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PubMed2004

Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans.

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

Biofilms↗

PubMed2001

Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials.

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.

Animals↗

PubMed2006

Biofilm formation by Staphylococcus epidermidis on intraocular lens material.

PURPOSE: To compare biofilm formation by Staphylococcus epidermidis on different intraocular lens (IOL) materials. METHODS: The S. epidermidis strains, ATCC 12228 (American Type Culture Collection) and ATCC 35984 (biofilm-producer) were used. Biofilms were cultivated on disks of different IOL materials: silicone, PMMA (polymethylmethacrylate), acrylic, or MPC (2-methacryloyloxyethyl phosphorylcholine) surface-modified acrylic. Biofilms were stained with crystal violet (CV) which served as an index of biofilm formation. The bacterial population was enumerated after biofilm homogenization. Biofilms were also examined by scanning electron microscopy (SEM). RESULTS: Among the four materials tested, the least amount of biofilm formed on silicone. Biofilm production was significantly different between acrylic and MPC surface-modified acrylic lenses at 48 hours (P<0.05-0.01). The bacterial populations were significantly different between acrylic and silicone over 72 hours (P<0.05-0.01). The population on acrylic and MPC surface-modified acrylic continued to increase over 72 hours. The biofilm was recognized after a 24-hour incubation. Rates of biofilm-positive SEM fields, which were defined as being occupied by biofilm over at least half of the area, were significantly different between acrylic and silicone at 72 hours (P<0.05-0.01). CONCLUSIONS: S. epidermidis formed biofilm most intensely on acrylic among the four IOL materials tested. MPC surface-modified acrylic has a preventive effect on biofilm formation.

Acrylic Resins↗

PubMed2006

Formation of biofilm at different nutrient levels by various genotypes of Listeria monocytogenes.

Strains of Listeria monocytogenes differ in their ability to form biofilms. The objectives of this study were to determine whether genetically related strains have similar biofilm-forming capacities and what effect nutrient concentration has on the ability of different strains to produce biofilms. Biofilms of 30 strains of L. monocytogenes, obtained from a variety of sources were grown on stainless steel in tryptic soy broth (TSB) or in a 1:10 dilution of TSB (DTSB) for 24 h at 32 degrees C. The amount of biofilm formed was determined with image analysis after cells were stained with bisBenzimide H 33258 (Hoechst 33258). The strains were genetically subtyped by repetitive element sequence-based PCR (rep-PCR) with the primer set rep-PRODt and rep-PROG5. Data were analyzed with an analysis of variance and Duncan's multiple range test. Eleven strains produced the same amount of biofilm in both media. Fourteen strains produced more biofilm in TSB than in DTSB. Five strains produced more biofilm in DTSB than in TSB. Serotype 4b strains produced more biofilm in TSB than did serotype 1/2a strains, whereas serotype 1/2a strains produced more biofilm in DTSB than did serotype 4b strains. Growth in DTSB resulted in decreased biofilm accumulation for serotype 4b strains. There was no correlation between genetic subtype and the amount of biofilm accumulation. These results indicate that strains of serotype 1/2a and serotype 4b differ in the regulation of their biofilm phenotype. The poor biofilm accumulation of serotype 4b isolates when grown in DTSB could be a factor in the predominance of serogroup 1/2 strains in food processing plants, where nutrients may be limited.

Analysis of Variance↗

PubMed1998

Activity and stability of a recombinant plasmid-borne TCE degradative pathway in biofilm cultures

The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term. Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

PubMed2001

Settlement of Pomatoceros lamarkii (Serpulidae) larvae on biofilmed surfaces and the effect of aerial drying.

The settlement responses of Pomatoceros lamarkii (Polychaeta: Serpulidae) larvae to biofilms of varying age on slate surfaces and to dried biofilms on slate surfaces were investigated in the laboratory. Settlement experiments were performed as multi-treatment, still water assays. Larvae did not settle on clean, non-biofilmed slates but settled on biofilms up to 28 days old. Settlement intensity was closely related to the bacterial density of a biofilm. Drying a biofilmed surface for 1-2 h at 20 degrees C to simulate a single tidal emersion completely negated the former inductive effect of the biofilm. Drying also negated the larval settlement-inducing effect that normally results from the presence of conspecific adults. The settlement inhibition lasted for approximately 5 days following a single drying event. Larvae settled readily on biofilms exposed to formalin and antibiotics. Treating biofilms with formalin or antibiotics before or after drying had no effect upon larval avoidance of dried biofilms. Freeze-drying a biofilm had the same effect as aerial drying. The biofilm drying effect could not be mimicked by exposing biofilms to hyper-saline seawater. The finding that P. lamarkii larvae do not settle on dried biofilms could have significance in explaining the natural distribution of this species in the intertidal.

Journal Article↗

PubMed2004

Bacterial biofilm presence in pediatric tracheotomy tubes.

OBJECTIVE: To determine whether bacterial biofilms are present on pediatric tracheotomy tubes. DESIGN: Prospective observational series. INTERVENTIONS: Eleven tracheotomy tubes removed during routine tracheotomy tube changes were analyzed for biofilm and live bacteria presence using confocal microscopy and vital stains. The external and internal surfaces of the tracheotomy tubes were studied in 3 locations: distal tip, midtracheotomy tube, and proximal opening. These data were correlated with tracheotomy site cultures and the reason for tracheotomy dependence. MAIN OUTCOME MEASURES: Microscopic images were analyzed for the presence of a biofilm (its morphological features and the presence of live and dead bacteria within the biofilm). RESULTS: Of 11 tracheotomy tubes, 10 had biofilm present on the internal surface of the distal tip. Externally, at the same location, 4 tubes had biofilms. On the internal surface of the midtracheotomy site, 8 had biofilm present, whereas only 1 had a biofilm on the internal surface of the proximal tracheotomy tube site. In the distal internal tracheotomy tube site, the biofilm was confluent in 5 tubes and patchy with evidence of microcolony formation in the remaining 5 tubes. Live bacteria were present in all biofilms. Control tracheotomy tubes did not have biofilms. All tracheotomy site cultures and disease states (chronic aspiration and bronchopulmonary dysplasia) were associated with tracheotomy tube biofilms. CONCLUSION: Bacterial biofilms containing live bacteria were demonstrated in most pediatric tracheotomy tubes, being most frequent and extensive on the internal surface of the distal tracheotomy tip.

Biofilms↗

PubMed2004

Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis.

OBJECTIVE: Oral streptococci are primary colonisers of the tooth surface and are abundant in dental plaque biofilms. Bacteria growing in these relatively dense, surface-associated communities are phenotypically quite distinct from their planktonic counterparts. The purpose of the present study was to develop a method to investigate biofilm-specific surface protein expression by Streptococcus sanguis to help provide a better understanding of the critical events in plaque development. DESIGN: Biofilm cells were grown on the surface of glass beads in a biofilm device fed with mucin-containing artificial saliva. Planktonic cells were grown in continuous culture at approximately the same growth rate. Surface hydrophobicity of biofilm and planktonic cells was determined by hexadecane partitioning, and expression of streptococcal fibronectin adhesin CshA was determined in ELISA using specific antiserum. Antisera raised to glutaraldehyde-fixed whole biofilm or planktonic grown cells were used to screen an expression library of S. sanguis genomic DNA, and isolated clones were sequenced. RESULTS: Phenotypic analysis of biofilm and planktonic cells confirmed that mode of growth affected surface properties of S. sanguis. Thus, hydrophobicity and CshA expression was significantly elevated in biofilm cells. Library screening with biofilm antiserum yielded 32 recombinant clones representing 21 different S. sanguis proteins involved in adhesion and colonisation, carbohydrate utilisation or bacterial metabolism. In differential analysis of four selected Escherichia coli clones, biofilm antiserum reacted five times stronger than planktonic antiserum with cell-free extracts of clones encoding homologues of CshA and Cna collagen adhesin of Staphylococcus aureus, suggesting that these surface proteins are up-regulated in biofilm cells. In contrast, both antisera reacted equally strongly with cell-free extracts of the remaining two clones (encoding dihydrofolate synthase and an unknown protein). CONCLUSIONS: The method described represents a useful means for determining bacterial protein expression in biofilms based on a combination of molecular and immunological techniques. Surface expression of putative fibronectin and collagen adhesins was up-regulated in biofilm cells.

Bacterial Adhesion↗

PubMed2004

Comparison of antibiotic susceptibility and plasmid content, between biofilm producing and non-producing clinical isolates of Pseudomonas aeruginosa.

Bacteria growing as biofilms are less susceptible to antimicrobial agents than free-living cells. Several infectious processes are now recognised to be related to biofilm formation. Pseudomonas aeruginosa is a known biofilm-producing organism and often resistant to several antibiotics. To assess the relationship between biofilm-forming capabilities and antibiotic resistance phenotypes in P. aeruginosa clinical isolates, we evaluated the ability of different phenotypes to form biofilms using 162 clinical isolates of P. aeruginosa. Only 14% of strains produced biofilm after an 8-h incubation and 8% after 24h; the latter group was slightly more multi-resistant, particularly to piperacillin-tazobactam and imipenem. The frequency of 8-h biofilm-formers was higher among isolates resistant to four agents, and that of 24-h biofilm-formers higher among isolates resistant to six antibiotics. Plasmid-content was not related to biofilm formation. In addition to the protection against antibiotics that bacteria gain by growing in a biofilm, clinical isolates forming biofilms seem to accumulate more resistance phenotypes. Bacteria in biofilms may survive antibiotic exposure not only because of the protective effect of biofilm itself, but also because of the increased frequency of resistance traits present.

Anti-Bacterial Agents↗

PubMed2006

The effect of a biofilm on solute diffusion in fractured porous media.

At sites in fractured rock where contamination has been exposed to the rock matrix for extended periods of time, the amount of contaminant mass residing in the matrix can be considerable. Even though it may be possible to diminish concentrations by the advection of clean water through the fracture features, back diffusion from mass held in the matrix will lead to a continuing source of contamination. In such an event, the development of a biofilm (a thin film of microbial mass) on the wall of the fractures may act to limit or prevent the back diffusion process. The objective of this preliminary study is to explore the influence imparted by the presence of a biofilm on the process of matrix diffusion. The investigation was conducted using radial diffusion cells constructed from rock core in which biofilm growth was stimulated in a central reservoir. Once biofilms were developed, forward diffusion experiments were conducted in which a conservative solute migrated from the central reservoir into the intact rock sample. Diffusion experiments were performed in a total of 11 diffusion cell pairs where biofilm growth was stimulated in one member of the pair and inhibited in the other. The effect of the presence of a biofilm on tracer diffusion was determined by comparison of the diffusion curves produced by each cell pair. A semi-analytical model that accounts for the presence of a biofilm was used to investigate the effect of the biofilm on mass transfer due to changes in the effective porosity, effective diffusion coefficient, and the depth of penetration of the biofilm into the intact rock. The results show that the biofilm acted to plug the rock matrix, rather than forming a discrete layer on the reservoir surface. The reduction in effective porosity due to the biofilm ranged from 6% to 52% with the majority of the samples in the 30% to 50% range. Based on the present results, with more efficient biofilm stimulation, it is reasonable to assume that a more complete plugging of the microcrack porosity might be possible, leaving a much thicker and efficient barrier than could be achieved via a surface biofilm.

Biofilms↗

PubMed2006

Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy.

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.

Bacteriological Techniques↗

PubMed2002

A photoacoustic technique for depth-resolved in situ monitoring of biofilms.

Biofilms occur in natural and engineered water systems. Biofouling in technical processes lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for removal of organic an inorganic pollutants. For improvement of antifouling strategies and for process optimization in wastewater treatments plants, an analytical technique for online monitoring of biofilms is needed. In this article, a new setup for in situ monitoring of biofilms by photoacoustic spectroscopy is presented. To produce a biofilm, a mixture of microorganisms was grown in a nutrient solution inside a tube reactor. The content of the tube reactor was pumped through a flow channel, and biofilms were generated at the inner surfaces. Three photoacoustic sensor heads were integrated at different positions into the base plate of the flow channel. By photoacoustic spectroscopy, growth, thickness, and detachment of biofilms can be monitored on-line and nondestructively. Experiments presented in this article showed that the flow conditions influence the structure and thickness of biofilms. By changing the pH value, electrostatic interactions inside the biofilm matrix were influenced, and the subsequent detachment processes were observed online. The interaction of iron(III) oxide particles with biofilms led to particle adsorption on the outer and inner surfaces of the biofilm. Afterwards, biofilm flocs were sloughed off from the base biofilm.

Acoustics↗

PubMed2004

Multipopulation model of membrane-aerated biofilms.

Biofilms cultivated on oxygen-filled gas-permeable membranes grow differently than conventional biofilms, as the chemical species required for growth diffuse from different sides of the biofilm. Oxygen is delivered directly to the base of the biofilm by the membrane, while organic substrates and other soluble nutrients are provided to the upper surface of the biofilm via the water in which the membranes are immersed. This counterdiffusion of nutrients results in a growth environment very different from that of conventional biofilms that receive both oxygen and other nutrients from the water. In recent years, membrane-supported biofilms have been shown to simultaneously remove chemical oxygen demand (COD) and inorganic nitrogen from wastewater in laboratory studies. Several investigators have developed computer models of these biofilms, but they have all focused on a single population of aerobic bacteria. While these models are useful in characterizing the behavior of these biofilms in pure cultures, they are not useful in modeling the behavior of the biofilms in mixed cultures such as those found in wastewater treatment. In this study, a multipopulation biofilm model was developed that includes aerobic heterotrophs, nitrifiers, denitrifiers, and acetoclastic methanogens. The model was constructed with Aquasim software and can predict the COD and inorganic nitrogen removal behavior observed previously in experimental studies. In this paper we present examples of predicted biofilm behavior and compare the results of this multiple-population model with the single-population models published previously. In addition, the behavior of the biofilm is discussed in terms of application to wastewater treatment.

Bacteria, Aerobic↗

PubMed2001

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms.

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.

Anti-Bacterial Agents↗

PubMed1999

Lactoferrin increases the susceptibility of S. epidermidis biofilms to lysozyme and vancomycin.

PURPOSE: The tear cationic protein lactoferrin increases the activity of various antimicrobial agents against suspended bacterial cultures including Staphylococcus epidermidis, the predominant causative agent of intraocular lens biofilm infections. We investigated the ability of lactoferrin to enhance the activity of vancomycin and lysozyme against biofilms of a clinical S. epidermidis isolate. METHODS: Biofilms were prepared on soft contact lenses and cells released from the biofilm surface were collected from the surrounding broth. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against the intact biofilm, released cells and suspended bacteria were evaluated using vancomycin with and without lactoferrin. RESULTS: Lactoferrin induced a two-fold reduction in the MBC of vancomycin for biofilm (p < 0.01) and biofilm-released cells from 64 microg/ml to 32 microg/ml and similarly decreased the MIC for biofilm-released cells (p < 0.01) from 32 microg/ml to 16 microg/ml. With or without lactoferrin, the MIC of vancomycin for suspended cells (2 and 4 microg/ml respectively) was less (p < 0.01) than that of both the biofilm (32 and 32 microg/ml) and biofilm-released cells (16 and 32 microg/ml). Lactoferrin did not reduce the MIC of lysozyme, but, at a lysozyme concentration of 16 mg/ml, did significantly (p < 0.05) reduce the number of viable biofilm and biofilm-released cells. CONCLUSIONS: Lactoferrin displays potential as an adjunctive agent to vancomycin in the treatment of S. epidermidis biofilm infections, such as endophthalmitis, associated with intraocular lenses.

Anti-Bacterial Agents↗

PubMed1995

Removal of Burkholderia cepacia biofilms with oxidants.

Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B. cepacia associated with both primary and secondary biofilms. The amount of biocide needed to inactivate 50% of planktonic B. cepacia in 10 min at 25 degrees C was 8.4, 0.5, and 0.2 mg l-1 for iodine, chlorine, and ozone, respectively. The data suggest that iodine maynot be the best chemical for treating of biofilms when removal of cellular material is required.

Bacterial Adhesion↗

PubMed2003

Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin.

OBJECTIVES: In Pseudomonas aeruginosa, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C12-HSL and C4-HSL. Previous studies suggested that C4-HSL plays no significant role in biofilm formation. However the wild-type PAO1 strain PAO-BI, used as a control in these studies is itself impaired in the production of C4-HSL. We wondered therefore whether the role of C4-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling. METHODS: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements. RESULTS: Wild-type strain PAO-BI, as a result of its reduced C4-HSL secretion, produced 40% less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a lasI mutant (deficient in 3-oxo-C12-HSL) produced similar amounts of biofilm to the wild-type, a rhlI mutant (deficient in C4-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C4-HSL. Azithromycin, known to reduce the production of both 3-oxo-C12-HSL and C4-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals. CONCLUSIONS: Our results indicate that C4-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formation.

4-Butyrolactone↗

PubMed2005

Mucosal biofilm formation on middle-ear mucosa in a nonhuman primate model of chronic suppurative otitis media.

BACKGROUND: An increased awareness of bacterial biofilms and their formation has led to a better understanding of bacterial infections that occur in the middle ear. Perhaps the best studied pathogen for its propensity toward biofilm formation is Pseudomonas aeruginosa, also the primary pathogen in chronic suppurative otitis media (CSOM). OBJECTIVE: The aim of this study was to determine whether P. aeruginosa forms a biofilm in the middle ear in the setting of CSOM in a nonhuman primate model. METHODS: Cynomolgus monkeys underwent perforation of the tympanic membrane and inoculation of the middle ear with a known biofilm-forming strain of P. aeruginosa. The contralateral ear was used as an internal control and was neither perforated nor infected. At the end of the study period, both ears were irrigated to remove planktonic bacteria, and the middle ear mucosa was removed and examined ultrastructurally using scanning electron microscopy (SEM) for determination of the presence or absence of biofilm formation. MAIN OUTCOME MEASURE: The identification of middle ear biofilm containing rod-shaped bacteria. RESULTS: SEM revealed that P. aeruginosa formed bacterial biofilm in vivo on the middle ear mucosal surface, seen only in the infected ear. Interestingly, biofilm formation caused by cocci was also seen in both the experimental as well as the control ear. CONCLUSION: P. aeruginosa forms biofilms in the middle ear in CSOM in primates. To our knowledge, this is the first report of disease-associated bacterial biofilm in a nonhuman primate model of CSOM. Such a model lays a foundation for much needed study into the role of biofilms in the pathophysiology of CSOM. Should CSOM be caused by biofilms, which is uncertain at this time, development of novel strategies for treatment and prevention may be possible. The finding of both rods and cocci forming biofilms also warrants further investigation.

Animals↗