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Uniaxial compression measurement device for investigation of the mechanical stability of biofilms.

The mechanical stability of biofilms is important for biotechnology, as sloughing of the biomass due to mechanical failure of the biofilm matrix can lead to severe interferences with biofilm processes. In cases of biofouling, biofilms have to be removed, in which case their mechanical stability must be overcome. The apparent modulus of elasticity and the yield strength as obtained from uniaxial compression experiments can be taken as parameters indicative for the mechanical stability of a biofilm. A film rheometer is presented which allows for the determination of these quantities, using model biofilms of Pseudomonas aeruginosa grown on membrane filters. The compressive stress-strain behaviour up to the point of failure is recorded at a compression speed of 1 microm s(-1). In accordance with the stress-strain curve, the investigated biofilm can be described as viscoelastic material, which demonstrates plastic flow properties. The extracellular polymeric substances (EPS), which keep biofilms together, form a temporary network of fluctuating junction points. Above the yield point, the gel structure fails and the system behaves as a highly viscous fluid. The apparent modulus of elasticity and the yield point are considered to be useful parameters for characterizing the mechanical properties of biofilms.

Biofilms↗

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.

Biofilms↗

Surface-induced and biofilm-induced changes in gene expression.

A biofilm is a community of microorganisms attached to a surface. Based on studies of single-species communities, biofilm formation follows a progression from initial attachment to a mature form composed of pillar-like multicellular structures interspersed with fluid-filled channels. The developmental progression leading to a mature biofilm requires changes in gene expression. With recent technological advances for visualizing biofilm growth, gene expression can be directly monitored during biofilm development. Hence, analyses of surface-induced and biofilm-induced changes in gene expression have begun in earnest. Recent studies have identified regulatory pathways that are important for biofilm formation and have focused on genetic responses to environmental stimuli in mature biofilms. These findings are providing new insights into biofilm development and physiology.

Biofilms↗

Growth rate and biofilm thickness of Streptococcus sobrinus and Streptococcus mutans on hydroxapatite.

Bacteria in biofilm and planktonic bacteria exhibit different properties. The objective of the present study was to compare the growth rates of Streptococcus sobrinus and Streptococcus mutans on different types of biofilm with their planktonic growth rate. Our experimental model consisted of hydroxyapatite beads coated with human saliva (sHA). Glucans or fructans were synthesized in situ on sHA by immobilized cell-free glucosyltransferase or fructosyltransferase isolated from oral bacteria. S. sobrinus or S. mutans was then adsorbed onto the glucan- or fructan-coated sHA and incubated for different time intervals. The depth of the developing biofilm was measured. Our results show that growth rates of S. sobrinus and S. mutans on both fructan- and glucan-coated sHA were similar during a 23 h period. In addition, the profile was similar to the growth profile of the same planktonic bacteria. The resemblance in growth rates between planktonic and biofilm bacteria may be attributed to the thin and non-dense biofilm formed in the initial stages of the biofilm formation. The thin biofilm coat, reaching a maximal depth of 11 microm, has only imposed limited diffusion restrictions, thus not affecting the growth of the bacteria in the biofilm. Our study shows that growth of bacteria on surfaces may resemble their growth in suspension if the bacteria are not embedded in a thick dense biofilm.

Biofilms↗

Gene expression in Pseudomonas aeruginosa biofilms.

Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.

Animals↗

Characterization of axenic Pseudomonas fragi and Escherichia coli biofilms that inhibit corrosion of SAE 1018 steel.

Corrosion inhibition of SAE 1018 steel by Pseudomonas fragi and Escherichia coli biofilms has been evaluated using batch cultures in rich medium (LB) and seawater-mimicking medium (VNSS) at 23 degrees C and 30 degrees C with or without daily medium replenishment. Biofilm components have been stained simultaneously for polysaccharide (calcofluor) and live and dead cells (Live/Dead Baclit viability kit) and visualized using confocal scanning laser microscopy (CSLM). Image analysis was used to quantify the relative proportions of live cells, dead cells, polysaccharide and void space in the biofilm. This staining technique and examination of the architecture of biofilms responsible for inhibiting metal corrosion revealed that both Ps. fragi and E. coli produce polysaccharide only in the seawater medium; in rich medium, the biofilm consisted mainly of a layer of sessile cells near the biofilm-metal interface and sparse thick clumps of cells at the biofilm-liquid interface. Biofilms of both strains had a higher proportion of live cells in the rich medium than in the seawater-mimicking medium at the higher temperature, and more live cells were present at the higher temperature for LB medium. The corrosion inhibition observed (2.3-6.9-fold in 8 d) was not significantly affected by medium type or replenishment. Increase in the cellular content of the biofilms, as a result of increasing temperature, led to a reduction in corrosion.

Biofilms↗

Analysis of biocide transport limitation in an artificial biofilm system.

An alginate gel bead artificial biofilm system was used to assay biofilm susceptibility to four biocides and to analyse the extent to which each agent penetrated the biofilm. Chlorine, glutaraldehyde, an isothiazolone, and a quaternary ammonium compound were tested on alginate-entrapped Enterobacter aerogenes in gel beads ranging from 1.8 to 6 mm in diameter. Gel-entrapped bacteria were less susceptible to all four antimicrobial agents than were planktonic micro-organisms. The degree of kill measured in artificial biofilm gel beads depended on the size of the gel bead and the cell density at which it was loaded. Disinfection efficacy decreased as gel bead radius or cell density increased. The manifest dependence of biofilm disinfection efficacy on the physical properties of the artificial biofilm (radius and cell density) suggests the impingement of transport limitation of biocide transport into the biofilm. A previously developed theory of biocide reaction and diffusion in biofilm was tested by calculating an appropriate Thiele modulus. In accordance with the theory, the efficacy of all four biocides decreased, albeit noisily, as the Thiele modulus exceeded 1. This result demonstrates that transport limitation can impact antimicrobial performance against biofilms not only of oxidizing biocides but also of nonoxidizing agents.

Biofilms↗

Limitation of adhesion and growth of Listeria monocytogenes on stainless steel surfaces by Staphylococcus sciuri biofilms.

The adhesion and subsequent development of Listeria monocytogenes on stainless steel was studied in the absence and in the presence of a Staphylococcus sciuri biofilm. In the three growth media studied, the percentage of adherent cells was reduced to nearly the same extent by the presence of 1-day biofilms of Staph. sciuri for the two strains of L. monocytogenes studied. One-day biofilms of Staph. sciuri exhibited the same exopolysaccharide content per square centimetre, although they colonized from 3.5 to 35% of the stainless steel depending on the growth media. This suggests that extracellular substances rather than cell-to-cell interactions were involved in the decreased adhesion. After 3 days of culture, Staphylococcus biofilms prevented the adherent L. monocytogenes population from increasing within the biofilm, leading to an average logarithmic cfu difference of 0.9-2.7 between the pure and mixed culture. A competition for nutrients by Staph. sciuri was observed in one of the three media. A role for extracellular polysaccharides produced by the Staphylococcus biofilm in preventing the adhesion of L. monocytogenes and in modifying the balance existing between its planktonic and biofilm phase is hypothesized. A higher proportion of L. monocytogenes cells was observed in the planktonic phase in mixed cultures, suggesting that the extracellular substances produced by Staph sciuri biofilms and involved in the decreased adhesion of L. monocytogenes could modify the balance existing between planktonic and biofilm populations. In addition, co-cultures of L. monocytogenes and Staph. sciuri in broth showed competition for nutrients for Staph. sciuri in one of the three media.

Bacterial Adhesion↗

Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants.

Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.

Biofilms↗

A versatile reactor for continuous monitoring of biofilm properties in laboratory and industrial conditions.

AIMS: The understanding of the dynamics of surface microbial colonization with concomitant monitoring of biofilm formation requires the development of biofilm reactors that enable direct and real-time evaluation under different hydrodynamic conditions. METHODS AND RESULTS: This work proposes and discusses a simple flow cell reactor that provides a means to monitoring biofilm growth by periodical removing biofilm-attached slides for off-line, both non-destructive and destructive biofilm analyses. This is managed without the stoppage of the flow, thus reducing the contamination and the disturbance of the biofilm development. With this flow cell, biofilm growth and respiratory activity can be easily followed, either in well-defined laboratory conditions or in an industrial environment. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The reproducible and typical biofilm development curves obtained, validated this flow cell and confirmed its potential for different biofilm-related studies, which can include biocidal treatment.

Biofilms↗

Biofilm loss in anaerobic immobilized fixed bed reactor system.

An anaerobic immobilized fixed bed bioreactor system was studied for treatment of herbal-based pharmaceutical wastewater. The biofilm loss rate (R(S)), shear stress (sigma), biofilm thickness (Lf) and biomass concentration (X,) were investigated for various flow rates applied to the bioreactor during the experimental studies. Some relationships applicable to a fixed bed reactor were obtained between biofilm loss rate, shear stress, biofilm thickness and specific flow rate (Q) with good accuracy. It was evident that shear stress and biofilm loss rate increased with increase in biofilm thickness. A similar trend was also observed with an increase in flow velocity. It was found that shear stress plays a major role in the biofilm detachment mechanism. The biofilm thickness determined in the present investigation was in the range of 0.5 mm to 5.5 mm, and the biofilm loss rate was estimated in the range of 1.7 x 10(-4) to 7.8 x 10(-4) kg COD m(-2) d(-1) at flow rate ranging between 5.0 and 50 1 d(-1).

Bacteria, Anaerobic↗

Quorum-sensing control of biofilm factors in Staphylococcus epidermidis.

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections, in which biofilm formation is considered to be the main virulence mechanism. Quorum-sensing systems have been recognized as important regulators of virulence and biofilm formation in many bacteria. There is a single quorum-sensing system in S. epidermidis encoded by the agr operon. To investigate quorum-sensing control of biofilm formation, we constructed an agr deletion mutant, assayed for the different stages of biofilm formation, and determined agr-dependent regulation of biofilm factors. The agr mutant showed increased biofilm formation, primary attachment, and expression of the autolysin AtlE, but lacked delta-toxin production. However, the level of polysaccharide intercellular adhesin expression was equivalent to the isogenic wild-type strain. In contrast to AtlE, which is known to influence primary attachment, delta-toxin appeared to exert its effect on attachment to polystyrene during later stages of biofilm formation. Importantly, addition of cross-inhibiting pheromones mimicked an agr mutation and significantly enhanced biofilm formation, which suggests that care should be used when treating S. epidermidis infections with cross-inhibiting peptides. Our data demonstrate the importance of quorum sensing in the establishment of a biofilm in this critical human pathogen.

Bacterial Adhesion↗

Early biofilm formation and the effects of antimicrobial agents on orthodontic bonding materials in a parallel plate flow chamber.

Decalcification is a commonly recognized complication of orthodontic treatment with fixed appliances. A technology, based on a parallel plate flow chamber, was developed to investigate early biofilm formation of a strain of Streptococcus sanguis on the surface of four orthodontic bonding materials: glass ionomer cement (Ketac Cem), resin-modified glass ionomer cement (Fuji Ortho LC), chemically-cured composite resin (Concise) and light-cured composite resin (Transbond XT). S. sanguis was used as it is one of the primary colonizers of dental hard surfaces. Artificial saliva was supplied as a source of nutrients for the biofilms. The effects of two commercially available mouthrinses (i.e. a fluoride containing rinse and chlorhexidine) were evaluated. Initial colonization of the bacterium was assessed after 6 hours of growth by the percentage surface coverage (PSC) of the biofilm on the disc surfaces. There were statistically significant differences in bacterial accumulation between different bonding materials (P < 0.05), Concise being the least colonized and Transbond XT being the most colonized by S. sanguis biofilms. All materials pre-treated with 0.05 per cent sodium fluoride mouthrinse showed more than 50 per cent reduction in biofilm formation. The 0.2 per cent chlorhexidine gluconate mouthrinse caused significant reduction of biofilm formation on all materials except Ketac Cem. This in vitro study showed that the use of a chemically-cured composite resin (Concise) reduced early S. sanguis biofilm formation. Also, fluoride had a greater effect in reducing the PSC by S. sanguis biofilms than chlorhexidine. Rinsing with 0.05 per cent sodium fluoride prior to placement of orthodontic appliances is effective in reducing early biofilm formation.

Acrylic Resins↗

Association of biofilm production of coagulase-negative staphylococci with expression of a specific polysaccharide intercellular adhesin.

An association between adherent biofilm production on tissue culture plates and expression of a specific polysaccharide intercellular adhesion (PIA), which is functionally involved in cell clustering, was investigated for 179 Staphylococcus epidermidis isolates. Of the S. epidermidis strains, 50.8% were biofilm producers (A570 of > 0.1). There was a significant positive association between biofilm production and PIA expression: 86.8% of biofilm-producing S. epidermidis strains produced PIA as detected with a specific coagglutination assay. In contrast, 88.6% of the biofilm-negative isolates did not express PIA (P < .001). A linear association between the amount of PIA produced as detected by inhibition ELISA and the amount of biofilm produced was established for 49 S. epidermidis strains, representing a continuum from biofilm-negative to strongly biofilm-producing (r = .81, P < .001). Apparently, PIA is important for biofilm accumulation in the majority of clinical S. epidermidis isolates.

Adhesins, Bacterial↗

Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis.

The in vitro effect of nine antibiotic combinations was investigated in Staphylococcus epidermidis biofilms using ATP-bioluminescence for viable bacterial cell quantification. Four slime-producing (SP) strains were used to form biofilms 6, 24 and 48 h old. These biofilms were exposed for 24 h to antibiotics at 4 x, 2 x, 1 x and 0.5 x MIC. Combinations involving tetracycline together with another antibiotic were the most efficient at the biofilm age and concentration range under study. The combination vancomycin-rifampicin produced the highest bactericidal effect on 6 h biofilms at 4 x MIC, but this effect decreased dramatically in older biofilms. To detect possible antibiotic synergy in combinations that had a significant killing effect, antibiotics were studied not only in combination but also individually. Synergic effects were observed in all the combinations tested. Differences between the effect in combination and the sum of individual antibiotic effects (degree of synergy) were significant (mostly P< 0.001) and exceeded 1 log10 cfu/mL in the majority of cases. In 48 h biofilms, antibiotics caused a significant bactericidal effect when applied in combination, but never when used individually. These results indicate that the biofilm test applied allows the detection of synergy between antibiotics and suggests that this assay could be useful in clinical and extensive synergy studies on S. epidermidis biofilms.

Anti-Bacterial Agents↗

Activity of amine oxide against biofilms of Streptococcus mutans: a potential biocide for oral care formulations.

AIMS: To assess the potential bactericidal activity of amine oxide (C10-C16-alkyldimethyl N-oxides) against Streptococcus mutans grown as planktonic suspension and as biofilm on hydroxyapatite discs, and its ability to control acidification of the media. METHODS: Amine oxide bacteriostasis was investigated using the Bioscreen C Microbiological Growth Analyser, while a standard suspension test was used to determine its bactericidal efficacy. In addition, the lethal activity of amine oxide was studied against sedimentation biofilms of S. mutans on hydroxyapatite (HA) discs and resuspended biofilms. Several parameters were considered such as the surfactant concentration, pH, the starting inoculum and the maturity of the biofilm. RESULTS: Amine oxide was bacteriostatic against planktonic S. mutans at a low concentration (0.006% v/v) and highly bactericidal against S. mutans in suspension or in a mature biofilm on hydroxyapatite, although the concentration required to achieve the latter effect was four times higher. The activity of amine oxide against biofilms depended upon its concentration and the age of the biofilm. In addition, amine oxide pre-treatment of the HA discs did not affect the growth of the biofilm. Finally, amine oxide did not prevent the acidification of the medium, although lower pHs had a potentiating effect on amine oxide activity. CONCLUSION: Amine oxide showed high potential for controlling early biofilms caused by periodontal bacteria. Further investigations should be carried out, particularly on the potential toxicity of amine oxide and its efficacy in complex formulations for oral care products.

Alkanes↗

Commensal isolates of methicillin-resistant Staphylococcus epidermidis are also well equipped to produce biofilm on polystyrene surfaces.

OBJECTIVES: To study biofilm production and to detect icaAD, atlE and aap genes in 137 isolates of methicillin-resistant Staphylococcus epidermidis (MRSE) obtained from healthy individuals from the community (35 isolates), from hospitalized patients at the Antônio Pedro University Hospital (25 isolates) and from individuals from a home-care system (HCS; 77 isolates). METHODS: Biofilm production was determined in vitro using polystyrene inert surfaces. icaAD, atlE and aap genes were detected using PCR. Hybridization experiments were also carried out to confirm some PCR results. Antimicrobial susceptibility testing was carried out using the NCCLS methods. RESULTS: Although many of the commensal MRSE isolates produced biofilms, the percentage of biofilm producers was significantly higher (P = 0.0107) among hospital isolates (76%) than among isolates from the community (60%) and from the HCS (57%). An association was observed between multiresistance and biofilm production for isolates obtained from healthy individuals from the community and from household contacts from the HCS (P < 0.0001). The concomitant presence of the ica operon and atlE and aap genes was associated with the strong biofilm-producer phenotype (P < 0.0001). CONCLUSION: Because many of the commensal MRSE isolates obtained from nares produced biofilms and carried icaAD, aap and atlE genes, biofilms or such genetic elements should not be used as markers for clinical significance. The biofilm environment seems to increase genetic exchanges and hence may contribute to multiresistance phenotypes.

Anti-Bacterial Agents↗

The resistance of maxillofacial reconstruction plates to biofilm formation in vitro.

OBJECTIVES/HYPOTHESIS: Bacterial biofilms, bacteria surrounded by a protective glycocalyx, have been demonstrated on bioimplants placed within and outside of the head and neck region. The presence of the biofilm often makes decontamination of an infected implant impossible, requiring removal of the implant. Infections attributable to biofilm formation within the facial skeleton after reconstruction with implants may result in delayed union, fibrous union, malunion, nonunion, and malocclusion. These complications often require removal of the implant and secondary surgery. Although the incidence of infections necessitating implant removal is relatively low, the increased numbers of implants being placed make this a growing problem. Previous work in the authors laboratory has demonstrated a resistance to biofilm formation on different types of pressure-equalizing tubes. The hypothesis evaluated in the study is that such resistance to biofilm formation is due to the inability of bacteria to adhere to the tubes because of the material's smoothness or surface charge. STUDY DESIGN: A controlled observational study. METHODS: Scanning electron microscopy was used to evaluate the formation of biofilms in vitro for a common strain of Staphylococcus aureus on four implantable materials. The implantable materials included titanium and polylactide resorbable plates. RESULTS: Consistent with the authors' prior findings, they were able to produce bacterial biofilm reliably on a silicone pressure equalizing tube but were unable to demonstrate biofilm formation on the titanium or resorbable implants. CONCLUSION: The absence of biofilm formation on these implants can best be explained by the surface charge or polarity properties of these materials. These findings are consistent with the relatively low incidence of infections among patients receiving these implants in maxillofacial applications.

Biofilms↗