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PubMed2003

Biofilm structure and function and possible implications for riverine DOC dynamics.

Biofilms are major sites of carbon cycling in streams and rivers. Here we elucidate the relationship between biofilm structure and function and river DOC dynamics. Metabolism (extracellular enzymatic activity) and structure (algae, bacteria, C/N content) of light-grown (in an open channel) and dark-grown (in a dark pipe) biofilms were studied over a year, and variations in dissolved organic carbon (DOC) and biodegradable DOC (BDOC) were also recorded. A laboratory experiment on 14C-glucose uptake and DOC dynamics was also performed by incubating natural biofilms in microcosms. On the basis of our field (annual DOC budget) and laboratory results, we conclude that light-grown biofilm is, on annual average, a net DOC consumer. This biofilm showed a high monthly variability in DOC uptake/release rates, but, on average, the annual uptake rate was greater than that of the dark-grown biofilm. The higher algal biomass and greater structure of the light-grown biofilm may enhance the development of the bacterial community (bacterial biomass and activity) and microbial heterotrophic activity. In addition, the light-grown biofilm may promote abiotic adsorption because of the development of a polysaccharide matrix. In contrast, the dark-grown biofilm is highly dependent on the amount and quality of organic matter that enters the system and is more efficient in the uptake of labile molecules (higher 14C-glucose uptake rate per mgC). The positive relationships between the extracellular enzymatic activity of biofilm and DOC and BDOC content in flowing water indicate that biofilm metabolism contributes to DOC dynamics in fluvial systems. Our results show that short-term fluvial DOC dynamics is mainly due to the use and recycling of the more labile molecules. At the river ecosystem level, the potential surface area for biofilm formation and the quantity and quality of available organic carbon might determine the effects of biofilm function on DOC dynamics.

Analysis of Variance↗

PubMed2004

Ultrasonic-enhanced gentamicin transport through colony biofilms of Pseudomonas aeruginosa and Escherichia coli.

The hypothesis that ultrasound increases antibiotic transport through biofilms of Escherichia coli and Pseudomonas aeruginosa was investigated using colony biofilms. Biofilms grown on membrane filters were transferred to nutrient agar containing 50 microg/ml gentamicin. A smaller filter was placed on top of the biofilm and a blank concentration disk was situated atop the filter. Diffusion of antibiotic through the biofilms was allowed for 15, 30, or 45 min at 37 degrees C. Some of these biofilms were exposed to 70-kHz ultrasound and others were not. Each concentration disk was then placed on a nutrient agar plate spread with a lawn of E. coli. The resulting zone of inhibition was used to calculate the amount of gentamicin that was transported through the biofilm into the disk. The E. coli and P. aeruginosa biofilms grown for 13 and 24 h were exposed to two different ultrasonic power densities. Ultrasonication significantly increased the transport of gentamicin through the biofilm. Insonation of biofilms of E. coli for 45 min more than doubled the amount of gentamicin compared to their noninsonated counterparts. For P. aeruginosa biofilms, no detectable gentamicin penetrated the biofilm within 45 min without ultrasound; however, when insonated (1.5 W/cm2) for 45 min, the disks collected more than 0.45 microg antibiotic. Ultrasonication significantly increased transport of gentamicin across biofilms that normally blocked or slowed gentamicin transport when not exposed to ultrasound. This enhanced transport may be partially responsible for the increased killing of biofilm bacteria exposed to combinations of antibiotic and ultrasound.

Anti-Bacterial Agents↗

PubMed2001

Artificial Pseudomonas aeruginosa biofilms and confocal laser scanning microscopic analysis.

Bacterial biofilms may be formed at various sites, including mucous membranes, teeth, and infectious lesions. To elucidate the structure and the function of biofilms, artificial biofilms of mucoid-type Pseudomonas aeruginosa organisms (strain PT1252) were made by centrifuging the organisms onto the surface of a coverglass and culturing further in broth media supplied continuously (45 ml/h). The biofilm structure at 4, 8, 12, and 24 h was visualized with fluorescent staining (SYTO9, propidium iodide [PI], and/or fluorescein isothiocyanate-concanavalin A [FITC-ConA]) by confocal laser scanning microscopy (CLSM). It was clearly demonstrated that the number of bacteria (10(4)--10(6)/ml) could be estimated by their fluorescence intensity. Sectional analysis of each biofilm layer (1-microm thickness) made it possible to demonstrate the three-dimensional development of biofilms, and revealed that the biofilms were 9 microm in height after 12 h. The live and dead organisms were differentiated by SYTO9 and PI, respectively, in situ in biofilms, and about 13% of the organisms were dead in 12-h-old biofilms. When 12-h-old biofilms were exposed to ciprofloxacin at minimum bactericidal concentration (6.26 microg/ml) for 90 min, all the organisms were killed, but some organisms (11 +/- 1.3%; n = 3) in 24-h-old biofilms with thicker and denser structure were still alive after exposure for 120 min. These results indicate that the CLSM analysis of artificial biofilms was useful for elucidating bacterial functions in biofilms, and may lead to a new quantitative system for estimating the bactericidal efficacy of antibacterial drugs in biofilms.

Anti-Infective Agents↗

PubMed2005

Effects of operating conditions on the adhesive strength of Pseudomonas fluorescens biofilms in tubes.

Understanding the mechanical properties of biofilms, especially the force required to disrupt them and remove them from substrata is very important to development of antibiofouling strategies. In this work, a novel micromanipulation technique with a specially designed T-shaped probe has been developed to serve as an experimental means to measure directly the adhesive strength of biofouling deposits on the surface of a glass test stud. The basic principle of this novel technique is to pull away a whole biofilm accumulated on the surface of a glass test stud with T-shaped probe, and to measure simultaneously the force imposed on the biofilm. The adhesive strength between the biofilms and the surface to which they are attached, is defined as the work per unit area required to remove the biofilms from the surface. The biofouling experiments were performed on an elaborate design of a simulated heat exchanger system. A monoculture of Pseudomonas fluorescens was chosen as the fouling microorganism for the laboratory studies. Results indicate that the adhesive strength of the biofilm was affected by the conditions of operation, such as biofilm age, nutrient concentration, suspended cell concentration, pH, surface roughness of the substratum and fluid velocity. As noted, the effect of fluid velocity on the biofilm adhesive strength seemed to overwhelm other factors. At the same operating conditions, the biofilm adhesive strength increased as the fluid velocity increased within the range of 0.6-1.6m/s. In addition, the flow-related biofilm structures were observed that biofilms generally grew as a more compact pattern at the higher fluid velocity. Apparently, the fluid velocity can affect the biofilm structure, which in turn determines the biofilm adhesive strength. The knowledge of the biofilm adhesive strength with associated influences of the operating conditions may be used to define better cleaning procedures.

Bacterial Adhesion↗

PubMed2006

Biofilm formation by field isolates and reference strains of Haemophilus parasuis.

The ability to form biofilms for a total of 80 field isolates and 15 reference strains of Haemophilus parasuis, the etiological agent of Glasser's disease, was tested by glass tube and polystyrene microtiter plate assays. A total 43% of field isolates, including strains representing 13 serovars (except serovars 3 and 8) and non-typable strains, exhibited the ability to form biofilms at different levels via polystyrene microtiter plate assays. Among the reference strains representing 15 serovars, only serovars 2, 9, 12, 13 and 15 could not form biofilms on the polystyrene surface. A total of 85% of the strains forming biofilms at air-liquid interfaces in glass tubes also formed biofilms on polystyrene surfaces. Generally, non-virulent serovars showed a higher degree of biofilm formation than virulent serovars. The biofilm formation phenotype of most strains was maintained when cultures were passaged on agar and in broth. H. parasuis from the nasal cavities of pigs experimentally infected with biofilm-positive bacteria maintained the biofilm formation phenotype, whereas bacteria recovered from the lung and brain lost the ability to form biofilms. The biofilm-negative strains did not recover the ability to form biofilms via experimental infection. Our data indicate that most serovars of H. parasuis could form biofilms in vitro, and the biofilm formation phenotype is associated with the recovery site of the strains and is maintained when bacteria are passaged in vitro and in the upper respiratory tract.

Animals↗

PubMed2001

Integration of Raman microscopy, differential interference contrast microscopy, and attenuated total reflection Fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in Candida albicans biofilms.

Two spectroscopic techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and Raman microscopy (RM), were used to characterize transport of chlorhexidine digluconate (CHG) in Candida albicans (CA) biofilms. Different (volumetric) regions of the biofilm are sampled by these two vibrational spectroscopies making them complementary techniques. Simple mathematical models were developed to analyze ATR-FTIR and RM data to obtain an effective diffusion coefficient describing transport through CA biofilms. CA biofilms were composed primarily of yeast and hyphal forms, with some pseudohyphae. Upper regions of biofilms that had become confluent, (i.e., biofilms that completely covered the germanium (Ge) substratum) were composed primarily of a tangled mass of hyphae with openings between germtubes about 10 to 50 microm across. Quantitative analysis of ATR-FTIR kinetic data curves indicated that the effective diffusion coefficient for transport of CHG through confluent biofilms about 200-microm thick was reduced 0.1 to 0.3 times compared to the diffusion coefficient for CHG in water. Effective diffusion coefficients obtained from analysis of RM data were consistently higher than those indicated by ATR-FTIR data suggesting that transport is more hindered in regions near the base of the biofilm than in the outer layers. Analysis of both ATR-FTIR and RM data obtained from thicker films indicated that adsorption of CHG to biofilm components was responsible for a substantial portion of the transport limitation imposed by the biofilm. Comparison of ATR-FTIR and RM data for both types of biofilms indicated that sites of CHG adsorption were more concentrated in the interfacial region than in the bulk biofilm. Comparison of results for ATR-FTIR and RM measurements suggests that these relatively thick CA biofilms can be modeled, for purposes of predicting transport, approximately as a homogeneous thin planar sheet. Thus, these biofilms offer a relatively tractable model system for initial investigations of the relation between antimicrobial transport and kinetics of antimicrobial action.

Anti-Bacterial Agents↗

PubMed2001

Growing reproducible biofilms with respect to structure and viable cell counts.

We have developed a new method of growing 4-day-old biofilms that are reproducible, with respect to viable cell number and biofilm structure. To demonstrate the utility of the method, we grew biofilms composed of Pseudomonas aeruginosa (ATCC#700829), P. fluorescens (ATCC#700830) and Klebsiella pneumoniae (ATCC#700831), 18 times in flat-plate reactors under well-defined conditions of: flow rate, nutrient concentration, temperature, inoculum and growth rate. The resulting 4-day-old biofilms were approximately 200-300 microm thick and exhibited a high degree of reproducibility. The number of viable cells that accumulated per unit surface area and the biofilm areal porosity were reproduced within 10% error. We have also quantified other parameters characterizing biofilm structure using biofilm-imaging techniques: fractal dimension, textural entropy and diffusion distance as auxiliary parameters characterizing the reproducibility of biofilm accumulation. As a result of analysis, we have introduced a new parameter to better quantify and characterize the number of viable cells in biofilms, "specific number of viable cells" (SNVC). This parameter is the viable cell number normalized with respect to the surface area covered by the biofilm and with respect to the biomass of the biofilm. This new descriptor represents the dynamics of biofilm accumulation better than the traditionally used colony-forming unit (CFU) per surface area covered by the biofilm because it accounts not only for the surface coverage but also for the biofilm thickness.

Bacteriological Techniques↗

PubMed2006

Biofilm formation on hydrophilic intraocular lens material.

PURPOSE: To estimate bacterial biofilm formation on the hydrophilic acrylic (hydrogel) intraocular lens (IOL) Meridian (HP60M, Baush & Lomb) and to investigate a preventive effect against biofilm formation of hydrogel IOLs presoaked in antibiotics. METHODS: Two Staphylococcus epidermidis strains, ATCC 12228 and ATCC 35984 (biofilm-producer), and an Enterococcus faecalis strain (KOS1, clinical isolate from an endophthalmitis patient) were used. Biofilms were cultivated on disks of different IOL materials: hydrogel, PMMA (polymethylmethacrylate), and acrylic. Biofilms were stained with crystal violet (CV), which served as an index of biofilm formation. The bacterial population was enumerated after biofilm homogenization. Biofilms were also examined by scanning electron microscopy (SEM). IOLs were presoaked in two antibiotics, levofloxacin (LVFX) and gatifloxacin (GFLX), and then the bacterial population was enumerated. As in vivo experiment, antibiotics-treated and nontreated Meridian IOLs were implanted in rabbit eyes, which served as an endophthalmitis model, and the bacterial population was enumerated. RESULTS: The amount of biofilm formed was the least on hydrogel from among the three materials tested after 48- and 72-hr incubation (p < 0.05 to 0.01). The bacterial population was the least on hydrogel from among the three materials with ATCC 12228 (p < 0.05 to 0.01), and the bacterial population was significantly different between hydrogel and acrylic after 72-hr incubation with ATCC 35984 (p < 0.05). Biofilm by the two S. epidermidis strains were recognized after 24-hr incubation. Rates of biofilm-positive SEM fields, which were defined as being occupied by biofilm over at least half of the area, were increased through 72 hr with ATCC 35984. While the E. faecalis strain showed no bacterial adherence on the antibiotics-treated hydrogel IOLs, adherence of the S. epidermidis strain, ATCC 35984 was recognized on the LVFX-treated IOLs after 48-hr incubation (103 to 104 CFU/ml). In the rabbit in vivo model, the bacterial populations in eyes with an antibiotics-treated Meridian IOL were significantly smaller than in eyes with a nontreated IOL for 72 hr after surgery (p < 0.05 to 0.01). CONCLUSIONS: The biofilm formation was less on hydrogel than on other two materials tested. Hydrogel presoaked in antibiotics exhibited a preventive effect against biofilm formation at least for 24 hr in vitro and against bacterial proliferation in the rabbit in vivo endophthalmitis model.

Acrylic Resins↗

PubMed1999

Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro.

Four slime-producing isolates of Staphylococcus aureus were used in an antibiotic susceptibility assay for biofilms developed on 96-well polystyrene tissue culture plates. The study involved 11 antibiotics, two biofilm ages (6 and 48 h), two biofilm growth media (tryptone soy broth (TSB) and delipidated milk) and three antibiotic concentrations (4 x MBC, 100 mg/L and 500 mg/L). ATP-bioluminescence was used for automated bacterial viability determination after a 24 h exposure to antibiotics, to avoid biofilm handling. Under the conditions applied, viability in untreated biofilms (controls) was lower when biofilm growth was attempted in milk rather than in TSB. Various antibiotics had a greater effect on viability when used at higher (> or =100 mg/L) antibiotic concentrations and on younger (6 h) biofilms. Increased antibiotic effect was observed in milk-grown rather than TSB-grown biofilms. Phosphomycin and cefuroxime, followed by rifampicin, cefazolin, novobiocin, vancomycin, penicillin, ciprofloxacin and tobramycin significantly affected biofilm cell viability at least under some of the conditions tested. Gentamicin and erythromycin had a non-significant effect on cell viability. Transmission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofilms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grown biofilms. A reduced matrix distribution and enhanced cell density were observed as the biofilm aged. The S. aureus biofilm test discriminated antibiotics requiring shorter (3 h or 6 h) from those requiring longer (24 h) exposure and yielded results which may be complementary to those obtained by conventional tests.

Adenosine Triphosphate↗

PubMed2001

In situ measurement of linezolid and vancomycin concentrations in intravascular catheter-associated biofilm.

We describe a new method for the measurement of antimicrobial concentrations in the biofilm associated with the endoluminal surface of intravascular catheters. We quantified endoluminal planktonic bacteria in haemodialysis catheters using the acridine orange method on catheter blood. After catheter removal, separate lumens were perfused in vitro with either vancomycin or linezolid to simulate in vivo antibiotic infusion. Biofilm was recovered using endoluminal brushes, weighed and assayed by fluoroimmunoassay for vancomycin and by bioassay for linezolid. Viable bacteria were counted by serial dilution and agar plating. Biofilm had measurable amounts of vancomycin in 11/11 catheter lumens post-infusion (0.3-18.2 mg biofilm per lumen, mean 6.8 mg; vancomycin concentration 0.2-89 mg/g biofilm, median 19 mg/g). By comparison, linezolid was detected in 4/11 catheter lumens post-infusion (0.5-18.1 mg biofilm per lumen, mean 5.9 mg; linezolid concentration 0.9-6.1 mg/g biofilm, median 1.5 mg/g). Percentage reductions in biofilm-associated bacterial counts post-antibiotic were 84-100%, median 95% (vancomycin) and 0-98%, median 91% (linezolid). We found a significant difference (P = 0.05; Wilcoxon rank sum test) in vancomycin concentrations in coagulase-negative staphylococcal biofilm (median 17.0 mg/g, mean 27.9 mg/g) compared with glycopeptide levels found in biofilm associated with other microorganisms (median 5.5 mg/g, mean 6.9 mg/g). Biofilm concentrations of vancomycin are generally higher than linezolid after antibiotic infusion, which can be explained partly by glycopeptide binding to glycocalyx. Neither antibiotic achieved consistent 100% kill of biofilm bacteria after single infusions, even when a very high concentration was present. The endoluminal brush technique can be used to measure antibiotic concentration in intravascular catheter-associated biofilm in situ. This approach can be exploited to measure biofilm antibiotic concentrations in vivo, without the need for catheter removal.

Acetamides↗

PubMed2004

Viridans streptococci in endocarditis and neutropenic sepsis: biofilm formation and effects of antibiotics.

OBJECTIVES: Viridans group streptococci (VGS) are a frequent cause of bacterial endocarditis or sepsis in patients with neutropenia. Endocarditis in particular, is associated with plaque formation on the endocardium and valve leaflets whereas VGS septicaemia in neutropenic patients is caused by the influx of oral flora bacteria through mucositic lesions. This study examined the in vitro potency for biofilm formation of clinical VGS bloodstream isolates, and the effects of antibiotics on these biofilms. METHODS: During the years 1998-2000, 40 VGS bloodstream isolates from 18 patients with endocarditis and 22 patients with severe sepsis and neutropenia were collected. The MICs of penicillin, teicoplanin and moxifloxacin were determined using the microdilution broth method according to NCCLS criteria. Biofilms were grown in microtitre plates, dyed with Crystal Violet, and the mean optical density (OD) was used for quantification. Biofilms were incubated with penicillin, teicoplanin and moxifloxacin at various concentrations starting with the MICs for the respective isolates tested. RESULTS: Isolates from eight out of 18 patients with endocarditis and six out of 22 patients with neutropenia formed biofilms (not significant). For the 14 isolates, the MIC(90)s (range) of penicillin, teicoplanin and moxifloxacin were 0.5 mg/L (0.001-0.5), 0.125 mg/L (0.025-0.125) and 0.5 mg/L (0.05-0.5), respectively. Generally, biofilms persisted although incubated with the antibiotics up to concentrations of 128 x MIC. However, the ODs of biofilms after incubation with an antibiotic were significantly lower than the ODs of biofilms without antibiotic (P<0.05). A significant decrease in the biofilms with increasing antibiotic concentrations was observed for teicoplanin and moxifloxacin, but not for penicillin G. CONCLUSIONS: VGS isolated from patients with endocarditis and patients with sepsis and neutropenia form biofilms. Biofilms persist even when exposed to antibiotics at concentrations up to 128 x MIC. Nevertheless, teicoplanin and moxifloxacin reduced the density of the biofilms at concentrations >/=16 x MIC. Thus, testing the effects of antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation is involved in the pathogenesis.

Adolescent↗

PubMed2001

Direct evidence of bacterial biofilms in otitis media.

OBJECTIVES/HYPOTHESIS: Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations. STUDY DESIGN: Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients. METHODS: Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes. RESULTS: Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes. CONCLUSIONS: Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in children's ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.

Animals↗

PubMed2005

Biofilms and antimicrobial resistance.

The pathogenesis of many orthopaedic infections is related to the presence of microorganisms in biofilms. I examine the emerging understanding of the mechanisms of biofilm-associated antimicrobial resistance. Biofilm-associated resistance to antimicrobial agents begins at the attachment phase and increases as the biofilm ages. A variety of reasons for the increased antimicrobial resistance of microorganisms in biofilms have been postulated and investigated. Although bacteria in biofilms are surrounded by an extracellular matrix that might physically restrict the diffusion of antimicrobial agents, this does not seem to be a predominant mechanism of biofilm-associated antimicrobial resistance. Nutrient and oxygen depletion within the biofilm cause some bacteria to enter a nongrowing (ie, stationary) state, in which they are less susceptible to growth-dependent antimicrobial killing. A subpopulation of bacteria might differentiate into a phenotypically resistant state. Finally, some organisms in biofilms have been shown to express biofilm-specific antimicrobial resistance genes that are not required for biofilm formation. Overall, the mechanism of biofilm-associated antimicrobial resistance seems to be multifactorial and may vary from organism to organism. Techniques that address biofilm susceptibility testing to antimicrobial agents may be necessary before antimicrobial regimens for orthopaedic prosthetic device-associated infections can be appropriately defined in research and clinical settings. Finally, a variety of approaches are being defined to overcome biofilm-associated antimicrobial resistance.

Animals↗

PubMed1999

Role of dimorphism in the development of Candida albicans biofilms.

Two model biofilm systems, involving growth on disks of catheter material or on cylindrical cellulose filters, were used to investigate the structure of Candida albicans biofilms. To assess the importance of dimorphism in biofilm development, biofilms produced by two wild-type strains were compared with those formed by two morphological mutants, incapable of yeast and hyphal growth, respectively. Scanning electron microscopy and thin sections of biofilms examined by light microscopy revealed that biofilms of the wild-type strains formed on catheter disks consisted of two distinct layers: a thin, basal yeast layer and a thicker, but more open, hyphal layer. The hypha- mutant produced only the basal layer, whereas the yeast- mutant formed a thicker, hyphal biofilm equivalent to the outer zone of the wild-type structures. Biofilms of the yeast- mutant were more easily detached from the catheter surface than the others, suggesting that the basal yeast layer has an important role in anchoring the biofilm to the surface. Biofilms formed on cylindrical cellulose filters were quite different in appearance. The hypha- mutant and both wild types produced exclusively yeast-form biofilms whereas the yeast- mutant generated a dense hyphal mat on the top of the filter. All these biofilms, irrespective of morphological form, were resistant to the antifungal agent, amphotericin B. Overall, these results indicate that the structure of a C. albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems.

Amphotericin B↗

PubMed2003

A three-dimensional, stochastic simulation of biofilm growth and transport-related factors that affect structure.

Biofilm structural heterogeneity affects a broad range of microbially catalysed processes. Solute transport limitation and autoinhibitor production, two factors that contribute to heterogeneous biofilm development, were investigated using BacMIST, a computer simulation model. BacMIST combines a cellular automaton algorithm for biofilm growth with Brownian diffusion for solute transport. The simulation represented the growth of microbial unit cells in a three-dimensional domain modelled after a repeating section of a constant depth film fermenter. The simulation was implemented to analyse the effects of various levels of transport limitation on a growing single-species biofilm. In a system with rapid solute diffusion, cells throughout the biofilm grew at their maximum rate, and no solute gradient was formed over the biofilm thickness. In increasingly transport-limited systems, the rapidly growing fraction of the biofilm population decreased, and was found exclusively at the biofilm-liquid interface. Trans-biofilm growth substrate gradients also deepened with increasing transport limitation. Autoinhibitory biofilm growth was simulated for various rates of microbially produced inhibitor transport. Inhibitor transport rates affected both the biofilm population dynamics and the resulting biofilm structures. The formation of networks of void spaces in slow-growing regions of the biofilm and the development of columns in the fast-growing regions suggested a possible mechanism for the microscopically observed evolution of channels in biofilms.

Biofilms↗

PubMed2006

Differences between single- and dual-species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine.

Streptococcus mutans, considered a primary pathogen in dental caries, thrives in dental plaque, which is a multispecies biofilm. Metabolic interactions between S. mutans and Veillonella parvula have been suggested. In this study we developed a biofilm model to quantify single-species (S. mutans or V. parvula) and dual-species (S. mutans and V. parvula) biofilm formation, and we identified the differences between the respective biofilms in terms of growth, acid formation, and response to chlorhexidine. Polystyrene 96-well microtiter plates were used for biofilm formation. These biofilms were exposed to various chlorhexidine concentrations (0.025-0.4 mg ml(-1)) and treatment conditions. Growth of the biofilms and the effects of chlorhexidine were evaluated by viable counts. Viability of the two species in all biofilm types was similar ( approximately 10(8) colony-forming units per well) after 72 h. Lactic acid accumulation of dual-species biofilms was significantly lower at 48 and 72 h than single-species biofilms of S. mutans. Dual-species biofilms were less susceptible to chlorhexidine than single-species biofilms when a neutralization step was included. These results indicate that bacteria in dual-species biofilms have different properties from bacteria in single-species biofilms.

Acetic Acid↗

PubMed1998

Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.

The role of oxygen availability in determining the local physiological activity of Pseudomonas aeruginosa growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 micron thick with biofilms grown in air, 2 micron thick with biofilms grown in pure nitrogen, and 46 micron thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 micron. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within P. aeruginosa biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 micron of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.

Adenosine Triphosphatases↗

PubMed2002

Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms.

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.

Analysis of Variance↗