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PubMed1999

Spatial distribution of respiratory activity in Pseudomonas putida 54G biofilms degrading volatile organic compounds (VOC).

All over the world, Microbial systems are used to clean soils, waters and air streams that have been contaminated with volatile organic compounds (VOC). Information about the structure and function of the microbes that metabolize these contaminants can be gained by studying these microbial systems. Here we describe the spatial patterns of respiratory activity in Pseudomonas putida 54G aerobic biofilms degrading two VOC, toluene and ethanol. Oxygen concentration profiles within the biofilm were measured using microsensors. These profiles are thought to be most accurate reflection of the structure and function of aerobic microbial biofilms. The degrading process certainly imposed a structural and functional patterns on the microbial biofilm community growing at the expense of the VOC substrate. Cryosectioning coupled with the staining of biofilm samples confirmed a high respiratory activity near the substratum, that decreased towards the biofilm/fluid interface. The accumulation of inactive cells in the outer biofilm layer protects the inner biofilm from high concentrations of toxic compounds and also limits the degradation rate. This stratification phenomenon appeared to be a general pattern for P. putida 54G biofilms degrading VOC.

Biofilms↗
PubMed2006

Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus epidermidis biofilms isolated from infected total hip arthroplasty cases.

BACKGROUND: It is difficult to treat implant-related infections because bacteria form biofilms on implants. Biofilms protect the organisms from the host's immune system and prevent penetration of antimicrobial agents. We investigated the susceptibility of six strains isolated from infected total hip arthroplasty (THA) cases to six antimicrobial agents. METHODS: Six Staphylococcus aureus and S. epidermidis strains isolated from infected THA cases and S. aureus ACTT 25923, positive strains of biofilms, were used in this study. Biofilms were developed on 96-well titer plates. After 7 days the nutrient solution was aspirated, and wells were rinsed three times with sterile water. A series of doubling dilutions of each antimicrobial agent (16, 32, 64, 128, 256, 512, and 1024 microg/ml) in trypticase soy broth were added to the wells. The plates were incubated at 37 degrees C. After 24 h, the contents were aspirated and wells were rinsed three times with sterile water. The surfaces of wells were vigorously scraped with a sterile swab and applied to sheep blood agar plates at 37 degrees C in an aerobic chamber for 24 h. The minimum bactericidal concentrations (MBCs) were determined as the lowest concentration of antibiotic resulting in 100% reduction of viable cells compared to the antibiotic-free control. Biofilms were produced on stainless steel washers, and the time-course changes were observed by scanning electron microscopy (SEM). RESULTS: Planktonic bacteria were sensitive to the antimicrobial agents, but biofilm bacteria were markedly resistant. SEM revealed that biofilms grew larger with time. CONCLUSIONS: The MBCs of antimicrobial agents for biofilms bacteria were higher than those of planktonic bacteria. Therefore, it is suggested that implant-related infection is difficult to treat with antimicrobial agents alone.

Adult↗
PubMed2006

Biofilms, a new approach to the microbiology of dental plaque.

Dental plaque has the properties of a biofilm, similar to other biofilms found in the body and the environment. Modern molecular biological techniques have identified about 1000 different bacterial species in the dental biofilm, twice as many as can be cultured. Oral biofilms are very heterogeneous in structure. Dense mushroom-like structures originate from the enamel surface, interspersed with bacteria-free channels used as diffusion pathways. The channels are probably filled with an extracellular polysaccharide (EPS) matrix produced by the bacteria. Bacteria in biofilms communicate through signaling molecules, and use this "quorum-sensing" system to optimize their virulence factors and survival. Bacteria in a biofilm have a physiology different from that of planktonic cells. They generally live under nutrient limitation and often in a dormant state. Such "sleepy" bacteria respond differently to antibiotics and antimicrobials, because these agents were generally selected in experiments with metabolically active bacteria. This is one of the explanations as to why antibiotics and antimicrobials are not as successful in the clinic as could be expected from laboratory studies. In addition, it has been found that many therapeutic agents bind to the biofilm EPS matrix before they even reach the bacteria, and are thereby inactivated. Taken together, these fundings highlight why the study of bacteria in the oral cavity is now taken on by studying the biofilms rather than individual species.

Bacteriological Techniques↗
PubMed2006

Protein expression in Escherichia coli S17-1 biofilms: impact of indole.

Bacteria undergo significant changes during adherence to surfaces and biofilm development. Cell-to-cell signalling molecules are known to be involved in these phenotypic adaptations to the sessile mode of life. We demonstrated previously that indole can act as an extracellular signal to regulate biofilm formation in E. coli. To identify proteins over- or under-expressed in response to E. coli biofilm formation and indole signalling, we compared the proteomes of the E. coli S17-1 wild-type and 3714 (S17-1 tnaA::Tn5) tryptophanase-negative mutant cells (which don't produce indole) grown as suspensions or biofilms in the presence or absence of exogenous indole. From computer-assisted image analysis, 407 spots were discriminated on two-dimensional electropherograms. Principal component analysis (PCA) of the electropherograms did not discriminate between the proteomes of the wild-type and mutant cells grown as suspensions indicating that indole has a limited impact onto protein expression of planktonic cells. The first principal component extracted by PCA, after standardization of the observations, opposed planktonic and biofilm cells confirming the existence of changes in protein expression during E. coli biofilm formation. Among proteins over- or under-expressed by both sessile wild-type and mutant cells, we identified metabolic enzymes, transporters, proteins involved in the translation and transcription machinery, stress response and regulation, and signalling proteins. The wild-type and mutant strains grown as biofilms in the presence of indole were discriminated by the second component. The role of some proteins whose expression was altered in biofilm bacteria compared to suspended counterparts is discussed.

Amino Acid Sequence↗
PubMed2005

The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in Candida albicans biofilms.

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.

Biofilms↗
PubMed2005

Characterization of switch phenotypes in Candida albicans biofilms.

The aim of this study was to characterize switch phenotypes in Candida albicans biofilms. Cells of Candida albicans 192887g biofilms (24 h) were resuspended and these together with their planktonic counterparts were separately inoculated on Lee's medium agar supplemented with arginine and zinc, at 25 degrees C for 9 days, for colony formation. The different switch phenotypes, as reflected by varying colony morphologies, were then examined for their (i) stability under various growth conditions, (ii) carbohydrate assimilation profiles, (iii) susceptibility to the polyene antifungal, nystatin, (iv) adhering and biofilm-forming ability, (v) filamentation, and (vi) growth rate in yeast nitrogen base medium supplemented with 100 mM glucose. Our data showed that the frequency of phenotypic switching in C. albicans biofilms was approximately 1%. Compared with the planktonic yeasts, cells derived from candidal biofilms generated one of the phenotypes less frequently (Chi-square-tests: P = 0.017). The five phenotypes derived from the biofilm growth demonstrated differing profiles for carbohydrate assimilation, adhesion, biofilm formation, filamentation, and growth rate. These findings reported here, for the first time, imply that phenotypic switching in the candidal biofilms differs from that in the planktonic growth, and affects multiple biological attributes.

Biofilms↗
PubMed2006

Effect of oral bacteria on growth and survival of Candida albicans biofilms.

The purpose of this study was to evaluate the effect of eight aerobic and anaerobic oral commensal bacterial species on in vitro Candida albicans biofilm development. A single isolate of C. albicans 2560 g, and eight different species of oral bacteria comprising, Actinomyces israelii, Lactobacillus acidophilus, Prevotella nigrescens, Porphyromonas gingivalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and Streptococcus intermedius were studied using an in vitro biofilm assay. Biofilm formation was quantified in terms of the ability of Candida to grow on polystyrene plastic surfaces co-cultured with the foregoing bacteria. A viable cell count was used to quantify the sessile yeast growth and scanning electron microscopy was employed to confirm and visualize biofilm formation. Co-culture with differing concentrations of bacteria had variable effects on Candida biofilm formation. Co-culture with the highest concentrations of each of the foregoing bacteria resulted in a consistent reduction in the yeast counts in the candidal biofilm (P<0.05), except for L. acidophilus, S. mutans, and, S. intermedius co-cultures. Further, on regression analysis a significant negative correlation between the co-culture concentration of either P. gingivalis or E. coli and viable yeast counts in the biofilm was noted (P<0.05) although this was not evident for the other bacterial species. Taken together, our data indicate that, quantitative and qualitative nature of the bacteria modulate C. albicans biofilm formation in mixed species environments such as the oral cavity.

Bacteria, Aerobic↗
PubMed2004

Effect of heterogeneous structure in mechanically unstressed biofilms on overall growth.

In contrast to their name, biofilms are not always flat and homogeneous but instead often exhibit complex structural heterogeneity. It has been suggested that nonhomogeneous geometry is selected in order to increase biofilm growth rate. A previous study (Dockery and Klapper (2002) SIAM J. Appl. Math., 62, 853-869) of a model biofilm system in a static bulk fluid demonstrated that under some circumstances a flat biofilm-bulk fluid interface is linearly unstable to perturbation due to growth induced forces. Computations indicated that subsequent nonlinear evolution results in fingers and mushrooms of biofilm similar to structures observed in actual biofilms. However, the important complementary issue of biological functionality was not considered. Here a weakly nonlinear analysis of the simple growing biofilm layer model in Dockery and Klapper (2002, SIAM J. Appl. Math., 62, 853-869) is presented. It is argued that, at least in the case of biofilms free of external mechanical stress, overall growth is in fact generally inhibited by the presence of growing perturbations in the linear stage. Hence a more complex explanation of function is necessary.

Biofilms↗
PubMed2006

Multiple combination antibiotic susceptibility testing of nontypeable Haemophilus influenzae biofilms.

Haemophilus influenzae is a cause of otitis media with effusion (OME). Animal models demonstrate growth of H. influenzae biofilms in OME, which may explain why OME does not respond well to conventional antibiotic therapy. Using a previously developed in vitro model, we performed H. influenzae susceptibility studies to see if H. influenzae biofilm cultures were more resistant to antibiotics than planktonic (broth) cultures, and to determine which antibiotics were most effective against H. influenzae biofilms. H. influenzae isolates were grown as biofilms on polystyrene pins. Biofilm and planktonic minimum inhibitory concentrations (MICs) were measured for 8 antibiotics, and multiple combination testing was performed with 66 groupings of 1, 2, or 3 antibiotics. We found that biofilm cultures were more resistant to antibiotics than planktonic ones. Antibiotic combinations containing rifampin and ciprofloxacin were most effective against biofilms. Biofilm testing reveals differences in effectiveness among antibiotics not apparent from conventional susceptibility testing, and suggests novel antibiotic regimens that could be studied for treatment of OME.

Anti-Bacterial Agents↗
PubMed2004

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

Biofilms↗
PubMed2006

Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa.

Clinical isolates of Pseudomonas aeruginosa were collected from hospitals in Tehran, Iran, and identified using biochemical tests. A modified microtitre plate test was used to determine the biofilm-forming capacity of the isolates, measured with an enzyme-linked immunosorbent assay (ELISA) reader. Results showed that P. aeruginosa strain 214 was the most efficient at producing biofilm compared with the other strains. Observation of the bacterial biofilm on Teflon sheets and on a catheter using a scanning electron microscope showed greater biofilm formation on the catheter than on Teflon sheets. In this study, we investigated the bactericidal activity of fluoroquinolones, beta-lactams, macrolides and aminoglycoside. The results showed differences in the antibiotic susceptibility of planktonic and biofilm cell populations. Fluoroquinolones showed more potent activity than the other antibiotics, and biofilms were completely eradicated by treatment with 16 x the minimum inhibitory concentration (MIC) of ciprofloxacin and 64 x MIC of ofloxacin, whereas all biofilms survived 2560 microg/mL of imipenem and ceftazidime. Production of an exopolysaccharide matrix is one of the distinguishing characteristics of biofilms. It has been suggested that this matrix prevents access of antibiotics to the bacterial cells embedded in the community. In this study, we also evaluated the permeation of antibiotics through alginate of P. aeruginosa strain 214 using a sandwich cup method. Macrolides were most efficient, showing 100% penetration; fluoroquinolones and beta-lactams had a high permeation rate > 75%, whereas the rates for aminoglycosides were low (amikacin = 59%; gentamicin = 73%).

Alginates↗
PubMed2006

Correlation between biofilm formation and production of polysaccharide intercellular adhesin in clinical isolates of coagulase-negative staphylococci.

The ability to form a biofilm seems to play an essential role in the virulence of coagulase-negative staphylococci (CoNS) by permitting them to cause persistent prosthetic device-related infections. The most clearly characterized component of staphylococcal biofilms is the polysaccharide intercellular adhesin (PIA) encoded by the icaADBC operon. In the present paper, we assess the link between the ability to form a biofilm (Bf+/-), to synthesize PIA (PIA+/-) and the presence of the ica locus (ica+/-). For this purpose, 66 CoNS strains were tested in vitro. Seventy three percent of all strains revealed presence of the ica locus (ica+), and therefore were potentially able to produce PIA and to form a biofilm. However, the characteristics observed indicated that 15% of all strains were biofilm forming without PIA production (Bf+, PIA-, ica+/-) while 8% were PIA producers without biofilm formation (Bf-, PIA+, ica+). On the basis of the obtained data we conclude that (i) PIA synthesis alone is not sufficient to produce a biofilm and (ii) staphylococci can also form a biofilm without producing PIA.

Biofilms↗
PubMed2004

A between-patient disinfection method to control water line contamination and biofilm inside dental units.

The aim of the present study was to evaluate the efficacy of a between-patient disinfection procedures to maintain low bacterial counts in dental unit water line (DUWL) effluents, and control dental water line biofilms. Six dental units already in use, that had never been cleaned, were monitored for three weeks. During the first week only baseline contamination levels were assessed with no treatment of the system. In the second week lines were flushed with water for 30 s before treating each patient. During the third week, a disinfection procedure with 0.26% peracetic acid, followed by a water flush, was implemented before treating each patient. DUWL samples were collected both at the beginning and at the end of 216 dental procedures (72 during each period), plated on R2A agar and incubated at room temperature for seven days to obtain total bacterial counts in colony forming units per millilitre. To assess biofilm control, nine dental units (five never used and four old dental units with established biofilm) were used for 30 days in routine dental practice undergoing five between-patient DUWL disinfecting cycles every day. Water line samples were removed at baseline and at the end of the study and examined by scanning electron microscopy to determine the presence or absence of biofilms. A significant difference (P < 0.01) in mean DUWL bacterial counts was found between the three sets of observations. Biofilms were not present in any of the new dental units and a demonstrable reduction in the biofilms from the four dental units with previous presence of established biofilms was observed at the end of the study. In this study, a between-patient disinfection procedure consisting of flushing DUWL with peracetic acid with use of water was efficacious in the control of both microbial contamination of dental treatment water and dental water line biofilms.

Biofilms↗
PubMed2005

Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler.

The fungal pathogen Candida albicans has the ability to grow as a biofilm on synthetic materials. This presents a significant problem in the clinical situation when the organism grows as a biofilm on medical devices resulting in infections which are resistant to antifungal agents. Determining the extent to which certain genes are involved in biofilm formation is an important aspect for the development of strategies to control pathogenic biofilms. ALS1 is a member of the ALS (agglutinin-like sequence) family, the protein products of which are implicated in attachment to endothelial cells and biofilm formation. The expression of ALS1 in biofilms grown on silicone elastomer, a material used in the manufacture of medical devices, and planktonically grown cells was investigated using a novel real-time quantitative reverse transcriptase PCR (q-RT PCR) on the LightCycler. This study demonstrates quantitatively that ALS1 is clearly up-regulated during biofilm growth. The real-time q-RT PCR assay described here has the potential to be used as an indicator of biofilm formation on medical devices.

Biofilms↗
PubMed2005

Lessons from DNA microarray analysis: the gene expression profile of biofilms.

DNA microarray technology has been used to identify the global gene expression profile of biofilm cells. This is an interesting case study in how DNA microarray technology has advanced the molecular understanding of an understudied research area. DNA microarray analyses have suggested that there may be common responses upon biofilm formation, such as the repression of flagella genes and hyper-expression of genes for adhesion and ribosomal protein. They have also assisted in the identification of transcription factors that affect the formation of biofilms and indicated that there may not be biofilm-specific genes, arguing against biofilm formation being a developmental process. Instead, the DNA microarray data suggest that biofilms may have a unique pattern of gene expression, in which sub-sets of genes expressed in biofilms are also expressed under different planktonic conditions, but only in the biofilm are they all expressed simultaneously.

Bacterial Physiological Phenomena↗
PubMed2006

Candida albicans biofilm development, modeling a host-pathogen interaction.

Medical device-associated infections involve the attachment of cells to a surface, production of an extracellular matrix and development of a mature biofilm. Many Candida albicans disease states involve biofilm growth. These infections have great impact on public health because organisms in biofilms exhibit dramatically reduced susceptibility to antifungal therapy. Progression to a mature biofilm is dependent on cell adhesion, extracellular matrix production and the yeast-to-hyphae transition. Numerous in vitro biofilm model systems have been successfully used to examine biofilm architecture, development, cell phenotypes and drug resistance. Although these studies have included a number of experimental variables to mimic infections in patients, it is difficult to accurately account for the multitude of host and infection-site variables that are probably important in humans. Recent studies have begun to explore C. albicans biofilms using animal biofilm infection models in order to more completely reflect the complexity of this host-fungal interaction.

Animals↗
PubMed2005

In situ and non-invasive detection of specific bacterial species in oral biofilms using fluorescently labeled monoclonal antibodies.

Noninvasive in situ detection of suspected cariogenic bacterial species within dental biofilms could facilitate monitoring of the dynamic change of oral microbial flora and assist in the assessment of the treatment efficacy of therapeutic agents. In this study, we explore the possibility to use three well-characterized monoclonal antibodies (MAbs) against Streptococcus mutans, Actinomyces naeslundii, and Lactobacillus casei to identify these three important members of the oral microbial community in the complex environment of oral biofilms. These MAbs, which were conjugated to different fluorescent labels and visualized with confocal laser scanning microscopy (CLSM), proved to be an useful tool to identify the three species of interest (S. mutans, A. naeslundii, and L. casei) under various experimental conditions including in vitro and in vivo derived oral biofilms. Manifold addition of the MAbs on consecutive days did not alter the biofilm structure thus allowing monitoring of the same biofilm over extended time periods. Using this MAb-based method the effect of sucrose challenge on the biofilm composition and the distribution of S. mutans, A. naeslundii, and L. casei were examined. S. mutans was found to be the predominant species under the various biofilm conditions tested. These studies indicate that MAbs based bacterial detection with CLSM is a versatile tool which permits new insights into the ecology of oral biofilm development.

Actinomyces↗
PubMed2006

Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device.

Knowledge of mechanical properties and failure mechanisms of biofilms is needed to determine how biofilms react on mechanical stress. Methods currently available cannot be used to determine mechanical properties of biofilms on a small scale with high accuracy. A novel microindentation apparatus in combination with a confocal microscope was used to determine the viscoelastic properties of Streptococcus mutans biofilms. The apparatus comprises a small glass indenter and a highly sensitive force transducer. It was shown that the present biofilm, grown under still conditions, behaves as a viscoelastic solid with a storage modulus of 1-8 kPa and a loss modulus of 5-10 kPa at a strain of 10%. Biofilm failure was investigated visually through a confocal microscope by dragging the indenter through the biofilm. It was shown that the tensile strength of the biofilm is predominantly determined by the tensile strength of the extracellular polysaccharide matrix. The combination of microindentation and confocal microscopy is a promising technique to determine and characterize the mechanical properties of soft materials in various fields of microbiology.

Bacteriological Techniques↗