders of magnitude. This minor biocidal effectiveness of ozone against bacterial biofilms is discussed in this paper, taking into account recent concepts on structure and dynamics of biofilms. Different metallic substrata were assayed to verify if there was any effect of metal nature on the biocidal action. Open circuit potentials vs. time experiments and potentiodynamic polarization curves were made for assessing the effect of dissolved ozone on the corrosion behavior of the metals tested.     

Plasma-Mediated Inactivation of Pseudomonas aeruginosa Biofilms Grown on Borosilicate Surfaces under Continuous Culture System

Biofilms are microbial communities attached to a surface and embedded in a matrix composed of exopolysaccharides and excreted nucleic acids. Bacterial biofilms are responsible for undesirable effects such as disease, prostheses colonization, biofouling, equipment damage, and pipe plugging. Biofilms are also more resilient than free-living cells to regular sterilization methods and therefore it is indispensable to develop better ways to control and remove them. The use of gas discharge plasmas is a good alternative since plasmas contain a mixture of reactive agents well-known for their decontamination potential against free microorganisms. We have previously reported that Pseudomonas aeruginosa biofilms were inactivated after a 1-min plasma exposure. We determined that the adhesiveness and the thickness of Pseudomonas biofilms grown on borosilicate were reduced. We also reported sequential morphological changes and loss of viability upon plasma treatment. However, the studies were carried out in batch cultures. The use of a continuous culture results in a more homogenous environment ensuring reproducible biofilm growth. The aim of this work was to study plasma-mediated inactivation of P. aeruginosa biofilms grown on borosilicate in a continuous culture system. In this paper we show that biofilms grown on glass under continuous culture can be inactivated by using gas discharge plasma. Both biofilm architecture and cell culturabilty are impacted by the plasma treatment. The inactivation kinetics is similar to previously described ones and cells go through sequential changes ranging from minimal modification without loss of viability at short plasma exposure times, to major structure and viability loss at longer exposure times. We report that changes in biofilm structure leading to the loss of culturability and viability are related to a decrease of the biofilm matrix adhesiveness. To our knowledge, there has been no attempt to evaluate the inactivation/sterilization of biofilms grown in a continuous system.     

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains

Abstract

In this study, the effect of three essential oils (EOs) – clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe – on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%–0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4?×?MIC), after 10?min, whereas GO completely controlled growth at 0.36% (v/v) (4?×?MIC) after treatment for 20?min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4?×?MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8?×?MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30?min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H₂O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4—used as an indicator strain, B. licheniformis T6-5, and B. firmus H₂O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H₂O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H₂O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H₂O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.     

Multi-species biofilms defined from drinking water microorganisms provide increased protection against chlorine disinfection

A model biofilm, formed of multiple species from environmental drinking water, including opportunistic pathogens, was created to explore the tolerance of multi-species biofilms to chlorine levels typical of water-distribution systems. All species, when grown planktonically, were killed by concentrations of chlorine within the World Health Organization guidelines (0.2–5.0?mg?l^?1). Higher concentrations (1.6–40-fold) of chlorine were required to eradicate biofilm populations of these strains, ～70% of biofilms tested were not eradicated by 5.0?mg?l^?1 chlorine. Pathogenic bacteria within the model multi-species biofilms had an even more substantial increase in chlorine tolerance; on average ～700–1100?mg?l^?1 chlorine was required to eliminate pathogens from the biofilm, 50–300-fold higher than for biofilms comprising single species. Confocal laser scanning microscopy of biofilms showed distinct 3D structures and multiple cell morphologies and arrangements. Overall, this study showed a substantial increase in the chlorine tolerance of individual species with co-colonization in a multi-species biofilm that was far beyond that expected as a result of biofilm growth on its own.     

A novel biofilm channel for evaluating the adhesion of diatoms to non-biocidal coatings

Laboratory assessment of the adhesion of diatoms to non-toxic fouling-release coatings has tended to focus on single cells rather than the more complex state of a biofilm. A novel culture system based on open channel flow with adjustable bed shear stress values (0–2.4?Pa) has been used to produce biofilms of Navicula incerta. Biofilm development on glass and polydimethylsiloxane elastomer (PDMSe) showed a biphasic relationship with bed shear stress, which was characterised by regions of biofilm stability and instability reflecting cohesion between cells relative to the adhesion to the substratum. On glass, a critical shear stress of 1.3–1.4?Pa prevented biofilm development, whereas on PDMS, biofilms continued to grow at 2.4?Pa. Studies of diatom biofilms cultured on zwitterionic coatings using a bed shear stress of 0.54?Pa showed lower biomass production and adhesion strength on poly(sulfobetaine methacrylate) compared to poly(carboxybetaine methacrylate). The dynamic biofilm approach provides additional information to supplement short duration laboratory evaluations.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

Influence of surface copper content on Stenotrophomonas maltophilia biofilm control using chlorine and mechanical stress

Abstract

This work aimed to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. Stenotrophomonas maltophilia isolated from drinking water was used as a model microorganism and biofilms were developed in a rotating cylinder reactor using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10?mg l^?1 of free chlorine for 10?min, an increased shear stress (a fluid velocity of 1.5?m s^?1 for 30s), and a combination of both treatments. These shock treatments were not effective in biofilm control. The benefits from the use of copper surfaces was found essentially in reducing the numbers of non-damaged cells. Copper materials demonstrated better performance in biofilm prevention than chlorine. In general, copper alloys may have a positive public health impact by reducing the number of non-damaged cells in the water delivered after chlorine exposure.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Abstract

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 μm h^?1 in the turbulent flow cell and 1.0 μm h^?1 in the laminar flow cell.     

Heterogeneity in the efficacy of dental chemical disinfectants on water-derived biofilms in vitro

Abstract

Conditions in dental unit waterlines are favourable for biofilm growth and contamination of dental unit water. The aim of this study was to assess the effect of several chemical disinfectants on bacteria in a biofilm model. Water-derived biofilms were grown in a static biofilm model (Amsterdam Active Attachment model), using two growth media. Biofilms were challenged with Alpron/Bilpron, Anoxyl, Citrisil, Dentosept, Green & Clean, ICX and Oxygenal in shock dose and maintenance doses. The concentration and the composition of the chemical disinfectants influenced the number of culturable bacteria in the biofilms. The application of a single shock dose followed by a low dose of the same chemical disinfectants resulted in the greatest suppression of viable bacteria in the biofilms. Exposure to Citrisil and ICX consistently resulted in failure to control the biofilms, while Alpron/Bilpron had a substantial and relevant effect on the number of bacteria in the biofilms.     

Surface properties,adhesion and biofilm formation on different surfaces by Scedosporium spp. and Lomentospora prolificans

Abstract

In the present work, some surface properties of the fungi Scedosporium apiospermum, S. aurantiacum, S. minutisporum, and Lomentospora prolificans and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4?h of fungi–surface contact. Additionally, after 72?h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72?h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that Scedosporium/Lomentospora spp. are capable of forming biofilms on diverse abiotic surfaces.     

An in vitro model for the growth and analysis of chronic wound MRSA biofilms

Aims: To develop an in vitro model (Colony/drip‐flow reactor – C/DFR) for the growth and analysis of methicillin‐resistant Staphylococcus aureus (MRSA) biofilms. Methods and Results: Using the C/DFR model, biofilms were grown on the top of polycarbonate filter membranes inoculated with a clinical isolate of MRSA, placed on absorbent pads in the DFR and harvested after 72 h. The biofilms varied from 256 to 308 μm in thickness with a repeatability standard deviation of 0·22. Testing of antimicrobial agents was also performed where C/DFR biofilms were grown in parallel with conventional colony biofilms. A saline solution (control), 1% silver sulfadiazine solution, and 0·25% Dakin’s solution were used to treat the biofilms for 15 min. Microscopic evaluation of biofilm morphology and thickness was conducted. The Dakins solution in both models produced statistically significantly higher log reductions than silver sulfadiazine treatment. Conclusions: The C/DFR biofilms were thick and repeatable and exhibited higher resistance to Dakins solution than the treated colony biofilms. Significance and Impact of the Study: The C/DFR can be used as a tool for examining complex biofilm physiology as well as for performing comparative experiments that test wound care products and novel antimicrobials.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l^?1 caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Roughness effects of diatomaceous slime fouling on turbulent boundary layer hydrodynamics

Abstract

Biofilm fouling significantly impacts ship performance. Here, the impact of biofilm on boundary layer structure at a ship-relevant, low Reynolds number was investigated. Boundary layer measurements were performed over slime-fouled plates using high resolution particle image velocimetry (PIV). The velocity profile over the biofilm showed a downward shift in the log-law region (ΔU⁺), resulting in an effective roughness height (k_s) of 8.8?mm, significantly larger than the physical thickness of the biofilm (1.7?±?0.5?mm) and generating more than three times as much frictional drag as the smooth-wall. The skin-friction coefficient, C_f, of the biofilm was 9.0?×?10^?3 compared with 2.9?×?10^?3 for the smooth wall. The biofilm also enhances turbulent kinetic energy (tke) and Reynolds shear stress, which are more heterogeneous in the streamwise direction than smooth-wall flows. This suggests that biofilms increase drag due to high levels of momentum transport, likely resulting from protruding streamers and surface compliance.     

Quantification of diatoms in biofilms: Standardisation of methods

Abstract

Benthic diatoms, which often dominate marine biofilms are mostly pennate along with a few centric species that have an attached mode of life. Even though the range of diatoms in biofilms is diverse, their ecology is poorly understood because of the difficulty in sampling and enumeration. Scraping or brushing are the traditional methods used for removal of diatoms from biofilms developed on solid substrata. The method of removal is the most critical step in enumerating the biofilm diatom community structure. In this study, a nylon brush and ceramic scraper were used as tools for the removal of diatoms from 1 – 4-day-old biofilms developed on fibreglass coupons and glass microscope slides. Standardisation of methods showed that the sample volume used in the analyses had the least influence on the quantification, whereas the method of removal was critical. The nylon brush was more efficient at recovering diatoms compared to a ceramic scraper. Direct microscopic enumeration of the community in the case of glass slides indicated that scraping resulted in between 30–50% underestimation. Heterogeneity in diatom community structure between replicate samples is one possible reason for such underestimation.