Effects of Quaternary Ammonium Silane Coatings on Mixed Fungal and Bacterial Biofilms on Tracheoesophageal Shunt Prostheses

Two quaternary ammonium silanes (QAS) were used to coat silicone rubber tracheoesophageal shunt prostheses, yielding a positively charged surface. One QAS coating [(trimethoxysilyl)-propyldimethyloctadecylammonium chloride] was applied through chemical bonding, while the other coating, Biocidal ZF, was sprayed onto the silicone rubber surface. The sprayed coating lost its stability within an hour, while the chemically bonded coating appeared stable. Upon incubation in an artificial throat model, allowing simultaneous adhesion and growth of yeast and bacteria, all coated prostheses showed significant reductions in the numbers of viable yeast (to 12% to 16%) and bacteria (to 27% to 36%) compared with those for silicone rubber controls, as confirmed using confocal laser scanning microscopy after live/dead staining of the biofilms. In situ hybridization with fluorescently labeled oligonucleotide probes showed that yeasts expressed hyphae on the untreated and Biocidal ZF-coated prostheses but not on the QAS-coated prostheses. Whether this is a result of the positive QAS coating or is due to the reduced number of bacteria is currently unknown. In summary, this is the first report on the inhibitory effects of positively charged coatings on the viability of yeasts and bacteria in mixed biofilms. Although the study initially aimed at reducing voice prosthetic biofilms, its relevance extends to all biomedical and environmental surfaces where mixed biofilms develop and present a problem.     

Microflora on explanted silicone rubber voice prostheses: taxonomy, hydrophobicity and electrophoretic mobility

Silicone rubber voice prostheses are implants which are inserted in a non-sterile environment and therefore become quickly colonized by micro-organisms. The micro-organisms exist on the medical grade silicone rubber as mixed biofilms of bacteria and yeasts. A total of 79 bacterial and 39 yeast strains were isolated from these biofilms by soft ultrasonic treatment. Gram-positive/catalase-negative and Gram-positive/catalase-positive cocci represented the dominant bacterial strains. The yeasts were mainly Candida species. Further characterization of cell surface properties such as hydrophobicity by microbial adhesion to hexadecane and electrophoretic mobility showed a distinct difference when the bacterial strains were compared with the yeasts. The bacterial hydrophobicities ranged from 0 to 100% adhesion to hexadecane, whereas the yeast strains, especially the Candida albicans strains, all had markedly hydrophilic cell surfaces. A comparison of the electrophoretic mobilities showed also differences between bacteria and yeast. The values for the bacteria were found to be between -2.5 to -0.5 (10^-8 m² V^-1 s^-1), whereas for the yeasts electrophoretic mobilities were more positive. Based on the adhesive properties of the isolated micro-organisms, strategies can now be developed to modify the properties of the silicone rubber to reduce biofilm formation on such prostheses.     

Microbial biofilms on facial prostheses

The composition of microbial biofilms on silicone rubber facial prostheses was investigated and compared with the microbial flora on healthy and prosthesis-covered skin. Scanning electron microscopy showed the presence of mixed bacterial and yeast biofilms on and deterioration of the surface of the prostheses. Microbial culturing confirmed the presence of yeasts and bacteria. Microbial colonization was significantly increased on prosthesis-covered skin compared to healthy skin. Candida spp. were exclusively isolated from prosthesis-covered skin and from prostheses. Biofilms from prostheses showed the least diverse band-profile in denaturing gradient gel electrophoresis (DGGE) whereas prosthesis-covered skin showed the most diverse band-profile. Bacterial diversity exceeded yeast diversity in all samples. It is concluded that occlusion of the skin by prostheses creates a favorable niche for opportunistic pathogens such as Candida spp. and Staphylococcus aureus. Biofilms on healthy skin, skin underneath the prosthesis and on the prosthesis had a comparable composition, but the numbers present differed according to the microorganism.     

Microbial biofilms on facial prostheses

The composition of microbial biofilms on silicone rubber facial prostheses was investigated and compared with the microbial flora on healthy and prosthesis-covered skin. Scanning electron microscopy showed the presence of mixed bacterial and yeast biofilms on and deterioration of the surface of the prostheses. Microbial culturing confirmed the presence of yeasts and bacteria. Microbial colonization was significantly increased on prosthesis-covered skin compared to healthy skin. Candida spp. were exclusively isolated from prosthesis-covered skin and from prostheses. Biofilms from prostheses showed the least diverse band-profile in denaturing gradient gel electrophoresis (DGGE) whereas prosthesis-covered skin showed the most diverse band-profile. Bacterial diversity exceeded yeast diversity in all samples. It is concluded that occlusion of the skin by prostheses creates a favorable niche for opportunistic pathogens such as Candida spp. and Staphylococcus aureus. Biofilms on healthy skin, skin underneath the prosthesis and on the prosthesis had a comparable composition, but the numbers present differed according to the microorganism.     

Streptococcus thermophilus and its biosurfactants inhibit adhesion by Candida spp. on silicone rubber.

The adhesion of yeasts, two Candida albicans and two Candida tropicalis strains isolated from naturally colonized voice prostheses, to silicone rubber with and without a salivary conditioning film in the absence and presence of adhering Streptococcus thermophilus B, a biosurfactant-releasing dairy isolate, was studied. Coverage of 1 to 4% of the surface of silicone rubber substrata with adhering S. thermophilus B gave significant reductions in the initial yeast adhesion regardless of the presence of a conditioning film. Mechanistically, this interference in yeast adhesion by S. thermophilus B was not due to direct physical effects but to biosurfactant release by the adhering bacteria, because experiments with S. thermophilus B cells that had released their biosurfactants prior to adhesion to silicone rubber and competition with yeasts did not show interference with initial yeast adhesion. The amounts of biosurfactants released were highest for mid-exponential- and early-stationary-phase bacteria (37 mg.g of cells-1 [dry weight]), but biosurfactants released by stationary-phase bacteria (14 mg.g of cells-1 [dry weight]) were the most surface active. The crude biosurfactants released were mixtures of various components, with a glycolipid-like component being the most surface active. A lipid-enriched biosurfactant fraction reduced the surface tension of an aqueous solution to about 35 mJ.m-2 at a concentration of only 0.5 mg.ml-1. The amount of biosurfactant released per S. thermophilus B cell was estimated to be sufficient to cover approximately 12 times the area of the cross section of the bacterium, making biosurfactant release a powerful defense weapon in the postadhesion competition of the bacterium with microorganisms such as yeasts. Preadsorption of biosurfactants to the silicone rubber prior to allowing yeasts to adhere was as effective against C. albicans GB 1/2 adhesion as covering 1 to 2% of the silicone rubber surface with adhering S. thermophilus B, but a preadsorbed biosurfactant layer was less effective against C. tropicalis GB 9/9.     

Adhesive interactions between voice prosthetic yeast and bacteria on silicone rubber in the absence and presence of saliva

Biofilms on silicone rubber voice prostheses are the major cause for frequent failure and replacement of these devices. The presence of both bacterial strains and yeast has been suggested to be crucial for the development of voice prosthetic biofilms. Adhesive interactions between Candida albicans, Candida krusei, and Candida tropicalis with 14 bacterial strains, all isolated from explanted voice prostheses were investigated in a parallel plate flow chamber. Bacteria were first allowed to adhere to silicone rubber, after which the flow chamber was perfused with yeast, suspended either in saliva or buffer. Generally, when yeast were adhering from buffer and saliva, the presence of adhering bacteria suppressed adhesion of yeast. In saliva, Rothia dentocariosa and Staphylococcus aureus enhanced adhesion of yeast, especially of C. albicans. This study shows that bacterial adhesion mostly reduces subsequent adhesion of yeast, while only a few bacterial strains stimulate adhesion of yeast, provided salivary adhesion mediators are present. Interestingly, different clinical studies have identified R. dentocariosa and S. aureus in biofilms on explanted prostheses of patients needing most frequent replacement, while C. albicans is one of the yeast generally held responsible for silicone rubber deterioration.     

Caffeinated soft drinks reduce bacterial prevalence in voice prosthetic biofilms

Laryngectomized patients use indwelling silicone rubber voice prostheses, placed in a surgically created fistula in between the trachea and the esophagus, for voice and speech rehabilitation. At the esophageal side, these voice prostheses rapidly become colonized by a thick biofilm consisting of a variety of oral and skin bacteria and yeasts, and on average, after 3–4 months a prosthesis has to be replaced. In this study, the influence of caffeinated soft drinks on biofilm formation on silicone rubber voice prostheses has been investigated in a modified Robbins device. Robbins devices were first inoculated with the total cultivable microflora from an explanted voice prosthesis for 3 d, after which the devices were perfused three times daily over a 12 day period with 650 ml of either phosphate buffered saline or carbonated mineral water (controls), caffeinated soft drinks (two types), or a decaffeinated and a sugar‐free version of one of the caffeinated soft drinks. At the end of a day, during the experimental period, the devices were filled with growth medium for 30 min. Both caffeinated soft drinks reduced bacterial prevalence in the biofilms to 1–5% of the control, while yeasts thrived in voice prosthetic biofilms exposed to caffeinated soft drinks. Neither the controls, nor the decaffeinated soft drink, nor the sugar‐free version of this showed these effects on bacterial prevalence.     

Influence of different combinations of bacteria and yeasts in voice prosthesis biofilms on air flow resistance

Laryngectomized patients use silicone rubber voice prostheses to rehabilitate their voice. However, biofilm formation limits the lifetime of voice prostheses. The presence of particular combinations of bacterial and yeast strains in voice prosthesis biofilms has been suggested to be crucial for causing valve failure. In order to identify combinations of bacterial and yeast strains causative to failure of voice prostheses, the effects of various combinations of bacterial and yeast strains on air flow resistances of Groningen button voice prostheses were determined. Biofilms were grown on Groningen button voice prostheses by inoculating so-called artificial throats with various combinations of clinically relevant bacterial and yeast strains. After 3 days, all throats were perfused three times daily with 250 ml phosphate buffered saline and at the end of each day the artificial throats were filled with growth medium for half an hour. After 7 days, the air flow resistances of the prostheses were measured. These air flow resistances were expressed relative to the air flow resistances of the same prostheses prior to biofilm formation. This study shows that biofilms causing strong increases in air flow resistance (26 to 28 cm water.s/l) comprised combinations of microorganisms, involving Candida tropicalis, Staphylococcus aureus and Rothia dentocariosa. This revised version was published online in June 2006 with corrections to the Cover Date.     

The development of marine biofilms on two commercial non-biocidal coatings: a comparison between silicone and fluoropolymer technologies

The antimicrobial performance of two fouling-release coating systems, Intersleek 700? (IS700; silicone technology), Intersleek 900? (IS900; fluoropolymer technology) and a tie coat (TC, control surface) was investigated in a short term (10 days) field experiment conducted at a depth of ca 0.5 m in the Marina Bandar Rawdha (Muscat, Oman). Microfouling on coated glass slides was analyzed using epifluorescence microscopy and adenosine-5'-triphosphate (ATP) luminometry. All the coatings developed biofilms composed of heterotrophic bacteria, cyanobacteria, seven species of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria sp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal spores and the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to the numbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density of the bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms on the TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuel consumption for those vessels that utilise the IS900 fouling-release coating.     

The development of marine biofilms on two commercial non-biocidal coatings: a comparison between silicone and fluoropolymer technologies

The antimicrobial performance of two fouling-release coating systems, Intersleek 700® (IS700; silicone technology), Intersleek 900® (IS900; fluoropolymer technology) and a tie coat (TC, control surface) was investigated in a short term (10 days) field experiment conducted at a depth of ca 0.5 m in the Marina Bandar Rawdha (Muscat, Oman). Microfouling on coated glass slides was analyzed using epifluorescence microscopy and adenosine-5′-triphosphate (ATP) luminometry. All the coatings developed biofilms composed of heterotrophic bacteria, cyanobacteria, seven species of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria sp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal spores and the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to the numbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density of the bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms on the TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuel consumption for those vessels that utilise the IS900 fouling-release coating.     

Inhibition of microbial adhesion to silicone rubber treated with biosurfactant from Streptococcus thermophilus A

Microbial adhesion of four bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber before and after conditioning with a biosurfactant obtained from the probiotic bacterium Streptococcus thermophilus A was investigated in a parallel plate flow chamber. The silicone rubber with and without an adsorbed biosurfactant layer was characterized using contact angle measurements. Water contact angles indicated that the silicone rubber surface with adsorbed biosurfactant was more hydrophilic (58 degrees) than bare silicone rubber (109 degrees). The results obtained showed that the biosurfactant was effective in decreasing the initial deposition rates, and the number of bacterial cells adhering after 4 h, for all microorganisms tested. A decrease in the initial deposition rate was observed for Rothia dentocariosa GBJ 52/2B and Staphylococcus aureus GB 2/1 from 1937+/-194 to 179+/-21 microorganisms cm(-2) s(-1) and from 1255+/-54 to 233+/-26 microorganisms cm(-2) s(-1), respectively, accounting for an 86% reduction of the initial deposition rate for both strains. The number of bacterial cells adhering to the silicone rubber with preadsorbed biosurfactant after 4 h was further reduced by 89% and 97% by the two strains, respectively. The two yeast strains tested showed less reduction in adhesion after 4 h, to values between 67% and 70%. Such a pretreatment with surface-active compounds may constitute a promising strategy to reduce the microbial colonization rate of silicone rubber voice prostheses.     

Gelatin-glutaraldehyde cross-linking on silicone rubber to increase endothelial cell adhesion and growth

Silicone is a biomaterial that is widely used in many areas because of its high optical clarity, its durability, and the ease with which it can be cast. However, these advantages are counterbalanced by strong hydrophobicity. Gelatin cross-linking has been used as a hydrophilic coating on many biomaterials but not on silicone rubber. In this study, two gelatin glutaraldehyde (GA) cross-linking methods were used to coat a hydrophilic membrane on silicone rubber. In method I, gelatin and GA were mixed in three different proportions (64:1, 128:1, and 256:1) before coating. In method II, a newly formed 5% gelatin membrane was cross-linked with a 2.5% GA solution. All coatings were hydrophilic, as determined from the measurement of contact angle for a drop of water on the surface. Bovine coronary arterial endothelial cells were shown to grow well on the surface modified by method II at 72 h. In method I, the cells grew well for gelatin-GA proportions of 64:1 and 128:1 at 72 h. No cell attachment on untreated silicone rubber was observed by the third d of seeding. The results indicated that both methods of gelatin-GA cross-linking provided a hydrophilic surface on silicone for endothelial cell adhesion and growth in vitro.     

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.     

Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants

AIMS: The effects and extent of adhesion of four different bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed rhamnolipid biosurfactant layer obtained from Pseudomonasaeruginosa DS10-129 was studied. METHODS AND RESULTS: The ability of rhamnolipid biosurfactant to inhibit adhesion of micro-organisms to silicone rubber was investigated in a parallel-plate flow chamber. The anti-adhesive activity of the biosurfactant at different concentrations was significant against all the strains and depended on the micro-organism tested. The results showed an effective reduction in the initial deposition rates, and the number of bacterial cells adhering after 4 h, for all micro-organisms tested at the 4 g l(-1) undiluted rhamnolipid solution. Maximum initial reduction of adhesion rate (an average of 66%) occurred for Streptococcus salivarius GB 24/9 and Candida tropicalis GB 9/9. The number of cells adhering after 4 h on silicone rubber conditioned with biosurfactant was reduced to 48% for Staphylococcus epidermidis GB 9/6, Strep. salivarius GB 24/9, Staphylococcus aureus GB 2/1 and C. tropicalis GB 9/9 in comparison to controls. Perfusing the flow chamber with biosurfactant containing solution followed by the passage of a liquid-air interface, to investigate detachment of micro-organisms adhering to silicone rubber, produced high detachment (96%) of adhered cells for all micro-organisms studied, except for Staph. aureus GB 2/1 (67%). SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that biosurfactant represent suitable compounds that should be considered in developing future strategies to prevent the microbial colonization of silicone rubber voice prostheses.     

Scanning electron microscopy of native biofilms on mung bean sprouts

Native biofilms present on the adaxial surface of cotyledons of mung bean sprouts (Vigna radiata) were studied by use of scanning electron microscopy. Biofilms were abundant on the cotyledon surfaces and were comprised of rod-shaped bacteria, cocci-shaped bacteria, or yeasts, often with one type of microbe predominant. In contrast to our earlier study of biofilms on green sprouts (alfalfa, clover, broccoli, and sunflower), yeast and cocci were abundant on mung bean. Filamentous fungi were not observed. Sheet-like or fibrillar material (presumably composed of secreted microbial polysaccharides, proteins, lipids, and nucleic acids) fully or partially covered the biofilms. Biofilms up to 5 mm in length were observed, and some biofilms were comprised of more than just a monolayer of microbial cells. Native biofilms on sprout surfaces undoubtedly play an important role in the ecology of plant epiphytic microbes and may also afford protected sites for plant and human bacterial pathogens.     

Biomolecules in multilayer film for antimicrobial and easy-cleaning stainless steel surface applications

Microorganisms are able to attach to, grow on, and ultimately form biofilms on a large variety of surfaces, such as industrial equipment, food contact surfaces, medical implants, prostheses and operating rooms. Once organized into biofilms, bacteria are difficult to remove and kill, which increases the risk of cross-contamination and infection. One way to address the problem may thus be to develop antibacterial, anti-adhesion, ‘easy cleaning’ surfaces. In this study, stainless steel (SS) surfaces with antibacterial properties were created by embedding several antimicrobial peptides in a multilayer film architecture. The biocidal effect of these surfaces was demonstrated against both Gram-positive and Gram-negative bacteria according to two ISO tests. Also, coating SS surfaces with either mucin or heparin led to a reduction of S. epidermidis adhesion of almost 95% vs the bare substratum. Finally, by combining both antibacterial and anti-adhesion biomolecules in the same multilayer film, SS surfaces with better cleanability were produced. This surface coating property may help to delay the buildup of a dead bacterial layer which is known to progressively reduce exposure of the coating, leading to an undesirable decrease in the antibacterial effect of the surface.     

Relating the Surface Properties of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) to Their Bactericidal Effect towards a Biofilm of Streptococcus mutans

This study was designed to determine the effects of superparamagnetic iron oxide nanoparticles (SPIONs) on the biological activity of a bacterial biofilm (Streptococcus mutans). Our hypothesis was that the diffusion of the SPIONs into biofilms would depend on their surface properties, which in turn would largely be determined by their surface functionality. Bare, positively charged and negatively charged SPIONs, with hydrodynamic diameters of 14.6 ± 1.4 nm, 20.4 ± 1.3 nm and 21.2 ± 1.6 nm were evaluated. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and electrophoretic mobility (EPM) measurements were used to confirm that carboxylic functional groups predominated on the negatively charged SPIONS, whereas amine functional groups predominated on the positively charged particles. Transmission electron microscopy (TEM) showed the morphology and sizes of SPIONs. Scanning electron microscopy (SEM) and EPM measurements indicated that the surfaces of the SPIONs were covered with biomolecules following their incubation with the biofilm. Bare SPIONs killed bacteria less than the positively charged SPIONs at the highest exposure concentrations, but the toxicity of the bare and positively charged SPIONs was the same for lower SPION concentrations. The positively charged SPIONs were more effective in killing bacteria than the negatively charged ones. Nonetheless, electrophoretic mobilities of all three SPIONs (negative, bare and positively charged) became more negative following incubation with the (negatively-charged) biofilm. Therefore, while the surface charge of SPIONS was important in determining their biological activity, the initial surface charge was not constant in the presence of the biofilm, leading eventually to SPIONS with fairly similar surface charges in situ. The study nonetheless suggests that the surface characteristics of the SPIONS is an important parameter controlling the efficiency of antimicrobial agents. The analysis of the CFU/mL values shows that the SPIONs have the same toxicity on bacteria in solution in comparison with that on the biofilm.     

Effect of the adhesive antibiotic TA on adhesion and initial growth of E. coli on silicone rubber

Catheter-associated urinary tract infection is the most common nosocomial infection, and contributes to patient morbidity and mortality. We investigated the effect that the TA adhesive antibiotic had on adhesion and initial growth in urine of Escherichia coli on silicone rubber. The TA antibiotic had reduced adhesion, and inhibited initial growth of the bacteria on the surface. Since adhesion and initial growth on the surface are an essential part of biofilm formation and subsequent infection, we speculate that the TA antibiotic coating might decrease the infection rate associated with indwelling urinary catheter.     

Electropositively charged filters for the recovery of yeasts and bacteria from beverages

The ability of electropositively charged filters to recover yeasts and lactic acid bacteria from a variety of beverages was evaluated. Filtration through 'Zeta plus', grade O5S, filters recovered nearly all of the yeast contaminants from table wines, sherry and port. Recovery of yeasts from cream liqueurs and egg-based beverages was also good but it was not possible to filter drinks containing orange juice, even through filters with nominal pore sizes of 2 to 10 micron. Lactic acid bacteria proved more difficult to recover than yeasts, even though smaller pore-sized filters (1 to 4 micron) were employed. However, a sufficiently high percentage of bacteria were recovered to justify use of these filters for quality assurance. The advantage of concentrating contaminants by using charged filters, and the influence of product composition on the efficiency of microbial adsorption are discussed. The growth of wine-spoiling yeasts and lactic acid bacteria were not inhibited by water- or ethanol-soluble extracts of the filter material.     

Electropositively charged filters for the recovery of yeasts and bacteria from beverages

The ability of electropositively charged filters to recover yeasts and lactic acid bacteria from a variety of beverages was evaluated. Filtration through 'Zeta plus', grade OSS, filters recovered nearly all of the yeast contaminants from table wines, sherry and port. Recovery of yeasts from cream liqueurs and egg-based beverages was also good but it was not possible to filter drinks containing orange juice, even through filters with nominal pore sizes of 2 to 10 μm. Lactic acid bacteria proved more difficult to recover than yeasts, even though smaller pore-sized filters (1 to 4 μm) were employed. However, a sufficiently high percentage of bacteria were recovered to justify use of these filters for quality assurance. The advantage of concentrating contaminants by using charged filters, and the influence of product composition on the efficiency of microbial adsorption are discussed. The growth of wine-spoiling yeasts and lactic acid bacteria were not inhibited by water- or ethanol-soluble extracts of the filter material.