Biofilms

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 102 - 7
{Aspects of Vibrio cholerae lipopolysaccharide}; Lobanov VV et al.; In this review information on the chemical structure, biosynthesis, antigenic and biological properties of V . cholerae lipopolysaccharide (LPS) is presented . The specific structural feature of this LPS is a small size of the polysaccharide chain of O-antigen . In vibrios of serogroup O 139 it is oligosaccharide . The modification of the O-chain (methylation of individual sugars, shortened chain, etc.) plays an essential role in the antigenic specificity of V . cholerae LPS . All these factors affect of endotoxin function, the microbial resistance to external influences . V . cholerae LPS takes part in the formation of microcapsules and biofilms . The evolutional development of V . cholerae in this direction determines, to some extent, their increased resistance in the environment . In human body the heterogeneity of the LPS composition permits the preservation of vibrios and ensures, together with cholerogen, their pathogenetic action.

Microb Ecol, 2002 May, 43(4), 416 - 23 Epub 2002 Apr 08.
Elongation correlates with nutrient deprivation in Pseudomonas aeruginosa-unsaturates biofilms; Steinberger RE et al.; Bacteria in nature frequently grow as biofilms, yet little is known regarding how biofilm bacteria morphologically adapt to low nutrient availability, which is common in unsaturated environments such as the terrestrial subsurface or on plant leaves . For unsaturated biofilms, in which the substratum may provide all nutrients, what are the relationships between nutrition and cell size and shape-the simplest metrics of cellular morphology? To address this question, we cultured Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium that is environmentally and medically important, on membranes overlaying solid media, and then measured cellular dimensions using atomic force microscopy (AFM) . Nutrition was controlled chemically by media composition and physically by stacking membranes to increase the path length for nutrient diffusion . Under conditions of carbon-nitrogen imbalance, low carbon bioavailability, or increased nutrient diffusional path length, cells elongated while maintaining constant width . A mathematical relationship suggests that, by elongating, biofilm bacteria strategically enlarge their nutrient collection surface without substantially changing the ratio of surface area to volume (SA/V) . We conclude that P . aeruginosa growing as unsaturated biofilm with a planar nutrient source morphologically adapt to starvation by elongating . This adaptation, if generalizable, differs from a better-understood starvation response (i.e., cell size decreases; thus SA/V in-creases) for planktonic bacteria in well-mixed environments.

J Antimicrob Chemother, 2002 Jun, 49(6), 973 - 80
Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms; Ramage G et al.; A main characteristic associated with microbial biofilms is their increased resistance to antimicrobial chemotherapies . However, at present very little is known about the phenotypic changes that occur during the transition from the planktonic to the biofilm mode of growth . Candida albicans biofilms displayed an organized three-dimensional structure, and consisted of a dense network of yeasts and filamentous cells deeply embedded in exopolymeric matrix . These biofilms were intrinsically resistant to fluconazole . Moreover, the resistance phenotype was maintained by sessile cells when resuspended as free-floating cells, thus demonstrating that biofilm integrity and the presence of exopolymeric material are not the sole determinants of biofilm resistance . Under planktonic conditions, one of the main mechanisms of azole resistance in C . albicans is through active efflux of these drugs mediated by ATP-binding cassette (ABC) transporters and major facilitators . In this study we used northern hybridization to monitor expression of genes belonging to two different types of efflux pump, the ABC transporters and major facilitators (encoded by CDR and MDR genes, respectively), in C . albicans populations under both planktonic and biofilm growth . It was demonstrated that expression of genes encoding both types of efflux pump were up-regulated during the course of biofilm formation and development . Moreover, antifungal susceptibilities of biofilms formed by a set of C . albicans mutant strains deficient in efflux pumps were investigated to determine their contribution to biofilm resistance . Remarkably, mutants carrying single and double deletion mutations in Delta(cdr)1, Delta(cdr)2, Delta(mdr)1, Delta(cdr)1/Delta(cdr)2 and Delta(mdr)1/Delta(cdr)1 were hypersusceptible to fluconazole when planktonic, but still retained the resistant phenotype during biofilm growth . These analyses demonstrate that C . albicans biofilm resistance is a complex phenomenon that cannot be explained by one mechanism alone, instead it is multifactorial and may involve different molecular mechanisms of resistance compared with those displayed by planktonic cells.

Appl Environ Microbiol, 2002 Jun, 68(6), 2972 - 81
Experimental study of interactions between purple and green sulfur bacteria in sandy sediments exposed to illumination deprived of near-infrared wavelengths; Masse A et al.; Sedimentary biofilms of the green sulfur bacterium Prosthecochloris aestuarii strain CE 2404, the purple sulfur bacterium Thiocapsa roseopersicina strain 5811, and a mixed culture of both were cultured in fine sand (100- to 300-microm grain size) within counter gradients of oxygen and sulfide . The artificial sediments were exposed to illumination deprived of near-infrared light (NIR) by filtering out the wavelengths longer than 700 nm to simulate the critical light conditions in submerged aquatic sediments . A 16 h of visible light-8 h of dark regimen was used . We studied the effects of these light conditions on the metabolisms of and interactions between both species by comparing the single species biofilms with the mixed biofilm . The photosynthesis rates of P . aestuarii were shown to be highly limited by the imposed light conditions, because the sulfide photooxidation rates were strongly stimulated when NIR was added . T . roseopersicina performed both aerobic chemosynthesis and photosynthesis, but the photosynthesis rates were low and poorly stimulated by the addition of NIR . This species decreased the penetration depth of oxygen in the sediment by about 1 mm by actively respiring oxygen . This way, the strict anaerobe P . aestuarii was able to grow closer to the surface in the mixed culture . As a result, P . aestuarii benefited from the presence of T . roseopersicina in the mixed culture, which was reflected by an increase in the biomass . In contrast, the density of the latter species was almost completely unaffected by the interaction . Both species coexisted in a layer of the same depth in the mixed culture, and the ecological and evolutionary implications of coexistence are discussed.

Appl Environ Microbiol, 2002 Jun, 68(6), 2950 - 8
Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation; Djordjevic D et al.; Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination . The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L . monocytogenes strains to form biofilms . A total of 31 coded L . monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells . Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution . Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences . The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains . The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy . Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.

Appl Environ Microbiol, 2002 Jun, 68(6), 2829 - 37
Species diversity improves the efficiency of mercury-reducing biofilms under changing environmental conditions; Von Canstein H et al.; Six mercury-resistant environmental proteobacterial isolates and one genetically modified mercury-resistant Pseudomonas putida strain were analyzed for physiological traits of adaptive relevance in an environment of packed-bed bioreactors designed for the decontamination of mercury-polluted chlor-alkali wastewater . The strains displayed characteristic differences in each trait (i.e., biofilm formation capability, growth rate in mercury contaminated wastewaters, and mercury reduction efficiency) . Subsequently, they were immobilized either as a monoculture or as a mixed culture on porous carrier material in packed-bed bioreactors through which different batches of filter-sterilized industrial chlor-alkali wastewater were pumped . In monospecies bioreactors, the mercury retention efficiency was sensitive to rapidly increasing mercury concentrations in the wastewater . Mixed culture biofilms displayed a high mercury retention efficiency that was not affected by rapid increases in mercury or continuously high mercury concentrations . The dynamic in the community composition of the mixed culture bioreactors was determined by ribosomal intergenic spacer polymorphism analysis . Mercury-mediated selective pressure decreased the number of prevalent strains . Microbial diversity was completely restored after easing of the selective pressure . Microbial diversity provides a reservoir of strains with complementary ecological niches that results in a superior bioreactor performance under changing environmental conditions.

Appl Environ Microbiol, 2002 Jun, 68(6), 2770 - 80
Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation; Oosthuizen MC et al.; Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods . B . cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface . Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool) . Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins . Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins . Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence . Among the proteins were catabolic ornithine carbamoyltransferase and L-lactate dehydrogenase . Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both sigma(B) and sigma(H), could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype . Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.

Adv Space Res, 2000, 26(12), 2015 - 9
Monitoring of biologically effective UV irradiance at El Arenosillo (INTA), Andalucia, in Spain; de la Torre R et al.; Biological UV (ultraviolet) dosimetry was applied using the biofilm-technique (DLR patent) to determine the UV levels weighted of biologically weighted UV radiation at the INTA Sounding Station of El Arenosillo at Huelva, Spain (37 degrees 06'N, 6 degrees 44'W, 50 m a s 1=above sea level) on 2 days in 1997 {correction of 1977} (April 1, and May 5) . Exposure periods were calculated for clear sky days using a radiative transfer model for erythemal doses to reach 1.3 to 1.5 MED (minimal erythemal dose) . Reliability of the radiative transfer model was demonstrated by the doses registered by a Yankee-UV biometer for the same exposure periods as used for the biosensor . This work presents the methodology employed (biofilm-technique utilized {correction of utiliced}, calculation of exposing periods with radiative transfer model, etc) and the results obtained with the Yankee biometer and the biofilm . At noon, the ratio of biofilm measurements (Ieff, W/m2=biological effective irradiance, in W/m2) to the UV Biometer data (in MED/h) was 3-4 . c2001 COSPAR Published by Elsevier Science Ltd . All rights reserved.

Adv Space Res, 2000, 26(12), 2005 - 14
Biologically weighted measurement of UV radiation in space and on Earth with the biofilm technique; Rettberg P et al.; Biological dosimetry has provided experimental proof of the high sensitivity of the biologically effective UVB doses to changes in atmospheric ozone and has thereby confirmed the predictions from model calculations . The biological UV dosimeter 'biofilm' whose sensitivity is based on dried spores of B . subtilis as UV target weights the incident UV radiation according to its DNA damaging potential . Biofilm dosimetry was applicated in space experiments as well as in use in remote areas on Earth . Examples are long-term UV measurements in Antarctica, measurements of diurnal UV profiles parallel in time at different locations in Europe, continuous UV measurements in the frame of the German UV measurement network and personal UV dosimetry . In space biofilms were used to determine the biological efficiency of the extraterrestrial solar UV, to simulate the effects of decreasing ozone concentrations and to determine the interaction of UVB and vitamin D production of cosmonauts in the MIR station . c2001 COSPAR Published by Elsevier Science Ltd . All rights reserved.

Nature, 2002 May 30, 417(6888), 552 - 5
A component of innate immunity prevents bacterial biofilm development; Singh PK et al.; Antimicrobial factors form one arm of the innate immune system, which protects mucosal surfaces from bacterial infection . These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent acute invasive infections . In many chronic infections, however, bacteria live in biofilms, which are distinct, matrix-encased communities specialized for surface persistence . The transition from a free-living, independent existence to a biofilm lifestyle can be devastating, because biofilms notoriously resist killing by host defence mechanisms and antibiotics . We hypothesized that the innate immune system possesses specific activity to protect against biofilm infections . Here we show that lactoferrin, a ubiquitous and abundant constituent of human external secretions, blocks biofilm development by the opportunistic pathogen Pseudomonas aeruginosa . This occurs at lactoferrin concentrations below those that kill or prevent growth . By chelating iron, lactoferrin stimulates twitching, a specialized form of surface motility, causing the bacteria to wander across the surface instead of forming cell clusters and biofilms . These findings reveal a specific anti-biofilm defence mechanism acting at a critical juncture in biofilm development, the time bacteria stop roaming as individuals and aggregate into durable communities.

Caries Res, 2002 Mar-Apr, 36(2), 108 - 15
Influence of antibody on the structure of glucans; Kopec LK et al.; Glucosyltransferase (GTF) plays an essential role in the formation of the biofilm known as dental plaque and in the pathogenesis of dental caries . Mutans streptococci produce at least three distinct GTFs (GtfB, C and D), each of which forms a glucan polymer from sucrose . Glucan is a major constituent of plaque biofilm . GTF adsorbed to a surface forms glucans that differ in structure from those formed by the same enzyme in solution . In the present study, activities of GtfB and GtfC in solution or adsorbed on a surface were inhibited in the presence of a polyclonal antiserum (DS-1) to a mixture of GTFs and by immunoglobulin G (IgG) prepared from DS-1; in contrast, enzyme activity was enhanced by normal rabbit serum (NRS) and IgG from NRS . GtfD activity on a surface was enhanced by both antiserum DS-1 and NRS, and IgG prepared from either serum; GtfD activity in solution was slightly inhibited by each of the sera . The structure of GtfB and GtfC glucans formed in the presence of antiserum differed from that of controls based on linkage analyses, and on their susceptibilities to the glucanohydrolases mutanase (alpha-1,3 hydrolase) and dextranase (alpha-1,6 hydrolase); soluble products from the enzymatic digestion also differed . The results show that the effects of antibody on enzyme activity are more complex than simple inhibition or enhancement and that the presence of antibody may influence glucan structure, which clearly could impact plaque formation . The results have implications for the formation and properties of biofilms formed in other environments .

Caries Res, 2002 Mar-Apr, 36(2), 93 - 100
An in vitro oral biofilm model for comparing the efficacy of antimicrobial mouthrinses; Shapiro S et al.; The ability of commercial mouthrinses to reduce total viable counts of mixed microbial populations was examined using a previously developed in vitro model of supragingival plaque . Exploratory experiments aimed at fine-tuning the model indicated that optimal correspondence between in vitro and clinical results for chlorhexidine-containing formulations were obtained at a saliva:medium ratio of 70:30 (v/v); moreover, expanding the microbial population from 5 bacterial species to 5 bacterial species + Candida albicans had no noticeable impact on overall results . The efficacies of 12 different mouthrinse proprietary products containing chlorhexidine, hexetidine, octenidine, Triclosan, plant extracts, or aminefluoride/stannous fluoride vis-a-vis biofilm clearance were compared . All mouthrinses promoted a statistically significant reduction in microbial load compared to distilled water . The herbal- and phenolic-based products were substantially less effective than most chlorhexidine-containing mouthrinses, or mouthrinses containing hexetidine or octenidine . No significant difference between the plaque-clearing plaque-clearing abilities of Listerine and Meridol was observed . This polyspecies biofilm model can be a valuable tool for preclinical testing of antiplaque formulations, particularly during the product development stage .

Caries Res, 2002 Mar-Apr, 36(2), 87 - 92
The effect of sucrose application frequency and basal nutrient conditions on the calcium and phosphate content of experimental dental plaque; Pearce EI et al.; A reduced pool of calcium in dental plaque would be expected to increase the ability of plaque fluid to dissolve the underlying enamel when the pH falls during sugar exposure . We have examined the relationship between frequency of sugar application and Ca and P(i) concentrations in artificial mouth plaque microcosm biofilms . Ten plaques were grown simultaneously from a human saliva inoculum using a continuous flow of simulated saliva, DMM, supplemented with either urea or glucose to modulate the resting pH . In addition the plaques received sucrose applications of varying frequency: 12-, 8-, 6-, or 4-hourly, or not at all . After 15 days the plaques were sampled by taking 4 full-thickness specimens of each, and acid-extractable Ca and P(i), and alkali-soluble protein and carbohydrate were determined . Ca and P(i) concentrations were in a range comparable with those in human plaque, except in the DMM + urea plaque receiving no sucrose, when concentrations were higher . Plaque Ca concentration decreased significantly as sucrose application frequency increased . Increasing sucrose application frequency also reduced the protein, i.e . the cell biomass, content of the plaques and, in the case of DMM + urea plaques, increased the water-insoluble hexose content, presumably extracellular polysaccharide . Reduced biomass was partly due to the bulking of plaque with extracellular polysaccharide, but the marked effect of urea on polysaccharide formation is not understood . This study shows that increasing frequency of sugar application alters dental plaque by reducing its mineral protection capacity .

Caries Res, 2002 Mar-Apr, 36(2), 81 - 6
Effects of glucose and fluoride on competition and metabolism within in vitro dental bacterial communities and biofilms; Bradshaw DJ et al.; Antimicrobial effects of fluoride in vivo remain contentious . Previous studies suggested that 1 mM NaF reduced acid production from glucose, and prevented the enrichment of bacteria associated with caries in a chemostat model . The present study examines the effects of a lower fluoride concentration (0.53 mM, 10 ppm NaF) in both biofilm and planktonic microbial communities . Nine oral species were grown at pH 7.0 and pulsed on 10 successive days with glucose; bacterial metabolism was allowed to reduce the pH for 6 h before being returned to neutrality, either in the presence or absence of NaF . In addition, 10-day-old mixed culture biofilms were overlaid with glucose, with or without NaF, and the pH change followed by microelectrode . After 10 days, chemostat pH dropped to ca . pH 4.5 following glucose pulses, and the community was dominated by Streptococcus mutans (rising from 4 to 23% of total CFU) and Veillonella dispar (16 to 73%) . In comparison, after 10 days pulsing with glucose + fluoride, the final pH was significantly higher (ca . pH 4.9) (paired t test, p < 0.0001) . The culture was predominated by V . dispar (70%) and Actinomyces naeslundii (13%), whereas S . mutans proportions were significantly lower (t test, p = 0.04), remaining <3% of the total flora, compared to the culture without fluoride . Biofilm pH fell to only pH 5.55 1 h after glucose/fluoride overlay, compared to 4.55 with glucose alone (paired t test, p < 0.000001) . Analysis of the data suggests that fluoride exerts dual antimicrobial modes of action . Fluoride prevents enrichment of S . mutans by inhibiting critical metabolic processes (direct effect) and, in an inter-related way, by reducing environmental acidification (indirect effect) in biofilms .

Microb Ecol, 2001 Jul, 42(1), 56 - 68
Assessment of Changes in Biodiversity when a Community of Ultramicrobacteria Isolated from Groundwater Is Stimulated to Form a Biofilm; Ross N et al.; The stimulation of groundwater bacteria to form biofilms, for the remediation of polluted aquifers, is subjected to environmental regulations that include measurement of effects on microbial biodiversity . Groundwater microorganisms contain a proportion of unidentified and uncharacterized ultramicrobacteria (UMB) that might play a major role in the bioclogging of geological materials . This study aimed to assess the changes in genetic and metabolic biodiversity when a community of UMB, isolated from groundwater, is stimulated to form biofilms on a ceramic surface . UMB were stimulated with aerobic conditions and injection of molasses, in reactors reproducing groundwater composition and temperature . Concentration of planktonic viable UMB, secretion of extracellular polymeric substances (EPS), and biofilm thickness were monitored . The assessment of changes in biodiversity was achieved by comparing the initial UMB community to the biofilm community, using the single strand conformational polymorphism (SSCP) method, the cloning and sequencing of 16S rRNA gene (16S rDNA) sequences, and the Biolog microplate system . The hypothesis stating that indigenous UMB would play a significant role of in the biofilm development was corroborated . Within 13 days of stimulation, the UMB produced 700 mg L?1 of planktonic EPS and formed a biofilm up to a thickness of 1100 mm . This stimulation led to a decrease in genetic diversity and an increase in metabolic diversity . The decrease in genetic diversity was shown by a reduced number of single strand DNA fragments in the SSCP profiles . As such, 16S rDNA sequences from the biofilm revealed the predominance of four bacterial groups: Zoogloea, Bacillus/Paenibacillus, Enterobacteriaceae, and Pseudomonads . A significant increase in metabolic diversity was shown by a highest substrate richness profile and a lower substrate evenness profile of the biofilm bacterial population (p = 0.0 and p = 0.09, respectively) . This higher metabolic diversity might be a consequence of the stimulation that seemed to favor the growth of bacteria having a high nutritional versatility . Stimulation of UMB, isolated from groundwater, was effective to form a biofilm having a high metabolic biodiversity . This combination of molecular-based and metabolic-based methods expanded the insight into monitoring the changes in bacterial biodiversity.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 8312 - 7 Epub 2002 May 28.
Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis; Ochsner UA et al.; A novel secretion pathway originally found in plants has recently been discovered in bacteria and termed TAT, for "twin-arginine translocation," with respect to the presence of an Arg-Arg motif in the signal sequence of TAT-secreted products . However, it is unknown whether the TAT system contributes in any way to virulence through the secretion of factors associated with pathogenesis or stress response . We found that the opportunistic pathogen Pseudomonas aeruginosa produces several virulence factors that depend on the TAT system for proper export to the periplasm, outer membrane, or extracellular milieu . We identified at least 18 TAT substrates of P . aeruginosa and characterized the pleiotropic phenotypes of a tatC deletion mutant . The TAT system proved essential for the export of phospholipases, proteins involved in pyoverdine-mediated iron-uptake, anaerobic respiration, osmotic stress defense, motility, and biofilm formation . Because all these traits have been associated with virulence, we studied the role of TAT in a rat lung model . A tatC mutant did not cause the typical multifocal pulmonary abscesses and did not evoke a heavy inflammatory host response compared with wild type, indicating that tatC mutant cells are attenuated for virulence . Because the TAT apparatus is well conserved among important bacterial pathogens yet absent in mammalian cells, it represents a potential target for novel antimicrobial compounds.

Microb Ecol, 2001 Feb, 41(2), 124 - 131
Population Dynamics of Two Toluene Degrading Bacterial Species in a Contaminated Stream; Tay ST et al.; Toluene uptake by a benthic biofilm community was previously shown to vary seasonally from 0.03 m hr?1 in winter to 0.2 m hr?1 in summer in a solvent-contaminated stream of the Aberjona watershed . We used quantitative PCR to estimate the population dynamics of previously isolated species of toluene-degrading Xanthobacter autotrophicus and Mycobacterium sp . in both toluene-contaminated and uncontaminated reaches of the stream, and to estimate their relative roles in overall biodegradation rate . Quantification using specific 16S rDNA primers forX . autotrophicus and Mycobacterium sp . showed that populations of both species were much larger in the toluene-contaminated than the toluene-free reach, in agreement with earlier culture-based investigations . A relatively brief bloom of X . autotrophicus occurred in the contaminated reach in the summer, while Mycobacterium sp . populations occurred at elevated densities for more than 5 months . Calculations showed that Mycobacterium, previously thought to be less important than Xanthobacter in annual toluene degradation based on single time-point CFU estimates, appears actually more important because of this longer persistence.

J Microbiol Methods, 2002 Aug, 50(3), 237 - 48
Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa; Strathmann M et al.; Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa . A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used . Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate . Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain . The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions . In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells . Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase . The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P . aeruginosa biofilms.

Can J Microbiol, 2002 Apr, 48(4), 326 - 32
Impact of pulsed Nd:YAG laser on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora: significance of physiological status; Nandakumar K et al.; The impact of pulsed Nd:YAG (neodymium-doped yttrium/aluminium garnet) laser irradiation on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora during two growth stages (log phase and stationary phase) and under two stresses (reduced temperature and nutrient limitation) was investigated . Bacteria were exposed to a laser fluence of 0.1 J x cm(-2) for 5, 10, and 15 min with a peak power of 20 MW x cm(-2), a pulse width of 5 ns, and an average power of 1 W x cm(-2) with a repetition rate of 10 Hz . The mortality of bacteria immediately after the irradiation as well as after a set period of time was determined . Mortality was higher among log-phase bacteria (72%) than bacteria in the stationary phase (51%) and those grown under nutrient limitation (51%) . Bacteria grown at reduced temperature had a mortality of 49% . However, the differences in cell density of log-phase, stationary-phase, nutrient-limited, and low-temperature irradiated samples compared with controls after 5 h of incubation were 96, 93, 94, and 86%, respectively . The mortality values suggest that the same laser fluence has different degrees of effectiveness, depending on the physiological state of the bacteria.

J Bacteriol, 2002 Jun, 184(12), 3406 - 10
Catabolite repression of Escherichia coli biofilm formation; Jackson DW et al.; Biofilm formation was repressed by glucose in several species of Enterobacteriaceae . In Escherichia coli, this effect was mediated at least in part by cyclic AMP (cAMP)-cAMP receptor protein . A temporal role for cAMP in biofilm development was indicated by the finding that glucose addition after approximately 24 h failed to repress and generally activated biofilm formation.

Lett Appl Microbiol, 2002, 34(6), 450 - 4
Detection of Helicobacter pylori DNA in drinking water biofilms: implications for transmission in early life; Bunn JE et al.; AIMS: To provide evidence of water quality as a risk factor for acquisition of Helicobacter pylori in early life, and to identify evidence for its presence within pots used to store drinking water . METHODS AND RESULTS: A prospective cohort study of 65 infants was conducted in the rural village of Keneba, The Gambia . Age of H . pylori colonization was determined and water pot biofilms were tested for H . pylori by sequencing of amplified DNA . Use of supplemental water was a strong risk factor for H . pylori colonization in infants (OR 4.71, 95% CI 1.17-22.5) . DNA with 95% homology to the 16S rRNA gene of H . pylori was isolated from biofilms of water pots . CONCLUSIONS: Drinking water may be a reservoir for H . pylori in areas of the developing world where water quality is poor . Early introduction of water, particularly if stored in, or collected from contaminated sources, may be associated with an increased rate of H . pylori colonization.

Microb Ecol, 2001 Oct, 42(3), 328 - 337
Effects of Photosynthesis on Bacterial Phosphatase Production in Biofilms; Espeland EM et al.; The fraction of bacteria displaying phosphatase activity within natural photosynthetic biofilms was examined in relation to phosphorus limitation and algal photosynthesis . An artificial substrate that forms a fluorescent precipitate was used in conjunction with the nucleic acid stain DAPI to enumerate extracellular phosphatase expression by biofilm bacteria exposed to different photosynthetic activities and phosphorus supplies . The proportion of bacteria displaying phosphatase activity changed in response to the presence or absence of algal photosynthesis . In general, phosphate-deprived biofilms had positive linear trends in bacterial phosphatase activity (p <0.001), with greater proportions of bacteria displaying phosphatase under photosynthetic inhibition compared to active photosynthesis . Under sufficient phosphate supplies, biofilms had negative linear trends (p <0.05) or were lower in the proportion of bacteria displaying phosphatase activity in the presence of algal photosynthesis, whereas bacterial phosphatase activity was generally maintained when photosynthesis was inhibited . it is suggested that the amount of extracellular organic carbon released within the biofilm matrix during photosynthesis indirectly affected bacterial phosphatase synthesis.

Microb Ecol, 2001 Dec, 42(4), 624 - 634
Long-Term Stability of Mercury-Reducing Microbial Biofilm Communities Analyzed by 16S-23S rDNA Interspacer Region Polymorphism; Canstein HF et al.; The composition of mercury-reducing communities in two bioreactors retaining Hg(II) from chloralkali electrolysis wastewater for 485 days was analyzed based on effluent community DNA . Packed bed bioreactors with lava chips as carrier of the biofilm were inoculated with nine Hg(II)-resistant isolates that belonged to the alpha and gamma subdivisions of the proteobacteria . A rapid DNA-fingerprinting method was applied, using the intergenic spacer region (ISR) of the 16S-23S rDNA for analysis of the community composition . This allowed discrimination of the inoculum strains down to subspecies level . A merA specific PCR permitted the discrimination of the community's merA genes . During the 485 days of operation, the bioreactors were exposed to various physical stresses (mixing, gas bubbles, temperature increase up to 41 degrees C, increased flow velocity) and repeated high mercury inflow concentrations, resulting in reduced bioreactor performance and decreased culturable cell numbers in the reactor effluent . Nevertheless, the composition of the microbial community remained rather stable throughout the investigated time period . Of the inoculum strains, two could be detected throughout, whereas three were sometimes present with varying periods of nondetection . Two inoculum strains were only detected within the first month . Two strains of gamma-proteobacteria that were able to reduce ionic mercury invaded the bioreactor community . They did not outcompete established strains and had no negative effect on the Hg(II)-retention activity of the bioreactors . The community comprised diverse merA genes . The abundance of merA genes matched the abundance of their respective strains as confirmed by ISR community analysis . The continuously high selection pressure for mercury resistance maintained a stable and highly active mercury-reducing microbial community within the bioreactors.

Microb Ecol, 2001 Dec, 42(4), 572 - 585
Complexation, Stabilization, and UV Photolysis of Extracellular and Surface-Bound Glucosidase and Alkaline Phosphatase: Implications for Biofilm Microbiota; Espeland EM et al.; Biofilm-produced and commercially-purified a- and b-glucosidase and alkaline phosphatase were subjected to different spectral portions of natural and artificial light and exposed to various humic substances to elucidate their impact on enzyme activities . Photochemical degradation of all enzymes occurred under different portions of the light spectrum . UVB irradiance produced the greatest overall photochemical degradation of enzymes, with significant rates occurring with UVA and PAR irradiance . The complexation of enzymes with humic substances resulted in inhibition, stabilization, and photochemical protection of the enzyme . Inhibition of enzyme activity occurred via reductions in overall enzyme activity in the presence of humic substances . However, humic-enzyme complexation also resulted in stabilization by restricting enzyme degradation while retaining high activities . Enzymes exposed to natural and artificial light sources had significantly lower reductions in enzyme activities in the presence of humic substances, which indicates that humic-enzyme complexes may protect enzymes from light-induced photochemical degradation . Bacterial surface-bound a- and b-glucosidase activities were significantly reduced in the presence of humic substances . Photosynthetically induced pH changes within biofilm communities can cause large reductions in a- and b-glucosidase activities while enhancing the hydrolytic activity of alkaline phosphatase.

Microb Ecol, 2001 Dec, 42(4), 524 - 530
Influence of Algal Photosynthesis on Biofilm Bacterial Production and Associated Glucosidase and Xylosidase Activities; Espeland EM et al.; Natural photosynthetic biofilms were incubated under light (100 mmol m-2 s-1) and dark conditions to elucidate the impact of photosynthesis on bacterial production, abundance, biovolume, biomass, and enzyme activities over 24 h . Use of organic carbon-free media limited carbon sources to algal photosynthesis and possibly the polysaccharides of the biofilm matrix . Bacterial production of biofilm communities was significantly higher in light incubations (p <0.001) . The greatest differences in production rates between light and dark incubations occurred between 8 and 24 h . Biomass-specific a- and b-glucosidase and b-xylosidase activities were stimulated by photosynthesis, with significantly greater activities occurring at hours 16 and 24 in the light treatment (p <0.01) . The results indicate that algal photosynthesis can have a significant impact on bacterial productivity, biomass, biovolume, and enzyme production over longer time periods at low photon flux densities (?100 mmol m-2 s-1).

Mol Cell Biol, 2002 Jun, 22(12), 3994 - 4000
Snf1 protein kinase and the repressors Nrg1 and Nrg2 regulate FLO11, haploid invasive growth, and diploid pseudohyphal differentiation; Kuchin S et al.; The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth . We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth . We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11 . We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes . Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation . Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes . These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

Microb Ecol, 2002 Mar, 43(2), 232 - 41 Epub 2002 Feb 21.
Large differences in the fraction of active bacteria in plankton, sediments, and biofilm; Haglund AL et al.; Generally, only a small fraction of free-living pelagic bacteria are metabolically active, while particle-associated bacteria usually exhibit a larger proportion of active bacteria . Most previous studies on the active fraction of bacteria focus on planktonic communities, and there are only a few studies on sediment and epiphytic biofilm bacteria . We compared the active fraction of the total number of bacteria in three different habitats of the littoral zone of Lake Erken, Sweden, including the sediments, the epiphytic biofilm on the submerged macrophyte Ranunculus circinatus, and the water column . Active bacteria were detected as those with an active electron transport system, identified by the capacity to reduce the tetrazolium salt CTC (5-cyano-2,3-ditolyltetrazolium chloride) into its fluorescent, water insoluble state . There were large differences between habitats . The active fraction of the total number of bacteria detected by fluorescence microscopy (annual mean +/- SD) in the sediments was 46 +/- 10%, on R . circinatus 37 +/- 18%, and in the water column 4 +/- 4% . The abundance of CTC-reducing cells was correlated with total bacterial abundance, and the fraction of CTC-reducing bacteria generally increased with total bacterial abundance, for all the habitats . Consequently, the difference in the fraction of CTC-reducing bacteria between the habitats could be attributed to different densities of bacteria, with a larger proportion of active bacteria at higher bacterial densities.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 25 - 31
Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis; Tremoulet F et al.; The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria . The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification . Thirty-one proteins varied significantly between the two growth conditions . Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified . The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology.

Genetics, 2002 May, 161(1), 33 - 46
Adaptive divergence in experimental populations of Pseudomonas fluorescens . I . Genetic and phenotypic bases of wrinkly spreader fitness; Spiers AJ et al.; A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known . When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge . Among the divergent morphs is a mutant type termed "wrinkly spreader" (WS) that colonizes a new niche through the formation of self-supporting biofilms . Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS(-)) mutants . Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP) . WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS . A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation . Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1773 - 80
Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins; Kuhn DM et al.; Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents . Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs . We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model . In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms . We also explored effects of preincubation of C . albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation . Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure . Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays . Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candida isolates examined when grown as biofilms, compared to planktonic forms . In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex {ABLC}) and echinocandins (caspofungin {Casp} and micafungin) showed activity against Candida biofilms . Preincubation of C . albicans cells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P < 0.0005) . CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent . In conclusion, our data show that Candida biofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 591 - 8
Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor: increase in sugar utilization; Lienhardt J et al.; Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101 . To achieve high reactor productivity, C . beijerinckii BA101 cells were immobilized by adsorption onto clay brick . The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration . Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%) . To increase sugar utilization, the reactor effluent was recycled . However, this approach is complicated by butanol toxicity . The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor . Recycling of butanol-free effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2 g/(L x h) at a dilution rate of 1.5 h(-1) . A dilution rate of 2.0 h(-1) resulted in a reactor productivity of 16.2 g/(L x h) and sugar utilization of 101.4% . It is anticipated that this reactor-recovery system would be economical for butanol production when using C . beijerinckii BA101.

J Dent Educ, 2002 Apr, 66(4), 549 - 55
How does time-dependent dental unit waterline flushing affect planktonic bacteria levels?
Cobb CM, Martel CR, McKnight SA 3rd, Pasley-Mowry C, Ferguson BL, Williams K.
The purpose of this study was to evaluate how time-dependent waterline flushing affects the presence of biofilm in otherwise-untreated dental unit waterlines (DUWLs) . Water samples were obtained from twelve highspeed handpiece DUWLs located in the undergraduate treatment clinic at the University of Missouri-Kansas City, School of Dentistry . Baseline water samples (50 cc) were collected prior to the start of continuous flushing . Additional 50 cc samples were collected after two-, three-, and four-minute flushing intervals from the baseline . The levels of planktonic bacteria in DUWLs were quantified by counting colony forming units (CFUs) . In addition, segments of water tubing from each of the highspeed handpiece waterlines were examined by scanning electron microscopy, which confirmed the presence of a residual biofilm in the lumen of each dental unit waterline . A one-factor repeated measures ANOVA showed a statistically significant (p<0.01) reduction in CFUs at all intervals compared to baseline and between each successive time interval . Indeed, after four minutes of continuous flushing, all waterlines still harbored CFU levels that exceed current American Dental Association (ADA) recommendations . It was concluded that water flushing of DUWLs produced a statistically significant reduction in planktonic bacteria at each time interval compared to the baseline and between each successive time interval . However, the level of CFUs after four minutes of continuous water flushing still exceeds the current ADA recommendations for acceptable levels of microorganisms.

Gen Dent, 2001 Jul-Aug, 49(4), 421 - 5
Effect of dental unit waterline biocides on enamel bond strengths; Taylor-Hardy TL et al.; This study evaluated the effects of chemical biocides used to control dental unit waterline biofilm on the bond strength of resin to enamel . Sixty bovine teeth were randomly assigned to six treatment groups . One-way ANOVA revealed a significant difference in means (p < 0.001) and Tukey's multiple range test indicated that three of the experimental groups had significantly lower mean shear bond strengths than the control (p < 0.05) . This finding suggests that dental unit waterline biocides may adversely affect adhesion of resin to enamel.

Appl Microbiol Biotechnol, 2002 May, 58(6), 853 - 9 Epub 2002 Mar 08.
Biodegradation of phenol in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1 immobilized in an air-stirred reactor with clarifier; Prieto MB et al.; Phenol biodegradation by suspended and immobilized cells of Rhodococcus erythropolis UPV-1 was studied in discontinuous and continuous mode under optimum culture conditions . Phenol-acclimated cells were adsorbed on diatomaceous earth, where they grew actively forming a biofilm of short filaments . Immobilization protected cells against phenol and resulted in a remarkable enhancement of their respiratory activity and a shorter lag phase preceding active phenol degradation . Under optimum operation conditions in a laboratory-scale air-stirred reactor, the immobilized cells were able to completely degrade phenol in synthetic wastewater at a volumetric productivity of 11.5 kg phenol m(-3) day(-1) . Phenol biodegradation was also tested in two different industrial wastewaters (WW1 and WW2) obtained from local resin manufacturing companies, which contained both phenols and formaldehyde . In this case, after wastewater conditioning (i.e., dilution, pH, nitrogen and phosphorous sources and micronutrient amendments) the immobilized cells were able to completely remove the formaldehyde present in both waters . Moreover, they biodegraded phenols completely at a rate of 0.5 kg phenol m(-3) day(-1) in the case of WW1 and partially (but at concentrations lower than 50 mg l(-1)) at 0.1 and 1.0 kg phenol m(-3) day(-1) in the cases of WW2 and WW1, respectively.

Clin Infect Dis, 2002 Jun 1, 34(11), 1500 - 7 Epub 2002 May 10.
An outbreak of Mycobacterium chelonae infection following liposuction; Meyers H et al.; Among 82 patients who underwent liposuction performed by a single practitioner in a 6-month period, 34 (41%) developed cutaneous abscesses . An organism identified as Mycobacterium chelonae by polymerase chain reaction restriction-enzyme analysis was recovered from cultures of samples from 12 of those patients . DNA large restriction-fragment pattern analysis by pulsed-field gel electrophoresis demonstrated that a strain of M . chelonae recovered from biofilm in the piped-water system in one of the physician's offices differed by only 2 restriction fragments from the 12 patient isolates, which differed from each other by 0 or 1 restriction fragment . A detailed retrospective cohort study that included interviews with former employees and statistical analysis of risk factors indicated that inadequate sterilization and rinsing of surgical equipment with tap water were likely sources of mycobacterial contamination . This is the first reported outbreak of nosocomial infection due to M . chelonae in which a source has been identified and the first to occur in association with liposuction in patients in the United States.

Nature, 2002 May 16, 417(6886), 243 - 4
Caenorhabditis elegans: plague bacteria biofilm blocks food intake; Darby C et al.; Bubonic plague is transmitted to mammals, including humans, by the bites of fleas whose digestive tracts are blocked by a mass of the bacterium Yersinia pestis . In these fleas, the plague-causing bacteria are surrounded by an extracellular matrix of unknown composition, and the blockage depends on a group of bacterial genes known as the hmsHFRS operon . Here we show that Y . pestis creates an hmsHFRS-dependent extracellular biofilm to inhibit feeding by the nematode Caenorhabditis elegans . Our results suggest that feeding obstruction in fleas is a biofilm-mediated process and that biofilms may be a bacterial defence against predation by invertebrates.

Periodontol 2000, 2002, 28, 240 - 55
Antifungal and antiviral chemotherapy; Pallasch TJ; Fungal and viral infections are difficult to treat, since fungal infections commonly rebound after suppression by the antifungal agent and current antiviral drugs are only virustatic, allowing the virus to reassert its pathogenicity if not eliminated by the host defenses . In addition, fungal infections commonly are associated with significant biofilms, retarding drug penetration, and the fluid nature of the oral cavity does not promote drug-fungus contact for long periods of time . Both mycotic and viral pathogens are developing sophisticated methods to elude the toxic effects of drugs intended to eliminate their existence . The drug therapy of oral fungal and viral infections is therefore limited but occasionally successful (more with fungal than viral infections) and is often relegated to palliative care . The specter of drug resistance and its promotion by prolonged, repetitive and frivolous use must always be foremost in the clinician's mind.

Sci STKE . 2002 May 14;2002(132):RE6.
An attractive surface: gram-negative bacterial biofilms; Schembri MA et al.; In nature, most bacteria live in close association with surfaces as complex communities referred to as biofilms . Community members within these compact microbial consortia show extraordinary resistance to conventional antibiotics, biocides, and hydrodynamic shear forces when compared to their planktonic counterparts . The buildup of these surface-associated bacterial communities is a highly organized and complex process that requires many signal transduction mechanisms to orchestrate the different stages of development . In this review, we describe several types of signal transduction that Gram-negative bacteria employ during the adhesion and expansion stages of biofilm formation, as well as discuss quorum-sensing in relation to the production of virulence factors.

Infect Immun, 2002 Jun, 70(6), 3180 - 6
Expression of the biofilm-associated protein interferes with host protein receptors of Staphylococcus aureus and alters the infective process; Cucarella C et al.; The adherence of Staphylococcus aureus to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections . S . aureus presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components . We examined the influence of biofilm-associated protein (Bap) expression on S . aureus adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model . Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556 . Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains . In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain . Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.

J Dermatol Sci, 2002 May, 29(1), 54 - 61
Confocal laser microscopic observation of glycocalyx production by Staphylococcus aureus in vitro; Akiyama H et al.; We used a scanning confocal laser microscope to study the effects of various agents on sugar production by Staphylococcus aureus in vitro . S . aureus cells attached to coverslips in Pl-TSB (plasma:tryptic soy broth=1:1) were stained with fluorescein isothiocyanate-conjugated concanavalin A (FITC-conA) and were more strongly stained over time . We considered that the materials that stained positive for FITC-conA consistent with S . aureus cells were sugars, probably glycocalyx, produced by the S . aureus cells . Since the cells in the stationary growth phase alone were strongly stained with FITC-conA, all S . aureus cells attached to the coverslips in Pl-TSB were considered to be in this phase (low growth rate) . The positive staining for FITC-conA was markedly reduced when fibrin was not formed in Pl-TSB with plasmin and sucrose, and was also markedly reduced when the fibrin in Pl-TSB was destroyed with plasmin . In conclusion, the results of the present study indicate that the existence of fibrin is essential for glycocalyx production and biofilm formation of S . aureus cells to aid in the attachment of S . aureus cells in vitro, because S . aureus cells attached on coverslips and fibrin alone produce glycocalyx . Of the antimicrobial agents tested, sulfadiazine silver most strongly inhibited the production of FITC-conA-positive materials by S . aureus cells at a sub-MIC concentration . Plasmin, sucrose, and sulfadiazine silver may be useful topical applications for use on clinical dermatology for the prevention and the treatment of staphylococcal biofilms . We consider that this simple method is very useful for the detection of S . aureus glycocalyx on dermatology field.

Clin Oral Implants Res, 2002 Feb, 13(1), 53 - 8
Particulate Bioglass reduces the viability of bacterial biofilms formed on its surface in an in vitro model; Allan I et al.; 45S5 Bioglass is a bioactive implant material which, in its particulate form, is used in the repair of periodontal defects . The surface reactions undergone by this material in an aqueous environment may exert an antibacterial effect that would be beneficial to periodontal surgical treatment . Biofilms of Streptococcus sanguis, an early plaque former, and mixed species biofilms from a salivary inoculum grown under conditions similar to those associated with periodontal implants, were grown on particulate Bioglass in a constant depth film fermenter (CDFF) . Control biofilms were grown on inert glass particulates . At sample times of 3, 24 and 48 hours the viability of biofilms of S . sanguis grown on Bioglass was significantly lower than for those grown on inert glass . In the experiments with subgingivally-modelled mixed species biofilms, the total anaerobic counts were significantly lower on Bioglass after 24 and 48 hours, but not 96 or 168 hours, compared to inert glass . Thus, particulate Bioglass has the potential to reduce bacterial colonisation of its surface in vivo, a feature relevant to post-surgical periodontal wound healing.

Gen Dent, 2002 Mar-Apr, 50(2), 190 - 5; quiz 196-7
Waterline biofilm and the dental treatment facility: a review; Pederson ED et al.; Biofilms are well-organized communities of cooperating microorganisms that can include bacteria, protozoa, diatoms, and fungi . Surveys of dental unit waterlines (DUWLs) indicate that biofilm formation is a universal problem and that environmental and human-derived opportunistic pathogens can be cultured consistently from biofilms retrieved from DUWLs and other dental devices . Although the health risks presented by waterline bacterial colonization have yet to be adequately addressed, professional and ethical considerations indicate that steps should be taken to improve the quality of DUWLs . To address these concerns, the Council on Scientific Affairs of the ADA recently published a list of products cleared by the FDA to control dental waterline contamination . The goal of this article is to increase the awareness of potential health risks posed by biofilm formation and provide information on techniques and devices designed to control the microbial contamination of DUWLs.

Gen Dent, 2000 Nov-Dec, 48(6), 682 - 8
A practical approach to improving the quality of water used for routine dental treatments; Plamondon T et al.; The water delivered through dental units connected to municipal water supplies often is contaminated with bacteria and other organisms as a result of biofilm formation in waterlines . Water and other solutions used for dental treatment should meet generally recognized standards for drinking water quality . Public concern over the safety of drinking water and media attention paid to dental water quality will continue to increase . The number of immunocompromised patients who are at risk of developing infectious diseases from exposure to contaminated water aerosolized from dental units also will continue to increase . Improving the quality of water used in routine dental treatment is a worthwhile goal and reasonable measures to improve water quality now are available.

J Antimicrob Chemother, 2002 May, 49(5), 867 - 70
Effect of clarithromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model; Yanagihara K et al.; Fourteen-membered macrolides (e.g . clarithromycin and erythromycin), but not 16-membered macrolides (e.g . josamycin), are effective in diffuse panbronchiolitis . However, there are no studies that have compared the effects of 14- and 16-membered macrolide antibiotics on biofilm formation . Treatment with high-dose clarithromycin (100 mg/kg) resulted in a significant decrease in the number of viable bacteria in an experimental murine model . Josamycin at a dose of up to 100 mg/kg had no effect on the number of viable bacteria in the lung . Our results may explain, at least in part, the clinical efficacy of 14-membered macrolide antibiotics in patients with chronic pneumonia caused by Pseudomonas aeruginosa.

J Antimicrob Chemother, 2002 May, 49(5), 769 - 75
Composition and antibiotic resistance profile of microcosm dental plaques before and after exposure to tetracycline; Ready D et al.; The aim of this study was to investigate the effects of tetracycline administration on the viability and antibiotic resistance profiles of microcosm dental plaques . A constant depth film fermenter was used to generate multi-species biofilms, which were grown for 216 h before tetracycline was added . The composition of the microcosm plaques was determined by viable counting on selective and non-selective media . The prevalence of antibiotic-resistant organisms was determined on antibiotic-containing media . Before administration of tetracycline, the biofilms had a total viable anaerobic count of 7 x 10(7) cfu per biofilm . They contained 7% lactobacilli, 19% streptococci and 2% Actinomyces spp . Immediately after pulsing with tetracycline, the composition of the biofilms changed and they consisted of 30% lactobacilli, 1.5% streptococci and 3% Actinomyces spp., with a total anaerobic count of 1 x 10(7) cfu per biofilm . The pre-valence and composition of the antibiotic-resistant microflora changed dramatically after the addition of tetracycline, with the proportion of the microflora displaying resistance to tetracycline increasing from 6% to 45% . Corresponding changes in the proportions of the microflora displaying resistance to other antibiotics were as follows: 5-28% for erythromycin, 1-5% for vancomycin and 0.4-3% for ampicillin . The results of this study have shown that the addition of tetracycline to microcosm dental plaques alters their composition and enriches for bacteria resistant to tetracycline and other unrelated agents.

Res Microbiol, 2002 Apr, 153(3), 181 - 5
Biofilm formation in Escherichia coli is affected by 3-(N-morpholino)propane sulfonate (MOPS); Corona-Izquierdo FP et al.; In most natural environments, association with a surface in a structure known as a biofilm is the prevailing microbial life-style . Escherichia coli has been a useful model for the study of biofilm formation . Here we analyzed the amounts of biofilm formed when E . coli was cultured in the presence of MOPS {3-(N-morpholino)propane sulfonatel . We used the "O'Toole and Kolter" method, which consisted of growing cells in PVC microtiter dishes and staining the formed biofilm with crystal violet . Our results showed that: 1) the addition of 100 mM MOPS to the rich Luria-Bertani (LB) medium increased the capacity of biofilm formation of several E . coli strains; and 2) the biofilm formed by cells growing in the presence of MOPS was more evident and well defined than that of cells cultured in LB-only medium . The improved ability of forming biofilms was maintained even for 60 h after removing MOPS from the medium, indicating that this improvement was due to a change in the metabolism of E . coli growing in the presence of MOPS or that, under these conditions, biofilm formation was favored.

J Appl Microbiol, 2002, 92 Suppl, 163S - 70S
Susceptibility of antibiotic-resistant Gram-negative bacteria to biocides: a perspective from the study of catheter biofilms; Stickler DJ; Bacteria resistant to both the agents deployed to prevent infections and those used to treat infections would be formidable nosocomial pathogens . The aim of this paper is to review the evidence that Gram-negative bacteria resistant to antibiotics and biocides have emerged and been responsible for catheter-associated urinary tract infection . A study of patients undergoing intermittent bladder catheterization revealed that the frequent application of the antiseptic chlorhexidine to the perineal skin prior to the insertion of the catheter was effective against the normal Gram-positive skin flora but not against the Gram-negative organisms that subsequently colonized this site . Organisms such as Providencia stuartii, Pseudomonas aeruginosa and Proteus mirabilis were repeatedly isolated from the skin of these patients and inevitably went on to cause urinary infections . The minimum inhibitory concentration (MIC) of chlorhexidine for many of these strains proved to be 200-800 microg ml(-1) compared with the 10-50 microg ml(-1) recorded for reference strains of Gram-negative species . A subsequent survey of over 800 Gram-negative isolates from urinary tract infections in patients from both hospitals and the community revealed that chlorhexidine resistance was not a widespread phenomenon, but was restricted to these species and to units where the care of catheterized patients involved the extensive use of chlorhexidine . Analysis of the antibiotic resistance patterns revealed that the chlorhexidine-resistant strains were also multidrug resistant . Other clinical studies also reported catheter-associated infections with chlorhexidine- and multidrug-resistant strains of Pr . mirabilis when chlorhexidine was being used extensively . This species poses particular problems to the catheterized patient . Chlorhexidine thus proved counterproductive in the care of catheters and its use in this context has been largely abandoned . Suggestions of reintroducing this agent in the form of biocide-impregnated catheters should be resisted.

J Appl Microbiol, 2002, 92 Suppl, 98S - 110S
Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?
Gilbert P, Allison DG, McBain AJ.
Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria . This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells . Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive . Such clones might include efflux mutants in addition to genotypes with modifications in single gene products . Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population . This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets . Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target . The latter might also confer resistance to antibiotics where the target is shared . Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis . Triclosan may, therefore, select for mutants in a target that is shared with the anti-E . coli diazaborine compounds and the antituberculosis drug isoniazid . Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents . Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants . Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics . It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities . Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed . In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.

J Appl Microbiol, 2002, 92 Suppl, 1S - 3S
Antibiotic and biocide resistance in bacteria: introduction; Russell AD; Drug resistance in bacteria is increasing and the pace at which new antibiotics are being produced is slowing . It is now almost commonplace to hear about methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-drug resistance in Mycobacterium tuberculosis (MDRTB) strains and multi-drug-resistant (MDR) Gram-negative bacteria . So-called new and emerging pathogens add to the gravity of the situation . Reduced susceptibility to biocides is also apparently increasing, but is more likely to be low level in nature and to concentrations well below those used in hospital, domestic an industrial practice . A particular problem, however, is found with bacteria and other micro-organisms present in biofilms, where a variety of factors can contribute to greater insusceptibility compared with cells in planktonic culture . Also of potential concern is the possibility that widespread usage of biocides is responsible for the selection and maintenance of antibiotic-resistant bacteria . The basic mechanisms of action of, and bacterial resistance to, antibiotics are generally well documented, although data continue to accumulate about the nature and importance of efflux systems . In contrast, the modes of action of most biocides are poorly understood and consequently, detailed evaluation of bacterial resistance mechanisms is often disappointing . During this Symposium, the mechanisms of bacterial resistance to antibiotics and biocides are discussed at length . It is hoped that this knowledge will be used to develop newer, more effective drugs and biocides that can be better and perhaps, on occasion, more logically used to combat the increasing problem of bacterial resistance.

Environ Microbiol, 2002 Mar, 4(3), 169 - 82
Enrichment versus biofilm culture: a functional and phylogenetic comparison of polycyclic aromatic hydrocarbon-degrading microbial communities; Stach JE et al.; The effect that culture methods have on the diversity of degradative microbial communities is not well understood . We compared conventional batch enrichment with a biofilm culture method for the isolation of polycyclic aromatic hydrocarbon (PAH)-degrading microbial communities from a PAH-contaminated soil . The two methods were assessed by comparing: (i) the diversity of culturable bacteria; (ii) the diversity of PAH-catabolic genes in isolated bacteria; (iii) the inter- and intraspecific diversity of active PAH-catabolic gene classes; (iv) the diversity of bacteria present in 16S rRNA gene libraries generated from RNA extracted from the two communities and soil; and (v) the estimated diversity of active bacteria in the soil and culture systems . Single-strand conformation polymorphism analysis showed that the biofilm culture yielded 36 bacterial and two fungal species compared with 12 bacterial species from the enrichment culture . Application of accumulation and non-parametric estimators to clone libraries generated from 16S rRNA confirmed that the biofilm community contained greater diversity . Sequencing of clones showed that only species from the Proteobacteria were active in the enrichment culture, and that these species were expressing an identical nahAc-like naphthalene dioxygenase . 16S rRNA clones generated from the biofilm community indicated that species from the Cytophaga/Flavobacterium, high G+C bacteria and Proteobacteria were active at the time of sampling, expressing cndA-, nahAc- and phnAc-like naphthalene dioxygenases . The diversity of active species in the biofilm culture system closely matched that in the PAH-contaminated source soil . The results of this study showed that biofilm culture methods are more appropriate for the study of community-level interactions in PAH-degrading microbial communities . The study also indicated that cultivation of microbial communities on solid media might be the primary source of bias in the recovery of diverse species.

Water Res, 2002 Mar, 36(6), 1423 - 8
Method for the measurement of the diffusion coefficient of benzalkonium chloride; Smith MJ et al.; Biofilm formation on the optical ports of cameras and underwater sensors is the primary cause of their reduced useful deployment time . The use of a transparent hydrogel coating containing the cationic surfactant benzalkonium chloride has been shown to extend the deployment times for up to 12 weeks for these instruments . In order to predict the effective lifetime of these coatings it was necessary to obtain the diffusion coefficient of the benzalkonium chloride used in the coatings . Benzalkonium chloride can have different alkyl chain lengths ranging from C8H17 to C18H37 with chain length greatly affecting its chemical properties . The benzalkonium chloride materials investigated here were mixtures of C12H25 and C14H29 as well as C14H29 on its own . These materials were selected for their proven biofilm resistant qualities . The diaphragm diffusion cell technique was investigated for its applicability to the measurement of diffusion coefficients of molecules with surfactant properties and the ability to form micelles . The method was found to be satisfactory for the cationic surfactant benzalkonium chloride . The average value of the membrane cell integral diffusion coefficient D was 7.78 x 10(-6) cm2 s(-1) at 25 degrees C and there was no significant effect of alkyl chain length on the measured value of D.

Water Environ Res, 2002 Jan-Feb, 74(1), 77 - 81
Effect of some environmental factors on mercury(II) reduction by suspended particulate matter-associated Kluvera cryocrescens biofilms in waterbodies; Wang W et al.; To resist the invasion of harmful external substances, microorganisms tend to attach to submerged surfaces in aquatic systems . The current study focused on the influence of environmental factors (e.g., pH value of the culture, oxidation-reduction condition, light, and cultured age of bacteria on the reduction of mercury(II) to elemental mercury (Hg0) that occurred on the surfaces of suspended particulate matter . A simulated biofilm system consisting of bacterium Kluvera cryocrescens separated from suspended particulate matter in the Yangtze River (China), its exopolysaccharide, and kaolinite (BEK) was used to study the transformation of mercury(II) in an aquatic system . The results showed that the reduction of mercury from mercury(II) to elemental mercury was favored in a neutral pH, light, and aerobic surrounding while a low pH, dark, and anaerobic condition inhibited the reaction . Moreover, the reduction capacity of both biofilms and free bacteria decreased with the increase of cultured age . Under all experimental conditions, the reduction capacity of biofilms was lower than that of the free bacteria, suggesting that biofilm, as a microecosystem, might function as a buffer to protect the effect of mercury(II) and the surrounding change on microorganisms.

J AOAC Int, 2002 Mar-Apr, 85(2), 479 - 85
Testing antimicrobials against biofilm bacteria; Hamilton MA; Standard laboratory methods are needed to assess the efficacy of antimicrobial agents that are applied to biofilm bacteria . Existing standard suspension tests and dried surface tests show much greater efficacy than antimicrobial agents applied to biofilms . The greater resistance of biofilm bacteria to antimicrobial agents can be attributed to a number of interacting factors, including reaction and diffusion processes that limit an agent's accessibility to bacteria, phenotypic changes in biofilm bacteria caused by stress, and adaptation of the bacteria . Because biofilm systems are so diverse, a variety of new biofilm tests with features that differ in important ways from existing tests will ultimately be required . For example, the biofilm test apparatus may include a pump and a continuous-flow stirred tank reactor . This report provides an overview of biofilm testing and suggests a strategy for creating standard test methods.

Water Sci Technol, 2002, 45(7), 51 - 6
Oil bio-degradation in permeable pavements by microbial communities; Newman AP et al.; This paper reports on continuing research at Coventry University into the improvement of highway water quality following flow through a permeable pavement . Such pavements have been shown elsewhere to be efficient in-situ bio-reactors, capable of degrading large quantities of clean motor oil . Further laboratory research, reported here, demonstrates that a commercially obtained oil degrading, microbial mixture was not significantly better at degrading clean motor oil than the indigenous microbial biomass established within the pavement over a 4-year period, when provided with an adequate nutrient supply . Scanning electron microscopy has been used to monitor biofilm development, which has also identified that the pavement has developed a complex community structure with high bio-diversity.

Can J Vet Res, 2002 Apr, 66(2), 86 - 92
Biofilm bacteria: formation and comparative susceptibility to antibiotics; Olson ME et al.; The Calgary Biofilm Device (CBD) was used to form bacterial biofilms of selected veterinary gram-negative and gram-positive pathogenic bacteria from cattle, sheep, pigs, chicken, and turkeys . The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of ampicillin, ceftiofur, cloxacillin, oxytetracycline, penicillin G, streptomycin, tetracycline, enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-sulfadoxine for gram-positive and -negative bacteria were determined . Bacterial biofilms were readily formed on the CBD under selected conditions . The biofilms consisted of micro-colonies encased in extracellular polysaccharide material . Biofilms composed of Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium renale, or Corynebacterium pseudotuberculosis were not killed by the antibiotics tested but as planktonic bacteria they were sensitive at low concentrations . Biofilm and planktonic Streptococcus dysgalactiae and Streptococcus suis were sensitive to penicillin, ceftiofur, cloxacillin, ampicillin, and oxytetracycline . Planktonic Escherichia coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and trimethoprim/ sulfadoxine . Enrofloxacin and gentamicin were the most effective antibiotics against E . coli growing as a biofilm . Salmonella spp . and Pseudomonas aeruginosa isolates growing as planktonic populations were sensitive to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these bacteria were only sensitive to enrofloxacin . Planktonic and biofilm Pasteurella multocida and Mannheimia haemolytica had similar antibiotic sensitivity profiles and were sensitive to most of the antibiotics tested . The CBD provides a valuable new technology that can be used to select antibiotics that are able to kill bacteria growing as biofilms.

Proteomics, 2002 May, 2(5), 571 - 9
Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae; Hommais F et al.; In this paper, we initiated the first two-dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease . In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine . By proteome analysis we investigated the effect of mild acid treatment on the physiology of V . cholerae . More than 50 proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database searching . Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope . Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide . Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6 . Our results suggest that V . cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.

J Ind Microbiol Biotechnol, 2002 May, 28(5), 268 - 79
Structure and on-site formation of biofilms in paper machine water flow; Mattila K et al.; Paper machine biofilms formed in situ on stainless steel surfaces were studied . A robust flow cell was fitted to side stream (1.8 m s(-1)) of the spray water circuit of a paper machine . This on-site tool allowed for assessing the efficacy of antifoulants and the adequacy of steel polishing under mill conditions . A rapid fluorescence-based assay was developed to quantify the biomass of shallow biofilms on machine steel . The fluorescence matched the ATP content measured for the same biofilms . Electrolytic polishing reduced the tendency of biofouling of 500 grit surface steel . Biofilm grew under machine conditions as clusters on the steels, showing uniformly coccoid, filaments or short rods; only one cell type in each cluster . The biofilm clusters excluded latex beads of 0.02 microm with hydrophilic or with hydrophobic surfaces from penetrating more than three to four layers of cells . Under the high hydraulic flow at the machine (1.8 m s(-1)), the biofilm grew in 7 days 6-10 microm thick . The high flow rate guided the shape of the biofilm clusters emerging after the primary attachment of cells . Adhered individual bacteria were the platform on steel to which solids such as paper machine fines then accumulated.

Mol Microbiol, 2002 Jan, 43(2), 383 - 97
Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation; Gavin R et al.; Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media . Such hyperflagellated cells demonstrate increased adherence . Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated . Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A . hydrophila and A . caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts . The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation . Mutation of the Aeromonas lafB, lafS or both A . caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation . Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels . Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A . hydrophila lafTlU genes resulted in non-motility . However, mutations that abolished polar flagellum production also inhibited lateral flagella expression . We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.

Int Endod J, 2002 Mar, 35(3), 268 - 74
An in vitro comparison of the bactericidal efficacy of lethal photosensitization or sodium hyphochlorite irrigation on Streptococcus intermedius biofilms in root canals; Seal GJ et al.; AIM: To compare the bacterial killing of Streptococcus intermedius biofilms in root canals using lethal photosensitization with various combinations of photosensitizer concentration and laser light dose or 3% sodium hypochlorite (NaOCl) irrigation . METHODOLOGY: Extracted teeth (n = 35) with single canals were selected and the canals prepared to apical size 25 with a 10% taper . The teeth were autoclaved and the canals inoculated with Streptococcus intermedius in brain heart infusion broth and were incubated for 48 h to allow a biofilm to form . The teeth were then subjected to 3% NaOCl irrigation (n = 4) or lethal photosensitization using combinations of a range of toluidine blue O (TBO) photosensitizer concentrations (12.5, 25, 50, 100 microgram/mL-1) and light doses (60, 90, 120, 300, 600 s equivalent to energy doses of 2.1-21 J) using a 35-mW helium-neon low power laser targeted at the access cavity (n = 4 for each combination) . Controls consisted of laser light only (TBO = 0 microgram/mL-1) (n = 4), TBO only (light dose = 0 s) (n = 4), and no treatment (positive control n = 17) . Following treatment the canal contents were sampled with sterile paper points, the sample was dispersed in transport medium, serially diluted and cultured on blood agar to determine the number of colony forming units (CFU) . RESULTS: The combination of 100 microgram/mL-1 TBO and 600 s (21 J) of laser energy achieved maximum reduction in recovered viable bacteria (5 log10 CFU) . TBO at low concentrations (< or =50 microgram/mL-1) was not bactericidal but treatment with 100 microgram/mL-1 TBO alone reduced recovered viable bacteria by 3 log10 CFU . Laser light alone had limited bactericidal effect . No viable bacteria were recovered following treatment with 3% NaOCl . CONCLUSIONS: The combined use of a photosensitizing agent and a low power laser directed at the access cavity was bactericidal to S . intermedius biofilms in root canals but was unable to achieve total kill, unlike 3% NaOCl.

Microb Ecol, 2002 Jan, 43(1), 181 - 8 Epub 2001 Oct 08.
Microhabitats and chemical microenvironments under saxicolous lichens growing on granite; de los Rios A et al.; Lasallia hispanica, Parmelia omphalodes, and Cornicularia normoerica, saxicolous thalli growing on granite, show a close relationship with other lichens and microorganisms living in the lithic substrate beneath them . The lithobiontic community is an accumulation of microorganisms at an interface forming a biofilm, which interacts with the lithic substrate both geophysically and geochemically . Because of their fruticose and foliose morphology, the saxicolous species examined here are mainly involved in geophysical processes, but in the proximity of their attachment structures, geochemical processes may also be observed . On the other hand, fungi, algae and cyanobacteria forming crustose lichens, as well as free-living lithobiontic microorganisms, are known to show combined geophysical and geochemical action, mainly on laminar minerals . The substrate zone where the saxicolous lichens are attached is most affected by weathering reactions and shows the highest co-occurrence of lithobiontic microorganisms . The physical and chemical properties of the substrate, along with lichen and microorganism activity, determine different microenvironments and microhabitats . The ecological functioning of these lithobiontic communities is not yet fully understood, and research efforts similar to the present are needed to confirm that their development is influenced by interrelations between different community members and the substrate, as suggested here.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 89 - 93
{Continuous biosynthesis of epoxypropane in a methanotrophic attached-films reactor}; Xin JY et al.; Using a fluidized bed as immobilization system, mixed culture methanotrophic attached-films were developed on diatomite particles . The Methane Monooxygenase (MMO) activity was found to increase obviously as soon as the lag phase ended . Greater than 90% of the MMO activity in the bed was attached . Biofilm concentration of 3.3-3.7 mg dry weight cell/g DS was observed . Batch experiments were performed to explore the possibility of producing epoxypropane by a cooxidation process . The effect of methane on the oxidation of propene to epoxypropane and the effect of propene on the growth of methanotroph were also studied . In continuous experiments, optimum mixed gaseous substrates (methane: 35%; propene: 20%; oxygen: 45%) were continuously circulated through the fluidized bed reactor to remove product . Initial epoxypropane productivity was 110-150 mumol/d . The bioreactor operated continuously for 25 d without obvious loss of epoxypropane productivity.

J Bacteriol, 2002 May, 184(10), 2699 - 708
A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation; Li YH et al.; In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J . Bacteriol . 183:897-908, 2001) . Here, we demonstrate that this system also plays a role in the ability of S . mutans to initiate biofilm formation . To test this hypothesis, S . mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation . The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy . The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm . The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass . Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms . The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP . Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea . We conclude that the quorum-sensing signaling system essential for genetic competence in S . mutans is important for the formation of biofilms by this gram-positive organism.

Appl Environ Microbiol, 2002 May, 68(5), 2509 - 18
Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand; Holden PA et al.; Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils . Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown . In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane . Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR . We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P . aeruginosa PG201 . GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media . Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface . Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P . aeruginosa biodegrading hexadecane in sand . Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production . Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms . Our findings suggest that P . aeruginosa likely produces surface-active compounds in sand culture . However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells . In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

Appl Environ Microbiol, 2002 May, 68(5), 2495 - 502
Metabolic commensalism and competition in a two-species microbial consortium; Christensen BB et al.; We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source . The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats . The numbers of CFU of P . putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers . When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P . putida R1 (500:1), whereas under similar growth conditions in biofilms, P . putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1) . In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms . Insertion into P . putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P . putida R1 cells at different positions in the biofilms . Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm . In the initial phase of biofilm development, the growth activity of P . putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6 . High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P . putida R1 . After a few days, Acinetobacter strain C6 colonies were overgrown by P . putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm . In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P . putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6 . We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P . putida R1 . In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.

Environ Microbiol, 2002 Feb, 4(2), 97 - 105
Microenvironments and microbial community structure in sediments; Tankere SP et al.; The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments . An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000 . There was a 0.2 mm overlap between the O2 and H2S profiles . pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface . The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm . Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S . The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment . Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm . Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.

Environ Microbiol, 2002 Feb, 4(2), 70 - 80
Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria; Springael D et al.; Pseudomonas putida BN210, carrying the self- transferable clc-element encoding degradation of 3-chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3-chlorobenzoate-contaminated model wastewater . Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3-chlorobenzoate mineralizing bacteria mainly belonging to the beta-proteobacteria . In contrast, in non-inoculated reactors, no 3-chlorobenzoate degradation was observed and no 3-chlorobenzoate degraders could be recovered . Southern blots hybridization of genomic DNA using clc-element-specific probes and FIGE analysis indicated the presence of the complete clc-element in one or more copies in the isolates . Moreover, the isolates could transfer the clc genes to Ralstonia metallidurans recipients . Two representative reactor isolates, Ralstonia sp . strains KP3 and KP9 demonstrated a higher growth rate on 3-chlorobenzoate than strain BN210 in batch cultures . When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3-chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3-chlorobenzoate degrading population in the reactor . Our data suggest that in situ horizontal transfer of the clc-element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3-chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.

Emerg Infect Dis, 2002 Apr, 8(4), 376 - 9
Biofilm on ventriculo-peritoneal shunt tubing as a cause of treatment failure in coccidioidal meningitis; Davis LE et al.; We describe a case of recurrent coccidioidal meningitis in which a fungal biofilm on the tip of ventriculo-peritoneal shunt tubing was likely responsible for a 4-year persistence of Coccidioides immitis, despite the patient's taking an adequate dosage of fluconazole . Fungal biofilms should be considered as a cause for treatment failure and fungal persistence, especially when artificial prostheses or indwelling catheters are present.

Intensive Care Med, 2002 Apr, 28(4), 426 - 31 Epub 2002 Mar 06.
Eradication of endotracheal tube biofilm by nebulised gentamicin; Adair CG et al.; OBJECTIVE: To compare the efficacy of gentamicin, nebulised via the endotracheal tube (ET), with that of parenteral cefotaxime or parenteral cefuroxime in preventing the formation of ET biofilm . SETTING: General intensive care units in two university teaching hospitals . DESIGN: The microbiology of ET biofilm from 36 ICU patients eligible to receive antibiotic prophylaxis was examined . Peak and trough tracheal concentrations of gentamicin, cefotaxime or cefuroxime were measured in each patient group, on the 2nd day of intubation . PATIENTS: Twelve patients received gentamicin (80 mg) nebulised in 4 ml normal saline every 8 h, 12 cefotaxime (1 g, 12 hourly) and 12 cefuroxime (750 mg, 8 hourly) . Prophylaxis was continued for the duration of intubation . MEASUREMENTS AND RESULTS: Samples of tracheal secretions were taken on the 2nd day of ventilation for determination of antibiotic concentrations . Following extubation, ETs were examined for the presence of biofilm . Pathogens considered to be common aetiological agents for VAP included Staphylococcus aureus, enterococci, Enterobacteriaceae and pseudomonads . While microbial biofilm was found on all ETs from the cephalosporin group, microbial biofilm of these micro-organisms was found on 7 of the 12 ET tubes from patients receiving cefotaxime { S . aureus (4), pseudomonads (1), Enterobacteriaceae (1), enterococcus (1)} and 8 of the 12 ET tubes from patients receiving cefuroxime {Enterobacteriaceae (6), P . aeruginosa (1) and enterococcus (1)} . While microbial biofilm was observed on five ETs from patients receiving nebulised gentamicin, none of these were from pathogens for ventilator-associated pneumonia (VAP) . Tracheal concentrations of both cephalosporins were lower than those needed to inhibit the growth of pathogens recovered from ET tube biofilm . The median (and range) concentrations for cefotaxime were 0.90 (<0.23-1.31) mg/l and 0.28 (<0.23-0.58) mg/l for 2 h post-dose and trough samples, respectively . Two hours post-dose concentrations of cefuroxime (median and range) were 0.40 (0.34-0.83) mg/l, with trough concentrations of 0.35 (<0.22-0.47) mg/l . Tracheal concentrations (median and range) of gentamicin measured 1 h post-nebulisation were 790 (352-->1250) mg/l and then, before the next dose, were 436 (250-1000) mg/l . CONCLUSION: Nebulised gentamicin attained high concentrations in the ET lumen and was more effective in preventing the formation of biofilm than either parenterally administered cephalosporin and therefore may be effective in preventing this complication of mechanical ventilation.

J Clin Periodontol, 2002 Apr, 29(4), 358 - 63
Effect of an amine-fluoride-triclosan mouthrinse on plaque regrowth and biofilm vitality; Arweiler NB et al.; BACKGROUND: The purpose of this double-blind, prospective, latin-square crossover randomised study was to examine the efficacy of a mouthrinse solution containing a combination of triclosan, amine fluoride and sodium fluoride on supragingival plaque regrowth compared to a placebo and a chlorhexidine solution . METHODS: 12 volunteers refrained after professional oral prophylaxis from all mechanical hygiene measures for the following 96 h and rinsed instead cross-over in a randomised order with either chlorhexidine digluconate (0.2%, positive control), a verum solution (0.5% amine fluoride, 0.028% sodium fluoride, 0.03% triclosan) or a placebo solution . The plaque index was assessed after 24 and 96 h (PI 1, PI 2) and the plaque area (PA) photographed and calculated after 96 h of undisturbed plaque regrowth . Moreover plaque samples were taken after 24 and 96 h and examined with the vital fluorescence technique to assess the vitality of the biofilm microbiota (VF 1, VF 2) . RESULTS: The amine-fluoride-triclosan solution reduced the clinical parameters (PI and PA) as well as the vitality of the plaque flora (VF) significantly when compared to the placebo solution . The verum reached a 36.5% (p<0.05) and a 39.8% reduction (p<0.0001) in PI 1 and PI 2, respectively, concomittant with a reduction of 23.8% and 32.2% (p<0.001) in VF 1 and VF 2 and of 46.9% (p<0.0001) in PA at day 4 . This was less than the reductions found with the positive control, i.e., the 0.2% chlorhexidine solution (54.2% and 71.1% reduction in PI 1 and PI 2, 40.0% and 53.4% in VF 1 and VF 2 and 71.5% in PA) . However, significant differences between both active solutions were only established for PI 2 and PA . CONCLUSION: During 4-day plaque regrowth the amine-fluoride-triclosan product displayed a significant antibacterial and plaque-reducing action in comparison to the control.

J Appl Microbiol, 2002, 92(4), 641 - 8
Combining chlorination and chloramination processes for the inhibition of biofilm formation in drinking surface water system models; Momba MN et al.; AIMS: The aim of this study was to investigate the inhibition of biofilm formation on stainless steel (SS) and galvanized mild steel (MS) in chlorine (AFC(1)) and chlorine-monochloramine treated waters (AFC(2)M) . METHODS AND RESULTS: Disinfection was carried out using 2.5 mg l(-1) free chlorine followed by 1.5 mg l(-1) monochloramine, with non-disinfected water used as control water . Results of the standard spread plate procedure, DAPI epifluorescence microscopy and scanning electron microscopy revealed bacterial colonization of SS and MS exposed to non-disinfected and chlorinated waters between 24 and 720 h, while no bacterial adhesion was detected on SS and MS exposed to AFC(2)M between 48 and 504 h . CONCLUSIONS: The inability of bacteria to grow on SS and MS was observed only when 0.35 mg l(-1) residual monochloramine was maintained throughout the system . SIGNIFICANCE AND IMPACT OF THE STUDY: This demonstrates the inability of chlorine alone to inhibit bacterial growth and suggests a combination of chlorine and monochloramine as a more effective treatment for drinking water, especially for rural communities with very poor source waters in Africa.

Environ Microbiol, 2002 Jan, 4(1), 18 - 28
The regulation of biofilm development by quorum sensing in Aeromonas hydrophila; Lynch MJ et al.; Aeromonas hydrophila is an opportunistic Gram-negative pathogen that readily attaches to stainless steel to produce a thin biofilm with a complex 3D structure covering 40-50% of the available surface and producing large microcolonies . As A . hydrophila possesses an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system based on the ahyRI locus, the presence of the AhyI protein and C4-HSL within the biofilm phase was first established by Western blot and AHL biosensor analysis respectively . The ability of the A . hydrophila AH-1 N strain to form biofilms in a continuous-flow chamber was compared with isogenic ahyI and ahyR mutants . The ahyI mutant, which cannot produce C4-HSL, failed to form a mature biofilm . In addition, the viable count of biofilm, but not planktonic phase ahyI mutants, was significantly lower that the parent or ahyR mutant . This defect in the differentiation of the ahyI mutant biofilm could be partially restored by the addition of exogenous C4-HSL . A mutation in ahyR increased coverage of the available surface to around 80% with no obvious effect upon biofilm microcolony formation . These data support a role for AHL-dependent quorum sensing in A . hydrophila biofilm development . Exposure of the A . hydrophila AH-1N biofilm to N-(3-oxodecanoyl)homoserine lactone, which inhibits exoprotease production in planktonic cells, however, had no effect on biofilm formation or architecture within the continuous-flow chamber.

Curr Opin Infect Dis, 2000 Jun, 13(3), 227 - 232
Periodontal disease: bacterial virulence factors, host response and impact on systemic health; Graves DT et al.; Teeth are coated with a biofilm that contains periodontal pathogens . Pathogens express virulence factors which enable them to invade and replicate within epithelial cells and to invade the underlying connective tissue . This stimulates production of prostaglandins and cytokines that induce tissue loss . In addition, these bacteria have the potential to modulate the course of systemic diseases such as atherosclerosis and to contribute to low birthweight and preterm labor.

Nature, 2002 Apr 18, 416(6882), 740 - 3
Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation; Drenkard E et al.; Colonization of the lungs of cystic fibrosis (CF) patients by the opportunistic bacterial pathogen Pseudomonas aeruginosa is the principal cause of mortality in CF populations . Pseudomonas aeruginosa infections generally persist despite the use of long-term antibiotic therapy . This has been explained by postulating that P . aeruginosa forms an antibiotic-resistant biofilm consisting of bacterial communities embedded in an exopolysaccharide matrix . Alternatively, it has been proposed that resistant P . aeruginosa variants may be selected in the CF respiratory tract by antimicrobial therapy itself . Here we report that both explanations are correct, and are interrelated . We found that antibiotic-resistant phenotypic variants of P . aeruginosa with enhanced ability to form biofilms arise at high frequency both in vitro and in the lungs of CF patients . We also identified a regulatory protein (PvrR) that controls the conversion between antibiotic-resistant and antibiotic-susceptible forms . Compounds that affect PvrR function could have an important role in the treatment of CF infections.

BMC Microbiol . 2002 Apr 15;2(1):7.
Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili; Murphy TF et al.; BACKGROUND: Nontypeable Haemophilus influenzae is an important cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD) . Several lines of evidence suggest that the bacterium grows as a biofilm in the human respiratory tract . RESULTS: Fifteen clinical isolates from middle ear fluid of children with otitis media and 15 isolates from sputum of adults with COPD were studied in an in vitro assay of biofilm formation . Striking variability among isolates was observed in their ability to form biofilms . Analysis of cell envelopes revealed minimal differences in banding patterns in polyacrylamide gels, alteration of expression of an epitope on lipooligosaccharide, and preservation of expression of selected epitopes on outer membrane proteins P2, P5 and P6 in biofilms compared to planktonically grown cells . A pilus-deficient variant showed a marked impairment in biofilm formation compared to its isogenic parent . CONCLUSIONS: Nontypeable H . influenzae forms biofilms in vitro . Clinical isolates show substantial variability in their ability to grow as biofilms . Three major outer membrane proteins (P2, P5 and P6) are expressed during growth as a biofilm . Expression of lipooligosaccharide is altered during growth as a biofilm compared to planktonic growth . Pili are important in biofilm formation . As the role of biofilms in human infection becomes better defined, characterization of biofilms may be important in understanding the pathogenesis of infection and immune response to nontypeable H . influenzae in children with otitis media and adults with COPD.

Antimicrob Agents Chemother, 2002 May, 46(5), 1492 - 502
Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin; Sakoulas G et al.; The majority of infections with glycopeptide intermediate-level resistant Staphylococcus aureus (GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm . Loss of function of the accessory gene regulator (agr) of S . aureus has been suggested to confer an enhanced ability to bind to polystyrene . We studied agr in GISA, hetero-GISA, and related glycopeptide-susceptible S . aureus isolates . All GISA strains from diverse geographic origins belong to agr group II . All GISA strains were defective in agr function, as demonstrated by their inability to produce delta-hemolysin . Hetero-GISA isolate A5940 demonstrated a nonsense mutation in agrA that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient . Various other agr point mutations were noted in several clinical GISA and hetero-GISA isolates . A laboratory-generated agr-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin . This was not seen in the isogenic agr group II parent strain in which agr was intact . The in vitro bactericidal activity of vancomycin was attenuated in the agr-null strain compared to the parent strain . These findings imply that compromised agr function is advantageous to clinical isolates of S . aureus toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.

Antimicrob Agents Chemother, 2002 May, 46(5), 1469 - 74
Interactions between biocide cationic agents and bacterial biofilms; Campanac C et al.; The resistance of bacterial biofilms to physical and chemical agents is attributed in the literature to various interconnected processes . The limitation of mass transfer alters the growth rate, and physiological changes in the bacteria in the film also appear . The present work describes an approach to determination of the mechanisms involved in the resistance of bacteria to quaternary ammonium compounds (benzalkonium chloride) according to the C-chain lengths of those compounds . For Pseudomonas aeruginosa CIP A 22, the level of resistance of the bacteria in the biofilm relative to that of planktonic bacteria increased with the C-chain length . For cells within the biofilm, the exopolysaccharide induced a characteristic increase in surface hydrophilicity . However, this hydrophilicity was eliminated by simple resuspension and washing . The sensitivity to quaternary ammonium compounds was restored to over 90% . Staphylococcus aureus CIP 53 154 had a very high level of resistance when it was in the biofilm form . A characteristic of bacteria from the biofilm was a reduction in the percent hydrophobicity, but the essential point is that this hydrophobicity was retained after the biofilm bacteria were resuspended and washed . The recovery of sensitivity was thus only partial . These results indicate that the factors involved in biofilm resistance to quaternary ammonium compounds vary according to the bacterial modifications induced by the formation of a biofilm . In the case of P . aeruginosa, we have underlined the involvement of the exopolysaccharide and particularly the three-dimensional structure (water channels) . In the case of S . aureus, the role of the three-dimensional structure is limited and drastic physiological changes in the biofilm cells are more highly implicated in resistance.

Can J Microbiol, 2002 Feb, 48(2), 132 - 7
Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase; Di Martino P et al.; Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation . The aim of this study was to gain a better understanding of the relationship between E . coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces . We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers . Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes . We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E . coli . Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E . coli . The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene . The possible mechanism of tryptophanase involvement in E . coli adherence and biofilm formation is discussed.

Appl Microbiol Biotechnol, 2002 Apr, 58(5), 690 - 4 Epub 2002 Feb 15.
Dynamic bioreactor operation: Effects of packing material and mite predation on toluene removal from off-gas; Woertz JR et al.; Recent studies have focused on using vapor-phase bioreactors for the treatment of volatile organic compounds from contaminated air streams . Although high removal capacities have been achieved in many studies, long-term operation is often unstable at high pollutant loadings due to biomass accumulation and drying of the packing medium . In this study, three bench-scale bioreactors were operated to determine the effect of packing material and fungal predation on toluene removal efficiency and pressure drop . Toluene elimination capacities (mass toluene removed per unit packing per unit time) above 100 g m(-3) h(-1) were obtained in the fungal bioreactors packed with light-weight, artificial medium, and submersion of the packing in mineral medium once per week was found to provide sufficient moisture and nutrients to the biofilm . The use of mites as fungal predators improved performance by increasing the overall mineralization of toluene to CO(2), and by dislodging biomass along the bioreactor.

Appl Microbiol Biotechnol, 2002 Apr, 58(5), 651 - 7 Epub 2002 Feb 14.
Pitting corrosion inhibition of aluminum 2024 by Bacillus biofilms secreting polyaspartate or gamma-polyglutamate; Ornek D et al.; Pitting corrosion of aluminum 2024 in Luria Bertani medium was reduced by the secretion of anionic peptides by engineered and natural Bacillus biofilms and was studied in continuous reactors using electrochemical impedance spectroscopy . Compared to sterile controls, pitting was reduced dramatically by the presence of the biofilms . The secretion of a 20 amino acid polyaspartate peptide by an engineered Bacillus subtilis WB600/pBE92-Asp biofilm slightly reduced the corrosion rate of the passive aluminum alloy at pH 6.5; however, the secretion of gamma-polyglutamate by a Bacillus licheniformis biofilm reduced the corrosion rate by 90% (compared to the B . subtilis WB600/pBE92 biofilm which did not secrete polyaspartate or gamma-polyglutamate) . The corrosion potential ( E(corr)) of aluminum 2024 was increased by about 0.15-0.44 V due to the formation of B . subtilis and B . licheniformis biofilms as compared to sterile controls . The increase of E(corr) and the observed prevention of pitting indicate that the pitting potential ( E(pit)) had increased . This result and the further decrease of corrosion rates for the passive aluminum alloy suggest that the rate of the anodic metal dissolution reaction was reduced by an inhibitor produced by the biofilms . Purified gamma-polyglutamate also decreased the corrosion rates of aluminum 2024.

Infect Immun, 2002 May, 70(5), 2640 - 9
Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp; Prouty AM et al.; Salmonellae can exist in an asymptomatic carrier state in the human gallbladder . Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against Salmonella enterica serovar Typhi in carriers with gallstones . Therefore, we hypothesized that Salmonella spp . form biofilms on the surfaces of gallstones, where the bacteria are protected from high concentrations of bile and antibiotics . A number of methods were utilized to examine biofilm formation on human gallstones and glass coverslips in vitro, including confocal, light, and scanning electron microscopy . In our assays, salmonellae formed full biofilms on the surfaces of gallstones within 14 days and appeared to excrete an exopolysaccharide layer that bound them to the surfaces and to other bacteria . Efficient biofilm formation on gallstones was dependent upon the presence of bile, as a biofilm did not form on gallstones within 14 days in Luria-Bertani broth alone . The biofilms formed by a Salmonella enterica serovar Typhi Vi antigen mutant, as well as strains with mutations in genes that eliminate production of four different fimbriae, were indistinguishable from the biofilms formed by the parents . Mutants with an incomplete O-antigen, mutants that were nonmotile, and mutants deficient in quorum sensing were unable to develop complete biofilms . In addition, there appeared to be selectivity in salmonella binding to the gallstone surface that did not depend on the topology or surface architecture . These studies should aid in the understanding of the Salmonella carrier state, an important but underresearched area of typhoid fever pathogenesis . If the basis of carrier development can be understood, it may be possible to identify effective strategies to prevent or treat this chronic infection.

Mol Microbiol, 2002 Mar, 43(6), 1367 - 78
Staphylococcus and biofilms; Gotz F; The genetic and molecular basis of biofilm formation in staphylococci is multifaceted . The ability to form a biofilm affords at least two properties: the adherence of cells to a surface and accumulation to form multilayered cell clusters . A trademark is the production of the slime substance PIA, a polysaccharide composed of beta-1,6-linked N-acetylglucosamines with partly deacetylated residues, in which the cells are embedded and protected against the host's immune defence and antibiotic treatment . Mutations in the corresponding biosynthesis genes (ica operon) lead to a pleiotropic phenotype; the cells are biofilm and haemagglutination negative, less virulent and less adhesive on hydrophilic surfaces . ica expression is modulated by various environmental conditions, appears to be controlled by SigB and can be turned on and off by insertion sequence (IS) elements . A number of biofilm-negative mutants have been isolated in which polysaccharide intercellular adhesin (PIA) production appears to be unaffected . Two of the characterized mutants are affected in the major autolysin (atlE) and in D-alanine esterification of teichoic acids (dltA) . Proteins have been identified that are also involved in biofilm formation, such as the accumulation-associated protein (AAP), the clumping factor A (ClfA), the staphylococcal surface protein (SSP1) and the biofilm-associated protein (Bap) . Concepts for the prevention of obstinate polymer-associated infections include the search for new anti-infectives active in biofilms and new biocompatible materials that complicate biofilm formation and the development of vaccines.

J Food Prot, 2002 Apr, 65(4), 627 - 35
Efficacy enhancement of trisodium phosphate against spoilage and pathogenic bacteria in model biofilms and on adipose tissue; Korber DR et al.; A two-step approach for enhancing the efficacy of trisodium phosphate (TSP) was evaluated using meat spoilage and pathogenic bacteria in flow cell biofilms and adipose tissue model systems . The process was based on the plasmolysis of attached bacteria (biofilms) with a hyperosmotic solution (1.5 M NaCl) and the subsequent deplasmolysis of cells with a low-osmotic-strength solution containing different concentrations of TSP (0.1, 0.25, 0.5, 0.625, and 1.0 % {wt/vol}) . Escherichia coli, Salmonella Enteritidis, Pseudomonas sp., Listeria monocytogenes, and Brochothrix thermosphacta strains were cultivated for 24 h as pure culture biofilms in glass flow cells with complex media and were then treated with either 0.1, 0.25, 0.5, 0.625, and 1.0% TSP, or the same TSP concentrations delivered in conjunction with plasmolysis-deplasmolysis (PDP) . Confocal scanning laser microscopy, a commercial fluorescent viability probe, and image analysis were then used to quantify the relative abundances of living and dead cells remaining after the different treatment regimes . With the exception of L . monocytogenes (which was resistant to TSP concentrations of up to 5%), the PDP process increased the sensitivity of the test strains to TSP . However, when similar experiments were conducted with pork adipose tissue, it became evident that higher TSP concentrations were necessary to produce significant decreases in the number of viable cells and that the PDP process generally failed to enhance TSP efficacy . An exception was L . monocytogenes, which exhibited an increase in sensitivity to TSP when inoculated tissue was pretreated with 1.5 M NaCl . It is thought that factors contributing to the failure of the PDP process to enhance the activity of TSP in meat systems involves the mode of TSP antimicrobial activity, alkaline pH stress, and the chemically complex, buffered nature of meats . It remains to be determined whether the PDP process is suitable for use with other food grade antimicrobial agents or can be used in nonfood biofilm control applications.

J Pharm Sci, 2002 Apr, 91(4), 1191 - 9
Preparation and properties of alginate lyase modified with poly(ethylene glycol); Sakakibara H et al.; Modification of the enzyme alginate lyase (AL) with poly(ethylene glycol) (PEG) was attempted for the degradation and removal of alginate biofilms in infectious diseases . The modification of AL with PEG was attempted with three kinds of N-succinimidyl succinate PEG (SS-PEG), which differed in molecular weight (i.e., 2000, 5000 and 12,000 Da) . The conjugation of PEG to free amino groups on AL was confirmed by gel permeation chromatography . Quantification of residual free amino groups revealed that PEG modification progressed further with a higher pH and a larger molar ratio of SS-PEG to AL . The reproducibility of the reaction was fairly good . The enzyme activity decreased with increasing PEG modification but the immunoreactivity toward anti-AL antibodies, as evaluated by an ELISA method, was much more remarkably reduced . The immunoreactivity was more reduced by the conjugated PEG with the larger molecular weight . In the reaction with PEG of molecular weight 12,000 Da, we obtained PEG-modified AL retaining approximately 40% enzyme activity but only 0.5% of the immunoreactivity of native AL .

J Bacteriol, 2002 May, 184(9), 2473 - 80
Firm but slippery attachment of Deinococcus geothermalis; Kolari M et al.; Bacterial biofilms impair the operation of many industrial processes . Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria . It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known . High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D . geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium . Extensive slime matrix was absent from the D . geothermalis E50051 biofilms . AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed . We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible . In spite of this, AFM imaging of living D . geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells . The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface . Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water . Biofilms were evaluated for their strength of attachment . D . geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47 . Deinococcus radiodurans strain DSM 20539(T) also formed tenacious biofilms . This paper shows that D . geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.

J Microbiol Methods, 2002 Jun, 50(1), 23 - 31
Modified enzyme activity assay to determine biofilm biomass; Butterfield PW et al.; An assay of potential exoproteolytic enzyme activity was modified to quantitatively measure the biomass of attached biofilm . The assay utilized the nonfluorescent compound L-leucine-beta-naphthylamide (LLbetaN) that becomes fluorescent when bacterial exoenzymes break the peptide bond, releasing the fluorochrome beta-naphthylamine . Fluorescence development was measured by pumping the liquid phase of a biofilm sample through a fluorescence detector and recording the detector output using a personal computer . A significant linear relationship was shown to exist between the rate of fluorescence development and the biofilm's biomass as carbon, determined using total direct cell counts, measured cell volumes and an existing relationship between cell volume and cell carbon . The technique was used to measure biofilm biomass for experiments where iron oxides were the substratum . Biofilm biomass measurements made using heterotrophic plate counts (HPCs) on R2A medium were shown to correlate well to biomass measurements made using the modified enzyme assay . The technique was shown to be sufficiently sensitive to measure biomass on samples containing little biofilm biomass, such as those exposed to free chlorine . While granular and porous media were used for the experiments presented, small biofilm coupons could easily be used to measure biofilm biomass, expanding the number of possible applications for the enzyme assay technique.

Rev Argent Microbiol, 2002 Jan-Mar, 34(1), 22 - 8
{Candida carriage in the oral mucosa of a student population: adhesiveness of the strains and predisposing factors}; Negroni M et al.; The aim of this study was to establish oral carriage of Candida and possible factors associated to their virulence in young adults and their relation with local and general situations considered as predisposing factors . Samples were obtained from dorsum tongue in 70 students attending the Faculty of Dentistry (University of Buenos Aires) average age: 23, all in healthy oral conditions . Of these, 21.42% were Candida positive . These samples were seeded in CHROMagar . Candida identification was completed in milk agar and Fungichrom 1 . The following species were identified: 11 Candida albicans (C.a), 2 Candida parapsilosis (C.p) and 1 Candida glabrata (C.g) . In one case, 2 species (C.a and C.g) were isolated in the same sample . Virulence was determined as adherence capacity by biofilm or in vitro plaque formation and hydrophobicity . Different host factors were analyzed statistically to establish their importance as predisposing factors to allow Candida colonization . Adherence of C.a . was found to be similar in all C.a . strains, whereas significant differences were found between C.a . and C.p . and between C.a . and C.g . Only the antiseptic mouthrinse and the diet were significant among the considered factors.

Planta, 2002 Apr, 214(6), 848 - 52 Epub 2002 Jan 18.
Chitinase production in pine callus (Pinus sylvestris L.): a defense reaction against endophytes?
Pirttila AM, Laukkanen H, Hohtola A.
In shoot tip-derived tissue cultures of Scots pine (Pinus sylvestris L.), browning and subsequent degeneration of the culture is accompanied by lipid peroxidation and lignification of cells, which are characteristic features of a plant defense reaction . Since chitinases are enzymes acting primarily in plant defense, their expression was studied in pine callus in order to elucidate the defense reaction . Chitinases were present diversely in tissue cultures originating from shoot tips and embryos of P . sylvestris, in contrast to Pinus nigra embryogenic callus, where production of chitinases or browning was not detected . Because endophytic microbes had earlier been detected in buds of Scots pine, their subsequent presence in the tissue cultures was considered a potential cause of the defense reaction . Therefore, the presence of endophytes in the tissue cultures was examined by in situ hybridization . Endophytes were found to colonize heavily in 45% of the tissue cultures of P . sylvestris and to form biofilms, while the P . nigra callus was not found to contain any microbes . The endophytes seemed to propagate uncontrollably once a tissue culture of P . sylvestris was initiated . Regardless of the high level of chitinase production in the callus, the control of the endophytes presumably becomes inadequate during the tissue culture of P . sylvestris.

Lett Appl Microbiol, 2002, 34(4), 293 - 9
Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone; Ren D et al.; AIMS: (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(furanone) of the marine alga Delisea pulchra was synthesized, and its inhibition of swarming motility and biofilm formation of Bacillus subtilis was investigated . METHODS AND RESULTS: Furanone was found to inhibit both the growth of B . subtilis and its swarming motility in a concentration-dependent way . In addition, as shown by confocal scanning laser microscopy, furanone inhibited the biofilm formation of B . subtilis . At 40 microg ml(-1), furanone decreased the biofilm thickness by 25%, decreased the number of water channels, and reduced the percentage of live cells by 63% . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Natural furanone has potential for controlling the multicellular behaviour of Gram-positive bacteria.

Lett Appl Microbiol, 2002, 34(4), 254 - 7
Specific layers in aerobically grown microbial granules; Tay JH et al.; AIMS: To determine the optimal size of aerobically grown granules for wastewater treatment by measuring specific layers within the granules . METHODS AND RESULTS: A variety of biological layers were detected by oligonucleotide probes, specific fluorochromes, and fluorescent microspheres . The channels in the granule matrix penetrated to depths of 900 microm . A layer of obligate anaerobic bacteria was detected at a depth of 800 microm below the granule surface . Dead cells were also observed in the granule interior . CONCLUSIONS: Aerobically grown granules contained layers of aerobic and anaerobic micro-organisms . SIGNIFICANCE AND IMPACT OF THE STUDY: The optimal diameter of the aerobic granule is less than 1600 microm . This is twice the distance from the granule surface to the anaerobic layer . This approach can be used to optimize the thickness of other microbial aggregates such as flocs, colonies and biofilms.

Mar Environ Res, 2002 Apr, 53(3), 311 - 26
Hydrocarbon effects on fouling assemblages: the importance of taxonomic differences, seasonal, and tidal variation; Banks PD et al.; Effects of hydrocarbon-contaminated substrata on recruitment of three species of fouling organisms were studied along the Louisiana gulf coast . Clay tiles (232 cm2) were exposed to crude oil, 10% water soluble fraction of crude oil, or 25 g/kg artificial seawater, and placed out at two locations, in two seasons, and at two tidal levels in an estuary near Port Fourchon, Louisiana . Bryozoan (Membranipora savartii) recruitment was significantly reduced in all experiments on crude oil-exposed tiles . However, oysters (Crassostrea virginica) and barnacles (Balanus eburneus) exhibited recruitment facilitation, and oysters grew to larger size, on crude oil-exposed tiles in 1-4 of the five experiments . When oyster larvae were exposed to the same treatments in the laboratory, settlement was, however, significantly depressed on crude oil-exposed tiles as compared with controls, although oyster size was larger on crude-oil exposed tiles . Recruitment on tiles exposed to the water soluble fraction of crude oil was similar to control tiles in nearly all experiments for all taxa . We suggest naturally occurring biofilms (which hydrocarbons facilitate) may promote or inhibit recruitment, depending on the taxon, because hydrocarbons facilitated recruitment only in field experiments, not in lab experiments without biofilms . However, stronger currents in the field experiments may have more rapidly diluted hydrocarbons, and hydrocarbon effects were not large in comparison with natural seasonal and tidal variation in recruitment.

Pesqui Odontol Bras, 2002 Jan-Mar, 16(1), 63 - 8
{Analysis of the point-counting and planimetric methods in the quantification of denture biofilm--a methodological validation study}; Fernandes RA et al.; Two methods of quantification of the biofilm (point-counting and planimetric) were tested and compared with the paper-weighing method and with the Prosthesis Hygiene Index . The internal surfaces of 62 complete dentures were stained and photographed . The slides were projected on a paper sheet . The total area and the area covered with biofilm were contoured using a black pencil . The point-counting method (experimental 1) was carried out on a mesh of equidistant points . For the planimetric method (experimental 2), the areas of interest were measured by means of a digital planimeter . In the paper-weighing method (control 1) the areas of interest were cut and weighted on a precision scale . In the determination of the Prosthesis Hygiene Index (control 2), the accumulation of biofilm was estimated by means of a scoring method . The results revealed an agreement rate of 82% between the paper-weighing method and the point-counting method, and an agreement rate of 95% between the former and the planimetric method, which was confirmed by high coefficients of correlation (0.98 and 0.99, respectively) . The comparison with the Prosthesis Hygiene Index resulted in 55% of agreement with the point-counting method and in 37% of agreement with the planimetric method . The experimental methods can be useful in clinical studies involving the evaluation of the performance of denture cleansers.

J Chemother, 2001 Nov, 13 Spec No 1(1), 251 - 62
A multicenter study on central venous catheter-associated infections in Italy; Donelli G et al.; In a 1-year multicenter study the microbial colonization of 1154 central venous catheters (CVCs) was investigated . Catheters explanted either from immunocompromised or immunocompetent patients were collected and analyzed by five clinical microbiology laboratories located in Ancona, Aviano, Catania, Pavia and Rome, Italy . A further aim was to investigate, by scanning electron microscopy, the features of currently used catheters, both new and explanted from patients, analyzing their surface quality, the influence of the host protein biofilm on their microbial colonization, the modifications caused by their permanence in the body and the relationship between these factors and the occurrence of infections.

J Chemother, 2001 Nov, 13 Spec No 1(1), 239 - 50
Novel strategies to prevent catheter-associated infections in oncology patients; Schierholz JM et al.; Aggressive cytotoxic treatment of cancer contributes to the growing number of life-threatening infections . Vascular catheters create predominant risks for staphylococcal, enterococcal and candida blood stream infections . Although the contaminating microorganisms may be few in number, the altered host immune response in the presence of such implants as well as disease-associated immunosuppression implies that even small bacterial counts have to be regarded as highly virulent species . Diagnosis of catheter-related infection (CRI) remains difficult before withdrawal of the suspected catheter . Positive culture of catheter surface, lumen and hub and positive peripheral blood probes (paired quantitative blood culture) are predictive for catheter related bacteremia (CRB) . Diligent catheter care and effective antimicrobial catheters may reduce prolonged hospital stay, increased morbidity or mortality and serious economical consequences . The most promising approach features the incorporation of antimicrobial drugs into the polymer matrices that entrap but do not bind the drugs, allowing for extended release . For the efficacious prevention of colonization in the microenvironment of the implantable device the concentration of the antimicrobial substances must exceed usual antibiotic concentrations by a thousand-fold . This is the desired effect--high concentration near the device surface and very low systemic concentration . Incorporation of antimicrobials in the bulk material that constitutes a device can be effective as shown in several in vitro and in vivo studies . In the future, modification of both short-term and long-term catheters by biofilm-active antimicrobials creating slow delivery systems may provide an effective method to protect patients from nosocomial infection in oncology.

Appl Microbiol Biotechnol, 2002 Mar, 58(3), 378 - 85 Epub 2002 Jan 15.
Responses of Mycobacterium sp . LB501T to the low bioavailability of solid anthracene; Wick LY et al.; Several recent reports have indicated that some bacteria may have adapted to the low bioavailability of hydrophobic environmental chemicals and that generalizations about the bioavailability of compounds such as polycyclic aromatic hydrocarbons (PAHs) may be inappropriate . Experimental evidence and theoretical considerations show that the utilization of PAHs requires bioavailability-enhancing mechanisms of the bacteria such as: (1) high-affinity uptake systems, (2) adhesion to the solid substrate, and (3) biosurfactant excretion . We examined possible specific physiological responses of anthracene-degrading Mycobacterium sp . LB501T to poorly water-soluble anthracene in batch cultures, using solid anthracene as a sole carbon source . Mycobacterium sp . LB501T exhibited a high specific affinity for anthracene (a(o)A=32,500 l g(-1) protein h(-1)) and grew as a confluent biofilm on solid anthracene present as sole carbon source . No biofilm formation on anthracene was observed when excess glucose was provided as an additional substrate . This difference could be attributed to a modification of the cell surface of the bacterium . Anthracene-grown cells were significantly more hydrophobic and more negatively charged than glucose-grown cells . In adhesion experiments, anthracene-grown cells adhered 1.5- to 8.0-fold better to hydrophobic Teflon and up to 70-fold better to anthracene surfaces than glucose-grown cells . However, no production of biosurfactants was observed . Our results thus indicate that attachment and biofilm formation may be a specific response of Mycobacterium sp . LB501T to optimize substrate bioavailability.

FEMS Microbiol Lett, 2002 Feb 19, 208(1), 9 - 13
A heteroduplex method for detection of targeted sub-populations of bacterial communities; Turner SJ et al.; We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities . A small (ca . 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers . Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group . The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology . A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected . The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.

Prep Biochem Biotechnol, 2002 Feb, 32(1), 51 - 63
Isolation and biochemical characterization of extracellular polymeric secretions (EPS) from modern soft marine stromatolites (Bahamas) and its inhibitory effect on CaCO3 precipitation; Kawaguchi T et al.; Bahamian soft marine stromatolites consist of cyanobacterial biofilms and carbonate sand grains (ooids) embedded in their extracellular polymeric secretions (EPS) . EPS were isolated from natural marine stromatolites and the laboratory cultured stromatolite forming cyanobacterium isolate Schizothix sp . Laboratory investigations were conducted to examine biochemical characteristics and the role of EPS in the inhibition of CaCO3 precipitation . EPS consisted of acid polysaccharides and proteins . SDS-PAGE and amino acid analysis suggested that EPS from both soft marine stromatolite and Schizothrix sp . mat contained small proteins (38 kD and 45 kD) enriched in aspartic acid and glutamic acid . Also, immuno blotting suggested that natural EPS contain high molecular weight acid polysaccharide (500 k) which may represent cross-linked products of laboratory cultured Schizothrix sp . acid polysaccharide (300 k) . EPS from both soft marine stromatolite and laboratory cultured Schizothrix sp . inhibited CaCO3 precipitation in vitro, as determined using pH drift assays examining pH decrease which occur in response to CaCO3 precipitation . PH drift assays of enzymatically and chemically modified EPS isolated from soft marine stromatolite and laboratory cultured Schizothrix sp . indicated that both uronic acids and protein fractions may be involved in the inhibition of CaCO3 precipitation.

Clin Microbiol Rev, 2002 Apr, 15(2), 194 - 222
Lung infections associated with cystic fibrosis; Lyczak JB et al.; While originally characterized as a collection of related syndromes, cystic fibrosis (CF) is now recognized as a single disease whose diverse symptoms stem from the wide tissue distribution of the gene product that is defective in CF, the ion channel and regulator, cystic fibrosis transmembrane conductance regulator (CFTR) . Defective CFTR protein impacts the function of the pancreas and alters the consistency of mucosal secretions . The latter of these effects probably plays an important role in the defective resistance of CF patients to many pathogens . As the modalities of CF research have changed over the decades from empirical histological studies to include biophysical measurements of CFTR function, the clinical management of this disease has similarly evolved to effectively address the ever-changing spectrum of CF-related infectious diseases . These factors have led to the successful management of many CF-related infections with the notable exception of chronic lung infection with the gram-negative bacterium Pseudomonas aeruginosa . The virulence of P . aeruginosa stems from multiple bacterial attributes, including antibiotic resistance, the ability to utilize quorum-sensing signals to form biofilms, the destructive potential of a multitude of its microbial toxins, and the ability to acquire a mucoid phenotype, which renders this microbe resistant to both the innate and acquired immunologic defenses of the host.

Clin Microbiol Rev, 2002 Apr, 15(2), 167 - 93
Biofilms: survival mechanisms of clinically relevant microorganisms; Donlan RM et al.; Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978 . We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health . Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels . Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells . Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms . Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices . Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood . Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process . Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms . Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient . In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.

Clin Microbiol Rev, 2002 Apr, 15(2), 155 - 66
Bacterial adhesion: seen any good biofilms lately?
Dunne WM Jr.
The process of surface adhesion and biofilm development is a survival strategy employed by virtually all bacteria and refined over millions of years . This process is designed to anchor microorganisms in a nutritionally advantageous environment and to permit their escape to greener pastures when essential growth factors have been exhausted . Bacterial attachment to a surface can be divided into several distinct phases, including primary and reversible adhesion, secondary and irreversible adhesion, and biofilm formation . Each of these phases is ultimately controlled by the expression of one or more gene products . Ultrastructurally, the mature bacterial biofilm resembles an underwater coral reef containing pyramidal or mushroom-shaped microcolonies of organisms embedded within an extracellular glycocalyx, with channels and cavities to allow the exchange of nutrients and waste . The biofilm protects its inhabitants from predators, dehydration, biocides, and other environmental extremes while regulating population growth and diversity through primitive cell signals . From a physiological standpoint, surface-bound bacteria behave quite differently from their planktonic counterparts . Recognizing that bacteria naturally occur as surface-bound and often polymicrobic communities, the practice of performing antimicrobial susceptibility tests using pure cultures and in a planktonic growth mode should be questioned . That this model does not reflect conditions found in nature might help explain the difficulties encountered in the management and treatment of biomedical implant infections.

Microbes Infect, 2002 Apr, 4(4), 481 - 9
Staphylococcus epidermidis infections; Vuong C et al.; The opportunistic human pathogen Staphylococcus epidermidis has become the most important cause of nosocomial infections in recent years . Its pathogenicity is mainly due to the ability to form biofilms on indwelling medical devices . In a biofilm, S . epidermidis is protected against attacks from the immune system and against antibiotic treatment, making S . epidermidis infections difficult to eradicate.

Mol Cell, 2002 Mar, 9(3), 685 - 94
Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing; Watson WT et al.; Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles . The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs . The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core . Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo . A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.

Oral Microbiol Immunol, 2002 Apr, 17(2), 108 - 12
Genetic exchange between Treponema denticola and Streptococcus gordonii in biofilms; Wang BY et al.; Some gram-positive oral bacteria such as Streptococcus gordonii are naturally transformable . We investigated the possibility of genetic exchange among oral bacteria using an erythromycin-resistant (Erm(r)) shuttle plasmid, pKMR4PE, as an indicator . S . gordonii Challis cells were co-cultivated with purified pKMR4PE or with Trepomena denticola harboring pKMR4PE either in broth or in artificial biofilms . Transformation of S . gordonii occurred both in broth and in biofilms with pKMR4PE or T . denticola harboring pKMR4PE as donor sources . These results provide direct experimental evidence that gene transfer can occur from T . denticola to S . gordonii.

Mol Microbiol, 2002 Feb, 43(3), 793 - 808
Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose; Solano C et al.; We report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development . The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcsABZC and bcsEFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase . Non-polar mutations of bcsC and bcsE genes and complementation experiments demonstrated that both operons are responsible for cellulose biosynthesis in both S . enteritidis and S . typhimurium . Using two different growth media, ATM and LB, we showed that the biofilm produced by S . enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions . Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose-deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S . enteritidis . However, cellulose-deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S . enteritidis on surface environments.

Acta Orthop Scand, 2002 Jan, 73(1), 11 - 9
Evaluation of four experimental osteomyelitis infection models by using precolonized implants and bacterial suspensions; Monzon M et al.; Staphylococcus aureus osteomyelitis, a major problem in orthopedic surgery, often involves biofilm bacteria adhering to implants and surrounding bone and tissues . The inadequacy of therapy or immunological surveillance has encouraged studies using animal models which simulate natural osteomyelitic infections, ensure the development of infections and avoid mortality . We evaluated 4 models for infection (8 animals/model) in rats, using stainless-steel implants in tibiae and a very adherent slime-producing bacterial strain . Each animal received: an implant containing a 12 h-biofilm with about 10(6) cfu (Model 1); an implant containing this biofilm and a suspension with about 10(4) cfu (Model 2): a sterile implant and a suspension with about 10(5) cfu (Model 3); or a sterile implant and a suspension with about 10(6) cfu (Model 4) . 63 days after surgery we found 100% rat survival, colonization of bone by implant biofilm bacteria in some animals and local, but not systemic infections . Model 1 (but not Models 2-4) reproduced an infection in both, tibiae and implants, most reliably (in 100% of the animals) . Model 3 was the least reliable (p < 0.01, 25% infected implants, 12% infected tibiae).

JAMA, 2002 Apr 3, 287(13), 1710 - 5
Mucosal biofilm formation on middle-ear mucosa in the chinchilla model of otitis media; Ehrlich GD et al.; CONTEXT: Chronic otitis media with effusion (OME) has long been considered to be a sterile inflammatory process . The previous application of molecular diagnostic technologies to OME suggests that viable bacteria are present in complex communities known as mucosal biofilms; however, direct imaging evidence of mucosal biofilms associated with OM is lacking . OBJECTIVE: To determine whether biofilm formation occurs in middle-ear mucosa in an experimental model of otitis media . DESIGN AND MATERIALS: A total of 48 research-grade, young adult chinchillas weighing 500 g were used for 2 series of animal experiments: one to obtain specimens for scanning electron microscopy and the other to obtain specimens for confocal laser scanning microscopy using vital dyes . In each series, 21 animals were bilaterally injected with viable Haemophilus influenzae bacteria and 1 was inoculated to account for expected mortality . Three served as negative controls . Effusions and mucosal specimens were collected from 2 infected animals that were euthanized at 3, 6, 12, and 24 hours and at days 2, 4, 5, 10, 16, and 22 after inoculation . MAIN OUTCOME MEASURES: Images were analyzed for biofilm morphology, including presence of microcolony formation and for presence of bacteria on tissue surfaces . RESULTS: Scanning electron microscopy demonstrated that biofilm formation was evident in all specimens from animals beginning 1 day after infection and was present through 21 days . Confocal laser scanning microscopy indicated that bacteria within the biofilms are viable . CONCLUSION: These preliminary findings provide evidence that mucosal biofilms form in an experimental model of otitis media and suggest that biofilm formation may be an important factor in the pathogenesis of chronic otitis media with effusion.

J Med Microbiol, 2002 Apr, 51(4), 344 - 9
Mixed species biofilms of Candida albicans and Staphylococcus epidermidis; Adam B et al.; A simple catheter disk model system was used to study the development in vitro of mixed species biofilms of Candida albicans and Staphylococcus epidermidis, two organisms commonly found in catheter-associated infections . Two strains of S . epidermidis were used: a slime-producing wild type (strain RP62A) and a slime-negative mutant (strain M7) . In mixed fungal-bacterial biofilms, both staphylococcal strains showed extensive interactions with C . albicans . The susceptibility of 48-h biofilms to fluconazole, vancomycin and mixtures of the drugs was determined colorimetrically . The results indicated that the extracellular polymer produced by S . epidermidis RP62A could inhibit fluconazole penetration in mixed fungal-bacterial biofilms . Conversely, the presence of C . albicans in a biofilm appeared to protect the slime-negative staphylococcus against vancomycin . Overall, the findings suggest that fungal cells can modulate the action of antibiotics, and that bacteria can affect antifungal activity in mixed fungal-bacterial biofilms.

Comp Med, 2001 Aug, 51(4), 361 - 8
High mortality in a large-scale zebrafish colony (Brachydanio rerio Hamilton & Buchanan, 1822) associated with Lecythophora mutabilis (van Beyma) W . Gams & McGinnis; Dykstra MJ et al.; Zebrafish (Brachydanio rerio) have become an important model system for studying vertebrate embryonic development and gene function through manipulation of genotype and characterization of resultant phenotypes . An established research zebrafish colony without substantial disease problems for more than 7 years of operation began experiencing appreciable mortalities in November of 1997 . Young fish (fry), from five to 24 days after hatching, spontaneously developed elongate strands of organic material protruding from the mouth, operculum, and anal pore, leading workers in the laboratory to describe the infected fish as "bearded." Unlike typical freshwater fish fungal infections, the skin surface did not have evidence of fungal colonization . The disease was associated with progressive lethargy, reduced feeding, and subsequent mortality . From 10 to 100% of the fry in a given tank were affected . Initial examination indicated that the biofilm around the head of affected fry consisted of bundles of septate fungal hyphae, large numbers of mixed bacterial populations, and protozoans . Environmental samples of air and water in the laboratory were obtained to ascertain the source of the infective agent and to isolate and identify the fungus . A fungus identified as Lecythophora mutabilis was isolated repeatedly from infected fish and water samples from infected fish tanks, and from the main laboratory water supply tanks, but not from laboratory air . Some biofilm beards on fish were found to consist of relatively pure bacterial populations, and beards on occasional fish examined in the later part of the study consisted of hyphae and spores of the oomycete genus Aphanomyces . Lecythophora mutabilis did not invade tissues; however, elimination of the epizootic correlated with reduction in the number of L . mutabilis conidia in the water following modification of the laboratory water system by use of new filtration and sterilization systems . We conclude that the dense hyphal strands of L . mutabilis composing the predominant biofilm type, along with mixed bacteria and protozoa, contributed to the die-off in young fry by occluding the oral cavity and/or gills, leading to starvation and/or asphyxiation.

J Clin Microbiol, 2002 Apr, 40(4), 1244 - 8
Biofilm production by isolates of Candida species recovered from nonneutropenic patients: comparison of bloodstream isolates with isolates from other sources; Shin JH et al.; Biofilm production has been implicated as a potential virulence factor of some Candida species responsible for catheter-related fungemia in patients receiving parenteral nutrition . We therefore compared clinical bloodstream isolates representing seven different Candida species to each other and to those from other anatomical sites for the capacity to form biofilms in glucose-containing medium . Potential associations between the capacity to form biofilms and the clinical characteristics of fungemia were also analyzed . Isolates included the following from nonneutropenic patients: 101 bloodstream isolates (35 C . parapsilosis, 30 C . albicans, 18 C . tropicalis, 8 C . glabrata, and 10 other Candida species isolates) and 259 clinical isolates from other body sites (116 C . albicans, 53 C . glabrata, 43 C . tropicalis, 17 C . parapsilosis, and 30 other Candida species isolates) . Organisms were grown in Sabouraud dextrose broth (SDB) containing a final concentration of 8% glucose to induce biofilm formation, as published previously . Biofilm production was determined by both visual and spectrophotometric methods . In this medium, biofilm production by C . albicans isolates was significantly less frequent (8%) than that by non-C . albicans Candida species (61%; P < 0.0001) . The overall proportion of non-C . albicans Candida species isolates from the blood that produced biofilms was significantly higher than that of non-C . albicans Candida isolates obtained from other sites (79% versus 52%; P = 0.0001) . Bloodstream isolates of C . parapsilosis alone were significantly more likely to be biofilm positive than were C . parapsilosis isolates from other sites (86% versus 47%; P = 0.0032) . Non-C . albicans Candida species, including C . parapsilosis, were more likely to be biofilm positive if isolates were derived from patients whose candidemia was central venous catheter (CVC) related (95%; P < 0.0001) and was associated with the use of total parenteral nutrition (TPN) (94%; P < 0.005) . These data suggest that the capacity of Candida species isolates to produce biofilms in vitro in glucose-containing SDB may be a reflection of the pathogenic potential of these isolates to cause CVC-related fungemia in patients receiving TPN.

Appl Environ Microbiol, 2002 Apr, 68(4), 2008 - 17
Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression; Heydorn A et al.; Four strains of Pseudomonas aeruginosa (wild type, Delta(pil)HIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy . The structural developments of the biofilms were quantified by the computer program COMSTAT (A . Heydorn, A . T . Nielsen, M . Hentzer, C . Sternberg, M . Givskov, B . K . Ersboll, and S . Molin, Microbiology 146:2395-2407, 2000) . Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P . aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments . The results showed that the wild type, the Delta(pil)HIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points . The wild type and the lasI mutant formed uniform, densely packed biofilms . The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the Delta(pil)HIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size . The results are discussed in relation to the current model of P . aeruginosa biofilm development.

Appl Environ Microbiol, 2002 Apr, 68(4), 1938 - 46
Spatially oscillating activity and microbial succession of mercury-reducing biofilms in a technical-scale bioremediation system; von Canstein H et al.; Mercury-contaminated chemical wastewater of a mercury cell chloralkali plant was cleaned on site by a technical-scale bioremediation system . Microbial mercury reduction of soluble Hg(II) to precipitating Hg(0) decreased the mercury load of the wastewater during its flow through the bioremediation system by up to 99% . The system consisted of a packed-bed bioreactor, where most of the wastewater's mercury load was retained, and an activated carbon filter, where residual mercury was removed from the bioreactor effluent by both physical adsorption and biological reduction . In response to the oscillation of the mercury concentration in the bioreactor inflow, the zone of maximum mercury reduction oscillated regularly between the lower and the upper bioreactor horizons or the carbon filter . At low mercury concentrations, maximum mercury reduction occurred near the inflow at the bottom of the bioreactor . At high concentrations, the zone of maximum activity moved to the upper horizons . The composition of the bioreactor and carbon filter biofilms was investigated by 16S-23S ribosomal DNA intergenic spacer polymorphism analysis . Analysis of spatial biofilm variation showed an increasing microbial diversity along a gradient of decreasing mercury concentrations . Temporal analysis of the bioreactor community revealed a stable abundance of two prevalent strains and a succession of several invading mercury-resistant strains which was driven by the selection pressure of high mercury concentrations . In the activated carbon filter, a lower selection pressure permitted a steady increase in diversity during 240 days of operation and the establishment of one mercury-sensitive invader.

Appl Environ Microbiol, 2002 Apr, 68(4), 1585 - 94
Temporal changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat; Huber JA et al.; The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles . To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000 . The archaeal population was divided into particle-attached (>3-microm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms . To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids . The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments . The particle-attached fraction consistently showed greater diversity than the free-living fraction . The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community . The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.

Ann Otol Rhinol Laryngol, 2002 Mar, 111(3 Pt 1), 200 - 3
Comparison of the microbial composition of voice prosthesis biofilms from patients requiring frequent versus infrequent replacement; Elving GJ et al.; This study was performed to establish a possible difference in biofilm composition in patients who require frequent versus infrequent prosthesis replacement . Only Groningen button voice prostheses that were removed because of increased airflow resistance or leakage of food or liquids through the prosthesis were considered for this study . These prostheses were selected from a total of 692 failed voice prostheses over a 2-year evaluation period . The failed voice prostheses were subdivided into a short-lifetime group, corresponding to an implantation period of less than 4 months (20 voice prostheses), and an extended-lifetime group, corresponding to an implantation period of greater than 9 months (18 voice prostheses) . The biofilm was removed from the valve sides of the prostheses . The bacterial strain Rothia dentocariosa and the yeast strains Candida albicans I and Candida tropicalis were the predominant strains isolated from the biofilms on the voice prostheses in the short-lifetime group, whereas in the extended-lifetime group, R dentocariosa was found with a fourfold lower isolation frequency and C albicans I was found with a twofold lower isolation frequency . Candida tropicalis was absent from the extended-lifetime group.

Curr Microbiol, 2002 Apr, 44(4), 262 - 6
Repressed respiration of oral streptococci grown in biofilms; Nguyen PT et al.; The respiratory activities of oral streptococci grown in biofilms were found to be markedly repressed compared with those of cells from aerobic culture, or for Streptococcus mutans GS-5, even for those grown in static culture . Respiration rates generally reflected levels of NADH oxidase activities in cell extracts . Superoxide dismutase levels were somewhat reduced in biofilm cells . However, sensitivities to oxidative damage caused by H2O2, t-butylhydroperoxide, or 8-hydroxyquinoline were not greatly different for cells from suspension cultures and those from either intact or dispersed biofilms . The capacities of S . sanguis and S . gordonii to produce H2O2 also were markedly repressed by biofilm growth, and presumably this repression would affect the ecology of dental plaque by reducing oxidative stresses under crowded conditions.

J Antimicrob Chemother, 2002 Apr, 49(4), 683 - 7
Alcoholic ingredients in skin disinfectants increase biofilm expression of Staphylococcus epidermidis; Knobloch JK et al.; The pathogenesis of Staphylococcus epidermidis is correlated with biofilm formation . We investigated the effect of three common alcoholic skin disinfectants, ethanol, n-propanol and isopropanol, on the biofilm formation of 37 clinical, icaADBC-positive S . epidermidis isolates . In alcohol-supplemented media 18 strains displayed increased biofilm expression . Sixteen of 19 strains were generally incapable of biofilm formation . In three representative isolates, the increase in biofilm formation was paralleled by increased polysaccharide intercellular adhesin synthesis . Regarding the widespread use of alcoholic skin disinfectants, it is possible that the alcohol-inducible biofilm phenotype of S . epidermidis could add to the development of foreign body-related infections.

Compend Contin Educ Dent, 2000 Oct, 21(10A), 906 - 14, 916-8
Assessment and management of periodontal infections: a medical-surgical approach; Oringer RJ et al.; Over the last 30 years, significant advances have been achieved in elucidating the etiology, pathogenesis, and treatment of periodontal diseases . A new paradigm has emerged that includes the initiation of disease by specific bacteria within a biofilm that stimulate an immunoinflammatory host response, resulting in host tissue destruction . Disease modifiers, which may be of genetic, environmental, or acquired origin, have been recognized as major determinants of disease severity and progression . Current treatment protocols include an assessment of risk factors to identify an individual's degree of susceptibility and therapeutic responsiveness . Basic and clinical research has resulted in the development of several strategies to identify specific bacteria and host-derived markers associated with disease progression . Preventive and therapeutic antimicrobial therapies, which use various delivery systems, have been devised to target drug placement to the infection site . More recently, host modulatory therapies have been created that inhibit disease progression through the reduction of inflammatory mediators . Finally, biological mediators, including growth and differentiation factors, have been used to enhance an individual's healing potential to achieve periodontal and bone regeneration . A combined medical-surgical approach is indicated for these new methods of diagnosis, prevention, and treatment of periodontal diseases, which will allow for earlier treatment interventions and improved patient outcomes.

Br J Dermatol, 2002 Feb, 146(2), 194 - 201
Epidermal barrier lipids in human vernix caseosa: corresponding ceramide pattern in vernix and fetal skin; Hoeger PH et al.; BACKGROUND: Vernix caseosa is a protective biofilm covering the fetus during the last trimester . Vernix and epidermal barrier lipids (i.e . cholesterol, free fatty acids and ceramides) appear to share protective functions for fetal and neonatal skin . OBJECTIVES: To analyse vernix samples for epidermal barrier lipid content, and to compare lipid profiles of vernix with those of fetal and postnatal epidermis . METHODS: Vernix samples were collected from 21 healthy term neonates . Skin samples were collected from 10 fetuses aborted between gestational week (GW) 16 and 25, nine infants and 11 older children . Lipids were extracted according to standard protocols and analysed by high-performance thin-layer chromatography . RESULTS: Vernix contained 196.5 +/- 70.1 microg barrier lipids mg-1 protein (mean +/- SD) . Cholesterol formed the major barrier lipid fraction (52.8%), followed by free fatty acids (27.7%) and ceramides (20.1%) . The ceramide composition of vernix resembled that of mid-gestational (GW 23-25) fetal epidermis both qualitatively and quantitatively, while there were major differences from postnatal epidermis . The total epidermal ceramide concentration increased significantly between prenatal and postnatal samples . CONCLUSIONS: The composition pattern of ceramides mirrors that of mid-gestational fetal epidermis . Vernix thus represents a 'homologous' substitute for the immature epidermal barrier in fetal skin . The differential role of individual ceramides in this process remains to be established.

Water Res, 2002 Mar, 36(5), 1385 - 91
Methane yield as a monitoring parameter for the start-up of anaerobic fixed film reactors; Michaud S et al.; This paper describes the variation of the methane yield during the start-up period of an anaerobic fluidized bed reactor . After a lag phase, with acclimatized sludge, the methane yield increased with time during biofilm development up to the theoretical steady yield value, reported to be around 0.351 CH4/g CODdeg . The establishment of the biofilm required a high consumption of organic material through the microbial synthesis (anabolism), thereby reducing the proportion of substrate converted to methane . As a result, this yield could be an indirect metabolic parameter for evaluating a start-up operation . It could provide vital information about bacterial fixation processes and is easy to be applied to any biofilm reactor, such as anaerobic filters, where biomass sampling is impracticable . Monitoring this parameter could also give useful dynamic information about the different steps of colonization and biomass attachment, which could be used to improve the start-up performance.

Water Sci Technol, 2002, 45(3), 35 - 44
Dissolved oxygen in gravity sewers--measurement and simulation; Gudjonsson G et al.; Dissolved oxygen (DO) concentrations were during 2 months continuously measured in an intercepting sewer . Measurements were made upstream and downstream in a 3.6 km gravity sewer . DO showed significant diurnal variations mainly caused by changes in the organic matter composition of the wastewater . At low temperatures the gravity sewer was strictly aerobic . However, towards the end of the measuring campaign, DO concentrations decreased as temperature increased and the sewer became anaerobic part of the day . A conceptual model that takes into account bulk water and biofilm DO uptake as well as reaeration was used to simulate the DO measured . Using measurements from the upstream station as input, the model was calibrated to yield good validation results of the DO at the downstream station.

Acta Cient Venez, 2001, 52 Suppl 1, 45 - 9
{Sliding motility and biofilm formation in mycobacteria}; Recht J et al.; Using as a model Mycobacterium smegmatis, a non-motile microorganism, we have studied for the first time in mycobacteria the phenomenon known as sliding motility, as well as the process of biofilm formation . A screen of random transposon mutants was performed in order to identify the genes required for mycobacterial sliding over the surface of motility plates . The genetic analysis described here has been published recently . The genes required for sliding and for biofilm formation (mps and tmtpC) are involved in the biosynthesis of the glycopeptidolipids (GPLs) and their transport to the mycobacterial capsule . Based on our results, we suggest a model for the role of the GPLs in both phenomena.

Rev Esp Anestesiol Reanim, 2002 Jan, 49(1), 17 - 33
{Prophylaxis of infective complications of central venous catheters}; Gomez Luque A et al.; Infections related to central venous cannulation present first-magnitude problems in recovery rooms and intensive care units . Catheter-related bloodstream infection (CRBSI) is the most serious complication because of its high frequency and a mortality rate that averages around 3% . Although infections arise for various reasons, point-of-insertion contamination is the main cause when catheters are implanted for periods of less than 10 days . Contaminating microorganisms (especially Staphyloccocus epidermidis) find refuge from the host's defenses in a biofilm that covers the catheter . Several factors participate in the formation of this biofilm, such as catheter composition, proteins of the host or type of microorganism . Biofilm bacteria are resistant to both antibiotics and the host's own defenses (e.g . phagocytes and antibodies) . The microorganism can then begin to reproduce, possibly leading to bloodstream infection . The measures designed to prevent this process include recommendations for both catheter insertion and maintenance . Recent meta-analyses have led to certain conclusions but no unanimity among authors . Thus, there is agreement on the adoption of strict aseptic technique during catheter insertion, on the use of chlorhexidine as a skin antiseptic and on choice of the subclavian vein . Such measures significantly decrease the frequency of CRBSI . Maintenance techniques that have been shown to be effective are the use of connectors impregnated with antiseptic, catheters impregnated with antiseptics or antibiotics, and permeable dressings . Additionally, building an experienced infusion-therapy team to insert and maintain central venous catheters has been shown to be one of the most effective measures for preventing CRBSI.

J Hazard Mater, 2002 Mar 29, 90(3), 279 - 95
Evaluation of microbial stability of simulated solid and liquid waste forms using a refined biofilm formation method; Idachaba MA et al.; A refined biofilm formation method was used to evaluate the stability of a simulated liquid waste form containing a simulated liquid waste (salts) and cement in three different proportions, and a simulated solid waste form containing a simulated solid waste (resin) and cement in three different proportions . The experimental samples of all the simulated liquid waste forms showed evidence of microbial growth on them after 3 days of evaluation as indicated by substantial increase in sulfate production, and exhibited considerable instability to microbial degradation as indicated by substantial leaching of calcium . The experimental samples of all the simulated solid waste forms showed evidence of inhibition of growth of Thiobacillus thiooxidans for about 18 days, after which the growth of the microbe became evident in two out of three . Within the growth inhibition period, the differences between experimental and control samples were minor . After the growth of T . thiooxidans became evident, comparatively higher degradations were observed for the experimental samples of the resin containing solid waste forms.

Lasers Surg Med, 2001, 29(5), 448 - 54
Laser-induced shock waves enhance sterilization of infected vascular prosthetic grafts; Nigri GR et al.; BACKGROUND AND OBJECTIVE: Bacteria that cause infection of vascular prosthetic grafts produce an exopolysaccharide matrix known as biofilm . Growth in biofilms protects the bacteria from leukocytes, antibodies and antimicrobial drugs . Laser-generated shock waves (SW) can disrupt biofilms and increase drug penetration . This study investigates the possibility of increasing antibiotic delivery and sterilization of vascular prosthetic graft . STUDY DESIGN/MATERIALS AND METHODS: Strains of Staphylococcus epidermidis and S . aureus were isolated from infected prosthetic grafts obtained directly from patients . Dacron grafts were inoculated with the isolated bacteria, which were allowed to form adherent bacterial colonies . The colonized grafts underwent the following treatments: (a) antibiotic (vancomycin) alone; (b) antibiotic and SW (c) saline only; and (d) saline and SW . Six hours after treatment, the grafts were sonicated, the effluent was cultured and the colony forming units (CFU) were counted . RESULTS: CFU recovered from control grafts colonized by S . epidermidis were comparable: saline, 3.05 x 10(8) and saline+SW 3.31 x 10(8) . The number of S . epidermidis CFU diminished to 7.61 x 10(6) after antibiotic treatment but the combined antibiotic+SW treatment synergistically decreased CFU number to 1.27 x 10(4) (P<0.001) . S . aureus showed a higher susceptibility to the antibiotic: 2.26 x 10(6) CFU; antibiotic +SW treatment also had an incremental effect: 8.27 x 10(4) CFU (P<0.001) . CONCLUSIONS: This study demonstrates that laser-generated shock waves have no effects alone, but can enhance the effectiveness of antibiotics against bacteria associated with prosthetic vascular graft biofilms, suggesting that this treatment may be of value as adjunctive therapy for prosthetic graft infections.

Semin Respir Infect, 2002 Mar, 17(1), 47 - 56
Pulmonary infections in patients with cystic fibrosis; Rajan S et al.; Cystic fibrosis (CF) is an autosomal-recessive disorder and affects about 60,000 people worldwide . The CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR), was found in 1989 and over 800 mutations have been sequenced . Although our understanding of the pathophysiology of CF has increased, pulmonary infections remain the major cause of morbidity and mortality . During the first decade of life, Staphylococcus aureus and nontypable Haemophilus influenza are most common, but Pseudomonas aeruginosa may be the first pathogen isolated in infants . By 18 years of age, 80% of patients harbor P . aeruginosa and 3.5% harbor Burkholderia cepacia . Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and nontuberculous mycobacteria may be newly emerging CF pathogens . The traditional approach to managing patients with CF is to treat acute pulmonary exacerbations with intravenous antimicrobial agents . However, prophylactic strategies to prevent initial infection or to delay chronic infection with P . aeruginosa or chronic maintenance therapy to slow deterioration of lung function may also improve clinical status . Recognition of the role of inflammation, even early in life, in the absence of clinical symptoms, has led to treatment with anti-inflammatory agents . Novel strategies to disrupt biofilm formation, stimulate chloride conductance, and replace abnormal CFTR are currently being studied.

Perit Dial Int, 2001, 21 Suppl 3, S357 - 8
Effect in a rat model of heparinized peritoneal dialysis catheters on bacterial colonization and the healing of the exit site; Kim YL et al.; We performed a prospective, double-blind, randomized study to evaluate whether stable surface heparinization of silicone peritoneal dialysis (PD) catheters prevents bacterial colonization or biofilm formation and improves healing of the exit site . Heparinized catheters were implanted in 20 Sprague-Dawley rats (group H) and non heparinized catheters in another 20 (group C) . The PD catheters, constructed of silicon tubing with two polyester cuffs, were patterned after the standard Tenckhoff catheter . A covalent multipoint method of attachment onto polymeric surfaces was used for stable, permanent chemical immobilization of heparin on the PD catheter . Dialysis exchanges (25-mL instillation volume) were performed twice daily for 4 weeks through the permanent catheter . Prophylactic antibiotics were not used . The exit sites were evaluated at 2-week intervals . The extent of biofilm coverage on the intraperitoneal portion of the catheter (obtained at the end of the experiment) was assessed, and sonicated fluid from the catheter tip was cultured for evaluating bacterial colonization of the catheter . Exit-site scores in group H were lower than in group C (p = 0.052) at the end of week 4 . Bacterial colonization tended to be less common in group H {2 of 12 catheters (17%)} than in group C {8 of 15 catheters (53%); p = 0.058}, but the extent of biofilm, the peritonitis rate, and the inflammation score of tissue adjacent to the cuff were not different between the groups . Those data suggest that heparinized PD catheters can be a practical approach to the prevention of bacterial colonization and can improve healing of the exit site.

Perit Dial Int, 2001, 21 Suppl 3, S213 - 7
Biofilms in peritoneal dialysis; Dasgupta MK et al.; Microbial adhesions and biofilm bacterial growth have been implicated in serious infections associated with the use of bioprosthetic medical devices and indwelling catheters in humans . Biofilm bacterial growth also commonly occurs on peritoneal dialysis (PD) catheters from skin bacteria . Mature biofilms develop high antibiotic resistance and cause recurrent peritonitis and catheter loss in a subgroup of PD patients . That subgroup of patients can be identified by comparing the antibiotic sensitivities {minimum inhibitory concentrations (MICs)} of a biofilm culture and a routine microbiologic (planktonic) culture of the same PD effluent.

Ann Periodontol, 2001 Dec, 6(1), 125 - 37
Periodontitis and diabetes interrelationships: role of inflammation; Iacopino AM; Diabetes mellitus is a systemic disease with several major complications affecting both the quality and length of life . One of these complications is periodontal disease (periodontitis) . Periodontitis is much more than a localized oral infection . Recent data indicate that periodontitis may cause changes in systemic physiology . The interrelationships between periodontitis and diabetes provide an example of systemic disease predisposing to oral infection, and once that infection is established, the oral infection exacerbates systemic disease . In this case, it may also be possible for the oral infection to predispose to systemic disease . In order to understand the cellular/molecular mechanisms responsible for such a cyclical association, one must identify common physiological changes associated with diabetes and periodontitis that produce a synergy when the conditions coexist . A potential mechanistic link involves the broad axis of inflammation, specifically immune cell phenotype, serum lipid levels, and tissue homeostasis . Diabetes-induced changes in immune cell function produce an inflammatory immune cell phenotype (upregulation of proinflammatory cytokines from monocytes/polymorphonuclear leukocytes and downregulation of growth factors from macrophages) . This predisposes to chronic inflammation, progressive tissue breakdown, and diminished tissue repair capacity . Periodontal tissues frequently manifest these changes because they are constantly wounded by substances emanating from bacterial biofilms . Diabetic patients are prone to elevated low density lipoprotein cholesterol and triglycerides (LDL/TRG) even when blood glucose levels are well controlled . This is significant, as recent studies demonstrate that hyperlipidemia may be one of the factors associated with diabetes-induced immune cell alterations . Recent human studies have established a relationship between high serum lipid levels and periodontitis . Some evidence now suggests that periodontitis itself may lead to elevated LDL/TRG . Periodontitis-induced bacteremia/endotoxemia has been shown to cause elevations of serum proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), which have been demonstrated to produce alterations in lipid metabolism leading to hyperlipidemia . Within this context, periodontitis may contribute to elevated proinflammatory cytokines/serum lipids and potentially to systemic disease arising from chronic hyperlipidemia and/or increased inflammatory mediators . These cytokines can produce an insulin resistance syndrome similar to that observed in diabetes and initiate destruction of pancreatic beta cells leading to development of diabetes . Thus, there is potential for periodontitis to exacerbate diabetes-induced hyperlipidemia, immune cell alterations, and diminished tissue repair capacity . It may also be possible for chronic periodontitis to induce diabetes.

Mol Microbiol, 2001 Dec, 42(5), 1223 - 32
Spontaneous sequence duplication within an open reading frame of the pneumococcal type 3 capsule locus causes high-frequency phase variation; Waite RD et al.; The molecular genetic basis of high-frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated . Pneumococci were grown in sorbarod biofilms at 34 degrees C to mimic nasopharyngeal carriage . Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11-239 bp segments within the cap3A gene of the type 3 capsule locus . These duplications alone were found to be responsible for high-frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wild-type cap3A genes, indicating the precise excision of the duplication . Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication . This apparently random duplication giving rise to phase variation is in stark contrast to the 'preprogrammed' contingency genes in many Gram-negative organisms that possess homopolymeric sequence repeats or motifs for site-specific recombination.

Mol Microbiol, 2001 Dec, 42(5), 1199 - 209
The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis; Hamon MA et al.; Biofilms are structured communities of cells encased in a polymeric matrix and adherent to a surface, interface or each other . We report here that the soil bacterium Bacillus subtilis forms biofilms . By confocal scanning laser microscopy, we observed that B . subtilis adhered to abiotic surfaces and formed a three-dimensional structure > or =30 microm in depth . These biofilms appeared to be at least partly encased in an extracellular polysaccharide matrix, as they could be stained with Calcofluor, a polysaccharide-binding dye . To understand the molecular mechanism of biofilm formation, we screened previously characterized mutants for a defect in biofilm formation . We found that mutations in spo0A, which encodes the major early sporulation transcription factor, caused a defect in biofilm formation . spo0A mutant cells adhered to a surface in a monolayer of cells rather than a three-dimensional biofilm . The requirement of Spo0A for biofilm development appears to result from its role in negatively regulating AbrB . Mutations in abrB suppressed the biofilm defect of a spo0A mutant, indicating that AbrB negatively regulates at least one gene that is required for the transition from a monolayer of attached cells to a mature biofilm . Implications of biofilm development for the ecology of B . subtilis are discussed.

Environ Toxicol Chem, 2002 Mar, 21(3), 626 - 32
Responses of biofilms to combined nutrient and metal exposure; Ivorra N et al.; Numerous studies have reported marked sensitivities of diatom species to phosphate and organic pollution but have ignored interactions with other common contaminants . The aim of the present study was to investigate the single and joint effects of increased phosphate and metal (cadmium, zinc) concentrations on benthic diatom communities . Microalgal biofilms from a relatively unpolluted stream were exposed in the laboratory to Zn, Cd, and P, separately and in combination, in concentrations found at a polluted stream in the same catchment . The Zn concentration reduced algal growth in biofilms more than the Cd concentration . Phosphate compensated for the single effect of each metal but not for their combined effects . Diatom community changes were evaluated using water quality indices based on the empirical sensitivities of taxa to nutrients (TDI) and organic pollution (%PTV) . Phosphate exposure resulted in an increase of the eutrophy rank and presumed pollution-tolerant taxa . In contrast, exposure to Zn, Zn+Cd, and Zn+Cd+P caused a marked reduction of the TDI and %PTV community values . The successional trends in the laboratory matched the observed differences in microphyte communities in the reference and polluted river stations . However, the autoecology of the species present also revealed that the resulting composition of diatom communities cannot be attributed solely to the direct toxic effects of metal and nutrients and their interaction . Observed changes in the relative abundance of species are also determined by their growth form and microdistribution in biofilms.

Eur J Oral Sci, 2002 Feb, 110(1), 48 - 53
The effect of dental restorative materials on dental biofilm; Auschill TM et al.; To investigate the arrangement of biofilms formed in vivo, volunteers wore splints with slabs of six different dental materials inserted to collect smooth surface plaque . After 5 d of undisturbed plaque accumulation, the specimens were vital stained and analyzed by the confocal laser scanning microscopy (CLSM) to evaluate the percentage of vital biofilm microflora (VF percentage) . Further parameters were the area of the specimens covered by plaque (surface coating; SC, %) and the height of the biofilms (BH, pm) . The metals amalgam and gold, the compomer, as well as the glass-ionomer cement harboured an almost entirely dead biofilm (VF <8%) . Resin composite led to vitality values between 4 and 21%, while a very thin biofilm on ceramic revealed the highest vitality values (34-86%) . SC varied from 6% on glass-ionomer cement to 100% on amalgam . BH reached its highest value on amalgam and gold of 17 and 11 microm, respectively, while heights of between 1 and 6 microm were found on the ceramic, resin composite, compomer and the glass-ionomer cement . Within their limits, the present findings indicate that amalgam, gold, compomer and glass-ionomer cement exert an influence against the adhering biofilm . No general relationship could be established between the different parameters VF percentage, SC percentage and BH (microm).

Lett Appl Microbiol, 2002, 34(3), 205 - 9
Immobilization of fructosyltransferase from Streptococcus mutans on hydroxyapatite surfaces induces the formation of multimeric complexes; Bronshteyn M et al.; AIMS: To investigate the formation of fructosyltransferase (FTF) complexes on hydroxyapatite (HA) surfaces . METHODS AND RESULTS: Cell-free extracellular FTF from Streptococcus mutans, purified from hyperproducing strain V-1995, was adsorbed onto HA and then eluted from the surface by means of a concentration gradient of potassium phosphate buffer . The FTF monomers loaded onto HA formed, upon adsorption, various complexes ranging from 200 to 700 kDa as demonstrated using native PAGE . All these complexes exhibited enzymatic activity . CONCLUSIONS: Adsorption of FTF onto HA induced the formation of stable and enzymatically-active complexes . SIGNIFICANCE AND IMPACT OF THE STUDY: The formation of these complexes may explain the change of FTF catalytic properties after adsorption onto HA . This study is another step in determining the properties of a-cellular constituents of the oral biofilm.

Lett Appl Microbiol, 2002, 34(3), 173 - 7
Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine; Nychas GJ et al.; AIMS: To establish the site of microbial growth on naturally black fermented table olives, and to monitor the population dynamics of yeasts and selected micro-organisms together with the changes in organic acid profile and pH in the cover brine during fermentation . METHODS AND RESULTS: During fermentation, the numbers of Enterobacteriaceae and Pseudomonas spp . in the brine decreased whilst lactic acid bacteria and yeast populations increased . Scanning electron microscopy showed that a yeast-rich biofilm developed on the epicuticular wax of the olive skin during fermentation . Yeasts also predominated in the stomatal openings, but bacteria were more numerous in intercellular spaces in the sub-stomatal flesh . Citric, malic and tartaric acids were the major organic acids accumulating in the brine during fermentation . CONCLUSIONS: Micro-organisms associated with the skin, stomata and flesh in fermenting black olives may experience different local conditions to those prevailing in the cover brine . SIGNIFICANCE AND IMPACT OF THE STUDY: These are the first observations of the micro-organisms associated with the fruit of naturally fermented black olives and of the accumulation of specific organic acids during fermentation.

J Lab Clin Med, 2002 Jan, 139(1), 28 - 34
Role of bile mucin in bacterial adherence to biliary stents; Zhang H et al.; Biliary stent placement is a well-established method of relieving obstructive jaundice . However, a frequent complication is occlusion of the stent caused by bacterial biofilm formation and sludge accumulation . In this study we investigated the possible effect of bile mucin on bacterial adherence to biliary stents at the initial stage of biofilm formation . By means of an in vitro bile-perfusion system, polyethylene stents were perfused with pig gallbladder bile infected with Escherichia coli . The concentrations of mucin in the pig bile were adjusted with purified mucin . The amount of bacteria adhering to the inner surface of the stents was measured and compared for stents perfused with bile containing various concentrations of mucin . Furthermore, we conditioned the stent inner surface with purified pig bile mucin and observed the effect of the conditioning on subsequent bacterial adherence . In addition, a common method for assaying bacterial adhesion with polystyrene microtiter plates was also used in this study . The results demonstrated that more bacteria adhered to the inner surface of stents perfused with bile containing 5 mg/mL mucin than of those perfused with bile containing 0.5 and 0 mg/mL mucin . Increased bacterial adherence was demonstrated on the stent surfaces conditioned with purified mucin compared with that seen on the nonconditioned stent surfaces . The optical densities indicating bacterial adhesion in the microtiter plate wells precoated with mucin were higher than those in non-coated plate wells . The in vitro results indicate that when a biliary stent is implanted in vivo, mucin in bile may condition the stent inner surface, modulate subsequent bacterial adherence to the surface, and participate in stent occlusion.

J Bacteriol, 2002 Mar, 184(6), 1678 - 84
An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus; Kearns DB et al.; An extracellular matrix connects bacteria that live in organized assemblages called biofilms . While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction . The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development . The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine . We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A) . Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family . Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors . However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed . Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils . The results suggest that this metalloprotease is involved in sensory transduction.

J Bacteriol, 2002 Mar, 184(6), 1630 - 9
A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation; Wang SY et al.; ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli . To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V . anguillarum serotype O1 were cloned . The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively . Deletion mutations were made in each gene and functional analyses were done . Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors . The toxR mutant but not the toxS mutant was 20% less motile than the wild type . Like many regulatory proteins, ToxR was shown to negatively regulate its own expression . Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP . The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V . cholerae, and which has an amino terminus 75% identical to that of OmpU . ToxR and to a lesser extent ToxS enhanced resistance to bile . Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR . Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.

J Bacteriol, 2002 Mar, 184(6), 1617 - 29
VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum; Croxatto A et al.; Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi . In this study, we have cloned an additional gene of this circuit, vanT, encoding a V . harveyi LuxR-like transcriptional regulator . A V . anguillarum Delta vanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence . Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ . Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the Delta vanT strain . SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway . HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production . V . anguillarum strains require an active VanT to produce high levels of an L-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production . Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation . A V . anguillarum Delta vanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms . Hence, a new member of the V . harveyi LuxR transcriptional activator family has been characterized in V . anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

J Bacteriol, 2002 Mar, 184(6), 1522 - 9
The global regulatory hns gene negatively affects adhesion to solid surfaces by anaerobically grown Escherichia coli by modulating expression of flagellar genes and lipopolysaccharide production; Landini P et al.; The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation . In this report we show that stationary-phase cultures of Escherichia coli W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28 degrees C in fully aerobic conditions . In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates . We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the fliC gene, encoding the major flagellar subunit, increases under oxygen-limited conditions . Inactivation of the global regulatory hns gene counteracts increased production of LPS and flagella in response to anoxia and allows E . coli W3100 to attach to sand columns even when it is grown under oxygen-limited conditions . We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of E . coli W3100 to adhere to hydrophilic surfaces . Indeed, overexpression of the fliC gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions . Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in E . coli.

Appl Environ Microbiol, 2002 Mar, 68(3), 1440 - 5
Microbial iron respiration can protect steel from corrosion; Dubiel M et al.; Microbiologically influenced corrosion (MC) of steel has been attributed to the activity of biofilms that include anaerobic microorganisms such as iron-respiring bacteria, yet the mechanisms by which these organisms influence corrosion have been unclear . To study this process, we generated mutants of the iron-respiring bacterium Shewanella oneidensis strain MR-1 that were defective in biofilm formation and/or iron reduction . Electrochemical impedance spectroscopy was used to determine changes in the corrosion rate and corrosion potential as a function of time for these mutants in comparison to the wild type . Counter to prevailing theories of MC, our results indicate that biofilms comprising iron-respiring bacteria may reduce rather than accelerate the corrosion rate of steel . Corrosion inhibition appears to be due to reduction of ferric ions to ferrous ions and increased consumption of oxygen, both of which are direct consequences of microbial respiration.

Appl Environ Microbiol, 2002 Mar, 68(3), 1392 - 402
Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions; Ito T et al.; A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 microM) and in the presence of nitrate . Microelectrode measurements showed that oxygen penetrated 200 microm from the surface during all stages of biofilm development . The first sulfide production of 0.32 micromol of H(2)S m(-2) s(-1) was detected below ca . 500 microm in the 3rd week and then gradually increased to 0.70 micromol H(2)S m(-2) s(-1) in the 8th week . The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time . This result coincided with an increase in SRB populations in the surface layer of the biofilm . The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 x 10(9) cells cm(-3) and 3.6 x 10(9) cells cm(-3), respectively, in the surface 400 microm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation . Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found . Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.

Appl Environ Microbiol, 2002 Mar, 68(3), 1196 - 203
Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans; Wen ZT et al.; Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium . The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation . In S . mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues . A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay . A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S . mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S . mutans to form biofilms . We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S . mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface . From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S . mutans.

Biotechnol Bioeng, 2002 Apr 20, 78(2), 179 - 89
Characterizing the adhesion of motile and nonmotile Escherichia coli to a glass surface using a parallel-plate flow chamber; McClaine JW et al.; A parallel-plate flow chamber was used to measure the attachment and detachment rates of Escherichia coli to a glass surface at various fluid velocities . The effect of flagella on adhesion was investigated by performing experiments with several E . coli strains: AW405 (motile); HCB136 (nonmotile mutant with paralyzed flagella); and HCB137 (nonmotile mutant without flagella) . We compared the total attachment rates and the fraction of bacteria retained on the surface to determine how the presence and movement of the flagella influence transport to the surface and adhesion strength in this dynamic system . At the lower fluid velocities, there was no significant difference in the total attachment rates for the three bacterial strains; nonmotile strains settled at a rate that was of the same order of magnitude as the diffusion rate of the motile strain . At the highest fluid velocity, the effect of settling was minimized to better illustrate the importance of motility, and the attachment rates of both nonmotile strains were approximately five times slower than that of the motile bacteria . Thus, different processes controlled the attachment rate depending on the parameter regime in which the experiment was performed . The fractions of motile bacteria retained on the glass surface increased with increasing velocity, whereas the opposite trend was found for the nonmotile strains . This suggests that the rotation of the flagella enables cells to detach from the surface (at the lower fluid velocities) and strengthens adhesion (at higher fluid velocities), whereas nonmotile cells detach as a result of shear . There was no significant difference in the initial attachment rates of the two nonmotile species, which suggests that merely the presence of flagella was not important in this stage of biofilm development .

Biotechnol Bioeng, 2002 Apr 20, 78(2), 164 - 71
Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm; Pereira MO et al.; A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented . The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope . The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume . Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions .

EDTNA ERCA J, 2001 Jul-Sep, 27(3), 129 - 31
Self-monitoring of microbiological levels in dialysis fluids; Trager H; Self monitoring of dialysis fluids is important . The dialysis technician has experience of the microbiological situation of the fluid systems and can make more tests for personnel safety . The tests are not expensive and are very easy to learn . It seems that the cells, which generate endotoxins, cannot be determined by the plate count system . No tanks or permanent water flow UV sterilisation in the pipeline to the dialysis machines can prevent the build up of biofilms and higher endotoxin amounts in water.

Curr Biol, 2002 Feb 19, 12(4), R132 - 4
Microbiology: breaking down biofilms; Stephens C; Biofilms--adherent bacterial communities embedded in a polymer matrix--are common in nature and can cause persistent human infections that are highly resistant to antibiotics . Recent work provides insight into how these communities develop and function, and offers clues to combating them.

Clin Microbiol Infect, 1997 Aug, 3(4), 432 - 439
Influence of macrolides on mucoid alginate biosynthetic enzyme from Pseudomonas aeruginosa; Nagino K et al.; OBJECTIVE: The long-term administration of erythromycin (EM), clarithromycin (CAM) or azithromycin (AZM) has generally resulted in a favorable outcome for patients with diffuse panbronchiolitis (DPB) infected with mucoid Pseudomonas aeruginosa . To elucidate the mechanism involved, the influence of macrolides on mucoid alginate production by P . aeruginosa was investigated in vitro . METHODS: The macrolides used in this study were EM with a 14-membered ring, AZM with a 15-membered ring, midecamycin (MDM) with a 16-membered ring, and CP-4305, which has had mycarose removed from MDM, The effects of macrolides on mucoid P . aeruginosa were investigated by quantitative assay of alginate production and inhibition of guanosine diphospho-D-mannose dehydrogenase activity . RESULTS: After incubation with EM, AZM and CP-4305, the structural material of P . aeruginosa biofilm was distorted, and the enzymatic activity of GDP-D-mannose dehydrogenase, the most important enzyme in mucoid alginate biosynthesis, was inhibited . However, these effects were not observed with the 16-membered macrolide MDM . CONCLUSIONS: The basic mechanism of clinical efficacy seen characteristically in 14- or 15-membered macrolides for patients with airway biofilm disease depends on the ability of such macrolides to inhibit alginate production by P . aeruginosa . Furthermore, this suggests that the inhibitory effect observed with 14-, 15- and 16-membered macrolides may depend on the sugar chain connected with the macrolide ring.

Microb Pathog, 2002 Mar, 32(3), 143 - 7
Detection of Pseudomonas aeruginosa cell-to-cell signals in lung tissue of cystic fibrosis patients; Favre-Bonte S et al.; Chronic Pseudomonas aeruginosa infections lead to progressive lung tissue destruction in cystic fibrosis (CF) patients . Two bacterial cell-to-cell signals, 3-oxo-C(12)-HSL and C(4)-HSL are required for the production of several extracellular virulence factors . 3-oxo-C(12)-HSL is also required for the development of a differentiated biofilm, induces IL-8 production by epithelial cells and possesses immunomodulatory activities . These two signalling molecules are therefore believed to play a role in the pathogenesis of P . aeruginosa infections, but have never been isolated from infected human tissues . We extracted and quantified the two P . aeruginosa cell-to-cell signals from lung tissues of two CF patients infected by P . aeruginosa . 3-oxo-C(12)-HSL and C(4)-HSL were detected in the lung tissues in fmol/gram, respectively pmol/gram concentrations; the ratio C(4)-HSL/3-oxo-C(12)-HSL exceeded 100 in all tissue samples . Random Amplified Polymorphism DNA genotyping revealed that one genotype was present per lung . In vitro the P . aeruginosa isolates from the two lungs produced 3-oxo-C(12)-HSL, whereas some isolates did not produce detectable C(4)-HSL . Our results suggest that both P . aeruginosa cell-to-cell signals were produced in the lung tissue of these two cystic fibrosis patients .

Biochem Biophys Res Commun, 2002 Mar 1, 291(3), 528 - 34
Use of gDNA as internal standard for gene expression in staphylococci in vitro and in vivo; Vandecasteele SJ et al.; An internal RNA standard proved less suitable in bacterial gene expression experiments . We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR . The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated . Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples . In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria . The average interassay variability of the in vitro expression is 20.1% . The in vivo intersample variability was 73.3% . This higher variability can be attributed to the biological variation of gene expression in vivo . This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro . (c)2002 Elsevier Science (USA).

Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 3129 - 34 Epub 2002 Feb 19.
Quorum-sensing regulators control virulence gene expression in Vibrio cholerae; Zhu J et al.; The production of virulence factors including cholera toxin and the toxin-coregulated pilus in the human pathogen Vibrio cholerae is strongly influenced by environmental conditions . The well-characterized ToxR signal transduction cascade is responsible for sensing and integrating the environmental information and controlling the virulence regulon . We show here that, in addition to the known components of the ToxR signaling circuit, quorum-sensing regulators are involved in regulation of V . cholerae virulence . We focused on the regulators LuxO and HapR because homologues of these two proteins control quorum sensing in the closely related luminous marine bacterium Vibrio harveyi . Using an infant mouse model, we found that a luxO mutant is severely defective in colonization of the small intestine . Gene arrays were used to profile transcription in the V . cholerae wild type and the luxO mutant . These studies revealed that the ToxR regulon is repressed in the luxO mutant, and that this effect is mediated by another negative regulator, HapR . We show that LuxO represses hapR expression early in log-phase growth, and constitutive expression of hapR blocks ToxR-regulon expression . Additionally, LuxO and HapR regulate a variety of other cellular processes including motility, protease production, and biofilm formation . Together these data suggest a role for quorum sensing in modulating expression of blocks of virulence genes in a reciprocal fashion in vivo.

Infect Immun, 2002 Mar, 70(3), 1113 - 20
Role of glutathione metabolism of Treponema denticola in bacterial growth and virulence expression; Chu L et al.; Hydrogen sulfide (H(2)S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis . Treponema denticola, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H(2)S . The metabolic pathway leading to H(2)S formation in periodontal pockets has not been determined . We used a variety of thiol compounds as substrates for T . denticola to produce H(2)S . Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H(2)S production by this microorganism . In addition to H(2)S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione . Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H(2)S, ammonia, and pyruvate . However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro . Moreover, the enzymatic activity(ies) in T . denticola responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56 degrees C) and freezing (-20 degrees C), by sonication, and by exposure to N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) . These treatments had no effect on degradation of cysteine by the purified enzyme . In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete T . denticola; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.

Ther Umsch, 2002 Jan, 59(1), 11 - 9
{Microbial resistance to antibiotics from the clinical viewpoint . Paradoxical therapy failures and the significance of pharmacokinetic-pharmacodynamic interactions in vivo}; Rossi M et al.; Antimicrobial efficacy in vivo is not exclusively defined by the activity of an antibiotic as determined in the in vitro susceptibility test where constant inocula of log-phase bacteria are exposed to a steady concentration of drug for short periods of time in a well defined biochemical environment tailored to optimize antimicrobial activity . In vivo, many additional factors come into play which may be decisive for the clinical result of antimicrobial therapy . Clinically, high numbers of bacteria in various states of growth and metabolic activity are to be eliminated in an environment which may be unfavorable for antimicrobial activity . Low pH, low pO2, the presence of proteins, cell debris and biofilms, are all known to interfere with antimicrobial activity in vivo . Moreover, drug concentrations in vivo are fluctuating and vary greatly depending on a variety of pharmacokinetic factors . Finally, clinical treatment usually lasts for days . The impact of the pharmacokinetic profile of an antibiotic on its in vivo activity may be decisive . It is dependent on both, the type of drug in question and its target organisms . The time of supra-MIC concentrations appears to be the main parameter for the activity of macrolides, clindamycin, linezolide and betalactam antibiotics, particularly against gram-negative organisms . In contrast, peak concentrations and the AUC/MIC relationship are key parameters for the activity of aminoglycosides and fluoroquinolones . Regarding upcoming resistant bacterial strains, an as yet ill-defined mutation-prevention concentration has recently been discussed . Thus, in addition to the aim of eliminating target bacteria at the site of infection, in vivo concentrations of antibiotics may be important for both, the suppression of antimicrobial resistance and its epidemic spread . We are looking forward to prospective clinical trials to better understand the clinical relevance of the discussed phenomena.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 900 - 3
Penetration of rifampin through Staphylococcus epidermidis biofilms; Zheng Z et al.; Rifampin penetrated biofilms formed by Staphylococcus epidermidis but failed to effectively kill the bacteria . Penetration was demonstrated by a simple diffusion cell bioassay and by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm.

Lett Appl Microbiol, 2002, 34(1), 22 - 6
A versatile reactor for continuous monitoring of biofilm properties in laboratory and industrial conditions; Pereira MO et al.; AIMS: The understanding of the dynamics of surface microbial colonization with concomitant monitoring of biofilm formation requires the development of biofilm reactors that enable direct and real-time evaluation under different hydrodynamic conditions . METHODS AND RESULTS: This work proposes and discusses a simple flow cell reactor that provides a means to monitoring biofilm growth by periodical removing biofilm-attached slides for off-line, both non-destructive and destructive biofilm analyses . This is managed without the stoppage of the flow, thus reducing the contamination and the disturbance of the biofilm development . With this flow cell, biofilm growth and respiratory activity can be easily followed, either in well-defined laboratory conditions or in an industrial environment . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The reproducible and typical biofilm development curves obtained, validated this flow cell and confirmed its potential for different biofilm-related studies, which can include biocidal treatment.

Kidney Int, 2002 Mar, 61(3), 1136 - 42
Management of hemodialysis catheter-related bacteremia with an adjunctive antibiotic lock solution; Krishnasami Z et al.; BACKGROUND: Tunneled dialysis catheters are complicated by frequent systemic infections . Standard therapy of catheter-associated bacteremia involves both systemic antibiotics and catheter replacement . Recent data suggest that biofilms in the catheter lumen are responsible for the bacteremia, and that instillation of an antibiotic lock (highly concentrated antibiotic solution) into the catheter lumen after dialysis sessions can eradicate the biofilm . METHODS: We analyzed prospectively the efficacy of an antibiotic lock protocol, in conjunction with systemic antibiotics, for treatment of patients with dialysis catheter-associated bacteremia without catheter removal . Protocol success was defined as resolution of fever and negative surveillance cultures one week following completion of the protocol . Protocol failure was defined as persistence of fever or surveillance cultures positive for any pathogen . In addition, infection-free catheter survival was compared to that observed in institutional historical control patients treated with catheter replacement . RESULTS: Blood cultures were positive in 98 of 129 of episodes (76%) in which patients dialyzing with a catheter had fever or chills . Protocol success occurred in 40 of 79 infected patients (51%) treated with the antibiotic lock . Protocol failure occurred in 39 cases (49%): 7 had persistent fever, 15 had positive surveillance cultures (9 for Candida and 6 for bacteria), and 17 required catheter removal due to malfunction . Each of the pathogens in the surveillance cultures was different from the original pathogen in that patient . Eight of the 9 secondary Candida infections and all 6 secondary bacterial infections resolved after catheter exchange and specific antimicrobial treatment . Overall catheter survival with the antibiotic lock protocol was similar to that observed among patients managed with catheter replacement (median survival, 64 vs . 54 days, P = 0.24) . CONCLUSIONS: Use of an antibiotic lock, in conjunction with systemic antibiotic therapy, can eradicate catheter-associated bacteremia while salvaging the catheter in about one half of cases . Moreover, this management approach offers clinical advantages over routine catheter exchange.

J Appl Microbiol, 2002, 92(2), 352 - 61
Differential efficacy of a chlorine dioxide-containing sanitizer against single species and binary biofilms of a dairy-associated Bacillus cereus and a Pseudomonas fluorescens isolate; Lindsay D et al.; AIMS: Daily exposure to 100 p.p.m . chlorine dioxide of single species and binary biofilms of dairy-associated Bacillus cereus DL5 and Pseudomonas fluorescens M2, attached to stainless steel surfaces in a laboratory flow system, was studied . METHODS AND RESULTS: Surfaces were sampled daily before and after sanitizer treatment and cells and spores dislodged and enumerated by standard methods . Duplicate surfaces were prepared for confocal scanning laser microscopy (CSLM) and scanning electron microscopy . Higher counts of Ps . fluorescens M2 were obtained in single species biofilms, microcolonies stained green (viable) in CSLM images and were closely packed on attachment surfaces . By contrast, higher counts of B . cereus DL5 were obtained in binary biofilms, microcolonies stained green in CSLM images, but were more spread out . Lower spore counts were obtained for B . cereus DL5 in binary biofilms . The survival of Ps . fluorescens M2 cells after exposure to chlorine dioxide was apparently enhanced by the presence of B . cereus DL5 in binary biofilms . By contrast, B . cereus DL5 showed increased susceptibility to sanitizer treatment in the presence of Ps . fluorescens M2 . CONCLUSIONS: Co-cultured bacteria in biofilms influence each other with respect to attachment capabilities and sanitizer resistance/susceptibility . SIGNIFICANCE AND IMPACT OF THE STUDY: Binary biofilms endemic in food-processing industries can survive sanitation regimes and may represent reservoirs of product contamination leading to subsequent spoilage and/or food safety risks.

J Appl Microbiol, 2002, 92(1), 81 - 9
Adaptation of a neutrophilic dairy-associated Bacillus cereus isolate to alkaline pH; Lindsay D et al.; AIMS: This study identified and studied the response of five Bacillus strains, isolated from alkaline cleaning in place (CIP) solutions, to alkaline conditions . METHODS AND RESULTS: Isolates were identified as B . cereus by 16S rDNA sequencing . External and internal cell pH and buffering capacity data of a representative strain, Bacillus DL5, were compared to B . cereus ATCC 10702 . Results indicated that a buffering system was induced when the pH of the growth medium increased to above pH 10, which was effective up to pH 12 and presumably cell wall associated . Volume measurements and confocal scanning laser microscope images of Bacillus DL5 cells showed that cells exhibited more pronounced stress symptoms when exposed to pH 10 than at pHs above 10 . Long-term exposure of Bacillus DL5 to pH 10 or 10.5 indicated that cells grew in planktonic form and formed biofilms at both pHs . CONCLUSIONS: Bacillus DL5 was a neutrophile with a growth pH range similar to B . cereus ATCC 10702, but tolerated alkaline pH . This may be a general trait of the B . cereus species rather than a specific phenomenon of isolates from alkaline ecosystems . SIGNIFICANCE AND IMPACT OF THE STUDY: Other neutrophilic B . cereus isolates may exhibit similar responses to alkaline conditions as the isolates studied here . These results may have important implications for dairy manufacturers.

Environ Microbiol, 2001 Dec, 3(12), 774 - 84
Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7; Kachlany SC et al.; The aromatic hydrocarbon-degrading bacterium, Pseudomonas putida G7, produces exopolymers of potential interest in biotechnological applications . These exopolymers have been shown to have significant metal-binding ability . To initiate the study of the metal-polymer interactions, we explored the physical and chemical nature of the P . putida G7 exopolysaccharide, a major component of the exopolymer . A capsular structure was observed by light microscopy surrounding both planktonic and attached cells in biofilms after immunofluorescence staining with polyclonal antiserum raised against planktonic cells . Further work with planktonic cells showed that the immunostained capsule remained associated with young (log phase) cells, whereas older (stationary phase) cells lost their capsular material to the external milieu . Visualization of frozen, hydrated stationary phase cells by cryo-field emission scanning electron microscopy (cryoFESEM) revealed highly preserved extracellular material . In contrast, conventional scanning electron microscopy (SEM) of stationary phase cells showed rope-like material that most probably results from dehydrated and collapsed exopolymer . Both capsular and released exopolymers were separated from cells, and the released extracellular polysaccharide (EPS) was purified . Deoxycholate-polyacrylamide gel electrophoresis (PAGE) and silver/alcian blue staining of the partially purified material showed that it contained both EPS and lipopolysaccharide (LPS) . Further purification of the EPS using a differential solubilization technique to remove LPS yielded highly purified EPS . Gas chromatography-mass spectrometry revealed that the purified EPS contained the monosaccharides, glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid . The structural and chemical properties of the P . putida EPS described here increase our understanding of the mechanisms of toxic metal binding by this well-known Proteobacterium.

Environ Microbiol, 2001 Nov, 3(11), 731 - 6
Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone; Ren D et al.; The quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was found to inhibit the swarming motility of Escherichia coli completely at 13 microg cm-2 (also at 20 microg ml-1) but did not inhibit its growth rate at 13-52 microg cm-2 or from 20 to 100 microg ml-1 . Swimming was not inhibited by the furanone at 20-40 microg ml-1 . In addition, confocal scanning laser microscopy revealed that this furanone at 60 microg ml-1 inhibited the biofilm formation of E . coli, as it decreased its thickness by 55%, reduced the number of water channels and decreased the percentage of live cells by 87% . This suggests that natural furanone may be used as a new method to control bacterial biofilms that does not involve toxicity . Furanone at 10 microg ml-1 also inhibited by 3300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1) and inhibited by 5500-fold that of V . harveyi via of autoinducer 2 (AI-2) as well as inhibited by 26-600-fold the quorum sensing of E . coli via AI-2; hence, this furanone is a non-specific intercellular signal antagonist.

Sci Total Environ, 2002 Jan 23, 282-283, 353 - 73
Phosphorus uptake into algal biofilms in a lowland chalk river; Jarvi HP et al.; This paper examines the growth and uptake of phosphorus into algal biofilms in the River Kennet, a lowland chalk (Cretaceous-age) stream in southern England . Algal biofilms were grown on artificial plastic substrates (templates) placed (i) on the riverbed and (ii) within the mid-water column . Experiments were set up to examine differences in growth rates of newly colonising biofilms compared with biofilms left to accumulate for periods of up to 6 months . Rates of algal biofilm production were measured by the chlorophyll a concentration that had accumulated per cm2 over the number of days that the biofilm template had been immersed in the river water . An algal biofilm bloom occurred in early spring, prior to peak suspended chlorophyll a concentrations within the water column . Biofilm samples collected in February and March had the highest chlorophyll a and total phosphorus concentrations . The biofilm bloom corresponded with increased solar radiation and declining river flow conditions . Periodic increases in soluble reactive phosphorus concentrations in the overlying river water did not correspond with any significant increase in biofilm production . These results suggest that light, rather than phosphorus is a key factor for biofilm growth in the River Kennet . Higher rates of chlorophyll a development in mid-water column biofilms may be linked to greater light exposure; however, maximum total-P concentrations were similar for both bed and water column biofilms . Newly colonising biofilms exhibited higher chlorophyll a and total-P concentrations than biofilms left to accumulate over longer terms, suggesting that fresh substrate availability promotes high rates of biofilm growth . Both 'condensed and organic' P (stored in biomass) and 'inorganic' (mineral) P fractions within the biofilms were present in varying proportions, although the early spring biofilm bloom resulted in maximum proportions and absolute concentrations of 'condensed and organic' P . Calcite was the only crystalline mineral detected within the biofilms . Ratios of Ca:inorganic P are largely consistent with the presence of CaCO3-P co-precipitates, although one very low value suggested that there may also be additional sources of inorganic P, possibly P adsorbed to clays or organics within the biofilm . However, poor linkages between CaCO3 and inorganic P concentrations suggest that, although the inorganic P fraction within the biofilm may be derived largely from CaCO3-P co-precipitation, the subsequent processes controlling overall CaCO3 and inorganic P concentrations in the biofilm are complex.

Adv Dent Res, 2000 Dec, 14, 29 - 39
Saliva and dental plaque; Rudney JD; Dental plaque is being redefined as oral biofilm . Diverse overlapping microbial consortia are present on all oral tissues . Biofilms are structured, displaying features like channels and projections . Constituent species switch back and forth between sessile and planktonic phases . Saliva is the medium for planktonic suspension . Several major functions can be defined for saliva in relation to oral biofilm . It serves as a medium for transporting planktonic bacteria within and between mouths . Bacteria in transit may be vulnerable to negative selection . Salivary agglutinins may prevent reattachment to surfaces . Killing by antimicrobial proteins may lead to attachment of dead cells . Salivary proteins form conditioning films on all oral surfaces . This contributes to positive selection for microbial adherence . Saliva carries chemical messengers which allow live adherent cells to sense a critical density of conspecifics . Growth begins, and thick biofilms may become resistant to antimicrobial substances . Salivary macromolecules may be catabolized, but salivary flow also may clear dietary substrates . Salivary proteins act in ways that benefit both host and microbe . All have multiple functions, and many do the same job . They form heterotypic complexes, which may exist in large micelle-like structures . These issues make it useful to compare subjects whose saliva functions differently . We have developed a simultaneous assay for aggregation, killing, live adherence, and dead adherence of oral species . Screening of 149 subjects has defined high killing/low adherence, low killing/high adherence, high killing/high adherence, and low killing/low adherence groups . These will be evaluated for differences in their flora.

Pharmacotherapy, 2002 Feb, 22(2), 227 - 39
Therapy with macrolides in patients with cystic fibrosis; Gaylor AS et al.; Cystic fibrosis affects 1/2500 individuals and is the most common lethal autosomal recessive disease in people of northern European descent . It is characterized by chronic infections with mucoid Pseudomonas aeruginosa and progressive deterioration of respiratory function . Much research has focused on the inflammatory component of the disease . Macrolide antibiotics are postulated to suppress inflammatory mediators and interfere with biofilm formation produced by P . aeruginosa . In vitro studies show promising results, and a limited number of human studies reported improvements in respiratory function with the drugs . Macrolide antibiotics are generally safe and well tolerated and may prove to be effective in patients with cystic fibrosis.

Comp Immunol Microbiol Infect Dis, 2002 Jan, 25(1), 43 - 8
The effects of prior growth as a biofilm on the virulence of Salmonella typhimurium for mice; Turnock LL et al.; Relatively little is known about how growth as a biofilm affects the virulence of pathogenic bacteria . In this study, the virulence of Salmonella typhimurium grown as a biofilm, or as planktonic cells, was compared in mice . Increased numbers of colony forming units were recovered from the spleens of mice 5 days after i.p . injection with S . typhimurium grown as a biofilm, as compared with planktonic cells (P < 0.05) . No significant difference in the CFU of S . typhimurium recovered from the liver was noted at the same time point, and no difference was noted in the CFU recovered from the spleen or liver at 5 days after i.v . or i.g . inoculation with 10(5) S . typhimurium . Nor were any differences noted at 7 days after i.p., i.v . or i.g . inoculation . Thus, any effect of growth as a biofilm has on the virulence of S . typhimurium seems to be limited to the first 5 days after i.p . inoculation.

Evolution Int J Org Evolution, 2001 Dec, 55(12), 2439 - 54
Foundations of gregariousness: a dispersal polymorphism among the planktonic larvae of a marine invertebrate; Toonen RJ et al.; Theory predicts that selection should favor genotypes that can vary their tendency to disperse in habitats that are spatially or temporally variable or those that remain near their carrying capacity . Although many marine habitats appear to fit these criteria, confirmed examples of dispersal polymorphism among marine invertebrates are exceedingly rare . Competent larvae of the gregarious tubeworm, Hydroides dianthus, settle specifically in response to living conspecific worms, but a small proportion of each spawn settle nonspecifically on uninhabited substrata concurrently with their gregarious siblings . Here, using a parental half-sib analysis, we show that the proportion of a spawn settling in response to uninhabited biofilm is highly heritable . When estimated as a continuous trait based on a one-way ANOVA, heritability is estimated to be 0.83 +/- 0.31 . When founder production was analyzed as a threshold trait, heritability was estimated to be 0.68 +/- 0.10 based on the breeding design experiment and 0.65 +/- 0.09 based on the artificial selection experiments . Realized heritability based on the selection experiments was considerably lower, however (0.17 per generation and 0.02 cumulative) . Artificial selection was ineffectual at sequentially increasing the proportion of founder larvae among inbred family lines, but after three generations of selection, the proportion of larvae settling in response to biofilm was significantly higher among inbred lines than among the field-collected parents . The obligate planktonic larval stage common among so many marine invertebrates is thought to preclude the evolution of dispersal polymorphisms in these animals . Theoretical expectations of variable dispersal may instead be realized through individual behavioral differences resulting in differential transport or settlement preference, but this possibility remains largely unexplored among marine invertebrates.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2287 - 92 Epub 2002 Feb 05.
Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway; Otto K et al.; Bacterial adhesion is an important initial step in biofilm formation, which may cause problems in medical, environmental, and industrial settings . In spite of obvious phenotypic differences between attached and planktonic cells, knowledge about the genetic basis for these differences and how adhesion-induced changes are mediated is limited . The Cpx two-component signal transduction pathway responds specifically to stress caused by disturbances in the cell envelope and activates genes encoding periplasmic protein folding and degrading factors . Here, we address the role of the Cpx-signaling pathway in sensing and responding to the physical change occurring during adhesion of Escherichia coli to surfaces . We present evidence that the expression of Cpx-regulated genes is induced during initial adhesion of E . coli to abiotic surfaces . This induction is specifically observed upon attachment of stationary-phase cells to hydrophobic surfaces . Moreover, surface-induced activity of the Cpx response requires NlpE, an outer membrane lipoprotein, which has previously been shown to induce the Cpx system when overproduced . The importance of a functional Cpx response during adhesion is further supported by the fact that a dramatically lower number of cells attach to the surface and dynamic cell-surface interactions as measured by a quartz crystal microbalance technique are altered when the CpxRA pathway is disrupted . The defects in adhesion exhibited by the cpxR and nlpE mutants were strikingly similar to those of wild-type cells in which protein synthesis was inhibited, suggesting that the Cpx pathway plays a key role in the regulation of adhesion-induced gene expression.

Water Res, 2002 Feb, 36(3), 551 - 60
Adsorption of p-chlorophenol by biofilm components; Wang W et al.; Through batch equilibrium experiments under the conditions of temperature 25 degrees C, pH values of 2.7, 5.3 and 6.1, the p-chlorophenol (4-cp) adsorption to biofilm components was investigated in this study . The contributions of biofilm components to 4-cp adsorption were discussed by comparing four adsorption systems, i.e . 4-cp adsorbed by model suspended particulate matter (kaolin) with biofilm coating, bacteria, bacterial exopolysaccharide (EPS) and kaolin, respectively . Langmuir and Freundlich isotherm equations were used to evaluate the experiment data . All the four adsorptions fitted for the two equations . Equilibrium isotherms were obtained for 4-cp adsorption on different adsorbents . The kinetic characteristics of 4-cp adsorption by biofilm components and the effect of pH on the kinetic process were investigated . The time to reach the highest adsorption amount and near equilibrium state in the four systems was different . The 4-cp adsorption by kaolin with biofilm coating reached near equilibrium at 60 min at pH 6.1 . The 4-cp adsorption by EPS and kaolin reached near equilibrium at 150 and 180 min, respectively . But the 4-cp adsorption by bacteria showed no evident near equilibrium during 3 h in the experiment . The impact of pH value on the adsorption was also examined . The adsorption amount slightly increased with increasing pH from 2.7 to 6.1 for the adsorption systems of bacterial EPS and kaolin, but it slightly decreased in the systems of kaolin with biofilm coating and bacteria.

Med Microbiol Immunol (Berl), 2001 Dec, 190(3), 105 - 12
Correlation of biofilm expression types of Staphylococcus epidermidis with polysaccharide intercellular adhesin synthesis: evidence for involvement of icaADBC genotype-independent factors; Rohde H et al.; IcaADBC-encoded proteins mediate synthesis of the intercellular polysaccharide adhesin (PIA), which is essentially involved in Staphylococcus epidermidis biofilm formation . Seventy S . epidermidis isolates were investigated for their ability to form biofilm and synthesize PIA in different growth media including trypticase soy broth obtained from Becton Dickinson (TSBBBL), or Oxoid (TSB(OXOID)), and TSB(OXOID) supplemented with 0.5% N-acetylglucosamine, and for the presence of icaADBC . Dependent on the medium used (TSB(BBL) or TSB(OXOID)), the isolates exhibited a differential expression of PIA and biofilm formation, with 51 (72.85%) and 34 (48.57%) being biofilm positive, respectively . Using these growth media four different expression phenotypes were differentiated: similar quantities of biofilm formation in both TSBBBL and TSB(OXOID) (11 isolates, type A), significantly reduced biofilm expression in TSB(OXOID) compared to TSB(BBL) (23 isolates, type B), biofilm negative in TSB(OXOID) but biofilm producing in TSB(BBL) (17 isolates, type C) and biofilm negative in both media (19 isolates, type D) . For all strains a biofilm-positive phenotype in a specific medium was closely linked to expression of PIA in that medium . All but one strain of expression type A-C and 7/19 expression type D strains were icaADBC positive . On the basis of restriction fragment length polymorphisms, the isolates were classified into two main icaADBC genotypes . There was no association between the observed biofilm-expression types and a defined icaADBC genotype . In the biofilm-negative S . epidermidis 5179, isolated from a ventriculo-atrial shunt infection, the insertion of IS257 interrupted the transcription of icaADBC, resulting in a PIA- and biofilm-negative phenotype . In all other icaADBC-positive, biofilm-negative isolates no major alterations of the icaADBC gene locus were identified . Obviously, expression of icaADBC, PIA synthesis and biofilm formation are integrated into a complex regulatory network involving other determinants independent of icaADBC genotype . Inactivation of icaADBC by IS elements is apparently a rare cause of a biofilm-negative phenotype in clinical S . epidermidis isolates.

J Clin Microbiol, 2002 Feb, 40(2), 382 - 8
The ica operon and biofilm production in coagulase-negative Staphylococci associated with carriage and disease in a neonatal intensive care unit; de Silva GD et al.; Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in the neonatal intensive care unit (NICU) . We evaluated the hypothesis that the ica operon and biofilm production are associated with CoNS disease in this setting . CoNS associated with bacteremia or blood culture contamination and from the skin of infants with CoNS bacteremia or healthy controls were obtained during a prospective case-control study on a busy NICU . A total of 180 strains were identified, of which 122 (68%) were Staphylococcus epidermidis and the remainder were S . capitis (n = 29), S . haemolyticus (n = 11), S . hominis (n = 9), S . warneri (n = 8), and S . auricularis (n = 1) . The presence of the genes icaA, icaB, icaC, and icaD was determined by PCR, and biofilm production was examined using qualitative (Congo red agar {CRA}) and quantitative (microtiter plate) techniques . There were no significant differences in the presence of the ica operon or CRA positivity among the four groups of strains . However, quantitative biofilm production was significantly greater in strains isolated from either the blood or the skin of neonates with S . epidermidis bacteremia . We conclude that the quantity of biofilm produced may be associated with the ability to cause CoNS infection . This conclusion suggests that the regulation of biofilm expression may play a central role in the disease process.

Nature, 2002 Jan 31, 415(6871), 545 - 9
Structural identification of a bacterial quorum-sensing signal containing boron; Chen X et al.; Cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers . This process, termed quorum sensing, allows bacterial populations to coordinate gene expression . Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development . Unlike other autoinducers, which are specific to a particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species . AI-2 has been proposed to serve as a 'universal' signal for inter-species communication . The chemical identity of AI-2 has, however, proved elusive . Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with autoinducer . The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized autoinducers . Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2 . Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.

Appl Environ Microbiol, 2002 Feb, 68(2), 901 - 9
Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms; Neu TR et al.; A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes . In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated . The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains . In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested . Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps . It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range . In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm) . The intensities of the emission signals for the nucleic acid-specific fluorochromes varied . For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI . In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples . Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents . Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm) . In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm . Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm . If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording . Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy . Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation . However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.

Appl Environ Microbiol, 2002 Feb, 68(2), 728 - 37
Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases; Chavant P et al.; The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents . The effect of these parameters on adhesion and biofilm formation by L . monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene {PTFE}) surfaces was assessed . The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature . The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature . However, biofilm formation was faster on the hydrophilic substratum . After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C . At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE . The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation . The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.

Appl Environ Microbiol, 2002 Feb, 68(2), 470 - 5
Occurrence and phenotypic characterization of Yersinia ruckeri strains with biofilm-forming capacity in a rainbow trout farm; Coquet L et al.; The presence of Yersinia ruckeri in a French fish farm was investigated . Y . ruckeri was isolated mainly from algae and sediment samples rather than from water . Twenty-two Y . ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification . These strains were able to adhere to solid supports . This characteristic was correlated with flagellum-mediated motility . Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts . Our results demonstrate that surface colonization of fish farm tanks by Y . ruckeri biofilms is a potential source of recurrent infection for extended periods of time.

Biotechnol Prog, 2002 Jan-Feb, 18(1), 55 - 61
Internal and external mass transfer in biofilms grown at various flow velocities; Beyenal H et al.; It appears that biofilms arrange their internal structure according to the flow velocity at which they are grown, which affects the internal mass transfer rate and microbial activity . In biofilms grown at various flow velocities we determined the vertical profiles of the local relative effective diffusivity (termed D(l)) at several locations within each biofilm . From these profiles we calculated the surface-averaged relative effective diffusivity (termed D(sa)) at various distances from the bottom and plotted it against these distances . The D(sa) decreased linearly toward the bottom, forming well-defined profiles that were different for each biofilm . The gradients of these profiles were multiplied by the diffusivity of oxygen, zeta = D(w) dD(sa)/dz, and plotted versus the flow velocity at which each biofilm was grown . The gradients were low at flow velocities below 10 cm/s, reached a maximum at a flow velocity of 10 cm/s, and decreased again at flow velocities exceeding 10 cm/s . The existence of a maximum indicates a possibility that two opposing forces were affecting the slope of the profiles . To explain these observations we hypothesized that biofilms, depending on the flow velocity at which they are grown, arrange their internal architecture to control (1) the nutrient transport rate and (2) the mechanical pliability needed to resist the shear stress of the water flowing past them . It appears that biofilms attempt to satisfy the second goal first, to increase their mechanical strength, and that they do so at the expense of the nutrient transfer rate to deeper layers . This strength increase is associated with an increase in biofilm density, which slows down the internal mass transport rate . Biofilms grown at low flow velocities exhibit low density and high effective diffusivity but cannot resist higher shear stress, whereas biofilms grown at higher flow velocities are denser and can resist higher shear stress but have a lower effective diffusivity.

Quintessence Int, 2001 Nov-Dec, 32(10), 755 - 61
Clearance of biofilms from dental unit waterlines through the use of hydroperoxide ion-phase transfer catalysts; Shepherd PA et al.; OBJECTIVES: The purpose of this research was to demonstrate the effectiveness of hydroperoxide ion-phase transfer catalyst (HPI-PTC) cleaners and disinfectants for maintaining dental unit waterlines free of planktonic organisms . METHOD AND MATERIALS: Water samples were taken from 117 sites, which included a variety of dental units and samples from the sink faucets of most operatories . Samples were plated on appropriate bacteriologic media and incubated . The presence or absence of biofilms was confirmed by scanning electron microscopy . Twenty-two of the dental units were retrofitted with independent water systems; the cleaning procedure involved an overnight application of an HPI-PTC cleaner followed by a 2-minute water rinse . RESULTS: Water from both the air-water syringe and the high-speed handpiece lines from all untreated units contained at least 6 x 10(2) colony-forming units per milliliter of planktonic or free-floating bacteria; the average was 1.4 x 10(5) CFU/mL . An initial 5% solution of HPI-PTC successfully cleared the lines of any apparent biofilm when applied for 3 consecutive days . Thereafter, once weekly use of the cleaner maintained the dental unit water supplies free of significant numbers of planktonic organisms . CONCLUSION: Routine weekly use of an HPI-PTC cleaner controlled dental unit waterline biofilm and reduced, with minimum effort, the microbial contamination level of water used for patient treatment to less than 200 CFU/mL.

Acta Orthop Scand, 2001 Dec, 72(6), 557 - 71
Infection of orthopedic implants and the use of antibiotic-loaded bone cements . A review; van de Belt H et al.; Infections by bacteria are a serious complication following orthopedic implant surgery, that can usually only be cured by removing the implant, since the biofilm mode of growth of infecting bacteria on an implant surface protects the organisms from the host immune system and antibiotic therapy . Over the past few decades, attempts have been made to prevent and cure orthopedic implant infections by incorporating antibiotics in polymethylmethacrylate bone cements, in primary and revision surgery . However, the clinical efficacy of antibiotic-releasing bone cements is not accepted by all and the long-term exposure to low doses from antibiotic-releasing bone cements in patients is strongly related to the emerging threat of antibiotic resistance in medicine today . In this article, we start by reviewing the mechanisms governing the formation of an infectious biofilm on orthopedic implant materials, the release mechanisms and properties of clinically-used, antibiotic-loaded bone cements . The clinical efficacy of antibiotic-loaded bone cements is evaluated analyzing separatedly the prophylactic and therapeutic uses of these products.

Environ Pollut, 2002, 116(1), 147 - 57
Metal-induced tolerance in the freshwater microbenthic diatom Gomphonema parvulum; Ivorra N et al.; The benthic diatom Gomphonema parvulum Kutzing is a common species in both clean and metal contaminated rivers . Our aim was to investigate whether metal-induced tolerance could explain the persistance of this taxon under metal polluted conditions . G . parvulum strains were isolated from a Zn- and Cd-contaminated stream and from a relatively clean ("reference") stream . The strains were cultured in synthetic medium as mono-specific biofilms to maintain their specific benthic growth features . Moreover, the strain from the metal polluted stream was cultured in plain and Zn- and Cd-enriched synthetic medium . Short-term (5 h) toxicity experiments with Zn were performed with the strains using pulse amplitude modulated (PAM) fluorometry . Zn lowered significantly the minimal chlorophyll fluorescence (F0) and the photon yield (phi(p)) of the exposed strains after 5 h exposure . The actual Zn concentrations that caused a 50% reduction (EC50's) of the phi(p) of the strain from the metal polluted stream were significantly higher than those of the isolate from the unpolluted stream . The absence of tolerance to Cu of the "polluted" strain indicated that Zn tolerance resulted from specific induction by chronic exposure to Zn in the field . Observations on field biofilms confirmed a higher tolerance of the G . parvulum population from the polluted stream than of the G . parvulum population from the reference stream . A genetic nature of this metal adaptation was supported by the persistance of the Zn tolerance of the polluted strain 2 years after isolation.

Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 337 - 43
Adhesive interactions between voice prosthetic yeast and bacteria on silicone rubber in the absence and presence of saliva; Millsap KW et al.; 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.

Yeast, 2002 Feb, 19(3), 269 - 76
HSP12 is essential for biofilm formation by a Sardinian wine strain of S . cerevisiae; Zara S et al.; Sardinian sherry strains of S . cerevisiae form a biofilm on the surface of wine at the end of the ethanolic fermentation, when grape sugar is depleted and when further growth becomes dependent on access to oxygen . A point mutation in HSP12 or deletion of the entire gene results in inability to form this film . HSP12 encodes a heat-shock protein previously foundby others to be active during stationary phase, in cells depleted for glucose, and in cells metabolizing ethanol and fatty acids, all conditions associated with sherry biofilms .

J Antimicrob Chemother, 2002 Feb, 49(2), 315 - 20
Phosphate deprivation is associated with high resistance to latamoxef of gel-entrapped, sessile-like Escherichia coli cells; Vilain S et al.; Viable Escherichia coli cells were entrapped in agar gel layers and incubated in a phosphate-limited glucose medium . Immobilized bacteria displayed enhanced alkaline phosphatase activity and overexpressed the outer membrane protein PhoE as compared with free-floating organisms . These observations highlighted the existence of high phosphate deprivation within biofilm-like structures . In addition, the antimicrobial efficacy of latamoxef against immobilized bacteria was partly recovered in the presence of a high phosphate concentration . From these data, a possible role of phosphate deprivation in the high resistance of sessile-like organisms to antibiotics may be considered.

J Antimicrob Chemother, 2002 Feb, 49(2), 309 - 14
Effects of quorum-sensing deficiency on Pseudomonas aeruginosa biofilm formation and antibiotic resistance; Shih PC et al.; Variations in biofilm formation by, and antibiotic resistance of, Pseudomonas aeruginosa PAO1 (wild type) and the quorum-sensing-deficient mutants PDO100 (Delta rhlI), JP1 (Delta lasI) and JP2 (Delta lasI Delta rhlI) were studied . For PAO1, the maximum-accumulation phase of biofilm formation began immediately and a plateau phase was reached after 24 h, whereas the quorum-sensing mutants showed 36-48 h lags before entering the maximum-accumulation phase . After 72 h, the cell density of the PAO1 biofilms was c . 0.8-1.2 log greater than for the mutants . On a unit protein basis, total polysaccharide production was similar for PAO1 and PDO100, whereas JP1 and JP2 biofilms accumulated only c . 36% of the PAO1 level after 72 h . Fluorescent micrographs revealed that the PAO1 biofilms were much thicker than those of the quorum-sensing-deficient mutants . In the case of the PAO1 and PDO100 biofilms, most cells were attached to the top of the biofilm layer, whereas the bottom layer consisted predominantly of polysaccharides . The JP1 and JP2 biofilms were closely packed with cells, and little polysaccharide was visible . Cells in PAO1 biofilms were little affected by kanamycin, even at 100 mg/L, whereas those in PDO100 biofilms were susceptible to the highest concentration of kanamycin (100 mg/L) but not to lower concentrations (10 and 50 mg/L) . In contrast, cells in JP1 and JP2 biofilms were susceptible to kanamycin at all three concentrations.

J Antimicrob Chemother, 2002 Feb, 49(2), 301 - 8
An investigation of the antimicrobial effects of linezolid on bacterial biofilms utilizing an in vitro pharmacokinetic model; Gander S et al.; Biofilms of methicillin-susceptible and -resistant Staphylococcus aureus, a strain of coagulase-negative staphylococcus and glycopeptide-intermediate strains of S . aureus (GISA) were exposed to the oxazolidinone linezolid, and four comparator antibiotics (quinupristin/ dalfopristin, vancomycin, teicoplanin and ciprofloxacin) using a Sorbarod model . The effects of these antibiotics were assessed by monitoring the reduction in the number of cells eluted from the biofilms . The biofilms were exposed to the antibiotics by two methods . The first was an exponentially decreasing drug concentration method, where the rate of dilution was matched to the half-lives of the antibiotics and the initial concentration matched peak serum levels . The second was a constant drug concentration method, in which biofilms were exposed to antibiotics for 2 h, with the concentration of the antibiotic equalling the total amount of drug used in the exponentially decreasing method . The results indicate that linezolid produces a greater reduction in the number of cells eluted with the exponentially decreasing method compared with the constant concentration exposure against all strains tested except for one of the GISA strains, Mu 50 . Overall, ciprofloxacin produced the greatest effects in the exponentially decreasing concentration experiments, but only against non-resistant strains . In the constant concentration exposure no one drug was responsible for the largest reductions in cell numbers observed . Linezolid and quinupristin/dalfopristin produced a reduction in the number of cells eluted from the biofilms of all of the strains tested in both methods of exposure and should be considered for further clinical studies of the treatment of staphylococcal biofilm-associated infections.

Front Biosci, 2002 Feb 01, 7, d442 - 57
Persistent infections and immunity in cystic fibrosis; Yu H et al.; Cystic fibrosis (CF) is the most common autosomal recessive lethal disease in the Caucasian population . Chronic respiratory infections with Pseudomonas aeruginosa, neutrophil-dominated airway inflammation and progressive lung damage are the major causes of morbidity and mortality in CF . Two persistent infection phenotypes expressed by this bacterium are biofilm and mucoidy . Biofilm, also called the microcolony mode of growth is the surface-associated adherent bacterial community, while mucoidy refers to a phenotype conducive to copious amounts of mucoid exopolysaccharide (MEP)/alginate that provides a matrix for mature biofilms conferring resistance to host defenses and antibiotics . Recent completion of the whole genomic sequence of the standard reference strain P . aeruginosa PAO1 has led to discoveries that many clinical isolates of this species possess unique genomic sequences (genomic islands) due to horizontal gene transfer . We propose this type of genetic exchange may play an important role in causing intrinsic genomic diversity of this organism . Therefore, the diversity, as revealed through profiles of restriction fragment length polymorphism (RFLP), may be linked to an array of novel and unexplored pathogenic mechanisms in P . aeruginosa . CF mouse models, while displaying many clinical similarities to human CF, have yet to demonstrate a chronic pulmonary disease phenotype . This review is intended to provide an overview of P . aeruginosa persistent infection phenotypes (biofilm and mucoidy) and an aerosol infection mouse model for CF . Genomic diversity of P . aeruginosa and its implications in the pathogenesis in CF will also be discussed.

Nephrologie, 2001, 22(8), 439 - 41
Bacterial biofilms: a review of current research; De Lancey Pulcini E; Biofilms provide bacterial cells with a protective environment that allows for survival from antibiotics and host defense mechanisms . In order to understand how to control biofilms, it is important to understand the complexity of the biofilm system . This is in overview of four areas of current biofilm research: biofilm resistance to antimicrobials and host defense mechanisms, the complexity of biofilm structure, the possible existence of a biofilm phenotype, and the ramifications of cell cell communication within the biofilm.

J Infect Chemother, 2000 Mar, 6(1), 45 - 50
Influence of macrolides on guanosine diphospho-D-mannose dehydrogenase activity in Pseudomonas biofilm; Mitsuya Y et al.; The formation of biofilm is regarded as a major cause of intractable infectious disease . Our studies were done to elucidate the action of a 14-membered-ring macrolide (erythromycin; EM) and a 16-membered-ring macrolide (midecamycin; MDM) on guanosine diphospho-d-mannose dehydrogenase (GDP-mannose dehydrogenase; GMD), one of the constituents of bacterial biofilm that is known to produce alginate . The mucoid type of Pseudomonas aeruginosa PT-1578 and the non-mucoid type of P . aeruginosa PAO1 were grown with nutrient-rich and nutrient-poor media . Comparative measurements were made of their GMD enzyme activities, with glucose-6-phosphate dehydrogenase (G6PDH), a cell membrane enzyme, used as a control . It was found that the GMD enzyme activity of mucoid type of Pseudomonas bacteria increased when they were grown on nutrient-poor media . Measurements were also made to determine the effects of EM and MDM against GMD and G6PDH enzyme activities . In media with either EM or MDM added, the production of G6PDH was not inhibited, irrespective of the concentration of EM or MDM . However, EM was effective against the production of GMD, showing a concentration-dependent effect . Scanning electron microscopy studies were also carried out to determine the effects of both macrolides on bacterial alginate production . It was found that reduction of alginate content occurred after the addition of EM . When environmental conditions for bacteria deteriorate, GMD enzyme is activated, production of alginate is initiated, and then biofilm is formed . Our results suggest that EM may have an inhibitory effect on the GMD production cycle, hence inhibiting the formation of biofilm . This may explain the differences in the clinical usefulness of 14-membered-and 16-membered-ring macrolides against biofilm disease.

J Infect Chemother, 1999 Mar, 5(1), 10 - 15
Inhibitory action of clarithromycin on glycocalyx produced by MRSA; Sano M et al.; We determined whether clarithromycin (CAM) had the ability to eliminate glycocalyx and biofilm produced by methicillin-resistant Staphylococcus aureus (MRSA) using an in-vitro experimental system (consisting of a bladder model and a kidney model) simulating complicated urinary tract infection (UTI) . We also examined whether a combination of CAM and vancomycin (VCM) was effective for eliminating the MRSA biofilm . VCM (urinary concentration simulating drip infusion of 500 mg twice a day for 5 days; minimum inhibitory concentration (MIC) 0.5 &mgr;g/ml) eliminated MRSA from the bladder model medium at 48 h, but reproliferation occurred immediately after withdrawal of the agent . No disappearance of MRSA biofilm was noted, and this appeared to be the cause of the bacterial regrowth . CAM (urinary concentration simulating oral administration of 200 mg twice a day for 5 days; MIC, 128 &mgr;g/ml) allowed microbial recovery to the initial level within 48 h, but led to the disappearance of the glycocalyx-forming biofilm . A combination of VCM and CAM caused microbial elimination from the bladder model medium at 46 h with no regrowth after withdrawal of the antimicrobial agents . Scanning electron microscopy confirmed that the MRSA biofilm disappeared completely and no microbial adhesion was noted . These results suggest that CAM has an inhibitory action on glycocalyx and biofilm of MRSA, and that the combined use of VCM and CAM may be efficacious for the treatment of MRSA UTI

J Bacteriol, 2002 Feb, 184(4), 1140 - 54
Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm; Sauer K et al.; Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis . Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations . Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion . Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development . The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins) . When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome) . This difference was higher than when planktonic P . aeruginosa were compared with planktonic cultures of Pseudomonas putida . Las quorum sensing was shown to play no role in early biofilm development but was important in later stages . Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria . These results demonstrate that P . aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.

Water Sci Technol, 2001, 44(11-12), 85 - 92
Removing filterable reactive phosphorus from highly coloured stormwater using constructed wetlands; Lund MA et al.; A constructed wetland design, consisting of 16 repeating cells was proposed for Henley Brook (Perth, Western Australia) to optimise the removal of FRP from urban stormwater . Three replicate experimental ponds (15 x 5 m), were constructed to represent at a 1:1 scale a single cell from this design . Three 5 m zones of each pond were sampled: shallow (0.3 m) vegetated (Schoenoplectus validus) inflow and outflow zones and a deeper (1 m), V-shaped central zone . In 1998/99, inflows and outflow waters were intensively sampled and analysed for FRP and Total P . In addition, all major pools of P (plants, sediment) within the ponds, and important P removal processes (benthic flux, uptake by biofilm and S . validus) were quantified . A removal efficiency of 5% (1998) and 10% (1999) was obtained for FRP . Initial uptake was mainly in plant biomass, although the sediment became an increasingly important sink . Benthic flux experiments showed that anoxia did not cause release of P from sediments, indicating that most of the P was bound as apatite rather than associated with Fe or Mn . The highly coloured waters were believed responsible for the very low biofilm biomass recorded (<1 g x m(-2)) . We have demonstrated that constructed wetlands can be effective for removing FRP immediately after construction, although their long-term removal capacity needs further research.

Water Sci Technol, 2001, 44(11-12), 441 - 8
Treatment of a molasses based distillery effluent in a constructed wetland in central India; Billore SK et al.; A field-scale 4-celled, horizontal subsurface constructed wetland (CW) was installed to evaluate removal efficiencies of wastewater constituents in an industrial distillery effluent . Total and dissolved solids, NH4-N, TKN, P and COD were measured . This CW design provides four serial cells with synthetic liners and a river gravel base . The first two unplanted cells provide preliminary treatment . Specific gravel depths and ensuing biofilm growth provides anaerobic treatment in Cell 1 and anaerobic treatment in Cell 2 . Cell 3 was planted with Typha latifolia with an inserted layer of brick rubble (for phosphorus removal) . Locally grown reed, Phragmites karka was planted in Cell 4 . COD was reduced from 8420 mg/l 3000 from Cell 1 to the outlet of Cell 4 . Likewise other parameters: total and dissolved solids, ammonium and total nitrogen, and total P, indicated declining trends at the 4-celled CW effluent . This study reveals how high strength distillery wastewater strongly impacts morphology, aeration anatomy in the chiseled plant tissues, reed growth; and composition of the biofilm in the specialized substratum . The reliability of a CW for organic and nutrients reduction, in association with a poorly performing conventional system is discussed . There is an immense potential for appropriately designed constructed wetlands to improve high strength wastewaters in India.

Zhonghua Jie He He Hu Xi Za Zhi, 2001 Jun, 24(6), 342 - 4
{Effects of erythromycin and fosfomycin on Pseudomonas aeruginosa biofilm in vitro}; Xu Z et al.; OBJECTIVE: To observe the effects on Pseudomonas aeruginosa biofilm of non-antipseudomonal drugs such as erythromycin and fosfomycin . METHODS: Clinical isolates of Pseudomonas aeruginosa in respiratory infection and ATCC27853 were cultivated in TSB-Teflon and NS-Teflon system respectively for biolfilm formation . Biofilm was observed under scanning electronic microscope (SEM) . MICs were determined by broth micro-dilution . RESULTS: SEM showed that biofilm of clinical isolate in TSB-Teflon system and ATCC27853 in NS-Teflon system increased as being cultivated for longer in 1, 3 and 7 days . And biofilm formation in the process of cultivating was inhibited by SubMIC of erythromycin and fosfomycin . MICs evaluation suggested that there were synergetic effects against biofilm Pseudomonas aeruginosa when SubMIC of erythromycin and fosfomycin were combined with ceftazidime or levofloxacin in vitro . CONCLUSION: SubMIC of erythromycin and fosfomycin could inhibit Pseudomonas aeruginosa biofilm in vitro.

Zhonghua Jie He He Hu Xi Za Zhi, 2001 Jun, 24(6), 339 - 41
{Correlation of endotracheal tube biofilm and recurrent ventilator-associated pneumonia with Pseudomonas aeruginosa}; Cai S et al.; OBJECTIVE: To determine the correlation of endotracheal tube biofilm (ETT-BF) and recurrent ventilator-associated pneumonia with Pseudomonas aeruginosa (PA-VAP) in long-term ventilation patients . METHODS: Pulsed-field gel electrophoresis (PFGE) of Spe I enzyme-restricted chromosomal DNA was performed to identify the DNA patterns of Pseudomonas aeruginosa from ETT-BF, pharynx and lower respiratory tract secretion in 15 VAP patients . The chromosomal DNA fingerprint profile of each strain was compared with those from all other strains to calculate the similarity by using the correlation coefficient . The strains were then grouped and groups were depicted as a dendrite using the average clustering algorithm . RESULTS: When bacteria has been isolated from ETT-BF, the same DNA pattern species are isolated from the lower respiratory tract secretion in 7 of 15 patients . Recurrent PA-VAP is caused by closely related and indistinguishable isolates in 6 of 15 patients . All PEGE patterns from 9 patients are different from one another (correlation coefficient < 31.2%) . CONCLUSIONS: The close pathogenic relationship between ETT-BF and lower airway secretion suggests that adhesive colonization of ETTs may be an important factor in the pathogenesis of VAP.

Laryngoscope, 2001 Dec, 111(12), 2083 - 94
Direct evidence of bacterial biofilms in otitis media; Post JC; OBJECTIVES/HYPOTHESIS: Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME . The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations . The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected . The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea . The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations . STUDY DESIGN: Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients . METHODS: Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H . influenzae . Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms . Adult and fetal chinchilla uninfected controls were similarly examined for comparison . In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes . RESULTS: Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H . influenzae otitis media had been induced . Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection . The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals . Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes . CONCLUSIONS: Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H . influenzae otitis media and can form on tympanostomy tubes placed in children's ears . Such biofilms can be directly observed by microscopy . These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.

Plasmid, 2002 Jan, 47(1), 10 - 7
Influence of Tn917 insertion on transcription of the icaADBC operon in six biofilm-negative transposon mutants of Staphylococcus epidermidis; Dobinsky S et al.; Insertion of Tn917 into the icaADBC operon determines a biofilm-negative phenotype in biofilm-producing Staphylococcus epidermidis due to the inactivation of the genes responsible for the synthesis of the polysaccharide intercellular adhesin . We previously characterized six isogenic biofilm-negative transposon mutants of S . epidermidis 1457 with Tn917 insertions in either icaA or icaC . Northern blot analysis using ica- and Tn917-specific probes revealed that ica sequences located upstream and downstream of the transposon insertion site were still transcribed in five mutants in which Tn917 was inserted in the same transcriptional orientation . Outward-directed transcription initiating from within the transposon resulted in the complete expression of individual ica genes . Our results indicate that not only the inactivation of the entire operon but the isolated interruption of individual icaA and icaC genes led to a biofilm-negative phenotype in S . epidermidis . Tn917 mutagenesis may also result in the activation of genes located downstream of the insertion site .

Infect Immun, 2002 Feb, 70(2), 878 - 88
Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces; Kuhn DM et al.; Little is known about fungal biofilms, which may cause infection and antibiotic resistance . In this study, biofilm formation by different Candida species, particularly Candida albicans and C . parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices . Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays . Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St . Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes . Although minimal variations in biofilm production among invasive C . albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted . C . albicans isolates produced more biofilm than C . parapsilosis, C . glabrata, and C . tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy . Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates . On microscopy, C . albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae . In contrast, C . parapsilosis biofilms had less volume than C . albicans biofilms and were comprised exclusively of clumped blastospores . Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml) . Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active . In conclusion, our data show that C . albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C . parapsilosis.

Water Res, 2001 Dec, 35(17), 4063 - 71
The potential for biofilm growth in water distribution systems; Hallam NB et al.; Biofilms on pipe walls in water distribution systems are composed of bacteria in a polymeric matrix, which can lead to chlorine demand, coliform growth, pipe corrosion and water taste and odour problems . The majority of previous studies have been laboratory or pilot plant based and few results are available for field conditions . In this study, field observations of biofilm were made using biofilm potential monitors . The monitor results were compared with pipe samples taken from the distribution system and with laboratory pipe reactors . An empirical equation quantified the inhibitory effects of free chlorine and decrease of temperature on biofilm growth . With water having total organic carbon concentrations in the range 1.5-3.9mg/1 a free chlorine residual of 0.2 mg/l was needed to reduce biofilm concentration to below 50 pg ATP cm2 . Pipe material influenced biofilm activity far less than chlorine with mean biofilm activity being ranked in the order glass (136 pg ATP/cm2) < cement (212 pg ATP/cm2) < MDPE (302 pg ATP/ cm2) < PVC (509 pg ATP/cm2).

Water Res, 2001 Dec, 35(17), 4011 - 8
Immunological methods for the study of Zoogloea strains in natural environments; Lu F et al.; Since Zoogloea ramigera has been considered to be important in aerobic wastewater treatment, we have evaluated several methods for detecting and enumerating Z . ramigera in water and wastewater samples . Indirect immunoassay methods for the detection of Zoogloea strains were developed using polyclonal antibodies against the cells or the isolated exocellular polymer (EP) of the neotype Zoogloea ramigera strain 106 (ATCC 19544) . The primary antibodies reacted with the cells and the exopolymer associated with finger-like zoogloeal projections, but not with other bacteria from natural samples . These antibodies allowed detection of Z . ramigera in environmental samples . Scanning electron microscopy (SEM) was used to show that the cells and the exocellular polymer of naturally occurring zoogloeal projections are antigenically and structurally related to those of Z . ramigera 106 . Both immunological procedures and probes complementary to regions on the 16S rRNA could detect Z . ramigera in natural samples but the immunological procedures were easier to use . RT-PCR was also used to detect Z . ramigera in natural samples . These methods were also used to identify Z . ramigera in biofilms that developed over wastewater samples as part of an MPN procedure that was used to quantitate Z . ramigera at different stages of the wastewater treatment process and in different lakes . Z . ramigera could be found in all stages of wastewa ter treatment processes, from raw wastewater to chlorinated effluent, The highest concentration of Z . ramigera was found in the mixed liquor stage of the a wastewater treatment plant . Additionally, Z . ramigera was found in all eutrophic and mesotrophic lakes and in some oligotrophic lakes.

J Bacteriol, 2002 Feb, 184(3), 621 - 8
Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence; Taylor CM et al.; We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface . Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction . The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria . The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp . Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L . monocytogenes in the host . Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L . monocytogenes for biofilm growth and pathogenesis.

Biotechnol Bioeng, 2002 Mar 5, 77(5), 495 - 516
Calculation of effective diffusivities for biofilms and tissues; Wood BD et al.; In this study we describe a scheme for numerically calculating the effective diffusivity of cellular systems such as biofilms and tissues . This work extends previous studies in which we developed the macroscale representations of the transport equations for cellular systems based on the subcellular-scale transport and reaction processes . A finite-difference model is used to predict the effective diffusivity of a cellular system on the basis of the subcellular-scale geometry and transport parameters . The effective diffusivity is predicted for a complex three-dimensional structure that is based on laboratory observations of a biofilm, and these numerical predictions are compared with predictions from a simple analytical solution and with experimental data . Our results indicate that, under many practical circumstances, the simple analytical solution can be used to provide reasonable estimates of the effective diffusivity .

Microbiology, 2002 Jan, 148(Pt 1), 277 - 88
Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp; Allan VJ et al.; A phosphatase-overproducing Citrobacter sp . (NCIMB 40259) was grown in an air-lift reactor in steady-state continuous culture under limitation of carbon, phosphorus or nitrogen . Substantial biofilm formation, and the highest phosphatase activity, were observed under lactose limitation . However, the total amount of biofilm wet biomass and the phosphatase specific activity were reduced in phosphorus- or nitrogen-limited cultures or when glucose was substituted for lactose as the limiting carbon source . Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) showed differences in cell and biofilm morphology in relation to medium composition . Electron microscopy suggested that the differences in biofilm formation may relate to differential expression of fimbriae on the cell surface.

Microbiology, 2002 Jan, 148(Pt 1), 87 - 102
Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound; Hentzer M et al.; Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms . The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp) . Gfp-based reporter technology has been applied for non-destructive, single-cell-level detection of quorum sensing in laboratory-based P . aeruginosa biofilms . It is reported that a synthetic halogenated furanone compound, which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra, is capable of interfering with AHL-mediated quorum sensing in P . aeruginosa . It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis . In addition, it reduces the production of important virulence factors, indicating a general effect on target genes of the las quorum sensing circuit . The furanone was applied to P . aeruginosa biofilms established in biofilm flow chambers . The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells . The compound did not affect initial attachment to the abiotic substratum . It does, however, affect the architecture of the biofilm and enhances the process of bacterial detachment, leading to a loss of bacterial biomass from the substratum.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 776 - 85
Analysis of the microbial communities on corroded concrete sewer pipes--a case study; Vincke E et al.; Conventional as well as molecular techniques have been used to determine the microbial communities present on the concrete walls of sewer pipes . The genetic fingerprint of the microbiota on corroded concrete sewer pipes was obtained by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments . The DGGE profiles of the bacterial communities present on the concrete surface changed as observed by shifts occurring at the level of the dominance of bands from non-corroded places to the most severely corroded places . By means of statistical tools, it was possible to distinguish two different groups, corresponding to the microbial communities on corroded and non-corroded surfaces, respectively . Characterization of the microbial communities indicated that the sequences of typical bands showed the highest level of identity to sequences from the bacterial strains Thiobacillus thiooxidans, Acidithiobacillus sp., Mycobacterium sp . and different heterotrophs belonging to the alpha-, beta- and gamma-Proteobacteria, Acidobacteria and Actinobacteria . In addition, the presence of N-acyl-homoserine lactone signal molecules was shown by two bio-assays of the biofilm on the concrete under the water level and at the most severely corroded places on the concrete surface of the sewer pipe.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 626 - 30
Continuous lactic acid fermentation using a plastic composite support biofilm reactor; Cotton JC et al.; An immobilized-cell biofilm reactor was used for the continuous production of lactic acid by Lactobacillus casei subsp . rhamnosus (ATCC 11443) . At Iowa State University, a unique plastic composite support (PCS) that stimulates biofilm formation has been developed . The optimized PCS blend for Lactobacillus contains 50% (wt/wt) agricultural products {35% (wt/wt) ground soy hulls, 5% (wt/wt) soy flour, 5% (wt/wt) yeast extract, 5% (wt/wt) dried bovine albumin, and mineral salts} and 50% (wt/wt) polypropylene (PP) produced by high-temperature extrusion . The PCS tubes have a wall thickness of 3.5 mm, outer diameter of 10.5 mm, and were cut into 10-cm lengths . Six PCS tubes, three rows of two parallel tubes, were bound in a grid fashion to the agitator shaft of a 1.2-1 vessel for a New Brunswick Bioflo 3000 fermentor . PCS stimulates biofilm formation, supplies nutrients to attached and suspended cells, and increases lactic acid production . Biofilm thickness on the PCS tubes was controlled by the agitation speed . The PCS biofilm reactor and PP control reactor achieved optimal average production rates of 9.0 and 5.8 g l(-1) h(-1), respectively, at 0.4 h(-1) dilution rate and 125-rpm agitation with yields of approximately 70%.

Orv Hetil, 2001 Nov 25, 142(47), 2621 - 5
{Molecular pathogenesis of oral candidiasis (candidosis)}; Hermann P et al.; Candida species are the most important pathogenic fungi in the oral cavity with the predominance of Candida albicans . In this review the authors summarise the most important cell-surface bound pathogenical factors such as fibrinogen, fibronectin, thrombin, collagen, laminin and vitronectin-binding proteins and extracellular virulence enzymes of Candida albicans and some microbiological aspects of oral candidiasis (candidosis) . Adherence to both artificial and mucosal surfaces is mediated by hydrophobic interactions and by ligand-receptor attachment . Surface bound proteins on Candida cells bind to mucosal surface proteins . Broad spectrum antibacterial treatment liberates binding sites for Candida colonisation by means of reducing the number of bacterial normal flora in the oral cavity . Non immune humoral factors such as iron, lysosyme, hystidine-rich-polypeptides, lactoferrin, lactoperoxidase and immune globulins such as s-IgA, moreover, elements of cellular immunity, especially polymorphonuclear leucocytes contribute to preventing the establishment of Candida infection . A disbalance in these constituents may result in colonisation and biofilm production of Candida . The biofilm consist of serum proteins mainly fibrin, desquamated epithelial cells, dead leukocytes, living and multiplying candida cells, pseudohyphae and extracellular matrix excreted by candida cells . Living candida cells are deeply embedded in the biofilm, thus protected from defence mechanisms of the host . Continuous destruction of mucosal surfaces beneath the biofilm may create a portal of entry for systematic candidal infections.

Zhonghua Jie He He Hu Xi Za Zhi, 2000 May, 23(5), 300 - 2
{Observation of biofilms inside tracheal tubes by electron microscopy and the relationship between biofilms and VAP}; Bo H et al.; OBJECTIVE: To observe the formation of biofilms inside tracheal tubes and to evaluate the effect of biofilm on the development of ventilator associated pneumonia (VAP) . METHODS: Biofilms inside tracheal tubes from intubated patients were observed by scanning electron microscopy and transmission electron microscopy, meanwhile bacteria were detected in the specimens from the lower respiratory tract and the inside of tracheal tubes . RESULTS: 76% (19/25) of inner surface of tracheal tubes was coated with a confluent amorphous matrix by scanning electron microscopy . The average duration of trach-intubation in this group {(10.7 +/- 7.9) days} was longer than that in the other group {(2.1 +/- 0.8) days} in which tracheal tubes were not coated with the confluent amorphous matrix (P < 0.05) . The presence of many bacteria(14/18) within these amorphous matrix was confirmed by transmission electron microscopy . Of the sixteen tracheal tubes, eleven tubes grew the same organisms that had been isolated from the secretions of the lower respiratory tract before extubation . In some VAP patients (7/8), organisms isolated from tracheal tubes were the same organisms which were pathogenic organisms of VAP . CONCLUSIONS: There was a close connection between the formation of bacterial BF and the long-term indwelling tracheal tubes . The presence of bacterial BF in the inner of tracheal tubes might be associated with the pathogenic organisms of VAP.

J Microbiol Methods, 2002 Feb, 48(2-3), 289 - 302
Direct interface of chemistry to microbiological systems: membrane inlet mass spectrometry; Lloyd D et al.; Direct measurement of dissolved gases and low molecular weight volatiles through permeable membranes (e.g . 50-microm-thick silicone rubber), provides an invaluable tool for the investigation of the activities of microorganisms in the laboratory and in their natural environments . Multiple molecular species are monitored at a single point . Fast response times (t(90%)<1 min) and long-term stability, (<1% week(-1)); high specificity and high sensitivity (e.g . 0.2 microM for O(2), <0.5 mM for ethanol), provides a technique that can provide information on the kinetics of processes over many decades (10(0)-10(6)) of minutes . Spatial resolution of <1 mm enables 3D mapping of gases in complex ecosystems (sediments, peat, soils, biofilms, foodstuffs) . Results with membrane inlet mass spectrometry (MIMS) when used in conjunction with confocal scanning laser microscopy, provides a powerful approach to the analysis of kinetic and spatial aspects of natural environments . Examples discussed are peat cores and cheese.

J Ind Microbiol Biotechnol, 2001 Dec, 27(6), 343 - 51
Mechanisms of biofilm formation in paper machine by Bacillus species: the role of Deinococcus geothermalis; Kolari M et al.; Mechanisms for the undesired persistence of Bacillus species in paper machine slimes were investigated . Biofilm formation was measured for industrial Bacillus isolates under paper machine wet-end-simulating conditions (white water, pH 7, agitated at 45 degrees C for 1-2 days) . None of the 40 tested strains of seven Bacillus species formed biofilm on polished stainless steel or on polystyrene surfaces as a monoculture . Under the same conditions, Deinococcus geothermalis E50051 covered all test surfaces as a patchy thick biofilm . The paper machine bacilli, however, formed mixed biofilms with D . geothermalis E50051 as revealed by confocal microscopy . Biofilm interactions between the bacilli and the deinococci varied from synergism to antagonism . Synergism in biofilm formation of D . geothermalis E50051 was strongest with Bacillus coagulans D50192, and with the type strains of B . coagulans, B . amyloliquefaciens or B . pumilus . Two B . licheniformis, one B . amyloliquefaciens, one B . pumilus and four B . cereus strains antagonized biofilm production by D . geothermalis . B . licheniformis D50141 and the type strain of B . licheniformis were the strongest antagonists . These bacteria inhibited deinococcal growth by emitting heat-stable, methanol-soluble metabolite(s) . We conclude that the persistence of Bacillus species in paper machine slimes relates to their ability to conquer biofilms formed by primary colonizers, such as D . geothermalis.

Appl Environ Microbiol, 2002 Jan, 68(1), 434 - 9
The microbial community structure of drinking water biofilms can be affected by phosphorus availability; Keinanen MM et al.; Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 microg of phosphorus liter(-1) and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs) . Phosphate increased the proportion of PLFAs 16:1 omega 7c and 18:1 omega 7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure . Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.

Appl Environ Microbiol, 2002 Jan, 68(1), 356 - 64
Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization; Ito T et al.; We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes . The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB . Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up {(14)C}propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments . Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions . SRB were also numerically important members of H(2)-utilizing and (14)CO(2)-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H(2)-utilizing bacteria hybridized with probe EUB338 . A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H(2) utilizers in this biofilm . The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date . These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.

Appl Environ Microbiol, 2002 Jan, 68(1), 201 - 10
New degenerate Cytophaga-Flexibacter-Bacteroides-specific 16S ribosomal DNA-targeted oligonucleotide probes reveal high bacterial diversity in River Taff epilithon; O'Sullivan LA et al.; River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors . In this study the bacterial diversity of River Taff epilithon in South Wales was investigated . A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes . The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones . Members of the Cytophaga-Flexibacter-Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria), gamma-Proteobacteria, gram-positive bacteria, Cyanobacteria, beta-Proteobacteria, delta-Proteobacteria, and the Prosthecobacter group . This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376 . The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division . CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b . The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced . They affiliated with the RDP-designated groups Cytophaga, Sphingobacterium, Lewinella, and Cytophaga aurantiaca . CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use . Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance.

Scanning, 2001 Nov-Dec, 23(6), 376 - 8
Rapid diagnosis of fungal infection of intravascular catheters in newborns by scanning electron microscopy; Sbarbati A et al.; Intravascular catheters carry a significant risk of becoming colonized with bacteria and fungi and are important risk factors of septicemia in premature neonates . The study was undertaken to evaluate whether scanning electron microscopy (SEM) examination of removed catheters can be useful in early diagnosis of plastic infection by Candida, providing information useful for initiation of an eventual therapy . The evolution of biofilms in 28 catheters (umbilical or central) implanted in 24 newborns for prematurity was studied by SEM and transmission electron microscopy (TEM) . In 4 of 24 patients, SEM examination revealed the presence of Candida in form of yeast or hyphae . In one of these patients, TEM confirmed the presence of organisms . In each case, hemoculture and culture of the catheter itself confirmed the diagnosis . The study demonstrates that SEM can identify fungi in the biomaterials covering the catheter surface in a few hours, allowing an early diagnosis of plastic infection.

Microbiol Res, 2001, 156(4), 383 - 6
Significance of physical attachment of fungi for bio-treatment of water; Jirku V et al.; The inhibitory effect of xenobiotics known to damage cell surface structures was studied . The sensitivity of suspended cells of the two fungi Candida maltosa and Fusarium proliferatum was compared with that of artificial or natural biofilms of these fungi . The results obtained indicate that the resistance of attached cell populations to model xenobiotics is increased compared with suspended cells . Only the attached fungal cells had the capacity to degrade acetone and phenol and to adapt to increasing concentrations of these substances, so they seem ideally suited for bioremediation of waste water.

Int Microbiol, 2001 Jun, 4(2), 73 - 80
Adaptation of bacterial communities to environmental transitions from labile to refractory substrates; Karthikeyan S et al.; The aim of this work was to assess the adaptation of bacterial communities to environmental transitions from labile to refractory substrates . This involved testing the hypothesis that bacteria self-organize and propagate not only as individual cellular systems, but also as functional sets of interacting organisms . A biofilm community was cultivated in a flow-cell irrigated with tryptic soy broth and subjected to a cyclic series of environmental transitions, from labile to refractory substrates, followed by a period of starvation (30 days) . The appearance and disappearance of specific colony morphotypes when the emigrants were plated onto tryptic soy agar was used to monitor the restructuring of the community . Confocal laser microscopy of flow cells showed that these transitions decreased the biofilm thickness and coverage . Substrate shifts also changed the architecture of the biofilm communities . Repeated inoculation of flow-cell communities with a composite inoculum increased the number and diversity of emigrants . Their biofilms were thicker and covered a wider area than those of communities that had been inoculated only at the beginning of the experiment . With repeated inoculation, the time required for the community to restructure and stabilize decreased during most transitions . This suggested that organismal recombination acted as a mechanism of adaptation, enhancing the growth of microbial communities exposed to environmental stresses . Changes in the profiles of emigrants during the adaptation of biofilm communities to environmental transitions showed the appearance and disappearance of discrete sets of organisms . This suggested that the biofilm communities responded to environmental stresses as sets of interacting organisms . Enhanced growth of biofilm communities due to repeated environmental cycling suggested that the functionality of cellular positioning accrued from one cycle to the next and was thus heritable, although it was not necessarily genetically encoded.

Eur J Oral Sci, 2001 Dec, 109(6), 425 - 7
Bactericidal effect of delmopinol on attached and planktonic Streptococcus sanguinis cells; Burgemeister S et al.; The aim of this investigation was to determine the antibacterial effect of varying concentrations of delmopinol-HCl on attached as well as on planktonic Streptooccus sanguinis cells in vitro . In addition, a possible antiadhesive effect on attached micro-organisms was to be investigated . S . sanguinis cells were allowed to attach to glass surfaces . These as well as planktonic cells were exposed to delmopinol-HCI in concentrations ranging from 0.2% to 0.00005% for 2 min . The percentage of vital bacteria was calculated by means of a fluorescence staining method . Total counts of attached bacteria were performed to determine any possible detaching effect by the delmopinol-HCl . The CFU were determined for the planktonic bacteria . Attached as well as planktonic bacteria showed a marked decrease in vitality following exposure to 0.2% delmopinol-HCl . After exposure to 0.05% this was only the case with the attached microorganisms . The total number of attached bacteria was not reduced by the delmopinol treatment . During initial dental biofilm formation, delmopinol-HCl causes a bactericidal effect when applied in concentrations of 0.05% and higher.

Environ Technol, 2001 Oct, 22(10), 1193 - 201
Biodegradation of isopropanol in a three phase fixed bed bioreactor: start up and acclimation using a previously-enriched microbial culture; Bustard MT et al.; The aerobic biodegradation of high liquid phase concentrations of 2-propanol (IPA) by a previously enriched solvent-tolerant bacterial consortium within a 1.9 l fed-batch three phase fixed bed bioreactor was investigated . Solvent concentrations of up to 7.9 g l(-1) were investigated . Previously enriched solvent-tolerant bacterial cells were immobilised onto porous glass cylinders as a means obioprocess intensification . Bioreactor start-up and acclimation was studied anacetone concentration tracked as an indicator of IPA utilization, as the sole carbon source within a minimal salts medium (MSM) . The initial batch treatment of IPA exhibited a biodegradation rate of 0.11 g l(-1) h(-1) prior to biofilm formation Biofilm growth during the second batch treatment was consistent with an increase in metabolic activity and an IPA biodegradation rate of 0.34 g l(-1), followed by a reduction of biodegradation rate to a constant value of 0.078 g l(-1) h(-1) after 650 h . A maximum acetone generation rate of 1.3 g l(-1) h(-1) was obtained during the fourth IPA addition although the maximum acetone biodegradation rate of 0.38 g l(-1) h(-1) was observed during the initial IPA addition . It is proposed that the metabolic lag resulting from switching from alcohol dehydrogenase to acetone carboxylase is a major rate-limiting step in the deep oxidation of IPA to acetone . The results demonstrate the potential of a previously enriched solvent-tolerant bacterial consortium in fixed bed bioreactor systems, for the aerobic treatment of concentrated solvent-containing wastestreams.

Environ Toxicol Chem, 2001 Dec, 20(12), 2690 - 8
Factors affecting the occurrence and enantiomeric degradation of hexachlorocyclohexane isomers in northern and temperate aquatic systems; Law SA et al.; Concentrations of (alpha- and gamma-hexachlorocyclohexane (HCH), alpha/gamma-HCH ratios, and enantiomer ratios (ER) of alpha-HCH were measured in lakes in the arctic, subarctic, Great Lakes, Canada, and temperate regions, and temperate and arctic wetlands and streams . The highest concentrations of alpha-HCH were found in cold, large, and oligotrophic lakes such as those in the arctic, subarctic, and the upper Great Lakes, which is attributed to greater inputs from atmospheric deposition and slower loss rates relative to warmer, temperate lakes . High alpha/gamma-HCH ratios in northern systems indicate aged HCH that has undergone long-range transport to high latitude areas, whereas low ratios in the lower Great Lakes and small temperate systems indicate recent gamma-HCH usage and residual alpha-HCH concentrations . Enantioselective degradation (ERs ranged from 0.31 to 0.7) was greatest in small, high arctic lakes and streams and in large lakes in the subarctic in which alpha-HCH concentrations and contact time between chemical and sediments are highest and nutrient concentrations are lowest . Low ERs were found in wetlands and streams in which contact between chemical and sediments was greatest . Conversely, minimal enantioselective degradation occurred in temperate small lakes and wetlands (ERs ranging from 0.77 to 1.06), despite the warmer temperatures, greater microbial populations, and nutrient availability . The results suggest that enantioselective degradation is optimized by maximal contact between chemical and sediment substrates in nutrient-poor waters in which, it is hypothesized, oligotrophic bacteria may act as biofilms.

Mar Pollut Bull, 2001 Nov, 42(11), 1103 - 13
Effects of chromated copper arsenate (CCA) wood preservative on early fouling community formation; Brown CJ et al.; The effects of the anti-marine-borer timber preservative CCA (a pressure impregnated solution of copper, chromium and arsenic compounds) on early fouling community formation were investigated during a number of field trials . The formation of a biofilm on the surface of CCA-treated and untreated timber panels of Scots pine was examined by scanning electron microscopy following submersion in Langstone Harbour, Portsmouth, UK for periods of 2, 7, 14 and 28 days . Results indicated a slightly faster rate of biofilm formation after 2 and 7 days of exposure on untreated timber compared to CCA-treated timber, although no differences were visible between panels after 14 and 28 days exposure, or between panels treated to different CCA loadings after all exposure periods . Settlement of the serpulid Ficopomatus enigmaticus and two species of barnacles (Elminius modestus and Balanus crenatus) onto the surface of untreated and CCA-treated panels of Scots pine was examined following 4 weeks exposure in a brackish water millpond at Emsworth, West Sussex . Numbers of individuals were higher on CCA-treated panels than on untreated panels, and in the case of F . enigmaticus abundance of individuals increased with increasing preservative loadings . Early colonization by macroalgal species on the surface of CCA-treated and untreated panels of Scots pine was examined following submersion of panels in Langstone Harbour for a period of 4 weeks . Percentage cover of most species of algae was similar on the surface of CCA-treated and untreated panels, with the exception of Hincksia granulosa and Ceramium nodulosum which had significantly higher percentage cover on untreated panels . Possible explanations for the recruitment patterns are discussed.

Water Res, 2001 Dec, 35(18), 4323 - 30
Degradation of aroclor 1242 in a single-stage coupled anaerobic/aerobic bioreactor; Tartakovsky B et al.; Degradation of Aroclor 1242 was studied in granular biofilm reactors with limited aeration . An aerobic biphenyl degrader, Rhodococcus sp . M5, was used to supplement a natural bacterial population present in a "bioaugmented" reactor, while the "non-bioaugmented" reactor only contained natural granular sludge . The bioaugmentation, however appeared to have no effect on the reactor performance . Aroclor measurements showed its disappearance in both reactors with only 16-19% of Aroclor recovered from the reactor biomass and effluent . Simultaneously, a chlorine balance indicated that dechlorination occurred at a specific rate of 1.43 mg PCB (g volatile suspended solids)(-1) d(-1), which was comparable to the observed rate of Aroclor disappearance . Intermediates detected in both reactors were biphenyl, benzoic acid, and mono-hydroxybiphenyls . This suggests that a near-complete mineralization of Aroclor can be achieved in a single-stage anaerobic/aerobic system due to a combination of reductive and oxidative degradation mechanisms.

Ann N Y Acad Sci, 2001 Nov, 946, 274 - 90
Catheter-related bloodstream infections in HIV-infected patients; Nicastri E et al.; Bloodstream infections (BSI) constitute a significant public health problem and represent an important cause of morbidity and mortality in hospitalized patients, with an approximate incidence of one episode per hundred hospital admissions . Studies on BSI in HIV+ patients have identified central venous catheters (CVC) as a risk factor, with an attributable mortality rate of 10-20% . The long-term CVC-related infection risk appeared to be 5 to 10-fold higher with respect to the infection rates among HIV- patients . CVC associated infection rate ranges from 1.3 to 12 infections per 1,000 catheter-days . Staphylococcus aureus is the most common etiologic agent causative of CVC-related BSI, likely the result of the high skin and nasal carriage of this organism among HIV+ patients, mostly intravenous drug users . Coagulase-negative staphylococci are also frequently identified as cause of CVC-related BSI, likely the result of breaches in infection control measures and in antiseptic technique during CVC management . Treating bacteremia without catheter removal would be optimal, but the reported efficacy of systemic antibiotic therapy alone is only 25-32% . Conversely, recent studies have shown that, using an antibiotic-lock procedure, up to 90% of HIV-infected and uninfected patients achieved complete eradication of catheter-related BSIs without catheter removal . Clinical trials using new materials such as covalently linked heparin on the CVC surface, electrically charged CVC, novel topical agents that interfere with bacterial colonization, antiadhesin molecules and agents that block the gene expression involved in the biofilm formation, are all needed to reduce the high catheter-related infection risk among HIV+ patients.

Plant Cell Environ, 2001 Jan, 24(1), 1 - 14
Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function; Newman IA; The transport of mineral ions into and out of tissues and cells is central to the life of plants . Ion transport and the plasma membrane transporters themselves have been studied using a variety of techniques . In the last 15 years, measurement of specific ion fluxes has contributed to the characterization of transport systems . Progress in molecular genetics is allowing gene identification and controlled expression of transporter molecules . However the molecular expression of transporter gene products must be characterized at the functional level . The ion-selective microelectrode technique to measure specific ion fluxes non-invasively is ideally suited to this purpose . This technique, its theory, its links with others and its application and prospects in plant science, are discussed . Ions studied include hydrogen, potassium, sodium, ammonium, calcium, chloride and nitrate . Applications discussed include: solute ion uptake by roots; gravitropism and other processes in the root cap, meristematic and elongation zones; Nod factor effect on root hairs; osmotic and salt stresses; oscillations; the effects of light and temperature . Studies have included intact roots, leaf mesophyll and other tissues, protoplasts and bacterial biofilms . A multi-ion capability of the technique will greatly assist functional genomics, particularly when coupled with imaging techniques, patch clamping and the use of suitable mutants.

Appl Microbiol Biotechnol, 2001 Oct, 57(3), 437 - 40
Phylogenetic diversity of a SRB-rich marine biofilm; Zhang T et al.; This study was conducted to characterize the phylogenetic diversity of a corrosive marine biofilm based on 16S rDNA . Results of phylogenetic analysis indicated that, out of the 112 clones developed, 52 clones (46.4%) were affiliated with two families of sulfate-reducing bacteria: Desulfovibrionaceae and Desulfobacteriaceae . Another 44 clones (39.3%) were affiliated with the Clostridiaceae family of low G+C, gram-positive bacteria . Three clones (2.7%) were closely related to Chlorobium vibrioforme, a green sulfur bacterium.

Appl Microbiol Biotechnol, 2001 Oct, 57(3), 419 - 26
A GAC biofilm reactor for the continuous degradation of 4-chlorophenol: treatment efficiency and microbial analysis; Carvalho MF et al.; Using a continuous enrichment technique, a bacterial consortium capable of degrading 4-chlorophenol (4-CP) was obtained from the rhizosphere of Phragmites australis . A granular activated carbon (GAC) biofilm reactor was established using this consortium, and the degradation of 4-CP was investigated under continuous flow operation using a feed of 20-50 mg l(-1) with a hydraulic residence time of 17 min over a 6-month period . Chloride liberation occurred throughout the operation, and the reactor had 4-CP removal efficiencies of 69-100% . Periods of lower performance were attributed to clogging of the column with biomass and the formation of channels . Subsequently, the immobilized biofilm was subjected to a starvation period of 5 months, after which its degradative capacity was still maintained . The microbial consortium was characterized during the continuous flow experiment and dynamic population changes were observed throughout . One isolate recovered from the biofilm was shown to be capable of degrading 4-CP as a sole carbon and energy source.

Antonie Van Leeuwenhoek, 2001 Oct, 80(2), 129 - 38
Effects of different nutrients on bacterial growth in a pilot distribution system; Frias J et al.; The growth of bacterial communities in drinking water distribution systems can lead to the development of problems incompatible with water quality requirements . This study was carried out in order to determine which factors promote bacterial growth in distribution networks . A pilot distribution system was used to perform these experiments . After addition of three different inorganic elements to the network (N, P and S) the results obtained show that they did not contribute to the growth or bacteria either in the circulating water or on the surface of the pipes of the distribution system . However, when organic carbon was added an increase in the number of circulating bacteria was observed though the number of bacteria in the biofilms of the network was constant . These results indicate that in the drinking water of the Barcelona distribution system the factor that controls the growth of bacteria is organic carbon . Moreover, bacteria from the biofilm growing on the surface of the pipes may cause the problems of bacterial growth.

J Dent Res, 2001 Nov, 80(11), 2005 - 10
The association of bacterial adhesion with dental caries; Stenudd C et al.; Saliva adhesion of bacteria is a key event in oral biofilm formation . Here, we used partial least-squares (PLS) analysis to correlate adhesion of cariogenic (Streptococcus mutans Ingbritt) and commensal (Actinomyces naeslundii LY7) model bacteria, and their agglutinin and acidic proline-rich protein ligands, respectively, with high and low caries experiences in 38 children reflecting today's skewed caries distribution . Adhesion of S . mutans was among the factors correlating strongest with high caries experience when PLS modeled together with traditional factors (e.g., sugar intake, lactobacilli counts) . Saliva phenotypes with high agglutinin levels and Db-s (an acidic PRP variant) coincided with both high caries experience and S . mutans adhesion . A . naeslundii adhesion correlated with low caries experience . Non-Db phenotypes (i.e., acidic PRP-1 and PRP-2 variants) coincided with both low caries experience and S . mutans, but high A . naeslundii, adhesion . Thus, bacterial adhesion may modulate susceptibility and resistance to dental caries.

Zhonghua Nei Ke Za Zhi, 2001 Sep, 40(9), 585 - 8
{The assay of beta-lactamase activity in Pseudomonas aeruginosa biofilm}; Gu X et al.; OBJECTIVE: To analyze the resistance mechanism of Pseudomonas aeruginosa biofilm bacteria . METHODS: An in vitro model of Pseudomonas aeruginosa bacterial biofilm was established in silicon disk with modified flat-board method and a rapid staining procedure of AgNO3 was used to verify it . Biofilm was observed under scanning electron microscopy . The test was carried out in three groups . Group A was planktonic bacteria of Pseudomonas aeruginosa, group B biofilm bacteria of Pseudomonas aeruginosa and group C biofilm bacteria induced by imipenem . The activity of beta-lactamase was quantitated with a spectrophotometric assay method and beta-lactamase quantitation was determined by using Bio-Rad protein assay . RESULTS: The activity and protein content of beta-lactamase in group B was higher than that in group A by 4.67 and 2.09 times . The activity of group C was 21.86 times as much as that in group A and 4.68 times as much as that in group B . The quantitation in group C was 6.28 times as much as that in group A and 3.00 times as much as that in group B . The activity and quantitation of the three groups were different significantly from each other (P < 0.01) . CONCLUSION: The production of beta-lactamase in Pseudomonas aeruginosa biofilm bacteria is one of the main reasons of its resistance.

Zhonghua Jie He He Hu Xi Za Zhi, 2001 Sep, 24(9), 537 - 8
{A study on beta-lactamase activity of biofilm Escherichia coli}; He P et al.; OBJECTIVE: To investigate the mechanism of drug resistance of biofilm escherichia coli . METHODS: The model of escherichia coli biofilm was established with the flat-board method . And the biofilm was confirmed by scanning electron microscopy . The beta-lactamase activities were quantitated, in escherichia coli, biofilm escherichia coli, biofilm escherichia coli induced by impenem or cefoxitin . RESULTS: The beta-lactamase activity of biofilm escherichia coli was 2.16 times as much as that of escherichia coli planctonically, and the beta-lactamase activities of biofilm escherichia coli induceded by impenem or cefoxitin were 1.30 and 1.05 times as much as those of biofilm escherichia coli, respectively . CONCLUSION: The drug resistance to antibiotics of biofilm escherichia coli was related to the production of beta-lactamase.

Perit Dial Int, 2001 Sep-Oct, 21(5), 480 - 6
Intraperitoneal ciprofloxacin and rifampicin versus cephradine as initial treatment of (C)APD-related peritonitis: a prospective randomized multicenter comparison (CIPPER trial); de Fijter CW et al.; OBJECTIVE: The initial treatment of peritonitis has evolved from single-agent to combination regimens . The initial response rates improved with these newer regimens but relapsing peritonitis continues to occur . For biofilm-embedded or intracellularly sequestrated bacteria, a combination of intracellularly- and biofilm-active agents such as ciprofloxacin and rifampicin might be beneficial . Many Dutch centers continue to use cephradine as initial treatment, claiming clinically adequate responses with this regimen . We compared the impact of these two regimens on outcome in patients who developed a new episode of peritonitis . DESIGN: Prospective randomized open trial . SETTING: Multicenter study including 14 Dutch dialysis units . PATIENTS AND INTERVENTIONS: From October 1996 to October 1999, 367 patients from 14 centers were randomized to be treated with ciprofloxacin + rifampicin (CR; each 50 mg/L) or cephradine (C; 250 mg/L) in case of peritonitis . Of these 367 patients, 98 developed peritonitis, 44 of whom were treated with CR and 54 with C . MAIN OUTCOME MEASURES: Clinical response, divided into early (during the 2 weeks of therapy) and late (including the following 4 weeks) response . Success was defined as disappearance of all signs and symptoms by days 4-6, through day 42 . Bacteriological response was either success (eradication) or failure (persistence, superinfection, or eradication with relapse/reinfection) . RESULTS: The groups were comparable for age, sex, duration of continuous ambulatory/automated peritoneal dialysis, and occurrence of diabetes . Bacteriological cultures in both groups revealed predominantly gram-positive micro-organisms . Initial and late clinical successes were obtained in 27/54 and 20/54 episodes (50% and 37%) in the C group, and 33/44 and 28/44 episodes (75% and 63.6%) in the CR group (p = 0.021 and p = 0.019) . Bacteriological success occurred in 29.6% in the C group, and in 59.1% in the CR group (p= 0.026), with failure in 46.3% and 18.2%, respectively . Peritonitis episodes were bacteriologically not evaluable in 24.1% of episodes in the C group and 22.7% of episodes in the CR group, due mostly to no growth in the initial culture . CONCLUSION: The CIPPER Trial showed ciprofloxacin + rifampicin to be superior to cephradine as empiric treatment of peritonitis.

Environ Sci Technol, 2001 Nov 15, 35(22), 4530 - 5
DGT as an in situ tool for measuring radiocesium in natural waters; Murdock C et al.; The application of diffusive gradients in thin-films (DGT) samplers for the measurement of cesium radionuclides in solution, using an ammonium molybdophosphate (AMP) binding agent, was tested under both laboratory and field conditions . In the former they proved able to reproduce known 134Cs concentrations (60 Bq L(-1)) with a high degree of accuracy and precision over periods up to approximately 1 d, in freshwaters over a wide range of pH and temperature, and in saline water . In field trials in a freshwater lake receiving nuclear power station discharges, mean concentrations of 137CS (47-61 mBq L(-1)) were measured over periods from 5 d to 1 month . These agreed, within error, with mean concentrations determined from grab samples but rigorous field validation of long-term (month) deployments of DGT devices proved impossible using conventional sampling procedures, due to loss of 137Cs to container walls . Identified limitations of the DGT technique included probable AMP degradation over longer periods and calibration problems if large changes in temperature and concentration occurred together . Potential limitations due to biofilm growth were considered not to be significant . Despite the limitations, the technique appears to measure concentrations accurately for deployment times of 1 month or less . It has several advantages over traditional sampling methods for monitoring radionuclides in the solution/dissolved phase, including its simplicity, provision of time-averaged mean concentrations, and automatic in-situ concentration onto a medium with ideal counting geometry for gamma spectrometry.

Aquat Toxicol, 2002 Jan, 56(2), 103 - 13
Effects of zinc on the phosphorus availability to periphyton communities from the river Göta Alv; Paulsson M et al.; It has been hypothesised that zinc additions in the phosphorus limited (12-15 microg l(-1)) River Gota Alv leads to an interaction between zinc and phosphate which causes a decreased availability of phosphate and a concomitant decrease in biomass production in the Gota Alv periphyton communities 'Aquat . Tox . 47 (2000) 243' . To test the hypothesis the experiment by Paulsson et al . 'Aquat . Tox . 47 (2000) 243' was repeated, now with focus on phosphorus status of the periphyton . Additional indicators of phosphorus deficiency, e.g . alkaline phosphatase activity (APA), surplus and total phosphorus, intracellular acid phosphatase activity, and nitrogen and carbon were measured in the communities . APA increased and surplus phosphorus decreased at about the same zinc exposure concentration as the dry weight started to decrease (> 0.1 microM), thus supporting the hypothesis of a zinc-induced phosphorus deficiency . Nitrogen and carbon concentrations in the biofilm also decreased with increasing zinc exposure, suggesting an influence on metabolism of these elements as well . It can be concluded that zinc might be an environmental hazard in phosphorus-limited environments at concentrations above 0.1-0.2 microM of total zinc.

Curr Opin Urol, 2002 Jan, 12(1), 45 - 9
Urinary tract infection in individuals with spinal cord lesion; Biering-Sorensen F; Urinary tract infection is the most frequently reported secondary impairment in individuals with spinal cord lesion . The most prevalent risk indicator is an indwelling catheter . Hydrophilic catheters for clean intermittent catheterization may induce lower rates of bacteriuria and long-term urethral complications . Due to chronic bacterial infection within biofilms, an antibacterial treatment based on a urinary culture of bacteria in the urine and its antimicrobial susceptibility may fail to eradicate catheter-associated urinary tract infection . No commercially available drugs are sufficiently active against the bacteria in a mature biofilm . Biomaterials may be modified to decrease the formation of a biofilm . Silver alloy catheters are effective in preventing urinary tract infection when indwelling urinary catheterization is necessary . The risk of systemic argyria in long-term use needs to be evaluated . Suprapubic cystostomy drainage in patients with neurogenic bladder is preferred to an indwelling urethral catheter . In cases of recurring urinary tract infection in patients with a permanent urinary catheter, it may be beneficial to change the catheter every 1 or 2 weeks . There is some evidence that cranberry products may prevent urinary tract infection . In the future, bacterial interference and vaccination may be a possibility for prevention of urinary tract infection.

Curr Opin Urol, 2002 Jan, 12(1), 33 - 8
Virulence factors of uropathogens; Oelschlaeger TA et al.; Urinary tract infections are among the most frequent infections encountered in developed countries . The vast majority of community-acquired urinary tract infections are caused by Escherichia coli . However, other bacterial species play an important role in nosocomial urinary tract infections . All these species are equipped with a variety of virulence factors . The best characterized are those from Escherichia coli . Among the first virulence factors that come into play during establishment of a urinary tract infection are adhesins . Besides their primary function as adhesin molecules several other additional functions can now be attributed to these organelles . Adhesins may also function as invasins, promote biofilm formation and transmit signals to epithelial cells resulting in inflammation . Furthermore, subunit proteins of adhesins seem to be promising vaccines . Later in infection, toxins seem to enhance virulence . However, for cytotoxic necrotizing factor type 1 this is controversial . Many virulence factors of uropathogenic bacteria are encoded by foreign DNA stretches inserted into the core genome . These pathogenicity islands or islets were obviously acquired via horizontal gene transfer creating new pathotypes more efficient in establishing infection . The role of new virulence factors and the new functions of already known virulence factors will be discussed as well as the concept of the composite genome of uropathogenic Escherichia coli.

J Bacteriol, 2002 Jan, 184(2), 547 - 55
Lateral flagella and swarming motility in Aeromonas species; Kirov SM et al.; Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence . In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility . Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h . Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure . Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution . Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies . We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp . The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site . Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface . Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement . Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.

Biol Bull, 2001 Dec, 201(3), 394 - 404
NO/cGMP signaling and HSP90 activity represses metamorphosis in the sea urchin Lytechinus pictus; Bishop CD et al.; Nitric oxide (NO) signaling repressively regulates metamorphosis in two solitary ascidians and a gastropod . We present evidence for a similar role in the sea urchin Lytechinus pictus . NO commonly signals via soluble guanylyl cyclase (sGC) . Nitric oxide synthase (NOS) activity in some mammalian cells, including neurons, depends on the molecular chaperone heat shock protein 90 (HSP90); this may be so in echinoid larvae as well . Pluteus larvae containing juvenile rudiments were treated with either radicicol L- or D-nitroarginine-methyl-ester (L-NAME and D-NAME), or IH-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), inhibitors of HSP90, NOS, and sGC, respectively . In all instances, drug treatment significantly increased the frequency of metamorphosis . SNAP, a NO donor, suppressed the inductive properties of L-NAME and biofilm, a natural inducer of metamorphosis . NADPH diaphorase histochemistry indicated NOS activity in cells in the lower lip of the larval mouth, the preoral hood, the gut, and in the tube feet of the echinus rudiment . Histochemical staining coincided with NOS immunostaining . Microsurgical removal of the oral hood or the pre-oral hood did not induce metamorphosis, but larvae lacking these structures retained the capacity to metamorphose in response to ODQ . We propose that the production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduces the production of NO, and consequently cGMP, to initiate metamorphosis.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 178 - 83
Differential expression of methicillin resistance by different biofilm-negative Staphylococcus epidermidis transposon mutant classes; Mack D et al.; Biofilm formation mediated by polysaccharide intercellular adhesin (PIA) is the major virulence factor of Staphylococcus epidermidis and is often associated with methicillin resistance . Transposon Tn917 insertions leading to a biofilm-negative phenotype in the biofilm-producing S . epidermidis strain 1457 (mecA-negative) were transferred into the methicillin-resistant, biofilm-producing S . epidermidis 1057 (mecA-positive) by transduction . According to their phenotypes and genotypes, the mutants could be separated into genetic classes I to IV (D . Mack, H . Rohde, S . Dobinsky, J . Riedewald, M . Nedelmann, J . K . M . Knobloch, H.-A . Elsner, and H . H . Feucht, Infect . Immun . 68:3799-3807, 2000) . All transductants of S . epidermidis 1057 had phenotypes for biofilm formation similar to those of the corresponding mutants of S . epidermidis 1457 . With a mecA-specific probe, identical hybridization patterns were observed for wild-type S . epidermidis 1057 and all the transductants . There were minor changes in oxacillin MICs for Class II and III transductants compared to those for wild-type S . epidermidis 1057 . On population analysis, S . epidermidis 1057 displayed a heterogeneous expression type of resistance with an oxacillin MIC of > or =6 microg/ml for more than 90% of the cells . An almost identical profile was observed with biofilm-negative class I mutants, where the transposon insertions inactivate the icaADBC gene locus essential for PIA synthesis . In contrast, class III mutants were more sensitive to oxacillin with a MIC of < or =1 microg/ml for more than 90% of the cells . The class IV mutant displayed homogeneous resistance with a MIC of > or =50 microg/ml for more than 90% of the cells . On oxacillin gradient plates, the class II mutant displayed decreased resistance . Apparently, different independent mutations leading to a biofilm-negative phenotype of S . epidermidis by influencing expression of icaADBC on the level of transcription significantly influence the expression of methicillin resistance . However, transcription of mecA was not significantly altered in the different transductants compared to the wild type, independent of mecA induction with oxacillin, indicating that other mechanisms influencing phenotypic expression of methicillin resistance are involved.

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 225 - 30
Effects of mutating putative two-component systems on biofilm formation by Streptococcus mutans UA159; Bhagwat SP et al.; Streptococcus mutans has at least six pairs of open reading frames that are homologous to bacterial two-component regulatory systems . Putative response regulators from five out of six of these pairs were successfully mutated by insertion of a kanamycin resistance marker and the effects of inactivation of the genes on the ability of the cells to form biofilms in an in vitro model were assessed . Disruption of the response regulators of four systems had no effect on biofilm formation, whereas disruption of one response regulator caused a substantial decrease in biofilm formation as compared to the wild-type S . mutans.

J Antimicrob Chemother, 2001 Dec, 48(6), 793 - 801
Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis; Monzon M et al.; The in vitro effect of nine antibiotic combinations was investigated in Staphylococcus epidermidis biofilms using ATP-bioluminescence for viable bacterial cell quantification . Four slime-producing (SP) strains were used to form biofilms 6, 24 and 48 h old . These biofilms were exposed for 24 h to antibiotics at 4 x, 2 x, 1 x and 0.5 x MIC . Combinations involving tetracycline together with another antibiotic were the most efficient at the biofilm age and concentration range under study . The combination vancomycin-rifampicin produced the highest bactericidal effect on 6 h biofilms at 4 x MIC, but this effect decreased dramatically in older biofilms . To detect possible antibiotic synergy in combinations that had a significant killing effect, antibiotics were studied not only in combination but also individually . Synergic effects were observed in all the combinations tested . Differences between the effect in combination and the sum of individual antibiotic effects (degree of synergy) were significant (mostly P< 0.001) and exceeded 1 log10 cfu/mL in the majority of cases . In 48 h biofilms, antibiotics caused a significant bactericidal effect when applied in combination, but never when used individually . These results indicate that the biofilm test applied allows the detection of synergy between antibiotics and suggests that this assay could be useful in clinical and extensive synergy studies on S . epidermidis biofilms.

J Biomed Mater Res, 2002 Jan, 59(1), 35 - 45
Biological characterization of a novel biodegradable antimicrobial polymer synthesized with fluoroquinolones; Woo GL et al.; Biomaterial-related infections continue to represent a significant challenge to the medical community . Several approaches have been utilized to incorporate antimicrobial agents at the surface of implant devices in attempts to delay or eliminate the formation of biofilms . To date, most of these strategies have focused on drug conjugation or diffusion-limited systems for the delivery of such pharmaceutical agents . More recently, work has been presented on the feasibility of incorporating drugs into the backbone of polymers as a main-chain monomer . When sequenced into the backbone of the polymer with other monomers that are hydrolytically sensitive to enzyme-catalyzed breakdown, it is thought that drugs may be able to be selectively released . Specifically, degradable polyurethanes have been synthesized with fluoroquinolone antibiotics and have shown an ability to kill bacteria when released following degradation of the polymer chains by the macrophage-derived enzyme cholesterol esterase . However, specificity of the cleavage sites in the polymer was difficult to control . Since cholesterol esterase has specificity for hydrophobic moieties, it is desirable to alter the formulation of the polyurethanes to incorporate long hydrophobic monomers immediately adjacent to the ciprofloxacin molecule . Hence, the current study focuses on evaluating the enzyme-catalyzed degradation of a degradable polyurethane synthesized with 1,12 diisocyanatododecane as a substitute for 1,6 diisocyanatohexane, which was used in previous work . Validation of specific ciprofloxacin release and the generation of antimicrobial are shown . A preliminary cell study to assess the cytotoxicity of this biodegradable antibiotic polymer shows that the material has no observable effects on cell proliferation or cell membrane structure .

Biotechnol Bioeng, 2001 Dec, 76(4), 341 - 50
Quantification of toluene dioxygenase induction and kinetic modeling of TCE cometabolism by Pseudomonas putida TVA8; Shingleton JT et al.; As measured by the toluene-induced bioluminescent response of Pseudomonas putida TVA8 in batch experiments, toluene dioxygenase (Tod) enzyme activities are dependent on toluene concentration between 0 and 30 mg/L . To provide a measure of the Tod activity for use in Michaelis-Menten competitive-inhibition kinetics, a correlation between toluene concentration and induced Tod activity as measured by an induced bioluminescent response of P . putida TVA8 is presented as a nondimensional Tod activity parameter . A packed-bed, radial-flow bioreactor (RFB) using the bioreporter P . putida TVA8A serves as the model system for studying the effect of the enzyme activity parameter on model predictions of vapor-phase toluene oxidation and trichloroethylene (TCE) cometabolism . Mass balances were performed on a differential section of the RFB to describe the radial transport of vapor-phase toluene and TCE through a bulk gas phase and the concomitant biological reaction in a stationary biofilm phase . The finite-element Galerkin weak-statement formulation with first-order basis functions was used to find the optimum solution to the highly nonlinear, coupled equations . For this RFB system with toluene concentrations less than 1 mg/L in the bulk gas phase, the Tod activity parameter enables accurate predictions of steady-state TCE degradation rate (0.27 microg TCE/min) .

J Bacteriol, 2002 Jan, 184(1), 290 - 301
Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli; Jackson DW et al.; The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms . Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites . While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown . Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions . Ectopic expression of the E . coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens . A csrA knockout mutation enhanced biofilm formation in E . coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation . In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant . Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism . Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor . We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E . coli biofilm development.

J Dent, 2002 Jan, 30(1), 47 - 51
Early formation of Streptococcus sobrinus biofilm on various dental restorative materials; Steinberg D et al.; OBJECTIVES: To examine the formation of dental biofilm by Streptococcus sobrinus on different types of restorative materials, using a model consisting of host and bacterial constituents . METHODS: The adsorption pattern of saliva to the restorative material was determined by means of gel electrophoresis coupled with computerized densitometry techniques . The amount of salivary proteins adsorbed onto the surfaces was measured using the Bradford method . Sucrose-dependent bacterial adhesion to the saliva-coated restorative material was tested by radioactive-labelled Streptococcus sobrinus, and viable counts of these bacteria in the biofilm was determined using bacterial culture techniques . RESULTS: Different adsorption patterns by salivary proteins to restorative materials were recorded . Durafil and acrylic dental materials demonstrated the most affinity to salivary proteins . A surface dependent adhesion profile was recorded, showing a high affinity of albumin and amylase to Acrylic and Durafil materials . Bacterial accumulation was the highest with Fuji LC and Fuji GC, which also demonstrated the highest bacterial viability . CONCLUSIONS: Our study demonstrates the specificity of biofilm formation on different brands of dental restorative materials . Formation of a variety of dental biofilms has a significant impact on the progression of dental diseases in the oral cavity.

J Hosp Infect, 2001 Dec, 49(4), 285 - 8
Use of chlorine dioxide to disinfect dental unit waterlines; Smith AJ et al.; This paper describes a trial of chlorine dioxide in dental unit waterlines to produce potable quality water . Four treatment protocols using 50 ppm activated chlorine dioxide solution were tested . Each caused a short-term (<48 h) decline in total viable counts but did not provide potable quality water . Intermittent use of chloride dioxide is thus not suitable for long-term decontamination of dental unit waterlines . Units should be redesigned to discourage biofilm formation, and more research into practical methods of achieving potable water is required in the interim .

Microbiology, 2001 Dec, 147(Pt 12), 3249 - 62
N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms; Riedel K et al.; Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients . Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the formation of biofilms . As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs . To address this issue, novel Gfp-based biosensors for non-destructive, in situ detection of AHLs were constructed and characterized . These sensors were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue . In both model systems B . cepacia was capable of perceiving the AHL signals produced by P . aeruginosa, while the latter strain did not respond to the molecules produced by B . cepacia . Measurements of extracellular proteolytic activities of defined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.

Int J Antimicrob Agents, 2001 Dec, 18(6), 575 - 8
Antibacterial effect of parabens against planktonic and biofilm Streptococcus sobrinus; Doron S et al.; Tooth decay is an infectious disease caused by bacteria immobilized on the tooth surfaces . Eradication of these bacteria, for example Streptococcus sobrinus (S . sobrinus), from the oral cavity is essential in the prevention and treatment of tooth decay . We have tested the antimicrobial effect of several paraben derivatives such as methyl (MP), ethyl (EP), propyl (PP) and butyl (BP) against immobilized and planktonic S . sobrinus . The antibacterial effect was as follows: MP>EP>PP=BP on immobilized bacteria and MP>EP=PP>BP on planktonic bacteria . An antibacterial synergistic effect was found between several combinations of parabens on immobilized and planktonic S . sobrinus . Our results indicate that parabens are potential antibacterial agents against immobilized or planktonic bacteria found in the oral cavity.

Biotechnol Prog, 2001 Nov-Dec, 17(6), 1180 - 2
Analysis of biofilm structure and gene expression using fluorescence dual labeling; Gilbert ES et al.; The use of biofilms for the degradation of recalcitrant environmental contaminants or for the production of secondary metabolites necessitates understanding and controlling gene expression . In this work, dual labeling with green fluorescent protein (GFP) variants was used to investigate inducible gene expression in a biofilm . Colocalization of GFP emissions was used to determine regions of attached cells and to correlate structure and activity within the biofilm . The labeling strategy reported here is unique in that the two GFP signals were distinguished by differential excitation rather than differential emission.

Sci Prog, 2001, 84(Pt 3), 235 - 54
Bacterial biofilms and human disease; Wilson M; The term biofilm is used to denote a polymer-encased community of microbes which accumulates at a surface . Biofilms are responsible for a number of diseases of man and, because of the intrinsic resistance of these structures to antibiotics and host defence systems, such diseases are very difficult to treat effectively . The application of new microscopic and molecular techniques to biofilms has revolutionised our understanding of their structure, composition, organisation and activities . This review will describe the role that biofilms play in human disease and will outline our new millennial view of these complex and fascinating bacterial communities.

Water Sci Technol, 2001, 44(8), 103 - 6
Corrosion protection by anaerobiosis; Volkland HP et al.; Biofilm-forming bacteria can protect mild (unalloyed) steel from corrosion . Mild steel coupons incubated with Rhodoccocus sp . strain C125 and Pseudomonas putida mt2 in an aerobic phosphate-buffered medium containing benzoate as carbon and energy source, underwent a surface reaction leading to the formation of a corrosion-inhibiting vivianite layer {Fe3(PO4)2} . Electrochemical potential (E) measurements allowed us to follow the buildup of the vivianite cover . The presence of sufficient metabolically active bacteria at the steel surface resulted in an E decrease to -510 mV, the potential of free iron, and a continuous release of ferrous iron . Part of the dissolved iron precipitated as vivianite in a compact layer of two to three microns in thickness . This layer prevented corrosion of mild steel for over two weeks, even in a highly corrosive medium . A concentration of 20 mM phosphate in the medium was found to be a prerequisite for the formation of the vivianite layer.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 139 - 46
Protein expression by planktonic and biofilm cells of Streptococcus mutans; Svensater G et al.; Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities . As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h) . Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis . Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells . Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells . The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced . The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.

Appl Environ Microbiol, 2001 Dec, 67(12), 5608 - 13
Growth and detachment of cell clusters from mature mixed-species biofilms; Stoodley P et al.; Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process . By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ . The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 microm . Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent . Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass . These results have significance in the establishment of an infectious dose and public health risk assessment.

Appl Environ Microbiol, 2001 Dec, 67(12), 5410 - 9
Motility of Marichromatium gracile in response to light, oxygen, and sulfide; Thar R et al.; The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients . The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system . M . gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide . The observed migration patterns could be explained solely by the various phobic responses . In the dark, M . gracile formed an approximately 500-microm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at approximately 10 microM O(2) . Flux calculations yielded a molar conversion ratio S(tot)/O(2) of 2.03:1 (S(tot) = {H(2)S} + {HS(-)} + {S(2-)}) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules . In the light, M . gracile spread into the anoxic zone while still avoiding regions with >10 microM O(2) . The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen . A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism . In darkness and anoxia, M . gracile lost its motility after ca . 1 h . In contrast, at oxygen concentrations of >100 microM with no sulfide present the cells remained viable and motile for ca . 3 days both in light and darkness . Oxygen was respired also in the light, but respiration rates were lower than in the dark . Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.

Curr Infect Dis Rep, 2001 Dec, 3(6), 483 - 486
Biofilms: A Clinical Perspective; Bell M; Biofilms play an increasingly recognized role in many aspects of human disease . Most of our understanding of infections is based on research that has examined free-living organisms . The results do not necessarily apply to biofilm organisms, since metabolic and synthetic characteristics of free-living organisms can change when they assume the biofilm mode of growth . Biofilms reduce our ability to eradicate infections, causing relapses after seemingly appropriate therapy . Awareness of biofilms, prevention of contamination of implanted or invasive devices, and use of appropriate antimicrobial dosing and treatment durations can limit the negative impact of biofilms while we strive for new technological solutions.

Can J Microbiol, 2001 Oct, 47(10), 949 - 54
Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia; Tomlin KL et al.; The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms . Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P . aeruginosa alone . The interactions between P . aeruginosa biofilms and B . cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship . Using the Calgary Biofilm Device (CBD), members of all genomovars of the B . cepacia complex were shown to form biofilms, including those isolated from CF lungs . Mixed species biofilm formation between CF isolates of P . aeruginosa and B . cepacia was readily achieved using the CBD . Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition . Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure . Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B . cepacia complex . Characterization of the soluble inhibitor suggested pyocyanin as the active compound.

J Bacteriol, 2001 Dec, 183(24), 7213 - 23
Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene; Prigent-Combaret C et al.; The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation . Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E . coli to grow as a biofilm community rather than in a planktonic state . In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription . The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion . Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E . coli is inhibited by increasing osmolarity in the growth medium . The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface . This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS . We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR; cpxR-mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.

Microbiol Res, 2001, 156(3), 225 - 38
Dynamics of protozoan and metazoan communities in a full scale wastewater treatment plant by rotating biological contactors; Martin-Cereceda M et al.; Performance of a full-scale wastewater treatment plant by rotating biological contactors (RBC) system was monitored during a year by physico-chemical and microbial characterisation . Six points along wastewater treatment were selected in the plant: three points along the water line (influent, sedimentation tank and effluent) and three points along RBC system (RBC1, RBC2 and RBC3) . Although a large seasonal change in the values of physico-chemical parameters was observed, operation of the plant was optimal during all year (90% of removal in BOD5 and SS influent content) . Microbial characterisation was approached by determining the structure and dynamics of protozoan and metazoan communities . Protozoa were the most abundant in all stages in the plant, heterotrophic flagellates being the most representative group in the water line and ciliates in the RBC system . The same seasonal preference was only observed for heterotrophic flagellates in the water line and green flagellates in the RBC system, both groups having highest abundances in summer and spring, respectively . Identification of ciliated protozoa populations rendered 58 species of ciliates in the plant . Most of these species are typical of aerobic wastewater treatment systems except three of them, which are cited for the first time in this type of ecosystems: Chaenea stricta, Holosticha mancoidea and Oxytricha lanceolata . Along the water line 34 species were identified, and half of them only appeared occasionally (once in all the study), while along the RBC system biofilms 55 species were observed, and the majority appeared permanently in this system . Our results indicate that the type of habitat, rather than the physico-chemical water parameters, was the primary factor in determining the different distribution of protozoan and metazoan communities in the plant . In RBC biofilms, the structure of ciliate protozoa community was found to be quite sensitive to changes in physico-chemical parameters, mainly to organic loading (BOD5) variations.

Zhonghua Yi Xue Za Zhi, 1999 Oct, 79(10), 757 - 60
{Influence of roxithromycin combined with fleroxacin on bacterial biofilm induced by Pseudomonas aeruginosa}; Chen Q et al.; OBJECTIVE: To investigate the influence of roxithromycin (RXM) on the production of glycocalyx (GLX) and the synergic effect of RXM and fleroxacin (FLRX) on P . Aeruginosa biofilms . METHODS: Bacterial biofilm (BF) was established and was influenced by RXM and FLRX with different concentrations . The samples were detected with scanning electron microscope and by a rapid method after BF was stained with AgNO3 solution . The synergism of antibacterial activities of RXM and FLRX to P . Aeruginosa was studied by computer image analysis and MTT method . RESULTS: GLX production was reduced significantly by both 1/16 MIC and 1/4 MIC RXM (P < 0.01) . 1/16 MIC and 1/4 MIC RXM showed no bactericidal activities to P . Aeruginosa in biofilms, while viable counts in biofilms almost had no difference . However, RXM could enhance the bactericidal activity of FLRX to P . Aeruginosa in biofilms . When FLRX of different concentrations was combined with 1/16 MIC and 1/4 MIC RXM respectively, the difference of the stained BF was counted up by computer image analysis system with significance of P < 0.05 and the viable counts were reduced significantly (P < 0.05) . The results was in accordance with SEM pictures . CONCLUSION: RXM has no direct bactericidal activities to P . Aeruginosa in biofilms, but it could inhibit GLX production . It can be considered that RXM could enhance antibacterial activities of FLRX by enhancing the permeability of FLRX into biofilms.

Cell Mol Life Sci, 2001 Oct, 58(11), 1562 - 71
Extracellular electron transfer; Hernandez ME et al.; Results from several laboratories indicate that extracellular electron transfer may be a general mechanism whereby microoorganisms generate energy for cell growth and/or maintenance . Specifically, bacteria can use redox-active organic small molecules, generated outside or inside the cells, to shuttle electrons between reduced and oxidized compounds . Electron shuttling has now been reported for several different bacterial species, and exchanges of shuttling compounds may even syntrophically link diverse organisms in nature . Biofilm systems in both geological and clinical settings are likely to be important environments for metabolisms that employ extracellular electron transfer . Both structural and functional analyses suggest that electron shuttles and some virulence factors may be related to one another.

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