Showing papers on "Biofilm published in 1997"

Biofilm Susceptibility to Antimicrobials

Abstract: Microbial biofilms, where organisms are intimately associated with each other and a solid substratum through binding and inclusion within an exopolymer matrix, are widely distributed in nature and disease. In the mouth, multispecies biofilms are associated not only with dental plaque and tooth decay but also with soft tissues of the buccal cavity and with most forms of periodontal disease. Organization of micro-organisms within biofilms confers, on the component species, properties which are not evident with the individual species grown independently or as planktonic populations in liquid media. While many of these properties relate to the establishment of functional, mixed-species consortia within the exopolymeric matrices, others relate to the establishment of physico-chemical gradients, within the biofilm, that modify the metabolism of the component cells. A consequence of biofilm growth that has profound implications for their control in the environment and in medicine is a markedly enhanced resistanc...

Interactions between biofilms and the environment

Abstract: The surfaces of bacteria are highly interactive with their environment. Whether the bacterium is Gram-negative or Gram-positive, most surfaces are charged at neutral pH because of the ionization of the reactive chemical groups which stud them. Since prokaryotes have a high surface area-to-volume ratio, this can have surprising ramifications. For example, many bacteria can concentrate dilute environmental metals on their surfaces and initiate the development of fine-grained minerals. In natural environments, it is not unusual to find such bacteria closely associated with the minerals which they have helped develop. Bacteria can be free-living (planktonic), but in most natural ecosystems they prefer to grow on interfaces as biofilms; supposedly to take advantage of the nutrient concentrative effect of the interface, although there must also be gained some protective value against predators and toxic agents. Using a Pseudomonas aeruginosa model system, we have determined that lipopolysaccharide is important in the initial attachment of this Gram-negative bacterium to interfaces and that this surface moiety subtly changes during biofilm formation. Using this same model system, we have also discovered that there is a natural tendency for Gram-negative bacteria to concentrate and package periplasmic components into membrane vesicles which bleb-off the surface. Since some of these components (e.g., peptidoglycan hydrolases) can degrade other surrounding cells, the vesicles could be predatory; i.e., a natural system by which neighboring bacteria are targeted and lysed, thereby liberating additional nutrients to the microbial community. This obviously would be of benefit to vesicle-producing bacteria living in biofilms containing mixed microbial populations.

A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces.

Abstract: Two distinct pathogenic mechanisms, adhesion to polymer surfaces and subsequent accumulation of sessile bacterial cells, are considered important pathogenic steps in foreign body infections caused by Staphylococcus epidermidis. By using mitomycin mutagenesis, we have recently generated a mutant, strain M7, from S. epidermidis RP62A which is unaffected in adhesion but deficient in accumulation on glass or polystyrene surfaces and lacks a 115-kDa extracellular protein (designated the 140-kDa antigen; F. Schumacher-Perdreau, C. Heilmann, G. Peters, F. Gotz, and G. Pulverer, FEMS Microbiol. Lett. 117:71-78, 1994). To evaluate the role of this protein in accumulation, we harvested extracellular proteins from S. epidermidis RP62A grown on dialysis membranes placed over chemically defined medium, purified the protein by using ion-exchange chromatography, determined its N-terminal amino acid sequence, and raised antiserum in rabbits. The antibody recognized only a single band in a Western immunoblot of the crude extracellular extract. With the microtiter biofilm test, antiserum at a dilution of < or =1:1,000 blocked accumulation of RP62A up to 98% whereas preimmune serum did not. The 140-kDa antigen was found only in extracellular products from bacteria grown under sessile conditions. Of 58 coagulase-negative clinical isolates, 32 strains were 140-kDa antigen positive and produced significantly larger amounts of biofilm than the 26 strains that were 140-kDa antigen negative. The 140-kDa protein appears to be biochemically and functionally unrelated to any previously described factors associated with biofilm formation. Thus, the 140-kDa antigen, referred to as accumulation-associated protein, may be a factor essential in S. epidermidis accumulation and, due to its immunogenicity, may allow the development of novel immunotherapeutic strategies for prevention of foreign body infection.

Evidence of autoinducer activity in naturally occurring biofilms

Abstract: N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria. Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens. In nature, bacteria often grow as surface-adherent biofilm communities. As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology. However, proof of AHL production within biofilms has heretofore been lacking. In this study we have employed a cross-feeding assay, using A. tumefaciens A136 (traI::lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones. AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm. This represents the first report of AHL activity in naturally occurring biofilms.

Enzymatic Removal and Disinfection of Bacterial Biofilms

Abstract: Model biofilms of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and Pseudomonas aeruginosa were made on steel and polypropylene substrata. Plaque-resembling biofilms of Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum were made on saliva-coated hydroxyapatite. The activity of enzymes against bacterial cells in biofilm was measured by fluorescence microscopy and an indirect conductance test in which evolution of carbon dioxide was measured. Glucose oxidase combined with lactoperoxidase was bactericidal against biofilm bacteria but did not remove the biofilm from the substrata. A complex mixture of polysaccharide-hydrolyzing enzymes was able to remove bacterial biofilm from steel and polypropylene substrata but did not have a significant bactericidal activity. Combining oxidoreductases with polysaccharide-hydrolyzing enzymes resulted in bactericidal activity as well as removal of the biofilm.

Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.

Abstract: The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.

Physico-Chemical Interactions in Initial Microbial Adhesion and Relevance for Biofilm Formation:

Abstract: This paper summarizes initial microbial adhesion events in dental plaque formation, including the physico-chemistry of the interaction between micro-organisms and solid substrata, detachment phenomena under the fluctuating shear of the oral cavity, co-adhesion between pairs of microbial strains, and biosurfactant release. A hypothesis is forwarded on how these initial events might influence the final microbial composition and structure of the plaque, although it is simultaneously emphasized that the necessary techniques for verification of the hypothesis have only recently become available, and supporting evidence is still to be collected.

Nutritional Influences on Biofilm Development

Abstract: The amounts and types of nutrients in the environment influence the development and final bacterial and chemical composition of biofilms. In oligotrophic environments, organisms respond to nutrient stress by alterations in their cell morphology and cell surfaces, which enhance adherence. Little is known of the responses to stress by bacteria in the animal oral cavity. The environment in the oral cavity is less extreme, and saliva provides a constant source of nutrients. Catabolic cooperation among oral bacteria allow carbon and nitrogen from salivary glycoproteins to be utilized. Modification of growth environments of oral bacteria can influence their cell surfaces and adhesion. Studies in experimental animals have shown that feeding either glucose or sucrose diets or fasting has little effect on the initial stages of development of oral biofilms. However, diet can influence the proportions of different bacterial species later in biofilm development. Studies of competition among populations in communities of oral bacteria in vitro and in vivo have shown the significance of carbon limitation and excess and changes in environmental pH. Relatively few studies have been made of the role of a nitrogen metabolism in bacterial competition in biofilms. In keeping with biofilms in nature, oral biofilms provide a sequestered habitat, where organisms are protected from removal by saliva and where interactions among cells generate a biofilm environment, distinct from that of saliva. Oral biofilms are an essential component in the etiologies of caries and periodontal disease, and understanding the biology of oral biofilms has aided and will continue to aid in the prevention and treatment of these diseases.

Stress-Induced Production of Biofilm in the Hyperthermophile Archaeoglobus fulgidus.

Abstract: Archaeoglobus fulgidus, an anaerobic marine hyperthermophile, forms a biofilm in response to environmental stresses The biofilm is a heterogeneous, morphologically variable structure containing protein, polysaccharide, and metals Production of the biofilm can be induced by nonphysiological extremes of pH and temperature, by high concentrations of metals, and by addition of antibiotics, xenobiotics, or oxygen Cells within the biofilm show an increased tolerance to otherwise toxic environmental conditions Metals sequestered within the biofilm stimulate growth of A fulgidus cells in metal-depleted medium These data suggest that cells may produce biofilm as a mechanism for concentrating cells and attaching to surfaces, as a protective barrier, and as a reserve nutrient Because similar biofilms are formed by Archaeoglobus profundus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum, biofilm formation might be a common stress response mechanism among the archaea

The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus

Abstract: Summary: Sensitivity of Staphylococcus aureus to the antibiotics tetracycline, benzylpenicillin and vancomycin was found to decrease by 2-10-fold when cells were grown adherent to silicone catheter surfaces. Sensitivity to rifampicin and fusidic acid was not significantly altered in adherent cells. Susceptibility further decreased with increased adherence time prior to antibiotic challenge. The resistance observed was not genotypic, or due to the presence of a specialized subpopulation of bacteria, as it disappeared when the bacteria were removed from the catheter, subcultured and retested. Also, adherent bacteria were found to grow more slowly than bacteria growing planktonically. It is concluded that the decrease in antibiotic susceptibility of adherent bacteria is a function of the physiological status of the individual cells rather than a function of biofilm formation or slime production. The decrease in growth rate of the adherent bacteria is a result of the adherence process rather than a result of nutrient depletion. The decrease in growth rate is implicated, but is not the sole factor, in the decreased antibiotic susceptibility of adherent bacteria.

Influence of electric fields and pH on biofilm structure as related to the bioelectric effect.

Abstract: Mixed species biofilms of Klebsiella pneumoniae, Pseudomonas fluorescens, and Pseudomonas aeruginosa were grown in a flow cell fitted with two platinum wire electrodes. The biofilm growing on the wires reached a thickness of approximately 50 microm after 3 days. When a voltage was applied with oscillating polarity, the biofilm attached to the wire expanded and contracted. The biofilm expanded by approximately 4% when the wire was cathodic but was reduced to 74% of the original thickness when the wire was anodic. The phenomenon was reproduced by alternately flushing the flow cell with media adjusted to pH 3 and pH 10 with no electric current. At pH 10 the biofilm was unaltered, but it became compacted to 69% of the original thickness at pH 3. We explained these phenomena in terms of the molecular interactions between charged acidic groups in the biofilm slime and the bacterial cell walls. Contraction of the biofilm under acidic conditions may be caused by (i) the elimination of electrostatic repulsion from neutralization of negatively charged carboxylate groups through protonation and (ii) subsequent hydrogen bonding between the carboxylic acids and oxygen atoms in the sugars. Electrostatic interactions between negatively charged groups in the biofilm and the charged wire may also be expected to cause biofilm expansion when the wire was cathodic and contraction when the wire was anodic. The consequences of the explanation of the increased susceptibility of biofilm cells to antibiotics in an electric field, the "bioelectric effect," are discussed.

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

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

Artificial Dental Plaque Biofilm Model Systems

Abstract: Difficulties with in vivo studies of natural plaque and its complex, heterogeneous structure have led to development of laboratory biofilm plaque model systems. Technologies for their culture are outlined, and the rationale, strengths, and relative uses of two complementary approaches to microbial models with a focus on plaque biodiversity are analyzed. Construction of synthetic consortia biofilms of major plaque species has established a variety of bacterial interactions important in plaque development. In particular, the 'Marsh' nine-species biofilm consortia systems are powerful quasi steady-state models which can be closely specified, modified, and analyzed. In the second approach, microcosm plaque biofilms are evolved in vitro from the natural oral microflora to the laboratory model most closely related to plaque in vivo. Functionally reproducible microcosm plaques are attainable with a biodiverse microbiota, heterogeneous structure, and pH behavior consistent with those of natural plaque. The resting pH can be controlled by urea supply. Their growth patterns, pH gradient formation, control of urease levels by environmental effectors, and plaque mineralization have been investigated. Microcosm biofilms may be the only useful in vitro systems where the identity of the microbes and processes involved is uncertain. Together, these two approaches begin to capture the complexity of plaque biofilm development, ecology, behavior, and pathology. They facilitate hypothesis testing across almost the whole range of plaque biology and the investigation of antiplaque procedures yielding accurate predictions of plaque behavior in vivo.

Physiological Approaches to the Control of Oral Biofilms

Abstract: Evidence that physiological strategies may be potential routes for oral biofilm control has come from (i) observations of the variations in the intra-oral distribution of members of the resident oral microflora, (ii) changes in plaque composition in health and disease, and (iii) data from laboratory model systems. Key physiological factors that were identified as significant in modulating the microflora included the local pH, redox potential (Eh), and nutrient availability. Increases in mutans streptococci and lactobacilli occur at sites with caries; growth of these species is selectively enhanced at low pH. In contrast, periodontal diseases are associated with plaque accumulation, followed by an inflammatory host response. The increases in Gram-negative, proteolytic, and obligately anaerobic bacteria reflect a low redox potential and a change in nutrient status due to the increased flow of gingival crevicular fluid (GCF). Consequently, physiological strategies for oral biofilm control should focus on red...

Corrosion inhibition by aerobic biofilms on SAE 1018 steel

Abstract: Carbon steel (SAE 1018) samples were exposed to complex liquid media containing either the aerobic bacterium Pseudomonas fragi or the facultative anaerobe Escherichia coli DH5α. Compared to sterile controls, mass loss was consistently 2- to 10-fold lower in the presence of these bacteria which produce a protective biofilm. Increasing the temperature from 23 °C to 30 °C resulted in a 2- to 5-fold decrease in corrosion inhibition with P. fragi whereas the same shift in temperature resulted in a 2-fold increase in corrosion inhibition with E. coli DH5α. Corrosion observed with non-biofilm-forming Streptomyces lividans TK24 was similar to that observed in sterile media. A dead biofilm, generated in situ by adding kanamycin to an established biofilm, did not protect the metal (corrosion rates were comparable to those in the sterile control), and mass loss in cell-free, spent Luria-Bertani (LB) medium was similar to that in sterile medium. Confocal laser scanning microscopy analysis confirmed the presence of a biofilm consisting of live and dead cells embedded in a sparse glycocalyx matrix. Mass-loss measurements were consistent with microscopic observations of the metal surface after 2 weeks of exposure, indicating that uniform corrosion occurred. The biofilm was also able to withstand mild agitation (60 rpm), provided that sufficient time was given for its development.

The effect of ultrasonic frequency upon enhanced killing of P. aeruginosa biofilms.

Abstract: It is widely recognized that the bacteria sequestered in a biofilm on a medical implant are much more resistant to antibiotics than their planktonic counterparts. Recent studies have shown that application of antibiotic along with low power ultrasound significantly increases the killing of planktonic bacteria by the antibiotic. Herein is reported a similar application of antibiotic and ultrasound to sessile bacteria in biofilms ofPseudomonas aeruginosa on a polyethylene substrate. Biofilm viability was measured after exposure to 12 μg/ml gentamicin sulfate and 10 mW/cm2 ultrasound at frequencies of 70 kHz, 500 kHz, 2.25 MHz, and 10 MHz. The results indicate that a significantly greater fraction of the bacteria was killed by gentamicin when they were subjected to ultrasound. However, ultrasound by itself did not have any deleterious effect on the biofilm viability. In addition, lower-frequency insonation is significantly more effective than higher frequency in reducing bacterial viability within the biofilm. The possible mechanisms of synergistic action are discussed.

The influence of cell surface properties of thermophilic streptococci on attachment to stainless steel

Abstract: The quality of milk products is threatened by the formation of biofilms of thermophilicstreptococci on the internal surfaces of plate heat exchangers used in milk processing. Althoughattachment to stainless steel surfaces is one of the first stages in the development of a biofilm, themechanisms involved in attachment have not been reported. The cell surface properties of 12strains of thermophilic streptococci were examined to determine their importance in attachment tostainless steel surfaces. Hydrophobicity, extracellular polysaccharide production and cell surfacecharge varied between the different strains but could not be related to numbers attaching. Treatingthe cells with sodium metaperiodate, lysozyme or trichloroacetic acid to disrupt cell surfacepolysaccharide had no effect on attachment. Treatment with trypsin or sodium dodecyl sulphate toremove cell surface proteins resulted in a 100-fold reduction in the number of bacteria attaching.This result suggests that the surface proteins of the thermophilic streptococci are important intheir attachment to stainless steel.

Chemical defenses of seaweeds against microbial colonization

Abstract: Any living or non-living surface immersed in seawaterrapidly acquires a bacterial biofilm. For living marineorganisms, biofilm formation can result in the death ofthe host, and thus there is strong evolutionary pressure formarine eukaryotes to evolve mechanisms which inhibit orcontrol the development of biofilms on their surfaces.Some marine eukaryotes are indeed successful incontrolling biofilms on their surfaces, and in manyinstances this control is achieved by the production ofinhibitory chemicals which act at or near the surface ofthe organism. In some cases these natural inhibitors aresimply toxic to bacteria. However, increasingly it appearsthat at least some of these compounds act by interferingspecifically with bacterial characteristics which effect theability of bacteria to colonize their hosts, such asattachment, surface spreading, or the production ofextracellular macromolecules. As an example, theAustralian seaweed Delisea pulchra appears tocontrol bacterial colonization by interfering with abacterial regulatory system (the acylated homoserinelactone system) that regulates several colonizationrelevant bacterial traits. Understanding how marineorganisms control specific bacterial colonization traitsshould provide us with insights into new technologies forthe control of biofilms on artificial surfaces.

Spatial Distribution and Coexistence of Klebsiella pneumoniae and Pseudomonas aeruginosa in Biofilms

Abstract: The heterotrophic bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa stably coexisted in laboratory-grown biofilms, even though the growth rate of K. pneumoniae was twice that of P. aeruginosa under planktonic growth conditions. The failure of K. pneumoniae to displace P. aeruginosa from the biofilm could not be attributed to concentration gradients of the limiting nutrient (glucose) arising from the interaction of reaction and diffusion. Comparisons of the growth rates of the two species in mono- and binary-population biofilms suggested partial segregation of the two species in the latter. We used a fluorescently labeled monoclonal antibody to examine the spatial distribution of K. pneumoniae in frozen cross sections of biofilm to confirm this segregation. K. pneumoniae microcolonies resided on top of, or intermixed with, a base film of P. aeruginosa. We hypothesize that microscale structural heterogeneity and differing rates of bacterial attachment and detachment of the two species are responsible for coexistence in this system.

Impact of nutrient composition on a degradative biofilm community.

Abstract: A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.

Comparison of fluorinated polymers against stainless steel, glass and polypropylene in microbial biofilm adherence and removal

Abstract: Biofilm formation is a long-standing problem in ultrapure water and bioprocess fluid transport lines. The standard materials used in these applications (316L stainless steel, polypropylene and glass) have long been known to be good surfaces for the attachment of bacteria and other biological materials. To compare the relative tenacity of biofilms grown on materials used in manufacturing processes, a model system for biofilm attachment was constructed that approximates the conditions in industrial process systems. New fluorinated polymers were compared to the above materials by evaluating the surface area coverage of bacterial populations on materials before and after mild chemical treatment. In addition, contact angle studies compared the relative hydrophobicity of surfaces to suspensions of bacteria in growth media, and scanning electron microscopy and atomic force microscopy studies were used to characterize surface smoothness and surface defects. Biofilm adherence to polymer-based substrata was determined to be a function of both surface finish and surface chemistry. Specifically, materials that are less chemically reactive, as indicated by higher contact angle, can have rougher surface finishes and still be amenable to biofilm removal.