Genetics and genomics of Candida albicans biofilm formation

Biofilm formation by the opportunistic fungal pathogen Candida albicans is a complex process with significant consequences for human health: it contributes to implanted medical device-associated infections. Recent advances in gene expression profiling and genetic analysis have begun to clarify the mechanisms that govern C. albicans biofilm development and acquisition of unique biofilm phenotypes. Such studies have identified candidate adhesin genes, and have revealed that biofilm drug resistance is multifactorial. Newly defined cell-cell communication pathways also have profound effects on biofilm formation. Future challenges include the elucidation of the structure and function of the extracellular exopolymeric substance that surrounds biofilm cells, and the extension of in vitro biofilm observations to newly developed in vivo biofilm models.     

Identification of Thiothrix unzii in two distinct ecosystems

AIMS: Molecular procedures were used to identify Thiothrix spp. in biofilms from sulphide-rich waters in two distinct ecosystems. METHODS AND RESULTS: Biofilm samples were obtained from two groundwater-fed systems in central and northern Florida, including an artesian spring and municipal water tank. The 16S rDNA in each sample was directly amplified by polymerase chain reaction. CONCLUSIONS: Clonal libraries of biofilm 16S rDNA from each site contained rDNA sequences that were 99-99.5% similar to Thiothrix unzii. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of T. unzii in a natural system. Biofilm formation by Thiothrix spp. can cause fouling in groundwater processing equipment, including municipal water-processing facilities, agricultural irrigation systems and spring water bottling plant filters. Biofouling can have severe economic and human health impacts as it will influence flow rates and related water treatments. Characterization of specific fouling bacteria and their molecular ecology is essential for their regulation.     

调节生物被膜化合物的研究进展

细菌的生物被膜是其在自然界中一种常见的生存状态。生物被膜的形成是细菌耐药性产生的主要机制之一,也是许多感染性疾病难以控制的重要原因。由于生物被膜在传染性疾病中的突出地位和细菌多重耐药性的蔓延,目前急需研制开发出能够调节生物被膜形成的新型抗菌药物。文中对调节生物被膜形成和发育的小分子抑制剂进行了详细的综述。     

Current and future trends for biofilm reactors for fermentation processes

Biofilms in the environment can both cause detrimental and beneficial effects. However, their use in bioreactors provides many advantages including lesser tendencies to develop membrane fouling and lower required capital costs, their higher biomass density and operation stability, contribution to resistance of microorganisms, etc. Biofilm formation occurs naturally by the attachment of microbial cells to the support without use of any chemicals agent in biofilm reactors. Biofilm reactors have been studied and commercially used for waste water treatment and bench and pilot-scale production of value-added products in the past decades. It is important to understand the fundamentals of biofilm formation, physical and chemical properties of a biofilm matrix to run the biofilm reactor at optimum conditions. This review includes the principles of biofilm formation; properties of a biofilm matrix and their roles in the biofilm formation; factors that improve the biofilm formation, such as support materials; advantages and disadvantages of biofilm reactors; and industrial applications of biofilm reactors.     

Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis

Bacterial biofilm formation on contact lenses (CLs), and CL storage cases may be a risk factor for CL-associated corneal infection and may explain the persistence of organisms in CL storage cases. This study evaluated biofilm formation on, and microbial contamination of, CLs and CL storage cases from patients with microbial keratitis. Contact lenses and CL storage cases from 20 wearers with microbial keratitis were sampled microbiologically and visualized using scanning electron microscopy (SEM). Culture results from the cornea were also noted. Bacterial biofilm was present more frequently ( P < 0·05) on CL storage case surfaces (17/20) compared with CL surfaces (11/20) and biofilm density was significantly greater on case surfaces ( P < 0·05). There was no association between poor compliance and microbial contamination of the CL storage case, nor between poor compliance and biofilm formation or density on the CL or CL storage case. Biofilm formation occurred equally frequently with hydrogen peroxide and chlorine release care systems. Microbial keratitis in CL wearers is frequently associated with bacterial biofilm in the CL storage case. Despite the use of current CL disinfection systems, the CL storage case is a favourable environment for proliferation of certain organisms. Biofilm on CLs may prolong the retention time of organisms at the ocular surface and increase their potential pathogenicity.     

Eap1p, an adhesin that mediates Candida albicans biofilm formation in vitro and in vivo

Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.     

Biofilms and biocides

Biofilm is a ubiquitous material generated by microorganisms proliferating on solid surfaces in water exposed to appropriate aqueous nutrients. It is suggested that model biofilm fermenters will be useful in investigating and in the end controlling biofilm formation. The Cardiff constant depth film fermenter is described. The growth of cutting fluid organisms on a model amine: carboxylate medium in this system is discussed. A simple film model based on a dominant metal-working fluid organism Pseudomonas aeruginosa was investigated and preliminary results using formaldehyde as a biocide are presented.     

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.     

Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.     

Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.     

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.     

Modelling biofilm‐induced formation damage and biocide treatment in subsurface geosystems

Biofilm growth in subsurface porous media, and its treatment with biocides (antimicrobial agents), involves a complex interaction of biogeochemical processes which provide non‐trivial mathematical modelling challenges. Although there are literature reports of mathematical models to evaluate biofilm tolerance to biocides, none of these models have investigated biocide treatment of biofilms growing in interconnected porous media with flow. In this paper, we present a numerical investigation using a pore network model of biofilm growth, formation damage and biocide treatment. The model includes three phases (aqueous, adsorbed biofilm, and solid matrix), a single growth‐limiting nutrient and a single biocide dissolved in the water. Biofilm is assumed to contain a single species of microbe, in which each cell can be a viable persister, a viable non‐persister, or non‐viable (dead). Persisters describe small subpopulation of cells which are tolerant to biocide treatment. Biofilm tolerance to biocide treatment is regulated by persister cells and includes ‘innate’ and ‘biocide‐induced’ factors. Simulations demonstrate that biofilm tolerance to biocides can increase with biofilm maturity, and that biocide treatment alone does not reverse biofilm‐induced formation damage. Also, a successful application of biological permeability conformance treatment involving geologic layers with flow communication is more complicated than simply engineering the attachment of biofilm‐forming cells at desired sites.     

Biofilm formation behaviour of marine filamentous cyanobacterial strains in controlled hydrodynamic conditions

Marine biofouling has severe economic impacts and cyanobacteria play a significant role as early surface colonizers. Despite this fact, cyanobacterial biofilm formation studies in controlled hydrodynamic conditions are scarce. In this work, computational fluid dynamics was used to determine the shear rate field on coupons that were placed inside the wells of agitated 12-well microtiter plates. Biofilm formation by three different cyanobacterial strains was assessed at two different shear rates (4 and 40 s⁻¹) which can be found in natural ecosystems and using different surfaces (glass and perspex). Biofilm formation was higher under low shear conditions, and differences obtained between surfaces were not always statistically significant. The hydrodynamic effect was more noticeable during the biofilm maturation phase rather than during initial cell adhesion and optical coherence tomography showed that different shear rates can affect biofilm architecture. This study is particularly relevant given the cosmopolitan distribution of these cyanobacterial strains and the biofouling potential of these organisms.     

Association of the hypha‐related protein Pra1 and zinc transporter Zrt1 with biofilm formation by the pathogenic yeast Candida albicans

Bloodstream infection by the pathogenic fungus Candida albicans is a major health problem. Candidemia is often associated with medical devices, which can act as substrates for biofilm development. Biofilm‐related infections are relatively difficult to treat because of their resistance to antimicrobial agents. It is therefore important to explore the mechanisms of biofilm formation. Dimorphism is a major contributor to biofilm formation in C. albicans. To determine whether the hypha‐related proteins Pra1 (pH‐regulated antigen) and Zrt1 (zinc transporter) are responsible for biofilm formation, the ability of pra1 and zrt1 deletion mutants to form biofilms was investigated. Biofilm formation by both deletion mutants was less than that of the wild‐type strain. Because Pra1 and Zrt1 are also related to the zinc homeostasis system, the effects of adding zinc on biofilm formation were also examined. Biofilm formation was increased in the presence of zinc. These data suggest that Pra1 and Zrt1 regulate biofilm formation through zinc homeostasis.

     

Ecological determinants of biofilm formation

Biofilm formation is discussed in terms of the ecological processes involved in their formation. It is emphasised that any cell responds to its environment i.e. to the composition of a liquid layer a few microns thick adjacent to the cell surface. The concept of multidimensional habitat domains defining the growth limits to every species is introduced, and put into the context of competition between different organisms. Activity domains of organisms are discussed and a definition for the niche of an organism introduced. The development of a biofilm on a clean surface is described. The constant depth film fermenter is introduced as an appropriate tool for investigating spatial and temporal sequence in biofilm formation. Finally, nutrient concentration is considered and the effects of diffusion limitation on film structure explored with the aid of a cellular automaton model.     

Bacterial biofilms: from the natural environment to infectious diseases

Biofilms--matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces--represent a significant and incompletely understood mode of growth for bacteria. Biofilm formation appears early in the fossil record (approximately 3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches. The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.     

Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection

Biofilm formation and function was studied in mixed culture using 20 bacterial strains isolated from a karst aquifer. When co-cultured in a glucose-limited chemostat, Vogesella indigofera and Pseudomonas putida were the dominant planktonic and biofilm organisms respectively. Biofilm formation and resistance to the iodine disinfectant betadine were then studied with monoculture and binary cultures of V. indigofera and P. putida and a 20-strain community. Biofilm population size [measured as colony-forming units (CFU) cm⁻²] increased with increasing species diversity. Significantly larger populations formed at dilution rates (DRs) of 0.0083 h⁻¹ than at 0.033 h⁻¹. P. putida populations were higher and V. indigofera lower in binary than in monoculture biofilms, suggesting that P. putida outcompeted V. indigofera . In binary biofilms, V. indigofera , a betadine-resistant organism, enhanced the survival of P. putida , a betadine-susceptible organism. In the 20-strain biofilms, this protective effect was not observed because of low concentrations of V. indigofera (< 1% of the total population), suggesting that resistant organisms contribute to overall biofilm disinfectant resistance. Growth at 0.033 h⁻¹ enhanced survival of V. indigofera biofilms against betadine. Although DR did influence survival of the other communities, its effects were neither consistent nor significant. All told, biofilm formation and betadine resistance are complex phenomena, influenced by community composition, growth rate and betadine concentration.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Efficacy of silver-releasing rubber for the prevention of Pseudomonas aeruginosa biofilm formation in water

The aim of the present study was to evaluate the efficacy of Elastoguard silver-releasing rubber in preventing Pseudomonas aeruginosa biofilm formation in water. Biofilm formation by P. aeruginosa under various conditions in an in vitro model system was compared for silver-releasing and conventional rubber. Under most conditions tested, the numbers of sessile cells attached to silver-releasing rubber were considerably lower with reference to conventional rubber, although the effect diminished with increasing volumes. The release of silver also resulted in a decrease in planktonic cells. By exposing both materials simultaneously to conditions for biofilm growth, it became obvious that the antibiofilm effect was due to a reduction in the number of planktonic cells, rather than to contact-dependent killing of sessile cells. The data demonstrate that the use of silver-releasing rubber reduces P. aeruginosa biofilm in water and reduces the number of planktonic cells present in the surrounding solution.     

Bacterial species in biofilm cultivated from the end of the Seoul water distribution system

AIMS: To investigate changes in the bacterial population and the safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), selective media and bacterial community analyses were applied to a semi-pilot galvanized iron pipe (GIP) model. METHODS AND RESULTS: No total coliforms or faecal streptococci were detected on m-Endo or m-Enterococcus agar. No Salmonella spp. and Shigella spp. were detected on bismuth sulphite agar or Hektoen enteric agar, respectively. The latter two media detected coliforms, where m-Endo was negative. Biofilm formation started within 1 week (ca 104 CFU cm(-2)) and exceeded 105 CFU cm(-2) within 6 weeks. Although the fatty acid methyl ester analysis revealed dynamic changes in bacterial composition, Micrococcus, Bacillus, and Pseudomonas spp. were persistent members of the biofilm community. Micrococcus spp. was detected most frequently and in high numbers. CONCLUSIONS: Coliforms and Enterococcus species can be recovered from biofilms in water distribution systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates the role of biofilms in the chronic deterioration of the water-distribution system in Seoul (Korea).