Biofilms

J Microbiol, 2004 Mar, 42(1), 64 - 7
Formation and dispersion of mycelial pellets of Streptomyces coelicolor A3(2); Kim YM et al.; The pellets from a culture of Streptomyces coelicolor A3(2) that were submerged shaken were disintegrated into numerous hyphal fragments by DNase treatment . The pellets were increasingly dispersed by hyaluronidase treatment, and mycelial fragments were easily detached from the pellets . The submerged mycelium grew by forming complexes with calcium phosphate precipitates or kaolin, a soil particle . Therefore, the pellet formation of Streptomyces coelicolor A3(2) can be considered a biofilm formation, including the participation of adhesive extracellular polymers and the insoluble substrates.

Lett Appl Microbiol, 2004, 39(4), 359 - 62
Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production; Ryu JH et al.; AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel . METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E . coli O157:H7 were studied . Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C . The number of cells attached to SSC was determined . To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C . Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms . CONCLUSIONS: Curli production by E . coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms . SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E . coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.

Water Res, 2004 Oct, 38(17), 3769 - 79
Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes; Lehtola MJ et al.; We studied the changes in water quality and formation of biofilms occurring in a pilot-scale water distribution system with two generally used pipe materials: copper and plastic (polyethylene, PE) . The formation of biofilms with time was analysed as the number of total bacteria, heterotrophic plate counts and the concentration of ATP in biofilms . At the end of the experiment (after 308 days), microbial community structure, viable biomass and gram-negative bacterial biomass were analysed via lipid biomarkers (phospholipid fatty acids and lipopolysaccharide 3-hydroxy fatty acids), and the numbers of virus-like particles and total bacteria were enumerated by SYBR Green I staining . The formation of biofilm was slower in copper pipes than in the PE pipes, but after 200 days there was no difference in microbial numbers between the pipe materials . Copper ion led to lower microbial numbers in water during the first 200 days, but thereafter there were no differences between the two pipe materials . The number of virus-like particles was lower in biofilms and in outlet water from the copper pipes than PE pipes . Pipe material influenced also the microbial and gram-negative bacterial community structure in biofilms and water.

Water Res, 2004 Oct, 38(17), 3671 - 84
Influence of growth history on sloughing and erosion from biofilms; Telgmann U et al.; The development of biofilms is determined by the balance of growth and detachment . But while the growth of biofilms is well studied, the influence of growth history and detachment on biofilm development is not . Here we report on laboratory scale experiments where heterotrophic biofilms were grown in a tubular reactor . Biofilm detachment was categorized based on particle size as erosion or sloughing . Erosion results in small particles and was approximated by the effluent suspended solids while sloughing was determined from the larger pieces of biomass that settled in a mixing tank . It was found that for all experiments, overall detachment was a combination of erosion and sloughing where erosion had a slightly larger contribution to the overall solids removal . However, sloughing had a significant influence on the biofilm morphology . Once the smooth biofilm surface was disturbed by a sloughing event (e.g., initiated through increasing liquid shear in the reactor), the biofilm became unstable resulting in spontaneous sloughing during subsequent operation . We propose that experimental investigations should consider sloughing events as an integral part of biofilm development rather than a disturbance.

Mol Cell, 2004 Sep 10, 15(5), 677 - 87
Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2; Miller ST et al.; Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence . This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers . While most autoinducers are species specific, autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi, is produced and detected by many Gram-negative and Gram-positive bacteria . The crystal structure of the V . harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester . Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium . We find that LsrB binds a chemically distinct form of the AI-2 signal, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), that lacks boron . Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling .

J Mater Sci Mater Med, 1998 Mar 1, 9(3), 147 - 57
In vitro and in vivo microbial adhesion and growth on argon plasma-treated silicone rubber voice prostheses; Everaert EP et al.; Patients who undergo a total laryngectomy usually receive a silicone rubber voice prosthesis for voice rehabilitation . Unfortunately, biofilm formation on the esophageal side of voice prostheses limits their lifetime to 3-4 mon on average . The effects of repeated argon plasma treatment of medical grade, hydrophobic silicone rubber on in vitro adhesion and growth of bacteria and yeasts isolated from voice prostheses, as well as in vivo biofilm formation are presented here . In vitro experiments demonstrated that initial microbial adhesion over a 4 h time span to plasma-treated, hydrophilized, silicone rubber was generally less than on original, hydrophobic silicone rubber, both in the absence and presence of a salivary conditioning film on the biomaterial . Growth studies over a time period of 14 d at 37 degrees C in a modified Robbins device, showed that fewer Candida cells adhered on plasma-treated, hydrophilized silicone rubber as compared to on original, hydrophobic silicone rubber . For the in vivo evaluation of biofilm formation on plasma-treated silicone rubber voice prostheses, seven laryngectomized patients received a partly hydrophilized "Groningen Button" voice prosthesis for a planned evaluation period of 4 wk . After removal of the voice prostheses, the border between the hydrophilized and the original, hydrophobic side of the prostheses was clearly visible . However, biofilm formation was, unexpectedly, less on the original, hydrophobic sides, although the microbial compositions of the biofilms on both sides were not significantly different . Summarizing, this study demonstrates that in vitro microbial adhesion and growth on silicone rubber can be reduced by plasma treatment, but in vivo biofilm formation on silicone rubber voice prostheses is oppositely enhanced by hydrophilizing the silicone rubber surface . Nevertheless, from the results of this study the important conclusion can be drawn that in vivo biofilm formation on voice prostheses is controlled by the hydrophobicity of the biomaterials surface used .

J Mater Sci Mater Med, 2002 Aug, 13(8), 717 - 22
Pathogenesis and prevention of biomaterial centered infections; Gottenbos B et al.; One of the major drawbacks in the use of biomedical materials is the occurrence of biomaterials centered infections . After implantation, the host interacts with a biomaterial by forming a conditioning film on its surface and an immune reaction towards the foreign material . When microorganisms can reach the biomaterials surface they can adhere to it . Adhesion of microorganisms to an implant is mediated by their physico-chemical surface properties and the properties of the biomaterials surface itself . Subsequent surface growth of the microorganisms will lead to a mature biofilm and infection, which is difficult to eradicate by antibiotics . The purpose of this review is to give an overview of the mechanisms involved in biomaterials centered infection and the possible methods to prevent these infections.

J Mater Sci Mater Med, 2003 Nov, 14(11), 967 - 72
Multi-technique characterization of retrieved bone cement from revised total hip arthroplasties; Eliades T et al.; The purpose of this study was to assess the chemical composition, structure and degree of double bond conversion of retrieved bone cement from 29 total hip replacement revision arthroplasties, employing a multi-technique approach . Scanning electron microscopy revealed a porous cement surface, which replicated the characteristics of bone or femoral stem surface irregularities . Fourier transform infrared spectroscopy indicated that the retrieved bone cement samples were covered by a well-organized proteinaceous film rich in amides and alcohols, probably because of the adsorption of species from body tissues and fluids . X-ray fluorescence spectrometry showed the presence of potassium, sodium, calcium and phosphorus, implying the development of a mineralization process of the adsorbed biofilm . X-ray microtomography demonstrated a dense porous network in the bulk material comprised of macropores with a mean diameter >1 mm . FTIR analysis of the degree of double bond conversion of retrieved samples was in the order of 70%, similar to that of samples prepared in vitro in air, but 30% lower relative to their counterparts mixed in vitro and set in water . The effect of the adsorption of species onto bone cement surface on the reactivity of the material with the surrounding tissues and materials, is currently unknown . The results of this investigation reveal that the in vivo aging pattern of bone cements may involve alterations, which cannot be simulated under current in vitro protocols, emphasizing the necessity for adopting in vivo approaches including retrieval studies in assessing bone cement properties .

Environ Technol, 2004 Jul, 25(7), 809 - 17
Development and analysis of anaerobic biofilms onto hydrophobic and hydrophilic surfaces; Araujo JC et al.; Fluorescent in situ hybridization (FISH) with domain and group specific probes that target intracellular 16S rRNA were used to investigate microbial composition of anaerobic biofilms developed on polypropylene (hydrophobic) and glass (hydrophilic) surfaces fitted inside a Modified Robbins Device (MRD) . Crushed anaerobic granular sludge was used as inoculum for biofilm development in the MRD . The inoculum and biofilms formed showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MB1174) . This group accounted for 44 to 90% of the DAPI-stained cells . Cells which hybridized to the Bacteria specific probe (EUB338) accounted for 3-18% of the DAPI-stained cells . After the first day of the biofilm formation experiment, a larger number of cells, 4.6 x 10(4) cells mm-2, could be seen colonizing the polypropylene coupon compared to the glass, 8.2 x 10(3) cells mm-2 . However, after 9 days these numbers were very similar, i.e . 6.3 x 10(5) cells mm-2 and 7.2 x 10(5) cells mm-2, for the glass and polypropylene coupons, respectively . Our data suggest that the hydrophobicity of the support material did not influence the initial development and the microbial composition of anaerobic biofilms developed in the MRD.

J Infect Dis, 2004 Oct 1, 190(7), 1245 - 53 Epub 2004 Aug 26.
Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis; Rogan MP et al.; Intractable formation of biofilm by and infection with the opportunistic pathogen Pseudomonas aeruginosa are hallmarks of cystic fibrosis (CF) . Lactoferrin, an innate immunity protein, has recently been shown to inhibit the formation of P . aeruginosa biofilm . Partial cleavage of lactoferrin by the proteases neutrophil elastase and Pseudomonas elastase has previously been described in CF . Here, we show that cathepsins in CF secretions are responsible for complete and rapid cleavage of lactoferrin . We demonstrate that levels of lactoferrin in P . aeruginosa-positive sputum samples are decreased when corrected for inflammatory burden and that P . aeruginosa-positive sputum samples have significantly higher cathepsin activity and significantly reduced ability to inhibit formation of biofilm, compared with P . aeruginosa-negative sputum samples . We also show that cleavage of lactoferrin by cathepsin results in loss of both its microbicidal and antibiofilm activity . Loss of such a vital innate immunity protein clearly has important implications for the pathogenesis of chronic P . aeruginosa lung infection in patients with CF.

Appl Environ Microbiol, 2004 Sep, 70(9), 5682 - 4
Effect of Escherichia coli morphogene bolA on biofilms; Vieira HL et al.; Biofilm physiology is established under a low growth rate . The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development . In order to verify this hypothesis, we tested the effect of bolA on biofilm formation . Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.

Appl Environ Microbiol, 2004 Sep, 70(9), 5094 - 101
Diversity and seasonal changes of uncultured Planctomycetales in river biofilms; Brummer IH et al.; Cell counts of planctomycetes showed that there were high levels of these organisms in the summer and low levels in the winter in biofilms grown in situ in two polluted rivers, the Elbe River and the Spittelwasser River . In this study 16S rRNA-based methods were used to investigate if these changes were correlated with changes in the species composition . Planctomycete-specific clone libraries of the 16S rRNA genes found in both rivers showed that there were seven clusters, which were distantly related to the genera Pirellula, Planctomyces, and Gemmata . The majority of the sequences from the Spittelwasser River were affiliated with a cluster related to Pirellula, while the majority of the clones from the Elbe River fell into three clusters related to Planctomyces and one deeply branching cluster related to Pirellula . Some clusters also contained sequences derived from freshwater environments worldwide, and the similarities to our biofilm clones were as high as 99.8%, indicating the presence of globally distributed freshwater clusters of planctomycetes that have not been cultivated yet . Community fingerprints of planctomycete 16S rRNA genes were generated by temperature gradient gel electrophoresis from Elbe River biofilm samples collected monthly for 1 year . Sixteen bands were identified, and for the most part these bands represented organisms related to the genus Planctomyces . The fingerprints showed that there was strong seasonality of most bands and that there were clear differences in the summer and the winter . Thus, seasonal changes in the abundance of Planctomycetales in river biofilms were coupled to shifts in the community composition.

Microbes Infect, 2004 Sep, 6(11), 965 - 71
Involvement of Mycobacterium smegmatis undecaprenyl phosphokinase in biofilm and smegma formation; Rose L et al.; We describe a Mycobacterium smegmatis mutant with impaired biofilm and smegma formation . A gene homologous to Escherichia coli bacA, which has been proposed to play a role as undecaprenyl phosphokinase (Upk) was unmarked in-frame deleted from M . smegmatis . Though Upk is involved in cell wall synthesis, the surface of the mutant strain appeared virtually comparable to that of the wild type by electron microscopy . The absence of Upk influenced colony morphology and bacitracin resistance . The M . smegmatis Deltaupk mutant developed a biofilm characterized by scattered islands of bacteria distinct from the completely covered biofilm surface observed for wild-type bacteria . We further demonstrate biological consequences of upk deletion for smegma development in an in vivo model . These results suggest the upk gene to be essential in biofilm and smegma development.

Environ Microbiol, 2004 Oct, 6(10), 1086 - 95
Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal Field, Mid-Atlantic Ridge; Schrenk MO et al.; The recently discovered Lost City Hydrothermal Field (LCHF) represents a new type of submarine hydrothermal system driven primarily by exothermic serpentinization reactions in ultramafic oceanic crust . Highly reducing, alkaline hydrothermal environments at the LCHF produce considerable quantities of hydrogen, methane and organic molecules through chemo- and biosynthetic reactions . Here, we report the first analyses of microbial communities inhabiting carbonate chimneys awash in warm, high pH fluids at the LCHF and the predominance of a single group of methane-metabolizing Archaea . The predominant phylotype, related to the Methanosarcinales, formed tens of micrometre-thick biofilms in regions adjacent to hydrothermal flow . Exterior portions of active structures harboured a diverse microbial community composed primarily of filamentous Eubacteria that resembled sulphide-oxidizing species . Inactive samples, away from regions of hydrothermal flow, contained phylotypes related to pelagic microorganisms . The abundance of organisms linked to the volatile chemistry at the LCHF hints that similar metabolic processes may operate in the subseafloor . These results expand the range of known geological settings that support biological activity to include submarine hydrothermal systems that are not dependent upon magmatic heat sources.

J Bacteriol, 2004 Sep, 186(18), 6327 - 31
Biofilm mode of growth of Streptococcus intermedius favored by a competence-stimulating signaling peptide; Petersen FC et al.; Gram-positive and gram-negative bacteria use quorum sensing to coordinate population behavior . In several streptococci, quorum sensing mediated by competence-stimulating peptides (CSP) is associated with development of competence for transformation . We show here that a synthetic CSP favored the biofilm mode of growth of Streptococcus intermedius without affecting the rate of culture growth.

J Bacteriol, 2004 Sep, 186(18), 6208 - 19
Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis; Conlon KM et al.; Expression of ica operon-mediated biofilm formation in Staphylococcus epidermidis RP62A is subject to phase variable regulation . Reversible transposition of IS256 into icaADBC or downregulation of icaADBC expression are two important mechanisms of biofilm phenotypic variation . Interestingly, the presence of IS256 was generally associated with a more rapid rate of phenotypic variation, suggesting that IS256 insertions outside the ica locus may affect ica transcription . Consistent with this, we identified variants with diminished ica expression, which were associated with IS256 insertions in the sigmaB activator rsbU or sarA . Biofilm development and ica expression were activated only by ethanol and not NaCl in rsbU::IS256 insertion variants, which were present in approximately 11% of all variants . sigmaB activity was impaired in rsbU::IS256 variants, as evidenced by reduced expression of the sigmaB-regulated genes asp23, csb9, and rsbV . Moreover, expression of sarA, which is sigmaB regulated, and SarA-regulated RNAIII were also suppressed . A biofilm-forming phenotype was restored to rsbU::IS256 variants only after repeated passage and was not associated with IS256 excision from rsbU . Only one sarA::IS256 insertion mutant was identified among 43 biofilm-negative variants . Both NaCl and ethanol-activated ica expression in this sarA::IS256 variant, but only ethanol increased biofilm development . Unlike rsbU::IS256 variants, reversion of the sarA::IS256 variant to a biofilm-positive phenotype was accompanied by precise excision of IS256 from sarA and restoration of normal ica expression . These data identify new roles for IS256 in ica and biofilm phenotypic variation and demonstrate the capacity of this element to influence the global regulation of transcription in S . epidermidis.

J Cataract Refract Surg, 2004 Sep, 30(9), 1972 - 6
Evaluation of biofilm formation on nylon sutures removed from clinically noninfected eyes after cataract surgery; Pan JC et al.; PURPOSE: To determine the presence of bacterial biofilm on nylon sutures removed from clinically noninfected eyes after cataract surgery . SETTING: The Eye Institute, Tan Tock Seng Hospital, Singapore, Singapore . METHODS: Sutures were removed from 10 eyes after cataract surgery at different time periods . Immediately after removal, the sutures were fixed and dehydrated . All sutures were viewed by scanning electron microscopy, and 6 were also viewed by transmission electron microscopy (TEM) . RESULTS: There was no evidence of bacterial biofilm formation on the nylon sutures . Significant cellular debris was seen, mainly at the knots . Clusters of coccoid-shaped structures were visible; however, examination by TEM showed they were not bacteria . CONCLUSION: There was no evidence of biofilm formation on sutures removed after cataract surgery from clinically noninfected eyes.

Mol Microbiol, 2004 Sep, 53(6), 1563 - 71
Quorum sensing and signal interference: diverse implications; Zhang LH et al.; Quorum sensing (QS) is a community genetic regulation mechanism that controls microbiological functions of medical, agricultural and industrial importance . Discovery of microbial QS signals and the signalling mechanisms led to identification of numerous enzymatic and non-enzymatic signal interference mechanisms that quench microbial QS signalling . Evidence is accumulating that such signal interference mechanisms can be developed as promising approaches to control microbial infection and biofilm formation . In addition, these mechanisms exist not only in microorganisms but also in the host organisms of bacterial pathogens, highlighting their potential implications in microbial ecology and in host-pathogen interactions . Investigation of QS and signal interference mechanisms might significantly broaden the scope of research in microbiology.

Sci Total Environ, 2004 Oct 1, 332(1-3), 253 - 60
Determination of biodegradable dissolved organic carbon in waters: comparison of batch methods; Trulleyova S et al.; An effect of different types of bacterial inocula upon the final biodegradable dissolved organic carbon (BDOC) result was investigated in samples of both low and high BDOC concentrations . Stream water and leaf leachate samples were incubated either with free, suspended bacteria or with bacteria attached to the stream sediment particles or attached to artificial substrata . The time course of dissolved organic carbon (DOC) decomposition was observed using absorbance analysis of DOC . BDOC determination by means of commonly used suspended bacteria as the inoculum made for an underestimation of BDOC between 5% and 25%, compared with attached bacterial community (biofilm) . The reason for these findings could be the higher microbial diversity, higher metabolic activity of attached bacteria and abiotic adsorption of organic molecules to inorganic support and biofilm matrix surfaces . Adsorbed DOC is easily hydrolyzed and utilized by biofilm bacteria.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 167 - 74
ComX activity of Streptococcus mutans growing in biofilms; Aspiras MB et al.; In many streptococci, including Streptococcus mutans, genetic competence is regulated by a quorum sensing system mediated by a competence stimulating peptide (CSP) pheromone, encoded by the comC gene . In Streptococcus pneumoniae, a central component of this system is ComX, which acts as an alternative sigma factor to activate competence genes involved in DNA uptake and processing . The quorum sensing system responsible for genetic competence induction in S . mutans has been linked to biofilm formation and the acid tolerance response . To examine the response of comX to CSP in S . mutans, a transcriptional fusion of the comX promoter (pcomX) with lacZ was constructed to generate reporter vector pcomx::pALH122 (replicative vector) and transformed into S . mutans UA159 comC-, which is unable to produce endogenous CSP . CSP was added and pcomX::lacZ relative expression index (REI) examined, revealing a 2-fold increase in maximal beta-gal activity 5 and 10 min after CSP addition . The effect of endogenous CSP on pcomX::lacZ expression was also examined by measuring REI in cells grown as a biofilm; peak pcomX activity was observed at 3 h . To determine the temporal pattern of transformation frequency, pMA2, a Spr shuttle vector, was transformed into biofilm-grown cells, with maximal transformation frequency observed at 3 h . Confocal microscopy was performed to examine pcomX activity using a similarly constructed green fluorescent protein reporter vector, pcomX::gfp, in a 4-h biofilm, revealing active pcomX activity in high cell density areas within the biofilm population . These results demonstrated a positive correlation between pcomX activity, natural transformation and competence development in biofilms.

Expert Opin Biol Ther, 2004 Sep, 4(9), 1519 - 31
Treatment and prevention of enterococcal infections--alternative and experimental approaches; Koch S et al.; Enterococci are one of the leading types of organisms isolated from infections of hospitalised patients and the third most common cause of nosocomial bloodstream infections . They contribute significantly to patient mortality and morbidity, as well as healthcare costs . The emergence of resistance against virtually all clinically available antibiotics and the ability to transfer these resistance determinants to other pathogens demonstrates the urgency for an improved understanding of enterococcal virulence mechanisms, and the development of alternative treatment and prevention options . This article reviews new antimicrobials, vaccine targets, bacteriophage therapy, as well as treatments targeting virulence factors and biofilm, for their potential to treat and/or prevent enterococcal infections . Although clinical isolates often cause serious infections, so-called 'non-pathogenic' strains are used as therapeutics in the form of probiotics . Understanding the differences between true pathogens and beneficial commensals may help to evaluate future treatment and prophylactic options.

Biotechnol Bioeng, 2004 Sep 30, 87(7), 874 - 83
Modeling hexavalent chromium removal in a Bacillus sp . fixed-film bioreactor; Nkhalambayausi-Chirwa EM et al.; A one-dimensional diffusion-reaction model was developed to simulate Cr(VI) reduction in a Bacillus sp . pure culture biofilm reactor with glucose as a sole supplied carbon and energy source . Substrate utilization and Cr(VI) reduction in the biofilm was best represented by a system of (second-order) partial differential equations (PDEs) . The PDE system was solved by the (fourth-order) Runge-Kutta method adjusted for mass transport resistance using the (second-order) Crank-Nicholson and Backward Euler finite difference methods . A heuristic procedure (genetic search algorithm) was used to find global optimum values of Cr(VI) reduction and substrate utilization rate kinetic parameters . The fixed-film bioreactor system yielded higher values of the maximum specific Cr(VI) reduction rate coefficient and Cr(VI) reduction capacity (kmc = 0.062 1/h, and Rc = 0.13 mg/mg, respectively) than previously determined in batch reactors (kmc = 0.022 1/h and Rc = 0.012 mg/mg) . The model predicted effluent Cr(VI) concentration well with 98.9% confidence (sigmay2 = 2.37 mg2/L2, N = 119) and effluent glucose with 96.4 % confidence (sigmay(w)2 = 5402 mg2/L2, N = 121, w = 100) over a wide range of Cr(VI) loadings (10-498 mg Cr(VI)/L/d) .

Biotechnol Bioeng, 2004 Sep 30, 87(7), 855 - 62
An impedance study on admiralty brass dezincification originated by microbiologically influenced corrosion; Ibars JR et al.; In this article we describe a field study of biofouling and microbiologically influenced corrosion (MIC) of admiralty brass heat exchanger tubes in contact with running fresh water on the river Tagus close to Almaraz nuclear power plant in Spain . Dezincification originated by biofouling and MIC was studied using impedance, polarization resistance, gravimetric, scanning electron microscopy (SEM), and X-ray diffraction (XRD) measurements . Close correlation was observed between the biofilms formed and the corrosion process (dezincification) using the different experimental techniques . Impedance data showed a capacitive behavior including two time constants . Kramers-Kronig (KK) transforms were used to validate impedance data . The admiralty tubes' impedance data satisfied the KK relations .

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(8), 2183 - 93
Mixing characteristics and whey wastewater treatment of a novel moving anaerobic biofilm reactor; Rodgers M et al.; A novel moving anaerobic biofilm reactor was used to treat whey wastewater . In this process, biofilm was grown on a plastic biofilm media module, which was vertically moved up and down in the bulk fluid . The objectives of the study were to investigate the mixing and performance characteristics of the new process in treating whey wastewater . The mixing efficiency was indicated by a dispersion number, D(L)/uL . D(L)/uL was up to 1.34, showing that the anaerobic reactor can be taken as a completely mixed reactor . At mesophilic conditions (35+/-2 degrees C), the admissible volumetric COD loading rate up to 11.6kg COD m(-3) day(-1) was achieved with the COD removal efficiency of 89% and the hydraulic retention time (HRT) of 1 day . When the HRT was 0.6 days, the volumetric COD loading rate was 15.2 kg COD m(-3) day(-1), but COD removal efficiency decreased to 81% . The percentage of methane (CH4) in the biogas was 63% on average and the yield of methane was 333.4 L CH4 kg(-1) COD removal at ambient conditions.

J Mater Sci Mater Med, 2004 Apr, 15(4), 403 - 6
Bacterial biosynthesis of a calcium phosphate bone-substitute material; Thackray AC et al.; A species of Serratia bacteria produces nano-crystalline hydroxyapatite (HA) crystals by use of a cell-bound phosphatase enzyme, located both periplasmically and within extracellular polymeric materials . The enzyme functions in resting cells by cleaving glycerol-2-phosphate (G-2-P) to liberate free phosphate ions which combine with calcium in solution to produce a cell-bound calcium phosphate material . Bacteria grown as a biofilm on polyurethane reticulated foam cubes were challenged with calcium and G-2-P in a bioreactor to produce a 3-D porous bone-substitute material . The scaffold has 1 mm macropores and 1 microm micropores . XRD showed the crystallites to be 25-28 nm in size, resembling HA before sintering and beta-tricalcium phosphate (beta-TCP, whitlockite) after . When biofilm was grown on titanium discs and challenged with calcium and G-2-P, a calcium phosphate layer formed on the discs . Biomineralisation is therefore a potential route to production of precursor nanophase HA, which has the potential to improve strength . The scaffold material produced by this method could be used as a bone-filler or as an alternative method for coating implants with a layer of HA.

Huan Jing Ke Xue, 2004 Jan, 25(1), 99 - 102
{Study on bio-contact oxidation A/O process of Guanting Reservior water under low temperature}; He XH et al.; The results of the experiments showed that biological contact A/O process could run steadily under low temperature and had better removal effect for ammonia nitrogen and had some removal effect for COD and TN from water of Guanting Reservoir . When the temperature dropped greatly, removal ratio of ammonia nitrogen dropped evidently, but gradually rose and remained at high removal level at last . Under the conditions of the experiment that the temperature was 0-5 degrees C and influent ammonia nitrogen was about 10 mg/L, ammonia nitrogen removal rate remained more than 95% at last . The change of temperature had no evident influence on removal effect of COD . And the conclusion could be got by microscope that under the temperature at 0-5 degrees C the microorganism in the biofilm still had a good activity.

J Chemother, 2004 Jun, 16(3), 244 - 7
Effects of antibiotics on Pseudomonas aeruginosa NK125502 and Pseudomonas fluorescens MF0 biofilm formation on immobilized fibronectin; Gagniere H et al.; The effect of subminimal inhibitory concentration (1/2 MIC) of antibiotics on the biofilm formation on immobilized fibronectin by Pseudomonas was investigated by examining the reference strains NK125502 P . aeruginosa and MF0 P . fluorescens in a microtiter plates assay . When the antibiotics were added during bacterial growth and biofilm development, gentamicin was the only antimicrobial agent tested which decreased significantly the biofilm formation by the two strains . Cefsulodin and chloramphenicol also decreased the P . aeruginosa biofilm development (P<0.01), whereas polymyxin B inhibited biofilm formation by P . fluorescens (p<0.05) . When the antibiotics were only present during bacterial growth and not during biofilm development, gentamicin was the only antibiotic tested to decrease significantly the biofilm formation by P . aeruginosa for incubation times of 20 and 72h (P<0.01), whereas P . fluorescens was not affected . This persistent inhibition of P . aeruginosa biofilm formation may be interesting in intermittent antibiotherapy treatments.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3358 - 66
Adherence of Candida albicans to silicone induces immediate enhanced tolerance to fluconazole; Mateus C et al.; Wild-type and efflux pump-deficient cells of Candida albicans adhering to silicone were compared with planktonic cells by flow cytometry for their relative resistance to fluconazole (FCZ) . Flow cytometry data on cells carrying a fusion of green fluorescent protein to efflux pump promoters confirmed that enhanced tolerance of attached cells to FCZ was due in part to increased expression of CaMDR1 and CDR1 promoters . Within 2 h of their attachment to silicone, the adherent cells demonstrated levels of FCZ tolerance shown by cells from 24-h biofilms . Following their mechanical detachment, this subset of cells retained a four- to eightfold increase in tolerance compared with the tolerance of planktonic cells for at least two generations . Enhanced efflux pump tolerance to FCZ appeared to be induced within the initial 15 min of attachment in a subset of cells that were firmly attached to the substrata.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3291 - 7
Penetration of Candida biofilms by antifungal agents; Al-Fattani MA et al.; A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing Candida . Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine . The rates of diffusion of either drug through biofilms of three strains of Candida albicans were similar . However, the rates of drug diffusion through biofilms of C . glabrata or C . krusei were faster than those through biofilms of C . parapsilosis or C . tropicalis . In all cases, after 3 to 6 h the drug concentration at the distal edge of the biofilm was very high (many times the MIC) . Nevertheless, drug penetration failed to produce complete killing of biofilm cells . These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms . The abilities of flucytosine, fluconazole, amphotericin B, and voriconazole to penetrate mixed-species biofilms containing C . albicans and Staphylococcus epidermidis (a slime-producing wild-type strain, RP62A, and a slime-negative mutant, M7) were also investigated . All four antifungal agents diffused very slowly through these mixed-species biofilms . In most cases, diffusion was slower with biofilms containing S . epidermidis RP62A, but amphotericin B penetrated biofilms containing the M7 mutant more slowly . However, the drug concentrations reaching the distal edges of the biofilms always substantially exceeded the MIC . Thus, although the presence of bacteria and bacterial matrix material undoubtedly retarded the diffusion of the antifungal agents, poor penetration does not account for the drug resistance of Candida biofilm cells, even in these mixed-species biofilms.

Oral Microbiol Immunol, 2004 Oct, 19(5), 322 - 6
Biofilm growth of Lactobacillus species is promoted by Actinomyces species and Streptococcus mutans; Filoche SK et al.; The ability of oral bacteria to integrate within a biofilm is pivotal to their survival . A dependence on the amount of biofilm growth by noncoaggregating Lactobacillus rhamnosus and Lactobacillus plantarum on coculture with Actinomyces naeslundii, Actinomyces gerencseriae, Streptococcus mutans and Veillonella parvula was investigated using an artificial-mouth culture system . Biofilm formation by the lactobacilli in mono-culture was poor . In coculture with Actinomyces species the amount of L . rhamnosus increased 7-20 times and L . plantarum 4-7 times compared to its mono-culture biofilm . S . mutans also promoted substantial biofilm growth of lactobacilli but V . parvula had no effect . We conclude that these Actinomyces species promoted growth of key Lactobacillus species in a biofilm, as did S . mutans to a smaller extent, and that the ability of individual bacteria to form mono-culture biofilms is not necessarily an indicator of their survival and pathogenic potential in a complex multispecies biofilm community.

Oral Microbiol Immunol, 2004 Oct, 19(5), 297 - 302
A simple approach to examine early oral microbial biofilm formation and the effects of treatments; Sreenivasan PK et al.; BACKGROUND/AIMS: A simple in vivo approach to examine early dental plaque formation in the human mouth and to determine the effects of common dietary and oral hygiene procedures on biofilm formation is reported . METHODS: A custom designed device that fits securely behind the teeth of the mandibular arch provides a surface for microbial colonization . This device is prepared with denture acrylic and can be repeatedly used by the subject, exposing a large and constant surface area for microbial accumulation . RESULTS: Large numbers of oral bacteria colonized the device by 2 h; these increased significantly by 4 h (P < 0.05) . Bacterial colonization increased significantly after rinsing with a sucrose solution (P < 0.05) but remained unaffected after rinsing with water, a commercially available fluoride mouthrinse without antimicrobial agents, or brushing with a fluoride dentifrice (P > 0.05) . Rinsing with mouthrinses formulated with chlorhexidine, cetylpyridinium chloride or triclosan/copolymer significantly inhibited colonization (P < 0.05) . A dose-dependent inhibition was noted with chlorhexidine rinses (P < 0.05) . Brushing with a triclosan/copolymer dentifrice significantly inhibited microbial colonization compared with a control (P < 0.05) . CONCLUSION: This simple approach was useful for examining the effects of common dietary and oral hygiene procedures . Significant biofilm inhibitory effects were noted with formulations that demonstrated efficacy in previous clinical studies.

Huan Jing Ke Xue, 2004 May, 25(3), 98 - 101
{The degradation characteristics of degrading bacterium of 2,6-di-tert-butylphenol}; Fang ZW et al.; A degrading bacterial strain F-3-4 for 2,6-Di-tert-butylphenol (2,6-DTBP) was isolated from biofilm in acrylic fiber wastewater treatment structures . By acclimation, its capacity for degradation of 2,6-DTBP was enhanced by 26% . It was identified as Alcaligenes sp . according to morphological, physiological and biochemical characters . By tests in shaking flasks, the effects of the conditions of growth was studied, and it was determined that the optimum conditions of growth for the strain was 37 degrees C, pH 7.0 and inoculum amount 0.1% . Under these conditions, the kinetics of degradation for 2,6-DTBP of initial concentration 100 mg/L was studied, and the result indicated that the removal rate of 2,6-DTBP within 11 days was 62.4%, and the degradation process followed Eckenfelder kinetics . The half life of 2,6-DTBP was 9.38 days . The effect of initial concentration on degradation ability of the strain also was investigated . The results showed that the optimum initial concentration was 200 mg/L . When the initial concentrations were below it, the growth of strain and removal of 2,6-DTBP increased with the increase of initial concentration, while when above the value, they were inhibited.

Arch Med Res, 2004 Jul-Aug, 35(4), 275 - 8
Neuraminidase produces dose-dependent decrease of slime production and adherence of slime-forming, coagulase-negative staphylococci; Sakarya S et al.; BACKGROUND: Slime is one of the important structures of certain bacterial strains involved in nonspecific adherence . This study was conducted to determine the role of neuraminidase on slime formation and adherence of slime-forming coagulase-negative staphylococci to inert surface . METHODS: Quantitative biofilm and qualitative bacterial adherence assays were performed with increasing concentrations of neuraminidase extracted from Clostridium perfringens-treated bacteria in polystyrene plates and polypropylene tubes . RESULTS: Slime production of slime-forming, coagulase-negative staphylococci was significantly decreased dose dependently at > or =100 mU/mL (p <0.001) . Bacterial adherence to smooth surface was impeded at > or =100 mU/mL of neuraminidase treatment and adherence results were comparable with slime production assay results . CONCLUSIONS: Sialic acid may be a constituent molecule of slime and involved in bacterial adherence to inert surface . These results represent new insight into the mechanism of slime production and adherence of slime-forming, coagulase-negative staphylococci to inert surface.

Int J Antimicrob Agents, 2004 Sep, 24(3), 234 - 40
Use of an oxonol dye in combination with confocal laser scanning microscopy to monitor damage to Staphylococcus aureus cells during colonisation of silver-coated vascular grafts; Strathmann M et al.; The antimicrobial silver-coating of medical prostheses is regarded as a means to reduce the risk of bacterial colonisation after implantation . The effect of a silver-coating of vascular grafts on biofilm formation was assessed in batch cultures of Staphylococcus aureus, using confocal laser scanning microscopy . Total cells in biofilms were analysed by staining with the DNA-binding fluorochrome SYTO 62 and the proportion of damaged cells was quantified with the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol . Both the extent of biofilm formation and the proportion of viable biofilm cells were significantly diminished on the surface of the silver-coated vascular grafts compared with uncoated controls, probably due to the antimicrobial activity of silver ions released from the silver-coated graft surface.

Water Res, 2004 Sep, 38(16), 3614 - 26
Interactions between laponite and microbial biofilms in porous media: implications for colloid transport and biofilm stability; Leon-Morales CF et al.; Quartz sand columns and sand-filled microscope flow cells were used to investigate the transport characteristics of the clay colloid laponite, and a biofilm-forming bacterium, Pseudomonas aeruginosa SG81 . Separate experiments were performed with each particle to determine their individual transport characteristics in clean sand columns . In a second set of experiments, bacterial biofilms were formed prior to introduction of the clay colloids . In the independent transport experiments, bacteria and laponite each conformed to known physicochemical principles . A sodium chloride concentration of 7 x 10(-2) M caused complete retention of the laponite within the sand columns . P . aeruginosa SG81 was generally less influenced by ionic strength effects; it showed relatively low mobility at all ionic strengths tested and some (albeit reduced) mobility when introduced to the columns in 1M NaCl, the highest concentration tested, but nevertheless showed reproducible trends . Under conditions favourable to laponite retention and biofilm stability (7 x 10(-2) MNaCl), laponite suspensions were able to remobilise a portion of the attached bacterial biomass . At low ionic strength, the profile of laponite elution was also altered in the presence of a P . aeruginosa biofilm . These observations suggest that while a reduction in ionic strength has a dominant influence on the mobilisation of biological and inorganic colloids, the presence of laponite and biomass can have a distinct influence on the mobility of both types of colloids . Since these events are likely to occur in subsurface environments, our results suggest that colloid-biofilm interactions will have implications for colloid-bound contaminant transport and the remobilisation of pathogens.

Langmuir, 2004 Aug 31, 20(18), 7753 - 9
Evaluating protein attraction and adhesion to biomaterials with the atomic force microscope; Wang MS et al.; Failure of implanted biomaterials is commonly due to nonspecific protein adsorption, which in turn causes adverse reactions such as the formation of fibrous capsules, blood clots, or bacterial biofilm infections . Current research efforts have focused on modifying the biomaterial interface to control protein reactions . Designing biomaterial interfaces at the molecular level, however, requires an experimental technique that provides detailed, dynamic information on the forces involved in protein adhesion . The goal of this study was to develop an atomic force microscope (AFM)-based technique to evaluate protein adhesion on biomaterial surfaces . In this study, the AFM was used to evaluate (i) protein-protein, (ii) protein-substrate, and (iii) protein-dextran interactions . The AFM was first used to measure the pull-off forces between bovine serum albumin (BSA) tips/BSA surfaces and BSA tips/anti-BSA surfaces . Results from these protein-protein studies were consistent with the literature . More importantly, the successful measurement of antibody-antigen binding interactions demonstrates that both the BSA and anti-BSA proteins retain their folded conformation and remain functional following our immobilization protocol . The AFM was also used to quantify the physiochemical interactions of proteins during adhesion to various self-assembled monolayers (SAMs) and dextran-coated substrates representative of potential biomaterial interface modifications . Dextran, which renders surfaces very hydrophilic, was the only surface coating that BSA protein did not adhere to . Hydrophobic interactions were not found to play a significant role in BSA adhesion . Therefore, the dextran molecules may resist protein adhesion by repulsive steric effects or hydration pressure . Moreover, the AFM-based methodology provides dynamic, quantitative information about protein adhesion at the nanoscale level.

Braz Dent J, 2004, 15(1), 41 - 5 Epub 2004 Aug 16.
Control of gingival inflammation in a teenager population using ultrasonic prophylaxis; Novaes Junior AB et al.; Gingival inflammation is clinically characterized by gingival redness, swelling and increased tendency of bleeding of the soft tissue . Bacterial biofilm is the etiological agent . If, at this stage, the bacterial biofilm is removed and appropriate control methods are applied, remission of gingival inflammation occurs . This study evaluated the effectiveness of a single session of ultrasonic prophylaxis for the reduction of gingivitis in an adolescent population using the Plaque Index (PI) and Gingival Index (GI) . The study sample consisted of 15 male adolescent students selected at a dentist's office of a public high school . Prior to treatment (baseline), plaque index (PI) and bleeding on probing (BOP) were recorded . The patients then received oral hygiene instructions and ultrasonic prophylaxis . Follow-up exams were made 15 and 30 days after the ultrasonic prophylaxis, again recording PI and BOP . The data were analyzed by the Student's t-test for dependent samples . Correlation analysis between presence of biofilm and bleeding on probing was also made using the Pearson correlation test . There was a statistically significant decrease in the plaque index and bleeding on probing between baseline and examinations at both 15 days and 30 days (p<0.05) . However, the difference between the means at 15 and 30 days was statistically similar . The correlation analysis showed correlation between both parameters (p<0.05) . The results indicate that a single session of ultrasonic prophylaxis associated to oral hygiene instructions is efficient to reverse gingivitis in adolescents.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 341 - 53
Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro; de Souza AA et al.; A biofilm is a community of microorganisms attached to a solid surface . Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses . Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen . It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity . In this study we utilized analysis of microarrays to specifically identify genes expressed in X . fastidiosa cells growing in a biofilm, when compared to planktonic cells . About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes . However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions . We also found a large number of genes from the pXF51 plasmid being differentially expressed . Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces . Other genes possibly confer advantages to the bacterium in the environment that it colonizes . This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X . fastidiosa is quite similar to other characterized systems.

J Bacteriol, 2004 Sep, 186(17), 5692 - 8
The Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica; Irie Y et al.; Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg+ phase, a nonvirulent Bvg- phase, and an intermediate Bvgi phase . Genes expressed in the Bvg+ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY) . Previous studies showed that in the Bvgi phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated . In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvgi phase . We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype . However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA . Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvgi phase in B . bronchiseptica.

ORL J Otorhinolaryngol Relat Spec, 2004, 66(3), 155 - 8
Evidence of bacterial biofilms in human chronic sinusitis; Cryer J et al.; The purpose of this study was to evaluate whether bacterial biofilms exist on the sinus mucosa surfaces of human subjects with recalcitrant chronic sinusitis . Scanning electron microscopy was used to evaluate patients with continued symptoms of chronic sinusitis despite prior appropriate medical and surgical management . Morphologic structures that confirm the presence of bacterial biofilms were identified on the sinus mucosa of patients infected with Pseudomonas aeruginosa, a known biofilm former . The presence of bacterial biofilms may explain the recalcitrant nature of some forms of chronic sinusitis .

Caries Res, 2004 Sep-Oct, 38(5), 448 - 53
A method for mapping the distribution pattern of cariogenic streptococci within dental plaque in vivo; Kato K et al.; This study was carried out to develop a method for mapping the distribution of cariogenic oral streptococci, Streptococcus mutans and Streptococcus sobrinus, from the outermost to the innermost plaque . Ten consenting subjects were asked to form plaque by abstaining from tooth brushing over 3 days within in situ plaque-generating devices, which were placed on the upper molars . The plaque formed in the devices was separated into 8-10 layered fractions (100 microm thick) . Genomic DNA was extracted from each plaque fraction by a commercial DNA purification kit and used for the amplification of the 16S ribosomal RNA gene sequences by polymerase chain reaction (PCR) with universal primers . The products were then amplified by PCR with S . mutans- or S . sobrinus-specific nested primers . The final products were separated on agarose gels, stained and photographed to confirm the existence of S . mutans and S . sobrinus . The results showed that S . mutans was detected in the plaque obtained from all of the 10 subjects and S . sobrinus in the plaque of 7 subjects . However, the distribution patterns of fractions positive for S . mutans and S . sobrinus varied among the subjects, with a tendency for frequent detection of both species in the outer to middle layers of dental plaque . There were no plaque fractions in which only S . sobrinus was found . This method could be useful to map the distribution of cariogenic microorganisms and to estimate the bacterial ecology for oral biofilm.

Caries Res, 2004 Sep-Oct, 38(5), 442 - 7
Individual vitality pattern of in situ dental biofilms at different locations in the oral cavity; Arweiler NB et al.; The aim of the study was to examine the three-dimensional vitality structure of dental biofilms grown simultaneously at different locations in the oral cavity over a 48-hour period . Eight healthy volunteers wore special acrylic appliances . On each buccal side of the upper and the lower jaw three glass slabs were inserted, allowing for growth of a biofilm mimicking approximal plaque . After 48 h, the specimens were removed and biofilms were stained using two fluorescent dyes which selectively stain vital bacteria green and dead bacteria red . Under the confocal laser scanning microscope optical sections of 1 microm throughout the biofilm were made . To assess the vitality values (proportion of vital bacteria) of the whole biofilm as well as the vitality distribution in the different plaque sections an image analysis program was used . Plaque from the different locations revealed mean vitality values between 64.4 and 75.7% in the upper jaw and between 64.3 and 76.8% in the lower jaw, which were not statistically different . However, a great variation of the vitality values for the different layers and among the 8 subjects was found . Nevertheless, the analysis of the data of each single volunteer revealed a very similar vitality pattern in all twelve locations.

J Med Microbiol, 2004 Sep, 53(Pt 9), 903 - 10
Effect of subinhibitory concentration of piperacillin/tazobactam on Pseudomonas aeruginosa; Fonseca AP et al.; Subinhibitory concentrations (sub-MICs) of antibiotics, although not able to kill bacteria, can modify their physico-chemical characteristics and the architecture of their outermost surface and may interfere with some bacterial functions . This study investigated the ability of sub-MIC piperacillin/tazobactam (P/T) to interfere with the bacterial virulence parameters of adhesiveness, cell-surface hydrophobicity, motility, biofilm formation and sensitivity to oxidative stress . Antimicrobial activity against five Pseudomonas aeruginosa clinical isolates, representative of clonal lineages of 96 strains of nosocomial origin, and six control strains (ATCC 27853, PAO1, AK1, MT1562, PT623, PAO1algC) was evaluated in vitro using the NCCLS microdilution method . The effects of sub-MIC on bacterial adhesion and biofilm formation were studied using a modified microtitre plate assay . The relative cell-surface hydrophobicity of P . aeruginosa strains was determined by measuring their ability to adhere to n-hexadecane . P . aeruginosa that had been exposed overnight to P/T and incubated with P/T in the plate were also screened for their ability to swim using flagella and to twitch and for their sensitivity to oxidative stress . The results obtained showed that the impact of sub-MIC P/T on bacterial characteristics was different for the various strains of P . aeruginosa . There was a change in bacterial morphology and hydrophobicity that could explain a significant decrease in adhesion values in all clinical isolates and controls tested, a decrease in biofilm formation, a significant increase in sensitivity to oxidative stress, a significant decrease in flagellum-mediated swimming and a decrease in type IV fimbriae-mediated twitching . The results obtained indicate that sub-MIC P/T interferes with the pathogenic potential of P . aeruginosa.

J Med Microbiol, 2004 Sep, 53(Pt 9), 841 - 53
Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa; Schaber JA et al.; Pseudomonas aeruginosa produces multiple virulence factors and causes different types of infections . Previous clinical studies identified P . aeruginosa isolates that lack individual virulence factors . However, the impact of losing several virulence factors simultaneously on the in vivo virulence of P . aeruginosa is not completely understood . The P . aeruginosa cell-to-cell communication system, or quorum sensing (QS), controls the production of several virulence factors . Animal studies using constructed QS mutants indicated that loss of the QS system severely impacts the virulence of P . aeruginosa . In this study, we tried to determine if deficiency within the QS system compromises the ability of P . aeruginosa to establish infections in humans . We have identified five QS-deficient strains through screening 200 isolates from patients with urinary tract, lower respiratory tract and wound infections . These strains lacked LasB and LasA activities and produced either no or very low levels of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone . PCR analysis revealed that three isolates contained all four QS genes (lasI, lasR, rhlI and rhlR) while two isolates lacked both the lasR and rhlR genes . We also examined the five isolates for other virulence factors . The isolates produced variable levels of exotoxin A and, with one exception, were deficient in pyocyanin production . One isolate produced the type III secretion system (TTSS) effector proteins ExoS and ExoT, two isolates produced ExoT only and two isolates produced no TTSS proteins . The isolates produced weak to moderate biofilms on abiotic surfaces . Analysis of the patients' data revealed that two of the isolates represented a single strain that was isolated twice from the same patient within a 1 month interval . One QS-deficient clinical isolate (CI-1) lacked all tested virulence factors and produced a weak biofilm . These results suggest that naturally occurring QS-deficient strains of P . aeruginosa do occur and are capable of causing infections; and, that besides the known virulence factors, additional factors may contribute to the ability of certain strains such as CI-1 to establish an infection.

J Mol Biol, 2004 Sep 3, 342(1), 195 - 205
Structural analysis of Escherichia coli OpgG, a protein required for the biosynthesis of osmoregulated periplasmic glucans; Hanoulle X et al.; Osmoregulated periplasmic glucans (OPGs) G protein (OpgG) is required for OPGs biosynthesis . OPGs from Escherichia coli are branched glucans, with a backbone of beta-1,2 glucose units and with branches attached by beta-1,6 linkages . In Proteobacteria, OPGs are involved in osmoprotection, biofilm formation, virulence and resistance to antibiotics . Despite their important biological implications, enzymes synthesizing OPGs are poorly characterized . Here, we report the 2.5 A crystal structure of OpgG from E.coli . The structure was solved using a selenemethionine derivative of OpgG and the multiple anomalous diffraction method (MAD) . The protein is composed of two beta-sandwich domains connected by one turn of 3(10) helix . The N-terminal domain (residues 22-388) displays a 25-stranded beta-sandwich fold found in several carbohydrate-related proteins . It exhibits a large cleft comprising many aromatic and acidic residues . This putative binding site shares some similarities with enzymes such as galactose mutarotase and glucodextranase, suggesting a potential catalytic role for this domain in OPG synthesis . On the other hand, the C-terminal domain (residues 401-512) has a seven-stranded immunoglobulin-like beta-sandwich fold, found in many proteins where it is mainly implicated in interactions with other molecules . The structural data suggest that OpgG is an OPG branching enzyme in which the catalytic activity is located in the large N-terminal domain and controlled via the smaller C-terminal domain.

Res Microbiol, 2004 Sep, 155(7), 514 - 21
Anaerobic growth does not support biofilm formation in Escherichia coli K-12; Colon-Gonzalez M et al.; Association with a surface in a structure known as biofilm is the prevailing microbial lifestyle . Here we show the kinetics of biofilm formation of Escherichia coli W3110 in static cultures growing under aerobic or anaerobic conditions . Aerobically growing cells in LB medium started to produce detectable amounts of biofilm after 4 to 8 h, displaying maximal accumulation of formed biofilm at 24 h, corresponding to the onset of stationary phase . Then an abrupt reduction in the biomass of the biofilm was observed . This decrease was not prevented by external addition of fresh nutrients and coincided with the depletion of oxygen as measured by the enzymatic activity of the AdhE protein . No biofilm formation was detected in cultures grown anaerobically in LB or LB supplemented with nitrate, nitrite, DMSO or fumarate, even after 72 h of incubation, well inside the stationary phase, suggesting that under anaerobic growth conditions E . coli cannot form biofilms.

J Clin Periodontol, 2004 Sep, 31(9), 749 - 57
The long-term effect of a plaque control program on tooth mortality, caries and periodontal disease in adults . Results after 30 years of maintenance; Axelsson P et al.; BACKGROUND: The biofilm that forms and remains on tooth surfaces is the main etiological factor in caries and periodontal disease . Prevention of caries and periodontal disease must be based on means that counteract this bacterial plaque . OBJECTIVE: To monitor the incidence of tooth loss, caries and attachment loss during a 30-year period in a group of adults who maintained a carefully managed plaque control program . In addition, a comparison was made regarding the oral health status of individuals who, in 1972 and 2002, were 51-65 years old . MATERIAL AND METHODS: In 1971 and 1972, more than 550 subjects were recruited . Three hundred and seventy-five subjects formed a test group and 180 a control group . After 6 years of monitoring, the control group was discontinued but the participants in the test group was maintained in the preventive program and was finally re-examined after 30 years . The following variables were studied at Baseline and after 3, 6, 15 and 30 years: plaque, caries, probing pocket depth, probing attachment level and CPITN . Each patient was given a detailed case presentation and education in self-diagnosis . Once every 2 months during the first 2 years, once every 3-12 months during years 3-30, the participants received, on an individual need basis, additional education in self-diagnosis and self-care focused on proper plaque control measures, including the use of toothbrushes and interdental cleaning devices (brush, dental tape, toothpick) . The prophylactic sessions that were handled by a dental hygienist also included (i) plaque disclosure and (ii) professional mechanical tooth cleaning including the use of a fluoride-containing dentifrice/paste . RESULTS: Few teeth were lost during the 30 years of maintenance; 0.4-1.8 in different age cohorts . The main reason for tooth loss was root fracture; only 21 teeth were lost because of progressive periodontitis or caries . The mean number of new caries lesions was 1.2, 1.7 and 2.1 in the three groups . About 80% of the lesions were classified as recurrent caries . Most sites, buccal sites being the exception, exhibited no sign of attachment loss . Further, on approximal surfaces there was some gain of attachment between 1972 and 2002 in all age groups . CONCLUSION: The present study reported on the 30-year outcome of preventive dental treatment in a group of carefully monitored subjects who on a regular basis were encouraged, but also enjoyed and recognized the benefit of, maintaining a high standard of oral hygiene . The incidence of caries and periodontal disease as well as tooth mortality in this subject sample was very small . Since all preventive and treatment efforts during the 30 years were delivered in one private dental office, caution must be exercised when comparisons are made with longitudinal studies that present oral disease data from randomly selected subject samples.

Arch Oral Biol, 2004 Oct, 49(10), 789 - 98
Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars; Jin Y et al.; The pathogenesis of both superficial and systemic candidiasis is closely dictated by properties of the yeast biofilms . Despite extensive investigations on bacterial biofilms, the characteristics of candidal biofilms, and various factors affecting this process remain to be determined . Therefore we examined the effect of human whole saliva and dietary sugars, glucose and galactose on the adhesion and biofilm formation of Candida albicans . Biofilms of C . albicans isolate 192 887 g were developed on polystyrene, flat-bottomed 96-well microtiter plates and monitored using ATP bioluminescence and tetrazolium (XTT) reduction assays as well as the conventional colony forming unit (CFU) evaluation . Our data showed that both the ATP and the XTT assays strongly correlated with the CFU assay (ATP versus CFU: r = 0.994, P = 0.006; XTT versus CFU: r = 0.985, P = 0.015) . Compared with a glucose-supplemented (100 mM) medium, galactose containing (500 mM) medium generated consistently lower levels of both candidal adhesion and biofilm formation (all P < 0.05), but a higher pace of biofilm development over time (96 h) . Whist the presence of an immobilised saliva coating had little effect on either the candidal adhesion or biofilm formation, the addition of saliva to the incubation medium quantitatively affected biofilm formation especially on day 3 and 4, without any significant effect on yeast adhesion . To conclude, biofilm formation of C . albicans within the oral milieu appears to be modulated to varying extents by dietary and salivary factors and, further investigations are required to elucidate these complex interactions.

Mol Microbiol, 2004 Aug, 53(4), 1075 - 87
Pseudomonas aeruginosa attachment and biofilm development in dynamic environments; Ramsey MM et al.; Biofilm formation by Pseudomonas aeruginosa is hypothesized to follow a developmental pattern initiated by attachment to a surface followed by microcolony formation and mature biofilm development . Swimming and twitching motility are important for attachment and biofilm development in P . aeruginosa . However, it is clear that many P . aeruginosa strains lacking swimming motility exist as biofilms in the lungs of cystic fibrosis patients . Consequently, we have developed a dynamic attachment assay to identify motility-independent attachment-defective mutants . Using transposon mutagenesis, we identified 14 novel dynamic attachment-deficient (dad) mutants including four mutants specific to dynamic assay conditions (dad specific) . Two of the dad-specific mutants contain insertions in genes involved in sensing and responding to external stimuli, implying a significant impact of external factors on the biofilm developmental pathway . Observations of initial attachment and long-term biofilm formation characterized our dad mutants into two distinct classes: biofilm delayed and biofilm impaired . Biofilm-delayed mutants form wild-type biofilms but are delayed at least 24 h compared with the wild type, whereas biofilm-impaired mutants never form wild-type biofilms in our assays . We propose a dynamic model for attachment and biofilm formation in P . aeruginosa including these two classes.

Mol Microbiol, 2004 Aug, 53(4), 1035 - 49
MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis; Ito M et al.; The stator-force generator that drives Na+-dependent motility in alkaliphilic Bacillus pseudofirmus OF4 is identified here as MotPS, MotAB-like proteins with genes that are downstream of the ccpA gene, which encodes a major regulator of carbon metabolism . B . pseudofirmus OF4 was only motile at pH values above 8 . Disruption of motPS resulted in a non-motile phenotype, and motility was restored by transformation with a multicopy plasmid containing the motPS genes . Purified and reconstituted MotPS from B . pseudofirmus OF4 catalysed amiloride analogue-sensitive Na+ translocation . In contrast to B . pseudofirmus, Bacillus subtilis contains both MotAB and MotPS systems . The role of the motPS genes from B . subtilis in several motility-based behaviours was tested in isogenic strains with intact motAB and motPS loci, only one of the two mot systems or neither mot system . B . subtilis MotPS (BsMotPS) supported Na+-stimulated motility, chemotaxis on soft agar surfaces and biofilm formation, especially after selection of an up-motile variant . BsMotPS also supported motility in agar soft plugs immersed in liquid; motility was completely inhibited by an amiloride analogue . BsMotPS did not support surfactin-dependent swarming on higher concentration agar surfaces . These results indicate that BsMotPS contributes to biofilm formation and motility on soft agar, but not to swarming, in laboratory strains of B . subtilis in which MotAB is the dominant stator-force generator . BsMotPS could potentially be dominant for motility in B . subtilis variants that arise in particular niches.

Water Sci Technol, 2004, 49(11-12), 371 - 7
Growth, structure and oxygen penetration in particle supported autotrophic biofilms; Boessmann M et al.; Particle supported autotrophic biofilms were cultivated in external-loop airlift reactors at two different pumice concentrations . Oxygen microelectrodes were used to investigate substrate transport and conversion . A special flow cell was designed for the measurement of oxygen concentration profiles in the particle supported biofilms under defined hydrodynamic conditions . The oxygen concentration profiles inside the biofilms were found to be steeper at high flow velocities in the bulk phase of the flow cell compared to those at low flow velocities . Furthermore, the oxygen flux increased and the thickness of the concentration boundary layer decreased with increasing flow velocity . This dependence was found to be more pronounced in less dense biofilms out of airlift reactors with lower pumice concentrations . In addition confocal laser scanning microscopy (CLSM) was used to visualize the biofilm structure . The volume fractions of bacteria and extracellular polymeric substances (lectin-specific EPS-glycoconjugates) were measured in living fully hydrated biofilms . Both the microelectrode and CLSM measurement showed the influence of shear stress on particle supported biofilms . A higher particle concentration led to dense biofilms with a homogeneous surface, lower thickness of the concentration boundary layer and steeper oxygen concentration profiles . The combination of both techniques allows a detailed and quantitative characterisation of particle associated biofilm structure and function.

Water Sci Technol, 2004, 49(11-12), 359 - 64
Reproducibility of biofilm processes and the meaning of steady state in biofilm reactors; Lewandowski Z et al.; The need for reproducing biofilm processes is undisputable - the quality of biofilm research depends on this reproducibility . However, as many biofilm researchers know, long-term biofilm processes are notoriously difficult to reproduce . To avoid problems related to biofilm reproducibility two strategies are used: (1) to study very young biofilms that have accumulated for a few hours to a few days only, and (2) to run biofilm experiments only once . The first approach trades reproducibility for relevance because natural biofilms are usually older, often much older than a few days . This approach can be applied to answer questions relevant to initial events of biofilm formation but not questions relevant to long-term biofilm accumulation . The second approach conceals the problem of biofilm reproducibility . To assure reproducibility of biofilm processes, we methodically followed a procedure for growing biofilms in terms of microbial makeup, media composition, temperature, surface preparation, etc . Despite all this effort the reproducibility of our results for long term growth is unimpressive . Consequently, the question had to be asked: Are biofilm processes reproducible? The experiments described in this paper address this question . Biofilms grown in two identical and identically operated biofilm reactors had comparable structure only until the first sloughing event . After that, biofilms had different patterns of accumulation.

Water Sci Technol, 2004, 49(11-12), 353 - 8
Microsensor measurement of oxygen concentration in biofilms: from one dimension to three dimensions; Yu T et al.; In this study, we measured oxygen concentration in biofilms in one dimension in field conditions and in three dimensions in laboratory conditions by using a robust oxygen microsensor in combination with an automation and data acquisition system . The biofilms were on the discs of rotating biological contactors treating domestic wastewater . The results of this study provide experimental evidence on oxygen distribution in wastewater biofilms and on biofilm structure . (1) The three dimensional measurements of oxygen concentration in biofilms revealed "pockets" of oxygen in deep sections of biofilms . In these isolated "pockets," located 600-760 microm from the biofilm surface, dissolved oxygen concentration was as high as 1 mg/L . This depth of oxygen diffusion is deeper than what was determined based on one dimensional measurements . (2) The heterogeneity of oxygen distribution was related to the surface structure of biofilms . The structure of the biofilm surface affected the diffusion boundary layer over the surface and, in turn, the oxygen diffusion and distribution inside biofilms . (3) Oxygen concentration in biofilms changed generally from a high degree of heterogeneity near the biofilm surface to a low degree of heterogeneity in deep sections of biofilms, indicating a cell-clusters-like structure near the surface and a more compact base layer close to the substratum.

Water Sci Technol, 2004, 49(11-12), 345 - 51
Behaviour of biofilm systems under varying hydrodynamic conditions; Leon Ohl A et al.; Heterotrophic biofilms were cultivated in long-term experiments in biofilm tube reactors . During the biofilm cultivation the substrate loading of glucose was kept constant while the hydrodynamic conditions were changed stepwise . To describe the behaviour of the biofilm structure under these varying flow conditions the mass transfer and transport at the bulk/biofilm interface and inside the biofilm was investigated with oxygen microelectrodes . Furthermore, the biofilm density was used to describe the biofilm compactness before and after the change of the hydrodynamic condition . The obtained results show that the biofilm density and also the substrate flux decreased with decreasing flow velocity in the bulk phase . Additionally the slope of the oxygen concentration profiles decreased and the thickness of the concentration boundary layer increased . On the other hand, increasing the flow velocity in the bulk phase led both to a higher biofilm density and a higher maximum substrate flux . The biofilm surface became more homogenous and the thickness of the concentration boundary layer decreased . The time for adaptation of the biofilm structure after changing the hydrodynamic conditions ranged between 1 and 3 weeks.

Water Sci Technol, 2004, 49(11-12), 337 - 44
Bioaugmentation of a sequencing batch biofilm reactor by horizontal gene transfer; Bathe S et al.; Bioaugmentation by introduction of catabolic genes residing on mobile genetic elements into the microbial community of a soil or wastewater environment might be an alternative to bioaugmentation by addition of bacterial cells with chromosomally encoded catabolic genes . This study investigates the possibility to enhance degradation of the xenobiotic model compound 2,4-dichlorophenoxyacetic acid in a sequencing batch biofilm reactor (SBBR) by using the conjugative plasmid pJP4 carrying genes for 2,4-D degradation . After introduction of a plasmid donor strain to a lab-scale SBBR operated without 2,4-D, the number of plasmid-carrying cells first dropped, and then increased after switching to 2,4-D as the sole carbon source . The donor cells were unable to grow in the applied synthetic wastewater with 2,4-D as the sole carbon source . Transconjugants could be detected both by culture-dependent and culture-independent methods in the 2,4-D degrading biofilm . In contrast to 90% 2,4-D degradation in the bioaugmented reactor within 40 h, a control reactor which had not received the plasmid still contained 60% of the initial 2,4-D concentration after 90 h . This experiment clearly demonstrates the introduction of 2,4-D degradative genes into a microbial biofilm and indicates that horizontal gene transfer is a promising tool for bioaugmentation of reactors treating wastewater.

Water Sci Technol, 2004, 49(11-12), 319 - 25
Full scale fluidized bed anaerobic reactor for domestic wastewater treatment: performance, sludge production and biofilm; Mendonca NM et al.; This paper describes the performance, sludge production and biofilm characteristics of a full scale fluidized bed anaerobic reactor (32 m3) for domestic wastewater treatment . The reactor was operated with 10.5 m x h(-1) upflow velocity, 3.2 h hydraulic retention time, and recirculation ratio of 0.85 and it presented removal efficiencies of 71+/-8% of COD and 77+/-14% of TSS . During the apparent steady-state period, specific sludge production and sludge age in the reactor were (0.116+/-0.033) kgVSS . kgCOD(-1) and (12+/-5)d, respectively . Biofilm formed in the reactor presented two different patterns: one of them at the beginning of the colonization and the other of mature biofilm . These different colonization patterns are due to bed stratification in the reactor, caused by the difference in local-energy dissipation rates along the reactor's height, and density, shape, etc . of the bioparticles . The biofilm population is formed mainly of syntrophic consortia among sulfate reducing bacteria, methanogenic archaea such as Methanobacterium and Methanosaeta-like cells.

Water Sci Technol, 2004, 49(11-12), 287 - 94
Treatment of leachate from the anaerobic fermentation of solid wastes using two biofilm support media; Comett I et al.; Biofilms growing on different carrier media have a different response to the nutrients contained in wastewater . Biofilms have proven to be an alternative to the treatment of wastewater containing higher concentrations of contaminants . The main objective of this research was to compare two biofilm support media for the treatment of leachate from the anaerobic fermentation of solid wastes . The removal of organic matter and ammonia was achieved in two fixed bed biofilm reactors containing Kaldnes and Linpor support materials with specific surface areas of 490 and 270 m2/m3, respectively, and operating under the sequencing batch procedure during 204 days . The Linpor reactor achieved higher total COD removal than the Kaldnes reactor (47% and 39%, respectively) . Linpor was shown to be less sensitive to influent COD changes than Kaldnes . The effluent total COD values of Kaldnes were higher than Linpor . The dissolved COD removal was 21% for both reactors . The average ammonia removal for Linpor was 72% and 42% for Kaldnes . The matrix of Linpor allows higher concentrations of microorganisms (as dry mass) than Kaldnes . The dry mass concentration was related to the "active" exposed surface area of the biofilm . This is considered to be the cause for the better performance of Linpor when compared with Kaldnes.

Water Sci Technol, 2004, 49(11-12), 277 - 86
Contamination potential of drinking water distribution network biofilms; Wingender J et al.; Drinking water distribution system biofilms were investigated for the presence of hygienically relevant microorganisms . Early biofilm formation was evaluated in biofilm reactors on stainless steel, copper, polyvinyl chloride (PVC) and polyethylene coupons exposed to unchlorinated drinking water . After 12 to 18 months, a plateau phase of biofilm development was reached . Surface colonization on the materials ranged between 4 x 10(6) and 3 x 10(7) cells/cm2, with heterotrophic plate count (HPC) bacteria between 9 x 10(3) and 7 x 10(5) colony-forming units (cfu)/cm2 . Established biofilms were investigated in 18 pipe sections (2 to 99 years old) cut out from distribution pipelines . Materials included cast iron, galvanized steel, cement and PVC . Colonization ranged from 4 x 10(5) to 2 x 10(8) cells/cm2, HPC levels varied between 1 and 2 x 10(5) cfu/cm2 . No correlation was found between extent of colonization and age of the pipes . Using cultural detection methods, coliform bacteria were rarely found, while Escherichia coli, Pseudomonas aeruginosa and Legionella spp . were not detected in the biofilms . In regular operation, distribution system biofilms do not seem to be common habitats for pathogens . However, nutrient-leaching materials like rubber-coated valves were observed with massive biofilms which harboured coliform bacteria contaminating drinking water.

Water Sci Technol, 2004, 49(11-12), 255 - 62
Engineering aspects of a mixed methanotrophic culture in a membrane-aerated biofilm reactor; Casey E et al.; Methanotrophic biodegradation using the membrane-aerated biofilm reactor (MABR) is a technology offering several advantages over both conventional biofilm reactors and suspended-cell processes . In this study the oxidation efficiency of a methanotrophic biofilm in a 1.5 litre MABR was investigated . Measurements of oxygen and methane uptake rates together with biofilm thickness were taken for developing biofilms . It was found that the specific rate of metabolic activity of the biofilm was unusually high as determined by the methane and oxygen uptake rates . Microbial activity stratification was evident and the location of stratified layers of oxygen consuming components of the consortium could be manipulated via the intra-membrane oxygen pressure.

Water Sci Technol, 2004, 49(11-12), 231 - 6
Methanotrophic biodegradation of cis-1,2-dichloroethylene in a continuously fed fixed-film bioreactor; Arcangeli JP et al.; Co-metabolic biodegradation of cis-dichloroethylene (cis-DCE) was investigated in a bench-scale fixed-film bioreactor inoculated with a mixed culture of methane oxidising bacteria . The aim of this work was to identify factors that affect the cis-DCE biodegradation . It was observed that the presence of methane was necessary to enhance the biodegradation of cis-DCE, but an excess of methane inhibited the cis-DCE removal . cis-DCE did not inhibit the methane biodegradation at concentrations up to 300 microg/L . Maximum cis-DCE removal was observed with a methane bulk concentration ranging from 0.2 to 0.7 mg/L . It was found that the activity of the biofilm was located in the upper 100 microm of the biofilm . On the basis of this study it is concluded that careful control of the oxygen and methane concentrations as well as of the biofilm thickness is necessary in order to optimise the biodegradation of cis-DCE in fixed film bioreactors.

Water Sci Technol, 2004, 49(11-12), 199 - 205
Increasing the capacity for treatment of chemical plant wastewater by replacing existing suspended carrier media with Kaldnes Moving Bed media at a plant in Singapore; Wessman FG et al.; The Kaldnes biomedia K1, which is used in the patented Kaldnes Moving Bed biofilm process, has been tested along with other types of biofilm carriers for biological pretreatment of a complex chemical industry wastewater . The main objective of the test was to find a biofilm carrier that could replace the existing suspended carrier media and at the same time increase the capacity of the existing roughing filter-activated sludge plant by 20% or more . At volumetric organic loads of 7.1 kg COD/m3/d the Kaldnes Moving Bed process achieved much higher removal rates and much lower effluent concentrations than roughing filters using other carriers . The Kaldnes roughing stage achieved more than 85% removal of organic carbon and more than 90% removal of BOD5 at the tested organic load, which was equivalent to a specific biofilm surface area load of 24 g COD/m2/d . Even for the combined roughing filter-activated sludge process, the Kaldnes carriers outperformed the other carriers, with 98% removal of organic carbon and 99.6% removal of BOD5 . The Kaldnes train final effluent concentrations were only 22 mg FOC/L and 7 mg BOD5/L . Based on the successful pilot testing, the full-scale plant was upgraded with Kaldnes Moving Bed roughing filters . During normal operation the upgraded plant has easily met the discharge limits of 100 mg COD/L and 50 mg SS/L . For the month of September 2002, with organic loads between 100 and 115% of the design load for the second half of the month, average effluent concentrations were as low as 9 mg FOC/L, 51 mg COD/L and 12 mg SS/L.

Water Sci Technol, 2004, 49(11-12), 193 - 8
Two-dimensional cellular automaton model for mixed-culture biofilm; Pizarro GE et al.; Structural and microbial heterogeneity occurs in almost any type of biofilm system . General approaches for the design of biofilm systems consider biofilms as homogeneous and of constant thickness . In order to improve the design of biofilms systems, models need to incorporate structural heterogeneity and the effect of inert microbial mass . We have improved a 2D biofilm model based on cellular automata (CA) and used it to simulate multidimensional biofilms with active and inert biomass including a self-organizing development . Results indicate that the presence of inert biomass within biofilm structures does not change considerably the substrate flux into the biofilm because the active biomass is located at the surface of the biofilm . Long-term simulations revealed that although the biofilm system is highly heterogeneous and the microstructure is continuously changing, the biofilm reaches a dynamic steady-state with prediction of biofilm thickness and substrate flux stabilizing on a delimited range.

Water Sci Technol, 2004, 49(11-12), 187 - 92
A new mathematical model for chemotactic bacterial colony growth; Alpkvist E et al.; A new continuum model for the growth of a single species biofilm is proposed . The geometry of the biofilm is described by the interface between the biomass and the surrounding liquid . Nutrient transport is given by the solution of a semi-linear Poisson equation . In this model we study the morphology of a chemotactic bacterial colony, which grows in the direction of increasing nutrient concentration . Numerical simulations using the level set method and finite difference schemes are presented . The results show rich heterogeneous morphology.

Water Sci Technol, 2004, 49(11-12), 169 - 76
Results from the multi-species benchmark problem 3 (BM3) using two-dimensional models; Noguera DR et al.; In addition to the one-dimensional solutions of a multi-species benchmark problem (BM3) presented earlier (Rittmann et al., 2004), we offer solutions using two-dimensional (2-D) models . Both 2-D models (called here DN and CP) used numerical solutions to BM3 based on a similar mathematical framework of the one-dimensional AQUASIM-built models submitted by Wanner (model W) and Morgenroth (model M1), described in detail elsewhere (Rittmann et al., 2004) . The CP model used differential equations to simulate substrate gradients and biomass growth and a particle-based approach to describe biomass division and biofilm growth . The DN model simulated substrate and biomass using a cellular automaton approach . For several conditions stipulated in BM3, the multidimensional models provided very similar results to the 1-D models in terms of bulk substrate concentrations and fluxes into the biofilm . The similarity can be attributed to the definition of BM3, which restricted the problem to a flat biofilm in contact with a completely mixed liquid phase, and therefore, without any salient characteristics to be captured in a multidimensional domain . On the other hand, the models predicted significantly different accumulations of the different types of biomass, likely reflecting differences in the way biomass spread within the biofilm is simulated.

Water Sci Technol, 2004, 49(11-12), 155 - 62
Modelling a spatially heterogeneous biofilm and the bulk fluid: selected results from benchmark problem 2 (BM2); Eberl HJ et al.; The numerical simulation of mass transfer and conversion in spatially heterogeneous biofilms on the meso-scale requires an accurate description of the hydrodynamics in the biofilm systems and of spatial effects . This leads to systems of three-dimensional nonlinear partial differential equations that are numerically very expensive to solve and to data requirements that are not easy to meet . In this paper several modeling approaches to reduce the physical complexity and, hence, accelerate the computation are compared . They range from a mere reduction of dimensionality by lumping the problem along a secondary flow direction to global mass balances or empirical correlations, at the core of which a one-dimensional boundary value problem must be solved . It is found that even strongly simplified models can describe the qualitative behaviour of the model with regard to variations in the geometrical and hydrodynamic model parameters quite well . In order to obtain also quantitatively reliable results the hydrodynamics must be considered in an appropriate manner.

Water Sci Technol, 2004, 49(11-12), 145 - 54
Comparing biofilm models for a single species biofilm system; Morgenroth E et al.; A benchmark problem was defined to evaluate the performance of different mathematical biofilm models . The biofilm consisted of heterotrophic bacteria degrading organic substrate and oxygen . Mathematical models tested ranged from simple analytical to multidimensional numerical models . For simple and more or less flat biofilms it was shown that analytical biofilm models provide very similar results compared to more complex numerical solutions . When considering a heterogeneous biofilm morphology it was shown that the effect of an increased external mass transfer resistance was much more significant compared to the effect of an increased surface area inside the biofilm.

Water Sci Technol, 2004, 49(11-12), 137 - 44
Biofilm modeling with AQUASIM; Wanner O et al.; AQUASIM is a computer program for the identification and simulation of aquatic systems . The program includes a one-dimensional multisubstrate and multispecies biofilm model and represents a suitable tool for biofilm simulation . The program can be used to calculate substrate removal in biofilm reactors for any user specified microbial system . One-dimensional spatial profiles of substrates and microbial species in the biofilm can be predicted . The program also calculates the development of the biofilm thickness and of the substrates and microbial species in the biofilm and in the bulk fluid over time . Detachment and attachment of microbial cells at the biofilm surface and in the biofilm interior can be considered, and simulations of sloughing events can be performed . Furthermore, AQUASIM allows pseudo two-dimensional modeling of plug flow biofilm reactors by a series of biofilm reactor compartments . The most significant limitation of the model is that it only considers spatial gradients of substrates and microbial species in the biofilm in the direction perpendicular to the substratum.

Water Sci Technol, 2004, 49(11-12), 131 - 6
Introduction to the IWA task group on biofilm modeling; Noguera DR et al.; An International Water Association (IWA) Task Group on Biofilm Modeling was created with the purpose of comparatively evaluating different biofilm modeling approaches . The task group developed three benchmark problems for this comparison, and used a diversity of modeling techniques that included analytical, pseudo-analytical, and numerical solutions to the biofilm problems . Models in one, two, and three dimensional domains were also compared . The first benchmark problem (BM1) described a monospecies biofilm growing in a completely mixed reactor environment and had the purpose of comparing the ability of the models to predict substrate fluxes and concentrations for a biofilm system of fixed total biomass and fixed biomass density . The second problem (BM2) represented a situation in which substrate mass transport by convection was influenced by the hydrodynamic conditions of the liquid in contact with the biofilm . The third problem (BM3) was designed to compare the ability of the models to simulate multispecies and multisubstrate biofilms . These three benchmark problems allowed identification of the specific advantages and disadvantages of each modeling approach . A detailed presentation of the comparative analyses for each problem is provided elsewhere in these proceedings.

Water Sci Technol, 2004, 49(11-12), 53 - 60
Difficulties in maintaining long-term partial nitritation of ammonium-rich sludge digester liquids in a moving-bed biofilm reactor (MBBR); Fux C et al.; Nitrogen can be eliminated effectively from sludge digester effluents by anaerobic ammonium oxidation (anammox), but 55-60% of the ammonium must first be oxidized to nitrite . Although a continuous flow stirred tank reactor (CSTR) with suspended biomass could be used, its hydraulic dilution rate is limited to 0.8-1 d(-1) (30 degrees C) . Higher specific nitrite production rates can be achieved by sludge retention, as shown here for a moving-bed biofilm reactor (MBBR) with Kaldnes carriers on laboratory and pilot scales . The maximum nitrite production rate amounted to 2.7 gNO2-Nm(-2)d(-1) (3 gO2m(-3)d(-1), 30.5 degrees C), thus doubling the dilution rate compared to CSTR operation with suspended biomass for a supernatant with 700 gNH4-Nm(-3) . Whenever the available alkalinity was fully consumed, an optimal amount of nitrite was produced . However, a significant amount of nitrate was produced after 11 months of operation, making the effluent unsuitable for anaerobic ammonium oxidation . Because the sludge retention time (SRT) is relatively long in biofilm systems, slow growth of nitrite oxidizers occurs . None of the selection criteria applied - a high ammonium loading rate, high free ammonia or low oxygen concentration - led to selective suppression of nitrite oxidation . A CSTR or SBR with suspended biomass is consequently recommended for full-scale operation.

Water Sci Technol, 2004, 49(11-12), 19 - 25
The effect of intermittent feeding on aerobic granule structure; McSwain BS et al.; Self-immobilized biofilms, or aerobic granules without the addition of carrier material, have only been reported in one suspended growth system, the Sequencing Batch Reactor (SBR) with a very short fill time (dump fill) . The SBR utilizes intermittent feeding which creates a period of high load followed by starvation (often referred to as feast-famine) . In this experiment, three identical SBRs were operated with different feeding conditions to determine the role of feast-famine on granule formation . All three SBRs were operated with a total volumetric load of 2.4 kg/m3 x d . The 90 minute Fill phase was altered for each reactor, providing an increasing time of Aerated Fill . A dump fill condition was applied for one reactor, while the other two reactors were aerated for different times during Fill, resulting in a smaller COD load at the beginning of each React phase . Aerobic granules formed in all reactors, but the structural properties and content of filamentous organisms were clearly dependent on a high feast condition . Only the reactor with dump fill formed compact, stable granules . It is concluded that intermittent feeding associated with the SBR affects the selection and growth of filamentous organisms and has a critical role in granule structure and composition.

Eukaryot Cell, 2004 Aug, 3(4), 1062 - 5
The two-component signal transduction protein Chk1p regulates quorum sensing in Candida albicans; Kruppa M et al.; Regulation of hyphal morphogenesis in Candida albicans can occur through quorum sensing (QS) . A QS signal, farnesol, is produced during high-density growth and inhibits morphogenesis . However, the signal transduction pathway that regulates QS is unknown . Here, we show that a C . albicans mutant lacking Chk1p but not either the Sln1p or the Nik1p histidine kinase is refractory to the inhibitory effect of farnesol both in cell suspension and during the formation of a biofilm . This study is the first to demonstrate a role for a two-component signal transduction protein in QS by a eukaryotic organism.

J Clin Microbiol, 2004 Aug, 42(8), 3827 - 30
Real-time TaqMan PCR for quantifying oral bacteria during biofilm formation; Suzuki N et al.; A TaqMan PCR was developed for quantifying early colonizer microorganisms in dental biofilms . To design species-specific primers and TaqMan probes, genomic subtractive hybridization was used . This quantitative assay in combination with subtractive hybridization may be of value in the study of microbial ecosystems consisting of related species that are involved in the formation and etiology of biofilms.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1082 - 90
Characteristics of a methanotrophic culture in a membrane-aerated biofilm reactor; Rishell S et al.; The membrane-aerated biofilm reactor (MABR) shows considerable potential as a bioprocess that can exploit methanotrophic biodegradation and offers several advantages over both conventional biofilm reactors and suspended-cell processes . This work seeks primarily to investigate the oxidation efficiency in a methanotrophic MABR . A mixed methanotrophic biofilm was immobilized on an oxygen-permeable silicone membrane in a single tube hollow fiber configuration . Under the conditions used the maximum oxygen uptake rate reached values of 16 g/m2.d, and the rate of biofilm growth achieved was 300 microm/d . Both indicators reflect a very high metabolic rate . It was shown that the biofilm was predominantly in a dual-substrate limitation regime but below about 250 microm was fully penetrated by both substrates . Oxygen limitation was not observed . Analysis indicated that microbial activity stratification was evident and the location of stratified layers of oxygen-consuming components of the consortium could be manipulated via the intramembrane oxygen pressure . The results confirm that an MABR can be employed to minimize substrate diffusion limitations in thick biofilms.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 147 - 50
Engineering the interaction between micro-organisms and construction materials; De Windt W et al.; The influence of micro-organisms on degradation of mineral materials, cement bound systems, wood and steel is a rather new subject of research slowly becoming recognised by the 'classical' technical disciplines . An increasing amount of literature appears on biodeterioration of construction materials and microbial activity can not be neglected as a determining factor in the deterioration process . Microbial communities interact in many different ways with mineral materials and their external environment . They can be present on the surface or in crevices and fissures within the material and often their actions become organized in a biofilm . The interaction with the material and its environment can give rise to biodeterioration . Yet recent findings show that in some cases the microbial interaction can lead to protection of materials . It is our mission for the future to engineer the microbiological processes with positive impact on construction materials with a view to practical applications.

J Infect Dis, 2004 Sep 1, 190(5), 957 - 66 Epub 2004 Jul 26.
Production of exopolysaccharide by Burkholderia cenocepacia results in altered cell-surface interactions and altered bacterial clearance in mice; Conway BA et al.; Despite the characterization of some Burkholderia cepacia complex exopolysaccharides (EPSs), little is known about the role of EPSs in the pathogenicity of B . cepacia complex organisms . We describe 2 Burkholderia cenocepacia (genomovar III) isolates obtained from a patient with cystic fibrosis (CF): the nonmucoid isolate C8963 and the mucoid isolate C9343 . Both isolates had identical random amplified polymorphic DNA patterns . C9343 produced a capsule composed of the EPSs PS-I and PS-II, as well as alpha -1,6-glucan . These isolates exhibited several phenotypic differences: C8963 synthesized octanoyl-homoserine lactone and produced biofilms, but C9343 did not; in a mouse model of pulmonary infection, C8963 was cleared more rapidly than was C9343; and C9343 interacted poorly with macrophages and neutrophils, compared with C8963, suggesting that the C9343 capsule interfered with cell-surface interactions . Overproduction of EPS by C9343 resulted in a mucoid appearance and interfered with cell-surface interactions and clearance in an animal model . This mucoid colonial appearance could enhance the persistence and virulence of this important CF-related pathogen.

Photochem Photobiol Sci, 2004 Aug, 3(8), 781 - 7 Epub 2004 Apr 20.
Biological UV dosimetry using the DLR-biofilm; Rettberg P et al.; Changes of environmental UV radiation as part of global atmospheric changes will influence the biosphere substantially . The determination of the biological effects of these changes requires accurate and reliable UV monitoring systems that weight the spectral irradiance according to the biological responses under consideration . Biological UV dosimeters, which directly weight the incident UV components of sunlight in relation to the effectiveness of the different wavelengths and the potential interactions between them, can complement weighted physical UV measurements . Up to now several UV-dependent endpoints in biomolecules (e.g . uracil, DNA, provitamin D3), bacteriophages (e.g . T7), bacteria (e.g.E . coli, B . subtilis) and cultured eukaryotic cells have been suggested as sensing elements in biological UV dosimeters . One example is the DLR-biofilm consisting of immobilised spores of the bacterium B . subtilis as a UV sensor . It weights per se the incident UV radiation according to its DNA-damaging effectiveness . In several examples the applicability of the DLR-biofilm technique for personal UV dosimetry as well as for the measurement of the biologically weighted irradiance of the sun and of artificial UV sources is demonstrated.

Pediatr Infect Dis J, 2004 Aug, 23(8), 774 - 8
Fungal biofilm formation on cochlear implant hardware after antibiotic-induced fungal overgrowth within the middle ear; Cristobal R et al.; Cochlear implantation in patients with chronic suppurative otitis media is managed with perioperative antibiotics; however, fungal overgrowth can occur . We present a child who received oral cefdinir and topical ofloxacin (Floxin) . After 6 weeks, a fungal (Candida) biofilm was demonstrated on the implant surface . In this clinical setting, an antimicrobial strategy using an oral antifungal to prevent fungal overgrowth is a possibility.

Appl Environ Microbiol, 2004 Aug, 70(8), 4980 - 8
Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time; Donlan RM et al.; Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation . A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S . pneumoniae in situ over time . A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer . Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods . ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS) . Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides . Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h . The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides . The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10(5) cells per cm(2), while viable counts decreased as the biofilm aged . This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S . pneumoniae over time by using multiple, corroborative techniques . This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.

Appl Environ Microbiol, 2004 Aug, 70(8), 4941 - 9
Differential gene expression to investigate the effect of (5Z)-4-bromo- 5-(bromomethylene)-3-butyl-2(5H)-furanone on Bacillus subtilis; Ren D et al.; (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming, and biofilm formation of gram-positive bacteria . Using the gram-positive bacterium Bacillus subtilis as a test organism, we observed cell killing by 20 microg of furanone per ml, while 5 microg of furanone per ml inhibited growth approximately twofold without killing the cells . To discover the mechanism of this inhibition on a genetic level and to investigate furanone as a novel antibiotic, full-genome DNA microarrays were used to analyze the gene expression profiles of B . subtilis grown with and without 5 microg of furanone per ml . This agent induced 92 genes more than fivefold (P < 0.05) and repressed 15 genes more than fivefold (P < 0.05) . The induced genes include genes involved in stress responses (such as the class III heat shock genes clpC, clpE, and ctsR and the class I heat shock genes groES, but no class II or IV heat shock genes), fatty acid biosynthesis, lichenan degradation, transport, and metabolism, as well as 59 genes with unknown functions . The microarray results for four genes were confirmed by RNA dot blotting . Mutation of a stress response gene, clpC, caused B . subtilis to be much more sensitive to 5 microg of furanone per ml (there was no growth in 8 h, while the wild-type strain grew to the stationary phase in 8 h) and confirmed the importance of the induction of this gene as identified by the microarray analysis.

Appl Environ Microbiol, 2004 Aug, 70(8), 4512 - 21
Analysis of structural and physiological profiles to assess the effects of Cu on biofilm microbial communities; Massieux B et al.; We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities . For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 microM) . Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria . The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects . Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates . The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria . However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control . Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations . Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration . Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.

J Environ Manage, 2004 Sep, 72(4), 241 - 7
Influence of organic loading on an anaerobic sequencing biofilm batch reactor (ASBBR) as a function of cycle period and wastewater concentration; Siman RR et al.; The effect of organic loading on the performance of a mechanically stirred anaerobic sequencing biofilm batch reactor (ASBBR) has been investigated, by varying influent concentration and cycle period . For microbial immobilization 1-cm polyurethane foam cubes were used . An agitation rate of 500 rpm and temperature of 30+/-2 degrees C were employed . Organic loading rates (OLR) of 1.5-6.0gCODl(-1)d(-1) were applied to the 6.3-l reactor treating 2.0 l synthetic wastewater in 8 and 12-h batches and at concentrations of 500-2000mgCODl(-1), making it possible to analyze the effect of these two operation variables for the same organic loading range . Microbial immobilization on inert support maintained approximately 60 gTVS in the reactor . Filtered sample organic COD removal efficiencies ranged from 73 to 88% for organic loading up to 5.4gCODl(-1)d(-1) . For higher organic loading (influent concentration of 2000mgCODl(-1) and 8-h cycle) the system presented total volatile acids accumulation, which reduced organics removal efficiency down to 55% . In this way, ASBBR with immobilized biomass was shown to be efficient for organic removal at organic loading rates of up to 5.4gCODl(-1)d(-1) and to be more stable to organic loading variations for 12-h cycles . This reactor might be an alternative to intermittent systems as it possesses greater operational flexibility . It might also be an alternative to batch systems suspended with microorganisms since it eliminates both the uncertainties regarding granulation and the time necessary for biomass sedimentation, hence reducing the total cycle period.

Mikrobiyol Bul, 2004 Jan-Apr, 38(1-2), 91 - 8
{Biofilm production and antifungal susceptibility patterns of Candida species}; Yucesoy M et al.; In this study, biofilm production and antifungal susceptibility of various Candida species were examined and compared . A total number of 156 Candida species (94 C . albicans, 21 C . tropicalis, 18 C . glabrata, 12 C . parapsilosis, 9 C . krusei, 1 C . guilliermondii and 1 C . kefyr) isolated from different clinical specimens were included in the study . The biofilm production of the strains was searched by modified tube adherence and microplate methods . Their antifungal susceptibilities against fluconazole and amphotericin B were determined by microdilution method, according to NCCLS M27-A2 standards . Forty three (27.6%) and 26 (16.7%) of the strains were found to be slime producing by tube adherence and microplate methods, respectively . The agreement between the two methods were detected as 65 percent . The rate of biofilm formation by different species ranged between 17% and 55% by tube adherence test and 0 and 48% by microplate method . No significant difference was found between the biofilm production of C . albicans and non-albicans species by tube adherence test (p=0.29) . However; a statistically important difference was found when microplate method was considered (p=0.04) . MIC50 and MIC90 values for fluconazole ranged between 4-64 microg/ml and 32-->64 microg/ml for different Candida species while these values changed between 0.25-1 microg/ml and 0.5-2 microg/ml for amphotericin B, respectively . Forty-five (28.8%) and 23 (14.7%) of the isolates were found to be dose dependent susceptible and resistant to fluconazole, respectively . Eleven (6.7%) of the strains had MIC values >1 microg/ml for amphotericin B . When the relation between the biofilm production and the susceptibility categories of the strains for amphotericin B were searched, no statistical differences were found by any of the two methods (p=0.12 and p=0.50) . A statistically important difference (p=0.03) was determined by tube adherence test and no important difference (p=0.11) was detected by microplate method when fluconazole susceptibility categories were considered . As a conclusion, it has been determined that biofilm production which is a potential virulence factor for Candida species seems to be in agreement with the antifungal susceptibility categories of the strains especially for amphotericin B when the planktonic cells were used for the susceptibility testing.

Aust Dent J, 2004 Jun, 49(2), 72 - 7
Morphological analysis of subgingival biofilm formation on synthetic carbonate apatite inserted into human periodontal pockets; Takeuchi H et al.; BACKGROUND: Details of the development of human subgingival biofilm are unknown due to the difficulties in conducting experiments and especially in obtaining undisturbed materials . METHODS: This study was performed using deposits on carbonate apatite that had been inserted into human periodontal pockets for up to three weeks . Scanning electron microscopy using the vertically sectioned method and transmission electron microscopy using the freeze-substitution method were adopted . RESULTS: The development of subgingival biofilm occurred in five sequential phases: pellicle formation, microbial adherence, initial colonization, microbial organization, and establishment . Certain species in each of the initial, secondary and tertiary colonizers were considered to have a predilection for biofilm formation . Gram-positive, bacillary initial colonizers and gram-negative, filamentous secondary colonizers organized one stable structure that served as the framework for biofilm formation, and gram-negative, rod-shaped tertiary colonizers with cell-surface vesicles showed multigeneric coaggregation . The microbiota in the tertiary colonizers underwent repeated microflora alteration . CONCLUSIONS: Subgingival biofilm is constituted by initial, secondary and tertiary colonizers . Microflora alteration which is suggested to be related to periodontal disease, frequently occurred in the tertiary colonizers.

J Dairy Sci, 2004 May, 87(5), 1536 - 44
The color of Brevibacterium linens depends on the yeast used for cheese deacidification; Leclercq-Perlat MN et al.; The color of smear cheeses (Muenster) is traditionally thought to be due to the bacterial flora, e.g., Brevibacterium linens . This study was carried out to evaluate indirect effects of yeast on the color of B . linens . A 60% cheese medium was desacidified with Debaryomyces hansenii or Kluyveromyces marxianus until pH 5.8 was reached . After inactivation of the yeast and addition of agar-NaCl, B . linens was inoculated on the medium surface and incubated at 12 degrees C from d 2 to 28 . For each bacterial biofilm, color was evaluated by L*C*h(degrees) (brightness, chroma, hue angle) spectrocolorimetry . After d 14 (D . hansenii deacidification) and d 21 (K marxianus desacidification), the color level (as a function of all 3 factors) of B . linens biofilms became maximal and remained so until d 28 . Debaryomyces hansenii 304 (LGMPA) was less efficient for deacidification than K . marxianus Laf5 . However, color intensity (function of chroma only) was higher when D . hansenii was used . The yeast used had an effect on the composition of the cheese medium in relation to production and consumption of metabolites during deacidification . The results concerning color are discussed with respect to this cheese medium composition.

Microbiology, 2004 Aug, 150(Pt 8), 2751 - 60
Biofilms promote altruism; Kreft JU; The origin of altruism is a fundamental problem in evolution, and the maintenance of biodiversity is a fundamental problem in ecology . These two problems combine with the fundamental microbiological question of whether it is always advantageous for a unicellular organism to grow as fast as possible . The common basis for these three themes is a trade-off between growth rate and growth yield, which in turn is based on irreversible thermodynamics . The trade-off creates an evolutionary alternative between two strategies: high growth yield at low growth rate versus high growth rate at low growth yield . High growth yield at low growth rate is a case of an altruistic strategy because it increases the fitness of the group by using resources economically at the cost of decreased fitness, or growth rate, of the individual . The group-beneficial behaviour is advantageous in the long term, whereas the high growth rate strategy is advantageous in the short term . Coexistence of species requires differences between their niches, and niche space is typically divided into four 'axes' (time, space, resources, predators) . This neglects survival strategies based on cooperation, which extend the possibilities of coexistence, arguing for the inclusion of cooperation as the fifth 'axis' . Here, individual-based model simulations show that spatial structure, as in, for example, biofilms, is necessary for the origin and maintenance of this 'primitive' altruistic strategy and that the common belief that growth rate but not yield decides the outcome of competition is based on chemostat models and experiments . This evolutionary perspective on life in biofilms can explain long-known biofilm characteristics, such as the structural organization into microcolonies, the often-observed lack of mixing among microcolonies, and the shedding of single cells, as promoting the origin and maintenance of the altruistic strategy . Whereas biofilms enrich altruists, enrichment cultures, microbiology's paradigm for isolating bacteria into pure culture, select for highest growth rate.

Waste Manag, 2004, 24(7), 723 - 38
Influence of landfill leachate suspended solids on clog (biorock) formation; VanGulck JF et al.; Laboratory column tests were performed to evaluate the role of leachate-suspended-solids in clogging a granular material permeated with Keele Valley Landfill leachate . The development of the clog material was a result of biological, chemical, and physical processes occurring within the column . The increase in volatile solids, which contributed to clog development over time, was primarily due to the retention of volatile suspended solids and growth of a biofilm capable of removing acetate, propionate, and butyrate from the leachate . Acetate fermentation was primarily responsible for precipitation of calcium within the column . The precipitated calcium and retention of inorganic suspended solids contributed to the increase in clog inorganic solids . Over the duration of the experiment, 3.7 times more calcium was precipitated in the column (due to acid fermentation) than was retained with inorganic suspended solids . Clogging resulted in a greater than 60% reduction in drainable porosity and a six-order magnitude decrease in hydraulic conductivity . The potential practical implications with respect to pipe cleaning and leachate recirculation were discussed .

Lett Appl Microbiol, 2004, 39(3), 226 - 31
In vitro new dialysis protocol to assay the antiseptic properties of a quaternary ammonium compound polymerized with denture acrylic resin; Pesci-Bardon C et al.; AIMS: To develop an in vitro protocol in order to assess the antiseptic properties of a quaternary ammonium compound polymerized with acrylic denture resin base, using experimental resin discs and dialysis membranes . METHODS AND RESULTS: Experimental acrylic resin discs were polymerized with Poly 202063A, an ammonium compound (2-50%) . Antiseptic properties were assayed against two reference strains (Escherichia coli, Staphylococcus aureus) and a laboratory strain (Candida albicans), using three different conditions (test A, B and C) . In test A, according to classical protocols the resin discs were first soaked in large volumes of microbial inoculum (45 ml) . An original dialysis protocol was then designed to recreate the small biofilm volume on the prosthetic surface . In test B, discs and bacterial inoculum (600 microl) were introduced in a dialysis bag and dialysed against a sterile buffer . A bactericidal effect was observed against E . coli and Staph . aureus (<0.1% viable cells in initial bacterial suspension) . A dose-dependent fungistatic effect was observed against C . albicans . Finally, in test C discs and sterile buffer (600 microl) were introduced in a dialysis bag and dialysed against microbial inoculum . Reduced activity was found outside the dialysis bag, demonstrating that free ammonium was able to diffuse through the dialysis membrane, displaying antiseptic properties . CONCLUSIONS: The present protocol demonstrated that a quaternary ammonium compound remains efficient after heat polymerization with an acrylic denture base resin, both in immediate and distant microbial environments . SIGNIFICANCE AND IMPACT OF THE STUDY: Such removable prosthetic devices with intrinsic antiseptic properties would contribute to improve the long-term management of denture stomatitis.

J Biosci, 2004 Jun, 29(2), 169 - 77
Microbial acetate oxidation in horizontal rotating tubular bioreactor; Slavica A et al.; The aim of this work was to investigate the possibility of conducting a continuous aerobic bioprocess in a horizontal rotating tubular bioreactor (HRTB) . Aerobic oxidation of acetate by the action of a mixed microbial culture was chosen as a model process . The microbial culture was not only grown in a suspension but also in the form of a biofilm on the interior surface of HRTB . Efficiency of the bioprocess was monitored by determination of the acetate concentration and chemical oxygen demand (COD) . While acetate inlet concentration and feeding rate influenced efficiency of acetate oxidation, the bioreactor rotation speed did not influence the bioprocess dynamics significantly . Gradients of acetate concentration and pH along HRTB were more pronounced at lower feeding rates . Volumetric load of acetate was proved to be the most significant parameter . High volumetric loads (above 2 g acetate l-1 h-1) gave poor acetate oxidation efficiency (only 17 to 50%) . When the volumetric load was in the range of 0.60-1.75 g acetate l-1 h-1, acetate oxidation efficiency was 50-75% . At lower volumetric loads (0.14-0.58 g acetate l-1 h-1), complete acetate consumption was achieved . On the basis of the obtained results, it can be concluded that HRTB is suitable for conducting aerobic continuous bioprocesses.

J Dent Res, 2004, 83 Spec No C, C35 - 8
What constitutes dental caries? Histopathology of carious enamel and dentin related to the action of cariogenic biofilms; Kidd EA et al.; Substantial pH fluctuations within the biofilm on the tooth surface are a ubiquitous and natural phenomenon, taking place at any time during the day and night . The result may be recordable in the dental tissues at only a chemical and/or ultrastructural level (subclinical level) . Alternatively, a net loss of mineral leading to dissolution of dental hard tissues may result in a caries lesion that can be seen clinically . Thus, the appearance of the lesion may vary from an initial loss of mineral, seen only in the very surface layers at the ultrastructural level, to total tooth destruction . Regular removal of the biofilm, preferably with a toothpaste containing fluoride, delays or even arrests lesion progression . This can occur at any stage of lesion progression, because it is the biofilm at the tooth or cavity surface that drives the caries process . Active enamel lesions involve surface erosion and subsurface porosity . Inactive or arrested lesions have an abraded surface, but subsurface mineral loss remains, and a true subsurface remineralization is rarely achievable, because the surface zone acts as a diffusion barrier . The dentin reacts to the stimulus in the biofilm by tubular sclerosis and reactionary dentin.

Trends Biotechnol, 2004 Aug, 22(8), 417 - 22
Towards microbial tissue engineering?
Markx GH, Andrews JS, Mason VP.
Tissue engineering involves the creation of multicellular tissues from individual cells . It was previously perceived that tissues were only formed by higher organisms such as plants and animals . However, it is now known that multicellular systems of microorganisms, such as microbial colonies, biofilms, flocs and aggregates, can also show extensive spatial organization . Here, we discuss methods that can be used to spatially organize microorganisms--bacteria, in particular--into tissue-like materials with defined internal architectures . Some potential uses of such "microbial tissues" are covered.

Int J Food Microbiol, 2004 Sep 1, 95(2), 189 - 204
Factors affecting production of extracellular carbohydrate complexes by Escherichia coli O157:H7; Ryu JH et al.; Production of extracellular carbohydrate complexes (ECC) by foodborne pathogens on raw fruits and vegetables may result in protection against removal or inactivation by sanitizers . The influence of environmental conditions on cell growth, the total amount of ECC produced, and the amount of ECC produced on a per cell basis by Escherichia coli O157:H7 strains ATCC 43895 (wild type) and 43895-exopolysaccharides (EPS) (natural mutant, extensive EPS producer) was studied . To determine the effects of pH on the production of ECC on a per cell basis, E . coli O157:H7 was grown aerobically at 12 and 22 degrees C on tryptic soy agar (TSA) acidified to pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, and 4.0 . Lettuce, alfalfa sprout, cantaloupe, tomato, and apple juice agars (pH 4.46-6.50) were also evaluated for their support of the ECC production . Conditions generally favorable for growth of E . coli O157:H7 were a rich nutrient medium (TSA) vs . heated lettuce juice agar (HLJA) or minimal salts medium (MSM), 22 degrees C compared to 12 degrees C, and an aerobic atmosphere compared to modified atmosphere (1% O(2), 10% CO(2), and 89% N(2)) . Conditions favorable for production of ECC on a per cell basis were HLJA, 12 degrees C, and an aerobic atmosphere . This suggests that modified atmosphere packaging of lettuce may not only decrease the growth of E . coli O157:H7 but also its propensity to form biofilm . There was a negative relationship between cell growth and production of ECC on a per cell basis, and environmental conditions that affected the total amount of ECC produced based on initial population reflected a combination of environmental conditions influencing both cell growth and ECC production on a per cell basis . A relative growth index factor (RGIF) was calculated to better understand ECC production as affected by various environmental conditions simultaneously . The production of ECC on a per cell basis by strain 43895-EPS showed a negative linear relationship with pH of TSA at both 12 and 22 degrees C . This strain generally produced a greater amount of ECC on fresh juice agar than on TSA at the same pH, but production of ECC on alfalfa sprout juice agar (FJA, pH 6.45) at 22 degrees C was significantly less than on TSA (pH 6.50) . This indicates that nutrient limitation is not based only on nutrient availability . There may be other factors that repress the production of ECC on FJA, and the effects of those factors may be temperature dependent . Further studies will be required to better understand the relationship between nutrient availability and other factors on the production of ECC by E . coli O157:H7 on raw produce.

Appl Spectrosc, 2004 Jul, 58(7), 804 - 10
On-line fermentation monitoring by mid-infrared spectroscopy; Mazarevica G et al.; A new method for on-line monitoring of fermentations using mid-infrared (MIR) spectroscopy has been developed . The method has been used to predict the concentrations of glucose and ethanol during a baker's yeast fermentations . A completely automated flow system was employed as an interface between the bioprocess under study and the Fourier transform infrared (FT-IR) spectrometer, which was equipped with a flow cell housing a diamond attenuated total reflection (ATR) element . By using the automated flow system, experimental problems related to adherence of CO(2) bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated . Gas bubbles were removed during sampling, and by using rinsing steps any biofilm could be removed from the ATR surface . In this way, constant measuring conditions could be guaranteed throughout prolonged fermentation times (approximately 8 h) . As a reference method, high-performance liquid chromatography (HPLC) with refractive index detection was used . The recorded data from different fermentations were modeled by partial least-squares (PLS) regression comparing two different strategies for the calibration . On the one hand, calibration sets were constructed from spectra recorded from either synthetic standards or from samples drawn during fermentation . On the other hand, spectra from fermentation samples and synthetic standards were combined to form a calibration set . Differences in the kinetics of the studied fermentation processes used for calibration and prediction, as well as the precision of the HPLC reference method, were identified as the main chemometric sources of error . The optimal PLS regression method was obtained using the mixed calibration set of samples from fermentations and synthetic standards . The root mean square errors of prediction in this case were 0.267 and 0.336 g/L for glucose and ethanol concentration, respectively.

J Appl Microbiol, 2004, 97(3), 590 - 7
Microbiological influences in 'blue water' copper corrosion; Critchley MM et al.; AIMS: To investigate the influence of micro-organisms associated with copper corrosion on 'blue water' corrosion in drinking water . METHODS AND RESULTS: Laboratory rigs comprising of polycarbonate containers attached to annealed copper plumbing tubes were filled with Melbourne drinking water and sterilized by autoclaving . The copper tubes were inoculated with sterile or nonsterile extracts obtained from corroding copper and allowed to stand for 7 days . The extracts were drained and the tubes flushed and filled with sterile water from the rig . The water within the tubes was removed weekly for analysis and the tubes were refilled with freshly aerated water . The tube water sampled was analysed for pH, total copper and the presence of micro-organisms . Sterile rigs and rigs containing nonsterile water, both without tube inoculums, were used as controls . The results demonstrated that tubes inoculated with nonsterile corrosion extracts showed statistically higher copper release compared with the other rigs . Copper release as blue water was only observed after a lag period of 9 weeks . The internal surfaces of tubes releasing copper showed significant amounts of corrosion products and the presence of biofilm . Bacteria isolated from the corroding tubes included Acidovorax spp . and Sphingomonas sp . CONCLUSIONS: The results demonstrate a microbial role in blue water, as corrosion was induced in new copper tubes by exposure to nonsterile copper corrosion products . SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for micro-organisms present in corrosion products to initiate blue water corrosion presents significant implications for the management of corrosion in distribution systems .

Carbohydr Res, 2004 Aug 23, 339(12), 2127 - 37
The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases; Hayacibara MF et al.; Glucanohydrolases, especially mutanase {alpha-(1-->3) glucanase; EC 3.2.1.59} and dextranase {alpha-(1-->6) glucanase; EC 3.2.1.11}, which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque . We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C {synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans} in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase) . The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored . The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan . The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan . The amount of (1-->6)-linked glucan was reduced in presence of dextranase . In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect . Glucan formed in the presence of dextranase provided fewer binding sites for S . sobrinus; mutanase was devoid of any effect . We also noted that the GTFs bind to dextranase and mutanase . Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque . Our data have relevance for the formation of polysaccharide matrix of other biofilms.

J Microbiol Methods, 2004 Sep, 58(3), 367 - 74
An optical microsensor to measure fluorescent light intensity in biofilms; Beyenal H et al.; We have developed an optical microsensor to quantify fluorescent light intensity distribution in biofilms . The optical system consisted of a beam splitter, light couplers, filters and a spectrophotometer able to accept the fiberoptic cable to measure fluorescent light intensity . The emitted light, fluorescence from the biofilm, was collected at the tip of the optical microsensor and was transferred to a spectrophotometer via a fiberoptic cable . The total fluorescent light intensity was evaluated from the emission spectrum by numerical integration . The newly developed fiberoptic microsensor was tested using a Staphylococcus aureus strain producing yellow fluorescent protein (YFP) grown as biofilm . We used a 405-nm violet laser diode for excitation, and measured the emission intensity between 480 nm and 540 nm . The optical microsensor that quantifies fluorescent light intensity is a promising tool in biofilm research which often requires detection and quantification of fluorescent light intensity distribution generated by various fluorescent proteins.

J Microbiol Methods, 2004 Sep, 58(3), 351 - 60
A simple screening protocol for the identification of quorum signal antagonists; McLean RJ et al.; Quorum sensing (QS) is a mechanism by which diverse microorganisms can control specific processes in response to population density . A relatively well-known form of QS among Proteobacteria involves production and subsequent response to acylated homoserine lactones (AHLs) . Quorum sensing inhibition (QSI), targeting AHL-dependent signaling, has been reported as a strategy for the control of biofilm formation used by several marine organisms . We developed a simple soft agar overlay protocol, based on pigmentation inhibition, to rapidly screen for the presence of potential QSI by bacteria and plants . For bacterial screens, test organisms are first streaked onto their appropriate media and incubated overnight . For plant screens, the plant material (leaf, stem, flower, etc.) is placed onto LB agar . The bacterial growth or plant samples are then covered with an overlay of LB soft agar containing an inoculum of either Pseudomonas aureofaciens 30-84 or Chromobacterium violaceum ATCC 12472 (indicator cultures) and then incubated overnight . These indicator bacteria regulate pigment production by N-hexanoyl-HSL (C6-HSL) QS and are readily inhibited by AHL analogues and other antagonists . QSI is indicated by the lack of pigment production of the indicator culture in the vicinity of the test sample . Growth inhibition of the indicator culture indicates possible antibiotic production . Two different biosensor organisms based on derivatives of Agrobacterium tumefaciens and C . violaceum, capable of detecting a range of AHLs were used to determine whether QSI is due to the production of interfering AHLs competing with the C6-HSL regulation of C . violaceum and P . aureofaciens pigment production . This simple protocol will facilitate the screening of multiple organisms for the production of potential antifouling compounds.

Appl Microbiol Biotechnol, 2004 Nov, 65(6), 747 - 53 Epub 2004 Jul 23.
Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms; Zuo R et al.; A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6 . When L . discophora SP-6 was introduced along with D . orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D . orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements . However, when a gramicidin-S-producing, protective B . brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D . orientis and L . discophora SP-6 . EIS data showed that the protective B . brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P . polymyxa ATCC 10401 biofilm control . The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease) . Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B . brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.

Water Res, 2004 Aug-Sep, 38(14-15), 3362 - 72
Evaluating trends in biofilm density using the UMCCA model; Laspidou CS et al.; We present a series of modeling cases that illustrate the trends described by the unique features of the unified multiple-component cellular automaton (UMCCA) model for a heterogeneous, two-dimensional biofilm . The outputs of the UMCCA model show five general trends . (1) The concentration profiles for the two soluble microbial products are opposite the profile for original substrate . (2) The top of the biofilm is dominated by active biomass and EPS, while the bottom is dominated by residual inert biomass . Within the top layers, active biomass has a much higher concentration than EPS . (3) The top of all biofilm is quite "fluffy," while the bottom is dense . (4) The peak of the composite density does not correspond to the peak of active biomass . (5) All biomass types show considerable local heterogeneity . The series of cases also indicate what conditions lead to particular characteristics observed in some biofilms . Biofilm clusters are promoted by substrate limitation, a high detachment rate, or strong consolidation . A high biofilm density is associated with an old biofilm, which is favored by a low substrate concentration, a high detachment rate, and strong consolidation . Old biofilms also can develop low-density pockets near the substratum, a possible cause of sloughing . Local heterogeneity is generally related to the same factors that cause a high density . We also solved the UMCCA model for conditions similar to the experiments of Bishop et al . (Water Sci . Technol . 31(1) (1995) 143), who measured the total biomass density in layers from the substratum . The model outputs captured all the major trends in the experimental data: the overall thickness and density of biofilms increase with time, and the total biomass density is 5-10 times greater near the substratum than near the top of the biofilm . Furthermore, the model indicates that the residual inert biomass becomes denser toward the substratum, a trend observed experimentally; the UMCCA model suggests that this trend is due to the combined effects of consolidation and inert biomass having a larger maximum density.

Water Res, 2004 Aug-Sep, 38(14-15), 3349 - 61
Modeling the development of biofilm density including active bacteria, inert biomass, and extracellular polymeric substances; Laspidou CS et al.; We present the unified multi-component cellular automaton (UMCCA) model, which predicts quantitatively the development of the biofilm's composite density for three biofilm components: active bacteria, inert or dead biomass, and extracellular polymeric substances . The model also describes the concentrations of three soluble organic components (soluble substrate and two types of soluble microbial products) and oxygen . The UMCCA model is a hybrid discrete-differential mathematical model and introduces the novel feature of biofilm consolidation . Our hypothesis is that the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time . Each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass . The UMCCA model also adds a cellular automaton algorithm that identifies the path of least resistance and directly moves excess biomass along that path, thereby ensuring that the excess biomass is distributed efficiently . A companion paper illustrates the trends that the UMCCA model is able to represent and shows a comparison with experimental results.

Water Res, 2004 Aug-Sep, 38(14-15), 3167 - 78
Anaerobic digestion of olive mill wastewaters in biofilm reactors packed with granular activated carbon and "Manville" silica beads; Bertin L et al.; Anaerobic digestion is one of the most promising technologies for disposing olive mill wastewaters (OMWs) . The process is generally carried out in the conventional contact bioreactors, which however are often unable to efficiently remove OMW phenolic compounds, that therefore occur in the effluents . The possibility of mitigating this problem by employing an anaerobic OMW-digesting microbial consortium passively immobilized in column reactors packed with granular activated carbon (GAC) or "Manville" silica beads (SB) was here investigated . Under batch conditions, both GAC- and SB-packed-bed biofilm reactors exhibited OMW COD and phenolic compound removal efficiencies markedly higher (from 60% to 250%) than those attained in a parallel anaerobic dispersed growth reactor developed with the same inoculum; GAC-reactor exhibited COD and phenolic compound depletion yields higher by 62% and 78%, respectively, than those achieved with the identically configured SB-biofilm reactor . Both biofilm reactors also mediated an extensive OMW remediation under continuous conditions, where GAC-reactor was much more effective than the corresponding SB-one, and showed a tolerance to high and variable organic loads along with a volumetric productivity in terms of COD and phenolic compound removal significantly higher than those averagely displayed by most of the conventional and packed-bed laboratory-scale reactors previously proposed for the OMW digestion.

Arch Oral Biol, 2004 Sep, 49(9), 727 - 38
Direct detection of cell surface interactive forces of sessile, fimbriated and non-fimbriated Actinomyces spp . using atomic force microscopy; Tang G et al.; Actinomyces species are predominant early colonizers of the oral cavity and prime mediators of inter-bacterial adhesion and coaggregation . Previous workers have evaluated the adhesion of Actinomyces spp . by quantitative assessment of sessile, as opposed to planktonic cells attached to substrates, but did not quantify the cell surface interactive forces . Therefore we used atomic force microscopy to directly detect the interactive force between an approaching silicon tip and sessile Actinomyces spp . adhering to a substrate, at nanonewton (nN) range force levels . A total of eight strains each belonging to fimbriated and non-fimbriated Actinomyces species were employed, namely A . bovis, A . gerencseriae, A . israelii, A . meyeri, A . naeslundii genospecies 1 and 2, A . odontolyticus and A . viscosus . The sterile mica discs, used as the adhesion substrate, were immersed in mono-species bacterial suspensions for five days to obtain a thin bacterial biofilm . Interactive forces were measured using a silicon nitride cantilever attached to a Nanoscope IIIA atomic force microscope . The interactive forces between the approaching silicon nitride tip and bacterial biofilm surfaces were randomly quantified at three different locations on each cell; namely, the cell surface proper, the periphery of the cell and the substrate and, the interface between two cells . When the interactive forces at these locations of the same species were compared, significantly higher force levels at the cell-cell interface than the other two locations were noted with A . gerencseriae (P < 0.001), A . viscosus (P < 0.01) and A . israelii (P < 0.05) . When the interactive forces of different Actinomyces spp . at an identical location were compared, fimbriated A . naeslundii genospecies 2 showed the greatest interactive force at the cell surface proper (-32.6 +/- 8.7 nN, P < 0.01) . A . naeslundii genospecies 1, 2 and A . viscosus demonstrated greater interactive force at the cell-mica periphery than the other five species (P < 0.05); A . viscosus (-34.6 +/- 10.5 nN) displayed greater interactive force at the cell-cell interface than the others (P < 0.01), except for A . gerencseriae (P > 0.05) . These data indicate that fimbriated Actinomyces spp., including A . naeslundii genospecies 1, 2 and A . viscosus exert higher cell surface interactive forces than those devoid of fimbriae and, such variable force levels may modulate their adhesion and coaggregation during biofilm formation.

J Infect Dis, 2004 Aug 15, 190(4), 783 - 92 Epub 2004 Jul 12.
Transmission of Yersinia pestis from an infectious biofilm in the flea vector; Jarrett CO et al.; Transmission of plague by fleas depends on infection of the proventricular valve in the insect's foregut by a dense aggregate of Yersinia pestis . Proventricular infection requires the Y . pestis hemin storage (hms) genes; here, we show that the hms genes are also required to produce an extracellular matrix and a biofilm in vitro, supporting the hypothesis that a transmissible infection in the flea depends on the development of a biofilm on the hydrophobic, acellular surface of spines that line the interior of the proventriculus . The development of biofilm and proventricular infection did not depend on the 3 Y . pestis quorum-sensing systems . The extracellular matrix enveloping the Y . pestis biofilm in the flea appeared to incorporate components from the flea's blood meal, and bacteria released from the biofilm were more resistant to human polymorphonuclear leukocytes than were in vitro-grown Y . pestis . Enabling arthropod-borne transmission represents a novel function of a bacterial biofilm.

Infect Immun, 2004 Aug, 72(8), 4905 - 10
Role of flagellum and motility in pathogenesis of Vibrio vulnificus; Lee JH et al.; To assess the role of the flagellum which was detected by immunoscreening of surface proteins of Vibrio vulnificus, an flgE-deleted mutant was constructed and tested for its pathogenicity . The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.

Infect Immun, 2004 Aug, 72(8), 4895 - 9
Inactivation of the ciaH Gene in Streptococcus mutans diminishes mutacin production and competence development, alters sucrose-dependent biofilm formation, and reduces stress tolerance; Qi F et al.; Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins . Mutacin production may play an important role in the ecology of S . mutans in dental plaque . In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production . Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.

Infect Immun, 2004 Aug, 72(8), 4888 - 90
Biofilm formation in vitro and virulence in vivo of mutants of Klebsiella pneumoniae; Lavender HF et al.; One of the early stages of Klebsiella pneumoniae airway infections may involve biofilm formation . Bacterial biofilm formation is frequently investigated using in vitro techniques that facilitate identification and analysis of individual genes . We investigated the correlation between K . pneumoniae biofilm formation in vitro and ability to cause infection in vivo following construction of a bank of mini-Tn5 mutants.

Infect Immun, 2004 Aug, 72(8), 4819 - 26
Molecular interactions of surface protein peptides of Streptococcus gordonii with human salivary components; Hamada T et al.; Oral streptococci play a large role in dental biofilm formation, and several types interact as early colonizers with the enamel salivary pellicle to form the primary biofilm, as well as to incorporate other bacteria on tooth surfaces . Interactions of surface molecules of individual streptococci with the salivary pellicle on the tooth surface have an influence on the etiological properties of an oral biofilm . To elucidate the molecular interactions of streptococci with salivary components, binding between surface protein (SspB and PAg) peptides of Streptococcus gordonii and Streptococcus sobrinus were investigated by utilizing BIAcore biosensor technology . The analogous peptide {change of T at position 400 to K in SspB(390-402), resulting in the SspB(390-T400K-402) peptide} from S . gordonii showed the greatest response for binding to salivary components and inhibited the binding of Streptococcus sanguis by more than 50% in a competitive inhibition assay in a comparison with other SspB and PAg peptides . This peptide also bound to the high-molecular-weight protein complex of salivary components and the agglutinin (gp340/DMBT1) peptide (scavenger receptor cysteine-rich domain peptide 2 {SRCRP 2}) . In addition, the SspB(390-T400K-402) peptide was visualized by two surface positive charges in connection with the positively charged residues, in which lysine was a key residue for binding . Therefore, the region containing lysine may have binding activity in S . gordonii and S . sanguis, and the SRCRP 2 region may function as a receptor for the binding . These findings may provide useful information regarding the molecular mechanism of early biofilm formation by streptococci on tooth surfaces.

J Food Prot, 2004 Jul, 67(7), 1438 - 43
Development of a laboratory scale clean-in-place system to test the effectiveness of "natural" antimicrobials against dairy biofilms; Dufour M et al.; A laboratory scale system, partially reproducing dairy plant conditions, was developed to quantify the effectiveness of chlorine and alternative sanitizers in reducing the number of viable bacteria attached to stainless steel surfaces . Stainless steel tubes fouled in a continuous flow reactor were exposed to a standard clean-in-place regime (water rinse, 1% sodium hydroxide at 70 degrees C for 10 min, water rinse, 0.8% nitric acid at 70 degrees C for 10 min, water rinse) followed by exposure to either chlorine (200 ppm) or combinations of nisin (500 ppm), lauricidin (100 ppm), and the lactoperoxidase system (LPS) (200 ppm) for 10 min or 2, 4, 8, 18, or 24 h . There was significant variation in the effectiveness of the alkaline-acid wash steps in reducing cell numbers (log reduction between 0 and 2) . Following a 10-min treatment, none of the sanitizers significantly reduced the number of attached cells . Two hours of exposure to chlorine, nisin + the LPS, or lauricidin + the LPS achieved 2.8, 2.2, and 1.6 log reductions, respectively . Exposure times > 2 h did not further decrease the number of viable bacteria attached to the stainless steel . The effectiveness of combinations of nisin, lauricidin, and the LPS was similar to that of chlorine (P > 0.05), and these sanitizers could be used to decontaminate the surfaces of small-volume or critical hard-to-clean milk processing equipment.

J Am Dent Assoc, 2004 Jun, 135(6), 799 - 805
A simulated-use evaluation of a strategy for preventing biofilm formation in dental unit waterlines; McDowell JW et al.; BACKGROUND: Prevention of biofilm formation is important in the maintenance of dental unit waterline systems . Without effective control measures, the waterlines will become contaminated with routine use . METHODS: The authors used a simulated-use dental unit waterline system to evaluate the ability of a test product, A-dec ICX (A-dec, Newburg, Ore.), to prevent biofilm formation . They evaluated buffered distilled water and hard water models versus mixed-challenge suspensions of Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa . RESULTS: The authors documented development of significant biofilm in untreated test units, while treated test units showed no indication of biofilm formation throughout the 16-week study . Student t tests and 95 percent confidence intervals performed on the plate count data confirmed that untreated test units had significantly greater bacterial populations than did treated test units (P < .05) . Qualitative images by scanning electron microscopy verified these findings . CONCLUSION: In this simulated clinical-use study, the test product effectively reduced bacterial counts in incoming water and produced water quality exceeding stated recommendations of the American Dental Association . CLINICAL IMPLICATIONS: The test product provides an approach to dental unit waterline maintenance that is simple to use and that, by continuously preventing biofilm formation, reduces concerns about bacterial contamination in dental unit water.

J Bacteriol, 2004 Aug, 186(15), 5087 - 92
A movable surface: formation of Yersinia sp . biofilms on motile Caenorhabditis elegans; Tan L et al.; Bubonic plague is transmitted by fleas whose feeding is blocked by a mass of Yersinia pestis in the digestive tract . Y . pestis and the closely related Y . pseudotuberculosis also block the feeding of Caenorhabditis elegans by forming a biofilm on the nematode head . C . elegans mutants with severe motility defects acquire almost no biofilm, indicating that normal animals accumulate the biofilm matrix as they move through a Yersinia lawn . Using the lectin wheat germ agglutinin as a probe, we show that the matrix on C . elegans contains carbohydrate produced by Yersinia . The carbohydrate is present in bacterial lawns prior to addition of nematodes, indicating that biofilm formation does not involve signaling between the two organisms . Furthermore, biofilm accumulation depends on continuous C . elegans exposure to a lawn of Yersinia bacteria.

J Bacteriol, 2004 Aug, 186(15), 4864 - 74
The sodium-driven flagellar motor controls exopolysaccharide expression in Vibrio cholerae; Lauriano CM et al.; Vibrio cholerae causes the life-threatening diarrheal disease cholera . This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence . Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype . We previously reported that the lack of a flagellum induces V . cholerae EPS expression . To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype . Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor . Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells . Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not . Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression . A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro . Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis . These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.

Water Res, 2004 Jul, 38(13), 3103 - 9
Evaluation of disinfectant efficacy against biofilm and suspended bacteria in a laboratory swimming pool model; Goeres DM et al.; Laboratory reactor systems designed to model specific environments enable researchers to explore environmental dynamics in a more controlled manner . This paper describes the design and operation of a reactor system built to model a swimming pool in the laboratory . The model included relevant engineering parameters such as filter loading and turn-overs per day . The water chemistry in the system's bulk water was balanced according to standard recommendations and the system was challenged with a bacterial load and synthetic bather insult, formulated to represent urine and perspiration . The laboratory model was then used to evaluate the efficacy of six chemical treatments against biofilm and planktonic bacteria . Results showed that the biofilm was able to accumulate on coupons and in the filter systems of reactors treated with either 1-3 mg/L free chlorine or 10 mg/L polyhexamethylene biguanide (PHMB) . All the treatments tested resulted in at least a 4 log reduction in biofilm density when compared to the control, but shock treatments were the most effective at controlling biofilm accumulation . A once weekly shock dose of 10 mg/L free chlorine resulted in the greatest log reduction in biofilm density . The research demonstrated the importance of studying a biofilm in addition to the planktonic bacteria to assess the microbial dynamics that exist in a swimming pool model.

Water Res, 2004 Jul, 38(13), 3083 - 91
UV disinfection in a model distribution system:; biofilm growth and microbial community; Pozos N et al.; Two model distribution systems were operated in parallel to investigate the impact of UV disinfection on water distribution system biofilms and microbial community composition . One system received an influent irradiated with UV light, whereas the control received the same influent with no treatment . The biofilm in the UV system, as compared to the control, was more responsive (i.e., had a greater increase in steady-state density of heterotrophic bacteria) to the increased nutrient availability afforded by a decrease in HRT from 12 to 2 h . However, the UV treatment did not have a consistent impact on the biofilm community, indicating the processes controlling HPC density were independent of the specific strains of bacteria forming the biofilm . There was evidence that particle shielding contributed to the survival of UV-susceptible bacteria . This hypothesis was consistent with the presence of UV-susceptible bacteria in the UV system, as well as the high similarity of the biofilm communities in the UV and control systems in one of the experiments . To simulate an intrusion event, opportunistic pathogens were added to each system after the biofilm community reached steady-state . Opportunistic pathogen attachment was not affected by the UV treatment, but was instead correlated to the biofilm density of heterotrophic bacteria.

Microb Ecol, 2004 Jan, 47(1), 87 - 95
Comparison of the phenotypes and genotypes of biofilm and solitary epiphytic bacterial populations on broad-leaved endive; Boureau T et al.; The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety . Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor . However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies . Broad-leaved endive (Cichorium endivia var . latifolia) harbors biofilms containing fluorescent pseudomonads . These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads . To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive . Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation . The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains . However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms . We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states . This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.

J Clin Periodontol, 2004 Aug, 31(8), 609 - 14
The effect of a chlorhexidine regimen on de novo plaque formation; Sekino S et al.; OBJECTIVE: To evaluate the effect of a pretreatment regimen that combined meticulous mechanical tooth cleaning with the daily use of chlorhexidine (rinse, gargle and tongue application) on de novo plaque formation and on the recolonization of various microbiological species in plaque and saliva during a 4-day period of no oral hygiene . MATERIAL AND METHODS: Ten subjects aged 24-36 years with gingivitis were recruited . The study was designed as a double blind cross-over clinical trial including two phases . Each experimental phase comprised one preparatory period of 7 days and one plaque accumulation period of 4 days . During the preparatory period, the volunteers (i) performed meticulous mechanical tooth cleaning using toothbrush and dentifrice and (ii) were, in addition, given two sessions of professional tooth cleaning (PTC) The final PTC was delivered after bacterial sampling had been made on Day 0 . In the Control group, no additional plaque control measures were included . In the Test group, the participants in addition to the mechanical measures (i) rinsed twice daily, for 60 s each time with a 0.2% chlorhexidine solution, (ii) gargled twice daily for 10 s with the chlorhexidine preparation, and finally (iii) brushed the dorsum of the tongue for 60 s, twice daily, with a 1.0% chlorhexidine gel . During the 4-day plaque accumulation period, the participants abstained from all mechanical and chemical plaque control measures . On Days 0, 1, 2 and 4 the quantity and quality of plaque formed was assessed by clinical means and by DNA probe techniques . The microbiota of the saliva was studied in samples obtained on Days 0 and 4 . RESULTS: It was demonstrated that chlorhexidine used as a mouthrinse combined with gargling and tongue application during the preparatory period significantly retarded the amount of plaque that formed on tooth surfaces during the following 4 days of no oral hygiene . Further, the number of microorganisms present in the biofilm representing Days 0, 1 and 2 of the "plaque accumulation period" was apparently affected by the use of the antiseptic . Among the microorganisms influenced by the chlorhexidine regimen, a substantial number belonged to the genus Actinomyces . It was also observed that the adjunctive use of chlorhexidine reduced the number of bacteria present in saliva at the end of the preparatory period (i.e . on Day 0) . After 4 days of no oral hygiene, the microbiota of the newly formed plaque in the Test and Control groups had many features in common . CONCLUSION: Habitat is critical in controlling the bacterial composition of the dental biofilm . The microbiota will tend to go back to the one that is characteristic of a given subject, once chemical antimicrobial means are withdrawn.

Shock, 2004 Aug, 22(2), 108 - 15
Polymorphonuclear neutrophils in posttraumatic osteomyelitis: cells recovered from the inflamed site lack chemotactic activity but generate superoxides; Wagner C et al.; The pathogenesis of posttraumatic osteomyelitis, one of the major complications after orthopedic surgery, is not yet understood . Formation of bacterial biofilms on the implant is presumed, conferring resistance to antibiotic therapy and probably also to the host defense mechanisms . In that context, the polymorphonuclear neutrophils (PMN) having infiltrated the infected site were recovered and characterized phenotypically and functionally . Loss of CD62L and upregulation of CD14 were seen, as was expression of CD83 . Expression of the latter is dependent on de novo protein synthesis and thus is indicative of an extended life span and a transdifferentiation of the PMN at the infected site . The infiltrated PMN had lost their chemotactic activity, whereas the capacity to produce superoxides was preserved and in some patients even enhanced . In vitro experiments done in parallel showed that long-term culture with interferon-gamma resulted in similar alterations of PMN: loss of chemotactic activity, whereas other functions of PMN, such generation of superoxides and phagocytosis of opsonized bacteria, were preserved or even enhanced . The loss of the migratory capacity of PMN having already emigrated from the blood vessel to the infected site is not expected to affect the host defense negatively . Assuming, however, that bacteria are organized as a biofilm and that infiltration into this biofilm is required for phagocytosis of the bacteria, our data could to some extent explain why despite being activated, the PMN are not able to control the infection . By releasing their cytotoxic, proteolytic, and collagenolytic potential, PMN might instead contribute to tissue destruction and eventually to osteolysis.

Microbiology, 2004 Jul, 150(Pt 7), 2401 - 7
Genetic analysis of Treponema denticola ATCC 35405 biofilm formation; Vesey PM et al.; Treponema denticola is a major aetiological organism implicated in periodontal disease . The interaction of T . denticola with other oral bacteria, in particular Porphyromonas gingivalis, in biofilm formation is thought to be an important step in the onset of periodontal disease . The interaction between T . denticola and P . gingivalis has been examined using a panel of T . denticola mutants and their effects on mixed biofilm formation tested in a static biofilm model . T . denticola ATCC 35405 did not form detectable biofilms on various inert surfaces . However, the spirochaete was demonstrated to form a biofilm with preattached P . gingivalis 381 . T . denticola cfpA, which lacks the cytoplasmic filament, was unable to produce a mixed biofilm with P . gingivalis . A T . denticola flgE mutant which lacks the flagella hook protein and is therefore non-motile displayed a reduced, but readily detectable, ability to form a mixed biofilm as did the T . denticola mutant which does not possess the major outer sheath protein (Msp) . The T . denticola lrrA mutant was only moderately defective in forming mixed biofilms with P . gingivalis . However, the T . denticola methyl-accepting chemotaxis protein (DmcA) did not appear to play a major role in mixed biofilm formation . In contrast, T . denticola lacking the PrtP protein for prolyl-phenylalanine-specific protease, showed an increased ability to form mixed biofilms and a prolonged viability in the biofilm.

Mol Microbiol, 2004 Aug, 53(3), 969 - 83
The Candida albicans CaACE2 gene affects morphogenesis, adherence and virulence; Kelly MT et al.; Morphogenesis between yeast and hyphal growth is a characteristic associated with virulence in Candida albicans and involves changes in the cell wall . In Saccharomyces cerevisiae, the transcription factor pair Ace2p and Swi5p are key regulators of cell wall metabolism . Here, we have characterized the CaACE2 gene, which encodes the only C . albicans homologue of S . cerevisiae ACE2 and SWI5 . Deleting CaACE2 results in a defect in cell separation, increased invasion of solid agar medium and inappropriate pseudohyphal growth, even in the absence of external inducers . The mutant cells have reduced adherence to plastic surfaces and generate biofilms with distinctly different morphology from wild-type cells . They are also avirulent in a mouse model . Deleting CaACE2 has no effect on expression of the chitinase gene CHT2, but expression of CHT3 and the putative cell wall genes CaDSE1 and CaSCW11 is reduced in both yeast and hyphal forms . The CaAce2 protein is localized to the daughter nucleus of large budded cells at the end of mitosis . C . albicans Ace2p therefore plays a major role in morphogenesis and adherence and resembles S . cerevisiae Ace2p in function .

Mol Microbiol, 2004 Aug, 53(3), 857 - 69
Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation; Tischler AD et al.; While studying virulence gene regulation in Vibrio cholerae during infection of the host small intestine, we identified VieA as a two-component response regulator that contributes to activating expression of cholera toxin . Here we report that VieA represses transcription of Vibrio exopolysaccharide synthesis (vps) genes involved in biofilm formation by a mechanism independent of its phosphorelay and DNA-binding activities . VieA controls the intracellular concentration of the cyclic nucleotide second messenger cyclic diguanylate (c-di-GMP) using an EAL domain that functions as a c-di-GMP phosphodiesterase . Two-dimensional thin layer chromatography of nucleotide extracts confirmed that VieA reduces the concentration of c-di-GMP, opposing the action of c-di-GMP synthetase proteins . Expression of unrelated V . cholerae c-di-GMP synthetase or phosphodiesterae proteins also modulated c-di-GMP concentration and vps gene expression . We propose that c-di-GMP synthetase and phosphodiesterase domain-containing proteins contribute to regulating biofilm formation by controlling c-di-GMP concentration .

Mol Microbiol, 2004 Aug, 53(3), 843 - 56
Investigating the role of secA2 in secretion and glycosylation of a fimbrial adhesin in Streptococcus parasanguis FW213; Chen Q et al.; Adhesion of Streptococcus parasanguis FW213, a primary colonizer, to the tooth surface is mediated mainly by peritrichous long fimbriae . The fimbrial structural unit, Fap1, is indispensable for fimbriae biogenesis, adhesion to an in vitro tooth model and biofilm formation . Mature Fap1 is a glycoprotein with an apparent molecular mass of 200 kDa . Glycosylated Fap1 is not present in some mutants screened from a transposon mutant library . Localization of the transposition sites revealed a gene determined to be secA2, which is distinct from the canonical secA gene . In FW213, glycosylated Fap1 was present in all the subcellular fractions including the cytoplasm . In VT1574, a non-polar mutant of secA2 generated by in frame deletion, Fap1 was not secreted . Glycosylated Fap1 was present in the membrane and cytoplasm of the mutant, although in greatly reduced amounts . Fap1 secretion and abundance were restored when VT1574 was complemented by a plasmid-borne secA2 . The secretion defect of the secA2 mutation appears to be limited to a small group of proteins such as Fap1 and FimA . These data suggested that Fap1 secretion rather than glycosylation was the major effect of the deletion of secA2; however, this deletion also had an impact on Fap1 abundance . Two more secA2 mutants with different regions deleted were tested for their ability to secrete Fap1 . One mutant was completely unable to secrete Fap1 while the other was able to secrete, but in a decreased amount . These data suggest that the region deleted in the latter mutant (nucleotides 2032-2337) is dispensable for Fap1 secretion .

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 313 - 8
A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence; Osiro D et al.; Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries . The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian . Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones . This indicates that high amounts of fastidian are not necessary for adhesion . In this paper we propose a kinetic model for X . fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls . Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 241 - 8
Antimicrobial effects of sanitizers against planktonic and sessile Listeria monocytogenes cells according to the growth phase; Chavant P et al.; This study was designed to investigate the individual or combined effects of sanitizers on survival of planktonic or sessile Listeria monocytogenes cells at different phase of growth . The sanitizers tested included: (i) acetic acid (pH 5.0), (ii) NaOH (pH 12.0), (iii) 10% Na2SO4, (iv) 10% Na2SO4 and acetic acid (pH 5.0), (v) 10% Na2SO4 and NaOH (pH 12.0), (vi) a quaternary ammonium (20 ppm) and (vii) glyceryl monolaurate (75 ppm) . Results revealed a great efficacy of alkaline treatments on both sessile and planktonic cells with a slightly higher resistance of 6 h biofilms . Quaternary ammonium appeared very effective in killing more than 98% of cells, but a resistance of 7 days biofilm was observed . Other sanitizers did not succeed in inhibiting totally the pathogen but acted in a similar way on both sessile and planktonic cells . Renewing the medium or not do not seem to be the major cause of a resistance emergence.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 163 - 73
What drives bacteria to produce a biofilm?
Jefferson KK.
Nearly 40 years ago, Dr . R.J . Gibbons made the first reports of the clinical relevance of what we now know as bacterial biofilms when he published his observations of the role of polysaccharide glycocalyx formation on teeth by Streptococcus mutans {Sci . Am . 238 (1978) 86} . As the clinical relevance of bacterial biofilm formation became increasingly apparent, interest in the phenomenon exploded . Studies are rapidly shedding light on the biomolecular pathways leading to this sessile mode of growth but many fundamental questions remain . The intent of this review is to consider the reasons why bacteria switch from a free-floating to a biofilm mode of growth . The currently available wealth of data pertaining to the molecular genetics of biofilm formation in commonly studied, clinically relevant, single-species biofilms will be discussed in an effort to decipher the motivation behind the transition from planktonic to sessile growth in the human body . Four potential incentives behind the formation of biofilms by bacteria during infection are considered: (1) protection from harmful conditions in the host (defense), (2) sequestration to a nutrient-rich area (colonization), (3) utilization of cooperative benefits (community), (4) biofilms normally grow as biofilms and planktonic cultures are an in vitro artifact (biofilms as the default mode of growth).

Res Microbiol, 2004 Jul-Aug, 155(6), 447 - 54
Reconstruction and computation of microscale biovolumes using geographical information systems: potential difficulties; Petrisor AI et al.; Biofilms are bacterial colonies enveloped in a matrix of extracellular polymeric secretions . Confocal scanning laser microscopy has been used in conjunction with different image analysis techniques to investigate the structure of biofilms . A major goal is to reconstitute the three-dimensional structure of biofilms, and compute or estimate the biovolumes . Our previous research focused on the utilization of remote sensing techniques and Geographical Information Systems for quantitative analyses of confocal images . The present study investigates potential problems in microbial imaging, and uses two approaches, the program COMSTAT and a Geographical Information Systems-based method, to reconstitute three-dimensional structures and estimate biovolumes . Volumes of thirty fluorescent polymeric microspheres with a known diameter were estimated and used as a ground truth, to statistically compare both methods . In a next step, the two approaches were used to estimate the biovolume of a section through a Pseudomonas aeruginosa biofilm . Difficulties were encountered in image acquisition due to the optical properties of the microbeads . Our results indicate that the Geographical Information Systems approach produced results consistent with the existing COMSTAT approach, and close to theoretical values, despite many problems inherent to each phase of this process . Also, the image classification process encountered several limitations . It is suggested that the unique constraints of the microscopic world may generate additional problems, especially related to image classification.

J Biol Chem, 2004 Sep 10, 279(37), 38749 - 54 Epub 2004 Jul 09.
Helix induction in antimicrobial peptides by alginate in biofilms; Chan C et al.; Bacterial exopolysaccharides provide protection against phagocytosis, opsonization, and dehydration and act as a major structural component of the extracellular matrix in biofilms . They contribute to biofilm-related resistance by acting as a diffusion barrier to positively charged antimicrobial agents including cationic antimicrobial peptides (CAPs) . We previously created novel CAPs consisting of a nonamphipathic hydrophobic core flanked by Lys residues and containing a Trp residue in the hydrophobic segment as a fluorescent probe . Peptides of this type above a specific hydrophobicity threshold insert spontaneously into membranes and have antimicrobial activity against Gram-positive and Gram-negative bacteria at micromolar concentrations . Here we show that alginate, a polymer of beta-d-mannuronate and alpha-l-guluronate secreted by the cystic fibrosis pathogen Pseudomonas aeruginosa, induces an alpha-helical conformation detected by circular dichroism spectroscopy and blue shifts in Trp fluorescence maxima in peptides above the hydrophobicity threshold, changes typically observed upon association of such peptides with nonpolar (membrane) environments . Parallel effects were observed in the archetypical CAPs magainin II amide and cecropin P1 . Fluorescence resonance energy transfer studies indicated that alginate induces peptide-peptide association only in peptides above the hydrophobicity threshold, suggesting that the hydrophilic alginate polymer behaves as an "auxiliary membrane" for the bacteria, demonstrating a unique protective role for biofilm matrices against CAPs.

J Photochem Photobiol B, 2004 Jul 19, 75(1-2), 21 - 5
delta-Aminolaevulinic acid mediated photodynamic antimicrobial chemotherapy on Pseudomonas aeruginosa planktonic and biofilm cultures; Lee CF et al.; To demonstrate photodynamic antimicrobial chemotherapy (PACT) against planktonic and biofilm cultures of Pseudomonas aeruginosa, using photoporphyrin IX which could be endogenously synthesized by administrating delta-aminolaevulinic acid (delta-ALA), and a light emitted diode (LED) array to photoactivate the photosensitizer . P . aeruginosa suspended cells or biofilms, grown on a rotating disk reactor, were treated by different concentrations of delta-ALA in the dark for 1 h, followed by LED irradiation for various time . Regrowth experiments were conducted by placed PACT-treated disks back to a sterile reactor . Viable cells were determined by serial dilution and plate counts . Both P . aeruginosa planktonic and biofilm cells were inhibited by PACT with light doses or photosensitizer concentrations increasing . Treatments of planktonic cells with 10 mM delta-ALA and incident dose 240 J cm(-2) or 7.5 mM ALA and incident dose 360 J cm(-2) led to completely photoinactivation . No viable biofilm cells were found after treatment of 20 mM delta-ALA and incident dose 240 J cm(-2) . However, regrowth was observed once PACT-treated biofilms were put back to a sterile reactor . Regrowth could be prevented only if biofilm samples were treated PACT twice . delta-ALA-mediated PACT on P . aeruginosa planktonic and biofilm cells was effective, though the detailed mechanism still required further investigation.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2004 Jul, 98(1), 53 - 9
Denture stomatitis: a role for Candida biofilms; Ramage G et al.; OBJECTIVE: To assess the contribution of Candida biofilms to the etiology of denture stomatitis . STUDY DESIGN: Samples of denture acrylic were retrieved from patients with denture stomatitis and subjected to scanning electron microscopy (SEM) analysis . Oral swab and swish samples were taken from the same group of patients and representative C albicans isolates recovered were used to investigate the kinetics of biofilm development in vitro . RESULTS: Candida biofilms could be visualized by SEM directly from denture samples from patients with denture stomatitis . These biofilms showed a propensity to adhere along cracks and imperfections of the denture acrylic . C albicans clinical isolates were able to form biofilms in vitro, although differences in the extent of biofilm formation were observed for different isolates recovered from the same patient . Susceptibility testing indicated that the resulting biofilms showed increased resistance to antifungal treatment . Presence of serum and saliva conditioning films increased the initial adherence of selected isolates but had little effect in overall biofilm formation . CONCLUSIONS: Candida biofilms play a role in denture stomatitis.

J Clin Microbiol, 2004 Jul, 42(7), 3073 - 6
Use of in vivo-generated biofilms from hemodialysis catheters to test the efficacy of a novel antimicrobial catheter lock for biofilm eradication in vitro; Kite P et al.; This biofilm study was conducted to assess the in vitro activity of tetrasodium EDTA on catheters that had been routinely removed from hemodialysis patients at Leeds Teaching Hospitals Trust due to maturation of fistula . Catheters were screened by culture of through-catheter flush, and isolates were identified by standard methodologies; 20 isolates were found to be biofilm positive . Initial biofilm cell count levels averaged above 10(5) CFU/1-cm catheter section . Bacteria identified in the biofilms were gram-negative (1 isolate), gram-positive (11 isolates), or mixed species (8 isolates) . After a 24-h lock, 40 mg of tetrasodium EDTA per ml was effective at eradicating the total biofilm viable count in almost all cases . The efficacy of tetrasodium EDTA as a catheter lock potentially shows that this agent could substantially reduce catheter-related infections and be used to treat patients with limited access.

J Clin Microbiol, 2004 Jul, 42(7), 3052 - 8
Studies on the involvement of the exopolysaccharide produced by cystic fibrosis-associated isolates of the Burkholderia cepacia complex in biofilm formation and in persistence of respiratory infections; Cunha MV et al.; Bacteria belonging to the Burkholderia cepacia complex (BCC) are important opportunistic pathogens that lead to respiratory infections in patients with cystic fibrosis (CF) . The clinical outcome following colonization with BCC bacteria is highly variable, and so far, unpredictable . A large percentage (80 to 90%) of BCC isolates from CF patients produce the exopolysaccharide (EPS) cepacian, which has been hypothesized to play a role in the colonization and persistence of these bacteria in the CF lung . In this work, we demonstrate that although it is not required for the initiation of biofilm formation, cepacian plays a role in the establishment of thick biofilms . This conclusion was based on a comparison of the abilities of EPS-defective mutants derived from a B . cepacia mucoid CF isolate by random plasposon insertion mutagenesis and the ability of the parental strain to form biofilms . However, the systematic characterization of 108 CF isolates, corresponding to 15 distinct strains, indicated that other strain-dependent factors are also involved in the development of thick, mature biofilms . The isolates examined belonged to the species B . cepacia, B . multivorans, B . cenocepacia, and B . stabilis and were obtained during a 7-year period of surveillance from 21 CF patients receiving care at the major Portuguese CF center . Most of them (90%) were serial isolates from 12 persistently infected patients . In spite of the concept that bacteria growing in biofilms display more resistance to antibiotics and to host phagocyte killing than do planktonically growing cells, no clear correlation could be established between the ability of the various strains examined to produce EPS and/or to form biofilms in vitro and the persistence or virulence of the respiratory infections they caused in different patients.

Lett Appl Microbiol, 2004, 39(2), 215 - 9
Isolation of bacteriophages from the oral cavity; Hitch G et al.; AIMS: To isolate bacteriophages lytic for oral pathogens from human saliva, dental plaque and mature biofilms constituted from saliva-derived bacteria . METHODS AND RESULTS: Saliva and dental plaque samples from healthy volunteers and from patients with gingivitis and periodontitis were examined for the presence of lytic bacteriophage using a panel of oral pathogens and bacteria isolated from the samples . Samples were also enriched for bacteriophage using static culture techniques and mature biofilms . A limited number of samples contained bacteriophage particles that were visualized using electron microscopy . Cultures yielded phage infecting non-oral bacteria (Proteus mirabilis) but no bacteriophage specific for recognized oral pathogens were found . Some micro-organisms from the oral microflora elaborated antibacterial substances that inhibited growth of other residents of the oral cavity . CONCLUSIONS: Unlike other ecosystems, the composition of the oral cavity does not appear to be heavily influenced by interactions between bacteriophages and their hosts . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage for control of oral infections may need to be obtained from other sources . Antibacterial substances derived from some members of the oral microflora warrant investigation as potential antibiotics.

Indian J Exp Biol, 2003 Sep, 41(9), 1023 - 9
Control of metallic corrosion through microbiological route; Maruthamuthu S et al.; Involvement of biofilm or microorganisms in corrosion processes is widely acknowledged . Although majority of the studies on microbiologically induced corrosion (MIC) have concentrated on aerobic/anaerobic bacteria . There are numerous aerobic bacteria, which could hinder the corrosion process . The microbiologically produced exopolymers provide the structural frame work for the biofilm . These polymers combine with dissolved metal ions and form organometallic complexes . Generally heterotrophic bacteria contribute to three major processes: (i) synthesis of polymers (ii) accumulation of reserve materials like poly-beta-hydroxy butrate (iii) production of high molecular weight extracellular polysaccharides . Poly-beta-hydroxy butyrate is a polymer of D(-)beta-hydroxy butrate and has a molecular weight between 60,000 and 2,50,000 . Some extracellular polymers also have higher molecular weights . It seems that higher molecular weight polymer acts as biocoating . In the present review, role of biochemistry on corrosion inhibition and possibilities of corrosion inhibition by various microbes are discussed . The role of bacteria on current demand during cathodic protection is also debated . In addition, some of the significant contributions made by CECRI in this promising area are highlighted.

Indian J Exp Biol, 2003 Sep, 41(9), 972 - 85
Bioremediation of chromium contaminated environments; Kamaludeen SP et al.; Bioremediation is the most promising and cost effective technology widely used nowadays to clean up both soils and wastewaters containing organic or inorganic contaminants . Discharge of chromium containing wastes has led to destruction of many agricultural lands and water bodies . Utilisation of chromium(Cr) reducing microbes and their products has enhanced the efficiency of the process of detoxification of Cr(VI) to Cr(III) . This review focuses mainly on the current technologies prevalent for remediation like natural attenuation, anaerobic packed bed bioreactors (using live cells, Cr(VI) reductases or their byproducts) and use of engineered microorganisms . Treatment of wastewaters by biosorption or using biofilms and immobilized microbial cells are also discussed.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004 Jul, 39(7), 1843 - 52
Attachment characteristics of biofilms in fixed-lock media for swine wastewater treatment; Rim JM et al.; A correlation between equilibrium biofilm thickness and biomass density in a laboratory-scale submerged media aeration system was evaluated . The system had lock-type media, and swine wastewater was used as a substrate . The influent wastewater had chemical oxygen demand and biochemical oxygen demand concentrations of 2940-3800 and 1310-1730 mg/L, respectively . The hydraulic retention time of the system was varied from 0.5 to 2 days . The following conclusions can be drawn from the operational results: (i) the maximum biofilm density was observed when the equilibrium biofilm thickness was 180-200 microm, (ii) the activated biomass represented as volatile suspended solids per unit area decreased after 10 days of operation, and (iii) the removal rate of chemical oxygen demand increased rapidly up for an equilibrium biofilm thickness up to 200 microm, but no further increase was observed for thicknesses of 200-1200 microm.

Appl Environ Microbiol, 2004 Jul, 70(7), 4340 - 8
Development of a multispecies oral bacterial community in a saliva-conditioned flow cell; Foster JS et al.; Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth . Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models . We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum . Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species . Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates . Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation . Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S . gordonii, regardless of the inoculation order . However, the representation of A . naeslundii and V . atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms . Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase . Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum . Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.

Appl Environ Microbiol, 2004 Jul, 70(7), 4187 - 92
Molecular analysis of shower curtain biofilm microbes; Kelley ST et al.; Households provide environments that encourage the formation of microbial communities, often as biofilms . Such biofilms constitute potential reservoirs for pathogens, particularly for immune-compromised individuals . One household environment that potentially accumulates microbial biofilms is that provided by vinyl shower curtains . Over time, vinyl shower curtains accumulate films, commonly referred to as "soap scum," which microscopy reveals are constituted of lush microbial biofilms . To determine the kinds of microbes that constitute shower curtain biofilms and thereby to identify potential opportunistic pathogens, we conducted an analysis of rRNA genes obtained by PCR from four vinyl shower curtains from different households . Each of the shower curtain communities was highly complex . No sequence was identical to one in the databases, and no identical sequences were encountered in the different communities . However, the sequences generally represented similar phylogenetic kinds of organisms . Particularly abundant sequences represented members of the alpha-group of proteobacteria, mainly Sphingomonas spp . and Methylobacterium spp . Both of these genera are known to include opportunistic pathogens, and several of the sequences obtained from the environmental DNA samples were closely related to known pathogens . Such organisms have also been linked to biofilm formation associated with water reservoirs and conduits . In addition, the study detected many other kinds of organisms at lower abundances . These results show that shower curtains are a potential source of opportunistic pathogens associated with biofilms . Frequent cleaning or disposal of shower curtains is indicated, particularly in households with immune-compromised individuals.

Appl Environ Microbiol, 2004 Jul, 70(7), 3996 - 4003
Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms; Zhao T et al.; Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites . A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L . monocytogenes, by two agar plate assays . Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity . All 24 isolates which produced metabolites inhibitory to L . monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE) . A five-strain mixture of 10(3) CFU of L . monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days . Substantial inhibition of L . monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C . The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp . lactis (two isolates), and Lactobacillus plantarum (one isolate) . The anti-L . monocytogenes activity of these isolates was evaluated in biofilms of L . monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C . Results revealed that two isolates (E . durans strain 152 and L . lactis subsp . lactis strain C-1-92) were highly inhibitory to L . monocytogenes (growth inhibition of >5 log(10) CFU of L . monocytogenes/cm(2)) . These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L . monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.

Int J Food Microbiol, 2004 Aug 15, 95(1), 29 - 39
Prevalence of microbial biofilms on selected fresh produce and household surfaces; Rayner J et al.; Investigations of biofilms in domestic environments are sparsely represented in the literature . In this study, samples of various household surfaces, including food, laundry and kitchen items, were analyzed for evidence of biofilm presence . Visualization of the surfaces was carried out using cryostage scanning electron microscopy (CSEM) and light microscopy . Qualitative evidence of the presence of biofilm formation was obtained from all of the sample groups analyzed, suggesting the widespread existence of microorganisms in biofilms on domestic surfaces . This suggests that biofilms may be important in household hygiene, and highlights the need for standardized, approved biofilm methods suitable for consumer products testing.

J Appl Microbiol, 2004, 97(2), 262 - 70
Impact of cleaning and disinfection agents on biofilm structure and on microbial transfer to a solid model food; Midelet G et al.; AIM: To determine how single cells and microcolonies transfer to food from open surfaces in the meat industry . METHODS AND RESULTS: Biofilms of four bacterial strains isolated from food processing surfaces were established on stainless steel substrates conditioned with meat exudate in the presence or absence of CaCl(2) . Image analysis of the biofilms showed that the addition of calcium resulted in an increase of the number and size of microcolonies with two strains: Staphylococcus sciuri and Pseudomonas fluorescens . Image analysis of the biofilms of those two strains grown in the presence of calcium was performed before and after contacts with tryptone soya agar as a solid model food . For the biofilms treated or not with a chlorinated alkaline agent, where a decrease in surface coverage occurred, it was accompanied by a decrease in the percentage of the coverage accounted for by microcolonies (P(m)) . Attachment strength was greater for P . fluorescens than for S . sciuri . When the P . fluorescens biofilms were treated with a solution containing glutaraldehyde, the contacts did not modify their structure . By contrast, their treatment with chlorinated alkaline resulted, after contacts, in the smallest coverage and P(m) . With S . sciuri, a decrease in coverage after contacts always occurred and was the greatest for the untreated biofilms . CONCLUSIONS: After contacts between biofilms and a solid model food, microcolonies were preferentially detached compared with single cells . A chlorinated alkaline product either decreased biofilm attachment strength (P . fluorescens) or unexpectedly increased it (S . sciuri), whereas a glutaraldehyde-based disinfectant increased both attachment strength and microcolony cohesion . SIGNIFICANCE AND IMPACT OF THE STUDY: The contaminating potential of a surface depends not only on the level of contamination but also on the nature, structure and history of the contamination.

Water Sci Technol, 2004, 49(9), 307 - 12
Geosmin occurrence in riverine cyanobacterial mats: is it causing a significant health hazard?
Blaha L, Sabater S, Babica P, Vilalta E, Marsalek B.
Toxicity endpoints (nonspecific cytotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, and mutagenicity) were studied in cyanobacterial mats obtained from a shallow river . Some of the cyanobacterial mats tested were known to be non-geosmin producers, while others were geosmin-producers . No microcystin-like compounds were detected by HPLC in any of the biofilm samples . The mutagenicity and neurotoxicity of biofilm metabolites was negligible, and generally weak adverse effects of biofilm extracts detected in a battery of in-vitro assays indicated relatively low human health risks associated with biofilm toxicity . While the toxicity responses detected in the studied biofilms were weak, effects were not related to production of geosmin . It was therefore concluded that the production of this metabolite cannot be taken as an indication per se of the existence of a health hazard.

Water Sci Technol, 2004, 49(9), 211 - 7
Biofilm in water pipelines; a potential source for off-flavours in the drinking water; Skjevrak I et al.; Volatile organic compounds (VOC) are identified in natural biofilm established in plastic pipes used at the drinking water supply . Odour potent VOCs such as ectocarpene, dictyopterene A and C', geosmin, beta-ionone, 6-methyl-5-hepten-2-one, menthol and menthone were prominent compounds in biofilm in the distribution network and at raw water test sites, and are associated with algae and cyanobacteria present in the raw water source.

Water Sci Technol, 2004, 49(9), 33 - 9
Periphyton: a primary source of widespread and severe taste and odour; Watson SB et al.; In the last decade, a late summer-fall taste and odour problem has been a prolonged and annual event in the St Lawrence River (SLR) . Earlier work identified the earthy/musty compounds geosmin and particularly, 2-methylisoborneol (GM-MIB), and ruled out Lake Ontario as a major source, but did not identify the biological origins . In 2000, we investigated the source(s) and underlying causes . We sampled littoral sites in the SLR near Cornwall, ON, and found that macrophytes (or associated biofilms) may be primary GM sources . Zebra mussel homogenate yielded low GM-MIB levels, but several associated actinomycetes generated high in vitro amounts . Periphyton from rocks showed significant yields, with cell-bound GM-MIB up to one hundred times the levels in overlying water . In 2001, we followed seasonal changes at some of these sites . Periphyton GM-MIB showed intriguing spatial and temporal patterns . Several cyanobacteria in these biofilms were identified as potential odour sources, notably Oscillatoriales . We conclude: i) periphyton is a major odour source in the SLR; ii) other biota such as macrophytes and mussels may also contribute; iii) seasonality in GM-MIB production and ratios indicate changes in cell production and/or taxa in response to environment . These results may account for the recent onset of the problematic odour events, which represent chemical signals of the increased water transparency and littoral surface area following the widespread dreissenid mussel invasion to the Great Lakes . Our data raise key questions about the processes that trigger the tremendous variability in biota and GM-MIB production in the SLR, the subject of our continued research.

Water Sci Technol, 2004, 49(9), 25 - 31
Nuisance odours produced by benthic cyanobacteria in a Mediterranean river; Vilalta E et al.; Geosmin dynamics in the Llobregat waters were related to the waxing and waning of benthic cyanobacterial mats developing in the river . Geosmin concentration in the water during 2002 reached a maximum of 204 ng L(-1), and coincided with an abundance of cyanobacteria in the river . Cyanobacterial mats were favoured by the high nutrient content of the waters . The cyanobacterial mats experienced a process of growth in thickness (attached forms), until they became unattached and drifted downstream (free-floating forms), accumulating in shallow areas of the river . Geosmin in the biofilm ranged from 0.55 +/- 0.97 ng geosmin per mg DW(-1) in the attached biofilms and 5.25 +/- 4.96 ng geosmin per mg DW(-1) in the free-floating biofilms . While the attached mats could be responsible for the local occurrence of geosmin at a given site, the free-floating mats became a relevant agent for the dispersion of the metabolite downstream . This impression was reinforced by the extremely high correlation between the geosmin content in the free-floating biofilm and in the water (r = 0.917, p = 0.00001) . In order to reduce the geosmin concentration and accumulation of the cyanobacterial mats in shallow river waters, the nutrient content should be controlled and the natural flow conditions restored, to prevent the growth and accumulation of the geosmin-producing cyanobacterial mats.

Ann Agric Environ Med, 2004, 11(1), 9 - 12
Risk of exposure to Legionella in dental practice; Szymanska J; Aerosols generated in dental operations are a source of exposure to microorganisms proliferated within dental unit waterlines (DUWL) biofilm . It has been suggested that presence of Legionella species in these aerosols may contribute to potential health hazards for dental staff and patients . The article attempts to provide a brief overview of the current knowledge about Legionella, its prevalence in DUWL, immunological reactions of the dentists and concepts for prophylaxis of Legionella in dentists' work place.

J Pharm Pharmacol, 2004 Jul, 56(7), 847 - 54
Biofilm formation and changes in bacterial cell surface hydrophobicity during growth in a CAPD model system; Hanlon GW et al.; Peritonitis is a frequent complication of continuous ambulatory peritoneal dialysis (CAPD), with patients suffering recurrent attacks . The microorganisms most frequently implicated in the infection are the skin microflora, in particular, the coagulase-negative staphylococci such as Staphylococcus epidermidis . These microorganisms gain access to the peritoneal cavity via the in-dwelling silicone rubber catheter in the abdominal wall and often persist as biofilms on the surface of the catheter . The surface characteristics of S . epidermidis were monitored during growth in a CAPD in-vitro model together with their ability to adhere to silicone rubber substrata . Fresh dialysis fluid exerted an injurious effect on the cells leading to a decrease in cell numbers but during the simulated dialysis period the cells adapted to the applied stresses . Over a 96-h period in the model both a clinical isolate and a skin isolate of S . epidermidis adopted a more hydrophobic phenotype . The data presented here show that the bacteria grown in this in-vivo reflective CAPD model continually adapt to their environment and become more tolerant to the stresses imposed . The adapted cells were seen to colonise silicone rubber substrata.

BMC Microbiol . 2004 Jul 02;4(1):25.
Effects of natural and chemically synthesized furanones on quorum sensing in Chromobacterium violaceum; Martinelli D et al.; BACKGROUND: Cell to cell signaling systems in Gram-negative bacteria rely on small diffusible molecules such as the N-acylhomoserine lactones (AHL) . These compounds are involved in the production of antibiotics, exoenzymes, virulence factors and biofilm formation . They belong to the class of furanone derivatives which are frequently found in nature as pheromones, flavor compounds or secondary metabolites . To obtain more information on the relation between molecular structure and quorum sensing, we tested a variety of natural and chemically synthesized furanones for their ability to interfere with the quorum sensing mechanism using a quantitative bioassay with Chromobacterium violaceum CV026 for antagonistic and agonistic action . We were looking at the following questions: 1) Do these compounds affect growth? 2) Do these compounds activate the quorum sensing system of C . violaceum CV026? 3) Do these compounds inhibit violacein formation induced by the addition of the natural inducer N-hexanoylhomoserine lactone (HHL)? 4) Do these compounds enhance violacein formation in presence of HHL? RESULTS: The naturally produced N-acylhomoserine lactones showed a strong non-linear concentration dependent influence on violacein production in C . violaceum with a maximum at 3.7*10-8 M with HHL . Apart from the N-acylhomoserine lactones only one furanone (emoxyfurane) was found to simulate N-acylhomoserine lactone activity and induce violacein formation . The most effective substances acting negatively both on growth and quorum sensing were analogs and intermediates in synthesis of the butenolides from Streptomyces antibioticus . CONCLUSION: As the regulation of many bacterial processes is governed by quorum sensing systems, the finding of natural and synthetic furanones acting as agonists or antagonists suggests an interesting tool to control and handle detrimental AHL induced effects.Some effects are due to general toxicity; others are explained by a competitive interaction for LuxR proteins . For further experiments it is important to be aware of the fact that quorum sensing active compounds have non-linear effects . Inducers can act as inhibitors and inhibitors might be able to activate or enhance the quorum sensing system depending on chemical structure and concentration levels.

J Bacteriol, 2004 Jul, 186(14), 4759 - 73
Evidence that the algI/algJ gene cassette, required for O acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer; Franklin MJ et al.; Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype . O acetylation of alginate plays an important role in the ability of mucoid P . aeruginosa to form biofilms and to resist complement-mediated phagocytosis . The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF) . The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm . Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria . In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis . The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis . The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer . AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ . The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3 . Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate . These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides .

J Bacteriol, 2004 Jul, 186(14), 4665 - 84
Global gene expression in Staphylococcus aureus biofilms; Beenken KE et al.; We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K . E . Beenken, J . S . Blevins, and M . S . Smeltzer, Infect . Immun . 71:4206-4211, 2003) . In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo . Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo . In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S . aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures . Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S . aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 {strain 252}, and MSSA-476) . The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture . Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions . Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions . A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions .

J Bacteriol, 2004 Jul, 186(14), 4492 - 501
Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system; Danhorn T et al.; The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces . Adherence of A . tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions . Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results . The increase in surface interactions under phosphate limitation was observed in both static culture and continuous-culture flow cells . Statistical analysis of confocal micrographs obtained from the flow cell biofilms revealed that phosphate limitation increased both the overall attached biomass and the surface coverage, whereas the maximum thickness of the biofilm was not affected . Functions encoded on the two large plasmids of A . tumefaciens C58, pTiC58 and pAtC58, were not required for the observed phosphate effect . The phosphate concentration at which increased attachment was observed triggered the phosphate limitation response, controlled in many bacteria by the two-component regulatory system PhoR-PhoB . The A . tumefaciens phoB and phoR orthologues could only be disrupted in the presence of plasmid-borne copies of the genes, suggesting that this regulatory system might be essential . Expression of the A . tumefaciens phoB gene from a tightly regulated inducible promoter, however, correlated with the amount of biofilm under both phosphate-limiting and nonlimiting conditions, demonstrating that components of the Pho regulon influence A . tumefaciens surface interactions .

J Bacteriol, 2004 Jul, 186(14), 4486 - 91
Detachment characteristics and oxacillin resistance of Staphyloccocus aureus biofilm emboli in an in vitro catheter infection model; Fux CA et al.; Catheter-related bloodstream infections due to Staphylococcus aureus are of increasing clinical importance . The pathophysiological steps leading to colonization and infection, however, are still incompletely defined . We observed growth and detachment of S . aureus biofilms in an in vitro catheter-infection model by using time-lapse microscopy . Biofilm emboli were characterized by their size and their susceptibility for oxacillin . Biofilm dispersal was found to be a dynamic process in which clumps of a wide range of diameters detach . Large detached clumps were highly tolerant to oxacillin compared with exponential-phase planktonic cultures . Interestingly, the degree of antibiotic tolerance in stationary-phase planktonic cultures was equal to that in the large clumps . The mechanical disruption of large clumps reduced the minimal bactericidal concentration (MBC) by more than 1,000 times . The MBC for whole biofilm effluent, consisting of particles with an average number of 20 bacteria was 3.5 times higher than the MBC for planktonic cultures . We conclude that the antibiotic resistance of detached biofilm particles depends on the embolus size and could be attributed to nutrient-limited stationary-phase physiology of cells within the clumps . We hypothesize that the detachment of multicellular clumps may explain the high rate of symptomatic metastatic infections seen with S . aureus .

J Bacteriol, 2004 Jul, 186(14), 4476 - 85
SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14; Caiazza NC et al.; Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state . Here we report a new class of P . aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation . The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P . aeruginosa PA14 and encodes a protein of unknown function . Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype . Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility . Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole . A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels . Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function . We discuss the results presented here in the context of microbial development as it applies to biofilm formation .

J Bacteriol, 2004 Jul, 186(14), 4466 - 75
Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation; Jackson KD et al.; Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents . Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute . Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P . aeruginosa strains PAO1 and PA14 . We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development . Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome . Reverse genetics was employed to generate mutations in genes from these clusters . We discovered that one group of genes, designated psl, are important for biofilm initiation . A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays . This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis . These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix . Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P . aeruginosa in cystic fibrosis and other infections involving biofilms .

J Bacteriol, 2004 Jul, 186(14), 4457 - 65
Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix; Friedman L et al.; Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix . Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix . The pel gene cluster is involved in the production of a glucose-rich matrix material in P . aeruginosa strain PA14 (L . Friedman and R . Kolter, Mol . Microbiol . 51:675-690, 2004) . Here we investigate the role of the pel gene cluster in P . aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material . The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing . P . aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials . Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P . aeruginosa strains PA14 and ZK2870 .

J Bacteriol, 2004 Jul, 186(14), 4449 - 56
Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development; Matsukawa M et al.; An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis . Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P . aeruginosa strain PAO1 . We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P . aeruginosa PAO1 biofilms . Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552 . The mutant with a PA2231 defect formed thin unstructured abnormal biofilms . The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent . Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA . We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P . aeruginosa PAO1 biofilms .

Mol Microbiol, 2004 Jul, 53(2), 497 - 515
Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant; Yildiz FH et al.; Reversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats . Microarray expression profiling studies of the rugose and smooth variants of the same strain led to the identification of 124 differentially regulated genes . Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPS(El Tor), a rugose-associated extracellular polysaccharide . The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation-specific gene expression . Bioinformatics analysis of these expression data shows that 'rugosity' and 'smoothness' are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.

Math Med Biol, 2004 Jun, 21(2), 147 - 66
The role of the biofilm matrix in structural development; Cogan NG et al.; Although the initiation, development and control of biofilms has been an area of experimental investigation for more than three decades, the role of extra-cellular polymeric substance (EPS) has not been well studied . We present a mathematical description of the EPS matrix to study the development of heterogeneous biofilm morphology . In developing the model, we assume that the biofilm is a biological gel composed of EPS and water . The bacteria are enmeshed in the network and are the producers of the polymer . In response to external conditions, gels absorb or expel solvent causing swelling or contraction due to osmotic pressure gradients . The physical morphology of the biofilm depends on the temperature, solvent composition, pH and ionic concentrations through osmotic pressure . This gives a physically based mechanism for the redistribution of biomass within the biofilm . Analysis of a reduced model indicates that biomass redistribution, through the mechanism of swelling, may induce the formation of isolated towers or mushroom clusters by spatial variation in EPS production which leads to gradients in osmotic pressure.

J Biomed Mater Res, 2004 Aug 1, 70A(2), 274 - 82
Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide; Maeyama R et al.; We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein-expressing Escherichia coli and red fluorescence staining of exopolysaccharides . Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three-dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides . These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time . Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time-dependent growth of biofilms . The curli (surface-located fibers specifically binding to fibronectin and laminin)-producing Escherichia coli strain, YMel, significantly proliferated on fibronectin-coated polyurethane, whereas the curli-deficient isogenic mutant, YMel-1, did not . The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential .

Clin Infect Dis, 2004 Jun 15, 38(12), 1690 - 9 Epub 2004 May 25.
Chemotaxonomic analysis of bacterial populations colonizing the rectal mucosa in patients with ulcerative colitis; Macfarlane S et al.; The etiology of ulcerative colitis (UC) is unknown, but evidence links it to bacteria belonging to the normal colonic microbiota . The aims of this study were to characterize bacteria colonizing the rectal epithelium, and to investigate whether significant differences existed in UC . Rectal biopsy specimens were obtained via endoscopy from 9 patients with active colitis and 10 patients without inflammatory bowel disease . Complex bacterial communities colonized the rectal mucosa in all subjects . Overall, 72 bacterial taxa (18 genera) were detected . Twenty species were common to both groups, but only differences in bifidobacteria were statistically significant (P=.005) . Peptostreptococci were only detected in patients with UC . Microscopy showed that bacteria in mucosal biofilms often occurred in microcolonies . Interindividual variations in mucosal biofilms made it difficult to assign a role for specific bacteria in UC etiology . However, differences in bifidobacteria and peptostreptococci may implicate these organisms in this disease.

Environ Sci Technol, 2004 Jun 1, 38(11), 3019 - 27
Uranium complexes formed at hematite surfaces colonized by sulfate-reducing bacteria; Neal AL et al.; Modeling uranium (U) transport in subsurface environments requires a thorough knowledge of mechanisms likely to restrict its mobility, such as surface complexation, precipitation, and colloid formation . In closed systems, sulfate-reducing bacteria (SRB) such as Desulfovibrio spp . demonstrably affect U immobilization by enzymatic reduction of U(VI) species (primarily the uranyl ion, UO2(2+), and its complexes) to U(IV) . However, our understanding of such interactions under chronic U(VI) exposure in dynamic systems is limited . As a first step to understanding such interactions, we performed bioreactor experiments under continuous flow to study the effect of a biofilm of the sulfate-reducing bacterium Desulfovibrio desulfuricans attached to specular hematite (alpha-Fe2O3) surfaces on surface-associated U(VI) complexation, transformation, and mobility . Employing real-time microscopic observation and X-ray photoelectron spectroscopy (XPS), we show that the characteristics of the U(VI) complex(es) formed at the hematite surface are influenced by the composition of the bulk aqueous phase flowing across the surface and bythe presence of surface-associated SRB . The XPS data further suggest higher levels of U associated with hematite surfaces colonized by SRB than with bacteria-free surfaces . Microscopic observations indicate that at least a portion of the U(VI) that accumulates in the presence of the SRB is exterior to the cells, possibly associated with the extracellular biofilm matrix . The U4f7/2 core-region spectrum and U5f2 valence-band spectrum provide preliminary evidence that the SRB-colonized hematite surface accumulates both U(VI) and U(IV) phases, whereas only the U(VI) phase(s) accumulates on uncolonized hematite surfaces . The results suggest that mineral surfaces exposed to a continuously replenished supply of U(VI)-containing aqueous phase will accumulate U phases that may be more representative of those that exist in U-contaminated aquifers than those which accumulate in closed experimental systems . These phases should be considered in models attempting to predict U transport through subsurface environments.

Water Res, 2004 Jul, 38(12), 2907 - 17
Anti-sized reed bed system for animal wastewater treatment: a comparative study; Zhao YQ et al.; This paper presents a comparative study on the treatment of high-strength animal wastewater in two parallel lab-scale constructed reed bed systems, namely progressively-sized system and anti-sized system, which have same configuration but different arrangement of bed media . The reed bed systems were operated in a tidal flow pattern to treat diluted pig slurry . Detailed analyses were carried out for the removal of some key pollutants including COD, BOD5, NH4-N, P and suspended solids . The results showed that both systems have considerable capacity for the removal of solids, organic matter and inorganic nutrients . The formation of biofilms on the surfaces of gravel media in both reed bed systems was monitored by scanning selected gravel samples using scanning electron microscopy . In general, no significant difference was detected with regard to the percentage pollutant removal in the systems . However, the anti-sized system demonstrated a clear advantage in its ability to slow down the clogging of bed media and avoid the impairment of long-term functioning and sustainability of the beds . A conceptual model was developed to predict the occurrence of the clogging . The validity of the model was tested using data from this study and from the literatures.

Water Res, 2004 Jul, 38(12), 2865 - 73
Indicators of biofilm development and activity in constructed wetlands microcosms; Ragusa SR et al.; Methods to measure protein, exopolysaccharide, viable cell number and INT reduction activity were tested on biofilm growing in a wastewater batch reactor . They were shown to be meaningful indicators of biofilm growth and correlated well with each other . Protein, exopolysaccharide, viable cells and INT reduction rates increased linearly over time . Viable cell number exhibited strong linear correlations with protein (R2= 0.98) and exopolysaccharide (R2= 0.99) while INT reduction rate was somewhat less well correlated (R2= 0.90) . Our results indicate production rates of 0.91 x 10(-7) microg EPS per viable cell and 1.0 x 10(-7) microg protein per viable cell . Protein and polysaccharide specific INT reduction rates decreased by approximately 50%, whereas viable cell specific INT reduction rates decreased by 65% and the protein to polysaccharide ratio stayed relatively constant at between 1.1 and 1.2 as the biofilm developed . Measurement of protein, polysaccharide, viable cells and INT reduction rate at depth within the bioreactor showed that they were concentrated in the top 1cm of the influent end of the reactor and each decreased to a base level within 4.5 cm of the inlet . Protein to polysaccharide ratios increased with depth in the reactor and the specific INT reduction rates were maximal at 4.5 cm depth . The results indicate that the biomass can take upwards of 100 days to stabilize during batch (fill and draw) operation of subsurface wetlands and that the relative ratios of biomass components remain relatively constant during biofilm growth . Also, it appears that filtration of suspended solids results in biomass concentration at the inlet to the wetland.

Biometals, 2004 Jun, 17(3), 271 - 8
Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans; Francesca B et al.; Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries . Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear . The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S . mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena . Our findings are consistent with the hypothesis that S . mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form . Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases . On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S . mutans . Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S . mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity . In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S . mutans free, aggregated or biofilm on abiotic surfaces . Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S . mutans pathogenicity.

Biometals, 2004 Jun, 17(3), 267 - 70
Iron sequestration by human lactoferrin stimulates P . aeruginosa surface motility and blocks biofilm formation; Singh PK; This paper summarizes a presentation at the 6th International Conference on Lactoferrin Structure and Function . Some of this data has been previously published.

Biometals, 2004 Jun, 17(3), 189 - 96
The antimicrobial activity of lactoferrin: current status and perspectives; Orsi N; Lactoferrin (Lf) is a multifunctional iron glycoprotein which is known to exert a broad-spectrum primary defense activity against bacteria, fungi, protozoa and viruses . Its iron sequestering property is at the basis of the bacteriostatic effect, which can be counteracted by bacterial pathogens by two mechanisms: the production of siderophores which bind ferric ion with high affinity and transport it into cells, or the expression of specific receptors capable of removing the iron directly from lactoferrin at the bacterial surface . A particular aspect of the problem of iron supply occurs in bacteria (e.g . Legionella) which behave as intracellular pathogens, multiplying in professional and non professional macrophages of the host . Besides this bacteriostatic action, Lf can show a direct bactericidal activity due to its binding to the lipid A part of bacterial LPS, with an associated increase in membrane permeability . This action is due to lactoferricin (Lfc), a peptide obtained from Lf by enzymatic cleavage, which is active not only against bacteria, but even against fungi, protozoa and viruses . Additional antibacterial activities of Lf have also been described . They concern specific effects on the biofilm development, the bacterial adhesion and colonization, the intracellular invasion, the apoptosis of infected cells and the bactericidal activity of PMN . The antifungal activity of Lf and Lfc has been mainly studied towards Candida, with direct action on Candida cell membranes . Even the sensitivity of the genus tricophyton has been studied, indicating a potential usefulness of this molecule . Among protozoa, Toxoplasma gondii is sensitive to Lf, both in vitro and in vivo tests, while Trichomonads can use lactoferrin for iron requirements . As to the antiviral activity, it is exerted against several enveloped and naked viruses, with an inhibition which takes place in the early phases of viral invection, as a consequence of binding to the viral particle or to the cell receptors for virus . The antiviral activity of Lf has also been demonstrated in in vivo model invections and proposed for a selective delivery of antiviral drugs . The new perspectives in the studies on the antimicrobial activity of Lf appear to be linked to its potential prophylactic and therapeutical use in a considerable spectrum of medical conditions, taking advantage of the availability of the recombinant human Lf . But the historical evolution of our knowledge on Lf indicates that its antimicrobial activity must be considered in a general picture of all the biological properties of this multifunctional protein.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 97 - 104 Epub 2004 Feb 19.
Colonization, biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus ruber; Orr IG et al.; A two-step enrichment procedure led to the isolation of a strain of Rhodococcus ruber (C208) that utilized polyethylene films as sole carbon source . In liquid culture, C208 formed a biofilm on the polyethylene surface and degraded up to 8% (gravimetrically) of the polyolefin within 30 days of incubation . The bacterial adhesion to hydrocarbon assay and the salt aggregation test both showed that the cell-surface hydrophobicity of C208 was higher than that of three other isolates which were obtained from the same consortium but were less efficient than C208 in the degradation of polyethylene . Mineral oil, but not nonionic surfactants, enhanced the colonization of polyethylene and increased biodegradation by about 50% . Fluorescein diacetate (FDA) hydrolysis and protein content analysis were used to test the viability and biomass density of the C208 biofilm on the polyethylene, respectively . Both FDA activity and protein content of the biofilm in a medium containing mineral oil peaked 48-72 h after inoculation and then decreased sharply . This finding apparently reflected rapid utilization of the mineral oil adhering to the polyethylene . The remaining biofilm population continued to proliferate moderately and presumably played a major role in biodegradation of the polyethylene . Fourier transform infrared spectra of UV-photooxidized polyethylene incubated with C208 indicated that biodegradation was initiated by utilization of the carbonyl residues formed in the photooxidized polyethylene.

Cornea, 2004 Jul, 23(5), 513 - 5
Case of a large, movable bacterial concretion with biofilm formation on the ocular surface; Mihara E et al.; OBJECTIVE: To report a case with a large movable bacterial concretion formed on the ocular surface without biomaterials . METHODS: Interventional case report . A 74-year-old woman with left eye pain and injection was referred to us . She had a past history of scleral patch graft for necrotizing scleritis after pterygium removal and mitomycin C instillation on her left eye 7 years before . On present examination, a 2.5- to 3.0-mm yellowish-white calcification-like mass was present on the nasal sclera and cornea, and it moved slightly with blinking . The anterior chamber was shallow, and cornea was suspected to be perforated under this object . RESULTS: This yellowish-white mass was surgically removed . Pathologic examination demonstrated that the specimen was not a calcification but a biofilm formation by many gram-positive bacilli with neutrophils . Corynebacterium was highly suspected as the causative agent of this unusual mass because of the earlier culture of the discharge before referral . CONCLUSION: The current case demonstrates that bacterial biofilms can be formed on the ocular surface without the involvement of biomaterials.

J Clin Gastroenterol, 2004 Jul, 38(6 Suppl), S127 - 9
Lactoferrin functions: current status and perspectives; Valenti P et al.; Lactoferrin, an iron-binding glycoprotein synthesized by neutrophils and exocrine glands, plays an important role in human innate defense mechanisms against bacteria, fungi, and viruses . First, a bacteriostatic activity of lactoferrin, depending on iron withholding to bacteria, and successively a bactericidal iron-independent effect, related to its binding on bacterial surfaces, was recognized . Many other functions have been ascribed to this cationic protein, including the inhibiting action toward bacterial adhesion and invasion of target host cells . Recent research also reported the lactoferrin influence on bacterial aggregation and biofilm development of Pseudomonas aeruginosa and Streptococcus mutans . The different lactoferrin functions can be justified by different physicochemical properties of the molecule, which include the iron-binding capability, the binding to anionic cell surfaces and molecules, and serine protease activity.

J Biotechnol, 2004 Jul 15, 111(2), 155 - 67
The impacts of the AOC concentration on biofilm formation under higher shear force condition; Tsai YP et al.; The logistic growth model was applied in the study to evaluate the impacts of assimilable organic carbon (AOC) concentration on the growth characteristics of biofilm and bulk bacteria under high flow velocity condition . The experimental results showed that there existed a growth and decline relation between biofilm and bulk bacteria at the low (0.05 mg/L) and medium (0.5 mg/L) AOC levels . Increasing the AOC concentration up to 1.0 mg/L, it resulted in high amounts of biofilm and bulk bacteria simultaneously . Although the carrying capacity of biofilm bacteria at the medium condition of AOC level was substantially reduced, the specific growth rate (GR) of biofilm bacteria was largest at this condition . It showed that the reduction of biofilm bacteria quantity did not represent the suppression of bacterial growth . The quantity of bulk water bacteria was obviously dependent with the quantity of biofilm bacteria and the increase of free bacteria with time in networks was mainly due to the growth and detachment of biofilm bacteria, not due to the growth of free bacteria themselves . The maximum growth rate of biofilm bacteria was increased upon increasing the AOC level . It indicated that the AOC level was an important factor affecting the growth of biofilm bacteria.

J Clin Dent, 2004, 15(1), 28 - 32
Effect of a dental unit waterline treatment solution on composite-dentin shear bond strengths; von Fraunhofer JA et al.; OBJECTIVE: The control of biofilm and effluent contamination of dental unit water lines (DUWL) includes additions of antimicrobial solutions, as well as automatic dosing units . There are, however, varying reports on the effects of such agents on the bond strength of restorative dental materials and, particularly, between these agents and dental hard tissues . METHODOLOGY: The possible effects of an antimicrobial DUWL treatment solution on the adhesion of composite resin to dentin was evaluated by shear bond strength (SBS) testing . A total of 20 caries-free human molar and premolar teeth were used as the test substrates . The teeth were divided into two sets of 10 teeth which, after appropriate cleaning with water and pumice, were embedded horizontally in dental die stone . The buccal surface of each tooth was ground flat to a 17 microns finish using water-lubricated SiC paper . The teeth were then etched for 15 seconds with 37% H3PO4 and rinsed with either water (control) or a proprietary DUWL treatment (ICX) solution . Thereafter, the teeth were lightly blown dry with clean dry air, and the dentin conditioned with Prime & Bond NT for 20 seconds . The excess solvent was then removed by gentle air drying for 5 seconds, and the conditioner cured with visible light for 10 seconds . A cylinder of composite was placed on the conditioned surface and cured . A second group of 20 caries-free human molar and premolar teeth were used as test substrates to evaluate the effect of the ICX DUWL treatment solution on a different dentin priming system (OptiBond Solo Plus) . The teeth in the second group were divided into two sets and after a 15 second etch with 37% H3PO4, were rinsed with water (control) or the proprietary ICX DUWL treatment solution . Thereafter, the teeth were lightly blown dry with clean, dry air and the dentin conditioned with OptiBond Solo for 20 seconds . The excess solvent was then removed by gentle air drying for 5 seconds, and the conditioner cured with visible light for 10 seconds . A cylinder of composite was placed on the conditioned surface and cured . Shear bond strength testing was performed with a universal test machine at the default cross-head speed of 0.1 mm/min . A set of teeth, sectioned, mounted and etched as above but rinsed with a 0.01% mineral oil/water mix prior to conditioning and bonding, was used as the negative control . A separate corrosion testing was performed by immersing brass coupons in water and ICX for 31 days and measuring the weight loss . The brass coupons were bright-dipped, electroless nickel-plated and bright nickel electroplated . RESULTS: The bonding studies indicated that the DUWL treatment solution applied to a cut and etched dentin surface prior to conditioning and bonding with an adhesive system has no effect (p > 0.05) on bond strength for either group of specimens, compared to water . Negative control specimens were found to have minimal bond strengths . The corrosion study indicated no difference in the behavior of the test specimens in ICX compared to those in water, although differences were noted between the different surface finishes applied to the brass substrate . CONCLUSION: The findings of this study demonstrate that exposure of an etched dentin surface to a water-based DUWL treatment mixture has no adverse effects on subsequent adhesion strength . Minimal corrosive attack was noted in the ICX solution and water for brass coupons provided with three different surface finishes.

J Chemother, 2004 Apr, 16(2), 134 - 8
Influence of acetylsalicylic acid (aspirin) on biofilm production by Candida species; Stepanovic S et al.; Candida spp . are important causative agents of infections associated with biofilm formation . Management of biofilm-related infections is extremely difficult and therefore new therapeutic solutions are needed . This study for the first time explored the possible effect of aspirin on Candida spp . Biofilm-producing capacity . Two strains of C . guilliermondii, and one strain per species of C . kefyr, C . glabrata, C . albicans, and C . parapsilosis were included in the study . The antifungal property of aspirin was tested by the broth microdilution method, while effect of aspirin on biofilm formation was determined by the microtiter-plate test . The minimal inhibitory concentrations of aspirin obtained ranged from 2.17 to 8.67 mM and minimal fungicidal concentrations were from 4.33 to 8.67 mM . The concentrations of aspirin which induced statistically significant decrease in biofilm formation ranged from 0.43 mM to 1.73 mM of aspirin, depending on the tested yeast strain . Therefore, the significant effects of aspirin on growth and biofilm formation of Candida spp . were achieved only with suprapharmacological concentrations of the drug . The influence of the inoculum size on the effect of aspirin on biofilm formation was determined for C . albicans only and a significant decrease was observed also at suprapharmacological concentrations of aspirin, irrespective of the inoculum size . The results obtained in the present study show aspirin to be a drug with the potential to affect and suppress biofilm formation by Candida spp., and provide support for further investigation.

J Infect Dis, 2004 Jul 15, 190(2), 318 - 20 Epub 2004 Jun 24.
Suppression of drug-resistant Staphylococcal Infections by the quorum-sensing inhibitor RNAIII-inhibiting peptide; Dell'Acqua G et al.; Staphylococcus aureus and S . epidermidis are major causes of infection related to biofilm formed on indwelling medical devices . Such infections are common causes of morbidity and mortality and, because of biofilm resistance to antibiotics, are difficult to treat . The RNAIII-inhibiting peptide (RIP) (YSPWTNF-NH2) inhibits the pathogenesis of staphylococci by disrupting bacterial cell-cell communication (known as "quorum sensing") . Using a vascular-graft rat model, we show that RIP, applied locally and systemically, can completely inhibit drug-resistant S . aureus and S . epidermidis biofilms . The present study provides the first direct demonstration that interfering with cell-cell communication by use of a quorum-sensing inhibitor can eliminate medical device-associated staphylococcal infections . We suggest that medical devices could be coated with RIP to prevent infections, including those by antibiotic-resistant staphylococcal strains.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2659 - 64
Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms; Borriello G et al.; The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro . Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h . It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions . Oxygen penetrated about 50 microm into the biofilms, which averaged 210 microm thick . The region of active protein synthesis was visualized by using an inducible green fluorescent protein . This zone was also limited to a narrow band, approximately 30 microm wide, adjacent to the air interface of the biofilm . The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h(-1) . These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state . In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air . When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically . Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested . Nitrate amendment stimulated the growth of untreated control P . aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics . Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P . aeruginosa biofilms causing infections in vivo.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2633 - 6
Enzymatic detachment of Staphylococcus epidermidis biofilms; Kaplan JB et al.; The gram-positive bacterium Staphylococcus epidermidis is the most common cause of infections associated with catheters and other indwelling medical devices . S . epidermidis produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces . We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans rapidly and efficiently removed S . epidermidis biofilms from plastic surfaces . The enzyme worked by releasing extracellular slime from S . epidermidis cells . Precoating surfaces with the enzyme prevented S . epidermidis biofilm formation . Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2544 - 50
A chimeric peptide composed of a dermaseptin derivative and an RNA III-inhibiting peptide prevents graft-associated infections by antibiotic-resistant staphylococci; Balaban N et al.; Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices . Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms . RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms . K(4)-S4(1-13)(a) is a 13-residue dermaseptin derivative (DD(13)) believed to kill bacteria via membrane disruption . We tested each of these peptides as well as a hybrid construct, DD(13)-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S . epidermidis (MRSE) . In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD(13)-RIP and DD(13) were equally effective, and that the chimeric peptide but not DD(13) was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis . In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose . In addition, each of the peptides acted synergistically with antibiotics . The data indicate that RIP and DD(13) act in synergy by attacking bacteria simultaneously by two different mechanisms . Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2350 - 4
Defined anaerobic growth medium for studying Candida albicans basic biology and resistance to eight antifungal drugs; Dumitru R et al.; The polymorphic fungus Candida albicans is one of the most versatile opportunistic pathogens in humans . Many organs of the human body are potential targets for infection by this pathogen, but infection is commonly localized in the gastrointestinal tract, an environment providing anaerobic growth conditions . We describe a chemically defined anaerobic growth medium for four strains of Candida albicans (A72, SC5314, MEN, and 10261) . It is a defined liquid glucose-phosphate-proline growth medium supplemented with oleic acid, nicotinic acid, and ammonium chloride . The cells did not require or respond to added ergosterol . Oleic acid and nicotinic acid are growth factors which are required only for the anaerobic growth of C . albicans . An important technical feature of this study was the use of anaerobically grown inocula to study anaerobic growth . Anaerobically, the cells grew exclusively as mycelia at 25, 30, and 37 degrees C . The doubling time at 30 degrees C was ca . 20 h . The cells did not produce farnesol and did not respond to exogenous farnesol, and they were resistant to the highest tested levels of amphotericin B and four of the azole antifungals . We suggest that the anaerobic growth of C . albicans may contribute to the trailing end point phenomenon and the resistance of C . albicans biofilms to antifungal drugs.

Syst Appl Microbiol, 2004 May, 27(3), 372 - 9
A simple method to isolate biofilm-forming Bacillus subtilis and related species from plant roots; Fall R et al.; A novel method was developed to isolate pure cultures of wild-type Bacillus subtilis and related species from plant roots, even roots washed free of adhering soil . The method uses casein digest-mannitol agarose (CM) media that promote rapid dendritic growth (low K+ ion) or profuse surface film formation (high K+ ion) of Bacillus species at 40 degrees C . Inoculation from the tips of surface growth on agarose leads to self-purification and streaking on CM agar plates (hard agar and high K+) leads to characteristic colony morphology . Phenotypic and 16S rDNA analysis revealed that most root isolates obtained by this method are spore-forming Bacillus species, with enrichment for B . subtilis and its close relatives . Of particular interest is the finding that the majority of these Bacillus isolates and the B . subtilis Marburg strain also form adhering biofilms on inert surfaces . Thus the methods presented may be useful in isolation of biofilm-forming Bacillus and investigation of their role on plant roots.

Infect Immun, 2004 Jul, 72(7), 4249 - 60
Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans; Greiner LL et al.; Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections . Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels . Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used . Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis . NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm . NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain . The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins . S . nigra TRITC-conjugated lectin bound to this biofilm, while M . amurensis FITC-conjugated lectin did not . S . nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase . Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms . Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

Infect Immun, 2004 Jul, 72(7), 3941 - 50
Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection; Jones BV et al.; Proteus mirabilis is a common cause of catheter-associated urinary tract infection (C-UTI) . It blocks indwelling urethral catheters through the formation of extensive crystalline biofilms . The obstruction of urine flow can induce episodes of pyelonephritis, septicemia, and shock . P . mirabilis exhibits a type of motility referred to as swarming, in which multicellular rafts of elongated, hyperflagellated swarmer cells form and move rapidly in concert over solid surfaces . It has been suggested that swarming is important in the pathogenesis of C-UTI . In this study we generated a set of stable transposon mutants deficient in swarming and used them to assess the role of swarming in the migration of P . mirabilis over urinary catheters . Swarming was found to be essential for migration over all-silicone catheters . Swarming-deficient mutants were attenuated in migration over hydrogel-coated latex catheters, but those capable of swimming motility were able to move over and infect these surfaces . A novel vapor fixation technique for the preparation of specimens and scanning electron microscopy were used to resolve the ultrastructure of P . mirabilis multicellular rafts . The flagellar filaments of P . mirabilis were found to be highly organized during raft migration and were interwoven in phase to form helical connections between adjacent swarmer cells . Mutants lacking these novel organized structures failed to swarm successfully . We suggest that these structures are important for migration and formation of multicellular rafts . In addition, the highly organized structure of multicellular rafts enables P . mirabilis to initiate C-UTI by migration over catheter surfaces from the urethral meatus into the bladder.

Infect Immun, 2004 Jul, 72(7), 3838 - 48
RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR; Knobloch JK et al.; Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis . However, the pathway of this regulatory mechanism was unknown . To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S . epidermidis strains 1457 and 8400 . The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted . Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus . Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S . epidermidis pathogenesis . However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations . Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR . Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR . This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S . epidermidis.

Pharmacogenomics, 2004 Jul, 5(5), 585 - 8
AutoGenomics, Inc; Vairavan R; AutoGenomics has created an automated multiplexing microarray platform to make genomic and proteomic analyses routine and efficient for clinical and research laboratories . While the emergence of microarrays has advanced genomic analyses, a number of underlying issues, such as cross-hybridization, poor spot morphology and intrinsic fluorescence of the solid substrate, have yet to be fully resolved . Current methods use discrete instrumentation, are manual and require highly skilled labor, which leads to inconsistent results . AutoGenomics' automated platform uses a three-dimensional BioFilmChip microarray to circumvent these issues, providing optimal spot morphology and utilizing solution-based hybridization with allele-specific primer extension to improve single-base discrimination . AutoGenomics is developing applications for the early detection and management of complex disease states in oncology, cardiology, and mental disorders . Customers include clinical reference laboratories, hospitals, academic institutions, and pharmaceutical and biotech companies . Founded in 1999, the company is headquartered in Carlsbad, California, USA.

J Periodontol, 2004 May, 75(5), 693 - 701
Estrogen suppression induces papillary gingival overgrowth in pregnant baboons; Reynolds MA et al.; BACKGROUND: Alterations in sex steroids during pregnancy are associated with the development and exacerbation of reactive lesions involving the gingiva . Currently, few experimental animal models similar to humans are available to examine regulatory pathways involving sex steroids and the periodontium . METHODS: In the present study, we used the baboon as a novel experimental model for the study of the regulatory actions of estrogen on the periodontium during pregnancy . Pregnant baboons (N = 5) were administered the potent, highly specific aromatase inhibitor CGS 20267 (2 mg/day subcutaneously) daily on days 60 through 165 of gestation (term = 184) . Untreated females (N = 10) and females (5) concomitantly administered aromatase inhibitor and estradiol benzoate (2.0 mg/day each subcutaneously) served as controls . Gingival biopsies were taken between days 145 and 165 of gestation . RESULTS: Administration of CGS 20267 in all females suppressed maternal serum concentrations of estradiol by 95% and induced the development of an exuberant papillomatous enlargement of the gingiva by gestational day 110, with the most prominent development involving the labial aspects of the anterior sextants . None of the untreated pregnant controls or females concomitantly administered aromatase inhibitor and estradiol benzoate developed gingival overgrowth . Thus, estradiol alone prevented the onset of gingival overgrowth induced by estrogen suppression . In all baboons, discontinuation of the aromatase inhibitor at time of cesarean section resulted in spontaneous regression and resolution of the papillomatous hyperplasia within 4 to 6 weeks . Clinically, the gingival papillary overgrowth was erythematous and edematous, with a propensity toward spontaneous subgingival hemorrhage . Histologically, the biopsy specimens demonstrated hyperplasia of the epithelium typified by mild hyperkeratosis, acanthosis, and elongation and isolated anastamoses of rete ridges . Subjacent to the intact epithelium was a loose connective tissue stroma with isolated areas of inflammatory cell infiltrate . Special stains verified the presence of isolated bacterial biofilms; however, no evidence of fungal filaments was present . Histological features suggestive of viral infection were notably absent in the epithelium . No evidence of viral particles or capsids was identified using transmission electron microscopy . Reverse transcription polymerase chain reaction analysis, using a panel of degenerate primers, was negative for papilloma family viruses . CONCLUSIONS: These results are consistent with a significant role for estrogen during primate pregnancy in the regulation of cellular proliferation and differentiation within the gingiva . The baboon represents an important experimental model for studying the regulatory actions of estrogen on the periodontium during pregnancy.

Biotechnol Bioeng, 2004 Jul 5, 87(1), 14 - 23
Performance of a substratum-irradiated photosynthetic biofilm reactor for the removal of sulfide from wastewater; Hurse TJ et al.; The performance of a sulfide-removal system based on biofilms dominated by green sulfur bacteria (GSB) has been investigated . The system was supplied with radiant energy in the band 720-780 nm, and fed with a synthetic wastewater . The areal net sulfide removal rate and the efficacy of the incident radiant energy for sulfide removal have been characterized over ranges of bulk sulfide concentration (1.6-11.5 mg L(-1)) and incident irradiance (0.21-1.51 W m(-2)) . The areal net sulfide removal rate increased monotonically with both increasing incident irradiance and increasing bulk sulfide concentration . The efficacy of the radiant energy for sulfide removal (the amount of sulfide removed per unit energy supplied) also increased monotonically with rising bulk sulfide concentration, but exhibited a maximum value with respect to incident irradiance . The maximum observed values of this net removal rate and this efficacy were, respectively, 2.08 g m(-2) d(-1) and 2.04 g W(-1) d(-1) . In-band changes in the spectral composition of the radiant energy affected this efficacy only slightly . The products of sulfide removal were sulfate and elemental-S . The elemental-S was scarcely released into the liquid, however, and reasons for this, such as sulfur reduction and polysulfide formation, are considered . Between 1.45 and 3.85 photons were needed for the net removal of one electron from S-species . Intact samples of the biofilm were characterized by microscopy, and their thicknesses lay between 39 +/- 9 and 429 +/- 57 microm . The use of the experimentally determined rates and efficacies for the design of a pilot-scale system is illustrated .

Chemotherapy, 2004 Jun, 50(2), 101 - 4
Moxifloxacin and biofilm production by coagulase-negative staphylococci; Perez-Giraldo C et al.; The in vitro activity of moxifloxacin against 41 strains of coagulase-negative staphylococci was determined . A relationship between the activity of moxifloxacin and biofilm formation was detected . Biofilm-producing strains were more resistant to moxifloxacin than biofilm-negative strains . Our global results obtained with six strains of Staphylococcus epidermidis showed that subinhibitory concentrations of moxifloxacin did not significantly modify biofilm formation . On the other hand, moxifloxacin concentrations of 2, 10, 50 and 100 x MIC produced a log decrease in viable count (included in a biofilm) of 0.20, 0.37, 1.10 and 1.69, respectively .

Bull Math Biol, 2004 Jul, 66(4), 809 - 24
Effect of heterogeneous structure in mechanically unstressed biofilms on overall growth; Klapper I; In contrast to their name, biofilms are not always flat and homogeneous but instead often exhibit complex structural heterogeneity . It has been suggested that nonhomogeneous geometry is selected in order to increase biofilm growth rate . A previous study (Dockery and Klapper (2002) SIAM J . Appl . Math., 62, 853-869) of a model biofilm system in a static bulk fluid demonstrated that under some circumstances a flat biofilm-bulk fluid interface is linearly unstable to perturbation due to growth induced forces . Computations indicated that subsequent nonlinear evolution results in fingers and mushrooms of biofilm similar to structures observed in actual biofilms . However, the important complementary issue of biological functionality was not considered . Here a weakly nonlinear analysis of the simple growing biofilm layer model in Dockery and Klapper (2002, SIAM J . Appl . Math., 62, 853-869) is presented . It is argued that, at least in the case of biofilms free of external mechanical stress, overall growth is in fact generally inhibited by the presence of growing perturbations in the linear stage . Hence a more complex explanation of function is necessary.

Oral Microbiol Immunol, 2004 Aug, 19(4), 270 - 6
Role of gene E2f1 in susceptibility to bacterial adherence of oral streptococci to tooth surfaces in mice; Matsumoto N et al.; Dental plaque is composed of a biofilm community of microorganisms on teeth that coats the oral cavity, including attaching to the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases . Oral streptococci are pioneering organisms that play an important role in biofilm formation on tooth surfaces as well as being primary causative agents of dental caries . The purpose of this study was to clarify the role of the E2f1 gene in susceptibility to dry mouth and bacterial adherence of oral streptococci to tooth surfaces in animal model experiments . A mutation of the E2f1 gene in mice is known to cause enhanced T-lymphocyte proliferation, leading to testicular atrophy, splenomegaly, salivary gland dysplasia, and other systemic and organ-specific autoimmunity . We found a decreased volume of saliva production and protein production rate, along with increased amylase activity, IgA concentration, and mucin 1 concentration in E2F-1(-/-) mice as compared with the control C57BL/6 mice . Further, we quantified the recolonization of oral streptococci in E2F-1(-/-) mice and found that a higher number of some oral streptococci were colonized on the teeth of these mice . In particular, following oral ingestion of 1% sucrose in water, the colonization of Streptococcus mutans increased in comparison with other streptococci . Our results suggest that the E2f1 gene may affect susceptibility for oral biofilm formation by streptococci in humans with dry mouth.

Oral Microbiol Immunol, 2004 Aug, 19(4), 252 - 6
Transcriptional analysis of mutacin I (mutA) gene expression in planktonic and biofilm cells of Streptococcus mutans using fluorescent protein and glucuronidase reporters; Kreth J et al.; Streptococcus mutans is implicated as the primary pathogen involved in the development of dental caries . The production of specific bacteriocins (called mutacins) by S . mutans is one of the major virulence factors which facilitate the dominance of the bacterium within dental plaque . While much has been revealed about the biochemical structures of mutacins, little is known about the expression and regulation of mutacin genes, largely due to the lack of proper methods to monitor mutacin gene expression, especially under biofilm conditions . In this study, a set of reporter systems with the green fluorescent protein (gfp), the monomeric red fluorescent protein (mrfp1), and the glucuronidase (gusA) are introduced to S . mutans to study the transcriptional activities of the mutacin I gene (mutA) . Although the mutA-reporter fusions are in single copy on the chromosome, these reporter systems display strong signals that allow us to effectively monitor mutA gene expression in S . mutans . Using these reporter systems, we show that mutA is expressed in both planktonic and biofilm cells, even though mutacin activities are normally detected only in biofilm cells . Furthermore, we confirm that mutR, the gene upstream of the mutacin operon, is required for mutacin I gene expression . The success of this study validates the feasibility of using these reporter systems to study gene expression and regulation in S . mutans.

Water Res, 2004 Jun, 38(11), 2726 - 36
Dynamics of lead immobilization in sulfate reducing biofilms; Beyenal H et al.; We have evaluated the effects of selected minerals present in subsoil environment on the efficiency of lead removal from contaminated groundwaters using biofilms composed of sulfate-reducing microorganisms, and examined the stability of metal deposits after the biofilms had been temporarily exposed to the air . To quantify the studied effects, lead was immobilized in biofilms of Desulfovibrio desulfuricans grown anaerobically in two flat-plate flow reactors, one filled with hematite and the other with quartz . While the biofilms in both reactors were heterogeneous and consisted of voids and channels, the biofilms grown on hematite were denser, thicker, and more porous than those grown on quartz . The average H2S concentrations, measured using microelectrodes, were higher in the biofilms grown on quartz than those measured in the biofilms grown on hematite . During 18 weeks of operation, iron was continuously released from the hematite . Lead was immobilized more efficiently in the biofilms grown on quartz than it was in the biofilms grown on hematite . Lead deposits were partially reoxidized, especially in biofilms grown on hematite, and the biofilms in both reactors responded to the presence of oxygen by lowering their density and increasing the H2S production rate.

Emerg Infect Dis, 2004 Jun, 10(6), 1074 - 81
Candida parapsilosis characterization in an outbreak setting; Kuhn DM et al.; Candida parapsilosis is an important non-albicans species which infects hospitalized patients . No studies have correlated outbreak infections of C . parapsilosis with multiple virulence factors . We used DNA fingerprinting to determine genetic variability among isolates from a C . parapsilosis outbreak and from our clinical database . We compared phenotypic markers of pathogenesis, including adherence, biofilm formation, and protein secretion (secretory aspartic protease {SAP} and phospholipase) . Adherence was measured as colony counts on silicone elastomer disks immersed in agar . Biofilms formed on disks were quantified by dry weight . SAP expression was measured by hydrolysis of bovine albumin; a colorimetric assay was used to quantitate phospholipase . DNA fingerprinting indicated that the outbreak isolates were clonal and genetically distinct from our database . Biofilm expression by the outbreak clone was greater than that of sporadic isolates (p < or =0.0005) . Adherence and protein secretion did not correlate with strain pathogenicity . These results suggest that biofilm production plays a role in C . parapsilosis outbreaks.

Appl Microbiol Biotechnol, 2004 Nov, 65(6), 664 - 70 Epub 2004 Jun 16.
Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using plastic composite support bioreactors; Urbance SE et al.; Continuous and repeat-batch biofilm fermentations using Actinobacillus succinogenes were performed with immobilized and suspended-cell systems . For the immobilized continuous system, plastic composite supports (PCS) containing 50% (w/w) polypropylene (PP), 35% (w/w) ground soybean hulls, 5% (w/w) dried bovine albumin, 2.5% (w/w) soybean flour, 2.5% (w/w) yeast extract, 2.5% (w/w) dried red blood cells, and 2.5% (w/w) peptone, or PP tubes (8.5 cm in length) were arranged around the agitator shaft in a grid formation . Agitation was controlled at 125 rpm and 150 rpm . Samples were taken at dilution rates of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 h(-1) and analyzed for succinic acid production and glucose consumption (g l(-1)) . For PCS bioreactors, the highest final succinic acid concentrations (10.1 g (-1), 10.4 g l(-1)) and percentage yields (62.6%, 71.6%) occurred at the dilution rate of 0.2 h(-1) . PCS disks were evaluated in a repeat-batch biofilm reactor . Suspended-cell batch fermentations were performed in flasks and a repeat-batch bioreactor . The maximum concentration of succinic acid produced was 40 g l(-1) . Peak succinic acid percentage yields in continuous and repeat-batch fermentations of A . succinogenes were observed in suspended-cell continuous fermentations at a dilution rate of 1.0 h(-1) (76.2%) and in PCS repeat-batch fermentations with an initial glucose concentration of 40 g l(-1) (86.7%).

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz, 2004 Apr, 47(4), 384 - 91
{Significance of the ordinance on the quality of water for human consumption (Drinking Water Ordinance 2001) for hospital hygiene}; Exner M et al.; Since January 2003, the new German Drinking Water Ordinance (DWO) has become operative . This paper briefly reviews some major consequences for hospitals . One of the main topics is the increased responsibility of operators of drinking water installation systems in hospitals concerning both the maintenance and control of good drinking water quality . Besides harmful chemical parameters, proliferation of pathogens can occur such as Pseudomonas aeruginosa, Legionella spec., Acinetobacter, and others which are mainly bound to biofilms and thus less affected by disinfectants . Recent epidemiological investigations point out that the relevance of waterborne pathogens is still considerably underestimated, particularly in hospitals . Local public health authorities play a central role in clinic inspection, supervision of water installations, surveillance, and risk assessment in cases of noncompliance with DWO requirements . For this reason, every irregularity detected must be reported to the local public health authority.

J Bacteriol, 2004 Jul, 186(13), 4246 - 53
Involvement of Streptococcus gordonii beta-glucoside metabolism systems in adhesion, biofilm formation, and in vivo gene expression; Kilic AO et al.; Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA) . To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed . To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions . Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides . Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants . Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci . Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium . Therefore, genes associated with beta-glucoside metabolism may regulate S . gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.

J Bacteriol, 2004 Jul, 186(13), 4159 - 67
Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis; Connelly MB et al.; Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming . A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface . As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria . While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria . To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility . Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm . While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function . These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.

Biofouling, 2004 Apr, 20(2), 81 - 5
Comparison of the efficacy of free residual chlorine and monochloramine against biofilms in model and full scale cooling towers; Turetgen I; The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion . Biofilms accumulate on all submerged industrial and environmental surfaces . The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm . The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower . The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers . A cooling tower and a laboratory model recirculating water system were used as biofilm reactors . Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers . Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal . Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons . Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.

Biofouling, 2004 Apr, 20(2), 71 - 9
Polyphasic detection of cyanobacteria in terrestrial biofilms; Gaylarde C et al.; Cyanobacterial populations detected on buildings by traditional methods are mainly filamentous, whereas direct microscopy shows that they are principally coccoid morphotypes that often cannot be isolated in culture, but may grow on artificial media when the spatial biofilm relationships are maintained . The polyphasic strategy described here was to select morphologically distinct colonies from rehydrated biofilms for direct DNA amplification, allowing uncultured organisms to be sequenced and their morphology to be characterized by microscopy . DNA data banks currently contain many entries for cyanobacteria of unrecorded morphology, which does not facilitate identification, although genetic variability in a population may be assessed . The sequence homologies of the present biofilm organisms (EMBL accession numbers AJ619681 to 619690) with those in DNA databanks were low, indicating differences between xerophytic cyanobacteria on walls and aquatic species comprising the majority in the databases . Further development of databases for the populations found in this environment, subject to temperature extremes, repeated desiccation and high UV and salt levels, is required.

J Urol, 2004 Jul, 172(1), 153 - 6
Penile prosthesis cultures during revision surgery: a multicenter study; Henry GD et al.; PURPOSE: Initial implantation of inflatable penile prosthesis has a 3% risk of infection . Reoperation of penile implants has a higher rate of infection, estimated between 10% and 18% . To explain the higher risk in revision surgery in this prospective study we cultured clinically uninfected prostheses requiring revision . Prosthesis pain was also investigated as a predictor of positive culture . MATERIALS AND METHODS: At 3 institutions cultures were prospectively obtained from 77 clinically uninfected penile prostheses at revision surgery . Immediately upon surgical exposure of the pump cultures were obtained . If a bacterial biofilm was noted on any component it was additionally cultured . All culture isolates positive for a staphylococcus species were tested for sensitivity to rifampin and tetracycline (minocycline) . An implant is now available that is coated with these antibiotics . Patient history of chronic prosthesis pain was ascertained . RESULTS: Culture positive bacteria were found in 54 of 77 (70%) patients with clinically uninfected penile prostheses . In some patients more than 1 organism grew and, occasionally, the pump culture was negative but the biofilm was positive . Of 54 patients 49 had positive (90%) culture for staphylococcus genus with 10 different species . All staphylococcal species were sensitive to rifampin and/or tetracycline . We did not find a significant association between prosthesis related pain and culture laboratory results . CONCLUSIONS: The majority of clinically uninfected penile prostheses have organisms growing in the implant spaces at reoperation . Most of these organisms are staphylococcal species that are sensitive to rifampin/minocycline.

Drugs, 2004, 64(12), 1359 - 73
Role of fluoroquinolones in the treatment of serious bacterial urinary tract infections; Carson C et al.; Serious urinary tract infections (UTIs) in adults--defined as acute complicated UTIs or pyelonephritis requiring initial intravenous antimicrobials and/or hospitalisation and nosocomial infections--cause significant morbidity and economic burden . In the US, UTIs are responsible for nearly 7 million outpatient physician office visits, 1 million emergency room visits and over 100 000 hospital admissions annually . Complicated UTIs often affect patients with underlying functional, metabolic or anatomical defects of the urinary tract, whereas most nosocomial UTIs (~80%) are related to short- or long-term catheterisation . Serious UTIs are often difficult to treat because infection involves a diverse array of Gram-negative and Gram-positive bacteria, coupled with increasing antimicrobial resistance in some uropathogens, and a higher rate of recurrent infections . Although Escherichia coli remains a common aetiology (< or =60%), other Enterobacteriaceae, Gram-negative bacilli (e.g . Pseudomonas aeruginosa), and Gram-positive bacteria (e.g . Staphylococcus aureus) are frequently isolated . Patients with long-term catheterisation have UTIs typically caused by organisms that produce biofilms making eradication even more difficult . Overall, aetiology and resistance patterns are not predictable for those with serious UTIs, necessitating confirmation by culture and susceptibility testing.Numerous intravenous and oral antimicrobial treatment options are available and the majority of patients with serious UTIs will need initial intravenous therapy because of the possibility of bacteraemia/sepsis or impaired gastrointestinal absorption . Many experts concur that empirical therapy for the institutionalised or hospitalised patient with a serious UTI should include an intravenous antipseudomonal agent because of an increased risk of urosepsis . While state-of-the-art treatment guidelines are lacking for these infections, targeted therapy should be initiated once susceptibility data are known.The use of targeted therapy--emphasising the "correct antibacterial spectrum" and pharmacodynamic superiority--is likely to provide important benefits (e.g . reduced morbidity and associated costs, reduced emergence of resistance) . Agents commonly prescribed include aminoglycosides, beta-lactam/beta-lactamase inhibitor combinations, imipenem, advanced-generation cephalosporins and fluoroquinolones . Fluoroquinolones are often recommended when conventional agents have failed or are less desirable (e.g . toxicity/hypersensitivity concerns), or when resistance is high . Several pivotal clinical trials support the use of fluoroquinolones for serious UTIs with most experience garnered with ciprofloxacin, including a new once-daily extended-release tablet formulation.Treatment of patients with serious UTIs remains challenging . Physicians should choose empirical therapy based on patient demographics/medical history, presumed aetiology and local resistance patterns until more definitive guidelines become available.

J Infect, 2004 Jul, 49(1), 20 - 2
Use of ethanol locks to prevent recurrent central line sepsis; Metcalf SC et al.; Catheter-related sepsis (CRS) is a common complication of long-term parenteral nutrition . Conventional antibiotic therapy is often effective in the short-term but, because of poor activity against intraluminal microbial biofilms, may not prevent relapse . Ethanol is an effective antiseptic . We describe a case of a patient with recurrent CRS successfully treated with 70% ethanol locks.

Curr Opin Biotechnol, 2004 Jun, 15(3), 181 - 6
Biocorrosion: towards understanding interactions between biofilms and metals; Beech IB et al.; The term microbially influenced corrosion, or biocorrosion, refers to the accelerated deterioration of metals owing to the presence of biofilms on their surfaces . The detailed mechanisms of biocorrosion are still poorly understood . Recent investigations into biocorrosion have focused on the influence of biomineralization processes taking place on metallic surfaces and the impact of extracellular enzymes, active within the biofilm matrix, on electrochemical reactions at the biofilm-metal interface.

J Antimicrob Chemother, 2004 Jul, 54(1), 21 - 8 Epub 2004 Jun 09.
Macrolide activities beyond their antimicrobial effects: macrolides in diffuse panbronchiolitis and cystic fibrosis; Schultz MJ; Diffuse panbronchiolitis (DPB) is a pulmonary disease characterized by chronic inflammation of the bronchioles and chronic infiltration of inflammatory cells in the lungs . DPB has several features in common with cystic fibrosis (CF) . Clinical trials in patients with DPB or CF suggest a potential role for maintenance (long-term and low-dose) macrolide therapy in the treatment of these chronic pulmonary conditions . Indeed, these studies demonstrate improved clinical and physiological states with macrolide therapy . The beneficial effects of long-term low-dose macrolides are not related to their antimicrobial properties, since levels of macrolides with low-dose treatment are too low to have sufficient antimicrobial effects . Data indicate that macrolides may have immunomodulatory activities: (1) in vitro and ex vivo studies clearly show that macrolides can influence cytokine production by several cell types; (2) furthermore, macrolides can alter polymorphonuclear cell functions in vitro and ex vivo . Although immunomodulation may serve as one explanation for the beneficial effects of macrolides in patients with chronic pulmonary inflammation, the effect of low-dose macrolide therapy on biofilm-formation may form a second explanation for the positive effects of long-term low-dose macrolide therapy . In the present paper, the clinical trials on maintenance macrolide therapy in patients with DPB or CF are reviewed . This is followed by a discussion on the immunomodulating effects of macrolides, and the effects of macrolides on biofilm formation.

Eukaryot Cell, 2004 Jun, 3(3), 567 - 78
Evidence that hsp90 is involved in the altered interactions of Acanthamoeba castellanii variants with bacteria; Yan L et al.; There are many similarities between the interactions of environmental protozoa with pathogenic bacterial species and those observed in mammalian macrophages . Since single-celled protozoa predate mammalian hosts, it is likely that interactions in environmental biofilms have selected for many of the bacterial virulence mechanisms responsible for human disease . In order to better understand bacterial-phagocyte interactions, we developed a selection for Acanthamoeba castellanii variants that are more resistant to killing by bacterial pathogens . We identified four amoebal clones that display decreased phagocytosis of bacteria but no difference in uptake of latex beads compared to wild-type amoebae . These amoebal variants display differences in cellular morphology, partial resistance to killing by bacteria, more bactericidal activity, and higher frequencies of lysosome fusion with the bacterial vacuole . Three proteins are present at lower levels in these variants than in wild-type amoebae, and matrix-assisted laser desorption ionization-time of flight mass spectrometry allowed identification of two of them as actin and hsp90 . We found that specific inhibitors of hsp90 produce a similar phenotypic effect in macrophages . These data suggest that hsp90 plays a role in phagocytic and, possibly, bactericidal pathways that affect interactions of phagocytic cells with bacteria .

Epidemiol Mikrobiol Imunol, 2004, 53(2), 66 - 9
{Differences in antibiotic sensitivity in biofilm-positive and biofilm-negative strains of Staphylococcus epidermidis isolated from blood cultures}; Hola V et al.; The adhering capability and biofilm growth facilitate staphylococcal colonization of surfaces of damaged tissues and foreign bodies . Biofilm-forming bacteria are more resistant to immune system activities, mechanical effects of blood flow and other adverse effects, e.g . those due to antibiotics . Minimal inhibitory concentrations (MICs) were compared for two groups of Staphylococcus epidermidis strains isolated from blood cultures . Group 1 included biofilm positive strains whose biofilm-forming potential was revealed by both phenotypic and genotypic methods . Group 2 included strains without biofilm-forming potential . The comparison of MICs for selected antibiotics showed higher resistance of biofilm positive compared to biofilm negative strains . The difference was evident particularly for oxacillin, tetracycline, co-trimoxazole and gentamicin.

Microbiology, 2004 Jun, 150(Pt 6), 1947 - 56
A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans; Shah DS et al.; Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose . These glucans are important in determining the permeability properties and adhesiveness of dental plaque . GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats . The S . mutans genome sequence was searched for ORFs containing 'A' repeats, and one novel gene, gbpD, which appears to be unique to the mutans group of streptococci, was identified . The GbpD sequence revealed the presence of three 'A' repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K(D) of 2-3 nM . Construction of truncated derivatives of GbpD confirmed that the 'A' repeat region was essential for binding . Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose . The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the alpha/beta hydrolase family (including lipases and carboxylesterases) . GbpD contains the two regions typical of these enzymes: a GxSxG active site 'lipase box' and an 'oxyanion hole' . GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity . The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid . The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S . mutans and another plaque bacterium, Streptococcus sanguinis . GbpD bound to and released FFA from lipoteichoic acid (LTA) of S . sanguinis, but had no effect on LTA from S . mutans . These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm.

J Med Microbiol, 2004 Jul, 53(Pt 7), 679 - 90
Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation; Stapper AP et al.; Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms . This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation . These strains, PAO1, Alg+ PAOmucA22 and Alg- PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy . Biofilm Image Processing (BIP) and Community Statistics (COMSTAT) software programs were used to provide quantitative measurements of the two-dimensional biofilm images . All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation . Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation . Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

Appl Environ Microbiol, 2004 Jun, 70(6), 3745 - 50
Noninvasive pigment identification in single cells from living phototrophic biofilms by confocal imaging spectrofluorometry; Roldan M et al.; A new imaging technique for the analysis of fluorescent pigments from a single cell is reported . It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods . The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages.

Appl Environ Microbiol, 2004 Jun, 70(6), 3736 - 41
Protein expression by Streptococcus mutans during initial stage of biofilm formation; Welin J et al.; Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents . In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface . In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface . Log-phase planktonic cells of S . mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture . Nonadhered cells were washed away, and the adhered cells were removed by sonication . The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis . Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished > or =1.3-fold in the biofilm cells . Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis . Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting . The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.

Appl Environ Microbiol, 2004 Jun, 70(6), 3449 - 56
Effects of quaternary-ammonium-based formulations on bacterial community dynamics and antimicrobial susceptibility; McBain AJ et al.; Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products . Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities . QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages) . A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD) . QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility . The susceptibility of Ralstonia sp . was considerably decreased following 14 consecutive QAC passages . Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis . Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions . DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD . Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility . These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.

Appl Environ Microbiol, 2004 Jun, 70(6), 3232 - 8
Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata; Mai-Prochnow A et al.; The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment . As part of our studies on the ecology of P . tunicata and its interaction with marine surfaces, we examined the ability of P . tunicata to form biofilms under continuous culture conditions within the laboratory . P . tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels . Remarkably, we observed a repeatable pattern of cell death during biofilm development of P . tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J . S . Webb et al., J . Bacteriol . 185:4585-4595, 2003) . Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures . A subpopulation of viable cells was always observed within the regions of killing in the biofilm . Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum . A novel 190-kDa autotoxic protein produced by P . tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment . A Delta alpP mutant derivative of P . tunicata was generated, and this mutant did not show cell death during biofilm development . We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P . tunicata and subsequent dispersal of surviving cells within the marine environment.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 357 - 62
Differences in biofilm and planktonic cell mediated reduction of metalloid oxyanions; Harrison JJ et al.; This study compares Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to the selenium and tellurium oxyanions selenite (SeO3(2-)), tellurate (TeO4(2-)), and tellurite (TeO3(2-)) . P . aeruginosa planktonic and biofilm cultures reduced the selenium and tellurium oxyanions to orange and black end-products (respectively) and were equally tolerant to killing by these metalloid compounds . S . aureus planktonic cell cultures processed these metalloid oxyanions in a similar way, but the corresponding biofilm cultures did not . S . aureus biofilms were approximately two and five times more susceptible to killing by tellurate and tellurite (respectively) than the corresponding planktonic cultures . Our data indicate that the means of reducing metalloid oxyanions may differ between the physiology displayed in biofilm and planktonic cultures of the same bacterial strain.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 265 - 71
The ypdI gene codes for a putative lipoprotein involved in the synthesis of colanic acid in Escherichia coli; Potrykus J et al.; In Escherichia coli, the synthesis of colanic acid, an extracellular polysaccharide imminent in biofilm development, is a complicated process involving numerous genes and not yet wholly elucidated . Using a plasmid-borne E . coli K-12 gene library, we have identified a clone whose presence conferred mucoid colony phenotype onto E . coli CM2555 strain . Our results indicate that overexpression of a gene previously catalogued as ypdI, which encodes a putative lipoprotein, is responsible for this phenotype . We show that the mucoidy of ypdI -overexpressing bacteria is due to increased production of colanic acid . This phenotype depends on the function of the rcsA gene, but not on that of rcsF . These results suggest that the ypdI gene product might be an additional factor playing a role in colanic acid synthesis, indicating that this process can be even more complicated than supposed to date . However, no obvious phenotype was observed in the DeltaypdI::kan mutant cultivated under standard laboratory conditions.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 221 - 8
Macrofilamentous microbial communities in the metal-rich and acidic River Tinto, Spain; Lopez-Archilla AI et al.; A novel type of macroscopic microbial community consisting of large dendritic filaments (up to 1.5 m) in a pH 2.0 dam of the River Tinto (South-western Spain) is described . The combined use of 16S rRNA-gene surveys and fluorescent in situ hybridisation (FISH) suggested that gamma-proteobacteria and a relative large diversity of alpha-proteobacteria dominated these structures . beta-Proteobacteria, Actinobacteria and Firmicutes were also detected . Whereas acidophilic bacteria of the genera Acidithiobacillus, Leptospirillum and Acidiphilium, and archaea belonging to the Thermoplasmatales dominate mine acid drainage waters and streamers (riverbed filamentous biofilms), none of the lineages identified in this study affiliate to typical acid mine drainage acidophilic bacteria . Bacteria of the Tinto macrofilaments might be heterotrophic, and could be feeding on the organic matter entrapped in the filamentous structure.

J Magn Reson, 2004 Jul, 169(1), 60 - 7
Combined relaxation and displacement experiment: a fast method to acquire T2, diffusion and velocity maps; Manz B; A fast method for quantitative imaging of T2 and displacement (flow and diffusion) is presented . The pulse sequence combines multi-PGSE NMR with multi-echo acquisition and compensates for flow effects in the read gradient and diffusion during multi-echo trains . The impact of the gradient pulses in a multi-echo train on the signal phase and amplitude is discussed . It is shown that separate T2 and displacement images with microscopic resolution can be obtained within minutes . The capability for 3D flow imaging is demonstrated . The sequence is then used to investigate forced detachment of a biofilm in a tube.

J Hosp Infect, 2004 Jun, 57(2), 175 - 8
Effectiveness of detergent-disinfecting agents on Escherichia coli 54127 biofilm; Henoun Loukili N et al.; Detergent-disinfecting agents (dD) are used daily for cleaning reused medical devices . We have devised a simple method to test dD detergent activity (DA) using an E . coli 54127 biofilm prepared in haemolysis glass tubes, which are cleaned with test dD, according to supplier's recommendations . Crystal violet 0.05% is used to colour the residual biofilm after dD (or tap water control) application . The biofilm quantification was made indirectly by measuring the absorbance of crystal violet at 585 nm . A measure of the detergent effectiveness called DA was calculated as the percentage reduction of colour from a tap water control . Fifteen products including enzymatic and non-enzymatic dDs were evaluated . Most enzymatic dDs gave a high DA, as did some non-enzymatic products . Thus, the view that enzymatic dDs are more effective than non-enzymatic dDs, put forward by some manufacturers, should be regarded with caution . The DA determination should help infection control teams choose, within the wide range of products available on the market, the most effective dD based on both its detergent and disinfecting activity .

Environ Pollut, 2004 Aug, 130(3), 437 - 43
Degradation of toxaphene in water during anaerobic and aerobic conditions; LacayoR M et al.; The degradation of technical toxaphene in water with two kinds of bioreactors operating in sequence was studied . One packed bed reactor was filled with Poraver (foam glass particles) running at anaerobic conditions and one suspended carrier biofilm reactor working aerobically . Chemical oxygen demand (COD), chloride, sulphate, pH, dissolved oxygen, total toxaphene and specific toxaphene isomers were measured . After 6 weeks approx . 87% of the total toxaphene was degraded reaching 98% by week 39 . The majority of the conversion took place in the anaerobic reactor . The concentrations of toxaphene isomers with more chlorine substituents decreased more rapidly than for isomers with less chlorine substituents.

Bioresour Technol, 2004 Sep, 94(3), 275 - 83
Evaluation of chlorines' impact on biofilms on scratched stainless steel surfaces; Lomander A et al.; Biofilms of a wild type Escherichia coli were grown on 316 stainless steel slides in a nutrient starved medium . The stainless steel surfaces were either polished to a smooth finish or scribed . The scribes consisted of lines and crosses . Biofilm samples were taken after 3, 6, 12, 24 and 48 h of growth . After sampling, the slides were soaked in deionized water or 50 or 200 ppm free chlorine prior to vital staining . Images were captured and the areas of viable and total biofilm were estimated . The individual biofilm patches, circularities, total percentage coverage, and viability percentage coverage were analyzed . The biofilms tended to increase in size between 6 and 24 h . A 3-6 h old biofilm on a polished stainless steel surface detached when 200 ppm sodium hypochlorite was applied . When grown in scribes, the circularity decreased up to 24 h, but thereafter increased . As the film grew older, it detached with or without a sodium hypochlorite treatment from the part of the surface that was polished, but remained in the neighborhood of the scribe . Based on the results, we recommend sanitizing at intervals of less than 12 h for this and similar strains of bacteria and protection of stainless steel surfaces to minimize scratching.

Bioprocess Biosyst Eng, 2004 Jul, 26(4), 259 - 70 Epub 2004 Jun 04.
Linearized kinetic model of Listeria monocytogenes biofilm growth; Takhistov P et al.; In this study the dynamics of biofilm formation on aluminum has been investigated . The process of cell growth has been observed using fluorescence microscopy . It has been confirmed that the process of biofilm formation can be represented as a sum of two separate processes: cell adhesion and colony proliferation . The derived set of equations describes kinetics of surface population growth and characteristic times for adsorption and combined growth processes, including characteristic time for the nutrient supply depletion . All equations contain variables based on the fundamental characteristics of bacterial population and can be easily determined from the experimental data or estimated theoretically . The developed theoretical model allows obtaining realistic values for population growth time and characteristic time for nutrient limitation occurrence during the biofilm development . Resulting equations qualitatively describe the biofilm formation process, and allow predicting microbial kinetics in the batch reactor system and determining critical values of the process parameters.

Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 291 - 7
Enhancement of metal bioremediation by use of microbial surfactants; Singh P et al.; Metal pollution all around the globe, especially in the mining and plating areas of the world, has been found to have grave consequences . An excellent option for enhanced metal contaminated site bioremediation is the use of microbial products viz . microbial surfactants and extracellular polymers which would increase the efficiency of metal reducing/sequestering organisms for field bioremediation . Important here is the advantage of such compounds at metal and organic compound co-contaminated site since microorganisms have long been found to produce surface-active compounds when grown on hydrocarbons . Other options capable of proving efficient enhancers include exploiting the chemotactic potential and biofilm forming ability of the relevant microorganisms . Chemotaxis towards environmental pollutants has excellent potential to enhance the biodegradation of many contaminants and biofilm offers them a better survival niche even in the presence of high levels of toxic compounds.

Carbohydr Res, 2004 Jun 1, 339(8), 1467 - 73
Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus epidermidis RP62A, a reference biofilm-positive strain; Sadovskaya I et al.; The ability to adhere to artificial surfaces and form biofilms is considered as a virulence factor of Staphylococcus epidermidis, one of the major causes of nocosomial infections, especially those related to implanted medical devices . Cell-wall teichoic acid is known to play an important role in biofilm formation of staphylococci . The structure of the cell wall and extracellular teichoic acids of S . epidermidis RP62A, a reference biofilm-positive strain, was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry . Their structures were found to be a (1-->3)-linked poly(glycerol phosphate), substituted at the 2-position of glycerol residues with alpha-Glc, alpha-GlcNAc, D-Ala and alpha-Glc6Ala . D-Alanyl acylation of a sugar hydroxyl group seems to be a novel structural feature of teichoic acids from staphylococci .

J Bacteriol, 2004 Jun, 186(12), 3970 - 9
Genes involved in formation of structured multicellular communities by Bacillus subtilis; Branda SS et al.; The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization . Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation . To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B . subtilis genome . This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation . This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media . In this way we identified six genes that are involved in the development of multicellular communities . These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function) . Further analysis revealed that these six genes play different roles in B . subtilis community development.

J Bacteriol, 2004 Jun, 186(12), 3837 - 47
Expression analysis of a highly adherent and cytotoxic small colony variant of Pseudomonas aeruginosa isolated from a lung of a patient with cystic fibrosis; von Gotz F et al.; The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms . In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P . aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population . Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins . This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type . The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS . Thus, the prevailing assumption that P . aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.

Med Sci Monit, 2004 Jun, 10(6), BR180 - 4 Epub 2004 Jun 01.
PER-1 type beta-lactamase production in Acinetobacter baumannii is related to cell adhesion; Sechi LA et al.; BACKGROUND: Recently, the dissemination of ESBL (PER-1) among Acinetobacter isolates was reported in Turkish hospitals . We investigated the presence and the association of various virulence determinants in 20 Acinetobacter baumannii isolates, of which 13 were blaPER-1 positive . MATERIAL/METHODS: Virulence tests were slime and hemolysin production, gelatinase and protease activity, biofilm formation, and Caco2 cell adhesion . RAPD analysis was also performed with ERIC primers . RESULTS: None of the strains was positive for slime or hemolysin production and gelatinase or protease activity . A total of 16 strains, five of which were PER-1 gene negative, formed a biofilm on polymer surfaces . There was no relation between the presence of the PER gene and biofilm formation . On the other hand, nine strains (all PER-1 gene positive) were positive in adhesion experiments to Caco2 cell lines . All PER-negative strains were negative for cell adhesion . CONCLUSIONS: This study indicates the existence of a relation between PER-1 gene and cell adhesion in Acinetobacter strains.

J Mol Microbiol Biotechnol, 2004, 7(1-2), 52 - 62
Pulling together with type IV pili; Nudleman E et al.; Type IV pili are an efficient and versatile device for bacterial surface motility . They are widespread among the beta-, gamma-, and delta-proteobacteria and the cyanobacteria . Within that diversity, there is a core of conserved proteins that includes the pilin (PilA), the motors PilB and PilT, and various components of pilus biogenesis and assembly, PilC, PilD, PilM, PilN, PilO, PilP, and PilQ . Progress has been made in understanding the motor and the secretory functions . PilT is a motor protein that catalyzes pilus retraction; PilB may play a similar role in pilus extension . Type IV pili are multifunctional complexes that can act as bacterial virulence factors because pilus-based motility is used to spread pathogens over the surface of a tissue, or to build multicellular structures such as biofilms and fruiting bodies .

Biotechnol Lett, 2004 Apr, 26(7), 559 - 62
Adaptive responses to static conditions in nutrient-rich cultures of luminous Ralstonia eutropha; Simkus R et al.; The lux-gene fused Ralstonia eutropha, when adapting to static conditions, causes stratification of air-exposed and nutrient-rich cultures at above 0.15 mg biomass ml(-1) . The O2 respiring biofilm (luminous neuston) phase, along with the dark sub-neustonic suspension phase, develops within 5-60 min . The instability of the biphasic static culture was identified as a reason for occasionally observable oscillatory bioluminescence.

Curr Opin Otolaryngol Head Neck Surg, 2004 Jun, 12(3), 185 - 90
The role of biofilms in otolaryngologic infections; Post JC et al.; PURPOSE OF REVIEW: Bacterial biofilms have recently been shown to be important in diseases of the head and neck . Because the concept of biofilms is novel to most practitioners, it is important to gain a basic understanding of biofilms and to recognize that strategies developed to treat planktonic bacteria are ineffective against bacteria in a biofilm . RECENT FINDINGS: Bacteria preferentially exist in complex, surface-attached organizations known as biofilms . Bacteria in biofilms express a different set of genes than their planktonic counterparts and have markedly different phenotypes . Biofilm bacteria communicate with each other, and have mechanisms to diffuse nutrients and dispose of waste . Biofilms provide bacteria with distinct advantages, including antimicrobial resistance and protection from host defenses . Thus, bacteria exist in a far more complex fashion than previously thought and can best be thought of as "self-assembling multicellular communities." Although a focus on the planktonic form of bacteria has been useful in understanding acute infections, chronic infections are much better understood as biofilm illnesses . Biofilms have been shown to be involved in chronic otitis media, chronic tonsillitis, cholesteatoma, and device-associated infections . SUMMARY: Now that basic research has demonstrated that the vast majority of bacteria exist in biofilms, the biofilm concept of disease is beginning to spread throughout the clinical world . Understanding that many of the infections that affect structures of the head and neck are actually biofilm related is fundamental to developing rational strategies for treatment and prevention.

Anesthesiology, 2004 Jun, 100(6), 1446 - 56
Endotracheal tubes coated with antiseptics decrease bacterial colonization of the ventilator circuits, lungs, and endotracheal tube; Berra L et al.; BACKGROUND: Formation of a bacterial biofilm within the endotracheal tube (ETT) after tracheal intubation is rapid and represents a ready source of lung bacterial colonization . The authors investigated bacterial colonization of the ventilator circuit, the ETT, and the lungs when the ETT was coated with silver-sulfadiazine and chlorhexidine in polyurethane, using no bacterial/viral filter attached to the ETT . METHODS: Sixteen sheep were randomized into two groups . Eight sheep were intubated with a standard ETT (control group), and eight were intubated with a coated ETT (study group) . Animals were mechanically ventilated for 24 h . At autopsy, the authors sampled the trachea, bronchi, lobar parenchyma, and ETT for quantitative bacterial cultures . Qualitative bacterial cultures were obtained from the filter, humidifier, inspiratory and expiratory lines, and water trap . ETTs were analyzed with light microscopy, scanning electron microscopy, and laser scanning confocal microscopy . RESULTS: In the control group, all eight ETTs were heavily colonized (10(5)-10(8) colony-forming units {cfu}/g), forming a thick biofilm . The ventilator circuit was always colonized . Pathogenic bacteria colonized the trachea and the lungs in five of eight sheep (up to 10(9) cfu/g) . In the study group, seven of eight ETTs and their ventilator circuits showed no growth, with absence of a biofilm; one ETT and the respective ventilator circuit showed low bacterial growth (10(3)-10(4) cfu/g) . The trachea was colonized in three sheep, although lungs and bronchi showed no bacterial growth, except for one bronchus in one sheep . CONCLUSIONS: Coated ETTs induced a nonsignificant reduction of the tracheal colonization, eliminated (seven of eight) or reduced (one of eight) bacterial colonization of the ETT and ventilator circuits, and prevented lung bacterial colonization.

Mol Microbiol, 2004 Jun, 52(5), 1495 - 511
The FNR-type transcriptional regulator SinR controls maturation of Agrobacterium tumefaciens biofilms; Ramey BE et al.; Agrobacterium tumefaciens is a plant pathogen that persists as surface-associated populations on plants or soil particles . A genetic screen for A . tumefaciens mutants deficient for surface interactions identified a mutant that forms thin, sparsely populated biofilms, but is proficient for initial attachment . The mutant is disrupted in a gene designated sinR, encoding a member of the DNR subfamily of FNR-type transcription regulators . SinR is required for normal maturation of A . tumefaciens biofilms on both inert surfaces and plant tissues, and elevated sinR expression results in accelerated biofilm formation . Expression of sinR is increased close to 30-fold in cultures grown in oxygen-limited environments and is also induced within biofilms grown under oxic conditions . A consensus FNR box, the presumptive binding site for FNR-type proteins, is located upstream of the sinR promoter . FnrN, a second A . tumefaciens FNR-like regulator, is required for induction of sinR in oxygen-limited cultures, whereas SinR negatively influences its own expression . FnrN influences biofilm formation, but its effects are less dramatic than those of SinR . We propose a model in which a signal cascade, responsive to oxygen limitation and initiated by FnrN, activates sinR expression in response to decreased oxygen levels, and influences the formation of A . tumefaciens biofilms.

J Antimicrob Chemother, 2004 Jul, 54(1), 86 - 9 Epub 2004 May 26.
Effect of a high-molecular-weight component of cranberry on constituents of dental biofilm; Steinberg D et al.; BACKGROUND: Previous studies have shown that high molecular-weight non-dialysable material derived from cranberry juice (NDM) inhibits co-aggregation of a variety of oral bacteria . OBJECTIVES: In the present study, we examined the effect of NDM on several constituents of the dental biofilm, glucosyltransferase (GTF) and fructosyltransferase (FTF), as well as on the adhesion of Streptococcus sobrinus . RESULTS: The activity of immobilized and soluble GTF and FTF was inhibited by NDM (P > 0.05) . NDM also inhibited adhesion of S . sobrinus to hydroxyapatite (P < 0.05) . CONCLUSIONS: Our results indicate that NDM may affect biofilm formation . One of the proposed mechanisms is via inhibition of extracellular polysaccharide synthesis, which promote the sucrose-dependent adhesion of oral bacteria as S . sobrinus.

Biotechnol Bioeng, 2004 Jun 30, 86(7), 729 - 36
In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength; Nandakumar K et al.; We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces . Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm . Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area) . Nonirradiated coupons with biofilm served as control . The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation . The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively . Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons) . The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells . The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm . The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag . The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass . Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces .

Can J Microbiol, 2003 Dec, 49(12), 775 - 9
Eight gram-negative bacteria are 10,000 times more sensitive to cationic detergents than to anionic detergents; Rajagopal S et al.; In liquid culture, eight typical gram-negative bacteria were ca . 10,000-fold more sensitive to cationic detergents than to the anionic detergent sodium dodecyl sulfate . Cetyltrimethylammonium bromide (CTAB) was inhibitory at concentrations ranging from 0.0006% to 0.01% . Four pseudomonads able to form biofilms were ca . 1000-fold more resistant to CTAB on Luria-Bertani agar plates than they were in liquid culture . A lasI mutant of Pseudomonas aeruginosa was only able to tolerate 0.1% CTAB on Luria-Bertani agar plates but could tolerate 5% CTAB when supplemented with homoserine lactone containing culture supernatants.

Can J Microbiol, 2003 Dec, 49(12), 741 - 53
Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization; Batte M et al.; Biofilms were grown in annular reactors supplied with drinking water enriched with 235 microg C/L . Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization . EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (alpha), beta (beta), and gamma (gamma) subclasses of Proteobacteria, respectively . The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days . From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria . Among these cells, 16.9%-24.7% were targeted with the BET42a probe, 1.8%-18.3% with the ALF1b probe, and <2.5% with the GAM42a probe . Phosphate treatment induced a significant enhancement to the proportion of gamma-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria . Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes . The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells . Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87% . However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment . Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at which the other bacterial groups were destroyed by chlorination . The results suggest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations involved.

Proc Natl Acad Sci U S A, 2004 Jun 1, 101(22), 8414 - 9 Epub 2004 May 24.
Programmable cells: interfacing natural and engineered gene networks; Kobayashi H et al.; Novel cellular behaviors and characteristics can be obtained by coupling engineered gene networks to the cell's natural regulatory circuitry through appropriately designed input and output interfaces . Here, we demonstrate how an engineered genetic circuit can be used to construct cells that respond to biological signals in a predetermined and programmable fashion . We employ a modular design strategy to create Escherichia coli strains where a genetic toggle switch is interfaced with: (i) the SOS signaling pathway responding to DNA damage, and (ii) a transgenic quorum sensing signaling pathway from Vibrio fischeri . The genetic toggle switch endows these strains with binary response dynamics and an epigenetic inheritance that supports a persistent phenotypic alteration in response to transient signals . These features are exploited to engineer cells that form biofilms in response to DNA-damaging agents and cells that activate protein synthesis when the cell population reaches a critical density . Our work represents a step toward the development of "plug-and-play" genetic circuitry that can be used to create cells with programmable behaviors.

Microbes Infect, 2004 May, 6(6), 623 - 9
Biofilm formation and dispersal in Xanthomonas campestris; Crossman L et al.; Xanthomonas campestris pathovar campestris is the causal agent of black rot disease of cruciferous plants . A cell-cell signalling system encoded by genes within the rpf cluster is required for the full virulence of this plant pathogen . This system has recently been implicated in regulation of the formation and dispersal of Xanthomonas biofilms.

Infect Immun, 2004 Jun, 72(6), 3489 - 94
Identification of a novel two-component system in Streptococcus gordonii V288 involved in biofilm formation; Zhang Y et al.; Streptococcus gordonii is a pioneer colonizer of the teeth, contributing to the initiation of the oral biofilm called dental plaque . To identify genes that may be important in biofilm formation, a plasmid integration library of S . gordonii V288 was used . After screening for in vitro biofilm formation on polystyrene, a putative biofilm-defective mutant was isolated . In this mutant, pAK36 was inserted into a locus encoding a novel two-component system (bfr {biofilm formation related}) with two cotranscribed genes that form an operon . bfrA encodes a putative response regulator, while bfrB encodes a receptor histidine kinase . The bfr mutant and wild-type strain V288 showed similar growth rates in Todd-Hewitt broth (THB) . A bfr-cat fusion strain was constructed . During growth in THB, the reporter activity (chloramphenicol acetyltransferase) was first detected in mid-log phase and reached a maximum in stationary phase, suggesting that transcription of bfr was growth stage dependent . After being harvested from THB, the bfr mutant adhered less effectively than did wild-type strain V288 to saliva-coated hydroxyapatite (sHA) . To simulate pioneer colonization of teeth, S . gordonii V288 was incubated with sHA for 4 h in THB with 10% saliva to develop biofilms . RNA was isolated, and expression of bfrAB was estimated . In comparison to that of cells grown in suspension (free-growing cells), bfr mRNA expression by sessile cells on sHA was 1.8-fold greater and that by surrounding planktonic cells was 3.5-fold greater . Therefore, bfrAB is a novel two-component system regulated in association with S . gordonii biofilm formation in vitro.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2283 - 7
Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo; Kadurugamuwa JL et al.; Eradication of Staphylococcus aureus biofilms after rifampin treatment was tested in a mouse model of device-related infection by using biophotonic imaging . Following treatment, the bioluminescent signals decreased to undetectable levels, irrespective of the age of the biofilm . After the final treatment, the signals rebounded in a time-dependent manner and reached those for the untreated mice . Readministration of rifampin was unsuccessful in eradicating reestablished infections, with the rifampin MICs for such bacteria being increased and with the bacteria having point mutations in the rpoB gene.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2251 - 9
Effect of erythromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model; Nagata T et al.; Diffuse panbronchiolitis (DPB) is a chronic lower respiratory tract infection commonly associated with persistent late-stage Pseudomonas aeruginosa infection . However, low-dose long-term therapy with certain macrolides is effective in most patients with DPB . The present study was designed to examine the effects of long-term erythromycin (ERY) therapy by using our established murine model of chronic respiratory P . aeruginosa infection . ERY or saline was administered from day 80 after intubation with a P . aeruginosa-precoated tube for the subsequent 10, 20, 40, and 80 days . Bacteriologic and histologic analyses of the murine lungs and electron microscopy of the intubated tube were performed . In the murine model, treatment with ERY for 80 days significantly reduced the number of viable P . aeruginosa organisms in the lungs (P < 0.05) . The biofilm formed in situ by P . aeruginosa on the inner wall of the inoculation tube placed into the murine bronchus became significantly thinner after 80 days of ERY treatment . We conclude that the clinical efficacy of macrolides in DPB may be due at least in part to the reduction in P . aeruginosa biofilm formation.

Pest Manag Sci, 2004 May, 60(5), 417 - 33
Transport and distribution of lindane and simazine in a riverine environment: measurements in bed sediments and modelling; Allan IJ et al.; Aquatic sediments often remove hydrophobic contaminants from fresh waters . The subsequent distribution and concentration of contaminants in bed sediments determines their effect on benthic organisms and the risk of re-entry into the water and/or leaching to groundwater . This study examines the transport of simazine and lindane in aquatic bed sediments with the aim of understanding the processes that determine their depth distribution . Experiments in flume channels (water flow of 10 cm s(-1)) determined the persistence of the compounds in the absence of sediment with (a) de-ionised water and (b) a solution that had been in contact with river sediment . In further experiments with river bed sediments in light and dark conditions, measurements were made of the concentration of the compounds in the overlying water and the development of bacterial/algal biofilms and bioturbation activity . At the end of the experiments, concentrations in sediments and associated pore waters were determined in sections of the sediment at 1 mm resolution down to 5 mm and then at 10 mm resolution to 50 mm depth and these distributions analysed using a sorption-diffusion-degradation model . The fine resolution in the depth profile permitted the detection of a maximum in the concentration of the compounds in the pore water near the surface, whereas concentrations in the sediment increased to a maximum at the surface itself . Experimental distribution coefficients determined from the pore water and sediment concentrations indicated a gradient with depth that was partly explained by an increase in organic matter content and specific surface area of the solids near the interface . The modelling showed that degradation of lindane within the sediment was necessary to explain the concentration profiles, with the optimum agreement between the measured and theoretical profiles obtained with differential degradation in the oxic and anoxic zones . The compounds penetrated to a depth of 40-50 mm over a period of 42 days.

Am J Infect Control, 2004 May, 32(3), 177 - 83
Role of biofilm in catheter-associated urinary tract infection; Trautner BW et al.; The predominant form of life for the majority of microorganisms in any hydrated biologic system is a cooperative community termed a "biofilm." A biofilm on an indwelling urinary catheter consists of adherent microorganisms, their extracellular products, and host components deposited on the catheter . The biofilm mode of life conveys a survival advantage to the microorganisms associated with it and, thus, biofilm on urinary catheters results in persistent infections that are resistant to antimicrobial therapy . Because chronic catheterization leads almost inevitably to bacteriuria, routine treatment of asymptomatic bacteriuria in persons who are catheterized is not recommended . When symptoms of a urinary tract infection develop in a person who is catheterized, changing the catheter before collecting urine improves the accuracy of urine culture results . Changing the catheter may also improve the response to antibiotic therapy by removing the biofilm that probably contains the infecting organisms and that can serve as a nidus for reinfection . Currently, no proven effective strategies exist for prevention of catheter-associated urinary tract infection in persons who are chronically catheterized.

Am J Infect Control, 2004 May, 32(3), 170 - 6
Removal of biofilm from endoscopes: evaluation of detergent efficiency; Vickery K et al.; BACKGROUND: Biofilm consisting of bacteria enclosed in a matrix of exopolysaccharide (EPS) forms on many medical devices such as catheters and implants . Nosocomial infection is, thus, a newly recognized scenario of biofilm development . Biofilm removal by physical methods such as ultrasound and mechanical cleaning is reasonably effective but difficult to supervise in practice . Chemical methods are often ineffective because of biofilm resistance to biocides . In this study, we compared the efficiency of different detergents used in endoscope reprocessing . METHODS: Escherichia coli biofilm was generated on Teflon and medical grade PVC tubing under low flow conditions . Sections of biofilm covered tubing were washed using test detergents and biofilm removal was assessed by counting remaining adherent bacteria after washing and by scanning electron microscopy to qualitatively assess the amount and nature of the remaining biofilm . RESULTS: Control tubing developed a multilayered biofilm consisting of >10(5) bacterial cells/cm(2) . Only Matrix (Whiteley Medical, Sydney, Australia) produced >4 log reduction in viable bacterial numbers . Matrix and Epizyme Rapid (3M Australia, Pymble, Australia) were able to remove up to 75% and 60% of the biofilm, respectively . CONCLUSIONS: Many commonly used enzymatic cleaners fail to reduce the viable bacterial load or remove the bacterial EPS . Cleaners with high enzyme activity, Epizyme Rapid, removed more biofilm but failed to reduce bacterial numbers more than 2 logs . The only cleaner containing no enzymes, Matrix, significantly reduced bacterial viability and residual bacterial EPS.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2004 May, 97(5), 632 - 41
Fungi in endodontic infections; Siqueira JF Jr et al.; Fungi are chemoorganotroph eukaryotic microorganisms that can take part in endodontic infections and thereby may participate in the etiology of periradicular diseases . They possess virulence attributes--including adaptability to a variety of environmental conditions, adhesion to a variety of surfaces, the production of hydrolytic enzymes, morphologic transition, biofilm formation, and evasion and immunomodulation of the host defense--that may play a role in the pathogenesis of periradicular diseases . Fungi have occasionally been found in primary root canal infections, but they seem to occur more often in the root canals of obturated teeth in which treatment has failed . Candida albicans is by far the fungal species most commonly isolated from infected root canals, and this species has been considered a dentinophilic microorganism because of its invasive affinity to dentin . C albicans has also been discovered to be resistant to some intracanal medicaments, such as calcium hydroxide . Its ability to invade dentinal tubules and resistance to commonly used intracanal medicaments may help to explain why C albicans has been associated with cases of persistent root canal infections . Some medicaments, such as chlorhexidine digluconate, calcium hydroxide combinations (with camphorated paramonochlorophenol or chlorhexidine), and EDTA, have the potential to be used as effective intracanal medications for patients in whom fungal infection is suspected.

Caries Res, 2004 May-Jun, 38(3), 305 - 13
How 'clean' must a cavity be before restoration?
Kidd EA.
The metabolic activity in dental plaque, the biofilm at the tooth surface, is the driving force behind any loss of mineral from the tooth or cavity surface . The symptoms of the process (the lesion) reflect this activity and can be modified by altering the biofilm, most conveniently by disturbing it by brushing with a fluoride-containing toothpaste . The role of operative dentistry in caries management is to restore the integrity of the tooth surface so that the patient can clean . Thus, the question, 'how clean must a cavity be before restoration?' may be irrelevant . There is little evidence that infected dentine must be removed prior to sealing the tooth . Leaving infected dentine does not seem to result in caries progression, pulpitis or pulp death . However, some of the bacteria survive . What is their fate and if they are not damaging, why is this?

Caries Res, 2004 May-Jun, 38(3), 212 - 22
Application of the Zürich biofilm model to problems of cariology; Guggenheim B et al.; The term biofilm is increasingly replacing 'plaque' in the literature, but concepts and existing paradigms are changing much more slowly . There is little doubt that biofilm research will lead to more realistic perception and interpretation of the physiology and pathogenicity of microorganisms colonizing plaques in the oral cavity . There is clear evidence that the genotypic and phenotypic expression profiles of biofilm and planktonic bacteria are different . Several techniques are available today to study multispecies biofilms of oral bacteria, each having its particular advantages and weaknesses . We describe a biofilm model developed in Zurich and demonstrate a number of applications with direct or indirect impact on prophylactic dentistry: spatial arrangement and associative behavior of various species in biofilms; multiplex fluorescent in situ hybridization analysis of oral bacteria in biofilms; use of the biofilm model to predict in vivo efficacy of antimicrobials reliably; mass transport in biofilms; de- and remineralization of enamel exposed to biofilms in vitro . The potential of biofilm experimentation in oral biology has certainly not yet been fully exploited and dozens of possible interesting applications could be investigated . The overall physiological parameters of multispecies biofilms can be measured quite accurately, but it is still impossible to assess in toto the multitude of interactions taking place in such complex systems . What can and should be done is to test hypotheses stemming from experiments with planktonic cells in monospecies cultures . In particular, it will be interesting to investigate the relevance to biofilm composition and metabolism of specific gene products by using appropriate bacterial mutants .

Caries Res, 2004 May-Jun, 38(3), 204 - 11
Dental plaque as a microbial biofilm; Marsh PD; New technologies have provided novel insights into how dental plaque functions as a biofilm . Confocal microscopy has confirmed that plaque has an open architecture similar to other biofilms, with channels and voids . Gradients develop in areas of dense biomass over short distances in key parameters that influence microbial growth and distribution . Bacteria exhibit an altered pattern of gene expression either as a direct result of being on a surface or indirectly as a response to the local environmental heterogeneity within the biofilm . Bacteria communicate via small diffusible signalling molecules (e.g . competence-stimulating peptide, CSP; autoinducer 2); CSP induces both genetic competence and acid tolerance in recipient sessile cells . Thus, rates of gene transfer increase in biofilm communities, and this is one of several mechanisms (others include: diffusion-reaction, neutralization/inactivation, slow growth rates, novel phenotype) that contribute to the increased antimicrobial resistance exhibited by bacteria in biofilms . Oral bacteria in plaque do not exist as independent entities but function as a co-ordinated, spatially organized and fully metabolically integrated microbial community, the properties of which are greater than the sum of the component species . A greater understanding of the significance of dental plaque as a mixed culture biofilm will lead to novel control strategies .

Caries Res, 2004 May-Jun, 38(3), 182 - 91
Changing paradigms in concepts on dental caries: consequences for oral health care; Fejerskov O; Kuhn proposed in his Structure of Scientific Revolutions (1962) that the theoretical framework of a science (paradigm) determines how each generation of researchers construes a causal sequence . Paradigm change is infrequent and revolutionary; thereafter previous knowledge and ideas become partially redundant . This paper discusses two paradigms central to cariology . The first concerns the most successful caries-preventive agent: fluoride . When it was thought that fluoride had to be present during tooth mineralisation to 'improve' the biological apatite and the 'caries resistance' of the teeth, systemic fluoride administration was necessary for maximum benefit . Caries reduction therefore had to be balanced against increasing dental fluorosis . The 'caries resistance' concept was shown to be erroneous 25 years ago, but the new paradigm is not yet fully adopted in public health dentistry, so we still await real breakthroughs in more effective use of fluorides for caries prevention . The second paradigm is that caries is a transmittable, infectious disease: even one caused by specific microorganisms . This paradigm would require caries prevention by vaccination, but there is evidence that caries is not a classical infectious disease . Rather it results from an ecological shift in the tooth-surface biofilm, leading to a mineral imbalance between plaque fluid and tooth and hence net loss of tooth mineral . Therefore, caries belongs to common 'complex' or 'multifactorial' diseases, such as cancer, cardiovascular diseases, diabetes, in which many genetic, environmental and behavioural risk factors interact . The paper emphasises how these paradigm changes raise new research questions which need to be addressed to make caries prevention and treatment more cost-effective .

J Food Prot, 2004 May, 67(5), 1053 - 70
Quorum sensing: a primer for food microbiologists; Smith JL et al.; Quorum sensing is a signaling mechanism through which bacteria modulate a number of cellular functions (genes), including sporulation, biofilm formation, bacteriocin production, virulence responses, as well as others . Quorum sensing is a mechanism of cell-to-cell communication and is mediated by extracellular chemical signals generated by the bacteria when specific cell densities are reached . When the concentration of the signal (and cell population) is sufficiently high, the target gene or genes are either activated or repressed . Quorum sensing increases the ability of the bacteria to have access to nutrients or to more favorable environmental niches and enhances bacterial defenses against eukaryotic hosts, competing bacteria, and environmental stresses . The physiological and clinical aspects of quorum sensing have received considerable attention and have been studied at the molecular level . Little is known, however, on the role of quorum sensing in food spoilage or in the growth and/or toxin production of pathogens present in food . A number of compounds have been isolated or synthesized that antagonize quorum sensors, and application of these antagonists may potentially be useful in inhibiting the growth or virulence mechanisms of bacteria in different environments, including food . It is important that food microbiologists have an awareness and an understanding of the mechanisms involved in bacterial quorum sensing, since strategies targeting quorum sensing may offer a means to control the growth of undesirable bacteria in foods.

Int J Food Microbiol, 2004 May 1, 92(3), 355 - 64
Involvement of humic substances in regrowth; Camper AK; There appear to be interactions in the distribution system that complicate the ability to use AOC/BDOC as an independent assessment of regrowth potential . Two such complications are the limitation of the assays themselves and the potential interaction between the organic carbon concentration with the presence of disinfectants and pipe materials . To address these interactions, a series of experiments spanning several years have been conducted in model distribution systems at the Center for Biofilm Engineering (CBE) using soil-derived humics . When compared to easily utilized organics, humic substances supported the same order of magnitude of biofilm organisms . As carbon concentration was increased from 500 to 1000 to 2000 ppb, there was no increase in growth rate of the organisms, suggesting zero-order kinetics . If the system was chlorinated, there was less biomass, but growth rates were higher . In the presence of corrosion products, humic-fed systems supported more organisms than a control system fed biologically treated water . When free chlorine was maintained at a residual of about 0.2 mg/l, biofilm numbers on the surfaces were reduced . Phosphate alone did not result in fewer bacteria, while a combination of chorine and phosphate had the best results (lowest biofilm numbers) . Adjustment to pH 9 was not effective . Recently completed work compared increasing levels of humic substances in the presence of free chlorine and monochloramine on biofilm growth on a number of surfaces (PVC, epoxy, cement, ductile iron) . As the concentration of humic substances was increased from 0, 0.5 to 2 mg/l, there was an increase in biofilm numbers on all surfaces . This effect was the most pronounced on iron surfaces . These results illustrate that carbon compounds not measured by the BDOC or AOC tests may profoundly influence biofilm numbers . In addition, iron surfaces are at much higher risk for elevated biofilm counts in the presence of humic substances, even if disinfection is practiced . However, corrosion control may mitigate this interaction .

J N Z Soc Periodontol, 2004, (87), 7 - 21
Dental plaque revisited: bacteria associated with periodontal disease; Lovegrove JM; Between 3-12 weeks after the beginning of supragingival plaque formation, a distinctive subgingival microflora predominantly made up of gram-negative, anaerobic bacteria and including some motile species, becomes established . In order to establish in a periodontal site, a species must be able to attach to one of several surfaces including the tooth (or host derived substances adhering to the tooth), the sulcular or pocket epithelium, or other bacterial species that are attached to these surfaces (Socransky and Haffajee 1991) . Bacterial adhesion has demonstrated specificity in the mechanisms involved and studies have shown that there is a diversity of receptors on tooth surfaces, epithelial or other host cells and other bacteria . Recent studies have described bacterial complexes that are present in subgingival plaque and these studies are likely to help in current understanding of the complex ecology observed in dental plaque biofilm (Socransky, Haffajee et al . 1998) . Bacterial interactions play important roles in species survival . Some interspecies relationships are favourable, in that one species produces growth factors for, or facilitates attachment of, another species . Other relationships are antagonistic due to competition for nutrients and binding sites, or to the production of substances that limit or prevent the growth of another species (Socransky and Haffajee 1991) . A number of different bacterial interactions within plaque biofilm have been discussed . In the last 30-40 years, a vast amount of evidence has been published to suggest that bacteria are the primary aetiological agents of periodontal diseases . In the 1950s and early 1960s, periodontal treatment was based on the non-specific plaque hypothesis . However, the non-specific plaque hypothesis gave way after studies suggested that not all organisms in plaque are equally capable of causing destructive periodontal disease . Thus the concept of specificity re-emerged . Criteria for defining periodontal pathogens have been developed and include association, elimination, host response, virulence factors, animal studies and risk assessment (Haffajee and Socransky 1994) . Until recently there were few consensus periodontal pathogens and trying to discriminate pathogenic from non-pathogenic species has been a difficult task for dental researchers for a variety of reasons . A discussion of the specific microbiota associated with gingivitis, chronic and aggressive periodontitis, NUG, HIV-associated periodontitis and implantitis has been presented . The bacteria associated with periodontal diseases are predominantly gram-negative anaerobic bacteria and may include A . actinomycetemcomitans, P . gingivalis, P . intermedia, B . forsythus, C . rectus, E . nodatum, P . micros, S . intermedius and Treponema sp . The bacterial numbers associated with disease are up to 10(5) times larger than those associated with health.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 681 - 4
Phylogeny of the ring-forming bacterium Arcicella aquatica gen . nov., sp . nov . (ex Nikitin et al . 1994), from a freshwater neuston biofilm; Nikitin DI et al.; Arcicella aquatica NO-502(T), obtained from a neuston film on a freshwater lake and belonging to the phylum Bacteroidetes, is characterized by ring-forming cells . The bacterium is a strict aerobe, with optimal growth between 28 and 30 degrees C . Carbohydrates, but no organic acids or amino acids, are used as substrates . The G+C content of strain NO-502(T) is 34.5 mol%; its genome size is 2.9 x 10(9) Da . The genus Arcicella and its type species Arcicella aquatica (type strain NO-502(T)=LMG 21963(T)=CIP 107990(T)) are proposed, and descriptions of this genus and species are given.

Environ Microbiol, 2004 Jun, 6(6), 546 - 51
Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa; Haussler S; Pseudomonas aeruginosa is an ubiquitous environmental bacterium and an opportunistic human pathogen . Not only in most natural habitats but also within the human host, e.g . within the chronically infected cystic fibrosis lung, P . aeruginosa is associated with surfaces in structures known as biofilms . These functional communities represent a unique mode of bacterial growth where bacteria display particular phenotypes that are fundamentally different from planktonic cells . In this review the issue of the molecular mechanisms underlying the emergence of small colony variant (SCV) P . aeruginosa morphotypes that are especially capable of forming biofilms is addressed . It is assumed that the expression of the chaperone usher pathway (cup) genes encoding putative fimbrial adhesins is responsible for the phenotypic switch to an autoaggregative SCV phenotype . The elucidation of phenotypic switching in response to environmental stimuli will significantly increase our understanding of regulatory processes during bacterial adaptation and might be the basis for the initiation of the development of new antimicrobial treatment strategies.

J Oral Rehabil, 2004 May, 31(5), 453 - 9
Efficacy of sodium hypochlorite and coconut soap used as disinfecting agents in the reduction of denture stomatitis, Streptococcus mutans and Candida albicans; Barnabe W et al.; This study evaluated the reduction of denture stomatitis and the antimicrobial activity of 0.05% sodium hypochlorite opposed to Candida albicans and Streptococcus mutans (SGM) when associated with brushing complete dentures with coconut soap . The mucosal characteristics were evaluated according to Newton's classification at baseline, after cleansing the dentures with coconut soap for 15 days in group 1 (nine patients) . In the other group (19 patients) the analysis were made before and after cleansing the dentures with coconut soap and with disinfection in a soak solution of 0.05% sodium hypochlorite for 10 min during 15 days . Microbiological tests were used to isolate C . albicans and SGM . Mann-Whitney and Wilcoxon tests were used to compare the mucosal characteristics and Fisher test and McNemar test to compare C . albicans and SGM levels . Statistical analysis at the 95% confidence level (P < 0.05) showed that: (i) the association of coconut soap and 0.05% sodium hypochlorite significantly reduced clinical signs of denture stomatitis, (ii) C . albicans did not reduce in counts, (iii) SGM were reduced but not significantly and (iv) the association of coconut soap and 0.5% sodium hypochlorite was effective in controlling denture biofilm.

J Appl Microbiol, 2004, 96(6), 1367 - 73
Influence of growth environment on coaggregation between freshwater biofilm bacteria; Rickard AH et al.; AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel . METHODS AND RESULTS: The ability of B . natatoria 2.1 and M . luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix . In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase . Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase . Conversely, M . luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase . Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation . CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer . Under all conditions, the B . natatoria 2.1 coaggregation adhesin and complementary receptor on M . luteus 2.13 were only expressed simultaneously during early stationary phase.

J Mol Recognit, 2004 May-Jun, 17(3), 180 - 5
Optimisation of polymeric surface pre-treatment to prevent bacterial biofilm formation for use in microfluidics; Davidson CA et al.; The production of a microfluidic device for microbial culture has necessitated the development of techniques for the prevention of bacterial adhesion to a range of polymeric substrates including fluoropolymers such as fluorinated ethylene polypropylene, and polyolefins such as low-density polyethylene . Treatment of such materials to increase hydrophilicity reduces the incidence of attachment of Escherichia coli during the first 4 h of cultivation, although no decrease in the number of biofilm initiation sites was detected after 16 h . The incorporation of a mannose analogue to block binding proteins on the F1 binding fimbriae was also investigated . The possibility of ensuring suspension culture of bacterial cells in high surface area to volume ratio nano-vessels is thus facilitated by the correct choice and pre-treatment of materials used in their construction .

J Biol Chem, 2004 Jul 23, 279(30), 31863 - 72 Epub 2004 May 10.
Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7: a novel alginate lyase with a beta-sandwich fold; Yamasaki M et al.; Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease . Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures . Family PL-7 enzymes are still poorly characterized especially in structural properties . Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P . aeruginosa genome . PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a beta-elimination reaction . The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 degrees C . The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P . aeruginosa . In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity . The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 A resolution . PA1167 was found to form a glove-like beta-sandwich composed of 15 beta-strands and 3 alpha-helices . The structural difference between the beta-sandwich PA1167 of family PL-7 and alpha/alpha-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics . Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel beta-helix, alpha/alpha-barrel, and alpha/alpha-barrel + antiparallel beta-sheet structures as basic frames . PA1167 is the fourth novel folding structure found among polysaccharide lyases.

Anal Biochem, 2004 Jun 1, 329(1), 120 - 30
Comparative proteomic analysis of planktonic and immobilized Pseudomonas aeruginosa cells: a multivariate statistical approach; Vilain S et al.; The protein maps of Pseudomonas aeruginosa cells from two natural (attached) and one artificial (gel-entrapped) immobilized-cell (IC) systems, together with their free (suspended) counterparts, were compared after incubation for 18 or 48 h in a minimal salt medium . Principal component analysis (PCA) was used to interpret the variations in protein spot densities that were observed on electropherogram obtained by two-dimensional electrophoresis (2-DE) . PCA of the 2-DE data, a matrix of 933 rows (observations, i.e., spot density values) and 12 columns (variables, i.e., incubation conditions), in which observations were standardized horizontally, extracted four principal components (PCs) accounting for 78.75% of the variability in the protein expression profiles . PC1 opposed the two modes of growth (planktonic and immobilized) while PC2 discriminated between the incubation times of free cell cultures . The incubation conditions of ICs, including the immobilization procedure (entrapment vs attachment) and the nature of the biofilm substratum, were fairly separated in PC3xPC4 . The dependence of the protein patterns on the cell immobilization process was further illustrated by the identification of a number of peptides whose amount remained unchanged or was altered in ICs compared to free bacteria . These results reinforce the topical assertion that bacteria in the immobilized state display a specific physiological behavior but also question the existence of a unique IC phenotype.

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