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PubMed2004

Influence of acetylsalicylic acid (aspirin) on biofilm production by Candida species.

Candida spp. are important causative agents of infections associated with biofilm formation. Management of biofilm-related infections is extremely difficult and therefore new therapeutic solutions are needed. This study for the first time explored the possible effect of aspirin on Candida spp. Biofilm-producing capacity. Two strains of C. guilliermondii, and one strain per species of C. kefyr, C. glabrata, C. albicans, and C. parapsilosis were included in the study. The antifungal property of aspirin was tested by the broth microdilution method, while effect of aspirin on biofilm formation was determined by the microtiter-plate test. The minimal inhibitory concentrations of aspirin obtained ranged from 2.17 to 8.67 mM and minimal fungicidal concentrations were from 4.33 to 8.67 mM. The concentrations of aspirin which induced statistically significant decrease in biofilm formation ranged from 0.43 mM to 1.73 mM of aspirin, depending on the tested yeast strain. Therefore, the significant effects of aspirin on growth and biofilm formation of Candida spp. were achieved only with suprapharmacological concentrations of the drug. The influence of the inoculum size on the effect of aspirin on biofilm formation was determined for C. albicans only and a significant decrease was observed also at suprapharmacological concentrations of aspirin, irrespective of the inoculum size. The results obtained in the present study show aspirin to be a drug with the potential to affect and suppress biofilm formation by Candida spp., and provide support for further investigation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Effects of antibiotics on Pseudomonas aeruginosa NK125502 and Pseudomonas fluorescens MF0 biofilm formation on immobilized fibronectin.

The effect of subminimal inhibitory concentration (1/2 MIC) of antibiotics on the biofilm formation on immobilized fibronectin by Pseudomonas was investigated by examining the reference strains NK125502 P. aeruginosa and MF0 P. fluorescens in a microtiter plates assay. When the antibiotics were added during bacterial growth and biofilm development, gentamicin was the only antimicrobial agent tested which decreased significantly the biofilm formation by the two strains. Cefsulodin and chloramphenicol also decreased the P. aeruginosa biofilm development (P<0.01), whereas polymyxin B inhibited biofilm formation by P. fluorescens (p<0.05). When the antibiotics were only present during bacterial growth and not during biofilm development, gentamicin was the only antibiotic tested to decrease significantly the biofilm formation by P. aeruginosa for incubation times of 20 and 72h (P<0.01), whereas P. fluorescens was not affected. This persistent inhibition of P. aeruginosa biofilm formation may be interesting in intermittent antibiotherapy treatments.

Anti-Bacterial Agents↗
PubMed2006

Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR.

BACKGROUND: Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. RESULTS: The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (beta-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase beta-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours. CONCLUSION: In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.

Biofilms↗
PubMed2006

Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species.

Biofilms are densely packed multicellular communities of microorganisms attached to a surface or interface. Bacteria seem to initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability. Biofilms undergo dynamic changes during their transition from free-living organisms to sessile biofilm cells, including the specific production of secondary metabolites and a significant increase in the resistivity to biological, chemical, and physical assaults. Bacillus subtilis is an industrially important bacterium exhibiting developmental stages. It forms rough biofilms at the air-liquid interface rather than on the surface of a solid phase in a liquid, due to the aerotaxis of the cells. Biofilm formation by B. subtilis and related species permits the control of infection caused by plant pathogens, the reduction of mild steel corrosion, and the exploration of novel compounds. Although it is obviously important to control harmful biofilm formation, the exploitation of beneficial biofilms formed by such industrial bacteria may lead to a new biotechnology.

Bacillus subtilis↗
PubMed2006

Involvement of N-acetyl-D-galactosamine-specific lectin in biofilm formation by the periodontopathogenic bacterium, Eikenella corrodens.

Eikenella corrodens is known not only as one of the periodontopathogenic bacteria but also as a pathogen associated with many infectious diseases of humans. Dental plaque is a complex biofilm community comprised of many bacterial species. E. corrodens has a lectin on its cell surface that is thought to be involved in its pathogenicity. In this study, we found that E. corrodens forms a biofilm on a polystyrene surface. A biofilm was formed at the bottom of the wells in microtiter plates after 24 h. Microcolonies were observed as the amount of biofilm became larger. When anaerobic respiration was repressed due to nitrate limitation, the biofilm formed only at the air-water interface. Strain 1073 and HU, which have higher lectin activity, formed a biofilm more effectively than other strains. Biofilm formation was repressed by the addition of N-acetyl-D-galactosamine. These results suggest that the lectin on the surface of E. corrodens might be involved in biofilm formation.

Acetylgalactosamine↗
PubMed2003

Bacterial biofilms of importance to medicine and bioterrorism: proteomic techniques to identify novel vaccine components and drug targets.

Biofilms are highly ordered microbial communities enmeshed in a carefully sculpted matrix designed for survival of organisms either in multi- or mono-genus/species in a specific microniche. In human disease, biofilm infections are some of the most recalcitrant to treat. Even with rigorous antibiotic regimens, some biofilms, such as those within the thick airway mucus of cystic fibrosis (CF) patients, persist throughout the course of the disease process. In this editorial, discussion will cover the utility of using advanced proteomic techniques to help identify potential weaknesses in the already impressive defensive armamentarium of biofilm bacteria. Two biofilm systems will be discussed herein, one of which is that of Pseudomonas aeruginosa biofilms within CF airway biofilms. The other is referred to as persistent 'bioterrorist agent biofilms' in which Francisella tularensis can grow on surfaces where environmental amoeba can phagocytose them, allowing for growth of F. tularensis within the amoebae.

Acanthamoeba↗
PubMed2002

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation.

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate-dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom-like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.

Bacteria↗
PubMed2005

Biofilm formation by algae as a mechanism for surviving on mine tailings.

Photosynthetic biofilms successfully colonize the sediments of a mine tailings reservoir (Guanajuato, Mexico) despite the high metal concentrations that are present. To elucidate the mechanisms of biofilm survival despite metal ores, experiments were performed to evaluate the response of seminatural biofilms to Cu, Zn, and a combination of both metals at concentrations observed in the field. The biofilms were composed mostly of the chlorophyte Chlorococcum sp. and the cyanobacterium Phormidium sp., and their response to the two added metals was described by measurements of extracellular polymeric substances (EPS) and in vivo fluorescence. The photosynthetic efficiency and the minimal chlorophyll fluorescence of dark-adapted cells were measured by multiwavelength pulse amplitude-modulated fluorometry. The photosynthetic efficiency of light-adapted cells (phi(PSII)) also was measured. Metal exposure increased the EPS production of biofilms, as visualized with confocal laser-scanning microscopy. Extracellular polymeric substances enhanced the extracellular metal accumulation from the first day of metal exposure. Metals provoked changes in the relative abundance of the dominant taxa because of a species-specific response to the metals when added individually. Metals affected the phi(PSII) less than the total biomass, suggesting ongoing activity of the surviving biofilms. Survival of individual biofilm photosynthetic cells was found to resume from the embedding in the mucilaginous structure, which immobilizes the metals extracellularly. The survival of biofilms under mixed-metal exposure has practical applications in the remediation of mine tailings.

Biodegradation, Environmental↗
PubMed2003

Effects of chlorhexidine, minocycline, and metronidazole on Porphyromonas gingivalis strain 381 in biofilms.

BACKGROUND: Porphyromonas gingivalis is found in subgingival biofilm and is associated with periodontal disease. Bacteria in biofilms are able to resist higher antimicrobial concentrations than in suspension. Little is known about the susceptibility of P. gingivalis in biofilms to antimicrobial agents. The effects of chlorhexidine gluconate, minocycline hydrochloride, and metronidazole on P. gingivalis biofilms were examined in vitro. METHODS: P. gingivalis strain 381 biofilms were prepared on 32 hydroxyapatite disks. At 0, 24, 72, and 144 hours after perfusion of the three antimicrobial agents, two disks from each device were used to assess the antimicrobial effects by adenosine triphosphate (ATP) bioluminescence, and for morphological investigation by scanning electron microscopy (SEM). RESULTS: Close relationships were found between the results of the ATP analyses and the SEM observations in all groups examined. A significant decrease (P < 0.001) in ATP content was found between the chlorhexidine-treated and control groups. The extracellular matrix structure and P. gingivalis cells were altered in the presence of chlorhexidine. Minocycline hydrochloride also caused a decrease (P < 0.05) in the ATP content and morphological change on P. gingivalis biofilms. Metronidazole showed no significant efficacy against P. gingivalis biofilms. CONCLUSION: Chlorhexidine gluconate was effective at reducing the viability of P. gingivalis 381 cells in biofilms, while minocycline hydrochloride and metronidazole exhibited weaker effects.

Adenosine Triphosphate↗
PubMed2006

Hydrogen limitation--a method for controlling the performance of membrane biofilm reactor for autotrophic denitrification of wastewater.

Hydrogen-driven denitrification using the fiber membrane biofilm reactor (MBfR) was evaluated for consistent operation in tertiary wastewater treatment. The possibility of controlling the process rates, as well as biofilm parameters by supplying limited amounts of electron donor (hydrogen), was tested. Limiting the hydrogen supply proved to be efficient in controlling the biofilm growth and performance of the MBfR. Denitrification rates remained unchanged for both synthetic wastewater (SWW) and real municipal wastewater (MWW) effluent as well through the fluctuations in the substrate (NO3-N) concentration. The average denitrification rates were 0.50 (+/- 0.02) g NO3-N per day per m2 for SWW and 0.59 (+/- 0.04) g NO3-N per day per m2 for MWW. Biofilm density rather than thickness was the determining factor in substrate diffusion and biofilm sloughing, ultimately determining operating stability. Limited hydrogen supply assured constant volatile solids (VS) concentration in the biofilm. It was determined that VS/TS ratio higher than 0.25 assured stable biofilm operation. Decrease of VS/TS ratio below 0.25 led to shearing of the nonbiological outer layers of the biofilm. The values of chemical oxygen demand (COD), volatile suspended solids (VSS) and total suspended solids (TSS) in the final effluent were stable and well below wastewater effluent guidelines. Substitutions of bicarbonate with gaseous carbon dioxide as the carbon source did not affect denitrification rates despite lower than optimum pH conditions.

Biofilms↗
PubMedDate not supplied

Monitoring of bacterial morphology for controlling filamentous overgrowth in an ultracompact biofilm reactor.

This research was part of a study of filamentous growth and control in an ultracompact biofilm reactor (UCBR). Morphologies of biofilm and filamentous bacteria in the UCBR were investigated. Ethanol was used as a substrate and sodium hypochlorite was applied as a toxicant to control filamentous growth. The results indicated that factors such as chemical oxygen demand, surface loading rate, pH, and dissolved oxygen could initiate filamentous overgrowth in the UCBR. Different biofilm and filamentous morphologies in the UCBR were observed under different operational conditions. Chlorination was an effective approach to control filamentous growth during and after biofilm formation. Proper chlorine dosing had no effect on biofilm, but killed filaments. Overdose of chlorine damaged biofilm and caused adverse effects such as low treatment efficiency, media clogging and washout, and biofilm color change in the reactor. Frequent monitoring of the morphologies of filaments and biofilm was needed during chlorination to prevent chlorine overdose.

Bacteria↗
PubMed2003

Detection of biofilm formation and nanobacteria under long-term cell culture conditions in serum samples of cattle, goats, cats, and dogs.

OBJECTIVE: To determine the prevalence of biofilm formation under long-term cell culture conditions in serum samples of dairy cattle, goats, cats, and dogs, and to determine whether there is an association between nanobacteria and biofilm formation. SAMPLE POPULATION: Serum samples of clinically normal animals (313 dairy cattle, 48 goats, 140 dogs, and 44 cats) and animals with various medical conditions (60 dogs and 116 cats). PROCEDURE: Serum was incubated under cell culture conditions and observed for biofilm formation by use of light microscopy, electron microscopy, and spectroscopy. A polymerase chain reaction assay was developed to identify 16S rRNA gene sequences of nanobacteria. RESULTS: Biofilm formation developed in serum samples of 304 of 313 (97%) cattle, 44 of 48 (92%) goats, 44 of 44 (100%) cats, and 126 of 140 (90%) dogs. Prevalence of serum samples with positive results for biofilm formation was not significantly different between cats or dogs with and without medical conditions associated with pathologic extraskeletal calcification processes. Scanning electron microscopy and spectroscopy of biofilm samples revealed small coccoid particles consisting mainly of calcium and phosphate. Polymerase chain reaction assay failed to amplify sequences of nanobacteria. CONCLUSIONS AND CLINICAL RELEVANCE: Under long-term cell culture conditions, biofilm made up of aggregates of calcium and phosphate crystals does form in serum samples of clinically normal dairy cattle, goats, cats, and dogs. Disease, however, does not predispose to biofilm formation in serum samples of dogs and cats. Our findings did not support the existence of nanobacteria in serum samples of cattle, goats, cats, and dogs.

Animals↗
PubMed2001

Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment.

Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening. Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.

Biofilms↗
PubMed2004

Phase variation of biofilm formation in Staphylococcus aureus by IS 256 insertion and its impact on the capacity adhering to polyurethane surface.

While ica gene of Staphylococcus epidermidis is known to undergo phase variation by insertion of IS256, the phenomenon in Staphylococcus aureus has not been evaluated. Six biofilm-positive strains were tested for the presence of biofilm-negative phase-variant strains by Congo red agar test. For potential phase-variant strains, pulsed-field gel electrophoresis was done to exclude the possibility of contamination. To investigate the mechanism of the biofilm-negative phase variation, PCR for each ica genes were done. Changes of ica genes detected by PCR were confirmed by southern hybridization, and their nucleotides were analyzed by DNA sequencing. Influence of ica genes and biofilm formation on capacity for adherence to biomedical material was evaluated by comparing the ability of adhering to polyurethane surface among a biofilm-negative phase-variant strain and its parent strain. A biofilm-negative phase-variant S. aureus strain was detected from 6 strains tested. icaC gene of the phase-variant strain was found to be inactivated by insertion of additional gene segment, IS256. The biofilm-negative phase-variant strain showed lower adhering capacity to polyurethane than its parent strain. This study shows that phase variation of ica gene occurs in S. aureus by insertion of IS256 also, and this biofilm-negative phase variation reduces adhering capacity of the bacteria.

Bacterial Adhesion↗
PubMed2006

Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods.

PURPOSE: The purpose of this study was to evaluate three methods for detection of biofilm formation in staphylococci. METHODS: For detection of biofilm formation, 152 clinical isolates of Staphylococcus spp. were screened by tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) method. RESULTS: Of the 152 Staphylococcus spp. 88(57.8%) displayed a biofilm-positive phenotype under the optimized conditions in the TCP method and strains were further classified as high 22 (14.47 %) and moderate 60 (39.4 %) while in 70 (46.0 %) isolates weak or no biofilm was detected. Though TM correlated well with the TCP test for 18 (11.8 %) strongly biofilm producing strains, weak producers were difficult to discriminate from biofilm negative isolates. Screening on CRA does not correlate well with either of the two methods for detecting biofilm formation in staphylococci. CONCLUSION: The TCP method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci on biomedical devices.

Agar↗
PubMed2003

A biofilm model for flowing systems in the food industry.

When bacteria attach to the walls of pipelines, they can form biofilms, which can cause the recontamination of food products. In order to quantify such recontamination, a one-dimensional biofilm model was developed taking into account adsorption, desorption, and the growth of cells. The model consisted of two mass balances describing increases in biofilm formation at the wall and the accumulation of cells in the liquid phase. The necessary parameters for the model were obtained in laboratory biofilm experiments. These experiments involved a flowing system and the use of Staphylococcus aureus as a model pathogen and silicon tubing as a testing material. S. aureus was inoculated into the system for 2 h, and then the system was changed to a sterile medium. Both biofilm formation and the release of cells into the flowing liquid were measured until steady-state conditions were reached (for up to 9 days). The experiments were performed in duplicate for different flow conditions (i.e., for Reynolds numbers of 3.2, 32, and 170). It was shown that at higher Reynolds numbers, the biofilm developed faster, probably owing to an increase in the transfer of nutrients to the surface. The proposed biofilm model was capable of describing the data obtained for the three different flow conditions with the use of the specific growth rate in the biofilm and the desorption coefficient as fit parameters. The specific growth rates were 0.16, 0.27, and 0.49 h(-1) for Reynolds numbers of 3.2, 32, and 170, respectively, and the desorption coefficients were about 1% of these values.

Adsorption↗
PubMed2000

Modeling biofilm antimicrobial resistance.

A computer model capable of integrating mechanisms of biofilm resistance to disinfection by antimicrobial agents was developed. Resistance mechanisms considered included retarded penetration due to a stoichiometric reaction between the antimicrobial agent and biomass, incomplete penetration due to a catalytic reaction between the antimicrobial agent and the biomass, and the existence of a fraction of the cells in a resistant phenotypic state. Mathematical models of these processes were derived and solved in the computer simulation package MATLAB. Four sets of fitted experimental data on the disinfection of Pseudomonas aeruginosa biofilms were fit to each of the three models. No one model fit all of the data sets adequately. Killing of a 2-day old biofilm by tobramycin was best described by the physiological limitation model. Killing by hypochlorite was best described by the stoichiometric transport model. Killing by hydrogen peroxide was best simulated by the catalytic transport model. These results suggest that multiple mechanisms of biofilm reduced susceptibility are manifested even in biofilms of the same species and that the particular resistance mechanism depends on the biofilm age, antimicrobial agent, and biofilm thickness. The models presented in this article may be useful for diagnosing mechanisms of biofilm resistance from experimental data.

Biofilms↗
PubMed2001

Extracellular polymeric substances in relation to nutrient removal from a sequencing batch biofilm reactor.

Experimental investigations were performed to determine the possibility of simultaneous biological nitrogen and phosphorus removal during various biofilm processes in conjunction with biofilm characterisation, especially extracellular polymeric substance (EPS). Since biologial nitrogen removal requires an alternating exposure of anaerobic-anoxic-oxic conditions in the bulk liquid that surrounds the biofilm growth media, a sequencing batch reactor (SBR)-type operation was used. Various materials including expanded clay, polystyrene, polyurethane, and acrylic materials were used as the biofilm growth support medium. Simultaneous nitrogen and phosphorus removal was possible with SBR, but it was postulated that nutrient removal efficiencies varied with film thickness. Thinner biofilm promoted nitrification and phosphorus removal, but thicker biofilm enhanced denitrification and reduced phosphorus removal. EPS contents were similar regardless of support media types or biofilm configuration, but EPS contents gradually increased as the film growth continued after backwashing. EPS contents were increased with increased nitrogen removal, but it was difficult to define its relation with phosphorus removal. In addition, suspended solids removal was correlated well with the EPS content in the biofilms.

Biofilms↗