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PubMed2003

Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions.

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.

Biofilms↗

PubMed1998

[Inhibitory activity of azithromycin on biofilm synthesis and synergism between azithromycin and fleroxacin on Pseudomonas aeruginosa in biofilms].

OBJECTIVE: The influence of azithromycin (AZT) on the production of glycocalyx (GLX) from P. aeruginosa biofilms and synergism of antibacterial activities between AZT and fleroxacin (FLX) on P. aeruginosa were investigated. METHOD: GLX production was measured by using a L-tryptophan method and viable counts in biofilms was determined by using a methylthiazolyldiphenyltetrazolium(MTT) method. RESULT: GLX production was reduced significantly from 28.0 +/- 8.1 micrograms/ml at 0MIC to 21.8 +/- 8.2 micrograms/ml at 1/16MIC and 16.7 +/- 7.9 micrograms/ml at 1/4MIC respectively (P = 0.0002). 1/16MIC and 1/4MIC AZT showed no bactericidal activities to P. Aeruginosa in biofilms. Viable counts in biofilms were 6.2 +/- 0.61 Lg/cm2 at 0MIC, 6.1 +/- 0.4 Lg/cm2 at 1/16MIC and 6.2 +/- 0.4 Lg CFU/cm2 at 1/4 MIC respectively (P = 0.63). However, AZT could enhance bactericidal activity of FLX on P. aeruginosa in biofilms. When 1/4MIC FLX was combined with 1/16MIC and 1/4 MIC AZT, viable counts were reduced significantly from 6.1 +/- 0.5 Lg CFU/cm2 to 5.9 +/- 0.3 Lg CFU/cm2 and 5.8 +/- 0.4 Lg CFU/cm2 respectively (P = 0.02). When 1/2MIC FLX was combined with 1/16MIC and 1/4MICAZT, viable counts were reduced significantly from 6.3 +/- 0.7 Lg CFU/cm2 to 5.8 +/- 0.5 LgCFU/cm2 and 5.7 +/- 0.6 Lg CFU/cm2 respectively (P = 0.03). CONCLUSION: Because AZT did not have direct bactericidal activities on P. aeruginosa in biofilms but could inhibit GLX production, We considered that AZT could enhance antibacterial activities of FLX by enhancing permeability of FLX into biofilms.

Anti-Bacterial Agents↗

PubMed2003

Elucidation and control of biofilm formation processes in water treatment and distribution using the Unified Biofilm Approach.

Controlling biological processes in water treatment and distribution is a major challenge to water supply companies. In the Netherlands, the use of chlorine-based disinfectants in water treatment is limited as much as possible and treated water is distributed without disinfectant residual in most cases. Biofilm formation processes in water treatment and distribution are studied using adenosinetriphosphate (ATP) as the parameter for active biomass. ATP measurements are applied to assess biofilm concentrations in distribution systems, in the biofilm monitor to determine the biofilm formation rate of treated water, in the biomass production potential test to determine the effect of pipe materials on microbial growth and in membrane systems to quantify biofouling. The use of a single parameter enables to compare biofilm concentrations in all situations and contributes to the understanding and control of biofilm formation processes in water treatment and distribution. This approach has been designated as the Unified Biofilm Approach.

Adenosine Triphosphate↗

PubMed2004

[Differences in antibiotic sensitivity in biofilm-positive and biofilm-negative strains of Staphylococcus epidermidis isolated from blood cultures].

The adhering capability and biofilm growth facilitate staphylococcal colonization of surfaces of damaged tissues and foreign bodies. Biofilm-forming bacteria are more resistant to immune system activities, mechanical effects of blood flow and other adverse effects, e.g. those due to antibiotics. Minimal inhibitory concentrations (MICs) were compared for two groups of Staphylococcus epidermidis strains isolated from blood cultures. Group 1 included biofilm positive strains whose biofilm-forming potential was revealed by both phenotypic and genotypic methods. Group 2 included strains without biofilm-forming potential. The comparison of MICs for selected antibiotics showed higher resistance of biofilm positive compared to biofilm negative strains. The difference was evident particularly for oxacillin, tetracycline, co-trimoxazole and gentamicin.

Biofilms↗

PubMed1994

Characterization of transposon mutants of biofilm-producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: genetic identification of a hexosamine-containing polysaccharide intercellular adhesin.

The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to production of Staphylococcus epidermidis biofilms, which are thought to contribute to virulence in biomaterial-related infections. We isolated Tn917 transposon mutants of biofilm-producing S. epidermidis 13-1, which were completely biofilm negative. In pulsed-field gel electrophoresis no obvious deletions of the mutants were noted. The Tn917 insertions of mutants M10 and M11 were located on different EcoRI fragments but on identical 60-kb SmaI and 17-kb BamHI chromosomal fragments. Linkage of transposon insertions of mutants M10 and M11 with the altered phenotype was demonstrated by phage transduction, whereas the several other mutants apparently represented spontaneous variants. In a primary attachment assay with polystyrene spheres, no significant difference between any of the mutants and the wild type could be detected. Cell clustering as an indication of intercellular adhesion, which is a prerequisite for accumulation in multilayered cell clusters, was not detected with any mutant. These results demonstrate that the mutants were impaired in the accumulative phase of biofilm production. Mutants M10 and M11 did not produce detectable amounts of a specific polysaccharide antigen (D. Mack, N. Siemssen, and R. Laufs, Infect. Immun. 60:2048-2057, 1992), whereas substantially reduced amounts of antigen were produced by the spontaneous variants. Hexosamine was determined as the major specific component of the antigen enriched by gel filtration of biofilm-producing S. epidermidis 1457 because almost no hexosamine was detected in material prepared from the isogenic biofilm-negative transductant 1457-M11, which differentiates the antigen from other S. epidermidis polysaccharide components. Our results provide direct genetic evidence for a function of the antigen in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.

Antigens, Bacterial↗

PubMed1997

Importance of biofilm formation for corrosion inhibition of SAE 1018 steel by axenic aerobic biofilms.

To investigate if corrosion inhibition by aerobic biofilms is a general phenomenon, carbon steel (SAE 1018) coupons were exposed to a complex liquid medium (Luria-Bertani) and seawater-mimicking medium (VNSS) containing fifteen different pure-culture bacterial suspensions representing seven genera. Compared to sterile controls, the mass loss in the presence of these bacteria (which are capable of developing a biofilm to various degrees) decreased by 2- to 15-fold. The extent of corrosion inhibition in LB medium depended on the nature of the biofilm: an increased proportion of live cells, observed with confocal scanning laser microscopy (CSLM) and image analysis, decreased corrosion. Corrosion inhibition in LB medium was greatest with Pseudomonas putida (good biofilm formation), while metal coupons exposed to Streptomyces lividans in LB medium (poor biofilm formation) corroded in a manner similar to the sterile controls. Pseudomonas mendocina KR1 reduced corrosion the most in VNSS. It appears that only a small layer of active, respiring cells is required to inhibit corrosion, and the corrosion inhibition observed is due to the attached biofilm.

Biofilms↗

PubMed2006

Role of quorum sensing and antimicrobial component production by Serratia plymuthica in formation of biofilms, including mixed biofilms with Escherichia coli.

We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

4-Butyrolactone↗

PubMed2003

Microbial characterization of biofilms in domestic drains and the establishment of stable biofilm microcosms.

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log(10) cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log(10) cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.

Bacteria↗

PubMed2006

Effect of formaldehyde on biofilm activity and morphology in an ultracompact biofilm reactor for carbonaceous wastewater treatment.

The effects of formaldehyde on biofilm morphology and biomass activity were investigated in an ultracompact biofilm reactor (UCBR) for carbonaceous wastewater treatment. The wastewater contained a fixed amount of glucose (with a chemical oxygen demand concentration of 600 mg/L) and an increasing concentration of formaldehyde (ranging from 21.4 to 271.1 mg/L). An influent formaldehyde concentration higher than 75 mg/L could facilitate filamentous growth (on biofilm) control and lead to a higher biofilm density, which is desirable as it enhanced the UCBR performance stability. However, at an influent formaldehyde concentration higher than 214.4 mg/L, biomass production was inhibited and deteriorations of biofilm morphology and biomass activity were observed. This study showed that it was desirable to maintain an influent formaldehyde concentration lower than 202.2 mg/L, as this concentration could achieve a good biofilm morphology while not inhibiting its microbial activity.

Biodegradation, Environmental↗

PubMed2002

Dental plaque as a biofilm and new research on biofilm removal by power toothbrushes.

Dental researchers have only recently begun to regard dental plaque as a biofilm. Dental plaque biofilm is a complex, heterogeneous structure of bacteria cells, a sticky extracellular matrix, and fluid channels. The biofilm must be modeled accurately for laboratory studies to be meaningful. To that end, researchers have compared the sonicare toothbrush to the Braun Oral-B 3D Excel Plaque Remover for the removal of interproximal dental plaque biofilm in an in vitro model. This article defines the concept of biofilms in the oral cavity and reviews how biofilm modeling is showing differences in toothbrush performance.

Bacterial Adhesion↗

PubMed2004

Comparing biofilm models for a single species biofilm system.

A benchmark problem was defined to evaluate the performance of different mathematical biofilm models. The biofilm consisted of heterotrophic bacteria degrading organic substrate and oxygen. Mathematical models tested ranged from simple analytical to multidimensional numerical models. For simple and more or less flat biofilms it was shown that analytical biofilm models provide very similar results compared to more complex numerical solutions. When considering a heterogeneous biofilm morphology it was shown that the effect of an increased external mass transfer resistance was much more significant compared to the effect of an increased surface area inside the biofilm.

Bacteria↗

PubMed2005

Biofilms in an urban water distribution system: measurement of biofilm biomass, pathogens and pathogen persistence within the Greater Stockholm Area, Sweden.

Distribution pipe biofilms can provide sites for the concentration of a wide range of microbial pathogens, thereby acting as a potential source of continual microbial exposure and furthermore can affect the aesthetic quality of water. In a joint project between Stockholm Water, the MISTRA "Sustainable Urban Water" program, the Swedish Institute for Infectious Disease Control and the Royal Technical University, Stockholm, the aim of the current study was to investigate biofilms formed in an urban water distribution system, and quantify the impact of such biofilms on potential pathogen accumulation and persistence within the Greater Stockholm Area, Sweden. When used for primary disinfection, ultra-violet (UV) treatment had no measurable influence on biofilm formation within the distribution system when compared to conventional chlorination. Biofilms produced within a model pilot-plant were found to be representative to those that had formed within the larger municipal water distribution system, demonstrating the applicability of the novel pilot-plant for future studies. Polystyrene microspheres (1.0 microm) and Salmonella bacteriophages demonstrated their ability to accumulate and persist within the model pilot-plant system, where the means of primary disinfection (UV-treatment, chlorination) had no influence on such phenomena. With the exception of aeromonads, potential pathogens and faecal indicators could not be detected within biofilms from the Stockholm water distribution system. Results from this investigation may provide information for water treatment and distribution management strategies, and fill key data gaps that presently hinder the refinement of microbial risk models.

Aeromonadaceae↗

PubMed2003

Nitrogen removal characteristics and biofilm analysis of a membrane-aerated biofilm reactor applicable to high-strength nitrogenous wastewater treatment.

A membrane-aerated biofilm reactor (MABR) capable of simultaneous nitrification and denitrification in a single reactor vessel was developed to investigate the characteristics of nitrogen removal from high-strength nitrogenous wastewater, and biofilm analysis using microelectrodes and the fluorescence in situ hybridization (FISH) technique was performed. Mean removal percentages of total organic carbon (TOC) and nitrogen were 96% and 83% at removal rates of 5.76 g-C m(-2) d(-1) and 4.48 g-N m(-2) d(-1), respectively. For stable removal efficiency, constant washing of the biofilm was needed. Dissolved oxygen microelectrode measurement revealed that the biofilm thickness was about 1600 microm, and that oxygen penetrated about 300 to 700 microm, from the outer surface of the membrane. Furthermore, FISH analysis revealed that ammonia-oxidizing bacteria (AOB) were located near the outer surface of the membrane, whereas other bacteria were located from the inner to the outer part of the biofilm. Combining these results demonstrated that simultaneous nitrification and denitrification occurred in the biofilm of the MABR system. In addition, stoichiometric analysis revealed that after 130 d(-1), the free ammonia (FA) concentration ranged within the concentration causing inhibition of the growth of nitrite oxidizing bacteria (NOB) and that AOB consumed 86% of the oxygen supplied through the intra-membrane. These results indicate that nitrogen removal not via nitrate but via nitrite was mainly achieved in the MABR system.

Journal Article↗

PubMed1996

A Novel Strategy for Control of Microbial Biofilms through Generation of Biocide at the Biofilm-Surface Interface.

Biofilms of a mucoid clinical isolate of Pseudomonas aeruginosa (24 h; ca. 10(sup6) CFU/cm(sup2)) were established by immersion of polymer discs in nutrient broth cultures at 37(deg)C. Biofilms exposed for 30 min to various concentrations (0 to 3 mg/ml) of hydrogen peroxide or potassium monopersulfate were rinsed and shaken vigorously in sterile saline to detach loosely associated cells, and the residual viable attached population was quantified by a blot succession method on agar plates. Incorporation of copper and cobalt phthalocyanine catalysts within the polymers significantly enhanced the activity of these oxidizing biocides towards biofilm bacteria by several orders of magnitude. Biofilms established on the control discs resisted treatment with concentrations of either agent of up to 3 mg/ml. Enhancement through incorporation of a catalyst was such that concentrations of potassium monopersulfate of as low as 20 (mu)g/ml gave no recoverable survivors either on the discs or within the washings. Catalysts such as these will promote the formation of active oxygen species from a number of oxidizing agents such as peroxides and persulfates, and it is thought that generation of these at the surface-biofilm interface concentrates the antimicrobial effect to the interfacial cells and generates a diffusion pump which further provides active species to the biofilm matrix. The survivors of low-concentration treatments with these agents were more readily removed from the catalyst-containing discs than from the control discs. This indicated advantages gained in hygienic cleansing of such modified surfaces.

Journal Article↗

PubMed2004

Interactions between laponite and microbial biofilms in porous media: implications for colloid transport and biofilm stability.

Quartz sand columns and sand-filled microscope flow cells were used to investigate the transport characteristics of the clay colloid laponite, and a biofilm-forming bacterium, Pseudomonas aeruginosa SG81. Separate experiments were performed with each particle to determine their individual transport characteristics in clean sand columns. In a second set of experiments, bacterial biofilms were formed prior to introduction of the clay colloids. In the independent transport experiments, bacteria and laponite each conformed to known physicochemical principles. A sodium chloride concentration of 7 x 10(-2) M caused complete retention of the laponite within the sand columns. P. aeruginosa SG81 was generally less influenced by ionic strength effects; it showed relatively low mobility at all ionic strengths tested and some (albeit reduced) mobility when introduced to the columns in 1M NaCl, the highest concentration tested, but nevertheless showed reproducible trends. Under conditions favourable to laponite retention and biofilm stability (7 x 10(-2) MNaCl), laponite suspensions were able to remobilise a portion of the attached bacterial biomass. At low ionic strength, the profile of laponite elution was also altered in the presence of a P. aeruginosa biofilm. These observations suggest that while a reduction in ionic strength has a dominant influence on the mobilisation of biological and inorganic colloids, the presence of laponite and biomass can have a distinct influence on the mobility of both types of colloids. Since these events are likely to occur in subsurface environments, our results suggest that colloid-biofilm interactions will have implications for colloid-bound contaminant transport and the remobilisation of pathogens.

Biofilms↗

PubMed2004

Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms.

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

Bacterial Proteins↗

PubMed2004

Use of in vivo-generated biofilms from hemodialysis catheters to test the efficacy of a novel antimicrobial catheter lock for biofilm eradication in vitro.

This biofilm study was conducted to assess the in vitro activity of tetrasodium EDTA on catheters that had been routinely removed from hemodialysis patients at Leeds Teaching Hospitals Trust due to maturation of fistula. Catheters were screened by culture of through-catheter flush, and isolates were identified by standard methodologies; 20 isolates were found to be biofilm positive. Initial biofilm cell count levels averaged above 10(5) CFU/1-cm catheter section. Bacteria identified in the biofilms were gram-negative (1 isolate), gram-positive (11 isolates), or mixed species (8 isolates). After a 24-h lock, 40 mg of tetrasodium EDTA per ml was effective at eradicating the total biofilm viable count in almost all cases. The efficacy of tetrasodium EDTA as a catheter lock potentially shows that this agent could substantially reduce catheter-related infections and be used to treat patients with limited access.

Anti-Bacterial Agents↗

PubMed1995

Microbial biofilms and biofilm reactors.

Scientists and engineers have realized the industrial and environmental significance of biofilm accumulation and activity. The ability to predict and control biofilm formation has led to less fouling and corrosion in industrial systems and a better understanding of biofilm importance in natural aquatic systems. Understanding the fundamental processes contributing to biofilm formation is beneficial to anyone involved with natural or industrial systems where biofilms may play a significant role in determining variables such as bulk water quality, toxic compound biodegradation, or product quality.

Adsorption↗