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PubMed2002

In situ identification of azo dye inhibition effects on nitrifying biofilms using microelectrodes.

In this study, the inhibitory effects of acid orange 7 (AO7), a common azo dye, on nitrification in biofilms were investigated in situ using microelectrodes. Biofilms were obtained from laboratory rotating drum biofilm reactor after the nitrification process reached a pseudo-steady state. Dissolved oxygen, pH, NH4+, NO3-, and redox potential microelectrodes, with tip diameters ranging from 3-15 microm, were used to monitor the spatial distribution and change of microbial activities within nitrifying biofilms. It was found that at lower concentration (1 mg/L), AO7 had only a slight impact on the NH4+-N concentration profiles. The ammonium consumption rate decreased as higher AO7 concentrations (15 mg/L and 25 mg/L) were exposed to the biofilms. A similar trend was observed for the NO3(-)-N microprofiles. The nitrate production rate decreased as the AO7 concentration in the bulk solution increased. The dissolved oxygen and pH microprofiles also showed oxygen and alkalinity utilization, but at lower rates throughout the biofilms when the nitrification process was inhibited. No significant redox potential differences were observed in the biofilms after AO7 was applied.

Azo Compounds↗
PubMed2002

Structure and function of nitrifying biofilms as determined by molecular techniques and the use of microelectrodes.

The phylogenetic diversity of a nitrifying bacterial community of two types of nitrifying biofilms, a domestic wastewater biofilm and an autotrophic nitrifying biofilm grown on rotating disk reactors (RDR), was characterized by 16S ribosomal DNA (rDNA)-cloning analysis. Thereafter, successional development of nitrifying the bacterial community within both biofilms was visualized in situ by fluorescent in situ hybridization (FISH) wih a set of fluorescently labeled 16S rRNA-targeted DNA probes. In situ hybridization revealed that Nitrosomonas ureae was the numerically dominant species of the ammonia-oxidizing population in the domestic wastewater biofilm and that a population shift from N. urea to N. europaea and N. eutropha occurred when the culture medium was switched to the synthetic media from the domestic wastewater. After reaching the steady-state condition, microprofiles of NH4+, NO2-, NO3-, and O2 in the biofilms were measured by use of microsensors, and the spatial distributions of in situ nitrifying activities were determined. The relationship between the spatial organization of nitrifying bacterial populations and the in situ activity of these populations within the biofilms was discussed. Microelectrode measurements revealed that the active ammonia-oxidizing zone was vertically separated from the active nitrite-oxidizing zone. This vertical separation became more evident with increase of the substrate C/N ratio, leading to deterioration of nitrification efficiency. The combined use of these techniques made it possible to relate in situ nitrifying activity directly to the occurrence of nitrifying bacterial populations.

Ammonia↗
PubMed2003

Establishment of a continuous model system to study Helicobacter pylori survival in potable water biofilms.

Close association of the pathogen Helicobacter pylori in drinking water biofilms has been suggested. Using a two-stage water model, the survival and development of the pathogen in potable water biofilms was monitored. Filter sterilized tap water was used as the growth medium and the inoculum consisted of a naturally occurring consortium of microorganisms. Biofilms were generated on removable stainless steel coupons that were placed in the second vessel. Novel technology peptide nucleic acid (PNA) molecular probes were used to detect and locate the pathogen in the biofilms. The PNA-labelled oligonucleotide probes were highly specific, and complementary to the helix 6 region of H. pylori 16S rRNA. The pathogen was tracked in the biofilms using epifluorescence microscopy and episcopic differential interference contrast microscopy. Results show that H. pylori can successfully incorporate within biofilms and its presence was detected for up to five days after inoculation. PNA probes provided an easy and quick way of performing fluorescence in situ hybridisation assays in heterogeneous biofilms.

Biofilms↗
PubMedDate not supplied

[Biofilms, tolerance and antimicrobial resistance].

Virtually every surface examined in natural, industrial and pathogenic ecosystems are colonized by biofilms consisting of adherent populations of microorganisms surrounded with a glycocalyx matrix. The development of biofilms has important economic and medical consequences. The development and use of a broad range of medical devices made us recognize a variety of infections caused by microorganisms that were regarded previously as harmless. Infections caused by biofilms are difficult to eradicate with antimicrobial treatment, and in-vitro susceptibility tests show significant resistance of biofilm cells to be killed. Advances in our understanding of biofilm formation can assist in the development of novel strategies for the prevention and treatment of infections caused by biofilms. In this review article, structures, general properties and antimicrobial resistance mechanisms of biofilms were discussed under the light of recent literature.

Biofilms↗
PubMed2002

[Architecture, influencing factors, and sensitivity to antifungal agents of Candida biofilm].

OBJECTIVE: To establish a model of Candida biofilm and to explore its characteristics, ultrastructure, influences by saliva and serum, and sensitivity to antifungal agents. METHODS: Evaluations of the in vitro growth kinetics, influences by saliva and serum, and sensitivity to antifungal agents of Candida biofilm were performed with the abated tetrazolium salt XTT method on a 96-well microtire petri dish. The ultrastructure of Candida biofilm was observed under Confocal Laser Scanning Microscope (CLSM). RESULTS: The bioactivity of Candida biofilm increased with culturing time and serum could obviously increase the action of biofilm. The Candida biofilm was significantly resistant to routine antifungal agents. CONCLUSION: The Candida cells adhered in biofilms are significantly different in morphology from those in suspension and are resistant to routine antifungal agents such as Amphotericine B, Fluconazole and Itraconazole.

Amphotericin B↗
PubMed2003

Microbial activity of biofilm during start-up period of anaerobic hybrid reactor at low and high upflow feeding velocity.

With an aim to shorten start-up time of an Anaerobic Hybrid Reactor (AHR), initial biofilm development was studied, particularly at different upflow feeding velocities. At a low (0.01 m x h(-1)) upflow velocity, initial biofilm was found to develop via the attachment of suspended biomass in the packed zone, while microbial growth on the film was insignificant. Contrarily, with higher (1.0 m x h(-1)) upflow velocity, initial biofilm development was from both microbial attachment and growth on supporting media. Biofilm thickness was determined using confocal laser scanning microscopy (CLSM), which indicated that the biofilm developed faster with the higher velocity, due to the contribution of the microbial growth on supporting media. When operated beyond the initial biofilm development with the lower velocity, both the activity of acetogens and the methanogens increased, although there was a lower amount of attached biomass on the supporting media. Whereas, both groups were found to decrease with higher upflow velocity, but acidogenic activity increased. It can be concluded that higher upflow velocity positively affected the initial stage of biofilm development and has the potential to accelerate attached biomass on supporting media during the initial phase. Subsequently, the upflow velocity should be reduced to the normal rate to enhance the methanogenic activity.

Bacteria, Anaerobic↗
PubMed2004

Quantification of biofilms in a sub-surface flow wetland and their role in nutrient removal.

Subsurface flow wetlands contain gravel or sand substrates through which the wastewater flows vertically or horizontally. The aims of this study were, firstly, to quantify biofilm development associated with different size gravel in sections of a subsurface flow wetland with and without plants, and secondly, to conduct laboratory experiments to examine the role of biofilms in nutrient removal. Techniques to quantify biofilm included: bacterial cell counts, EPS and total protein extraction. Based on comparative gravel sample volume, only EPS was greater on the smaller 5 mm gravel particles. There was no significant difference between biofilm growth in sections with and without plants. Two vertical flow laboratory-scale reactors, one containing fresh wetland gravel, the other containing autoclaved gravel, were constructed to determine nutrient transformations. The autoclaved gravel in the "sterile" reactor rapidly became colonised with biofilm. Both reactors were dosed with two types of influent. Initially the influent contained 7.25 mg/L NO3-N and 0.3 mg/L NH4-N; the biofilm reactor removed most of the ammonium and nitrite but nitrate concentrations were only reduced by 20%. In the "sterile" reactor there was negligible removal of ammonium and nitrite indicating little nitrification, however nitrate was reduced by 72%, possibly due to assimilatory nitrate reduction associated with new biofilm development. When the influent contained 3 mg/L NO3-N and 16 mg/L NH4-N almost 100% removal and transformation of NH4-N occurred in both reactors providing an effluent high in NO3-N. Organic P was reduced but inorganic soluble P increased possibly due to mineralisation.

Biofilms↗
PubMed2004

Introduction to the IWA task group on biofilm modeling.

An International Water Association (IWA) Task Group on Biofilm Modeling was created with the purpose of comparatively evaluating different biofilm modeling approaches. The task group developed three benchmark problems for this comparison, and used a diversity of modeling techniques that included analytical, pseudo-analytical, and numerical solutions to the biofilm problems. Models in one, two, and three dimensional domains were also compared. The first benchmark problem (BM1) described a monospecies biofilm growing in a completely mixed reactor environment and had the purpose of comparing the ability of the models to predict substrate fluxes and concentrations for a biofilm system of fixed total biomass and fixed biomass density. The second problem (BM2) represented a situation in which substrate mass transport by convection was influenced by the hydrodynamic conditions of the liquid in contact with the biofilm. The third problem (BM3) was designed to compare the ability of the models to simulate multispecies and multisubstrate biofilms. These three benchmark problems allowed identification of the specific advantages and disadvantages of each modeling approach. A detailed presentation of the comparative analyses for each problem is provided elsewhere in these proceedings.

Bacteria↗
PubMed2005

Biodynamics of biofilm formation on nasogastric tubes in elderly patients.

BACKGROUND: We previously reported on the high propensity of pathogenic oral flora in the oropharynx of nasogastric tube-fed patients, and subsequently showed biofilm formation on the NGTs of these patients. There is a close relationship of biofilm and oropharyngeal colonization with pathogenic bacteria, aspiration pneumonia and antibiotic resistance. OBJECTIVES: To investigate the time relation between the insertion of a new NGT and formation of the biofilm. METHODS: We examined sequential samples on NGTs that were forcibly pulled out by the patients themselves during any of the 7 days after insertion. Scanning electron micrography and confocal laser scanning microscopy were used for biofilm detection. RESULTS: Biofilm was identified on 60% of the five samples of day 1 and on all the samples of the following days, by both microscopic methods. CONCLUSIONS: Biofilms form within a single day on most NGTs inserted for the feeding of elderly patients with dysphagia. Further research should be devoted to prevention of biofilm formation on NGTs.

Aged↗
PubMedDate not supplied

[The effectiveness of antibiotics on bacteria in biofilms].

Bacteria can form different types of communities, united by common notion: biofilms. The aim of the present study was to determine the capacity of different antibiotics to penetrate into biofilms and act on unrelated bacteria. The study revealed that the formation of barriers between the community and the environment on artificial biofilms occurred in all strains of unrelated Gram-positive and Gram-negative bacteria used in this investigation. The capacity of antibiotics to penetrate into biofilms varied in different strains of the same species. For certain antibiotics similarity in their penetrating capacity was found to exist with respect to biofilms of unrelated bacteria. The penetration of antibiotics into mixed biofilms depended on the strain which determined its minimal value, so that the protection of one microorganism by another was thus observed. The method for the evaluation of the effectiveness of antibiotic penetration into bacterial biofilms, suitable for use in bacteriological laboratories, is proposed.

Anti-Bacterial Agents↗
PubMed1991

An in vivo model to study the pathobiology of infectious biofilms on biomaterial surfaces.

This study examines the morphology, ultrastructure, and microbiology of the intact biofilm developing on an implant surface. Silastic subdermal implant material was colonized with P. aeruginosa and surgically inserted into the peritoneal cavity of adult rabbits. After 4, 8, 28, and 42 days implants were recovered and the intact biofilms examined. P. aeruginosa colonized the implant throughout the entire experimental time. Microcolonies of glycocalyx-coated bacteria were observed within the biofilm. However, the bulk of the biofilm was host-generated and typically contained phagocytes trapped within a thick mesh of fibrin. Polymorphonuclear neutrophils were the predominant cell type. Isolated erythrocytes, macrophages, and fibroblasts were also observed. By day 28, the biofilm was enclosed in a fibrous capsule of vascularized connective tissue. The low numbers of neutrophils seen in biofilms from sterile Silastic sheets implanted into control animals suggested that neutrophilia may represent a specific cellular response to the bacterial colonization. The results indicate that the cell-mediated immune response provides for most of the biofilm mass on colonized implant surfaces. Inactivated phagocytes trapped in fibrin may "wall-off" the embedded bacterial microcolonies and thus shield them from live phagocytic leucocytes. Such a mechanism may play an important role in the pathogenesis of prosthetic device infections.

Animals↗
PubMed1996

The Use of Microelectrodes to Study the Precipitation of Calcite upon Algal Biofilms

Calcium and pH ion-selective microelectrodes are used for the first time to measure chemical activity profiles above laboratory-grown algal biofilms (green alga, Chlorococcum sp). The results are used to calculate the solution composition changes above an actively photosynthesizing biofilm. These changes include the total calcium concentration, degree of supersaturation of the solution with respect to calcite, and the partial pressure of carbon dioxide. Measurements are made in a flow-cell, which was designed to hold the templates on which the biofilms are grown. Measurements show pronounced gradients of pH and calcium concentration, within a distance of approximately 500 μm above the biofilm. The high degree of supersaturation caused by the low concentrations of carbon dioxide near the biofilm surface leads to the precipitation of calcite on the biofilm. SEM micrographs illustrate the close association of calcite crystals with the algal cells. Preliminary results are presented of the precipitation rates estimated from the diffusion flux of calcium at the biofilm surface. The results are in broad agreement with the rates expected from calcite seed growth experiments in similar solution conditions.

Journal Article↗
PubMed2000

Apparent surface associated lag time in growth of primary biofilm cells.

The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean'' surface are referred to as ``primary'' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary'' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions.

Journal Article↗
PubMed2000

Influence of algal biomass on extracellular enzyme activity in river biofilms.

Relationships between biofilm structural components (algal and bacterial biomass) and the activities of some extracellular enzymes that contribute to the ability to degrade organic matter) were explored for six Atlantic and three Mediterranean streams and rivers. The biofilms in these fluvial systems accounted for a wide range of bacterial and algal biomass and colonized the most common benthic habitats. Ratio of bacteria/algae biomass was lower in Atlantic than in Mediterranean streams, but enzymatic activities (β-glucosidase, β-xylosidase, phosphatase) were in general greater in the Mediterranean stream biofilms. Climatic characteristics (especially temperature) may explain the differences in enzymatic activities between biofilms of similar structure but different flow regime. The ratio β-xylosidase: β-glucosidase was similar (around 0.5) for all streams and substrata considered, showing that there is a general higher utilization of cellobiosic than xylobiosic molecules in fluvial systems. In general, highly heterotrophic biofilms showed lower extracellular enzymatic activities than more autotrophic biofilms. Maximum enzymatic activity is achieved when the algal biomass is two- to threefold higher than the bacterial biomass. The relevance of algal biomass on the heterotrophic ability of biofilms may be related to the physical proximity between the two, but also to the high proportion of polymeric carbohydrates included in algal exudates and lysis products, whose use is enzyme-mediated.

Journal Article↗
PubMed1993

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection.

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

Amines↗
PubMed2003

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms.

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.

Alginates↗
PubMed1994

Iron chelator, exopolysaccharide and protease production in Staphylococcus epidermidis: a comparative study of the effects of specific growth rate in biofilm and planktonic culture.

The growth rate of Staphylococcus epidermidis was controlled for populations growing as a biofilm and perfused with supplemented, simple-salts medium. Production of iron chelators, extracellular protease and exopolysaccharide (EPS) by these populations was assessed as a function of specific growth rate and compared to that by planktonic populations grown in the same medium within a chemostat. Perfused biofilms increased their iron chelator and protease production with increasing growth rate. Chemostat populations decreased their production of iron chelators with increasing growth rate, whilst showing much enhanced production of proteases at intermediate growth rates (mu 0.15-0.25 h-1). Production of iron chelator and protease was generally 2-50 times higher by biofilms than by planktonic populations. EPS production was low and relatively unaffected by growth rate for the chemostat cultures (about 0.2 micrograms per unit cell mass) but high for the attached biofilms, particularly at slow growth rates (about 4 micrograms per unit cell mass). EPS production within the biofilms decreased markedly with increasing growth rate. At growth rates of 0.35 h-1 and above, the levels of EPS for biofilms and planktonic populations were equivalent. The results of this study clearly indicate that growth as a biofilm markedly influences extracellular virulence factor production by S. epidermidis.

Animals↗
PubMedDate not supplied

Bacterial biofilm, brown pigment stone and blockage of biliary stents.

Bacterial pathogens gain access into the biliary system by descending via the portal venous circulation or ascending through the sphincter of Oddi in duodenal-biliary reflux. Bacteria thrive as glycocalyx-enclosed microcolonies, coalescing to form an adherent biofilm. The establishment of biofilm is a key event in the formation of biliary sludge and pigment gallstones, and the blockage of biliary stents. The biofilm mode of growth is very effective because it provides bacteria with a measure of protection from antibacterial agents and phagocytic leucocytes. Calcification of the matrix confers further protection for the micro-organisms living inside the biofilm. To date, attempts to prevent blockage of biliary stent have employed physical methods by using large self-expandable stents and stents without side hole. Incorporation of antibiotics within stents has not been successful presumably because bacteria once living in their biofilm are quite resistant to antimicrobial agents. Even the most toxic bile salts have no effect on the biofilm bacteria. Yet, hydrophobic bile salts reduce bacterial adhesion on biomaterial, suggesting that incorporation of such bile salts might prevent the formation of bacterial biofilm.

Animals↗