Assessment of three-dimensional biofilm structure using an optical microscope

A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the calculation of the biofilm thickness. The method is intended to overcome the need for expensive microscopes to study biofilms.     

Quantification of viable biomass in biofilm reactors by extractable lipid phosphate

 Lipid phosphate, which is a measure of viable biomass, was determined using biofilm samples from three different laboratory-scale reactors. The analysis procedure proposed in the literature was modified and tested for suitability in experiments with biofilm reactors. The microbial contents of the biofilms studied are compared in three types of reactor. Received: 2 November 1994/Accepted: 23 January 1995     

A novel approach to investigate biofilm accumulation and bacterial transport in porous matrices

Knowledge of bacterial transport through, and biofilm growth in, porous media is vitally important in numerous natural and engineered environments. Despite this, porous media systems are generally oversimplified and the local complexity of cell transport, biofilm formation and the effect of biofilm accumulation on flow patterns is lost. In this study, cells of the sulphate-reducing bacterium, Desulfovibrio sp. EX265, accumulated primarily on the leading faces of obstructions and developed into biofilm, which grew to narrow and block pore throats (at a rate of 12 micro m h(-1) in one instance). This pore blocking corresponded to a decrease in permeability from 9.9 to 4.9 Darcy. Biofilm processes were observed in detail and quantitative data were used to describe the rate of biofilm accumulation temporally and spatially. Accumulation in the inlet zone of the micromodel was 10% higher than in the outlet zone and a mean biofilm height of 28.4 micro m was measured in a micromodel with an average pore height of 34.9 microm. Backflow (flow reversal) of fluid was implemented on micromodels blocked with biofilm growth. Although biofilm surface area cover did immediately decrease (approximately 5%), the biofilm quickly re-established and permeability was not significantly affected (9.4 Darcy). These results demonstrate that the glass micromodel used here is an effective tool for in situ analysis and quantification of bacteria in porous media.     

Quantification of biofilm exopolysaccharides using an in situ assay with periodic acid–Schiff reagent

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid–Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins).     

Evaluation of three indices for biofilm accumulation on complete dentures

doi:10.1111/j.1741‐2358.2009.00285.x
Evaluation of three indices for biofilm accumulation on complete dentures Objectives: The objective of this study was to evaluate the accuracy and reproducibility of three complete denture biofilm indices (Prosthesis Hygiene Index; Jeganathan et al. Index; Budtz‐Jørgensen Index) by means of a computerised comparison method. Background: Clinical studies into denture hygiene have employed a large number of biofilm indices among their outcome variables. However, the knowledge about the validity of these indices is still scarce. Materials and methods: Sixty‐two complete denture wearers were selected. The internal surfaces of the upper complete dentures were stained (5% erythrosine) and photographed. The slides were projected on paper, and the biofilm indices were applied over the photos by means of a scoring method. For the computerised method, the areas (total and biofilm‐covered) were measured by dedicated software (Image Tool). In addition, to compare the results of the computerised method and Prosthetic Hygiene Index, a new scoring scale (including four and five graded) was introduced. For the Jeganathan et al. and Budtz‐Jørgensen indices, the original scales were used. Values for each index were compared with the computerised method by the Friedman test. Their reproducibility was measured by means of weighed κ. Significance for both tests was set at 0.05. Results: The indices tested provided similar mean measures but they tended to overestimate biofilm coverage when compared with the computerised method (p < 0.001). Agreement between the Prosthesis Hygiene Index and the computerised method was not significant, regardless of the scale used. Jeghanathan et al. Index showed weak agreement, and consistent results were found for Budtz‐Jorgensen Index (κ = 0.19 and 0.39 respectively). Conclusion: Assessment of accuracy for the biofilm indices showed instrument bias that was similar among the tested methods. Weak inter‐instrument reproducibility was found for the indices, except for the Budtz‐Jørgensen Index. This should be the method of choice for clinical studies when more sophisticated approaches are not possible.     

Quantification of Pseudomonas aeruginosa hydrogen cyanide production by a polarographic approach

Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous infections acquired in hospital especially in persons whose immune systems are weakened, such as with patient suffering from AIDS or cystic fibrosis. This bacterium produces a great diversity of virulence factors among them hydrogen cyanide (HCN) which is one of the most potent and toxic. A precise quantification of HCN or CN(-) ion is essential to understand the involvement of this toxin in the pathogenesis of P. aeruginosa. In the present study, we present a new technique based on a polarographic approach to measure the production kinetics of HCN/CN(-) by P. aeruginosa strains, in several media commonly used in microbiology labs. The method was validated using mutants (hcnB- and hcnC-) which are unable to produce detectable HCN/CN(-). The kinetics of HCN/CN(-) production by P. aeruginosa in Luria Bertani (LB) medium showed a parabolic shape with a peak observed at 4, 5 and 8h for strains PA14, PAO1 and MPAO1, respectively. When bacteria were grown in ordinary nutrient broth (ONB) 2.5% medium, a less adapted medium for bacterial growth, the general profile of the kinetics was conserved but peak production was delayed (10 and 12h for PAO1 and MPAO1, respectively). When the bacteria were cultured in minimum medium MMC, bacterial growth was particularly slow and HCN/CN(-) production was markedly reduced. Taken together, this new polarographic method appears as a useful technique to detect and quantify HCN/CN(-) in routine media where the bacteria can express and regulate high amounts of toxins. With this method, we demonstrate that HCN/CN(-) production by P. aeruginosa is maximal at the end of the exponential growth phase and depends on the richness of the growth medium used.     

Nickel accumulation by immobilized biofilm of Cirobacter sp. containing cell-bound polycrystalline hydrogen uranyl phosphate

Hydrogen uranyl phosphate (HUO2PO4: HUP), deposited enzymatically on Citrobacter N14 cells immobilized as biofilm on ceramic Raschig rings in a flow-through column, removed nickel quantitatively from dilute aqueous solution in the form of nickel uranyl phosphate, via intercalative ion exchange. Nickel-loaded columns were regenerated by washing either with citrate buffer or with buffer containing UO22 and phosphate donor (glycerol 2-phosphate), this giving additional crystalline HUP deposit for subsequent improvement of nickel removal. No uranium release occurred during selective desorption of Ni, proving the integrity of the biofilm within the column. The use of ceramic supports to manufacture an artificial, bioinorganic, ion exchanger is novel and the use of solid matrices overcomes the problems of mechanical stability which limit the applications of gel-immobilized cells for large-scale processes. © Rapid Science Ltd. 1998     

Indole is an inter-species biofilm signal mediated by SdiA

Background  

As a stationary phase signal, indole is secreted in large quantities into rich medium by Escherichia coli and has been shown to control several genes (e.g., astD, tnaB, gabT), multi-drug exporters, and the pathogenicity island of E. coli; however, its impact on biofilm formation has not been well-studied.     

Utilisation of a packed-bed biofilm reactor for the determination of the potential of biofilm accumulation in water systems

An experimental system has been developed that allows the monitoring of biofilm development on supports exposed to water of different characteristics. The system consists of a series of packed-bed reactors filled with glass beads, and by periodically removing biofilm attached to these beads for off-line analyses this provides a means for monitoring biofilm development. Despite its reduced dimensions (6.9 cm long and 1.58 cm in diameter), the experimental system used has a sampling surface of 90.3 cm2 (including only the surface of the glass beads). This allows reproducible and representative samples to be taken from different water systems, providing a reliable and economic method for evaluating in situ the formation of biofilms from different environments. The set-up of the entire experimental system was constructed to meet the demands of field experiments in a well-defined hydrodynamic environment and to allow easy removal of samples for biomass quantification and microscopic observation. Data obtained using this device can be used as an indicator of the risk of biofilm formation in different water systems. This indicator, "the biofilm accumulation potential", represents an effective and representative tool for the monitoring of biofilm development in an integrated antifouling strategy, in order to help keep biofouling, scaling and microbial risks under control. According to the experiments with the packed-bed reactors used with a high flow regime, the ratio TCN/HPC could provide an indication of the state of the biofilm, and lower ratios could indicate a higher biofilm accumulation potential.     

In vitro and in vivo inhibition of Streptococcus mutans biofilm by Trachyspermum ammi seeds: an approach of alternative medicine

The aim of this study was to evaluate the influence of the crude and active solvent fraction of Trachyspermum ammi on S. mutans cariogenicity, effect on expression of genes involved in biofilm formation and caries development in rats. GC-MS was carried out to identify the major components present in the crude and the active fraction of T. ammi. The crude extract and the solvent fraction exhibiting least MIC were selected for further experiments. Scanning electron microscopy was carried out to observe the effect of the extracts on S. mutans biofilm. Comparative gene expression analysis was carried out for nine selected genes. 2-Isopropyl-5-methyl-phenol was found as major compound in crude and the active fraction. Binding site of this compound within the proteins involved in biofilm formation, was mapped with the help of docking studies. Real-time RT-PCR analyses revealed significant suppression of the genes involved in biofilm formation. All the test groups showed reduction in caries (smooth surface as well as sulcal surface caries) in rats. Moreover, it also provides new insight to understand the mechanism influencing biofilm formation in S. mutans. Furthermore, the data suggest the putative cariostatic properties of T. Ammi and hence can be used as an alternative medicine to prevent caries infection.     

Quantifying dental biofilm growth using cross-polarization optical coherence tomography

Aims: Quantifying the ex vivo growth of complex multispecies dental biofilms using cross‐polarization 1310‐nm optical coherence tomography (CP‐OCT) system was investigated. Methods and Results: Bacterial microcosms, which were derived from plaque samples of paediatric subjects, were incubated in a biofilm reactor system containing discs of different dental materials for 72 h with daily sucrose pulsing (5×). CP‐OCT analysis of biofilm mass was validated with crystal violet (CV) assays at various growth stages of these complex biofilms. CP‐OCT was able to filter out the back‐reflected signals of water layers in the hydrated biofilm and allowed for direct biofilm quantification. The overall depth‐resolved scattering intensity of the biofilm showed very strong positive correlation with CV assay quantification (Spearman’s ρ = 0·92) during the growth phase of the biofilm. Conclusion: CP‐OCT was able to quantify the mass of the biofilm by measuring the overall depth‐resolved scattering of the biofilm. Significance and Impact of the Study: CP‐OCT has the ability to nondestructively monitor biofilm growth and elucidate the growth characteristics of these microcosms on different dental material compositions.     

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

AIMS: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18:2) via conjugated 18:2 metabolites (mainly cis-9,trans-11-18:2, conjugated linoleic acid) to vaccenic acid (trans-11-18:1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18:0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. METHODS AND RESULTS: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. CONCLUSIONS: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. SIGNIFICANCE AND IMPACT OF THE STUDY: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.     

Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.     

Inhibition of Streptococcus mutans biofilm accumulation and development of dental caries in vivo by 7-epiclusianone and fluoride

7-Epiclusianone (7-epi), a novel naturally occurring compound isolated from Rheedia brasiliensis, effectively inhibits the synthesis of exopolymers and biofilm formation by Streptococcus mutans. In the present study, the ability of 7-epi, alone or in combination with fluoride (F), to disrupt biofilm development and pathogenicity of S. mutans in vivo was examined using a rodent model of dental caries. Treatment (twice-daily, 60s exposure) with 7-epi, alone or in combination with 125 ppm F, resulted in biofilms with less biomass and fewer insoluble glucans than did those treated with vehicle-control, and they also displayed significant cariostatic effects in vivo (p < 0.05). The combination 7-epi + 125 ppm F was as effective as 250 ppm F (positive-control) in reducing the development of both smooth- and sulcal-caries. No histopathological alterations were observed in the animals after the experimental period. The data show that 7-epiclusianone is a novel and effective antibiofilm/anticaries agent, which may enhance the cariostatic properties of fluoride.     

Inhibition of Streptococcus mutans biofilm accumulation and development of dental caries in vivo by 7-epiclusianone and fluoride

7-Epiclusianone (7-epi), a novel naturally occurring compound isolated from Rheedia brasiliensis, effectively inhibits the synthesis of exopolymers and biofilm formation by Streptococcus mutans. In the present study, the ability of 7-epi, alone or in combination with fluoride (F), to disrupt biofilm development and pathogenicity of S. mutans in vivo was examined using a rodent model of dental caries. Treatment (twice-daily, 60s exposure) with 7-epi, alone or in combination with 125 ppm F, resulted in biofilms with less biomass and fewer insoluble glucans than did those treated with vehicle-control, and they also displayed significant cariostatic effects in vivo (p < 0.05). The combination 7-epi + 125 ppm F was as effective as 250 ppm F (positive-control) in reducing the development of both smooth- and sulcal-caries. No histopathological alterations were observed in the animals after the experimental period. The data show that 7-epiclusianone is a novel and effective antibiofilm/anticaries agent, which may enhance the cariostatic properties of fluoride.     

Biodegradation by immobilized bacteria in an airlift-loop reactor-influence of biofilm diffusion limitation

Naphthalene-2-sulfonate was degraded by submerse growing Pseudomonads in a chemostat culture. The kinetic parameters for the Monod equation, including Pirts maintenance energy, were calculated from these experiments regarding naphthalene-2-sulfonate as substrate and oxygene as cosubstrate. By immobilizing the bacteria on sand particles, the degradation of naphthalene-2-sulfonate was carried out in a specialy designed three-phase airlift-loop reactor in a completely fluidized state. From these experiments, the influence of biofilm diffusion limitation on reaction kinetics and criteria for stable biofilm formation on sand particles were obtained.     

A proteomic approach for exploring biofilm in Streptococcus mutans

Biofilm formation by Streptococcus mutans is considered as its principal virulence factor, causing dental caries. Mutants of S. mutans defective in biofilm formation were generated and analyzed to study the collective role of proteins in its formation. Mutants were characterized on the basis of adherence to saliva-coated surface, and biofilm formation. The confocal laser microscopy and scanning electron microscopy images showed that the control biofilms had cluster of cells covered by layer of exo-polysaccharide while the biofilms of mutants were thin and spaced. Two-dimensional protein electrophoresis data analysis identified 57 proteins that are either up (44 proteins) or down (13 proteins) regulated. These data points to the importance of up and down regulated proteins in the formation of biofilm in Streptococcus mutans.     

Metabolic blocking of exopolysaccharides synthesis: effects on microbial adhesion and biofilm accumulation

A blocking agent of polysaccharide synthesis (5 x 10-4 M 2,4-dinitrophenol) was continuously added to a reaction system, where a heterogeneous microbial population was cultivated at a dilution rate of 0.1 h-1. The results indicate that adhesion and biofilm accumulation were severely reduced when exopolysaccharydes synthesis was blocked.