Showing papers on "Biofilm published in 2006"

Effect of material characteristics and/or surface topography on biofilm development

Abstract: Background: From an ecological viewpoint, the oral cavity, in fact the oro-pharynx, is an ‘open growth system’. It undergoes an uninterrupted introduction and removal of both microorganisms and nutrients. In order to survive within the oro-pharyngeal area, bacteria need to adhere either to the soft or hard tissues in order to resist shear forces. The fast turn-over of the oral lining epithelia (shedding 3 ×/day) is an efficient defence mechanism as it prevents the accumulation of large masses of microorganisms. Teeth, dentures, or endosseous implants, however, providing non-shedding surfaces, allow the formation of thick biofilms. In general, the established biofilm maintains an equilibrium with the host. An uncontrolled accumulation and/or metabolism of bacteria on the hard surfaces forms, however, the primary cause of dental caries, gingivitis, periodontitis, peri-implantitis, and stomatitis. Objectives: This systematic review aimed to evaluate critically the impact of surface characteristics (free energy, roughness, chemistry) on the de novo biofilm formation, especially in the supragingival and to a lesser extent in the subgingival areas. Methods: An electronic Medline search (from 1966 until July 2005) was conducted applying the following search items: ‘biofilm formation and dental/oral implants/surface characteristics’, ‘surface characteristics and implants’, ‘biofilm formation and oral’, ‘plaque/biofilm and roughness’, ‘plaque/biofilm and surface free energy’, and ‘plaque formation and implants’. Only clinical studies within the oro-pharyngeal area were included. Results: From a series of split-mouth studies, it could be concluded that both an increase in surface roughness above the Ra threshold of 0.2 μm and/or of the surface-free energy facilitates biofilm formation on restorative materials. When both surface characteristics interact with each other, surface roughness was found to be predominant. The biofilm formation is also influenced by the type (chemical composition) of biomaterial or the type of coating. Direct comparisons in biofilm formation on different transmucosal implant surfaces are scars. Conclusions: Extrapolation of data from studies on different restorative materials seems to indicate that transmucosal implant surfaces with a higher surface roughness/surface free energy facilitate biofilm formation.

A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

Abstract: Pseudomonas aeruginosa produces extracellular DNA which functions as a cell-to-cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular ?-galactosidase released from lacZ-containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI, pqsA, pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber-grown wild-type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria and the cap-forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm

Biofilm and nanowire production leads to increased current in geobacter sulfurreducens fuel cells

Abstract: Geobacter sulfurreducens developed highly structured, multilayer biofilms on the anode surface of a microbial fuel cell converting acetate to electricity. Cells at a distance from the anode remained viable, and there was no decrease in the efficiency of current production as the thickness of the biofilm increased. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili. These pili may represent an electronic network permeating the biofilm that can promote long-range electrical transfer in an energy-efficient manner, increasing electricity production more than 10-fold.

Dental plaque as a biofilm and a microbial community - implications for health and disease.

Abstract: Dental plaque is a structurally- and functionally-organized biofilm. Plaque forms in an ordered way and has a diverse microbial composition that, in health, remains relatively stable over time (microbial homeostasis). The predominant species from diseased sites are different from those found in healthy sites, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift toward community dominance by acidogenic and acid-tolerating species such as mutans streptococci and lactobacilli, although other species with relevant traits may be involved. Strategies to control caries could include inhibition of biofilm development (e.g. prevention of attachment of cariogenic bacteria, manipulation of cell signaling mechanisms, delivery of effective antimicrobials, etc.), or enhancement of the host defenses. Additionally, these more conventional approaches could be augmented by interference with the factors that enable the cariogenic bacteria to escape from the normal homeostatic mechanisms that restrict their growth in plaque and out compete the organisms associated with health. Evidence suggests that regular conditions of low pH in plaque select for mutans streptococci and lactobacilli. Therefore, the suppression of sugar catabolism and acid production by the use of metabolic inhibitors and non-fermentable artificial sweeteners in snacks, or the stimulation of saliva flow, could assist in the maintenance of homeostasis in plaque. Arguments will be presented that an appreciation of ecological principles will enable a more holistic approach to be taken in caries control.

Involvement of Nitric Oxide in Biofilm Dispersal of Pseudomonas aeruginosa

Abstract: Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, ΔnirS) did not disperse, whereas a NO reductase mutant (ΔnorCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

Antibiotics as intermicrobial signaling agents instead of weapons

Abstract: It has been widely assumed that the ecological function of antibiotics in nature is fighting against competitors. This made them a good example of the Darwinian struggle-for-life in the microbial world. Based on this idea, it also has been believed that antibiotics, even at subinhibitory concentrations, reduce virulence of bacterial pathogens. Herein, using a combination of genomic and functional assays, we demonstrate that specific antibiotics (namely tobramycin, tetracycline, and norfloxacin) at subinhibitory concentrations trigger expression of determinants influencing the virulence of the major opportunistic bacterial pathogen Pseudomonas aeruginosa. All three antibiotics induce biofilm formation; tobramycin increases bacterial motility, and tetracycline triggers expression of P. aeruginosa type III secretion system and consequently bacterial cytotoxicity. Besides their relevance in the infection process, those determinants are relevant for the ecological behavior of this bacterial species in natural, nonclinical environments, either by favoring colonization of surfaces (biofilm, motility) or for fighting against eukaryotic predators (cytotoxicity). Our results support the notion that antibiotics are not only bacterial weapons for fighting competitors but also signaling molecules that may regulate the homeostasis of microbial communities. At low concentrations, they can even be beneficial for the behavior of susceptible bacteria in natural environments. This is a complete change on our vision on the ecological function of antibiotics with clear implications both for the treatment of infectious diseases and for the understanding of the microbial relationships in the biosphere.

A major protein component of the Bacillus subtilis biofilm matrix

Abstract: Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

Abstract: Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.

Flocculation, adhesion and biofilm formation in yeasts

Abstract: Yeast cells possess a remarkable capacity to adhere to abiotic surfaces, cells and tissues. These adhesion properties are of medical and industrial relevance. Pathogenic yeasts such as Candida albicans and Candida glabrata adhere to medical devices and form drug-resistant biofilms. In contrast, cell-cell adhesion (flocculation) is a desirable property of industrial Saccharomyces cerevisiae strains that allows the easy separation of cells from the fermentation product. Adhesion is conferred by a class of special cell wall proteins, called adhesins. Cells carry several different adhesins, each allowing adhesion to specific substrates. Several signalling cascades including the Ras/cAMP/PKA and MAP kinase (MAPK)-dependent filamentous growth pathways tightly control synthesis of the different adhesins. Together, these pathways trigger adhesion in response to stress, nutrient limitation or small molecules produced by the host, such as auxin in plants or NAD in mammals. In addition, adhesins are subject to subtelomeric epigenetic switching, resulting in stochastic expression patterns. Internal tandem repeats within adhesin genes trigger recombination events and the formation of novel adhesins, thereby offering fungi an endless reservoir of adhesion properties. These aspects of fungal adhesion exemplify the impressive phenotypic plasticity of yeasts, allowing them to adapt quickly to stressful environments and exploit new opportunities.

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

Abstract: The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-μm) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 μM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).

The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional

Abstract: Summary The role of quorum sensing in Pseudomonas aeruginosa biofilm formation is unclear. Some researchers have shown that quorum sensing is important for biofilm development, while others have indicated it has little or no role. In this study, the contribution of quorum sensing to biofilm development was found to depend upon the nutritional environment. Depending upon the carbon source, quorum-sensing mutant strains (lasIrhlI and lasRrhlR) either exhibited a pronounced defect early in biofilm formation or formed biofilms identical to the wild-type strain. Quorum sensing was then shown to exert its nutritionally conditional control of biofilm development through regulation of swarming motility. Examination of pilA and fliM mutant strains further supported the role of swarming motility in biofilm formation. These data led to a model proposing that the prevailing nutritional conditions dictate the contributions of quorum sensing and swarming motility at a key juncture early in biofilm development.

The Role of Sucrose in Cariogenic Dental Biofilm Formation—New Insight

Abstract: Dental caries is a biofilm-dependent oral disease, and fermentable dietary carbohydrates are the key environmental factors involved in its initiation and development. However, among the carbohydrates, sucrose is considered the most cariogenic, because, in addition to being fermented by oral bacteria, it is a substrate for the synthesis of extracellular (EPS) and intracellular (IPS) polysaccharides. Therefore, while the low pH environment triggers the shift of the resident plaque microflora to a more cariogenic one, EPS promote changes in the composition of the biofilms’ matrix. Furthermore, it has recently been shown that the biofilm formed in the presence of sucrose presents low concentrations of Ca, Pi, and F, which are critical ions involved in de- and remineralization of enamel and dentin in the oral environment. Thus, the aim of this review is to explore the broad role of sucrose in the cariogenicity of biofilms, and to present a new insight into its influence on the pathogenesis of dental caries.

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

Abstract: 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 (β-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 β-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

Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo.

Abstract: The fungal pathogen Candida albicans is frequently associated with catheter-based infections because of its ability to form resilient biofilms. Prior studies have shown that the transcription factor Bcr1 governs biofilm formation in an in vitro catheter model. However, the mechanistic role of the Bcr1 pathway and its relationship to biofilm formation in vivo are unknown. Our studies of biofilm formation in vitro indicate that the surface protein Als3, a known adhesin, is a key target under Bcr1 control. We show that an als3/als3 mutant is biofilm-defective in vitro, and that ALS3 overexpression rescues the biofilm defect of the bcr1/bcr1 mutant. We extend these findings with an in vivo venous catheter model. The bcr1/bcr1 mutant is unable to populate the catheter surface, though its virulence suggests that it has no growth defect in vivo. ALS3 overexpression rescues the bcr1/bcr1 biofilm defect in vivo, thus arguing that Als3 is a pivotal Bcr1 target in this setting. Surprisingly, the als3/als3 mutant forms a biofilm in vivo, and we suggest that additional Bcr1 targets compensate for the Als3 defect in vivo. Indeed, overexpression of Bcr1 targets ALS1, ECE1, and HWP1 partially restores biofilm formation in a bcr1/bcr1 mutant background in vitro, though these genes are not required for biofilm formation in vitro. Our findings demonstrate that the Bcr1 pathway functions in vivo to promote biofilm formation, and that Als3-mediated adherence is a fundamental property under Bcr1 control. Known adhesins Als1 and Hwp1 also contribute to biofilm formation, as does the novel protein Ece1.

Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells

Abstract: 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

Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm.

Abstract: Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.

Messing with Bacterial Quorum Sensing

Abstract: Quorum sensing is widely recognized as an efficient mechanism to regulate expression of specific genes responsible for communal behavior in bacteria. Several bacterial phenotypes essential for the successful establishment of symbiotic, pathogenic, or commensal relationships with eukaryotic hosts, including motility, exopolysaccharide production, biofilm formation, and toxin production, are often regulated by quorum sensing. Interestingly, eukaryotes produce quorum-sensing-interfering (QSI) compounds that have a positive or negative influence on the bacterial signaling network. This eukaryotic interference could result in further fine-tuning of bacterial quorum sensing. Furthermore, recent work involving the synthesis of structural homologs to the various quorum-sensing signal molecules has resulted in the development of additional QSI compounds that could be used to control pathogenic bacteria. The creation of transgenic plants that express bacterial quorum-sensing genes is yet another strategy to interfere with bacterial behavior. Further investigation on the manipulation of quorum-sensing systems could provide us with powerful tools against harmful bacteria.

Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel

Abstract: The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.

Endocarditis and biofilm-associated pili of Enterococcus faecalis

Abstract: Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E. faecalis cell wall-anchored proteins; of these, we have now characterized an in vivo-expressed locus of 3 genes and an associated sortase gene (encoding sortase C; SrtC). Here, using mutation analyses and complementation, we demonstrated that both the ebp (encoding endocarditis and biofilm-associated pili) operon and srtC are important for biofilm production of E. faecalis strain OG1RF. In addition, immunogold electron microscopy using antisera against EbpA-EbpC proteins as well as patient serum demonstrated that E. faecalis produces pleomorphic surface pili. Assembly of pili and their cell wall attachment appeared to occur via a mechanism of cross-linking of the Ebp proteins by the designated SrtC. Importantly, a nonpiliated, allelic replacement mutant was significantly attenuated in an endocarditis model. These biologically important surface pili, which are antigenic in humans during endocarditis and encoded by a ubiquitous E. faecalis operon, may be a useful immunotarget for studies aimed at prevention and/or treatment of this pathogen.

Analysis of Pseudomonas aeruginosa Conditional Psl Variants Reveals Roles for the Psl Polysaccharide in Adhesion and Maintaining Biofilm Structure Postattachment

Abstract: The ability to form biofilms in the airways of people suffering from cystic fibrosis is a critical element of Pseudomonas aeruginosa pathogenesis. The 15-gene psl operon encodes a putative polysaccharide that plays an important role in biofilm initiation in nonmucoid P. aeruginosa strains. Biofilm initiation by a P. aeruginosa PAO1 strain with disruption of pslA and pslB (ΔpslAB) was severely compromised, indicating that psl has a role in cell-surface interactions. In this study, we investigated the adherence properties of this ΔpslAB mutant using biotic surfaces (epithelial cells and mucin-coated surfaces) and abiotic surfaces. Our results showed that psl is required for attachment to a variety of surfaces, independent of the carbon source. To study the potential roles of Psl apart from attachment, we generated a psl-inducible P. aeruginosa strain (Δpsl/pBAD-psl) by replacing the psl promoter region with araC-pBAD, so that expression of psl could be controlled by addition of arabinose. Analysis of biofilms formed by the Δpsl/pBAD-psl strain indicated that expression of the psl operon is required to maintain the biofilm structure at steps postattachment. Overproduction of the Psl polysaccharide led to enhanced cell-surface and intercellular adhesion of P. aeruginosa. This translated into significant changes in the architecture of the biofilm. We propose that Psl has an important role in P. aeruginosa adhesion, which is critical for initiation and maintenance of the biofilm structure.

Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth

Abstract: 4,5-Dihydroxy-2,3-pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). AI-2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by genetic or chemical (synthetic AI-2 in the form of DPD) complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100-fold lower than the detection limit of the currently accepted AI-2 assay. Thus, AI-2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity.

Function of Candida albicans Adhesin Hwp1 in Biofilm Formation

Abstract: Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1 mutant is defective in retention of nonadherent bcr1/bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1/bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.

Biofilm Formation by Streptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial Accretion

Abstract: Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 μm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.

Abstract: The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.