Showing papers on "Biofilm matrix published in 2005"

Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

Abstract: We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this "Fe-bead" system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.

BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enterica serovar Enteritidis

Abstract: In environmental settings, biofilms represent the common way of life of microorganisms. Salmonella enterica serovar Enteritidis, the most frequent cause of gastroenteritis in developed countries, produces a biofilm whose matrix is mainly composed of curli fimbriae and cellulose. In contrast to other bacterial biofilms, no proteinaceous compound has been reported to participate in the formation of this matrix. Here, we report the discovery of BapA, a large cell-surface protein required for biofilm formation by S. Enteritidis. Deletion of bapA caused the loss of the capacity to form a biofilm whereas the overexpression of a chromosomal copy of bapA increased the biofilm biomass formation. We provide evidence that overproduction of curli fimbriae and not cellulose can compensate for the biofilm deficiency of a bapA mutant strain. BapA is secreted through a type I protein secretion system (BapBCD) situated downstream of the bapA gene and was found to be loosely associated with the cell surface. Experiments with mixed bacterial populations positive or negative for BapA showed that BapA minus cells are not recruited into the biofilm matrix. The expression of bapA is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of csgD. Studies on the contribution of BapA to S. Enteritidis pathogenesis revealed that orally inoculated animals with a bapA-deficient strain survived longer than those inoculated with the wild-type strain. Also, a bapA mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild-type strain. Taken together, these data demonstrate that BapA contributes both to biofilm formation and invasion through the regular Salmonella infection route.

Use of Confocal Microscopy To Analyze the Rate of Vancomycin Penetration through Staphylococcus aureus Biofilms

Abstract: When bacteria assume the biofilm mode of growth, they can tolerate levels of antimicrobial agents 10 to 1,000 times higher than the MICs of genetically equivalent planktonic bacteria. The properties of biofilms that give rise to antibiotic resistance are only partially understood. Inhibition of antibiotic penetration into the biofilm may play a role, but this has not been proven directly. In this report, penetration of the glycopeptide antibiotic vancomycin into viable Staphylococcus aureus biofilms was analyzed by confocal scanning laser microscopy using a fluorescently labeled derivative of the drug. We found that while vancomycin bound to free-floating bacteria in water within 5 min, it took more than 1 h to bind to cells within the deepest layers of a biofilm. These results indicate that the antibiotic is transported through the depth of the biofilm but that the rate is significantly reduced with respect to its transport through flowing water. This suggests that, whereas planktonic bacteria were rapidly exposed to a full bolus of vancomycin, the bacteria in the deeper layers of the biofilm were exposed to a gradually increasing dose of the drug due to its reduced rate of penetration. This gradual exposure may allow the biofilm bacteria to undergo stress-induced metabolic or transcriptional changes that increase resistance to the antibiotic. We also investigated the role of poly-N-acetylglucosamine, an important component of the S. aureus biofilm matrix, and found that its production was not involved in the observed decrease in the rate of vancomycin penetration.

Microbe-surface interactions in biofouling and biocorrosion processes

Abstract: The presence of microorganisms on material surfaces can have a profound effect on materials performance. Surface-associated microbial growth, i.e. a biofilm, is known to instigate biofouling. The presence of biofilms may promote interfacial physico-chemical reactions that are not favored under abiotic conditions. In the case of metallic materials, undesirable changes in material properties due to a biofilm (or a biofouling layer) are referred to as biocorrosion or microbially influenced corrosion (MIC). Biofouling and biocorrosion occur in aquatic and terrestrial habitats varying in nutrient content, temperature, pressure and pH. Interfacial chemistry in such systems reflects a wide variety of physiological activities carried out by diverse microbial populations thriving within biofilms. Biocorrosion can be viewed as a consequence of coupled biological and abiotic electron-transfer reactions, i.e. redox reactions of metals, enabled by microbial ecology. Microbially produced extracellular polymeric substances (EPS), which comprise different macromolecules, mediate initial cell adhesion to the material surface and constitute a biofilm matrix. Despite their unquestionable importance in biofilm development, the extent to which EPS contribute to biocorrosion is not well-understood. This review offers a current perspective on material/microbe interactions pertinent to biocorrosion and biofouling, with EPS as a focal point, while emphasizing the role atomic force spectroscopy and mass spectrometry techniques can play in elucidating such interactions. [Int Microbiol 2005; 8(3):157-168]

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa.

Abstract: In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.

Biofilm-control strategies based on enzymic disruption of the extracellular polymeric substance matrix: a modelling study

Abstract: A kinetic model is proposed to assess the feasibility of strategies for the removal of biofilms by using substances that induce detachment by affecting the cohesiveness of the matrix of extracellular polymeric substances (EPSs). The model uses a two-state description of the EPS (natural EPS and compromised EPS) to provide a unified representation of diverse mechanisms of action of detachment-promoting agents (DPAs), which include enzymes that degrade the EPS and other agents described in the literature. A biofilm-cohesiveness factor describes local increases in detachment rates resultant from losses in cohesive strength. The kinetic model was implemented in an individual-based biofilm-modelling framework, including detachment rates dependent on local cohesiveness. The efficacy of treatments with DPAs was assessed by three-dimensional model simulations. Changes in treatment efficacy were evaluated quantitatively by using a Thiele modulus, which quantifies the relationship between diffusion of the DPA through the biofilm matrix and DPA decay rate, and a Damkohler number relating the rate of EPS reaction with a DPA and the rate of EPS production by the micro-organisms in the biofilm. This study demonstrates the feasibility and limits of implementing biofilm-control strategies based on attacking the EPS.

Staphylococcus aureus Develops an Alternative, ica-Independent Biofilm in the Absence of the arlRS Two-Component System

Abstract: The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation.

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

Abstract: An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the x,y and x,z planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.

Role of Silver Ions in Destabilization of Intermolecular Adhesion Forces Measured by Atomic Force Microscopy in Staphylococcus epidermidis Biofilms

Abstract: In this paper, we report on the potential use of atomic force microscopy (AFM) as a tool to measure the intermolecular forces in biofilm structures and to study the effect of silver ions on sessile Staphylococcus epidermidis cell viability and stability. We propose a strategy of destabilizing the biofilm matrix by reducing the intermolecular forces within the extracellular polymeric substances (EPSs) using a low concentration (50 ppb) of silver ions. Our AFM studies on the intermolecular forces within the EPSs of S. epidermidis RP62A and S.epidermidis 1457 biofilms suggest that the silver ions can destabilize the biofilm matrix by binding to electron donor groups of the biological molecules. This leads to reductions in the number of binding sites for hydrogen bonds and electrostatic and hydrophobic interactions and, hence, the destabilization of the biofilm structure.

Biofilm Formation by Neisseria gonorrhoeae

Abstract: Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 microM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection.

Bacterial biofilm within diseased pancreatic and biliary tracts.

Abstract: Background: Bacterial community structures in human pancreatic and biliary tracts were evaluated. Methods: Gall bladder stones from 153 patients, 20 gall bladder walls, six common duct stones, 52 biliary stents, 21 duodenal biopsies, nine pancreatic duct biopsies, and five bile ducts were investigated using fluorescence in situ hybridisation (FISH) with ribosomal RNA targeted Cy3/Cy5 (carbocyanine) labelled oligonucleotide probes. Result: Duodenal, gall bladder, and bile duct walls were free of bacteria. A dense multispecies bacterial biofilm was present within the pancreatic duct of patients with calcific pancreatitis and within biliary stents, irrespective of diagnosis. The concentration, density, and amenability of the biofilm to FISH and DNA staining declined progressively with the grade of stent occlusion. The lowest detectable bacterial concentrations were found by FISH in completely occluded stents and brown/mixed gall stones. Bacteria were not detectable with FISH in cholesterol gall stones. Conclusions: A wide range of different branches and groups of bacteria participate in the development of biofilms on the surfaces of foreign bodies, such as biliary stents, mixed gall stones, or calcific pancreatic ducts, but not on the surface of pure cholesterol gall stones. Occlusion of stents leads to progressive extinction of the biofilm and mummification of its components. Deposition of cholesterol or other substances within the biofilm matrix may be a novel mechanism of host defence against bacteria present in these biofilms.

Effects of Growth in the Presence of Subinhibitory Concentrations of Dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus Biofilms

Abstract: Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions ofbiofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.

Capsular Polysaccharide Surrounds Smooth and Rugose Types of Salmonella enterica serovar Typhimurium DT104

Abstract: The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37°C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37°C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25°C than at 37°C. Since the CP was shown to be produced at both 37°C and 25°C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.

Chlorhexidine-induced ultrastructural alterations in oral biofilm.

Abstract: Chlorhexidine, the most used biocide in periodontology, alters the permeability of the bacterial cell membrane. However, the chlorhexidine-induced morphological alterations in the oral biofilm have not been studied. To examine the effects of chlorhexidine on oral biofilm on an electron microscopic level, gingival epithelial cells with attached biofilm were collected from 10 volunteers, subjected to 0.1% chlorhexidine for 1 or 5 min, stained with ruthenium red-tetroxide, and analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM visualized the bacterial glycocalyces and the biofilm matrix on the biofilm surface; however, no chlorhexidine-induced alterations were observed. TEM revealed loss of bacterial membrane integrity and fimbrial disintegration in a few bacteria. In the proximity of these alterations, a restricted matrix disintegration was also observed. However, the chlorhexidine-induced alterations only effected a minor part of the oral biofilm and did not cause its disintegration. These findings suggest the insufficient efficiency of chlorhexidine against oral biofilm.

The curdlan-type exopolysaccharide produced by Cellulomonas flavigena KU forms part of an extracellular glycocalyx involved in cellulose degradation.

Abstract: The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (beta-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.

Ex vivo gingival-biofilm consortia

Abstract: Aims: To develop a protocol for harvesting ex vivo samples of gingival-biofilm consortia and to investigate their basic characteristics. Methods and Results: Gingival epithelial cells with attached biofilm were collected from healthy subjects by taking a smear. The bacterial viability was estimated via the alteration of the membrane permeability and metabolic activity via the double/single-stranded nucleic acid ratio using a confocal laser-scanning microscope. Morphological analysis was performed by scanning and transmission electron microscopy. Additionally, microbiological estimations were made. The electron microscopy revealed fimbriae-mediated adhesion and the formation of a biofilm matrix. Most bacteria were viable and had a high metabolic activity. Conclusions: The presented study offers an easy to follow approach for harvesting samples of gingival-biofilm consortia. The latter differs considerably from the supragingival plaque in viability and zonal distribution. Related to free-living and in vitro-grown biofilms, the gingiva-associated biofilm revealed an atypically high metabolic activity. Significance and Impact of the Study: Biofilm fragments should possess the basic features of the entire gingiva-associated biofilm; which as yet cannot be simulated in vitro. Thus, samples of ex vivo gingival-biofilm consortia can be used to investigate the resistance of oral biofilms against antibiotics and biocides.

Ex vivo gingival-biofilm consortia.

Abstract: Aims: To develop a protocol for harvesting ex vivo samples of gingival-biofilm consortia and to investigate their basic characteristics. Methods and Results: Gingival epithelial cells with attached biofilm were collected from healthy subjects by taking a smear. The bacterial viability was estimated via the alteration of the membrane permeability and metabolic activity via the double/single-stranded nucleic acid ratio using a confocal laser-scanning microscope. Morphological analysis was performed by scanning and transmission electron microscopy. Additionally, microbiological estimations were made. The electron microscopy revealed fimbriae-mediated adhesion and the formation of a biofilm matrix. Most bacteria were viable and had a high metabolic activity. Conclusions: The presented study offers an easy to follow approach for harvesting samples of gingival-biofilm consortia. The latter differs considerably from the supragingival plaque in viability and zonal distribution. Related to free-living and in vitro-grown biofilms, the gingiva-associated biofilm revealed an atypically high metabolic activity. Significance and Impact of the Study: Biofilm fragments should possess the basic features of the entire gingiva-associated biofilm; which as yet cannot be simulated in vitro. Thus, samples of ex vivo gingival-biofilm consortia can be used to investigate the resistance of oral biofilms against antibiotics and biocides.

13 Minimal Biofilm Eradication Concentration (MBEC) Assay: Susceptibility Testing for Biofilms

Abstract: 13.1 Biofilms and Their Properties 257 13.2 The Relationship of Biofilm Formation and Disease 258 13.2.1 Abiotic Surfaces 258 13.2.2 Biotic Surfaces 259 13.3 Biofilm Susceptibility 260 13.3.1 Antimicrobial Penetration of the Biofilm Matrix 260 13.3.2 Growth Rate of Biofilms 261 13.3.3 Phenotypic Plasticity in the Biofilm 261 13.3.4 Persister Cells 262 13.4 Biofilm Susceptibility Assays: The Need for a Minimal Biofilm Eradication Concentration (MBEC) Value 262 References 265

Cytochemical Differences in Bacterial Glycocalyx

Abstract: To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid-metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances-probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

Biopelículas y salud pública

Abstract: We now understand that biofilms are universal, occurring in aquatic and industrial water system 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, biofilms 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. 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 and to colonize a wide variety of medical devices. 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.