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PubMed2006

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms.

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.

Aerobiosis↗
PubMed1996

Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes.

Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage-infected host cells of Escherichia coli and Pseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rII-LacZ fusion), within a lac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded beta-galactosidase activity. Fluorescent antibodies were used to detect non-labeled progeny phage. Phage T4 infected both surface-attached and surface-associated E. coli while phage E79 adsorbed to P. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.

Bacteriophage T4↗
PubMed2003

Process analysis of biofilms by photoacoustic spectroscopy.

Biofilms are aggregates of microorganisms and biopolymers which occur at aqueous interfaces. Biofilms play an important role in the degradation of pollutants in natural water systems as well as in wastewater treatment plants. In this communication, the use of photoacoustic spectroscopy (PAS) as a new biofilm monitoring technique is presented. PAS combines features of optical spectroscopy and ultrasonic tomography and allows a depth-resolved analysis of optically and acoustically inhomogeneous media. For the first time, both biofilm and bulk liquid were monitored by photoacoustic sensor heads. In this way, sorption of suspended iron(III) oxide particles on the outer and inner surfaces of the biofilm could be observed on-line and in situ. Colloids can act as carriers of pollutants and influence stability and degradation efficiency of biofilms.

Acoustics↗
PubMed2002

Movement, replication, and emigration rates of individual bacteria in a biofilm.

Single-cell behavior within a biofilm was observed over a period of several hours. The observations were converted into quantitative stochastic rules governing the behavior of individual cells within a biofilm. Such a quantitative summary provides not only a concise description of the results but also information helpful when constructing computer models of dynamic biofilm systems. The time to division, emigration, and rate of motility of individual green fluorescent protein labeled Pseudomonas aeruginosa PAO1 cells in a 3-10 microm thick biofilm containing predominantly non-GFP labeled cells were calculated based on images of individual cells collected at 15-min time intervals. The biofilms were grown in flow cells and the images captured with a confocal laser microscope. Cells destined to emigrate are more active than those that remain; the geometric means for velocities in the biofilm are 1.0 microm/h for remaining cells and 1.5 microm/h for emigrating cells. The median time to emigration was 2.0 h. During the experimental observation period, the estimated probability for emigration is 0.44, illustrating that a substantial number of bacteria leave the field of view. Cells emigrate at a median time one-third that of the median time to replication. Specifically, the median time for cells to divide was 6.9 h, and it was estimated that 10% of the cells had a time to division greater than 10 h.

Biofilms↗
PubMed2004

Sulfate-reducing bacteria-dominated biofilms that precipitate ZnS in a subsurface circumneutral-pH mine drainage system.

The microbial diversity of ZnS-forming biofilms in 8 degrees C, circumneutral-pH groundwater in tunnels within the abandoned Piquette Zn, Pb mine (Tennyson, Wisconsin, USA) has been investigated by molecular methods, fluorescence in situ hybridization (FISH), and cultivation techniques. These biofilms are growing on old mine timbers that generate locally anaerobic zones within the mine drainage system. Sulfate-reducing bacteria (SRB) exclusively of the family Desulfobacteriaceae comprise a significant fraction of the active microbiota. Desulfosporosinus strains were isolated, but could not be detected by molecular methods. Other important microbial clusters belonged to the beta-, gamma-, and epsilon-Proteobacteria, the Cytophaga/Flexibacter/Bacteroides-group (CFB), Planctomycetales, Spirochaetales, Clostridia, and green nonsulfur bacteria. Our investigations indicated a growth dependence of SRB on fermentative, cellulolytic, and organic acid-producing Clostridia. A few clones related to sulfur-oxidizing bacteria were detected, suggesting a sulfur cycle related to redox gradients within the biofilm. Sulfur oxidation prevents sulfide accumulation that would lead to precipitation of other sulfide phases. FISH analyses indicated that Desulfobacteriaceae populations were not early colonizers in freshly grown and ZnS-poor biofilms, whereas they were abundant in older, naturally established, and ZnS-rich biofilms. Gram-negative SRB have been detected in situ over a period of 6 months, supporting the important role of these organisms in selective ZnS precipitation in Tennyson mine. Results demonstrate the complex nature of biofilms responsible for in situ bioremediation of toxic metals in a subsurface mine drainage system.

Bacteria↗
PubMed2002

Inhibition of lotic biofilms by Diclofenac.

Diclofenac, a common drug, was subjected to degradation studies using river biofilms grown in rotating annular reactors. Degradation of diclofenac was possible after acclimatisation as confirmed by liquid chromatography-mass spectrometry analyses. Adapted biofilms showed that degradation down to 10-25% of the initial concentration could be achieved within 4 days. In situ observation by confocal laser scanning microscopy, however, revealed slow biofilm development in the presence of diclofenac compared with control experiments grown in river water only. This was substantiated by low cell counts and isolation of fewer kinds of microorganisms from diclofenac-grown biofilms. Fluorescent in situ hybridisation analyses confirmed the presence of various bacterial groups, especially those belonging to the Cytophaga-Flavobacterium and gamma-Proteobacteria groups, in the biofilms. Quantification of image data indicated a negative effect of diclofenac on the growth of bacteria and algae. This is the first report on degradation of diclofenac by lotic biofilms.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms.

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.

Anti-Bacterial Agents↗
PubMed2005

Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy.

Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200-900 cm(-1) region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm(-1)) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.

Bacterial Adhesion↗
PubMed2003

Biofilm formation and biocide susceptibility testing of Mycobacterium fortuitum and Mycobacterium marinum.

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC trade mark assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state.

Anti-Bacterial Agents↗
PubMed2006

Multiple exposures to chlorhexidine and xylitol: adhesion and biofilm formation by Streptococcus mutans.

Growing evidence from clinical studies suggests that mothers using xylitol gums or lozenges have decreased levels of Streptococcus mutans (SM) and do not transmit these cariogenic bacteria as readily to their children. To begin to determine mechanisms for these clinical findings and to explore potential synergism of antimicrobial combinations, we studied the effect of multiple exposures of chlorhexidine (CHX) combined with copper gluconate (CG) or zinc gluconate (ZG) followed by xylitol (XYL) on the ability of SM to adhere and form biofilms. Cell suspensions of SM were exposed two times to CHX; CG; CHX plus CG; ZG; and CHX plus ZG, and then four times to XYL. Control cells were exposed six times to water or XYL or received no treatment. For biofilm assessment, glass slides were inoculated with treated cells, and numbers of bacteria were enumerated after 48 hours of incubation. To assess the ability of SM to adhere, microtiter plate wells coated with primary S. sanguinis biofilms grown in sucrose were inoculated with treated SM, and adhesion was determined. Cells exposed to CHX-XYL combinations exhibited significant but transient inhibition of growth. The multiple-exposure regimen groups showed significant decreases in the ability of SM to form biofilms (P < 0.05). However, the CHX-XYL group exhibited a much greater effect than the other treatment groups (P < 0.001). Adhesion studies revealed that none of the multiple-exposure regimens had a significant effect on adhesion of SM to primary biofilms of S. sanguinis. We concluded that significant inhibition of SM growth and subsequent inability to grow as biofilms in the presence of sucrose occurs after a staggered exposure regimen to CHX initially and then to XYL. This may help explain the clinical data showing the decreased levels of SM in mothers treated with CHX and XYL.

Anti-Infective Agents, Local↗
PubMed2006

The role of biofilm infection in urology.

In the process of endourological development a great variety of foreign bodies have been invented besides urinary catheters on which biofilm can be formed. Bacteria in the biofilm are less sensible to antibiotics. An additional problem of medical biomaterials in the urinary tract environment is the development of encrustation and consecutive obstruction. In this review, we tried to sum up the conditions where biofilm formation has a great impact on the development or maintenance of urological infections and on treatment success. Modification of the biomaterial surface seems to be the most promising prevention strategy for bacterial biofilms. Easier methods for diagnosing and quantifying biofilm infection, to develop more specific antimicrobial agents and ideal device surfaces would surely help the fight against biofilm formation.

Anti-Bacterial Agents↗
PubMed1998

The effect of buttermilk consumption on biofilm formation on silicone rubber voice prostheses in an artificial throat.

Biofilm formation on indwelling silicone rubber voice prostheses in laryngectomized patients is still the main reason for dysfunction of the valve, leading to frequent replacements. Within patient support groups in The Netherlands, laryngectomees have suggested that the consumption of buttermilk prolongs the life-time of indwelling silicone rubber voice prostheses. The aim of the present study was to compare biofilm formation on Groningen button voice prostheses in a so-called artificial throat. Ten prostheses were placed in a simulated control group and ten other prostheses in a group with a simulated consumption of 700 ml buttermilk three times a day. Biofilms were allowed to grow on the prostheses by inoculating two artificial throats with the total cultivable microflora (bacteria and yeasts) isolated from an explanted Groningen button voice prosthesis. After 3 days, one artificial throat was perfused three times daily with phosphate buffer (control group) for 8 days, while the other artificial throat was perfused with buttermilk. Prostheses removed from the artificial throat in the control group were covered with a thick biofilm. Scanning electron microscopy showed microcolonies growing into the silicone rubber, similar to the ingrowth observed on explanted Groningen buttons. The simulated consumption of buttermilk in the other artificial throat almost fully prevented the formation of a biofilm on the prostheses during the experimental period. These in vitro experiments in the artificial throat demonstrate that the deterioration of voice prostheses can be lessened by the daily intake of buttermilk through its inhibitory effects on biofilm formation.

Animals↗
PubMed2002

Evaluation of different detection methods of biofilm formation in Staphylococcus aureus.

The icaADBC gene locus of Staphylococcus aureus and its polysaccharide intercellular adhesin (PIA/PNSG) were recently identified, but biofilm formation has rarely been detected in vitro. In this study we evaluated a tissue culture plate (TCP) assay and a tube test, as well as Congo red agar, using the two basic media trypticase soy broth (TSB) and brain heart infusion (BHI) broth with different sugar supplements for detection of biofilm formation in 128 ica-positive S. aureus isolates. Of the S. aureus strains, 57.1% displayed a biofilm-positive phenotype under optimized conditions in the TCP test. The tube test correlated well with the TCP test for strongly biofilm-producing strains, whereas weak producers were not safely discriminated from biofilm-negative strains. Screening on Congo red agar displayed a strong correlation with the TCP and the tube test for only 3.8%, and is therefore not recommended for investigation of biofilm formation in S. aureus.

Agar↗
PubMed2002

Role of fosfomycin in a synergistic combination with ofloxacin against Pseudomonas aeruginosa growing in a biofilm.

We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa biofilms of four clinical isolates with different susceptibilities to ofloxacin. A clear synergistic effect was detected in all four strains in accordance with their susceptibilities to ofloxacin. To clarify the mechanism of this synergistic action, changes in cellular accumulation of ofloxacin into fosfomycin-pretreated cells and morphological changes in cells treated with fosfomycin, ofloxacin, or fosfomycin plus ofloxacin were investigated. Pretreatment with fosfomycin significantly enhanced cellular uptake of labeled or unlabeled ofloxacin in biofilm cells as well as in floating cells. The accumulation of ofloxacin into fosfomycin-pretreated biofilm cells was further enhanced by treating cells simultaneously with ofloxacin and fosfomycin. Morphological studies using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) demonstrated that fosfomycin induced dramatic changes in cell shape and the outer membrane structure responsible for the altered membrane permeability of both surface and embedded biofilm cells. The resulting increased accumulation of ofloxacin in multilayers of biofilm cells was correlated with the kinetics of biofilm cell eradication, and this synergistic killing effect was confirmed by a combined study using SEM, TEM, and CLSM.

Anti-Bacterial Agents↗
PubMed2006

A microplate spectrofluorometric assay for bacterial biofilms.

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r (2)=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.

Acetylglucosamine↗
PubMed2006

Biofilm formation in an ice cream plant.

The sites of biofilm formation in an ice cream plant were investigated by sampling both the production line and the environment. Experiments were carried out twice within a 20-day period. First, stainless steel coupons were fixed to surfaces adjacent to food contact surfaces, the floor drains and the doormat. They were taken for the analysis of biofilm at three different production stages. Then, biofilm forming bacteria were enumerated and also presence of Listeria monocytogenes was monitored. Biofilm forming isolates were selected on the basis of colony morphology and Gram's reaction; Gram negative cocci and rod, Gram positive cocci and spore forming isolates were identified. Most of the biofilm formations were seen on the conveyor belt of a packaging machine 8 h after the beginning of the production, 6.5 x 10(3) cfu cm(-2). Most of the Gram negative bacteria identified belong to Enterobacteriaceae family such as Proteus, Enterobacter, Citrobacter, Shigella, Escherichia, Edwardsiella. The other Gram negative microflora included Aeromonas, Plesiomonas, Moraxella, Pseudomonas or Alcaligenes spp. were also isolated. Gram positive microflora of the ice cream plant included Staphyloccus, Bacillus, Listeria and lactic acid bacteria such as Streptococcus, Leuconostoc or Pediococcus spp. The results from this study highlighted the problems of spread of pathogens like Listeria and Shigella and spoilage bacteria. In the development of cleaning and disinfection procedures in ice cream plants, an awareness of these biofilm-forming bacteria is essential for the ice cream plants.

Biofilms↗
PubMed2006

Biomonitoring in the Boulder River Watershed, Montana, USA: metal concentrations in biofilm and macroinvertebrates, and relations with macroinvertebrate assemblage.

Portions of the Boulder River watershed contain elevated concentrations of arsenic, cadmium, copper, lead, and zinc in water, sediment, and biota. We measured concentrations of As, Cd, Cu, Pb, and Zn in biofilm and macroinvertebrates, and assessed macroinvertebrate assemblage and aquatic habitat with the objective of monitoring planned remediation efforts. Concentrations of metals were generally higher in downstream sites compared with upstream or reference sites, and two sites contained metal concentrations in macroinvertebrates greater than values reported to reduce health and survival of resident trout. Macroinvertebrate assemblage was correlated with metal concentrations in biofilm and macroinvertebrates. However, macroinvertebrate metrics were significantly correlated with a greater number of biofilm metals (8) than metals in invertebrates (4). Lead concentrations in biofilm appeared to have the most significant impact on macroinvertebrate assemblage. Metal concentrations in macroinvertebrates were directly proportional to concentrations in biofilm, indicating biofilm as a potential surrogate for monitoring metal impacts in aquatic systems.

Animals↗
PubMed2006

The diabetic foot: the importance of biofilms and wound bed preparation.

Biofilms are ubiquitous and medically important complex structures consisting of microbial-associated cells embedded in self-produced extracellular matrix of hydrated extrapolymeric substances, which are irreversibly attached to a biological or nonbiological surface. Bacteria that reside as biofilms are resistant to traditional therapy. This alternative community in which microbes exist has recently attracted interest as a potential reason why chronic wounds do not heal. This may be especially important for diabetic foot ulcers, which are often characterized by their refractory nature, their predisposition to have associated underlying infection, and their improvement with débridement. Animal and in vitro models have been developed to better study biofilms, which will allow a venue for therapeutic intervention. Potential opportunities exist that include prevention of bacterial attachment, prevention of biofilm formation, disruption of the biofilm to allow penetration of topical antimicrobial agents, interference with quorum sensing, and enhancement of bacteria dispersion from biofilms to a more easily destroyed planktonic state.

Animals↗