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PubMed2005

Three-dimensional mapping of oxygen distribution in wastewater biofilms using an automation system and microelectrodes.

The three-dimensional oxygen distribution in wastewater biofilms was evaluated using combined oxygen microelectrodes and an automation system. The biofilms were sampled from rotating biological contactors treating domestic wastewater. The samples studied were mature biofilms with a thickness from 630 to 1600 microm. It was demonstrated that the dissolved oxygen concentration could be depleted at the biofilm surface. The heterogeneity of the dissolved oxygen distribution was high in sections further away from the biofilm surface in the water layer. The study showed that the concentration and level of heterogeneity of dissolved oxygen inside the biofilms decreased with depth, forming stratification. The oxygen concentration in biofilms changed generally from a high degree of heterogeneity near the biofilm surface to a low degree of heterogeneity in deep sections of biofilms, indicating a cell--cluster-like structure near the surface and more compact base layer close to the substratum. The three-dimensional oxygen distribution maps revealed pockets of dissolved oxygen in deep sections of biofilms. The dissolved oxygen concentrations of these pockets in the biofilm samples ranged from 0.4 to 1.0 mg/L at 760 microm depth. The three-dimensional oxygen distribution maps produced relevant knowledge of functional and structural characteristics of biofilms used for the treatment of wastewater.

Automation↗

PubMed2001

Metabolic response of biofilm to shear stress in fixed-film culture.

AIMS: In a biofilm reactor, detachment force resulting from hydraulic shear is a major factor that determines the formation and structure of steady state biofilm. The metabolic response of biofilm to change in shear stress was therefore investigated. METHODS AND RESULTS: A conventional annular reactor made of PVC was used, in which shearing over the rotating disc surface was strictly defined. Results from the steady state aerobic biofilm reactor showed that the biofilm structure (density and thickness) and metabolic behaviour (growth yield and dehydrogenase activity) were closely related to the shear stress exerted on the biofilm. Smooth, dense and stable biofilm formed at relatively high shear stress. Higher dehydrogenase activity and lower growth yield were obtained when the shear stress was raised. Growth yield was inversely correlated with the catabolic activity of biofilm. The reduced growth yield, together with the enhanced catabolic activity, suggests that a dissociation of catabolism from anabolism would occur at high shear stress. CONCLUSION: Biofilms may respond to shear stress by regulating metabolic pathways associated with the substrate flux flowing between catabolism and anabolism. A biological phenomenon, besides a simple physical effect, is underlying the observed relation between the shear stress and resulting biofilm structure. SIGNIFICANCE AND IMPACT OF THE STUDY: A hypothesis is proposed that the shear-induced energy spilling would be associated with a stimulated proton translocation across the cell membrane, which favours formation of a stronger biofilm. This research may provide a basis for experimental data on biofilm obtained at different shear stresses to be interpreted in relation to energy.

Biofilms↗

PubMed2002

Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biofilms.

AIMS: The objective of this study was to investigate the antagonistic interactions between bacteriocin-producing enteric bacteria in dual species biofilms and the interspecies interactions correlated with sensitivity to biocides. METHODS AND RESULTS: When compared with their single species counterparts, the dual species biofilms formed by bacteriocin-producing strains exhibited a decrease in biofilm size and an increase in sensitivity to the antimicrobial agents hypochlorite, triclosan and benzalkonium chloride. The five dual species biofilms studied all resulted in biofilms containing a mixture of the two strains. This was attributed to the spatial distribution of cells within the biofilm, with each strain forming its own microcolonies. The production of a bacteriocin also gave a strain a competitive advantage when interacting with a bacteriocin-sensitive strain within a biofilm, both in gaining a foothold in a new environment and in preventing the colonization of a potential competitor into a pre-established biofilm. CONCLUSIONS: It was concluded that bacteriocins might be used specifically for interacting with competing strains within a biofilm, as opposed to a planktonic, environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike planktonically grown bacteriocin-producing populations, where one strain will always be out-competed, bacteriocin-producing and bacteriocin-sensitive strains can coexist in biofilm communities, clearly demonstrating major differences between biofilm and planktonic competition. This paper highlights the importance of bacteriocin production in the development of biofilm communities.

Animals↗

PubMed1997

In-vitro studies of the activity of glycopeptide combinations against Enterococcus faecalis biofilms.

Perfused biofilms of Enterococcus faecalis reached a pseudo-steady state, with respect to bioburden and the numbers of cells liberated within the perfusates. Biofilm growth rate was slow (approximately 0.006 doublings/h) relative to batch culture (approximately 0.01 doublings/h). MICs were determined for vancomycin (0.12 mg/L) and teicoplanin (1.2 mg/L) in batch culture. Steady-state biofilms (24 h and 48 h) were perfused continuously for 96 h with medium containing antibiotic at 4 X MIC. Susceptibility was assessed as cfu in the perfusate, indicating growth inhibition, and as cfu for the parent biofilm. Vancomycin at these levels had little or no effect on either parameter, whilst teicoplanin produced a temporary (30 h) reduction in growth rate (99.99% for 24 h biofilms, 50% for 48 h biofilms). Antibiotic concentrations were raised to therapeutic (trough) levels (vancomycin, 5 mg/L; teicoplanin, 12 mg/L) and applied continuously to 24 h old biofilms. Neither agent affected viability of the biofilm over 96 h. Biofilm growth rate, however, was decreased markedly over the first 8-10 h of antibiotic treatment and was maintained at the reduced level for approximately 40 h. Thereafter growth of the biofilms gradually returned to pre-exposure levels. The pattern of recovery was different for the two agents suggesting that different mechanisms might be involved. Accordingly biofilms were exposed to successive and concurrent 24 h treatments with vancomycin (5 mg/L) and teicoplanin (12 mg/L). This led not only to a further 2-3 log reduction in the growth rate but also to a 3 log reduction in the viability of the parent biofilm. Such synergy between the glycopeptide agents might have therapeutic implications for the treatment of E. faecalis infection.

Anti-Bacterial Agents↗

PubMed2002

Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.

A main characteristic associated with microbial biofilms is their increased resistance to antimicrobial chemotherapies. However, at present very little is known about the phenotypic changes that occur during the transition from the planktonic to the biofilm mode of growth. Candida albicans biofilms displayed an organized three-dimensional structure, and consisted of a dense network of yeasts and filamentous cells deeply embedded in exopolymeric matrix. These biofilms were intrinsically resistant to fluconazole. Moreover, the resistance phenotype was maintained by sessile cells when resuspended as free-floating cells, thus demonstrating that biofilm integrity and the presence of exopolymeric material are not the sole determinants of biofilm resistance. Under planktonic conditions, one of the main mechanisms of azole resistance in C. albicans is through active efflux of these drugs mediated by ATP-binding cassette (ABC) transporters and major facilitators. In this study we used northern hybridization to monitor expression of genes belonging to two different types of efflux pump, the ABC transporters and major facilitators (encoded by CDR and MDR genes, respectively), in C. albicans populations under both planktonic and biofilm growth. It was demonstrated that expression of genes encoding both types of efflux pump were up-regulated during the course of biofilm formation and development. Moreover, antifungal susceptibilities of biofilms formed by a set of C. albicans mutant strains deficient in efflux pumps were investigated to determine their contribution to biofilm resistance. Remarkably, mutants carrying single and double deletion mutations in Delta(cdr)1, Delta(cdr)2, Delta(mdr)1, Delta(cdr)1/Delta(cdr)2 and Delta(mdr)1/Delta(cdr)1 were hypersusceptible to fluconazole when planktonic, but still retained the resistant phenotype during biofilm growth. These analyses demonstrate that C. albicans biofilm resistance is a complex phenomenon that cannot be explained by one mechanism alone, instead it is multifactorial and may involve different molecular mechanisms of resistance compared with those displayed by planktonic cells.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

Susceptibility of Candida albicans biofilms grown in a constant depth film fermentor to chlorhexidine, fluconazole and miconazole: a longitudinal study.

OBJECTIVES: The aim of this study was to assess the resistance of Candida albicans biofilms to both antifungal and antimicrobial agents in vitro. METHODS: Biofilms of C. albicans were grown on denture acrylic discs in a constant depth film fermentor and maintained with artificial saliva. The MIC of fluconazole, miconazole and chlorhexidine for C. albicans was first determined. Using these data, 72 h biofilms were exposed to these agents at different MIC levels. In order to assess growth, biofilms were removed from the fermentor, incubated in the test agent for various periods, the biofilms disrupted and the viable yeast cells present determined. The MIC for these cells was then also determined. In a separate experiment, biofilms of various ages (2-72 h) were exposed to sub-biofilm MIC concentrations for two different periods. RESULTS: C. albicans biofilms were found to be highly resistant to fluconazole and miconazole compared with the same cells grown in suspension (>/=1024 x MIC). In contrast, chlorhexidine inhibited the growth of C. albicans biofilms at a concentration up to 8 x MIC. When the susceptibility of biofilms over time was investigated, higher reductions were observed for chlorhexidine and miconazole than fluconazole for biofilms of 2 and 6 h. CONCLUSIONS: We have shown in this study that the susceptibility of C. albicans to antifungal and antimicrobial agents changes throughout biofilm development.

Antifungal Agents↗

PubMed2005

Anti-metabolic activity of caspofungin against Candida albicans and Candida parapsilosis biofilms.

OBJECTIVES: Candidiasis can be associated with the formation of biofilms on bioprosthetic surfaces and the intrinsic resistance of Candida albicans biofilms to the most commonly used antifungal agents has been demonstrated. In this study, we report on the antifungal activity of caspofungin at two different concentrations, on C. albicans and Candida parapsilosis biofilms with different ages of maturation. METHODS: Fifteen strains of C. albicans (10 strains susceptible to fluconazole in vitro and five strains resistant to this antifungal agent) and six strains of C. parapsilosis (all were susceptible to fluconazole in vitro) were studied. The antifungal activity of caspofungin was assessed by looking for a significant inhibition of the metabolic activity of yeasts within biofilms. Biofilms of Candida were produced in vitro, on silicone catheters. RESULTS: Caspofungin used at MIC did not modify the metabolic activity of C. albicans, whatever the maturation age of the biofilms. The same concentration of caspofungin significantly reduced the metabolism (P<or=0.001) of 25% (biofilms of 48 h) to 50% (biofilms of 2 h) of the C. parapsilosis yeasts. The use of a therapeutic concentration of caspofungin (2 mg/L) significantly decreased (P<or=0.001) the metabolism of all the strains of C. albicans and C. parapsilosis tested, independently of the biofilm maturation age. This potent antifungal activity of caspofungin on C. albicans biofilms was observed independently of the yeast susceptibility to fluconazole. CONCLUSIONS: This study demonstrated that caspofungin used at MIC was not sufficient to reduce C. albicans biofilms, but it suggested an activity on C. parapsilosis biofilms depending on their maturation age. This study also indicated that caspofungin used at 2 mg/L could be a good candidate in the prevention of candidiasis associated with silicone medical devices. Our results also suggested that fluconazole resistance of yeasts did not affect caspofungin activity.

Antifungal Agents↗

PubMed2006

Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces.

BACKGROUND: Microbial biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus biofilm formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro biofilm formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS). METHODS: Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on biofilm formation on polystyrene, polyurethane and silicon elastomer. RESULTS: Sodium citrate at concentrations above 0.5% efficiently inhibits biofilm formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate biofilm formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of biofilm formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated biofilm formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus biofilm formation. CONCLUSIONS: This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the formation of S. aureus biofilms, suggesting that they may reduce the risk of biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates biofilm formation by S. aureus.

Anticoagulants↗

PubMed2005

Bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis.

OBJECTIVES: Biofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processes. We hypothesize that biofilms play a role in chronic rhinosinusitis (CRS). Our goal is to demonstrate biofilms in mucosal specimens of patients undergoing surgery for CRS. STUDY DESIGN: A prospective study of the presence of biofilms in patients undergoing endoscopic sinus surgery for CRS compared with control patients without CRS. METHODS: There were a total of 30 subjects and 4 controls enrolled. The samples of 24 subjects and 4 controls were cultured and then prepared using standard methods for scanning electron microscopy (SEM). The remaining six subjects' samples were treated using advanced cryofixation methods as preparation to preserve structure for SEM and transmission electron microscopy (TEM). RESULTS: Using strict SEM morphologic criteria, 24 (80%) of the 30 patients were found to have micrographic evidence of biofilms. All controls had healthy appearing cilia and goblet cells without biofilms. The six cryofixation samples showed biofilm structures on SEM micrographs that were correlated with bacterial structures seen at the mucosal surface on the corresponding TEM cross sections. Bacterial cultures were positive on all patients. CONCLUSIONS: Biofilms were demonstrated to be present in patients undergoing surgery for CRS; none of the patients without CRS had any evidence of biofilms. Although SEM is capable of demonstrating the biofilms' three-dimensional structure, glycocalyx, and water channels, it cannot clearly demonstrate the presence of bacteria within the biofilm. We were able to demonstrate evidence of bacteria in the biofilms on the subjects tested using TEM.

Adolescent↗

PubMed2006

Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis.

INTRODUCTION: Chronic rhinosinusitis (CRS) is a common disease poorly controlled by antibiotics. Postulated etiologies of CRS include allergy, fungi, functional factors, and biofilm. OBJECTIVES: We presented a preliminary study demonstrating bacterial biofilms' presence on the sinus mucosa of patients with CRS using fluorescent in situ hybridization (FISH). The advantage of FISH in biofilm identification is that it is the only method that identifies the specific bacteria creating the biofilm matrix. We now present the results of a larger series of patients. METHODS: Patients with CRS scheduled for sinus surgery were enrolled in the study. Biopsies of the sinus mucosa and cultures were taken at the time of surgery. Control samples were taken from patients undergoing septoplasty. Specimens underwent FISH testing for Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenza, and Pseudomonas aeruginosa. RESULTS: Bacterial biofilms were present on 14 of 18 specimens. The predominant species were H. influenzae, S. pneumoniae, and S. aureus. P. aeruginosa biofilm was not identified on any specimens. The intraoperative cultures of the planktonic bacteria present in the sinuses did not correlate with the biofilms identified. Two of the five control samples were positive for biofilm. CONCLUSION: The presence of biofilms on the mucosa of patients with CRS offers a possible cause of antimicrobial therapy failure and could change the approach to treatment. However, the presence of biofilms on healthy control samples implies that biofilms may simply be colonizers. The precise role that biofilms play in CRS still remains to be determined. Further studies with larger sample sizes are needed.

Biofilms↗

PubMed1995

Diffusion and binding measurements within oral biofilms using fluorescence photobleaching recovery methods.

Numerous studies have postulated that bacteria which reside in a biofilm differ from planktonic bacteria. These differences are thought to affect biofilm permeability and, indirectly, the susceptibility of biofilm bacteria to antibacterial agents. In this study fluorescence recovery after photobleaching (FRAP) was used to monitor the diffusion and binding characteristics of a set of size fractionated fluorescein isothiocyanate (FTTC)-conjugated dextrans over small areas (ca. 10 micron) in bacterial biofilms. From these measurements it was straightforward to calculate apparent diffusion rates. Initial studies on the concentration dependence of dextran interaction with planktonic bacteria showed that no irreversible interaction was occurring, however, anomalous faster than free solution diffusion rates were obtained. This phenomenon was modelled using novel analytical and numerical methods which incorporate reversible binding with associated fluorescence changes. Apparent diffusion rates measured in biofilms were highly dependent on biofilm preparation. Sucrose starved biofilms produced an apparent slow-down of two- to fivefold depending on dextran molecular mass and location within the biofilm, indicating that diffusion within the biofilm is hindered. Sucrose supplemented biofilms produced apparent diffusion rates close to those in free solution, suggesting less hindered diffusion. Ex vivo plaque showed diffusion and binding similar to the sucrose supplemented biofilms. The FRAP approach provides a fast and convenient method for determining diffusion rates over small areas within bacterial biofilms. This study reinforces the importance of considering the influence of reversible binding and associated fluorescence changes, as these may have a marked effect on the measured apparent diffusion rate.

Bacteria↗

PubMed2001

Individual-based modelling of biofilms.

Understanding the emergence of the complex organization of biofilms from the interactions of its parts, individual cells and their environment, is the aim of the individual-based modelling (IbM) approach. This IbM is version 2 of BacSim, a model of Escherichia coli colony growth, which was developed into a two-dimensional multi-substrate, multi-species model of nitrifying biofilms. It was compared with the established biomass-based model (BbM) of Picioreanu and others. Both models assume that biofilm growth is due to the processes of diffusion, reaction and growth (including biomass growth, division and spreading). In the IbM, each bacterium was a spherical cell in continuous space and had variable growth parameters. Spreading of biomass occurred by shoving of cells to minimize overlap between cells. In the BbM, biomass was distributed in a discrete grid and each species had uniform growth parameters. Spreading of biomass occurred by cellular automata rules. In the IbM, the effect of random variation of growth parameters of individual bacteria was negligible in contrast to the E. coli colony model, because the heterogeneity of substrate concentrations in the biofilm was more important. The growth of a single cell into a clone, and therefore also the growth of the less abundant species, depended on the randomly chosen site of attachment, owing to the heterogeneity of substrate concentrations in the biofilm. The IbM agreed with the BbM regarding the overall growth of the biofilm, due to the same diffusion-reaction processes. However, the biofilm shape was different due to the different biomass spreading mechanisms. The IbM biofilm was more confluent and rounded due to the steady, deterministic and directionally unconstrained spreading of the bacteria. Since the biofilm shape is influenced by the spreading mechanism, it is partially independent of growth, which is driven by diffusion-reaction. Chance in initial attachment events modifies the biofilm shape and the growth of single cells because of the high heterogeneity of substrate concentrations in the biofilm, which again results from the interaction of diffusion-reaction with spreading. This stresses the primary importance of spreading and chance in addition to diffusion-reaction in the emergence of the complexity of the biofilm community.

Algorithms↗

PubMed2006

Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance.

Matrix material was extracted from biofilms of Candida albicans and Candida tropicalis and analysed chemically. Both preparations contained carbohydrate, protein, hexosamine, phosphorus and uronic acid. However, the major component in C. albicans matrix was glucose (32%), whereas in C. tropicalis matrix it was hexosamine (27%). Biofilms of C. albicans were more easily detached from plastic surfaces by treatment with the enzyme lyticase (beta-1,3-glucanase) than were those of C. tropicalis. Biofilms of C. albicans were also partially detached by treatment with proteinase K, chitinase, DNase I, or beta-N-acetylglucosaminidase, whereas C. tropicalis biofilms were only affected by lipase type VII or chitinase. To investigate a possible role for the matrix in biofilm resistance to antifungal agents, biofilms of C. albicans were grown under conditions of continuous flow in a modified Robbins device (MRD). These biofilms produced more matrix material than those grown statically, and were significantly more resistant to amphotericin B. Biofilms of C. tropicalis synthesized large amounts of matrix material even when grown statically, and such biofilms were completely resistant to both amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and a slime-producing strain of Staphylococcus epidermidis (RP62A), when grown statically or in the MRD, were also completely resistant to amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and a slime-negative mutant of S. epidermidis (M7), on the other hand, were completely drug resistant only when grown under flow conditions. These results demonstrate that the matrix can make a significant contribution to drug resistance in Candida biofilms, especially under conditions similar to those found in catheter infections in vivo, and that the composition of the matrix material is an important determinant in resistance.

Amphotericin B↗

PubMed2005

The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity.

The wrinkly spreader (WS) isolate of Pseudomonas fluorescens SBW25 forms a substantial biofilm at the air-liquid interface. The biofilm is composed of an extracellular partially acetylated cellulose-fibre matrix, and previous mutagenesis of WS with mini-Tn5 had identified both the regulatory and cellulose-biosynthetic operons. One uncharacterized WS mutant, WS-5, still expressed cellulose but produced very weak biofilms. In this work, the mini-Tn5 insertion site in WS-5 has been identified as being immediately upstream of the tol-pal operon. Like Tol-Pal mutants of other Gram-negative bacteria, WS-5 showed a "leaky-membrane" phenotype, including the serendipitous ability to utilize sucrose, increased uptake of the hydrophilic dye propidium iodide, and the loss of lipopolysaccharide (LPS) expression. WS-5 cells were altered in relative hydrophobicity, and showed poorer recruitment and maintenance in the biofilm than WS. The WS-5 biofilm was also less sensitive to chemical interference during development. However, growth rate, cellulose expression and attachment were not significantly different between WS and WS-5. Finally, WS-5 biofilms could be partially complemented with WS-4, a biofilm- and attachment-deficient mutant that expressed LPS, resulting in a mixed biofilm with significantly increased strength. These findings show that a major component of the WS air-liquid biofilm strength results from the interactions between LPS and the cellulose matrix of the biofilm--and that in the WS biofilm, cellulose fibres, attachment factor and LPS are required for biofilm development, strength and integrity.

Bacterial Adhesion↗

PubMed2004

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities.

AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF). METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B. cepacia. Reverse challenges were also conducted. Both B. cepacia and P. aeruginosa were able to form steady-state monoculture biofilms after 48 h growth. When steady-state biofilms of B. cepacia NTCT 10661 were challenged with planktonically grown P. aeruginosa PAO1 known to produce a B. cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B. cepacia. By contrast, established biofilms of P. aeruginosa PAO1 resisted immigration of B. cepacia 10661. Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P. aeruginosa 313113 and B. cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances. Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm. Similar observations were made for biofilms generated in CDFFs. In such instances following pan-swapping between two monoculture CDFFs, B. cepacia 313113 was able to integrate into an established P. aeruginosa 313113 biofilm to form a stable binary biofilm. CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P. aeruginosa predisposes uncolonized sections of the surface to permit B. cepacia colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species. Disinfection of a surface might well increase the probability of pathogen harbourage.

Bacteriological Techniques↗

PubMed2005

Protozoan grazing and its impact upon population dynamics in biofilm communities.

AIMS: To determine the impact of protozoan grazing on the population dynamics of a multispecies bacterial biofilm community. METHODS AND RESULTS: Grazing by Acanthamoeba castellanii and the ciliate Colpoda maupasi upon biofilm and planktonic communities, composed of Klebsiella pneumoniae, Pseudomonas fluorescens and Staphylococcus epidermidis was investigated. Biofilms were formed using glass coverslips, held in a carousel device, as substrata for biofilm formation or in glass flow cells. The predatory effects of the amoeba were generally confined to the biofilm, where grazing rates corresponded to losses from the biofilm equivalent to ca 30,000 biofilm cells cm(-2) h(-1), with the amoeba becoming an integral part of the community. C. maupasi reduced the thickness of mature multispecies biofilms at steady-state from 500 to <200 microm. CONCLUSIONS: We report that the presence of the protozoa A. castellanii and C. maupasi markedly influence population dynamics within defined biofilm communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study dispels the popular opinion that biofilms are protected against predation by protozoa. A. castellanii clearly has the capacity to graze mixed biofilm communities and to become integrally associated with them, whereas the ciliate C. maupasi reduced biofilm thickness by up to 60%.

Acanthamoeba↗

PubMed1998

Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents.

A perfused biofilm fermentor, which allows growth-rate control of adherent microbial populations, was used to assess whether the susceptibility of Candida albicans biofilms to antifungal agents is dependent on growth rate. Biofilms were generated under conditions of glucose limitation and were perfused with drugs at a high concentration (20 times the MIC). Amphotericin B produced a greater reduction in the number of daughter cells in biofilm eluates than ketoconazole, fluconazole, or flucytosine. Similar decreases in daughter cell counts were observed when biofilms growing at three different rates were perfused with amphotericin B. In a separate series of experiments, intact biofilms, resuspended biofilm cells, and newly formed daughter cells were removed from the fermentor and were exposed to a lower concentration of amphotericin B for 1 h. The susceptibility profiles over a range of growth rates were then compared with those obtained for planktonic cells grown at the same rates under glucose limitation in a chemostat. Intact biofilms were resistant to amphotericin B at all growth rates tested, whereas planktonic cells were resistant only at low growth rates (</=0.13 h-1). Cells resuspended from biofilms were less resistant than intact biofilm populations but more resistant than daughter cells; the susceptibilities of both these cell types were largely independent of growth rate. Our findings indicate that the amphotericin B resistance of C. albicans biofilms is not simply due to a low growth rate but depends on some other feature of the biofilm mode of growth.

Amphotericin B↗

PubMed2002

Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins.

Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms. We also explored effects of preincubation of C. albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candida isolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candida biofilms. Preincubation of C. albicans cells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P < 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candida biofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

Amphotericin B↗