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