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Pseudomonas aeruginosa biofilms react with and precipitate toxic soluble gold.

The interaction between biofilms of Pseudomonas aeruginosa PAO1 and 0.01-5 mM gold chloride was investigated using flow-cells. Scanning confocal laser microscopy (SCLM) of these biofilms revealed the formation of two distinct structural features: (i) confluent areas of uniform thickness and (ii) cell clusters which often emerged as 30-40 micro m, tall narrow pillars (or pedestals) of cells and exopolymeric substance (EPS). When 5-day-old, quasi-steady state biofilms (as indicated by the stability of film thickness and overall structure) were exposed to relatively high AuCl3 (i.e. 0.5-5 mM) for 30 min at 20 degrees C, reduction of the auric ion resulted in the formation of both extracellular and intracellular metallic gold colloids, as revealed by transmission electron microscopy (TEM). Most mineralization occurred on cell surfaces with lesser amounts within cells and little throughout the EPS. Little to no mineralization of gold was seen at 0.01-0.1 mM concentrations. As initial AuCl3 concentrations approached 0.5 mM or greater, more gold particles were seen and cell viability, as determined by a BacLight live/dead viability probe, approached zero. At an intermediate concentration of 0.1 mM, the live:dead ratio increased to 4:1. However, when planktonic cells were exposed to this same 0.1 mM concentration, it resulted in a 4-log reduction in viable counts as determined by plating. The higher resistance of biofilm cells to 0.1 mM gold can be attributed to its binding to the EPS and cell surfaces of the biofilm which ensured a (presumably) low effective cytoplasmic concentration of gold (i.e. no gold crystals were seen in cells by TEM). In addition, SCLM revealed the formation of larger extracellular gold crystals at the substratum (coverslip) level of the biofilms, with a higher proportion of crystals detected beneath pillars (cell cluster structures), suggesting the possibility of unique cell types, more reduced microenvironments at the base of each cluster, or a combination of both. These results suggest that the biomineralization of gold is impacted by biofilm structure.

Biofilms↗

Leitbakteria of microbial biofilm communities causing occlusion of biliary stents.

Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single-strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single-strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture-independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.

Aged↗

Evaluating biofilm growth of two oral pathogens.

AIMS: To determine the expediency of a microtitre assay system for establishing, quantifying and antimicrobial testing of two representative oral pathogens. METHODS AND RESULTS: Streptococcus mutans and Porphyromonas gingivalis were used. Morphological characteristics of the attached population were evaluated. Biofilm growth was evaluated spectrophotometrically (undisturbed and 1 N NaOH dissipated biofilm). The minimum concentration of chlorhexidine gluconate that inhibited biofilm growth was determined. Growth of the biofilms was successfully monitored by direct optical density measurements or those re-suspended in 1 N NaOH. The latter was necessary when glucans were present in Strep. mutans biofilms. The minimum concentration of chlorhexidine gluconate that inhibited biofilm growth was 1.25 microg ml(-1) for both species. The attached bacteria exhibited common biofilm characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay system developed was especially useful for monitoring the growth of adherent Strep. mutans in the presence of glucans, which is particularly significant for the study of anti-plaque chemicals.

Anti-Infective Agents, Local↗

Prevention of biofilm formation in dialysis water treatment systems.

BACKGROUND: Biofilm formations in dialysis systems may be relevant because they continuously release bacterial compounds and are resistant against disinfection. The aim of the study was to compare the development of biofilm between a water treatment system based on a single reverse osmosis unit producing purified dialysate water [bacterial count, 350 colony-forming unit (CFU)/L] (center A) and a water treatment system based on double reverse osmosis and electric deionization, which is continuously disinfected with ultraviolet light and treated with ozone once a week (bacterial count, 1 CFU/L) (center B). METHODS: During a period of 12 weeks, biofilm formation was studied in the tubing segment between the water piping and the dialysis module, using four dialysis monitors in each center. On a weekly basis, tubing samples of 5 cm length (N = 96) were taken under aseptic conditions and investigated for microbiologic contamination [cystine lactose electrolyte-deficient (CLED) Agar], endotoxin levels [limulus amoeben lysate (LAL) gel test, cutoff value, 0.0125 EU/mL], and biofilm formation [electron scanning microscopy (SEM)]. RESULTS: In center A, tube cultures were positive (>100 CFU/mL) in 16% of samples at 22 degrees C and 37 degrees C, compared to 3% of samples of center B (P < 0.05; chi-square). Endotoxin levels were positive in 76% of the tubing samples of center A and negative in all of the samples of center B (P < 0.05). Biofilm was present in 91.7% of the samples of center A (Fig. 1), and only present in one sample (taken after 9 weeks) of center B (P < 0.05) (Fig. 2). In center A, biofilm formation was already observed after 1 week. CONCLUSION: In contrast to a standard water treatment system producing purified water, the use of a system producing highly purified water, which is also treated with regular disinfection procedures, leads to a significant reduction in biofilm formation, bacterial growth, and endotoxin levels in a highly vulnerable part of a water treatment system.

Biofilms↗

Evidence of bacterial biofilm in tubing from hydraulic pathway of hemodialysis system.

Biofilms consist of microorganisms immobilized at a substratum surface embedded in an organic polymer matrix of bacterial origin. Tubing drawn from the fluid pathways within dialysis machines of various models were investigated for biofilm. Scanning electron microscopy (SEM), performed on approximately 2 cm2 samples of the tubing inner surfaces revealed that the inner surfaces of the tubing were covered with biofilms consisting of numerous deposits and glycocalix at different stages of formation with components containing bacteria and algae. Evaluations of biomass were performed from tubing sections of various lengths and inner diameters put in tubes containing water for injection and immersed in an ultrasound washtub for 1 h to ensure sloughing of the biofilm. Living bacteria were identified by plating on nutrient agar media and incubation for 48 h at 37 degrees C. Epifluorescent stains were used for the total bacteria count. Lipopolysaccharide levels were determined by the endotoxin activity measurements. Polyoside contents were determined by the colometric method, and the chemical oxygen demand was measured to evaluate the amount of organic substance. Biofilms detached from tubing samples drawn from the water path, bicarbonate path, and fresh dialysate path within dialysis machines contained approximately 1.10(3)-1.10(6) total bacteria/cm2, yet only some living bacteria were found. Endotoxin levels ranged from 1 to 12 EU/cm2. In contrast in the dialysate fluid, no bacteria were found, and the endotoxin content was under the detection level of the method. The polyoside content and chemical oxygen demand of the biomass ranged from 11 to 83 microg/cm2 and from 53 to 234 mg/cm2, respectively. It is concluded that a germ- and endotoxin-free dialysate does not exclude the risks and hazards of bacteria and endotoxin discharge from biofilm developed on the fluid pathway tubing, acting as a reservoir for continuous contamination, and efforts in the optimization of cleaning and disinfection procedures used for hemodialysis systems should aim to detach and neutralize biofilm when necessary.

Bacillus↗

In vitro resistance to bacterial biofilm formation on coated fluoroplastic tympanostomy tubes.

Bacterial biofilm formation has been implicated in persistent posttympanostomy otorrhea and irreversible tube contamination. The use of a tympanostomy tube with a resistance to biofilm formation by the most common organisms associated with persistent infection may decrease the incidence of chronic otorrhea and the need for tube removal. In this investigation, scanning electron microscopy was used to compare a phosphorylcholine-coated fluoroplastic tympanostomy tube to plain fluoroplastic and silver oxide-impregnated fluoroplastic for resistance to biofilm formation after in vitro incubation with Staphylococcus aureus or Pseudomonas aeruginosa. Only a biofilm from Pseudomonas formed on the untreated fluoroplastic tubes, whereas the silver oxide-impregnated tubes developed biofilms from both S aureus and P aeruginosa. In contrast, the coated fluoroplastic tube showed resistance to both staphylococcal and pseudomonal biofilm adhesion. This is the first study to demonstrate the effect of a surface treatment of fluoroplastic as a method to inhibit biofilm formation by both S aureus and P aeruginosa. This reinforces our previous studies showing that surface-adherence properties such as charge or slickness or both may be more beneficial than antibacterial treatments in preventing film adhesion.

Biofilms↗

Biofilm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae.

Persistence of the opportunistic bacterial pathogen Vibrio cholerae in aquatic environments is the principal cause for seasonal occurrence of cholera epidemics. This causality has been explained by postulating that V. cholerae forms biofilms in association with animate and inanimate surfaces. Alternatively, it has been proposed that bacterial pathogens are an integral part of the natural microbial food web and thus their survival is constrained by protozoan predation. Here, we report that both explanations are interrelated. Our data show that biofilms are the protective agent enabling V. cholerae to survive protozoan grazing while their planktonic counterparts are eliminated. Grazing on planktonic V. cholerae was found to select for the biofilm-enhancing rugose phase variant, which is adapted to the surface-associated niche by the production of exopolymers. Interestingly, grazing resistance in V. cholerae biofilms was not attained by exopolymer production alone but was accomplished by the secretion of an antiprotozoal factor that inhibits protozoan feeding activity. We identified that the cell density-dependent regulator hapR controls the production of this factor in biofilms. The inhibitory effect of V. cholerae biofilms was found to be widespread among toxigenic and nontoxigenic isolates. Our results provide a mechanistic explanation for the adaptive advantage of surface-associated growth in the environmental persistence of V. cholerae and suggest an important contribution of protozoan predation in the selective enrichment of biofilm-forming strains in the out-of-host environment.

Biofilms↗

A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels.

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.

Bacterial Proteins↗

Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide.

The development of surface-attached biofilm bacterial communities is considered an important source of nosocomial infections. Recently, bacterial interference via signaling molecules and surface active compounds was shown to antagonize biofilm formation, suggesting that nonantibiotic molecules produced during competitive interactions between bacteria could be used for biofilm reduction. Hence, a better understanding of commensal/pathogen interactions within bacterial community could lead to an improved control of exogenous pathogens. To reveal adhesion or growth-related bacterial interference, we investigated interactions between uropathogenic and commensal Escherichia coli in mixed in vitro biofilms. We demonstrate here that the uropathogenic strain CFT073 and all E. coli expressing group II capsules release into their environment a soluble polysaccharide that induces physicochemical surface alterations, which prevent biofilm formation by a wide range of Gram-positive and Gram-negative bacteria. We show that the treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens. These findings identify capsular polymers as antiadhesion bacterial interference molecules, which may prove to be of significance in the design of new strategies to limit biofilm formation on medical in dwelling devices.

Bacterial Adhesion↗

Cooperation and conflict in microbial biofilms.

Biofilms, in which cells attach to surfaces and secrete slime (polymeric substances), are central to microbial life. Biofilms are often thought to require high levels of cooperation because extracellular polymeric substances are a shared resource produced by one cell that can be used by others. Here we examine this hypothesis by using a detailed individual-based simulation of a biofilm to investigate the outcome of evolutionary competitions between strains that differ in their level of polymer production. Our model includes a biochemical description of the carbon fluxes for growth and polymer production, and it explicitly calculates diffusion-reaction effects and the resulting solute gradients in the biofilm. An emergent property of these simple but realistic mechanistic assumptions is a strong evolutionary advantage to extracellular polymer production. Polymer secretion is altruistic to cells above a focal cell: it pushes later generations in their lineage up and out into better oxygen conditions, but it harms others; polymer production suffocates neighboring nonpolymer producers. This property, analogous to vertical growth in plants, suggests that polymer secretion provides a strong competitive advantage to cell lineages within mixed-genotype biofilms: global cooperation is not required. Our model fundamentally changes how biofilms are expected to respond to changing social conditions; the presence of multiple strains in a biofilm should promote rather than inhibit polymer secretion.

Biofilms↗

The Vibrio cholerae O139 O-antigen polysaccharide is essential for Ca2+-dependent biofilm development in sea water.

Vibrio cholerae is both an inhabitant of estuarine environments and the etiologic agent of the diarrheal disease cholera. Previous work has demonstrated that V. cholerae forms both an exopolysaccharide-dependent biofilm and a Ca2+-dependent biofilm. In this work, we demonstrate a role for the O-antigen polysaccharide of V. cholerae in Ca2+-dependent biofilm development in model and true sea water. Interestingly, V. cholerae biofilms, as well as the biofilms of several other Vibrio species, disintegrate when Ca2+ is removed from the bathing medium, suggesting that Ca2+ is interacting directly with the O-antigen polysaccharide. In the Bay of Bengal, cholera incidence has been correlated with increased sea surface height. Because of the low altitude of this region, increases in sea surface height are likely to lead to transport of sea water, marine particulates, and marine biofilms into fresh water environments. Because fresh water is Ca2+-poor, our results suggest that one potential outcome of an increase is sea surface height is the dispersal of marine biofilms with an attendant increase in planktonic marine bacteria such as V. cholerae. Such a phenomenon may contribute to the correlation of increased sea surface height with cholera.

Bacterial Proteins↗

Mechanisms of antibiotic resistance in bacterial biofilms.

Bacteria that attach to a surface and grow as a biofilm are protected from killing by antibiotics. Reduced antibiotic susceptibility contributes to the persistence of biofilm infections such as those associated with implanted devices. The protective mechanisms at work in biofilms appear to be distinct from those that are responsible for conventional antibiotic resistance. In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multi-layered defense. The genetic and biochemical details of these biofilm defenses are only now beginning to emerge. Each gene and gene product contributing to this resistance may be a target for the development of new chemotherapeutic agents. Disabling biofilm resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to current treatments.

Bacterial Adhesion↗

Solute size effects on the diffusion in biofilms of Streptococcus mutans.

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.

Biofilms↗

Biofilm diatom community structure: influence of temporal and substratum variability.

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.

Biofilms↗

The effect of solid surface tension and exposure to elevated hydrodynamic shear on Pseudomonas fluorescens biofilms grown on modified titanium surfaces.

The solid surface tension of titanium was varied by using organosilane monolayers of various terminations, minimising differences in other material properties. Both the quantity of Pseudomonas fluorescens biofilms grown on the modified surfaces, and the percentage of biofilm remaining after exposure to hydrodynamic shear stress, varied significantly as a function of solid surface tension. The quantity of biofilm was less on chloropropyl-terminated surfaces than on an alkyl-terminated surfaces. However, the percentage of biofilm remaining after exposure to hydrodynamic shear stress (which depends on the adhesion and cohesion strengths of the biofilm) was less for the alkyl-terminated surface than for the chloropropyl-terminated surface, for one of the two sample sets analysed. These results demonstrate the importance of differentiating between the quantity of biofilm on a surface and the adhesion and cohesion strength of the biofilm, and may help explain discrepancies in the existing literature regarding the effect of solid surface tension on the propensity of a surface for microfouling.

Biofilms↗

Studies on the behaviour of Pseudomonas fluorescens biofilms after Ortho-phthalaldehyde treatment.

A relatively novel biocide, ortho-phthalaldehyde (OPA), was tested to control biofilms formed by Pseudomonas fluorescens on stainless steel surfaces. The toxic action of OPA was assessed in terms of inactivation and removal of the biofilm by means of, respectively, the determination of the respiratory activity and the variation in the dry weight of the biofilms. For comparison, the activity of OPA against suspended bacteria was also evaluated. The results showed that higher concentrations of OPA and longer exposure times are needed to inactivate P. fluorescens biofilms than planktonic populations, thus denoting that sessile bacteria have a reduced susceptibility to OPA. This appears to be associated with the reaction with the proteins of the matrix, as demonstrated by the reduction of the antimicrobial action of OPA in the presence of a protein (bovine serum albumin). The application of OPA appeared to cause little effect in the removal of biofilms from the metal slides since the mass of biofilm that remained on the surfaces, after biocide treatment, was within the same range as those observed in the control tests. These results suggest that, with OPA application, biofilms can be inactivated but stay attached to the surfaces, decreasing thereby the success of the chemical treatment.

Biofilms↗

"Escherichia coli-milk" biofilm removal from stainless steel surfaces: synergism between ultrasonic waves and enzymes.

Three different methods to standardize biofilm removal for in situ sanitary control of closed surfaces in the food industry have been developed and compared, i.e. sonication, enzymatic treatment and a combined treatment which involved the application of ultrasound to enzyme preparations. The biofilm studied was an Escherichia coli model biofilm, made with milk on stainless steel sheets. Plate counting and epifluorescence microscopy were used to assess the efficiency of each treatment. The results are expressed in percentages, 100% denoting total removal, obtained with a flat ultrasonic transducer (T1) developed and presented in a previous study. The application of ultrasound by a patented curved transducer, T2 (10 s, 40 kHz), specifically devised for closed surfaces, was not sufficient to completely remove the biofilm (30 +/- 7%). This biofilm was dislodged by two proteolytic enzyme preparations tested by immersion, viz. a 15-min application of protease (84 +/- 1%) and a 30-min trypsin application (95 +/- 8%). Using a combined treatment, the results showed a synergism between ultrasonic waves and proteolytic or glycolytic enzyme preparations, with removal of a significant amount of biofilm, i.e. 61-96% depending on the conditions tested, i.e. two to three times greater compared to sonication alone (30%). This application was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in 10 s compared with 30 or 15 min with the enzyme alone.

Biofilms↗

The application of confocal microscopy to the study of liposome adsorption onto bacterial biofilms.

Confocal laser scanning microscopy has been used to visualise the adsorption of fluorescently labelled liposomes on immobilised biofilms of the bacterium Staphylococcus aureus. The liposomes were prepared with a wide range of compositions with phosphatidylcholines as the predominant lipids using the extrusion technique. They had weight average diameters of 125 +/- 5 nm and were prepared with encapsulated carboxyfluorescein. Cationic liposomes were prepared by incorporating dimethyldioctadecylammonium bromide (DDAB) or 3, beta [N-(N1,N1 dimethylammonium ethane)-carbamoyl] cholesterol (DC-chol) and anionic liposomes were prepared by incorporation of phosphatidylinositol (PI). Pegylated cationic liposomes were prepared by incorporation of DDAB and 1,2-dipalmitoylphosphatidylethanolamine-N-[polyethylene glycol)-2000]. Confocal laser scanned images showed the preferential adsorption of the fluorescent cationic liposomes at the biofilm-bulk phase interface which on quantitation gave fluorescent peaks at the interface when scanned perpendicular (z-direction) to the biofilm surface (x-y plane). The biofilm fluorescence enhancement (BFE) at the interface was examined as a function of liposomal lipid concentration and liposome composition. Studies of the extent of pegylation of the cationic liposomes incorporating DDAB, on adsorption at the biofilm-bulk phase interface were made. The results demonstrated that pegylation inhibited adsorption to the bacterial biofilms as seen by the decline in the peak of fluorescence as the mole% DPPE-PEG-2000 was increased in a range from 0 to 9 mole%. The results indicate that confocal laser scanning microscopy is a useful technique for the study of liposome adsorption to bacterial biofilms and complements the method based on the use of radiolabelled liposomes.

Adsorption↗