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PubMed2004

Quantifying biofilm structure: facts and fiction.

There is no doubt among biofilm researchers that biofilm structure is important to many biofilm processes, such as the transport of nutrients to deeper layers of the biofilm. However, biofilm structure is an elusive term understood only qualitatively, and as such it cannot be directly correlated with any measurable parameters characterizing biofilm performance. To correlate biofilm structure with the parameters characterizing biofilm performance, such as the rate of nutrient transport within the space occupied by the biofilms, biofilm structure must first be quantified and expressed numerically on an appropriate scale. The task of extracting numerical parameters quantifying biofilm structure relies on using biofilm imaging and image analysis. Although defining parameters characterizing biofilm structure is relatively straightforward, and multiple parameters have been described in the computer science literature, interpreting the results of such analyses is not trivial. Existing computer software developed by several research groups, including ours, for the sole purpose of analyzing biofilm images helps quantify parameters from biofilm images but does nothing to help interpret the results of such analyses. Although computing structural parameters from biofilm images permits correlating biofilm structure with other biofilm processes, the meaning of the results is not obvious. The first step to understanding the quantification of biofilm structure, developing image analysis, methods to quantify information from biofilm images, has been made by several research groups. The next step is to explain the meaning of these analyses. This presentation explains the meaning of several parameters commonly used to characterize biofilm structure. It also reviews the authors' research and experience in quantifying biofilm structure and their attempts to quantitatively relate biofilm structure to fundamental biofilm processes.

Anti-Bacterial Agents↗

PubMed2006

Putative role of beta-1,3 glucans in Candida albicans biofilm resistance.

Biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface. Nearly all device-associated infections involve growth in the biofilm life style. Biofilm communities have characteristic architecture and distinct phenotypic properties. The most clinically important phenotype involves extraordinary resistance to antimicrobial therapy, making biofilm infections very difficulty to cure without device removal. The current studies examine drug resistance in Candida albicans biofilms. Similar to previous reports, we observed marked fluconazole and amphotericin B resistance in a C. albicans biofilm both in vitro and in vivo. We identified biofilm-associated cell wall architectural changes and increased beta-1,3 glucan content in C. albicans cell walls from a biofilm compared to planktonic organisms. Elevated beta-1,3 glucan levels were also found in the surrounding biofilm milieu and as part of the matrix both from in vitro and in vivo biofilm models. We thus investigated the possible contribution of beta-glucans to antimicrobial resistance in Candida albicans biofilms. Initial studies examined the ability of cell wall and cell supernatant from biofilm and planktonic C. albicans to bind fluconazole. The cell walls from both environmental conditions bound fluconazole; however, four- to fivefold more compound was bound to the biofilm cell walls. Culture supernatant from the biofilm, but not planktonic cells, bound a measurable amount of this antifungal agent. We next investigated the effect of enzymatic modification of beta-1,3 glucans on biofilm cell viability and the susceptibility of biofilm cells to fluconazole and amphotericin B. We observed a dose-dependent killing of in vitro biofilm cells in the presence of three different beta-glucanase preparations. These same concentrations had no impact on planktonic cell viability. beta-1,3 Glucanase markedly enhanced the activity of both fluconazole and amphotericin B. These observations were corroborated with an in vivo biofilm model. Exogenous biofilm matrix and commercial beta-1,3 glucan reduced the activity of fluconazole against planktonic C. albicans in vitro. In sum, the current investigation identified glucan changes associated with C. albicans biofilm cells, demonstrated preferential binding of these biofilm cell components to antifungals, and showed a positive impact of the modification of biofilm beta-1,3 glucans on drug susceptibility. These results provide indirect evidence suggesting a role for glucans in biofilm resistance and present a strong rationale for further molecular dissection of this resistance mechanism to identify new drug targets to treat biofilm infections.

Amphotericin B↗

PubMed2002

Phosphorus uptake into algal biofilms in a lowland chalk river.

This paper examines the growth and uptake of phosphorus into algal biofilms in the River Kennet, a lowland chalk (Cretaceous-age) stream in southern England. Algal biofilms were grown on artificial plastic substrates (templates) placed (i) on the riverbed and (ii) within the mid-water column. Experiments were set up to examine differences in growth rates of newly colonising biofilms compared with biofilms left to accumulate for periods of up to 6 months. Rates of algal biofilm production were measured by the chlorophyll a concentration that had accumulated per cm2 over the number of days that the biofilm template had been immersed in the river water. An algal biofilm bloom occurred in early spring, prior to peak suspended chlorophyll a concentrations within the water column. Biofilm samples collected in February and March had the highest chlorophyll a and total phosphorus concentrations. The biofilm bloom corresponded with increased solar radiation and declining river flow conditions. Periodic increases in soluble reactive phosphorus concentrations in the overlying river water did not correspond with any significant increase in biofilm production. These results suggest that light, rather than phosphorus is a key factor for biofilm growth in the River Kennet. Higher rates of chlorophyll a development in mid-water column biofilms may be linked to greater light exposure; however, maximum total-P concentrations were similar for both bed and water column biofilms. Newly colonising biofilms exhibited higher chlorophyll a and total-P concentrations than biofilms left to accumulate over longer terms, suggesting that fresh substrate availability promotes high rates of biofilm growth. Both 'condensed and organic' P (stored in biomass) and 'inorganic' (mineral) P fractions within the biofilms were present in varying proportions, although the early spring biofilm bloom resulted in maximum proportions and absolute concentrations of 'condensed and organic' P. Calcite was the only crystalline mineral detected within the biofilms. Ratios of Ca:inorganic P are largely consistent with the presence of CaCO3-P co-precipitates, although one very low value suggested that there may also be additional sources of inorganic P, possibly P adsorbed to clays or organics within the biofilm. However, poor linkages between CaCO3 and inorganic P concentrations suggest that, although the inorganic P fraction within the biofilm may be derived largely from CaCO3-P co-precipitation, the subsequent processes controlling overall CaCO3 and inorganic P concentrations in the biofilm are complex.

Biofilms↗

PubMed2001

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates.

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.

Animals↗

PubMed2006

Phenotypic characterization of Streptococcus pneumoniae biofilm development.

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.

Bacterial Proteins↗

PubMed2007

Temporal gene-expression in Escherichia coli K-12 biofilms.

Analysis of the temporal development of Escherichia coli K-12 biofilms in complex medium indicates the greatest differential gene expression between biofilm and suspension cells occurred in young biofilms at 4 and 7 h (versus 15 and 24 h). The main classes of genes differentially expressed (biofilm versus biofilm and biofilm versus suspension cells) include 42 related to stress response (e.g. cspABFGI), 66 related to quorum sensing (e.g. ydgG, gadABC, hdeABD), 20 related to motility (e.g. flgBCEFH, fliLMQR, motB), 13 related to fimbriae (e.g. sfmCHM, fimZ, csgC), 24 related to sulfur and tryptophan metabolism (e.g. trpLBA, tnaLA, cysDNCJH), 80 related to transport (e.g. gatABC, agaBC, ycjJ, ydfJ, phoU, phnCJKM), and six related to extracellular matrix (e.g. wcaBDEC). Of the 93 mutants identified and studied, 76 showed altered biofilm formation. Biofilm architecture changed from thin and dense to globular and dispersed to dense and smooth. The quorum-sensing signal AI-2 controls gene expression most clearly in mature biofilms (24 h) when intracellular AI-2 levels are highest. Sulfate transport and metabolism genes (cysAUWDN) and genes with unknown functions (ymgABCZ) were repressed in young (4, 7 h) biofilms, induced in developed biofilms (15 h), and repressed in mature (24 h) biofilms. Genes related to both motility and fimbriae were induced in biofilms at all sampling time points and colanic acid genes were induced in mature biofilms (24 h). Genes related to dihydroxyacetone phosphate synthesis from galactitol and galactosamine (e.g. gatZABCDR, agaBCY) were highly regulated in biofilms. Genes involved in the biosynthesis of indole and sulfide (tnaLA) are repressed in biofilms after 7 h (corroborated by decreasing intracellular indole concentrations in biofilms). Cold-shock protein transcriptional regulators (cspABFGI) appear to be positive biofilm regulators, and deletions in respiratory genes (e.g. hyaACD, hyfCG, appC, narG) increased biofilm formation sevenfold.

Biofilms↗

PubMed2006

Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces.

Candida albicans ALS3 encodes a large cell-surface glycoprotein that has adhesive properties. Immunostaining of cultured C. albicans germ tubes showed that Als3p is distributed diffusely across the germ tube surface. Two-photon laser scanning microscopy of model catheter biofilms grown using a PALS3-green fluorescent protein (GFP) reporter strain showed GFP production in hyphae throughout the biofilm structure while biofilms grown using a PTPI1-GFP reporter strain showed GFP in both hyphae and yeast-form cells. Model catheter biofilms formed by an als3 Delta/als3 Delta strain were weakened structurally and had approximately half the biomass of a wild-type biofilm. Reintegration of a wild-type ALS3 allele restored biofilm mass and wild-type biofilm structure. Production of an Als3p-Ag alpha 1p fusion protein under control of the ALS3 promoter in the als3 Delta/als3 Delta strain restored some of the wild-type biofilm structural features, but not the wild-type biofilm mass. Despite its inability to restore wild-type biofilm mass, the Als3p-Ag alpha 1p fusion protein mediated adhesion of the als3 Delta/als3 Delta C. albicans strain to human buccal epithelial cells (BECs). The adhesive role of the Als3p N-terminal domain was further demonstrated by blocking adhesion of C. albicans to BECs with immunoglobulin reactive against the Als3p N-terminal sequences. Together, these data suggest that portions of Als3p that are important for biofilm formation may be different from those that are important in BEC adhesion, and that Als3p may have multiple functions in biofilm formation. Overexpression of ALS3 in an efg1 Delta/efg1 Delta strain that was deficient for filamentous growth and biofilm formation resulted in growth of elongated C. albicans cells, even under culture conditions that do not favour filamentation. In the catheter biofilm model, the ALS3 overexpression strain formed biofilm with a mass similar to that of a wild-type control. However, C. albicans cells in the biofilm had yeast-like morphology. This result uncouples the effect of cellular morphology from biofilm formation and underscores the importance of Als3p in biofilm development on silicone elastomer surfaces.

Biofilms↗

PubMed1997

Substratum topography influences susceptibility of Salmonella enteritidis biofilms to trisodium phosphate.

Established (48- and 72-h) Salmonella enteritidis biofilms grown in glass flow cells with or without artificial crevices (0.5-, 0.3-, and 0.15-mm widths) were subjected to a 10% trisodium phosphate (TSP) solution under different flow regimens (0.3, 0.6, 1.2, and 1.8 cm s-1). The abundance of biofilm remaining after TSP treatment, the biocidal efficacy of TSP, and the factors which contributed to bacterial survival were then evaluated by using confocal laser microscopy and a fluorescent viability probe. Biofilm age affected the amount of biofilm which remained following a 15-s exposure to TSP. After TSP treatment of 48-h biofilms, 29% of the original biofilm remained at the biofilm-liquid interface, whereas 75% of the biofilm remained at the base (the attachment surface). Following TSP treatment of 72-h biofilms, 27% of the biofilm material remained at the biofilm-liquid interface, 73% remained at the 5-micron depth, and 91% remained at the biofilm base. Results obtained using the BacLight viability probe indicated that TSP exposure killed all the cells in 48-h biofilms, whereas in the thicker 72-h biofilms, surviving bacteria (approximately 2% of the total) were found near the 5- and 0-micron depths. In the presence of artificially constructed crevices, an inverse relationship was shown to exist between bacterial survival (ranging from approximately 13 to 83% of total biofilm material) and crevice width. This relationship was further influenced by the velocity of TSP flow; high TSP flow velocities (1.8 cm s-1) resulted in the lowest number of surviving bacteria at the base of crevices (approximately 42% survival). Extended time courses demonstrated that after TSP stress was relieved, biofilms continued to grow within crevices but not in systems without crevices. It is suggested that advective TSP flux into crevices and through the biofilm matrix was enhanced under conditions of high flow. These results suggest that the inherent roughness of the substratum on which the biofilm was grown and the timing of TSP application are important factors controlling the efficacy of TSP treatment.

Animals↗

PubMed2001

Determination of nutrients limiting biofilm formation and the subsequent impact on disinfectant decay.

Understanding the contribution of both organic and inorganic nutrients to biofilm development and the subsequent impact of developed biofilms on disinfectant decay are important requirements for distribution system management strategies. Nutrient limitation may be one way to control biofilm development without increasing disinfectant dosing. Little is known, however, of the nutrient requirements of biofilms in distribution systems. Indeed, the effects on biofilm development due to the addition of nutrients to distribution systems and what impact biofilm development may have on disinfectant decay is still poorly understood. This study used annular reactors to determine the nutrients limiting for biofilm development in drinking water from two different Sydney sources and the subsequent effects of biofilm development on disinfectant decay. It was found that biofilm development in Sydney water was limited by organic carbon and that biofilm development promoted chloramine decay. Moreover, biofilm development occurred in the presence of chloramine. The ability of biofilms to respond to increases in disinfectant concentrations was dependent on the biomass of the biofilms. In a comparative study using chlorinated drinking water containing very low levels of organic carbon, biofilm development was not detected. Removal of organic carbon resulted in greater persistence of chlorine, which led to greater biofilm control. It was also shown that biofilms could contribute cells to the aqueous phase. The results of the study indicate that treatment and system management strategies should incorporate organic carbon removal to limit biofilm development through a combination of retarding bacterial growth and enhancing disinfectant persistence.

Australia↗

PubMed2006

Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm.

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-L-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.

Anti-Infective Agents↗

PubMed2005

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy.

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).

Biofilms↗

PubMed2001

Correlation of biofilm expression types of Staphylococcus epidermidis with polysaccharide intercellular adhesin synthesis: evidence for involvement of icaADBC genotype-independent factors.

IcaADBC-encoded proteins mediate synthesis of the intercellular polysaccharide adhesin (PIA), which is essentially involved in Staphylococcus epidermidis biofilm formation. Seventy S. epidermidis isolates were investigated for their ability to form biofilm and synthesize PIA in different growth media including trypticase soy broth obtained from Becton Dickinson (TSBBBL), or Oxoid (TSB(OXOID)), and TSB(OXOID) supplemented with 0.5% N-acetylglucosamine, and for the presence of icaADBC. Dependent on the medium used (TSB(BBL) or TSB(OXOID)), the isolates exhibited a differential expression of PIA and biofilm formation, with 51 (72.85%) and 34 (48.57%) being biofilm positive, respectively. Using these growth media four different expression phenotypes were differentiated: similar quantities of biofilm formation in both TSBBBL and TSB(OXOID) (11 isolates, type A), significantly reduced biofilm expression in TSB(OXOID) compared to TSB(BBL) (23 isolates, type B), biofilm negative in TSB(OXOID) but biofilm producing in TSB(BBL) (17 isolates, type C) and biofilm negative in both media (19 isolates, type D). For all strains a biofilm-positive phenotype in a specific medium was closely linked to expression of PIA in that medium. All but one strain of expression type A-C and 7/19 expression type D strains were icaADBC positive. On the basis of restriction fragment length polymorphisms, the isolates were classified into two main icaADBC genotypes. There was no association between the observed biofilm-expression types and a defined icaADBC genotype. In the biofilm-negative S. epidermidis 5179, isolated from a ventriculo-atrial shunt infection, the insertion of IS257 interrupted the transcription of icaADBC, resulting in a PIA- and biofilm-negative phenotype. In all other icaADBC-positive, biofilm-negative isolates no major alterations of the icaADBC gene locus were identified. Obviously, expression of icaADBC, PIA synthesis and biofilm formation are integrated into a complex regulatory network involving other determinants independent of icaADBC genotype. Inactivation of icaADBC by IS elements is apparently a rare cause of a biofilm-negative phenotype in clinical S. epidermidis isolates.

Bacterial Proteins↗

PubMed2005

Effect of mechanical stress on biofilms challenged by different chemicals.

In this study a methodology was applied in order to ascertain the mechanical stability of biofilms, by using a stainless-steel (SS) rotating device immersed in a biological reactor where biofilms formed by Pseudomonas fluorescens were allowed to grow for 7 days at a Reynolds number of agitation of 2400. The biofilms developed with this system were characterised in terms of amount of total, extracellular and intracellular proteins and polysaccharides, amount of mass, metabolic activity and mechanical stability, showing that the biofilms were active, had a high content of extracellular constituents and an inherent mechanical stability. In order to assess the role of chemical agents on the mechanical stability, the biofilms were exposed to chemical agents followed by mechanical treatments by submission to increase Reynolds number of agitation. Seven different chemical agents were tested (two non-oxidising biocides, three surfactants and two oxidising biocides) and their effects on the biofilm mechanical stability were evaluated. The increase in the Reynolds number increased the biofilm removal, but total biofilm removal was not found for all the conditions tested. For the experiment without chemical addition (only mechanical treatment), the biofilm remaining on the surface was about 76%. The chemical treatment followed by the subsequent mechanical treatment did not remove all the biofilms from the surface. The biofilm remaining on the SS cylinder ranged from 3% to 62%, depending on the chemical treatment, showing that the chemical treatment is far from being a cause that induces massive biofilm detachment and even the synergistic chemical and mechanical treatments did not promote biofilm removal. Some chemical agents promoted an increase in the biofilm mechanical stability such as glutaraldehyde (GTA), benzalkonium chloride (BC), except for the lower concentration tested, and sodium dodecyl sulphate (SDS), except for the higher concentration tested. Treatments that promoted biofilm removal, to an extent similar to the control experiment (without chemical treatment), were BC, for the lower and the higher concentration of SDS. Cetyltrimethyl ammonium bromide (CTAB), ortho-phthalaldehyde (OPA), sodium hydroxide (NaOH) and sodium hypochlorite (SHC) promoted the weakening of the biofilm mechanical stability.

Bacterial Proteins↗

PubMed2006

Candida albicans biofilms produce antifungal-tolerant persister cells.

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.

Antifungal Agents↗

PubMed2004

Penetration of Candida biofilms by antifungal agents.

A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing Candida. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. The rates of diffusion of either drug through biofilms of three strains of Candida albicans were similar. However, the rates of drug diffusion through biofilms of C. glabrata or C. krusei were faster than those through biofilms of C. parapsilosis or C. tropicalis. In all cases, after 3 to 6 h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms. The abilities of flucytosine, fluconazole, amphotericin B, and voriconazole to penetrate mixed-species biofilms containing C. albicans and Staphylococcus epidermidis (a slime-producing wild-type strain, RP62A, and a slime-negative mutant, M7) were also investigated. All four antifungal agents diffused very slowly through these mixed-species biofilms. In most cases, diffusion was slower with biofilms containing S. epidermidis RP62A, but amphotericin B penetrated biofilms containing the M7 mutant more slowly. However, the drug concentrations reaching the distal edges of the biofilms always substantially exceeded the MIC. Thus, although the presence of bacteria and bacterial matrix material undoubtedly retarded the diffusion of the antifungal agents, poor penetration does not account for the drug resistance of Candida biofilm cells, even in these mixed-species biofilms.

Amphotericin B↗

PubMed2004

Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time.

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10(5) cells per cm(2), while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.

Bacteriological Techniques↗

PubMed2002

Biofilms: survival mechanisms of clinically relevant microorganisms.

Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.

Bacteria↗

PubMed2006

Alcohol dehydrogenase restricts the ability of the pathogen Candida albicans to form a biofilm on catheter surfaces through an ethanol-based mechanism.

Candida biofilms formed on indwelling medical devices are increasingly associated with severe infections. In this study, we used proteomics and Western and Northern blotting analyses to demonstrate that alcohol dehydrogenase (ADH) is downregulated in Candida biofilms. Disruption of ADH1 significantly (P = 0.0046) enhanced the ability of Candida albicans to form biofilm. Confocal scanning laser microscopy showed that the adh1 mutant formed thicker biofilm than the parent strain (210 microm and 140 microm, respectively). These observations were extended to an engineered human oral mucosa and an in vivo rat model of catheter-associated biofilm. Inhibition of Candida ADH enzyme using disulfiram and 4-methylpyrazole resulted in thicker biofilm (P < 0.05). Moreover, biofilms formed by the adh1 mutant strain produced significantly smaller amounts of ethanol, but larger amounts of acetaldehyde, than biofilms formed by the parent and revertant strains (P < 0.0001), demonstrating that the effect of Adh1p on biofilm formation is mediated by its enzymatic activity. Furthermore, we found that 10% ethanol significantly inhibited biofilm formation in vitro, with complete inhibition of biofilm formation at ethanol concentrations of >/=20%. Similarly, using a clinically relevant rabbit model of catheter-associated biofilm, we found that ethanol treatment inhibited biofilm formation by C. albicans in vivo (P < 0.05) but not by Staphylococcus spp. (P > 0.05), indicating that ethanol specifically inhibits Candida biofilm formation. Taken together, our studies revealed that Adh1p contributes to the ability of C. albicans to form biofilms in vitro and in vivo and that the protein restricts biofilm formation through an ethanol-dependent mechanism. These results are clinically relevant and may suggest novel antibiofilm treatment strategies.

Alcohol Dehydrogenase↗