Biofilms

Biomaterials, 2004 Aug, 25(18), 4117 - 25
Search for the insertion element IS256 within the ica locus of Staphylococcus epidermidis clinical isolates collected from biomaterial-associated infections; Arciola CR et al.; Staphylococcus epidermidis biofilm-forming strains produce a polysaccharide intercellular adhesin (PIA), which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants . PIA synthesis is regulated by the icaADBC locus . In vitro, by repeated subcultures of a biofilm-producing strain, the loss of the ability to produce biofilm appears associated with the insertion of the IS256 element into the ica locus . This study was aimed (i) to investigate if the five genes of ica locus are always all present in different strains of S . epidermidis, and (ii) to search if IS256 insertion naturally occurs in ica locus without making recourse to the experimental procedure of repeated subcultures of strains . 120 S . epidermidis clinical isolates from peri-prosthesis infections were investigated both by an original multiplex PCR analysis of the ica genes and by PCR amplification of the IS256 element . Also two reference strains (the biofilm-negative S . epidermidis ATCC 12228 and the biofilm-forming ATCC 35984 {RP62A}) and two biofilm-negative RP62A-derived acriflavin mutants (D9 and HAM892) were analysed . D9 e HAM892 were for the first time shown to contain in ica locus, at the base 3319, a 1300-bp insertion with a DNA sequence corresponding to IS256 . Among the 120 clinical isolates, 51 (43%) turned out completely ica-positive, 69 completely ica-negative (57%) . The genes of the ica locus appear, in all cases of the present collection, strictly linked each other, so they are either all present or all absent . In this collection, IS256 was present in eight out of the 69 ica-negative strains and in 34 out of the 51 ica-positive strains . IS256, also when present in bacterial genomic DNA, was never found inside the ica locus, thus suggesting that insertion/excision of this element is not a natural occurring mechanism for off/on switching of biofilm production.

Biochem Soc Trans, 2004 Apr, 32(Pt 2), 188 - 92
Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms; Johnson MR et al.; Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures . Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats . Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.

Am J Gastroenterol, 2004 Feb, 99(2), 383 - 9
Enteroaggregative Escherichia coli: an emerging enteric pathogen; Huang DB et al.; Enteroaggregative Escherichia coli (EAEC) represents an emerging pathogen that causes enteric and food-borne infectious diseases . Subgroups in many populations throughout the world are susceptible to EAEC infection . EAEC pathogenesis involves adherence to the intestinal mucosa; increased production and deposition of a mucus biofilm; and mucosal toxicity due to inflammation and cytokine release . Due to the heterogeneity of EAEC strains and differing host immune responses, not all EAEC infections are symptomatic . Recent data suggest that individuals with a homozygous genotype -251 AA single nucleotide polymorphism (SNP), in the IL-8 promoter region, are more susceptible to EAEC diarrhea . The HEp-2 cell adherent assay allows identification of EAEC's characteristic aggregative or "stacked brick" adherence pattern . Antimicrobial treatment of individuals who develop EAEC diarrhea should be individually based . Ciprofloxacin and rifaximin, compared to placebo, have been shown to significantly shorten the course of diarrhea in patients who developed EAEC infection . The objective of this review is to increase awareness of this important emerging pathogen and to discuss the epidemiology, pathogenesis, and host-pathogen factors associated with EAEC infection.

Microb Pathog, 2004 May, 36(5), 237 - 45
The ability of biofilm formation does not influence virulence of Staphylococcus aureus and host response in a mouse tissue cage infection model; Kristian SA et al.; The virulence of Staphylococcus aureus Sa113 (SA113) and an isogenic ica deletion mutant (ica-), deficient in the production of polysaccharide intercellular adhesin (PIA), which is crucial for biofilm formation, was compared in a mouse tissue cage infection model . The minimal infective doses for the induction of persistent tissue infections in C57BL/6 mice were 10(3) CFU for both SA113 and the ica- mutant . Bacterial growth, initial adherence to surfaces within the implants and the course of inflammation including growth-dependent host TNF and MIP-2 release, influx of phagocytes and an accumulation of dead leukocytes were similar as well . Since SA113 expressed PIA in vivo, we could demonstrate that PIA and the lack of biofilm formation did not influence the capacity of S . aureus to induce persistent infections and did not modulate host responses in the mouse tissue cage model.

Curr Opin Investig Drugs, 2004 Feb, 5(2), 186 - 97
Candida biofilms: antifungal resistance and emerging therapeutic options; Kuhn DM et al.; Intravascular catheter infections are a major cause of morbidity and mortality in hospitalized patients, accounting for the majority of the 200,000 nosocomial bloodstream infections occurring in the US annually . Of the intravenous lines that are culture-positive for Candida, 40% actually represent fungemia, which generally necessitates systemic treatment and line removal to affect cure . Until recently, the reason for the need for device removal was unclear . However, our research group and others have demonstrated a near-total resistance to antifungals by biofilm-associated Candida . Similar to bacterial species, Candida biofilm formation proceeds through early, intermediate and maturation phases . This process is associated with the generation of a polysaccharide extracellular matrix (ECM) . Mature C . albicans biofilms have a heterogeneous architecture, in terms of distribution of fungal cells and ECM, and exhibit broad antimicrobial resistance . The mechanisms causing such profound antifungal resistance are beginning to be understood . Recent data indicate that resistance is phase-specific and multifactorial, involving efflux pumps and sterol synthesis (at early and mature biofilm phases, respectively) . Neither metabolic quiescence nor the ECM appear to contribute substantially . Susceptibility testing and confocal scanning laser microscopy demonstrated that azoles failed to exert activity against mature Candida biofilms . However, sub-inhibitory concentrations of voriconazole impaired biofilm formation and caused cell morphological aberrations . In contrast, lipid-formulation amphotericins and the echinocandins uniquely exhibited activity against mature biofilms . The mechanisms underlying this ability are unknown . The role of other pharmacological (eg, catheter coatings, antimicrobial peptides and antibiotic locks) and non-pharmacological methods in the prevention and treatment of device-related biofilms is discussed in this review.

J Magn Reson, 2004 Apr, 167(2), 322 - 7
Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor; Seymour JD et al.; Microorganisms that colonize surfaces, biofilms, are of significant importance due to their role in medical infections, subsurface contaminant remediation, and industrial processing . Spatially resolved data on the distribution of biomass within a capillary bioreactor, the heterogeneity of the biofilm itself and the impact on transport dynamics for a Staphylococcus epidermidis biofilm in the natural growth state are presented . The data demonstrate the ability of magnetic resonance microscopy to study spatially resolved processes in bacterial biofilms, thus providing a basis for future studies of spatially resolved metabolism and in vivo clinical detection.

Nat Rev Microbiol, 2004 Feb, 2(2), 95 - 108
Bacterial biofilms: from the natural environment to infectious diseases; Hall-Stoodley L et al.; Biofilms--matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces--represent a significant and incompletely understood mode of growth for bacteria . Biofilm formation appears early in the fossil record (approximately 3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree . It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments . Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems . Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches . The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.

Infect Immun, 2004 Apr, 72(4), 2177 - 85
Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus; Cucarella C et al.; Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm . Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation . We have classified bovine mastitis S . aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica(+) bap(+)), group 2 (ica(+), bap negative), and group 3 (ica negative, bap negative) . Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3 . In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro . Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S . aureus isolates obtained under field conditions throughout the animal's life . The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S . aureus infections indicated the production of Bap during infection . Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide) . Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S . aureus.

Infect Immun, 2004 Apr, 72(4), 1939 - 45
Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces; Kirov SM et al.; Aeromonas spp . (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces . Only approximately 60% of mesophilic strains can produce lateral flagella . To evaluate flagellar contributions to Aeromonas intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of Aeromonas caviae for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes . Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by approximately 60% in comparison to the wild-type level . Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of approximately 50% of wild-type values, irrespective of their flagellar expression . Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for Aeromonas spp . (broth inoculum, with a 16- to 20-h incubation at 37 degrees C) . All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type) . For the chemotactic motility mutant cheA, biofilm formation decreased >80% from the wild-type level . The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels . We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.

Infect Immun, 2004 Apr, 72(4), 1929 - 38
Immunoglobulin-mediated agglutination of and biofilm formation by Escherichia coli K-12 require the type 1 pilus fiber; Orndorff PE et al.; The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to Escherichia coli is thought to be dependent on type 1 pili . Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the fimA gene . A minor component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described . We found that the aggregation of E . coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM alpha-CH(3)Man . The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin . IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin . These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes . Under conditions in which E . coli was agglutinated by SIgA, the binding of SIgA to E . coli was not increased by the presence of the pili, with or without adhesin . This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin.

Int J Antimicrob Agents, 2004 Mar, 23 Suppl 1, S67 - 74
Bacterial biofilm formation on urologic devices and heparin coating as preventive strategy; Tenke P et al.; In the process of endourological development a variety of foreign bodies have been invented besides urinary catheters, on which biofilm can be formed . Bacteria in the biofilm are less susceptible to antibiotics . An additional problem of medical biomaterials in the urinary tract environment is the development of encrustation and consecutive obstruction . The most promising prevention strategy for bacterial biofilms is the production of materials with anti-adhesive surfaces such as heparin . Although heparin-coated ureteral stents are expensive, they justify their cost . Our studies show that such devices are protected against incrustation and biofilm formation for a longer period of time: 6-12 months, both in vitro and in vivo.

Int J Antimicrob Agents, 2004 Mar, 23 Suppl 1, S24 - 9
Emergence of antibiotic resistance and prudent use of antibiotic therapy in nosocomially acquired urinary tract infections; Wagenlehner FM et al.; Nosocomially acquired urinary tract infections (NAUTI) are common . The reported rates, however, depend very much on the definitions used and the number of investigations requested . In a prospective study on a surgical intensive care unit and adhering closely to the CDC criteria, NAUTI was diagnosed in about 17% of the patients . The urinary catheter associated UTI rate per 1000 catheter days was 14.5 much higher than otherwise reported . Whereas the rates of symptomatic NAUTI and other nosocomially acquired infections were similar, the main difference was found for asymptomatic UTI which depends very much on the effort to search for it systematically . In a prospective study on a urological ward it could be demonstrated that cross-transmission probably plays a much greater role than so far suggested . Continuous surveillance of the bacterial spectrum and resistance is necessary not only on a global but also on a local level . Selection of an appropriate agent for empirical antibacterial therapy can be better tailored if not only the total bacterial spectrum is considered but if all information already available during the identification process is used, such as Gram stain and other simple and rapid tests for stratification of the pathogens . Since in NAUTI usually some kind of biofilm infection is involved, the fluoroquinolones can be considered agents of choice . Only those substances with high antibacterial activity, good bioavailability and those that are mainly excreted by the kidneys should be chosen and they have to be administered at sufficiently high doses.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 718 - 25 Epub 2004 Mar 19.
Membrane-aerated biofilm reactor for the removal of 1,2-dichloroethane by Pseudomonas sp . strain DCA1; Hage JC et al.; A membrane-aerated biofilm reactor (MBR) with a biofilm of Pseudomonas sp . strain DCA1 was studied for the removal of 1,2-dichloroethane (DCA) from water . A hydrophobic membrane was used to create a barrier between the liquid and the gas phase . Inoculation of the MBR with cells of strain DCA1 grown in a continuous culture resulted in the formation of a stable and active DCA-degrading biofilm on the membrane . The maximum removal rate of the MBR was reached at a DCA concentration of approximately 80 micro M . Simulation of the DCA fluxes into the biofilm showed that the MBR performance at lower concentrations was limited by the DCA diffusion rate rather than by kinetic constraints of strain DCA1 . Aerobic biodegradation of DCA present in anoxic water could be achieved by supplying oxygen solely from the gas phase to the biofilm grown on the liquid side of the membrane . As a result, direct aeration of the water, which leads to undesired coagulation of iron oxides, could be avoided.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2003 Nov, 147(1), 27 - 35
Pathogenesis of prosthesis-related infection; Gallo J et al.; In spite of its incidence decreasing to 1% nowadays, prosthesis-related infection remains a research, diagnostic, therapeutic and cost-related problem . It can be defined as a presence of bacteria in the artificial joint space, which is significantly associated with evident laboratory and/or tissue markers, and clinical signs of running infection . We believe that the more precise understanding of pathogenesis, the more effective preventative and therapeutic measures, and the lower infection rate . The implants are colonized by airborne, skin-, and/ or surgeon-related bacteria during surgery despite being operated in closely respected operating regime . Some prosthetic characteristics are advantageous and may play important roles in the process of bacterial adherence . After successful attachment on the biomaterial surface bacteria multiply and physiologically transform into a "biofilm" community, making them much more resistant to antibiotic therapy and host immunity . Bacterial resistance is a complex phenomenon influenced by intrinsic and extrinsic factors, including the cell configuration in the biofilm community . So the cure of periprosthetic sepsis without removing of all foreign bodies and necrotic bone fragments is often ineffective . Acute hematogenous sepsis is suggestive of a distortion of a previously aseptic joint space by invasion of bacteria through the vessels.

J Ind Microbiol Biotechnol, 2004 Mar, 31(3), 109 - 14 Epub 2004 Mar 16.
Biodegradation of acid blue-15, a textile dye, by an up-flow immobilized cell bioreactor; Sharma DK et al.; Acid blue-15, a complex and resonance-stabilized triphenylmethane (TPM) textile dye, resistant to transformation, was decolorized/degraded in an up-flow immobilized cell bioreactor . A consortium comprised of isolates belonging to Bacillus sp., Alcaligenes sp . and Aeromonas sp . formed a multispecies biofilm on refractory brick pieces used as support material . The TPM dye was degraded to simple metabolic intermediates in the bioreactor with 94% decolorization at a flow rate of 4 ml h(-1).

Arch Otolaryngol Head Neck Surg, 2004 Mar, 130(3), 339 - 43
Bacterial biofilm presence in pediatric tracheotomy tubes; Perkins J et al.; OBJECTIVE: To determine whether bacterial biofilms are present on pediatric tracheotomy tubes . DESIGN: Prospective observational series . INTERVENTIONS: Eleven tracheotomy tubes removed during routine tracheotomy tube changes were analyzed for biofilm and live bacteria presence using confocal microscopy and vital stains . The external and internal surfaces of the tracheotomy tubes were studied in 3 locations: distal tip, midtracheotomy tube, and proximal opening . These data were correlated with tracheotomy site cultures and the reason for tracheotomy dependence . MAIN OUTCOME MEASURES: Microscopic images were analyzed for the presence of a biofilm (its morphological features and the presence of live and dead bacteria within the biofilm) . RESULTS: Of 11 tracheotomy tubes, 10 had biofilm present on the internal surface of the distal tip . Externally, at the same location, 4 tubes had biofilms . On the internal surface of the midtracheotomy site, 8 had biofilm present, whereas only 1 had a biofilm on the internal surface of the proximal tracheotomy tube site . In the distal internal tracheotomy tube site, the biofilm was confluent in 5 tubes and patchy with evidence of microcolony formation in the remaining 5 tubes . Live bacteria were present in all biofilms . Control tracheotomy tubes did not have biofilms . All tracheotomy site cultures and disease states (chronic aspiration and bronchopulmonary dysplasia) were associated with tracheotomy tube biofilms . CONCLUSION: Bacterial biofilms containing live bacteria were demonstrated in most pediatric tracheotomy tubes, being most frequent and extensive on the internal surface of the distal tracheotomy tip.

Curr Opin Infect Dis, 2004 Apr, 17(2), 91 - 6
Bacteria and wound healing; Edwards R et al.; PURPOSE OF REVIEW: Wound healing is a complex process with many potential factors that can delay healing . There is increasing interest in the effects of bacteria on the processes of wound healing . All chronic wounds are colonized by bacteria, with low levels of bacteria being beneficial to the wound healing process . Wound infection is detrimental to wound healing, but the diagnosis and management of wound infection is controversial, and varies between clinicians . RECENT FINDINGS: There is increasing recognition of the concept of critical colonization or local infection, when wound healing may be delayed in the absence of the typical clinical features of infection . The progression from wound colonization to infection depends not only on the bacterial count or the species present, but also on the host immune response, the number of different species present, the virulence of the organisms and synergistic interactions between the different species . There is increasing evidence that bacteria within chronic wounds live within biofilm communities, in which the bacteria are protected from host defences and develop resistance to antibiotic treatment . SUMMARY: An appreciation of the factors affecting the progression from colonization to infection can help clinicians with the interpretation of clinical findings and microbiological investigations in patients with chronic wounds . An understanding of the physiology and interactions within multi-species biofilms may aid the development of more effective methods of treating infected and poorly healing wounds . The emergence of consensus guidelines has helped to optimize clinical management.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 39 - 43
Streptococcus mutans fructosyltransferase interactions with glucans; Rozen R et al.; Streptococcus mutans utilizes sucrose to synthesize glucans by glucosyltransferase and fructans by fructosyltransferase (FTF) . Antibodies raised against a recombinant FTF were used to study S . mutans FTF secretion . Low amounts of cell-free FTF were found in culture of S . mutans grown with sucrose, while an increase in bacteria displaying cell surface FTF was detected . FTF added to S . mutans cultures was adsorbed to bacteria grown with sucrose but not to bacteria grown with glucose or fructose or to a gtf inactivated mutant grown with sucrose . Recombinant FTF was found to have high affinity for glucans suggesting that fructans and glucans are an integral part of the polysaccharide matrix of oral biofilms.

J Pharm Biomed Anal, 2004 Mar 1, 34(4), 803 - 10
Potential use of solid phase immunoassays in the diagnosis of coagulase-negative staphylococcal infections; Lamari FN et al.; Staphylococcus epidermidis is a major nosocomial pathogen, even though it is a member of the normal bacterial flora of skin and the mucous membranes . A major complication is the development of biofilms on implanted medical devices . Diagnosis of coagulase-negative staphylococcal infections relies on the presence of clinical manifestation of infections and on microbiologic evidence, usually obtained after the removal of the biomaterial . Solid-phase immunoassays have not yet been used for routine diagnosis of coagulase-negative staphylococcal infections and distinction between pathogenic and normal cocci . The enzyme immunoassays developed in the last decade are presented in this review article . Serodiagnosis has been attempted by determining antibodies against bacterial cells, mixtures of S . epidermidis slime antigens and discrete slime antigens . Detection or typing of staphylococcal cells has been performed by specific antibodies and lectins . There is still a long way until the application of such assays in the routine clinical laboratory and large clinical studies are necessary.

Water Res, 2004 Mar, 38(6), 1626 - 32
Effect of O2 exposure on perchlorate reduction by Dechlorosoma sp . KJ; Song Y et al.; Anaerobic bioreactors have been developed to remove perchlorate from water, but backwashing and operational interruptions can expose biofilms to oxygen . While it is well known that oxygen is a preferential electron acceptor to perchlorate for perchlorate-respiring bacteria, little is known about the effect of oxygen exposure or redox potentials on perchlorate reduction . Four different dissolved oxygen scavengers were tested for their ability to quickly restore anaerobic conditions and allow perchlorate reduction by a facultative, perchlorate respiring bacterium Dechlorosoma sp . KJ . Of the four different oxygen scavengers tested (Oxyrase trade mark, L-cysteine, Na2S and FeS), only Oxyrase trade mark was able to rapidly (<30 min) scavenge dissolved oxygen and allow cell growth . There was no cell growth after addition of Na2S and FeS, and l-cysteine produced a long lag in cell growth . To investigate the effect of dissolved oxygen on perchlorate reduction, anaerobically grown cultures Dechlorosoma sp . KJ, were exposed to dissolved oxygen for various periods ranging from 1 to 32 h . Perchlorate reduction and redox potential were then measured for cells returned to an anaerobic environment containing an oxygen scavenger . It was determined that cells exposed to dissolved oxygen for more than 12h were incapable of reducing perchlorate . Cells exposed to dissolved oxygen for less than 12h quickly reduced the redox potential to negative values (-127 mV to -337 mV) and were able to reduce perchlorate or chlorite . Our results suggest that aeration during backwashing of biofilm reactors, or exposure of perchlorate-degrading cell suspensions to dissolve oxygen for less than 12h, will not be detrimental to the ability of perchlorate-degrading bacteria to use perchlorate as an electron acceptor.

Water Res, 2004 Mar, 38(6), 1530 - 41
Overcoming oxygen limitations in membrane-attached biofilms--investigation of flux and diffusivity in an anoxic biofilm; Emanuelsson EA et al.; The possibility of overcoming oxygen limitations in membrane-attached biofilms has been investigated by using nitrate as an electron acceptor instead of oxygen in an extractive membrane bioreactor (EMB) degrading toluene . The effect of nitrate concentration on toluene flux, the effective diffusivity in the biofilm and the biofilm activity has been investigated . A counter-diffusion-reaction model is also presented, describing the pollutant flux versus biofilm thickness . The toluene flux decreased with increasing biofilm thickness under excess nitrate concentrations, similar to the experiment with low nitrate . Mathematical modelling indicated that this was either due to decreasing activity, and/or different diffusivities in the biofilm . The effective diffusivity was investigated by using an inert tracer molecule . It remained constant for biofilm thicknesses up to 1.8mm, with a value twice that in water . The biofilm activity was investigated by inactivating a mature biofilm using sodium azide . The toluene flux remained the same before and after the addition of sodium azide, suggesting that the activity in the biofilm is very low . We conclude that the decreasing toluene flux with increasing biofilm thickness is due to the diffusional resistance of the inactive biofilm.

Water Res, 2004 Mar, 38(6), 1457 - 66
Survival of Mycobacterium avium in a model distribution system; Norton CD et al.; A pilot study was designed to examine the impact of nutrient levels, pipe materials, and disinfection on the survival of M . avium in model drinking water distribution system biofilms . Studies showed that the survival of the organism was dependant upon a complex interaction between pipe surface, nutrient levels, and disinfectants . The findings showed that when no disinfection was applied, M . avium could be recovered from biofilms at nutrient levels of 50microg/L assimilable organic carbon . M . avium concentrations were lower on copper pipe surfaces following disinfection with free chlorine as compared to monochloramine . However, due to the interference of corrosion products, chloramination of iron pipe surfaces controlled M . avium levels better than free chlorine . These data demonstrate the significance of pipe materials on the survival of M . avium complex in biofilms . Elimination of competitive heterotrophic bacteria on copper pipe surfaces by the application of disinfection resulted in a population of nearly 100% M . avium . Heat treatment of M . avium biofilms was affected by the pipe composition and organic content of the water . Effluent temperatures >53 degrees C were required to control the occurrence of M . avium in the pipeline system . Although additional studies are required using improved detection methods, the results of this investigation suggest that reducing the biodegradable organic material in drinking water, control of corrosion, maintenance of an effective disinfectant residual, and management of hot water temperatures can help limit the occurrence of M . avium complex in drinking water biofilms.

Water Res, 2004 Mar, 38(6), 1390 - 404
The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass; Batstone DJ et al.; The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors . The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength . Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies . In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries . Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM) . The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters . The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics . Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations . The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater . Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates.

Water Res, 2004 Mar, 38(6), 1376 - 89
Anaerobic sludge granulation; Hulshoff Pol LW et al.; This paper reviews different theories on anaerobic sludge granulation in UASB-reactors that have been proposed during the past two decades . The initial stages of the formation of anaerobic granules follow the same principles as biofilm formation of bacteria on solid surfaces . There exist strong evidence that inert carriers play an important positive role in granulation . Most researchers conclude that Methanosaeta concilii is a key organism in granulation . Only the Cape Town Hypothesis presumes that an autotrophic hydrogenotrophic organism, i.e., Methanobacterium strain AZ, growing under conditions of high H(2)-pressures, is the key organism in granulation . Many authors focus on the initial stage of granulation, and only a few contributions discuss the latter stages in granulation: granule maturation and multiplication . Granule enhancing factors in the latter stages predominantly rely on manipulation of the selection pressure, through which selectively heavier sludge particles are retained in the UASB reactor.

Water Res, 2004 Mar, 38(6), 1355 - 67
Retention and removal of pathogenic bacteria in wastewater percolating through porous media: a review; Stevik TK et al.; Properly designed biological filters or infiltration systems have the capacity to significantly reduce effluent concentrations of pathogenic microorganisms in wastewater . The retention and elimination of microbial cells in biological wastewater filter systems is influenced by several factors . In this review, these factors are discussed . Immobilization of microbial cells moving through a porous media is influenced by mechanisms such as physical straining as well as adsorption to porous media . The grain size of porous media and bacterial cell size are important factors affecting the straining of bacteria, as are the hydraulic loading rate or the extent of clogging layer development in the filter . Adsorption of cells to the porous media is influenced by the content of organic matter, degree of biofilm development, and electrostatic attraction due to ion strength of the solution or electrostatic charges of cell- and particle surfaces . The rate of inactivation of pathogenic microorganisms, in adsorbed or liquid phases, has been shown to be affected by abiotic and biotic factors such as moisture content, pH, temperature, organic matter, bacterial species, predation, and antagonistic symbiosis between microorganisms in the system.

Clin Diagn Lab Immunol, 2004 Mar, 11(2), 379 - 86
Establishment of an animal model using recombinant NOD.B10.D2 mice to study initial adhesion of oral streptococci; Abdus Salam M et al.; An oral biofilm is a community of surface-attached microorganisms that coats the oral cavity, including the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases in patients with dry mouth or Sjogren's syndrome (SS) . The purpose of this study was to establish an animal model for studying the initial adhesion of oral streptococci that cause biofilm formation in patients with dry mouth and SS in an attempt to decrease the influence of cariogenic organisms and their substrates . In nonobese diabetogenic (NOD) mice that spontaneously develop insulin-dependent diabetes mellitus (IDDM) and SS, we replaced major histocompatibility complex (MHC) class II (A(g7) E(g7)) and class I D(b) with MHC class II (A(d) E(d)) and class I D(d) from nondiabetic B10.D2 mice to produce an animal model that inhibited IDDM without affecting SS . The adhesion of oral streptococci, including Streptococcus mutans, onto tooth surfaces was then investigated and quantified in homologous recombinant N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice . We found that a higher number of oral streptococci adhered to the tooth surfaces of N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice than to those of the control C57BL/6 and B10.D2 mice . On the basis of our observation, we concluded that these mouse models might be useful as animal models of dry mouth and SS for in vivo biological studies of oral biofilm formation on the tooth surfaces.

Infect Dis Clin North Am, 2003 Dec, 17(4), 679 - 95
Hospital-acquired pneumonia: etiologic considerations; Alcon A et al.; The development of pneumonia requires the pathogen to reach the alveoli and the host defenses to be overwhelmed, either by microorganism virulence or by inoculums size . The endogenous sources of microorganisms are nasal carriers, sinusitis, mouth, oropharynx, gastric, or tracheal colonization, and hematogenous spread . The exogenous sources of microorganisms are biofilm of the tracheal tube, ventilator circuits, nebulizers, and humidifiers . Health care workers may also play a role in this setting . Different microorganisms can be found depending on the onset time of pneumonia and on the local pattern variation encountered between different institutions and countries . Healthy patients may be chronically colonized . A very important, unresolved issue is the definition of early and late-onset pneumonia; it still remains uncertain from the literature whether the given threshold refers to the number of days in hospital or to the number of days following intubation . Noninvasive ventilation is demonstrating that the term "ventilator-associated pneumonia" is perhaps inaccurate and should be referred to as "intubation-associated pneumonia."

Appl Environ Microbiol, 2004 Mar, 70(3), 1698 - 707
Molecular evidence for the evolution of metal homeostasis genes by lateral gene transfer in bacteria from the deep terrestrial subsurface; Coombs JM et al.; Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities . To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface . Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria . A nested PCR approach was employed to obtain DNA sequences encoding P(IB)-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall . Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P(IB)-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the beta subclass of the Proteobacteria (beta-proteobacterium) that may have predated the diversification of the genus PSEUDOMONAS: Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of beta- and gamma-proteobacteria . Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P(IB)-type ATPase amplicons . This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.

Appl Environ Microbiol, 2004 Mar, 70(3), 1528 - 36
High diversity among environmental Escherichia coli isolates from a bovine feedlot; Yang HH et al.; Approximately 280 Escherichia coli isolates were isolated from a bovine feedlot at the University of Connecticut campus via enrichment in lauryl tryptose broth and random selection from MacConkey plates . The E . coli subspecies diversity was estimated by employing whole-cell BOX-PCR genomic fingerprints . A total of 89 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 85% fingerprint similarity as a surrogate for an OTU, while the Chao1 index estimated the E . coli population richness at 128 OTUs . One genotype (at a similarity level of 60%) dominated the population at 66% regardless of sampling depth or location, while no significant vertical distribution pattern was observed in terms of genotype, mobility, antibiotic resistance profile, or biofilm-forming ability . Motility, measured by a soft agar assay, had a very broad range among the E . coli population and was positively correlated with biofilm-forming ability in minimal medium (Spearman's rank correlation coefficient r = 0.619, P < 10(-4)) but not in Luria broth . Only an estimated 48% of the population possessed gene agn43, which encodes Ag43, a phase-variable outer membrane protein that has been implicated in biofilm formation in minimal medium . We observed significantly more biofilm formation in both minimal medium and Luria broth for agn43(+) strains, with a larger effect in minimal medium . This study represents an exhaustive inventory of extant E . coli population diversity at a bovine feedlot and reveals significant subspecies heterogeneity in interfacial behavior.

Appl Environ Microbiol, 2004 Mar, 70(3), 1321 - 7
Characterization of the arginine deiminase operon of Streptococcus rattus FA-1; Griswold A et al.; The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology . The ADS consists of three enzymes . Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia . Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB) . Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP . Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells . The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci . Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed . Results showed that the genes encoding the AD pathway in S . rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR) . The arcA transcriptional start site was identified by primer extension, and a sigma(70)-like promoter was mapped 5' to arcA . Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed . Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.

J Microbiol Methods, 2004 Apr, 57(1), 95 - 106
Green and red fluorescent protein vectors for use in biofilm studies of the intrinsically resistant Burkholderia cepacia complex; Tomlin KL et al.; Cystic fibrosis isolates of the Burkholderia cepacia complex (BCC) have demonstrated a propensity to associate intimately with Pseudomonas aeruginosa in mixed community biofilms, which may impact on their overall pathogenicity during infection of the lungs in cystic fibrosis . Here, we describe the construction and use of novel green and red fluorescent protein expression vectors suitable for labeling biofilm cells of multi-resistant clinical isolates of the BCC for microscopic analysis of both single species biofilms and mixed community associations with P . aeruginosa . Antimicrobial susceptibility testing established that tetracycline and/or trimethoprim were suitable selective agents for widespread use in BCC . The green and red fluorescent protein genes, driven by constitutively active promoters, were cloned into two mobilizable plasmids pBBR1MCS-3 and pBBR1Tp, carrying tetracycline and trimethoprim resistance cassettes, respectively . The fluorescence of transformed BCC and P . aeruginosa planktonic cells was detectable using fluorescence microscopy and/or fluorometry . The plasmids were stable in the absence of selection for at least 3 days in planktonic and biofilm cultures, and fluorescence was still visible in a 4-day glass coverslip flow cell biofilm . The plasmids functioned well to distinguish the two species in a mixed community biofilm, with no indications of plasmid transfer between species or cross-talk of the fluorescent signals . These vectors represent the first green and red fluorescent vectors to be constructed and analyzed specifically for wide spread use in BCC and P . aeruginosa single and mixed biofilm cultures.

J Microbiol Methods, 2004 Apr, 57(1), 55 - 64
Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy; Kamper M et al.; Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques . A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale . By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.

Arch Oral Biol, 2004 Apr, 49(4), 295 - 304
Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis; Black C et al.; OBJECTIVE: Oral streptococci are primary colonisers of the tooth surface and are abundant in dental plaque biofilms . Bacteria growing in these relatively dense, surface-associated communities are phenotypically quite distinct from their planktonic counterparts . The purpose of the present study was to develop a method to investigate biofilm-specific surface protein expression by Streptococcus sanguis to help provide a better understanding of the critical events in plaque development . DESIGN: Biofilm cells were grown on the surface of glass beads in a biofilm device fed with mucin-containing artificial saliva . Planktonic cells were grown in continuous culture at approximately the same growth rate . Surface hydrophobicity of biofilm and planktonic cells was determined by hexadecane partitioning, and expression of streptococcal fibronectin adhesin CshA was determined in ELISA using specific antiserum . Antisera raised to glutaraldehyde-fixed whole biofilm or planktonic grown cells were used to screen an expression library of S . sanguis genomic DNA, and isolated clones were sequenced . RESULTS: Phenotypic analysis of biofilm and planktonic cells confirmed that mode of growth affected surface properties of S . sanguis . Thus, hydrophobicity and CshA expression was significantly elevated in biofilm cells . Library screening with biofilm antiserum yielded 32 recombinant clones representing 21 different S . sanguis proteins involved in adhesion and colonisation, carbohydrate utilisation or bacterial metabolism . In differential analysis of four selected Escherichia coli clones, biofilm antiserum reacted five times stronger than planktonic antiserum with cell-free extracts of clones encoding homologues of CshA and Cna collagen adhesin of Staphylococcus aureus, suggesting that these surface proteins are up-regulated in biofilm cells . In contrast, both antisera reacted equally strongly with cell-free extracts of the remaining two clones (encoding dihydrofolate synthase and an unknown protein) . CONCLUSIONS: The method described represents a useful means for determining bacterial protein expression in biofilms based on a combination of molecular and immunological techniques . Surface expression of putative fibronectin and collagen adhesins was up-regulated in biofilm cells.

J Proteome Res, 2004 Jan-Feb, 3(1), 132 - 6
Biofilm proteome: homogeneity or versatility?
Vilain S, Cosette P, Zimmerlin I, Dupont JP, Junter GA, Jouenne T.
The problems associated with biofilm infections in humans result from the distinct characteristics of biofilms, in particular their high level of resistance to antibiotics . One of the hypotheses that have been advanced to explain this resistance to antimicrobials is the phenotypic differentiation of biofilm cells . Although many studies on biofilms have highlighted physiological alterations following the attachment of bacteria to a surface, no studies have explicitly demonstrated a "biofilm" physiology . To contribute to this topical debate, we used principal component analysis to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis of crude protein extracts from planktonic and sessile Pseudomonas aeruginosa cells . These analyses showed that the proteome of attached P . aeruginosa cells differs from that of their planktonic counterparts . Furthermore, we found that the proteome of sessile P . aeruginosa is strongly dependent on the nature of the biofilm substratum.

J Chemother, 2003 Dec, 15(6), 536 - 42
Device-related infections in critically ill patients . Part II: Prevention of ventilator-associated pneumonia and urinary tract infections; Di Filippo A et al.; Device utilization in critically ill patients is responsible for a high risk of complications such as catheter-related bloodstream infections (CRBSI), ventilator-associated pneumonia (VAP) and urinary tract infections (UTI) . In this article we will review the current status of data regarding the prevention of VAP and UTI . The results of the more recent (5 years) randomized controlled trials are reviewed and discussed . General recommendations include staff education and use of a surveillance program with a restrictive antibiotic policy . Adequate time must be allowed for hand washing and barrier precautions must always be used during device manipulation . Specific measures for VAP prevention are: 1) use of multi-use, closed-system suction catheters; 2) no routine change of the breathing circuit; 3) lubrication of the cuff of the endotracheal tube (ET) with a water-soluble gel; 4) maintenance of patient in semi-recumbent position to improve chest physiotherapy in intubated patients . Specific measures for UTI prevention include: 1) use of a catheter-valve instead of a standard drainage system; 2) use of a silver-alloy, hydro gel-coated latex urinary catheter instead of uncoated catheters . Biofilm represents a new variable: the capacity of bacteria to organize a biofilm on a device surface can explain the difficulty in preventing and eradicating an infection in a critically ill patient . More clinical trials are needed to verify the efficacy of prevention measures of ICU infections.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 726 - 34 Epub 2004 Mar 02.
High-diversity biofilm for the oxidation of sulfide-containing effluents; Ferrera I et al.; In the present work, we describe for the first time the utilization of a complex microbial biofilm for the treatment of sulfide-containing effluents . A non-aerated packed-column reactor was inoculated with anoxic lake sediment and exposed to light . A biofilm developed in the column and showed a stable oxidation performance for several weeks . Microbial species composition was analyzed by microscopy, pigment analysis and a bacterial 16S rRNA gene clone library . Colorless sulfur bacteria, green algae and purple sulfur bacteria were observed microscopically . Pigment composition confirmed the presence of algae and purple sulfur bacteria . The clone library was dominated by alpha-Proteobacteria (mostly Rhodobacter group), followed by gamma-Proteobacteria (Chromatiaceae-like and Thiothrix-like aerobic sulfur oxidizers) and the Cytophaga- Flavobacterium- Bacteroides group . Plastid signatures from algae were also present and a few clones belonged to both the beta- ( Rhodoferax sp., Thiobacillus sp.) and delta-Proteobacteria ( Desulfocapsa sp.) and to the low G+C Gram-positive bacteria (Firmicutes group) . The coexistence of aerobic, anaerobic, phototrophic and chemotrophic microorganisms in the biofilm, the species richness found within these metabolic groups (42 operational taxonomic units) and the microdiversity observed within some species could be very important for the long-term functioning and versatility of the reactor.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 659 - 64 Epub 2004 Mar 02.
A new non-aerated illuminated packed-column reactor for the development of sulfide-oxidizing biofilms; Ferrera I et al.; This paper describes an illuminated reactor that allows the spontaneous development of biofilms aimed at the treatment of sulfide-containing streams . The reactor operates as a sulfidostat and is composed of an illuminated packed-column, in which microorganisms are exposed to constant low substrate concentrations, thereby avoiding inhibition due to high sulfide concentrations . The control system allows highly polluted streams to be oxidized by the microbial biofilm while ensuring the quality of the effluent produced . Both monospecies and multispecies biofilms have been developed . Biofilms undergo changes in light irradiance and sulfide load while providing a consistent reduction of the sulfide levels, down to micromolar concentrations . Both types of biofilm developed differ from stirred reactors in that their specific activities are lower, constituting systems with a slow dynamic behavior and, therefore, they are less sensitive to sudden disturbances.

J Bacteriol, 2004 Mar, 186(6), 1838 - 50
Quorum sensing in Staphylococcus aureus biofilms; Yarwood JM et al.; Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role . We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms . Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation . Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin . Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time . In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm . Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions . We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.

Oral Dis, 2004 Jan, 10(1), 5 - 12
Involvement of periodontopathic biofilm in vascular diseases; Okuda K et al.; Oral bacteria inhabit biofilms, which are firm clusters adhering in layers to surfaces and are not easily eliminated by immune responses and are resistant to antimicrobial agents . Dental plaque is one such biofilm . In the past 10 years, subgingival plaque bacteria forming biofilms have been increasingly reported to be involved in systemic diseases . A close relationship between microbial infections and vascular disease has also been reported in the past two decades . The present review discusses the significance of the ecologic characteristics of biofilms formed by periodontopathic bacteria in order to further clarify the associations between periodontal disease and systemic disease . We focus on the relationships between periodontal disease-associated bacteria forming biofilms and vascular diseases including atherosclerosis and carotid coronary stenotic artery disease, and we discuss the direct and indirect effects on vascular diseases of lipopolysaccharides as well as heat shock proteins produced by periodontopathic bacteria.

Oral Dis, 2004 Mar, 10(2), 106 - 12
Candida yeasts in chronic periodontitis tissues and subgingival microbial biofilms in vivo; Jarvensivu A et al.; The frequency of Candida infection in periodontal tissues of chronic periodontitis (CP) patients and the extent of candidal penetration into gingival tissues was studied . Tissue specimens collected from 25 CP patients during periodontal flap operations of initial periodontal therapy were examined by immunohistochemistry using Candida albicans-specific antibodies . Sections were also stained with periodic acid-Schiff (PAS) and subgingival plaque samples from 17 patients were cultured . Immunoreactivity for Candida was present in four of the 25 CP specimens (16%) . Only one yeast-positive specimen was found when PAS-staining was used (4%) and two yeast-positive specimens were found with plaque culture (8%) . Hyphal formation seemed essential and candidal hyphae were found to extend into the periodontal connective tissue . The degree and type of inflammation adjacent to the hyphae varied from a site and a patient to another . The sensitivity of specific antibodies was superior to PAS-stain or plaque culture in detection of Candida in tissues.

Microb Ecol, 2004 Apr, 47(3), 205 - 17 Epub 2004 Mar 04.
Sulfate-reducing bacteria-dominated biofilms that precipitate ZnS in a subsurface circumneutral-pH mine drainage system; Labrenz M et al.; The microbial diversity of ZnS-forming biofilms in 8 degrees C, circumneutral-pH groundwater in tunnels within the abandoned Piquette Zn, Pb mine (Tennyson, Wisconsin, USA) has been investigated by molecular methods, fluorescence in situ hybridization (FISH), and cultivation techniques . These biofilms are growing on old mine timbers that generate locally anaerobic zones within the mine drainage system . Sulfate-reducing bacteria (SRB) exclusively of the family Desulfobacteriaceae comprise a significant fraction of the active microbiota . Desulfosporosinus strains were isolated, but could not be detected by molecular methods . Other important microbial clusters belonged to the beta-, gamma-, and epsilon-Proteobacteria, the Cytophaga/Flexibacter/Bacteroides-group (CFB), Planctomycetales, Spirochaetales, Clostridia, and green nonsulfur bacteria . Our investigations indicated a growth dependence of SRB on fermentative, cellulolytic, and organic acid-producing Clostridia . A few clones related to sulfur-oxidizing bacteria were detected, suggesting a sulfur cycle related to redox gradients within the biofilm . Sulfur oxidation prevents sulfide accumulation that would lead to precipitation of other sulfide phases . FISH analyses indicated that Desulfobacteriaceae populations were not early colonizers in freshly grown and ZnS-poor biofilms, whereas they were abundant in older, naturally established, and ZnS-rich biofilms . Gram-negative SRB have been detected in situ over a period of 6 months, supporting the important role of these organisms in selective ZnS precipitation in Tennyson mine . Results demonstrate the complex nature of biofilms responsible for in situ bioremediation of toxic metals in a subsurface mine drainage system.

Microb Ecol, 2004 Apr, 47(3), 284 - 92 Epub 2004 Mar 04.
Utilization of microbial biofilms as monitors of bioremediation; Peacock AD et al.; A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix . Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S . Department of Energy Natural and Accelerated Bioremediation Research Program's Field Research Center at the Oak Ridge National Laboratory . During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces . Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH . Biofilms that formed on Bio-Sep beads had 2-13 times greater viable biomass; however, the bead communities were less metabolically active {higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios} and had a lower aerobic respiratory state (lower total respiratory quinone/ PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool . Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions . Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions . DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter . Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis.

Microbiology, 2004 Mar, 150(Pt 3), 735 - 42
Effect of acid stress on the physiology of biofilm cells of Streptococcus mutans; McNeill K et al.; Streptococcus mutans is a component of the dental plaque biofilm and an important aetiological agent in dental caries . Although this organism growing in the suspended (planktonic) state has been well characterized, relatively little is known about its physiology in biofilms, particularly in the acidic environments associated with caries development . The authors determined the effect of biofilm age (1-5 days) and cell density on selected metabolic properties under conditions of glucose limitation in a biofilm-chemostat at pH 7.5 and compared these baseline values with those of 3 day biofilms subjected to acid stress . Biofilm cell biomass more than doubled over the 5 day experimental period under baseline conditions, with the glycolytic rate, glucose uptake, glucose-PTS (phosphotransferase system) activity and protein synthesis maximum at 1-2 days . DNA and RNA synthesis increased for the first 3 days before decreasing in the 5 day biofilms, while H(+)/ATPase activity was higher in 5 day biofilms than 1 day biofilms, with overall activity 5-13-fold higher per cell unit than in the associated planktonic cells . Glucose pulsing (50 mM final concentration) for three consecutive days without pH control for 5 h (pH 4.39+/-0.02) resulted in a progressive decrease in planktonic cell numbers; however, the rate of acid formation and glucose utilization in the chemostat by these cells increased per cell unit . Assays for carbohydrate metabolism in the latter cells showed increased activity, as did an assay for H(+)/ATPase (8-fold); however, DNA, RNA and protein synthesis were repressed (0.3-0.7-fold) . Although the 3 day biofilm viable cell counts declined by 51 % on glucose pulsing, all the physiological parameters measured by cell unit increased in activity, with notable increases in RNA and protein synthesis (4.6-7.6-fold) . The results indicate that the maintenance of intracellular pH homeostasis is the basis of the enhanced physiological status and acid tolerance of biofilm cells.

Biotechnol Bioeng, 2004 Mar 30, 85(7), 697 - 705
High-rate ferric sulfate generation by a Leptospirillum ferriphilum-dominated biofilm and the role of jarosite in biomass retention in a fluidized-bed reactor; Kinnunen PH et al.; The aims of this work were to develop a high-rate fluidized-bed bioprocess for ferric sulfate production, to characterize biomass retention, and to determine the phylogeny of the enrichment culture . After 7 months of continuous enrichment and air aeration at 37 degrees C, the iron oxidation rate of 8.2 g Fe(2+) L(-1)h(-1) (4.5.10(-12) g Fe(2+) cell(-1) h(-1)) was obtained at a hydraulic retention time (HRT) of 0.6 h . However, oxygen supply became the rate-limiting factor . With gas mixture (99.5% O(2)/0.5% CO(2) (vol/vol)) aeration and HRT of 0.2 h, the iron oxidation rate was 26.4 g Fe(2+) L(-1)h(-1) (1.0.10(-11) g Fe(2+) cell(-1) h(-1)) . Leptospirillum sp . was predominant in the mesophilic fluidized-bed reactor (FBR) enrichment culture as determined by fluorescent in situ hybridization, while Acidithiobacillus ferrooxidans was not detected . Denaturing gradient gel electrophoresis (DGGE) of the amplified partial 16S rDNA showed only three bands, indicating a simple microbial community . DGGE fragment excision and sequencing showed that the populations were related to L . ferriphilum (100% similarity in sequence) and possibly to the genus Ferroplasma (96% similarity to F . acidiphilum) . Jarosite precipitates accumulated on the top of the activated carbon biomass carrier material, increasing the rate of iron oxidation . The activated carbon carrier material, jarosite precipitates, and reactor liquid contained 59% (or 3.71.10(9) cells g(-1)), 31% (or 3.12.10(10) cells g(-1)) and 10% (or 1.24.10(8) cells mL(-1)) of the total FBR microbes, respectively, demonstrating that the jarosite precipitates played an important role in the FBR biomass retention .

Bioprocess Biosyst Eng, 2004 Apr, 26(3), 169 - 75 Epub 2004 Feb 19.
Bioprocess kinetics in a horizontal rotating tubular bioreactor; Ivancic M et al.; A horizontal rotating tubular bioreactor (HRTB) is a plug flow bioreactor whose interior is provided with O-ring-shaped partition walls that serve as carriers for microbial biomass . During this investigation, microbial biomass was grown in suspension and on the bioreactor inner surface as a microbial biofilm with average mass that was considerably higher than suspended biomass . The dynamics of bioprocess in HRTB was studied by different combinations of process parameters (bioreactor rotation speed and mean residence time) and it was monitored by withdrawing the samples from five positions along the bioreactor . During this investigation it was also observed that mean residence time had a more pronounced effect on the bioprocess dynamics than bioreactor rotation speed . For the description of bioprocess kinetics in HRTB an unstructured kinetic model was established that defines biomass growth, product formations and substrate consumption rate by using a modified Monod (Levenspiel) model . This kinetic model defines changes in suspension and in microbial biofilm, and it shows relatively good agreement with experimental data.

Clin Oral Investig, 2004 Jun, 8(2), 97 - 101 Epub 2004 Feb 19.
Impact of the intraoral location on the rate of biofilm growth; Auschill TM et al.; The aims of the present study were: a) to assess the impact of the intraoral location on the rate of biofilm growth, and b) to establish an in vivo biofilm model to examine intraoral biofilm growth . Eight healthy volunteers wore acrylic splints with 15 glass slabs each in the upper and lower jaws to build up plaque . After 48 h, the specimens were removed and stained using the vital fluorescence technique . Biofilm thickness was evaluated by confocal laser scanning microscopy (CLSM) . The mean plaque thickness amounted to 77.6 +/- 29.1 microm on the buccal sites of the upper jaw and 71.9 +/- 26.3 microm on the buccal sites of lower jaw . On the palatal site a biofilm of 52.1 +/- 26.2 microm thickness was grown, which was significantly less compared with the other locations evaluated (p < 0.001) . The results demonstrate that the in situ biofilm thickness on the buccal sites was similar irrespective of the location in the oral cavity . The new splint system described may be a useful tool for further standardised experimental studies regarding influences on growth and structure of intraoral biofilms.

Shanghai Kou Qiang Yi Xue, 2002 Mar, 11(1), 37 - 9
{The role of dead bacteria in streptococcus sanguis biofilm reformation}; Ma R et al.; OBJECTIVE: To investigate the role of dead bacteria in Streptococcus sanguis biofilm reformation . METHODS: Vital fluorescent staining technique and Confocal laser Scanning Microscopy (CLSM) were combined to observe Streptococcus sanguis biofilm on the saliva-coated glass (SCG) in an artificial mouth model, the thickness and bacterial density were compared between biofilm reformed on saliva covered slide with dead cells (test group) and biofilm reformed on saliva coated slide without dead cells (control group) at different times . RESULTS: At 30 min, the bacterial density rather than biofilm thickness of test group was significantly higher than that of control group (P<0.05), whereas at 60 min, 120min, 240min, the biofilms were significantly thicker in test group than that (P<0.05) . CONCLUSION: The dead cells may help biofilm reformation during the formation of biofilm.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 890 - 6
Killing by ampicillin and ofloxacin induces overlapping changes in Escherichia coli transcription profile; Kaldalu N et al.; The basis of bactericidal versus bacteriostatic action of antibiotics and the mechanism of bacterial cell death are largely unknown . Related to this important issue is the essential invulnerability to killing of persisters: cells forming a small subpopulation largely responsible for the recalcitrance of biofilms to chemotherapy . To learn whether death is accompanied by changes in expression of particular genes, we compared transcription profiles of log-phase Escherichia coli treated with bactericidal concentrations of two unrelated antibiotics: ampicillin and ofloxacin . Massive changes in transcription profile were observed in response to either agent, and there was a significant overlap in genes whose transcription was affected . A small group of mostly uncharacterized genes was induced and a much larger set was transcriptionally repressed by both antibiotics . Among the repressed genes were those required for flagellar synthesis, energy metabolism, transport of small molecules, and protein synthesis.

Water Sci Technol, 2004, 49(2), 107 - 13
Impact of localised dissolved iron concentrations on the biofouling of environmental wells; Stuetz RM et al.; Iron biofouling of wells can significantly impact the performance of a groundwater extraction system . A subsurface drainage scheme (Wakool, Australia) designed to reduce waterlogging was used to identify some of the relationships between aquifer properties and well biofouling . Piezometers drilled radially one metre from two biofouled wells showed that during normal well operation the concentration of dissolved iron (Fe2+) entering the groundwater well was highly localised around the site and with depth . CCTV survey of the biofouling on the well screens supported these findings of localised iron concentrations . Dissolved oxygen (DO) measured during pumping and under non-pumping conditions (aquifer DO) showed that oxygen was not a limiting factor, whereas stalked bacteria (Gallionella sp.) were only found in the biofouled wells . The wellhead water therefore represents only a composite of all the waters entering the well and does not indicate the possibility of localised iron concentrations in a shallow aquifer . The degree of iron biofouling within a groundwater well is therefore related directly to the presence of dissolved iron in the groundwater, as well as various oxidative processes occurring as the groundwater enters the well screen and its subsequent extraction . The distribution of iron biofilms on the well screen reflects these processes; however, the presence of well biofouling cannot always be linked to a decrease in well screen performance, but can have an impact on the overall performance of the groundwater extraction system.

Water Sci Technol, 2004, 49(2), 91 - 8
The physico-chemistry of biofilm-mediated pitting corrosion of copper pipe supplying potable water; Keevil CW; Copper is a generally robust material that has beneficial properties to reduce biofilm formation and pathogen colonisation of pipes supplying potable water . However, a rare pitting corrosion can occur in soft, poorly buffered waters that can lead to pipe failure . This has been shown to be mediated by a copper-tolerant biofilm whose physical and chemical heterogeneity can establish microenvironments for corrosion potentials, causing micro pits that eventually coalesce into large perforations through the pipe wall . Control of the biofilm, for example through reduced cold water or elevated hot water temperatures, can suppress this corrosion phenomenon.

Infect Immun, 2004 Mar, 72(3), 1431 - 40
Effects of RelA on key virulence properties of planktonic and biofilm populations of Streptococcus mutans; Lemos JA et al.; Streptococcus mutans is a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity . Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism . Here, we investigated the role of the S . mutans relA gene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate {(p)ppGpp} synthetase/hydrolase, in biofilm formation and acid tolerance . Two mutants in which relA was insertionally inactivated or replaced by an antibiotic resistance determinant were constructed . Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar . The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks . There was no difference in the sensitivities to acid killing of the parent and relA strains grown in planktonic cultures . However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain . Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants . Expression of the luxS gene was increased as much as fivefold in the relA mutants, suggesting a link between AI-2 quorum sensing and the stringent response.

Front Biosci, 2004 May 01, 9, 1267 - 77
Virulence properties of Streptococcus mutans; Banas JA; Streptococcus mutans is considered one of the primary causative agents of dental caries and can also be a source of infective endocarditis . The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance . Each of these properties works coordinately to alter dental plaque ecology . The ecological changes are characterized by increased proportions of S . mutans and other species that are similarly acidogenic and aciduric . The selection for a cariogenic flora increases the magnitude of the drop in pH following the fermentation of available carbohydrate and increases the probability of enamel demineralization . This review focuses on the bacterial components that contribute to each of the major virulence properties . Further understanding of how these components work together in the development of dental caries will be aided by the recent completion of the sequence of the S . mutans genome and experimental designs that model the dental plaque biofilm.

Water Res, 2004 Mar, 38(5), 1237 - 47
Quantification of oxygen fluxes in a long gravity sewer; Huisman JL et al.; Quantification of the oxygen fluxes in the sewer system is at present the optimal methodology to obtain information about the influence of sewers on transformations and mass balances in the urban drainage system . However, the relative and absolute values of these fluxes are practically unknown . In this work, the oxygen fluxes were quantified experimentally in a full-scale aerobic main sewer . The sewer biofilm respiration was determined with an in situ flow cell, a method that has not been used before in the sewer . The surface reaeration was determined with a gas tracer method based on the inert, non-radio-active and non-toxic gas tracer sulphur hexafluoride . In addition, the wastewater biomass respiration rate was measured . The validity of the applied methods was verified with redundant oxygen balances over a 2-km-long section . Measurement campaigns under different hydrodynamic conditions showed that the relative contribution of the biofilm, the wastewater, the reaeration and the in- and outflow with the water, all contributed significantly . However, the absolute contributions varied extensively and depended especially on the discharge . The COD conversion in the sewer could be estimated from the aerobic activity . The aerobic total degradation in the study reach was 3% . However, when extrapolated to the entire sewer net of the catchment area with 5000 PE, the COD conversion was estimated as high as 30% of the dissolved COD during the night . This indicates that the wastewater composition at the treatment plant will be strongly affected by the sewer system.

Water Res, 2004 Mar, 38(5), 1197 - 206
Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration; Emtiazi F et al.; Populations of bacteria in biofilms from different sites of a drinking water production system were analysed . Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks . In addition, common DNA bands that were attributed to ubiquitous bacteria were found . Biofilms even developed directly after UV disinfection (1-2m distance) . Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system . Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH) . Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system . In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp . were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically . Enterococci were not found in any biofilm . Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased . beta-glucosidase activity was not found in the biofilms . Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms . This suggested an increased tolerance of biofilm bacteria against toxic compounds.

Water Res, 2004 Mar, 38(5), 1189 - 96
Investigation of biocide efficacy by photoacoustic biofilm monitoring; Schmid T et al.; The undesired growth of biofilms on solid surfaces is often termed biofouling . Biofilms consist mainly of water and microbial cells which are embedded in a biopolymer matrix . Biofouling lowers the water quality and increases the frictional resistance in tubes . Further, biofilms increase the pressure differences in membrane processes and can clog filtration membranes, valves, and nozzles . For investigation and improvement of biocide efficacy and anti-fouling strategies, on-line and in situ monitoring of the biofilm is necessary . In this study, photoacoustic spectroscopy (PAS) was employed for biofilm monitoring . PAS allows the depth-resolved investigation of growth and detachment processes of biofilms . Strategies based on the oxidant hydrogen peroxide were compared to popular isothiazolinone biocides . Hydrogen peroxide allowed a very fast and efficient removal of attached biofilms, whereas no effect on the biofilm matrix was observed in most cases when isothiazolinone biocides were used.

Recenti Prog Med, 2003 Dec, 94(12), 537 - 9
{Biofilms in infectious diseases}; Rossi G; Microorganisms attack and grow upon living as well inorganic surfaces, forming a "biofilm" . Biofilms are a serious problem of public health, because biofilm-associated microorganisms exhibit increased resistance to antibiotics . The mechanisms of biofilm formation, the mechanisms of resistance and the possibilities of therapy are briefly summarized.

J Bacteriol, 2004 Mar, 186(5), 1574 - 8
VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor; Casper-Lindley C et al.; Vibrio cholerae switches between smooth and rugose colonial variants . The rugose variant produces more vibrio polysaccharides (VPS(El Tor)) and forms well-developed biofilms . Both phenotypes depend on expression of vps biosynthesis genes . We identified a positive transcriptional regulator of vps gene expression, VpsT, which is homologous to response regulators of two-component regulatory systems . Disruption of vpsT in the rugose variant yields smooth colonies, prevents formation of mature biofilms, and decreases vps gene expression . The interaction between VpsT and VpsR, a previously identified positive regulator of vps genes, was also investigated.

J Bacteriol, 2004 Mar, 186(5), 1249 - 57
Capsule shields the function of short bacterial adhesins; Schembri MA et al.; Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors . Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding . The phenomenon is not restricted to Escherichia coli but can occur in other gram-negative bacteria . Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule . The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins . This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms.

J Colloid Interface Sci, 2004 Mar 15, 271(2), 351 - 8
The measurement temperature: an important factor relating physicochemical and adhesive properties of yeast cells to biomaterials; Gallardo-Moreno AM et al.; Flow chambers applied to the study of the initial adhesion process of Candida parapsilosis are rarely found in the literature . The ability of these microorganisms to proliferate and form biofilms in environments at temperatures around 22 or 37 degrees C is reflected in the contamination of laboratory instruments and material or in human implant infections, respectively . The initial interaction between yeasts and substrata is mediated by physicochemical forces, which in turn originate from the physicochemical surface properties of both interacting phases . In this context, this work aims to relate the initial rates of adhesion rates to glass and silicone of Candida parapsilosis, strains 294 and 289, grown at 22 and 37 degrees C with the theoretical predictions of the adhesion process, expressed by the interaction free energies and calculated through the physicochemical parameters, which are also measured at 22 and 37 degrees C . The results indicate that physicochemical parameters of yeasts are changed not only by the culture temperature but also by the measurement temperature; only when the measurement temperature is equal to the growth temperature a coherent relation between in vitro adhesion data and interaction free energies can be established . In this sense, the adhesion to glass is mediated by long-range forces or, what amounts to the same thing, by Lifshitz-van der Waals interaction free energy . On the other hand, the adhesion to silicone rubber seems to be moderated by acid-base interaction free energy, which involves the presence of short-range forces . Based on these results, it can be assumed that the substratum surface properties are directly related to the kind of force acting on the initial microbial adhesion process, while cell surface properties dictate the changes in the strength of the force between different samples.

J Colloid Interface Sci, 2004 Mar 15, 271(2), 342 - 50
Study of bioadhesion on a flat plate with a yeast/glass model system; Mercier-Bonin M et al.; The attachment of microorganisms to a surface is a critical first step of biofilm fouling in membrane processes . The shear-induced detachment of baker's yeast in adhesive contact with a plane glass surface was thus experimentally studied, using a specially designed shear stress flow chamber . The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast) . A complete series of experiments for measuring the shear stress necessary to detach a given proportion of individual yeast cells of type I or II was performed under different environmental conditions (ionic strength, contact time) . In parallel, the surface physicochemical properties of the cells (surface charge, hydrophobicity, and electron donor and electron acceptor components) were determined . For the first type of yeast cells, which were rather hydrophilic, adhesion to the glass plate was weak . This was due to both electrostatic effects and hydrophilic repulsion . Furthermore, adhesion was not sensitive to any variation of the ionic strength . For yeast of the second type, adhesion was drastically increased . This could be explained by their physicochemical surface properties and especially their hydrophobic and electron acceptor components, which caused strong attractive van der Waals and Lewis acid-base interactions, counterbalancing the electrostatic repulsion . For increasing ionic strengths, adhesion was greater, due to lower electrostatic repulsion . The results were quantified through the definition of a critical wall shear stress ( tau w 50% ) required to detach 50% of the yeast cells initially deposited on the glass surface . The influence of the contact time was also evaluated and it was shown that, whatever the type of yeast, macromolecules such as proteins were released into the extracellular medium due to cell lysis and could contribute to the formation of a conditioning film . As a result, the cells were more strongly stuck to the glass plate.

J Environ Sci (China), 2004, 16(1), 79 - 85
Lead adsorption capacities of different components in natural surface coatings; Dong DM et al.; Pb adsorption capacities of Fe oxide, Mn oxide and organic materials in natural surface coatings( biofilms and associated minerals) collected in three lakes, two ponds and a river in Jilin Province, China and Cayuga Lake in US were studied . A novel extraction technique was employed to remove one or more component(s) from the surface coatings . Pb adsorption to surface coatings before and after extraction was performed to determine the adsorptive properties of the extracted component(s) . The statistical analysis of observed Pb adsorption was carried out using nonlinear least squares fitting(NLSF) to estimate the Pb adsorption capacity of each component of surface coatings . For each body of water, the estimated Pb adsorption capacity of Mn oxide(mol Pb/mol Mn) was significantly higher than that of Fe oxide(mol Pb/ mol Fe) . The value of estimated adsorption capacities of organic materials with the unit mol Pb per kg COD was similar to or less than that of Fe oxides with the unit mol Pb per mol Fe . Comparison of components of surface coatings in different waters showed that the estimated Pb adsorption capacities of components in surface coatings developed in different natural waters were different, especially for Mn oxides.

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3587 - 90 Epub 2004 Feb 17.
Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia; Chun CK et al.; Mammalian airways protect themselves from bacterial infection by using multiple defense mechanisms including antimicrobial peptides, mucociliary clearance, and phagocytic cells . We asked whether airways might also target a key bacterial cell-cell communication system, quorum-sensing . The opportunistic pathogen Pseudomonas aeruginosa uses two quorum-sensing molecules, N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL), to control production of extracellular virulence factors and biofilm formation . We found that differentiated human airway epithelia inactivated 3OC12-HSL . Inactivation was selective for acyl-HSLs with certain acyl side chains, and C4-HSL was not inactivated . In addition, the capacity for inactivation varied widely in different cell types . 3OC12-HSL was inactivated by a cell-associated activity rather than a secreted factor . These data suggest that the ability of human airway epithelia to inactivate quorum-sensing signal molecules could play a role in the innate defense against bacterial infection.

J Med Microbiol, 2004 Mar, 53(Pt 3), 259 - 61
Enterococcus durans endocarditis in a patient with transposition of the great vessels; Stepanovic S et al.; A case of native valve endocarditis caused by Enterococcus durans in a patient with transposition of the great vessels is reported . The patient was treated initially with gentamicin and ceftriaxone; after isolation of enterococci, ceftriaxone was switched to ampicillin . The only virulence factors established in the strain were haemolytic activity and biofilm formation.

J Food Prot, 2004 Feb, 67(2), 322 - 7
Behavior of Listeria monocytogenes in a Pseudomonas putida biofilm on a condensate-forming surface; Hassan AN et al.; Listeria monocytogenes has been isolated from condensate-forming surfaces in food processing plants . The objective of this research was to observe the behavior of L . monocytogenes on condensate-covered stainless steel with a Pseudomonas putida biofilm . L . monocytogenes-containing biofilms, either with or without added chicken protein, were incubated in a high humidity chamber at 12 degrees C to allow formation of condensate . Samples were analyzed for attached and unattached L . monocytogenes and total plate count periodically for 35 days . Samples were also taken for microscopic observation of Listeria and bacterial extracellular polymeric substances (EPS) . L . monocytogenes attached in significantly greater numbers (> 3-log difference) to surfaces with preexisting P . putida biofilms than to Pseudomonas-free surfaces . L . monocytogenes survived in the presence or absence of P . putida with no added nutrients for 35 days, with numbers of survivors in the range of 3 to 4 log CFU/cm2 in the presence of P . putida and less than 2.9 log CFU/cm2 in pure culture . Attached and unattached L . monocytogenes were at similar levels throughout the incubation under all conditions studied . The addition of protein to the biofilms allowed growth of L . monocytogenes in pure culture during the first 7 days of incubation . Numbers of L . monocytogenes were not affected by the presence of P . putida when protein was present . Unattached L . monocytogenes were at levels of 3.6 to 6.7 log CFU/cm2 on the protein-containing surfaces . Microscopic observation of the condensate-covered biofilms indicated that L . monocytogenes formed microcolonies embedded within an EPS matrix over a 28-day period . This research demonstrates that L . monocytogenes can survive on condensate-forming stainless steel in low and high nutrient conditions, with or without the presence of Pseudomonas biofilm . The Listeria can detach and, therefore, have the potential to contaminate product.

Ultrastruct Pathol, 2004 Jan-Feb, 28(1), 23 - 7
Microbial biofilms in the gut: visualization by electron microscopy and by acridine orange staining; Palestrant D et al.; The expression of colonization factors by gut bacteria, the growth rate of gut bacteria, and the rate of plasmid exchange by gut bacteria indicate that biofilms are a normal component of bacterial growth in the large bowel . Further, in vitro experiments demonstrate that growth of normal enteric bacteria in biofilms can be facilitated by secretory IgA (SIgA) and by mucins, 2 major components of the gut milieu . However, biofilms have not been previously observed in the normal gut . In this study, bacterial colonies characteristic of biofilms were observed by electron microscopy in normal rat, baboon, and human gut by electron microscopy . Confirming these results, acridine orange staining of flash-frozen tissues revealed biofilms in the mucus lining along normal gut epithelium . Immunofluorescenct microscopy supported this finding and demonstrated an association between IgA and the biofilms . These findings provide direct evidence that biofilms are present and may play an important role in the commensal relationship between enteric bacteria and their hosts . Hematoxylin and eosin staining of formalin-fixed tissues resulted in dissociation of the luminal contents from the epithelium, suggesting that the association between biofilms and the gut epithelium is sensitive to some conditions used to preserve tissue for histologic evaluation.

J Appl Microbiol, 2004, 96(3), 455 - 63
Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities; Al-Bakri AG et al.; AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF) . METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B . cepacia . Reverse challenges were also conducted . Both B . cepacia and P . aeruginosa were able to form steady-state monoculture biofilms after 48 h growth . When steady-state biofilms of B . cepacia NTCT 10661 were challenged with planktonically grown P . aeruginosa PAO1 known to produce a B . cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B . cepacia . By contrast, established biofilms of P . aeruginosa PAO1 resisted immigration of B . cepacia 10661 . Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P . aeruginosa 313113 and B . cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances . Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm . Similar observations were made for biofilms generated in CDFFs . In such instances following pan-swapping between two monoculture CDFFs, B . cepacia 313113 was able to integrate into an established P . aeruginosa 313113 biofilm to form a stable binary biofilm . CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P . aeruginosa predisposes uncolonized sections of the surface to permit B . cepacia colonization . SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species . Disinfection of a surface might well increase the probability of pathogen harbourage.

Lett Appl Microbiol, 2004, 38(3), 211 - 6
Minimizing prion risk without compromising the microbial composition of biofilms grown in vivo in a human plaque model; Watson PS et al.; AIMS: To determine whether the stringency of sterilization procedures for biological components of in vivo dental plaque-generating devices based on enamel can be increased to minimize prion risk without compromising natural biofilm composition . METHODS AND RESULTS: The composition of in vitro biofilms, grown on hypochlorite-treated and untreated autoclaved enamel surfaces, was determined using culture-based methods and checkerboard DNA: DNA hybridization analysis . No differences were found between biofilms recovered from either substrate . SIGNIFICANCE: Several in situ models allow generation of plaque in the oral cavity, followed by recovery of intact biofilms for experimentation . Approaches allowing plaque formation on natural tooth surfaces are most valuable, but present a possible infection risk to volunteers wearing plaque-collecting devices, particularly with respect to prions . Hypochlorite treatment of biological material, as an adjunct to autoclaving, reduces infection risk without compromising biofilm composition and should be adopted in all future studies using plaque-generating devices incorporating enamel, where there is a potential prion threat, and further investigated in other biological hard tissues.

Clin Otolaryngol, 2004 Feb, 29(1), 38 - 46
Is otitis media with effusion a biofilm infection?
Fergie N, Bayston R, Pearson JP, Birchall JP.
Recent attention has focused on the possibility that otitis media with effusion (OME) may represent a chronic infective state such as those evidenced in conditions secondary to biofilms or small colony variants . This review discusses the evidence suggesting that this may indeed be the case and explains why this may prove to be important in the future management of this condition by discussing recent advances in understanding these bacterial phenotypic variants.

Mar Biotechnol (NY), 2002 Sep, 4(4), 356 - 66
Surface attachment induced production of antimicrobial compounds by marine epiphytic bacteria using modified roller bottle cultivation; Yan L et al.; A modified roller bottle culture method elicited the production of antimicrobial compounds from 2 epibiotic marine bacterial strains, EI-34-6 and II-111-5, isolated from the surface of the marine alga Palmaria palmata . These isolates, tentatively identified as Bacillus species, were grown as a biofilm on the surface of nutrient glycerol ferric agar (NGFA) and marine Columbia glycerol agar (MCGA) on the inside of a rolling bottle . The biofilm was shown to be stable, and the cells were difficult to remove from the agar surface . The culture supernatant exhibited a different antibiotic spectrum when the strains were grown using the agar roller bottle method compared with shake flask cultures or nonagar roller bottle cultures . These results suggest that biofilm formation is an important factor in the production of antimicrobial compounds by these 2 strains, and roller bottle cultivation also allowed production of these compounds to be increased . The methodology used here has the potential to allow increased production of useful secondary metabolites such as antibiotics from marine epibiotic bacteria.

Nature, 2004 Mar 4, 428(6978), 37 - 43 Epub 2004 Feb 01.
Community structure and metabolism through reconstruction of microbial genomes from the environment; Tyson GW et al.; Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear . Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes . This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low . Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level . The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants . The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes . Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

Chest, 2004 Feb, 125(2 Suppl), 62S - 69S; quiz 69S
Effects of subinhibitory concentrations of macrolide antibiotics on Pseudomonas aeruginosa; Wozniak DJ et al.; Biofilm-forming bacteria such as Staphylococcus, Haemophilus, and Pseudomonas species resist phagocytosis by host immune cells and the actions of antimicrobial agents . In susceptible individuals, such as patients with cystic fibrosis (CF) or diffuse panbronchiolitis (DPB), strains of Pseudomonas aeruginosa produce a number of virulence determinants that permit colonization and infection of the respiratory tract . P aeruginosa strains isolated from CF and DPB patients typically have a mucoid colony morphology . This is due to the overproduction of alginate, an exopolysaccharide capsule that is composed of D-mannuronic and L-guluronic acids . In addition, the P aeruginosa type IV pilus mediates cell surface translocation by a process known as twitching motility . Both alginate production and twitching motility contribute to the virulence of P aeruginosa, as does the formation of biofilms . Biofilms bind cells and organic and inorganic materials to each other, and to a variety of substrata . Their tightly formed structure reduces antimicrobial activity, promotes bacterial adhesion to lung epithelia, and prevents bacterial dehydration . Prior work has suggested that macrolides have therapeutic value in patients with DPB and CF . We hypothesized that the improved clinical status of these patients was due, in part, to macrolides inhibiting the production of P aeruginosa virulence determinants . Traditionally, macrolides have not been considered to exhibit antipseudomonal activity, as their mean inhibitory concentration (MIC) values for clinical and environmental strains of the microbe range from 50 to 550 microg/mL . In this study, we found that sub-MIC levels of clarithromycin substantially inhibited twitching motility . In addition, the incubation of biofilm-grown P aeruginosa with clarithromycin altered the structure and architecture of the biofilm . Investigating the potential nonribosomal effects of macrolides on opportunistic pathogens such as P aeruginosa and elucidating the molecular mechanisms that underlie the inhibition of twitching motility may lead to more effective treatments of pulmonary infections in patients with CF and DPB.

Environ Microbiol, 2004 Mar, 6(3), 218 - 26
Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing; Matz C et al.; This study was based on the hypothesis that biofilms of the opportunistic pathogen Pseudomonas aeruginosa are successfully adapted to situations of protozoan grazing . We tested P . aeruginosa wild type and strains that were genetically altered, in structural and regulatory features of biofilm development, in response to the common surface-feeding flagellate Rhynchomonas nasuta . Early biofilms of the wild type showed the formation of grazing resistant microcolonies in the presence of the flagellate, whereas biofilms without the predator were undifferentiated . Grazing on biofilms of quorum sensing mutants (lasR and rhlR/lasR) also resulted in the formation of microcolonies, however, in lower numbers and size compared to the wild type . Considerably fewer microcolonies than the wild type were formed by mutant cells lacking type IV pili, whereas no microcolonies were formed by flagella-deficient cells . The alginate-overproducing strain PDO300 developed larger microcolonies in response to grazing . These observations suggest a role of quorum sensing in early biofilms and involvement of flagella, type IV pili, and alginate in microcolony formation in the presence of grazing . More mature biofilms of the wild type exhibited acute toxicity to the flagellate R . nasuta . Rapid growth of the flagellate on rhlR/lasR mutant biofilms indicated a key role of quorum sensing in the upregulation of lethal factors and in grazing protection of late biofilms . Both the formation of microcolonies and the production of toxins are effective mechanisms that may allow P . aeruginosa biofilms to resist protozoan grazing and to persist in the environment.

Eur J Oral Sci, 2004 Feb, 112(1), 42 - 7
The effects of substratum on the pH response of Streptococcus mutans biofilms and on the susceptibility to 0.2% chlorhexidine; Deng DM et al.; Antimicrobial agents are now being studied in bacterial biofilms rather than in suspensions . However, no data are available whether the choice of substratum influences biofilm properties or the assessment of antimicrobial efficacy . This study compared dentin and polyacrylate (PA) grooves as substratum for biofilm formation on pH profiles after sugar challenge, as well as biofilm susceptibility to chlorhexidine . Streptococcus mutans biofilms were formed in a constant depth film fermenter (CDFF) by alternate pulsing with sucrose solutions and bacterial growth medium . Spatial and temporal pH profiles were measured using a pH microelectrode outside the CDFF in a flow reactor . Also, 15-d biofilms were treated with either water or 0.2% chlorhexidine and tested for total viable counts and organic acid content . The results show that in PA grooves, compared with dentin grooves, minimum pH was lower but recovered faster after a sucrose pulse . The pH response to sugar application decreased with the age of the biofilm more in PA than in dentin grooves . Chlorhexidine treatment in PA grooves gave greater reductions in viable counts and acid formation than in dentin . We conclude that the nature of the substratum not only affects metabolic activity of biofilms but also their susceptibility to antimicrobials.

Biofouling, 2003 Dec, 19(6), 371 - 80
Patterns of biofilm succession on a sheltered rocky shore in Hong Kong; Chan BK et al.; Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment . Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers . In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong . In the succession, bacteria were followed by diatoms and cyanobacteria . Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter) . Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period . The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer . Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.

Biofouling, 2003 Dec, 19(6), 347 - 53
The influence of antimicrobial peptides and mucolytics on the integrity of biofilms consisting of bacteria and yeasts as affecting voice prosthetic air flow resistances; Oosterhof JJ et al.; The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech . Biofilm integrity is ensured by extracellular polymeric substances (EPS) . This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms . Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan) . Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses afterm biofilm formation . Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance . Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.

Microbiology, 2004 Feb, 150(Pt 2), 267 - 75
RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms; Green CB et al.; An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family . Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C . albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm . RT-PCR analysis of total RNA samples extracted from C . albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed . Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern . The expression pattern of ALS genes in C . albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C . albicans with buccal RHE does little to alter ALS gene expression . RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens . Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C . albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells . The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models . The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C . albicans and its host.

J Clin Microbiol, 2004 Feb, 42(2), 554 - 62
Characterization of cell-to-cell signaling-deficient Pseudomonas aeruginosa strains colonizing intubated patients; Denervaud V et al.; Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa . In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P . aeruginosa pulmonary isolates from 13 patients . Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids . We identified nine genotypically distinct P . aeruginosa strains . Six of these strains produced AIs {N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone} and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro . Three of the nine strains were defective in the production of both AIs and extracellular virulence factors . Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively . The third of these AI-deficient strains was only mutated in the lasR gene . Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

Appl Environ Microbiol, 2004 Feb, 70(2), 1213 - 21
Metamorphosis of a scleractinian coral in response to microbial biofilms; Webster NS et al.; Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species . However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA) . Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral . Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films . There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities . Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis . A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time . Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition . A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms . No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not . Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred . This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.

Appl Environ Microbiol, 2004 Feb, 70(2), 1097 - 103
Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbor these bacteria in the environment; Marsollier L et al.; Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans . Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown . We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M . ulcerans and promoted biofilm formation . Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M . ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic . Genotype analysis showed that plant-associated M . ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens . These observations implicate aquatic plants as a reservoir of M . ulcerans and add a new potential link in the chain of transmission of M . ulcerans to humans.

Front Biosci, 2004 Jan 01, 9, 841 - 63
Virulence factors of the coagulase-negative staphylococci; Otto M; Coagulase-negative staphylococci (CNS) have gained substantial interest as pathogens involved in nosocomial, particularly catheter-related infections . The pathogenic potential of CNS is mainly due to their capacity to form biofilms on indwelling medical devices . In a biofilm, the bacteria are protected against antibiotics and from attacks by the immune system . The factors contributing to biofilm formation are among the best-studied virulence factors of CNS and comprise factors involved in the adhesion to a catheter surface and in cell accumulation . CNS usually persist in the host in relative silence, but may cause sepsis, for which the recently found inflammatory peptides called phenol-soluble modulins are prime candidates . Many CNS also produce several lipases, proteases, and other exoenzymes, which possibly contribute to the persistence of CNS in the host and may degrade host tissue . We are also beginning to understand how regulators of virulence trigger the expression of virulence factors in CNS . A better conception of the mechanisms underlying the pathogenicity and the frequently encountered antibiotic resistance of CNS may help to develop novel, efficient anti-staphylococcal therapeutics.

Cell Microbiol, 2004 Mar, 6(3), 269 - 75
Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system; Vuong C et al.; The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections . Little is known about the mechanisms by which S . epidermidis protects itself against the innate human immune system during colonization and infection . We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion . Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain . The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human beta-defensin 3 and LL-37, and anionic dermcidin . PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.

Adv Perit Dial, 2003, 19, 195 - 7
Strategies for intervention in and prevention of biofilm-related infection in peritoneal dialysis patients; Dasgupta MK; Bacteria from the exit site of the peritoneal dialysis (PD) catheter and from contaminated dialysis fluids can grow into microcolonies in biofilm . Those bacteria are implicated in recurrent peritonitis in patients undergoing treatment with PD . The present article highlights new strategies that are designed to alter the major factors regulating biofilm formation and that thereby reduce biofilm-related infections in PD patients.

Crit Rev Oral Biol Med, 2004, 15(1), 4 - 12
THE BIOFILM CONCEPT: CONSEQUENCES FOR FUTURE PROPHYLAXIS OF ORAL DISEASES?
Scheie AA, Petersen FC.
Biofilm control is fundamental to oral health . Existing oral prophylactic measures, however, are insufficient . The main reason is probably because the micro-organisms involved organize into complex biofilm communities with features that differ from those of planktonic cells . Micro-organisms have traditionally been studied in the planktonic state . Conclusions drawn from many of these studies, therefore, need to be revalidated . Recent global approaches to the study of microbial gene expression and regulation in non-oral micro-organisms have shed light on two-component and quorum-sensing systems for the transduction of stimuli that allow for coordinated gene expression . We suggest interference with two-component and quorum-sensing systems as potential novel strategies for the prevention of oral diseases through control of oral biofilms . Information is still lacking, however, on the genetic regulation of oral biofilm formation . A better understanding of these processes is of considerable importance.

FEMS Microbiol Lett, 2004 Jan 30, 230(2), 283 - 90
Colony aggregation and biofilm formation in xylem chemistry-based media for Xylella fastidiosa; Leite B et al.; Two chemically defined media based on xylem fluid chemistry were developed for Xylella fastidiosa . These media were tested and compared to chemically defined media XDM2, XDM4 and XF-26 . New media were evaluated for the Pierce's disease (PD) strain UCLA-PD . Our media either was similar to the concentration of some amino acids found in the xylem fluid of the PD-susceptible Vitis vinifera cv . Chardonnay (medium CHARD2) or incorporated the tripeptide glutathione found in xylem fluid composition (medium 3G10-R) . CHARD2 and 3G10-R are among the simplest chemically defined media available . Xylem fluid chemistry-based media supported X . fastidiosa growth and especially stimulated aggregation and biofilm formation.

Environ Microbiol, 2004 Feb, 6(2), 183 - 7
A novel approach to investigate biofilm accumulation and bacterial transport in porous matrices; Dunsmore BC et al.; Knowledge of bacterial transport through, and biofilm growth in, porous media is vitally important in numerous natural and engineered environments . Despite this, porous media systems are generally oversimplified and the local complexity of cell transport, biofilm formation and the effect of biofilm accumulation on flow patterns is lost . In this study, cells of the sulphate-reducing bacterium, Desulfovibrio sp . EX265, accumulated primarily on the leading faces of obstructions and developed into biofilm, which grew to narrow and block pore throats (at a rate of 12 micro m h(-1) in one instance) . This pore blocking corresponded to a decrease in permeability from 9.9 to 4.9 Darcy . Biofilm processes were observed in detail and quantitative data were used to describe the rate of biofilm accumulation temporally and spatially . Accumulation in the inlet zone of the micromodel was 10% higher than in the outlet zone and a mean biofilm height of 28.4 micro m was measured in a micromodel with an average pore height of 34.9 microm . Backflow (flow reversal) of fluid was implemented on micromodels blocked with biofilm growth . Although biofilm surface area cover did immediately decrease (approximately 5%), the biofilm quickly re-established and permeability was not significantly affected (9.4 Darcy) . These results demonstrate that the glass micromodel used here is an effective tool for in situ analysis and quantification of bacteria in porous media.

Curr Microbiol, 2003 Dec, 47(6), 485 - 91
Effect of Porphyromonas gingivalis vesicles on coaggregation of Staphylococcus aureus to oral microorganisms; Kamaguchi A et al.; Vesicles from the outer membrane of Porphyromonas gingivalis have the ability to aggregate a wide range of Streptococcus spp., Fusobacterium nucleatum, Actinomyces naeslundii, and Actinomyces viscosus . We found that in the presence of P . gingivalis vesicles, Staphylococcus aureus coaggregated with Streptococcus spp., and the mycelium-type Candida albicans, but not the yeast type . Autoaggregation of S . aureus in the presence of P . gingivalis vesicles is inhibited by L-arginine, L-lysine, and L-cysteine . Both the methicillin-sensitive (MSSA) and -resistant (MRSA) strains of S . aureus were able to coaggregate with Streptococcus spp., A . naeslundii, and A . viscosus when they were treated with P . gingivalis vesicles . P . gingivalis vesicle-treated mycelium-type C . albicans coaggregated with S . aureus, but the yeast-type did not . These results indicate that strains of S . aureus, including MRSA, could adhere to oral biofilms in dental plaque on the tooth surface or in the gingival crevice when P . gingivalis is present.

J Environ Sci (China), 2003 Nov, 15(6), 757 - 61
Biodegradation kinetic of organic compounds of acrylic fiber wastewater in biofilm; Zhang YL et al.; A group function relation curve between flux (J) and bulk phase concentration of substrate (S) was set up . The biodegradation kinetic of organic compounds of acrylic fiber wastewater in biofilm is studied (the treatment technology is coagulation/sedimentation-anoxic/aerobic biofilm process), and the results showed that the concentration of non-degradation pollutants in effluent is 77 mg/L . In aerobic zone, the half-rate constant is 72.84 mg/L, the maximum removal rate of organic compounds at unit area filler is very low, 0.089 g/(m2 x d), which corresponds to the fact that there are some biorefractory compounds in the wastewater.

Laryngoscope, 2004 Feb, 114(2), 364 - 7
Microbiology of stents in laryngotracheal reconstruction; Simoni P et al.; OBJECTIVES: Granulation tissue often forms around a laryngotracheal stent, tracheostomy tube, or other airway prosthesis, especially if infection occurs . We studied the types and frequency of organisms colonizing stents used in pediatric laryngotracheal reconstruction . STUDY DESIGN: This prospective study included 21 patients undergoing 23 consecutive laryngotracheal reconstructions with stents between 1991 and 1999 . METHODS: After endoscopic removal, each laryngotracheal stent was placed immediately in a sterile container and transported to the laboratory . Specimens for culture were obtained from biofilms on the stents and plated on agars for growth of aerobic, anaerobic, and fungal organisms . Culture results were analyzed with regard to patient age, duration of stenting, and graft type . RESULTS: All stents were colonized with more than one pathogen (range 2-7) . The most frequent aerobic isolates were Streptococcus viridians, Pseudomonas aeruginosa, Staphylococcus aureus,Haemophilus influenza, and Neisseria species . Anaerobic organisms were isolated in 26% of cases . Candida species were isolated in 57% of the cases; patients whose stents were colonized with Candida were significantly (P =.007) older (mean 77.5 months) than those not colonized with this organism (mean 26.1 months) CONCLUSIONS: The antibiotic agents currently used for children undergoing laryngotracheal reconstruction target mainly aerobic organisms . Despite prophylactic measures, the incidence of granulation tissue formation is clinically significant, and the prevalence of anaerobic, including fungal, pathogens is high . Antibiotic therapy directed toward controlling anaerobic and fungal organisms could help in controlling local inflammation and thus granulation tissue formation.

Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1737 - 42 Epub 2004 Jan 30.
Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants; Newman KL et al.; Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission . Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes . Recently available X . fastidiosa genomic sequences and molecular tools have provided new routes for investigation . Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants . We constructed strains of X . fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity . In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants . This signal therefore directs interaction of X . fastidiosa with both its insect vector and plant host . Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X . fastidiosa gene expression . This article reports a cell-cell signaling requirement for vector transmission . Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Dec;68(6 Pt 1):061904 . Epub 2003 Dec 17.
Hierarchical population model with a carrying capacity distribution for bacterial biofilms; Indekeu JO et al.; In order to describe biological colonies with a conspicuous hierarchical structure, a time- and space-discrete model for the growth of a rapidly saturating local biological population N(x,t) is derived from a hierarchical random deposition process previously studied in statistical physics . Two biologically relevant parameters, the probabilities of birth, B, and of death, D, determine the carrying capacity K . Due to the randomness the population depends strongly on position x and there is a distribution of carrying capacities, Pi(K) . This distribution has self-similar character owing to the exponential slowing down of the growth, assumed in this hierarchical model . The most probable carrying capacity and its probability are studied as a function of B and D . The effective growth rate decreases with time, roughly as in a Verhulst process . The model is possibly applicable, for example, to bacteria forming a "towering pillar" biofilm, a structure poorly described by standard Eden or diffusion-limited-aggregation models . The bacteria divide on randomly distributed nutrient-rich regions and are exposed to a random local bactericidal agent (antibiotic spray) . A gradual overall temperature or chemical change away from optimal growth conditions reduces bacterial reproduction, while biofilm development degrades antimicrobial susceptibility, causing stagnation into a stationary state.

Water Sci Technol, 2003, 48(11-12), 463 - 72
Biofilm bacteria inactivation by citric acid and resuspension evaluations for drinking water production systems; Tsai YP et al.; The study investigates the inactivation of biofilm bacteria colonized on the surface of polyvinyl chloride (PVC) pipes delivering either groundwater or treated wastewater . It does so using a citric acid (C6H8O7) solution . The results of the study showed that the optimal conditions of the biofilm bacteria inactivation were over 10,000 mg/L citric acid concentration and 60 minutes of contact time at least . Under these conditions, the removal efficiency could reach above 99.999% for heterotrophic plate count (HPC) bacteria and 99.95% for coliform bacteria . The study also showed that the biofilm bacteria were the major sources of planktonic bacteria resuspended into water purified by drinking water production systems (DWPS).

Water Sci Technol, 2003, 48(11-12), 299 - 307
Experimental study on sequencing batch biofilm reactor with biological filtration (SBBR-BF) for wastewater treatment; Li TW et al.; A novel wastewater treatment technology combining a sequencing batch biofilm reactor and biological filtration in an SBBR-BF system was presented . Elastic plastic filaments were fixed as biofilms carrying media . Particle materials (sand or anthracite) and the settled sludge constituted the filtration layer . In the laboratory studies, operating results of SBR, SBBR and SBBR-BF were compared . Better quality and stable water quality of effluent could be achieved in SBBR-BF because the fixed film and filtration layer were added in the reactor . Other laboratory experiment results indicated that slow filtration, cycle water stirring and backwashing making use of the settled supernatant are successful methods for preventing clogging and saving energy . The velocity and headloss of filtration were significantly impacted by different MLSS concentration . The MLSS concentration in the reactor must be less than 1,400 mg/L for optimal results . The average velocity of filtration ranging from 0.6 to 1.0 m/h, the backwash velocity of 10-15 m/h and the backwash time of 20 seconds are recommended according to the laboratory experiment . On-site experiment and study showed that SBBR-BF is a stable and efficient system for domestic wastewater treatment, and is particularly suited for small wastewater treatment plants, because of the simple operation and compact installation.

Water Sci Technol, 2003, 48(11-12), 169 - 77
Treatment of mountain refuge wastewater by fixed and moving bed biofilm systems; Andreottola G et al.; Tourists visiting mountain refuges in the Alps have increased significantly in the last decade and the number of refuges and huts at high altitude too . In this research the results of an intensive monitoring of a wastewater treatment plant (WWTP) for a tourist mountain refuge located at 2,981 m a.s.l . are described . Two biofilm reactors were adopted: (a) a Moving Bed Biofilm Reactor (MBBR); (b) a submerged Fixed Bed Biofilm Reactor (FBBR) . The aims of this research were: (i) the evaluation of the main parameters characterising the processes and involved in the design of the wastewater plants, in order to compare advantages and disadvantages of the two tested alternatives; (ii) the acquisition of an adequate knowledge of the problems connected with the wastewater treatment in alpine refuges . The main results have been: (i) a quick start-up of the biological reactors obtainable thanks to a pre-colonization before the transportation of the plastic carriers to the refuge at the beginning of the tourist season; (ii) low volume and area requirement; (iii) significantly higher removal efficiency compared to other fixed biomass systems, such as trickling filters, but the energy consumption is higher.

J Antimicrob Chemother, 2004 Mar, 53(3), 522 - 5 Epub 2004 Jan 28.
Caspofungin modulates in vitro adherence of Candida albicans to plastic coated with extracellular matrix proteins; Soustre J et al.; OBJECTIVES: Some manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces and the intrinsic resistance of Candida albicans biofilms to the most commonly used antifungal agents has been demonstrated . In this study, we report on the influence of the growth of C . albicans in medium containing a sub-inhibitory concentration (MIC/2) of caspofungin, on subsequent fungal adherence to plastic coated with extracellular matrix (ECM) proteins . METHODS: Eleven strains of C . albicans were studied: six strains were susceptible to fluconazole in vitro and five strains were resistant to this antifungal agent . RESULTS: Caspofungin induced a decrease in the adherence of all the tested strains that were susceptible to fluconazole but induced a decrease in the adherence of only 60% of the fluconazole-resistant strains . CONCLUSIONS: This study demonstrated the anti-adherent activity of caspofungin but indicated a reduced effect in the case of in vitro fluconazole resistance . These results indicated a possible relationship between the efficiency of caspofungin to inhibit the first step of the development of C . albicans biofilm and the resistance of C . albicans to fluconazole in vitro.

J Microbiol Methods, 2004 Feb, 56(2), 161 - 72
Three-dimensional differentiation of photo-autotrophic biofilm constituents by multi-channel laser scanning microscopy (single-photon and two-photon excitation); Neu TR et al.; A simple microscopic method to three-dimensionally differentiate between various members in photo-autotrophic biofilm systems is described . By dual-channel single-photon (confocal) and two-photon laser scanning microscopy, the signals in the red and far red channels as well as their combination can be simultaneously recorded . The method takes advantage of the autofluorescent signal of cyanobacteria-recorded in the red and far red channel and the autofluorescent signal of the green algae-recorded in the far red channel only . The differentiation is based on the specific pigment composition of cyanobacteria and green algae in combination with the appropriate filter settings for detection of the autofluorescent emission signals . The method allows the non-destructive, three-dimensional examination of fully hydrated interfacial microbial communities at high resolution as well as the clear separation between autofluorescent signals of cyanobacteria and green algae . Furthermore, there is a third option to record additional signals simultaneously such as nucleic acid stained bacteria, bacteria labeled with phylogenetic probes or glycoconjugates stained by using lectins . With state of the art laser scanning microscopes, even a fourth channel is available for recording yet another parameter, e.g . in the reflection (single-photon only) or fluorescence (single- and two-photon) mode . Thus the approach represents a convenient tool to study multiple parameters of complex photo-autotrophic biofilm systems.

J Dent Res, 2004 Feb, 83(2), 175 - 9
Dental caries, oral hygiene, and oral clearance in children with craniofacial disorders; Ahluwalia M et al.; The reason that children with cleft palates tend to have a greater prevalence of tooth decay than normal children is unclear . We hypothesized that children with cleft palates would have increased oral clearance times for foods and, consequently, higher levels of caries and caries-associated micro-organisms than control children . Children aged 6-16 yrs, with (n = 81) or without (n = 61) cleft palates, were studied . Children with cleft palates had DMFT and dmft scores greater (p < 0.01) than those of the control group . The number of caries-associated organisms was greater in the saliva of the cleft palate children (all p < 0.001) . The oral hygiene, plaque and gingival index scores were greater (p < 0.0001), oral clearance was longer (p < 0.01), and levels of sucrose and starch-derived saccharides higher (p < 0.01) in the cleft palate group . However, salivary concentrations of organic acids were lower in the children with craniofacial disorders, probably reflecting the altered physiology of the more mature dental biofilm . The longer oral clearance times of foods and the consequent generation of fermentable sugars from starches may contribute to the higher caries prevalence observed in children with cleft palates.

Infect Immun, 2004 Feb, 72(2), 1210 - 5
The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides; Kozitskaya S et al.; Staphylococcus epidermidis is a normal constituent of the healthy human microflora, but it is also the most common cause of nosocomial infections associated with the use of indwelling medical devices . Isolates from device-associated infections are known for their pronounced phenotypic and genetic variability, and in this study we searched for factors that might contribute to this flexibility . We show that mutator phenotypes, which exhibit elevated spontaneous mutation rates, are rare among both pathogenic and commensal S . epidermidis strains . However, the study revealed that, in contrast to those of commensal strains, the genomes of clinical S . epidermidis strains carry multiple copies of the insertion sequence IS256, while other typical staphylococcal insertion sequences, such as IS257 and IS1272, are distributed equally among saprophytic and clinical isolates . Moreover, detection of IS256 was found to be associated with biofilm formation and the presence of the icaADBC operon as well as with gentamicin and oxacillin resistance in the clinical strains . The data suggest that IS256 is a characteristic element in the genome of multiresistant nosocomial S . epidermidis isolates that might be involved in the flexibility and adaptation of the genome in clinical isolates.

Infect Immun, 2004 Feb, 72(2), 651 - 8
Porphyromonas gingivalis genes involved in fimA regulation; Xie H et al.; Porphyromonas gingivalis is an important component of the complex plaque biofilm that is a direct precursor of periodontal disease . The major fimbriae are required for attachment to oral surfaces and are an important virulence factor . Fimbrillin (FimA) expression in P . gingivalis is inhibited by surface molecule of Streptococcus cristatus, an early colonizer of dental plaque . In this study, differential display PCR was used to identify P . gingivalis genes that are regulated in response to S . cristatus . Of several differentially expressed genes, pg2131 and pg2167 were upregulated by S . cristatus signaling molecules . A null mutant of pg2167 did not transcriptionally regulate fimA following exposure to S . cristatus . In fact, fimA transcription was enhanced in the pg2167 mutant, suggesting that pg2167 may act to repress fimA expression . In contrast, a mutation in pg2131 did not affect transcription of fimA in the presence of S . cristatus . However, production of fimbrillin was significantly diminished in the pg2131 mutant, implicating involvement in posttranscriptional regulation in fimbriation . These data suggest that P . gingivalis fimbriation is controlled by more than one regulation mechanism, involving both transcriptional and posttranscriptional processes.

Proc Natl Acad Sci U S A, 2004 Feb 3, 101(5), 1333 - 8 Epub 2004 Jan 22.
Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis; Justice SS et al.; Uropathogenic Escherichia coli (UPEC) are capable of forming complex intracellular bacterial communities (IBC) within the superficial umbrella cells of the bladders of C3H and BALB/c mice . By using time-lapse fluorescence videomicroscopy to observe infected mouse bladder explants, we discovered that IBCs formed by uropathogenic E . coli progressed through four distinct developmental stages that differed with respect to growth rate, bacterial length, colony organization, motility, and its eventual dispersal . In the first phase, bacteria in the IBC were nonmotile, rod shaped, and grew rapidly in loosely organized colonies free in the cytoplasm of the bladder superficial umbrella cells . In the second phase, the loose collection of bacteria in the IBC matured into a slower growing, highly organized biofilm-like community consisting of coccoid bacteria that ultimately filled most of the cytoplasm . In the third phase, bacteria in the biofilm-like state in the IBC switched to a motile rod-shaped phenotype allowing detachment from the community and eventual fluxing out of the host cell . During the fourth phase, the bacteria filamented . Filamentation appeared to be in response to a Toll-like receptor 4-mediated innate defense mechanism . Bacteria that fluxed out of the superficial umbrella cells were able to reenter the IBC developmental cascade but with slower kinetics and ultimately a quiescent reservoir was established . Intracellular growth and filamentation provided an advantage to the bacteria in evading infiltrating polymorphonuclear leukocytes . This work has developed a technique to observe live infected organs and revealed a complex differentiation pathway that facilitates bacterial persistence in the urinary tract.

Eur Respir J, 2004 Jan, 23(1), 146 - 58
New concepts of the pathogenesis of cystic fibrosis lung disease; Boucher RC; Although there has been impressive progress in the elucidation of the genetic and molecular basis of cystic fibrosis (CF), the pathogenesis of CF lung disease remains obscure . The elucidation of the pathogenesis of CF lung disease requires both a full description of normal innate airway defence and how absent function of the cystic fibrosis transmembrane regulator protein (CFTR) adversely perturbs this activity . Recent data have linked the abnormal ion transport properties of CF airway epithelia to depleted airway surface liquid (ASL) volume, reflecting the combined defects of accelerated Na+ transport and the failure to secrete Cl- . Depletion of a specific compartment of the ASL, i.e . the periciliary liquid (PCL), appears to abrogate both cilia-dependent and cough clearance . Subsequent to PCL depletion, mucus adheres to airway surfaces and persistent mucin secretion generates the formation of "thickened" mucus plaques and plugs, which become the nidus for bacterial infection . The paucity of liquid in these plaques/plugs, and the hypoxia in this environment, appear to promote biofilm bacterial infection . Therapeutic agents that restore airway surface liquid volume, i.e . blockers of Na+ transport, initiators of Cl- transport and osmolytes, are reviewed, as are strategies that may be required to use volume-restoring agents safely in patients with cystic fibrosis.

FEMS Microbiol Lett, 2004 Jan 15, 230(1), 105 - 13
Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae; Rashid MH et al.; Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS) . Some strains can also phenotypically switch from a smooth to a 'rugose' phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions . High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology . VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS) . However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood . We report that rugose strains of both classical and El Tor biotypes of epidemic V . cholerae are defective in the in vitro production of extracellular collagenase activity . In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity . Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis . Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis . Additionally, VpsR might regulate genes with roles in virulence . Rugose strains appear to be a subpopulation of cells that might act as a 'helper' phenotype promoting the pathogenesis of certain strains . Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V . cholerae.

FEMS Microbiol Lett, 2004 Jan 15, 230(1), 13 - 8
Persister cells and tolerance to antimicrobials; Keren I et al.; Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics . Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology . Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters . The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase . Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin) . This shows that production of persisters depends on growth stage . Maintaining a culture of E . coli at early exponential phase by reinoculation eliminated persisters . This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics . Our data indicate that persisters are specialized survivor cells.

Environ Technol, 2003 Nov, 24(11), 1431 - 43
Anaerobic digestion of dairy wastewater by inverse fluidization: the inverse fluidized bed and the inverse turbulent bed reactors; Arnaiz C et al.; This paper describes the application of the inverse fluidization technology to the anaerobic digestion of dairy wastewater . Two reactors were investigated: the inverse fluidized bed reactor and the inverse turbulent reactor . In these reactors, a granular floating solid is expanded by a down-flow current of effluent or an up-flow current of gas, respectively . The carrier particles (Extendospheres) were chosen for their large specific surface area (20,000 m2m(-3)) and their low energy requirements for fluidization (gas velocity of 1.5 mm s(-1), 5.4 m h(-1)) . Organic load was increased stepwise by reducing hydraulic retention time from more than 60 days to 3 days, while maintaining constant the feed COD concentration . Both reactors achieved more than 90% of COD removal, at an organic loading rate of 10-12 kgCOD m(-3) d(-1), respectively . The performances observed were similar or even higher than that of other previously tested fluidized bed technologies treating the same wastewater . It was found that the main advantages of this system are: low energy requirement, because of the low fluidization velocities required; there is no need of a settling device, because solids accumulate at the bottom of the reactor, so they can be easily drawn out and particles with high-biomass content can be easily recovered . Lipid phosphate concentration has been revealed as a good method for biomass estimation in biofilms since it only includes living biomass.

Blood Purif, 2004, 22(1), 124 - 9
When good water goes bad: how it happens, clinical consequences and possible solutions; Lonnemann G; Dialysis fluid produced by state-of-the-art water preparation and distribution is contaminated with gram-negative bacteria and cytokine-inducing substances (CIS) derived from these microorganisms . The presence of a biofilm increases the risk of continuous contamination of dialysis fluid . Depending on the type of dialyzer membrane (cellulosic vs . synthetic) and the mode of dialysis (low flux vs . high flux with backfiltration), CIS may penetrate intact dialyzer membranes, induce cytokine production in the patient's blood and contribute to chronic inflammation associated with long-term hemodialysis therapy . Measures to improve the microbiological quality of dialysis fluid are: (1) the awareness of the problem and regular testing of dialysate samples using adequate methods; (2) disinfection of the entire water preparation and distribution system on a regular basis, replacement of biofilm-containing tubings, and (3) installation of ultrafilters in the dialysate circuit in particular when high-flux hemodialysis modalities are performed .

Mol Microbiol, 2004 Feb, 51(3), 675 - 90
Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms; Friedman L et al.; Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments . One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures . We screened for transposon insertion mutants of P . aeruginosa PA14 that were unable to form pellicles . Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix . In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms . Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing . Microscopic and macroscopic observations revealed that wild-type P . aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants . A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material . Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P . aeruginosa PA14.

Mol Microbiol, 2004 Feb, 51(3), 659 - 74
Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression; Beloin C et al.; The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons . We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm . We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up- or downregulation by a factor greater than two, and that 10% of the E . coli genome is significantly differentially expressed . The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions . We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E . coli mature biofilms . We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes . Using gene disruption of 54 of the most biofilm-induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm . This group includes 11 genes of previously unknown function . These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E . coli biofilms and provide insights into its physiological signature.

J Antimicrob Chemother, 2004 Feb, 53(2), 383 - 5 Epub 2004 Jan 16.
Susceptibility of Candida albicans biofilms grown in a constant depth film fermentor to chlorhexidine, fluconazole and miconazole: a longitudinal study; Lamfon H et al.; OBJECTIVES: The aim of this study was to assess the resistance of Candida albicans biofilms to both antifungal and antimicrobial agents in vitro . METHODS: Biofilms of C . albicans were grown on denture acrylic discs in a constant depth film fermentor and maintained with artificial saliva . The MIC of fluconazole, miconazole and chlorhexidine for C . albicans was first determined . Using these data, 72 h biofilms were exposed to these agents at different MIC levels . In order to assess growth, biofilms were removed from the fermentor, incubated in the test agent for various periods, the biofilms disrupted and the viable yeast cells present determined . The MIC for these cells was then also determined . In a separate experiment, biofilms of various ages (2-72 h) were exposed to sub-biofilm MIC concentrations for two different periods . RESULTS: C . albicans biofilms were found to be highly resistant to fluconazole and miconazole compared with the same cells grown in suspension (>/=1024 x MIC) . In contrast, chlorhexidine inhibited the growth of C . albicans biofilms at a concentration up to 8 x MIC . When the susceptibility of biofilms over time was investigated, higher reductions were observed for chlorhexidine and miconazole than fluconazole for biofilms of 2 and 6 h . CONCLUSIONS: We have shown in this study that the susceptibility of C . albicans to antifungal and antimicrobial agents changes throughout biofilm development.

J Antimicrob Chemother, 2004 Feb, 53(2), 180 - 4 Epub 2004 Jan 16.
Biofilms, homoserine lactones and biocide susceptibility; MacLehose HG et al.; AIMS: To investigate the susceptibility, to a range of different biocides, of Pseudomonas aeruginosa strains variously deficient in N-acyl homoserine lactone systems, grown either as planktonic or biofilm populations . METHODS AND RESULTS: Biocide susceptibility data were generated for strains of P . aeruginosa deficient in N-acyl homoserine lactone production, grown planktonically or as biofilm populations using a poloxamer hydrogel construct . Component cells from the biofilm constructs were also tested for their susceptibility . Significant differences in susceptibility were noted between the wild-type strain, a mutant defective in the long chain (C-12) homoserine lactone and a mutant defective in the short chain (C-4) homoserine lactone which could not be related to the biofilm mode of growth . Moreover, differences in susceptibility appeared to be dependent upon the nature of the homoserine lactone deletion and type of biocide rather than the mode of growth . CONCLUSIONS: No general trend exists between homoserine lactone deficiency and biocide susceptibility regardless of mode of growth.

J Bacteriol, 2004 Feb, 186(3), 889 - 93
Expression of Vibrio vulnificus capsular polysaccharide inhibits biofilm formation; Joseph LA et al.; Vibrio vulnificus is a human pathogen that produces lethal septicemia in susceptible persons, and the primary virulence factor for this organism is capsular polysaccharide (CPS) . The role of the capsule in V . vulnificus biofilms was examined under a variety of conditions, by using either defined CPS mutants or spontaneous CPS expression phase variants derived from multiple strains . CPS expression was shown to inhibit attachment and biofilm formation, which contrasted with other studies describing polysaccharides as integral to biofilms in related species.

J Bacteriol, 2004 Feb, 186(3), 722 - 9
Control of glucose- and NaCl-induced biofilm formation by rbf in Staphylococcus aureus; Lim Y et al.; Both Staphylococcus aureus and S . epidermidis are capable of forming biofilm on biomaterials . We used Tn917 mutagenesis to identify a gene, rbf, affecting biofilm formation in S . aureus NCTC8325-4 . Sequencing revealed that Rbf contained a consensus region signature of the AraC/XylS family of regulators, suggesting that Rbf is a transcriptional regulator . Insertional duplication inactivation of the rbf gene confirmed that the gene was involved in biofilm formation on polystyrene and glass . Phenotypic analysis of the wild type and the mutant suggested that the rbf gene mediates the biofilm formation of S . aureus at the multicellular aggregation stage rather than at initial attachment . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the mutation resulted in the loss of an approximately 190-kDa protein . Biofilm production by the mutant could be restored by complementation with a 2.5-kb DNA fragment containing the rbf gene . The rbf-specific mutation affected the induction of biofilm formation by glucose and a high concentration of NaCl but not by ethanol . The mutation did not affect the transcription of the ica genes previously shown to be required for biofilm formation . Taken together, our results suggest that the rbf gene is involved in the regulation of the multicellular aggregation step of S . aureus biofilm formation in response to glucose and salt and that this regulation may be mediated through the 190-kDa protein.

J Bacteriol, 2004 Feb, 186(3), 692 - 8
Quorum sensing-controlled biofilm development in Serratia liquefaciens MG1; Labbate M et al.; Serratia liquefaciens MG1 contains an N-acylhomoserine lactone-mediated quorum-sensing system that is known to regulate swarming motility colonization . In this study, we describe for S . liquefaciens MG1 the development of a novel biofilm consisting of cell aggregates and differentiated cell types, such as cell chains and long filamentous cells . Furthermore, quorum sensing is shown to be crucial for normal biofilm development and for elaborate differentiation . A mutant of S . liquefaciens MG1 that was incapable of synthesizing extracellular signal formed a thin and nonmature biofilm lacking cell aggregates and differentiated cell chains . Signal-based complementation of this mutant resulted in a biofilm with the wild-type architecture . Two quorum-sensing-regulated genes (bsmA and bsmB) involved in biofilm development were identified, and we propose that these genes are engaged in fine-tuning the formation of cell aggregates at a specific point in biofilm development.

Appl Microbiol Biotechnol, 2004 May, 64(4), 515 - 24 Epub 2004 Jan 16.
Gene expression in Escherichia coli biofilms; Ren D et al.; DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms . Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms . For the biofilms grown on glass wool, 22 genes were induced significantly (p< or =0.05) compared to suspension cells, including several genes for the stress response ( hslS, hslT, hha, and soxS), type I fimbriae ( fimG), metabolism ( metK), and 11 genes of unknown function ( ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836) . The DNA microarray results were corroborated with RNA dot blotting . For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h . The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E . coli JM109 biofilm formation .

Clin Microbiol Rev, 2004 Jan, 17(1), 98 - 106
Health impacts of environmental mycobacteria; Primm TP et al.; Environmental mycobacteria are emerging pathogens causing opportunistic infections in humans and animals . The health impacts of human-mycobacterial interactions are complex and likely much broader than currently recognized . Environmental mycobacteria preferentially survive chlorination in municipal water, using it as a vector to infect humans . Widespread chlorination of water has likely selected more resistant environmental mycobacteria species and potentially explains the shift from M . scrofulaceum to M . avium as a cause of cervical lymphadenitis in children . Thus, human activities have affected mycobacterial ecology . While the slow growth and hydrophobicity of environmental mycobacteria appear to be disadvantages, the unique cell wall architecture also grants high biocide and antibiotic resistance, while hydrophobicity facilitates nutrient acquisition, biofilm formation, and spread by aerosolization . The remarkable stress tolerance of environmental mycobacteria is the major reason they are human pathogens . Environmental mycobacteria invade protozoans, exhibiting parasitic and symbiotic relationships . The molecular mechanisms of mycobacterial intracellular pathogenesis in animals likely evolved from similar mechanisms facilitating survival in protozoans . In addition to outright infection, environmental mycobacteria may also play a role in chronic bowl diseases, allergies, immunity to other pulmonary infections, and the efficacy of bacillus Calmette-Guerin vaccination.

Int Dent J, 2003 Dec, 53(6 Suppl 1), 371 - 8
Antimicrobial effects of a novel Triclosan/zinc citrate dentifrice against mixed culture oral biofilms; Finney M et al.; AIM: To compare the antimicrobial efficacy in vitro of a new fluoride system containing 0.3% Triclosan and 2.0% zinc citrate with a standard fluoride toothpaste against bacteria commonly isolated from dental plaque . METHOD: Mature biofilms of fixed depth containing ten oral bacterial species were developed in a model system and then exposed to the formulations both in situ and ex situ . RESULTS: The standard fluoride toothpaste had limited activity against the Gram positive species following 1 and 5 minutes ex situ exposure (p = 0.004 and p = 0.02, respectively) but none against the Gram negative species; also, no overall effect following repeated pulsing in situ was observed . In contrast, viable counts of Gram positive and Gram negative species were markedly reduced following ex situ exposure for 1 and 5 minutes to the new formulation (Gram positive: p = 0.0007 and p = 0.04; Gram negative: p = 0.04 and p = 0.00001, respectively) . Counts of Gram positive bacteria were reduced by 99.9% over a 4-day in situ pulsing period, while no Gram negative species were recoverable from the biofilm by day 4 . CONCLUSION: The new toothpaste formulation had a broad spectrum of antimicrobial activity against biofilm consortia containing bacteria associated with both dental caries and periodontal diseases.

Int Dent J, 2003 Dec, 53(6 Suppl 1), 363 - 70
Anti-microbial efficacy and mode of action studies on a new zinc/ Triclosan formulation; Brading MG et al.; OBJECTIVE: To test in vitro the anti-plaque/ antimicrobial efficacy of a new toothpaste formulation containing a 2% zinc citrate/ 0.3% Triclosan anti-microbial system compared with a 0.75% zinc citrate/ 0.3% Triclosan system and where appropriate, against controls of a standard fluoride paste and a 0.3% Triclosan/ 2% copolymer product . METHODS: The anti-metabolic activity was assessed using a range of assays measuring the ability of the active systems to inhibit bacterial glycolysis . The antibacterial/ anti-plaque activity was assessed in an in vitro multispecies biofilm assay . RESULTS: Both zinc formulations were shown to have significantly superior activity at inhibiting glycolysis compared with the 0.3% Triclosan/ 2% copolymer formulation and the standard fluoride paste, particularly in reducing the pH drop after sugar challenge, the new formulation having the greatest activity . Likewise, in the antibacterial assay, both zinc formulations were found to have significantly superior activity over a standard fluoride paste and the 2% zinc citrate/ 0.3% Triclosan formulation was shown to be significantly better than 0.75% zinc citrate/ 0.3% Triclosan formulation . CONCLUSION: These data provide support for the enhanced performance of the 2% zinc citrate/ 0.3% Triclosan formulation.

Int Dent J, 2003 Dec, 53(6 Suppl 1), 353 - 62
The oral environment: the challenge for antimicrobials in oral care products; Brading MG et al.; For any antibacterial/ anti-plaque system from an oral care product to be effective, it must firstly be delivered and retained at relevant sites in the oral cavity and secondly, remain active within the chosen formulation to successfully target the biofilm cells within dental plaque . This must include inhibition of the growth and metabolism of relevant organisms associated with disease . This review will concentrate on understanding the environmental conditions in which such oral care products must work and summarise the activity within the oral cavity of the main antibacterial and anti-plaque agents in common oral care products, namely chlorhexidine, essential oils, metal salts and Triclosan . Routes to further enhance the activity of these products, together with the use of relatively novel formats such as confectionery products to provide added consumer oral health benefits will be considered.

Chemosphere, 2004 Apr, 55(1), 45 - 50
Degradation of phenol and cresols at low temperatures using a suspended-carrier biofilm process; Perron N et al.; Degradation of phenol and o-, m- and p-cresol at a concentration of 150 mg l(-1) of each compound was studied in a suspended-carrier biofilm process consisting of two aerobic stages . The fungus Mortierella sarnyensis Mil'ko dominated the microflora in the first reactor, while bacteria dominated in the second reactor . The process was studied at 4, 7, 11 and 15 degrees C . The results from the experiments showed the process to be relatively efficient even at 4 degrees C . The degradation rate was 33% of that at 15 degrees C for o-cresol . Both phenol and the cresols were degraded in the first reactor and a new peak appeared in the HPLC-chromatograms indicating the formation of one or more intermediate compounds in the first stage . These compounds were however degraded to below the detection limit in the second reactor . Small new peaks appeared in the chromatograms of the outlet from the second reactor at the maximum loading rates.

Microbiol Res, 2003, 158(4), 327 - 35
Development and morphological features of biofilms formed by transgenic and wild type strains of Bacillus subtilis; Mogilnaya OA et al.; The study addressed the ability of the transgenic strain (TM) B . subtilis 2335/pBMB 105 (KmrInf+) to form biofilms on the surface of liquid media of various compositions, inoculated with vegetative cells and spores . The morphological features of these biofilms do not differ from those of the films formed by the recipient strain (WT) B . subtilis 2335 (Kms) . However . the TM and the natural one differ in the dynamics of biofilm formation and the cellular composition of the films . Biofilms of the TM are formed earlier, develop at a higher rate, but decompose later than the films of the WT . When the medium is inoculated with vegetative cells, sporulation in the biofilms of both strains undergoes glucose repression; no such effect is observed when the medium is inoculated with spores . The TM does not form films when the medium is inoculated with spores and supplemented with glycerin and kanamycin.

Environ Sci Technol, 2003 Dec 15, 37(24), 5823 - 8
Trichloroethylene degradation in a coupled anaerobic/aerobic reactor oxygenated using hydrogen peroxide; Tartakovsky B et al.; In this work, trichloroethylene (TCE) degradation under combined anaerobic-aerobic conditions was studied in an ethanol-fed biofilm reactor oxygenated using hydrogen peroxide . The reactor was inoculated with a biomass originating from an anaerobic digestor . Granulated peat was added to the reactor as a substratum for biofilm development . Extensive characterization of reactor populations using activity tests and PCR analysis revealed the development of a mutualistic consortium, particularly methanotrophic and methanogenic microorganisms . This consortium was shown to degrade TCE by a combination of reductive and oxidative pathways . A near complete degradation of TCE at a load of 18 mg L(R)(-1) day(-1) was evidenced by a stoichiometric release of inorganic chloride.

Environ Sci Technol, 2003 Dec 15, 37(24), 5617 - 24
Pore-scale analysis of anaerobic halorespiring bacterial growth along the transverse mixing zone of an etched silicon pore network; Nambi IM et al.; The anaerobic halorespiring microorganism, Sulfurospirillum multivorans, was observed in the pore structure of an etched silicon wafer to determine how flow hydrodynamics and mass transfer limitations along a transverse mixing zone affect biomass growth . Tetrachloroethene (PCE, an electron acceptor, 0.2 mM) and lactate (an electron donor, 2 mM) were introduced as two separate and parallel streams that mixed along a reaction line in the pore structure . The first visible biomass occupied a single line of pores in the direction of flow, a few pore bodies from the micromodel centerline . This growth was initially present as small aggregates; over time, these grew and fused to form finger-like structures with one end attached to downgradient ends of the silicon posts and the other end extending into pore bodies in the direction of flow . Biomass did not grow in pore throats as expected, presumably because shear forces were not favorable . Over the next few weeks, the line of growth migrated upward into the PCE zone and extended over a width of up to five pore spaces . When the PCE concentration was increased to 0.5 mM, the microbial biomass increased and growth migrated down toward the lactate side of the micromodel . A new analytical model was developed and used to demonstrate that transverse hydrodynamic dispersion likely caused the biomass to move in the direction observed when the PCE concentration was changed . The model was unable, however, to explain why growth migrated upward when the PCE concentration was initially constant . We postulate that this occurred because PCE, not lactate, sorbed to biofilm components and that biomass on the lactate side of the micromodel was limited in PCE . A fluorescent tracer experiment showed that biomass growth changed the water flow paths, creating a higher velocity zone in the PCE half of the micromodel . These results contribute to our understanding of biofilm growth and will help in the development of new models to describe this complex process.

Appl Microbiol Biotechnol, 2004 Jan, 63(4), 452 - 9 Epub 2003 Mar 05.
Evaluation of bacterial strategies to promote the bioavailability of polycyclic aromatic hydrocarbons; Johnsen AR et al.; Polycyclic aromatic hydrocarbon (PAHs)-degrading bacteria may enhance the bioavailability of PAHs by excreting biosurfactants, by production of extracellular polymeric substances, or by forming biofilms . We tested these hypotheses in pure cultures of PAHs-degrading bacterial strains . Most of the strains did not substantially reduce the surface tension when grown on PAHs in liquid shaken cultures . Thus, pseudo-solubilization of PAHs in biosurfactant micelles seems not to be a general strategy for these isolates to enhance PAHs-bioavailability . Three semi-colloid Sphingomonas polysaccharides all increased the solubility of PAHs (Gellan 1.3- to 5.4-fold, Welan 1.8- to 6.0-fold and Rhamsan 2.4- to 9.0-fold) . The increases were most pronounced for the more hydrophobic PAHs . The polysaccharide-sorbed PAHs were bioavailable . Mineralization rates of 9-{14C}-phenanthrene and 3-{14C}-fluoranthene by Sphingobium EPA505, were similar with and without sphingans, indicating that mass-transfer rates from PAHs crystals to the bulk liquid were unaffected by the polysaccharides . Biofilm formation on PAHs crystals may favor the diffusive mass transfer of PAHs from crystals to the bacterial cells . A majority of the PAHs-degraders tested formed biofilms in microtiter wells coated with PAHs crystals . For strains capable of growing on different PAHs; the more soluble the PAHs, the lower the percentage of cells attached . Biofilm formation on PAHs-sources was the predominant mechanism among the tested bacteria to overcome mass transfer limitations when growing on poorly soluble PAHs.

Z Naturforsch {C}, 2003 Nov-Dec, 58(11-12), 879 - 84
Influence of polyphenols on bacterial biofilm formation and quorum-sensing; Huber B et al.; Many bacteria utilize sophisticated regulatory systems to ensure that some functions are only expressed when a particular population density has been reached . The term 'quorum-sensing' has been coined to describe this form of density-dependent gene regulation which relies on the production and perception of small signal molecules by bacterial cells . As in many pathogenic bacteria the production of virulence factors is quorum-sensing regulated, it has been suggested that this form of gene regulation allows the bacteria to remain invisible to the defence systems of the host until the population is sufficiently large to successfully establish the infection . Here we present first evidence that polyphenolic compounds can interfere with bacterial quorum-sensing . Since polyphenols are widely distributed in the plant kingdom, they may be important for promoting plant fitness.

J Microbiol Methods, 2004 Jan, 56(1), 37 - 47
Multiplex FISH analysis of a six-species bacterial biofilm; Thurnheer T et al.; Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria . The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria . Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degrees C on hydroxyapatite disks preconditioned with saliva . Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme . After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A . naeslundii), EUK116 (C . albicans), FUS664 (F . nucleatum), MIT447 and MIT588 (S . oralis), SOB174 (S . sobrinus), and VEI217 (V . dispar) . Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively . Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized . Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible . In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.

Biotechnol Bioeng, 2004 Jan 5, 85(1), 47 - 55
Reconsidering the use of photosynthetic bacteria for removal of sulfide from wastewater; Hurse TJ et al.; The feasibility of using photosynthetic sulfide-oxidizing bacteria to remove sulfide from wastewater in circumstances where axenic cultures are unrealistic has been completely reconsidered on the basis of known ecophysiological data, and the principles of photobioreactor and chemical reactor engineering . This has given rise to the development of two similar treatment concepts relying on biofilms dominated by green sulfur bacteria (GSB) that develop on the exterior of transparent surfaces suspended in the wastewater . The GSB are sustained and selected for by radiant energy in the band 720-780 nm, supplied from within the transparent surface . A model of one of these concepts was constructed and with it the reactor concept was proven . The dependence of sulfide-removal rate on bulk sulfide concentration has been ascertained . The maximum net areal sulfide removal rate was 2.23 g m-(2) day-(1) at a bulk sulfide concentration of 16.5 mg L(-1) and an incident irradiance of 1.51 W m(-2) . The system has a demonstrated capacity to mitigate surges in sulfide load, and appears to use much less radiant power than comparable systems . The efficacy with which this energy was used for sulfide removal was 1.47 g day(-1) W(-1) . The biofilm was dominated by GSB, and evidence gathered indicated that other types of phototrophs were not present .

Biotechnol Bioeng, 2004 Jan 20, 85(2), 185 - 9
Recolonization of laser-ablated bacterial biofilm; Nandakumar K et al.; The recolonization of laser-ablated bacterial monoculture biofilm was studied in the laboratory by using a flow-cytometer system . The marine biofilm-forming bacterium Pseudoalteromonas carrageenovora was used to develop biofilms on titanium coupons . Upon exposure to a low-power pulsed irradiation from an Nd:YAG laser, the coupons with biofilm were significantly reduced both in terms of total viable count (TVC) and area cover . The energy density used for a pulse of 5 ns was 0.1 J/cm(2) and the durations of irradiation exposure were 5 and 10 min . When placed in a flow of dilute ZoBell marine broth medium (10%) the laser-destructed bacterial film in a flow-cytometer showed significant recovery over a period of time . The flow of medium was regulated at 3.2 ml/min . The increase in area cover and TVC, however, was significantly less than that observed for nonirradiated control (t-test, P< 0.05) . The coupons were observed for biofilm area cover and TVC at different intervals (3, 6, and 9 h) after irradiation . While the biofilm in the control coupon at the end of 9 h of exposure showed 95.6 +/- 4.1% cover, the 5- and 10-min irradiated samples after 9 h showed 60.3 +/- 6.5 and 37.4 +/- 12.1% area cover, respectively . The reduced rate of recolonization compared to control was thought be due to the lethal and sublethal impacts of laser irradiation on bacteria . This observation thus provided data on the online recolonization speed of biofilm, which is important when considering pulsed laser irradiation as an ablating technique of biofilm formation and removal in natural systems .

Microbiology, 2004 Jan, 150(Pt 1), 229 - 40
Deletion of the NOT4 gene impairs hyphal development and pathogenicity in Candida albicans; Krueger KE et al.; The Candida albicans NOT4 gene was disrupted in order to investigate the role of Not4p in growth, morphogenesis and pathogenicity . Heterozygote (NOT4/not4), null (not4/not4) and reconstructed heterozygote ({NOT4}/not4) strains of C . albicans, as well as CAF2-1, the parental strain, were grown under conditions that promote hyphal formation . When cultured in liquid medium 199 the heterozygote, reconstructed and wild-type strains began the yeast-to-hyphal transition within 3 h and continued hyphal growth for the duration of experiments . The null mutant also began hyphal growth within 3-5 h but hyphae tended to be shorter and distorted . Subsequently, hyphal growth was arrested and growth returned predominantly to the yeast form . Similar differences were observed when strains were grown on solid Spider medium and medium 199 . The parental, heterozygote and reconstructed strains formed normal filamentous networks emanating from colonies . In contrast, the null mutant failed to form hyphae on all solid media tested . The ability of the NOT4 null strain to form biofilms was also investigated, and it was observed that biofilm development does not readily occur for this strain . Virulence of each strain was examined utilizing the mouse model of systemic candidiasis . Mice infected with CAF2-1 succumbed to infection within 3-7 days . All mice infected with the null strain survived for the duration of experiments, while the heterozygote and reconstructed heterozygote strains showed an intermediate level of virulence . These findings suggest that NOT4 may play a role in affecting strain pathogenicity, possibly by regulating expression of certain genes that effect cellular morphogenesis and virulence.

Plant Physiol, 2004 Jan, 134(1), 320 - 31 Epub 2003 Dec 30.
Pseudomonas aeruginosa-plant root interactions . Pathogenicity, biofilm formation, and root exudation; Walker TS et al.; Pseudomonas aeruginosa is an opportunistic human pathogen capable of forming a biofilm under physiological conditions that contributes to its persistence despite long-term treatment with antibiotics . Here, we report that pathogenic P . aeruginosa strains PAO1 and PA14 are capable of infecting the roots of Arabidopsis and sweet basil (Ocimum basilicum), in vitro and in the soil, and are capable of causing plant mortality 7 d postinoculation . Before plant mortality, PAO1 and PA14 colonize the roots of Arabidopsis and sweet basil and form a biofilm as observed by scanning electron microscopy, phase contrast microscopy, and confocal scanning laser microscopy . Upon P . aeruginosa infection, sweet basil roots secrete rosmarinic acid (RA), a multifunctional caffeic acid ester that exhibits in vitro antibacterial activity against planktonic cells of both P . aeruginosa strains with a minimum inhibitory concentration of 3 microg mL(-1) . However, in our studies RA did not attain minimum inhibitory concentration levels in sweet basil's root exudates before P . aeruginosa formed a biofilm that resisted the microbicidal effects of RA and ultimately caused plant mortality . We further demonstrated that P . aeruginosa biofilms were resistant to RA treatment under in vivo and in vitro conditions . In contrast, induction of RA secretion by sweet basil roots and exogenous supplementation of Arabidopsis root exudates with RA before infection conferred resistance to P . aeruginosa . Under the latter conditions, confocal scanning laser microscopy revealed large clusters of dead P . aeruginosa on the root surface of Arabidopsis and sweet basil, and biofilm formation was not observed . Studies with quorum-sensing mutants PAO210 (DeltarhlI), PAO214 (DeltalasI), and PAO216 (DeltalasI DeltarhlI) demonstrated that all of the strains were pathogenic to Arabidopsis, which does not naturally secrete RA as a root exudate . However, PAO214 was the only pathogenic strain toward sweet basil, and PAO214 biofilm appeared comparable with biofilms formed by wild-type strains of P . aeruginosa . Our results collectively suggest that upon root colonization, P . aeruginosa forms a biofilm that confers resistance against root-secreted antibiotics.

Eur J Orthod, 2003 Dec, 25(6), 615 - 9
An in vitro study into the corrosion of intra-oral magnets in the presence of dental amalgam; Noar JH et al.; The aim of this investigation was to study the corrosion behaviour and products of uncoated neodymium-iron-boron magnets in the presence of dental amalgam . Microcosm plaques were grown on discs of neodymium-iron-boron magnets or amalgam in a constant depth film fermentor . The biofilms were supplied with artificial saliva and growth was determined by viable counting . The results showed that the neodymium-iron-boron magnets corroded with an average daily weight loss of 0.115 +/- 0.032 per cent . However, when the magnets were in close proximity to the amalgam the amount of corrosion was reduced to a daily loss of 0.066 +/- 0.023 per cent . The highest loss of constituent elements from the corrosion products of the magnets was observed for iron . The composition of the microcosm plaques altered markedly between the two materials with less streptococci and more Veillonella spp . present in the biofilms grown on magnets in the presence of amalgam . The corrosion of neodymium-iron-boron magnets is limited and in the presence of amalgam is reduced further . This suggests that amalgam present in the mouth will not cause an increased clinical risk in terms of biocompatibility with neodymium-iron-boron magnets.

Antimicrob Agents Chemother, 2004 Jan, 48(1), 151 - 60
Biofilm formation by Stenotrophomonas maltophilia: modulation by quinolones, trimethoprim-sulfamethoxazole, and ceftazidime; Di Bonaventura G et al.; We investigated the in vitro effects of seven fluoroquinolones (ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, and rufloxacin), compared to those of trimethoprim-sulfamethoxazole (SXT) and ceftazidime on total biomass and cell viability of Stenotrophomonas maltophilia biofilm . S . maltophilia attached rapidly to polystyrene, within 2 h of incubation, and then biofilm formation increased over time, reaching maximum growth at 24 h . In the presence of fluoroquinolones at one-half and one-fourth the MIC, biofilm biomass was significantly (P < 0.01) reduced to 55 to 70% and 66 to 76% of original mass, respectively . Ceftazidime and SXT did not exert any activity . Biofilm bacterial viability was significantly reduced by all antibiotics tested at one-half the MIC . At one-fourth the MIC all antibiotics, except levofloxacin, significantly reduced viability . Treatment of preformed biofilms with bactericidal concentrations (500, 100, and 50 micro g/ml) of all fluoroquinolones caused, except for norfloxacin, significant reduction of biofilm biomass to 29.5 to 78.8, 64.1 to 83.6, and 70.5 to 82.8% of original mass, respectively . SXT exerted significant activity at 500 micro g/ml only . Ceftazidime was completely inactive . Rufloxacin exhibited the highest activity on preformed biofilm viability, significantly decreasing viable counts by 0.6, 5.4, and 17.1% at 500, 100, and 50 micro g/ml, respectively . Our results show that (i) subinhibitory (one-half and one-fourth the MIC) concentrations of fluoroquinolones inhibit adherence of S . maltophilia to polystyrene and (ii) clinically achievable concentrations (50 and 100 micro g/ml) of rufloxacin are able to eradicate preformed S . maltophilia biofilm.

Antimicrob Agents Chemother, 2004 Jan, 48(1), 48 - 52
Modeling antibiotic tolerance in biofilms by accounting for nutrient limitation; Roberts ME et al.; A mathematical model of biofilm dynamics was used to investigate the protection from antibiotic killing that can be afforded to microorganisms in biofilms based on a mechanism of localized nutrient limitation and slow growth . The model assumed that the rate of killing by the antibiotic was directly proportional to the local growth rate . Growth rates in the biofilm were calculated by using the local concentration of a single growth-limiting substrate with Monod kinetics . The concentration profile of this metabolic substrate was calculated by solving a reaction-diffusion problem . The model predicted the following features: stratified patterns of growth with zones of no growth in the biofilm interior, slow killing of biofilm microorganisms that was further retarded as the initial biofilm thickness increased, nonuniform spatial patterns of killing inside the biofilm, biofilm killing rates that decrease in a nonlinear way as the concentration of the growth-limiting substrate feeding the biofilm is decreased, and heightened tolerance when external mass transfer resistance is manifested . This modeling study also provides motivation for further investigation of a hypothetical cell state in which damaged cells score as nonviable but continue to consume substrate . The existence of such a cell state can further retard biofilm killing, according to the simulations . The results support the important contributions of nutrient limitation and slow growth to the antibiotic tolerance of microorganisms in biofilms.

Antimicrob Agents Chemother, 2004 Jan, 48(1), 41 - 7
Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans; Alem MA et al.; Prostaglandins are now known to be produced by Candida albicans and may play an important role in fungal colonization . Their synthesis in mammalian cells is decreased by inhibitors of the cyclooxygenase isoenzymes required for prostaglandin formation . In the present study, a catheter disk model system was used to investigate the effects of nonsteroidal anti-inflammatory drugs (all cyclooxygenase inhibitors) on biofilm formation by three strains of C . albicans . Seven of nine drugs tested at a concentration of 1 mM inhibited biofilm formation . Aspirin, etodolac, and diclofenac produced the greatest effects, with aspirin causing up to 95% inhibition . Celecoxib, nimesulide, ibuprofen, and meloxicam also inhibited biofilm formation, but to a lesser extent . Aspirin was active against growing and fully mature (48-h) biofilms; its effect was dose related, and it produced significant inhibition (20 to 80%) at pharmacological concentrations . Simultaneous addition of prostaglandin E(2) abolished the inhibitory effect of 25 or 50 micro M aspirin . At 1 mM, aspirin reduced the viability of biofilm organisms to 1.9% of that of controls . Surviving cells had a wrinkled appearance, as judged by scanning electron microscopy, and consisted of both yeasts and hyphae . Treatment with other cyclooxygenase inhibitors, such as etodolac, resulted in biofilms that consisted almost entirely of yeast cells . In conventional assays for germ tube formation, these drugs produced significant inhibition, whereas aspirin had little effect . Our findings suggest that cyclooxygenase-dependent synthesis of fungal prostaglandin(s) is important for both biofilm development and morphogenesis in C . albicans and may act as a regulator in these physiological processes . Our results also demonstrate that aspirin possesses potent antibiofilm activity in vitro and could be useful in combined therapy with conventional antifungal agents in the management of some biofilm-associated Candida infections.

Appl Environ Microbiol, 1997 Mar, 63(3), 978 - 82
Poliovirus-1 inactivation and interaction with biofilm: a pilot-scale study; Quignon F et al.; A pilot-scale study was initiated to examine the behavior of viruses pulse injected into a distribution system . The influence of a free-chlorine residual and that of virus preadsorption to clay particles was evaluated by tracing the viruses both in the water flow and after elution from the biofilm . These experiments demonstrated, first, that virus preadsorption on 40 mg of Na-montmorillonite per liter increased the residence time of the viruses within the pilot plant by roughly three times and, second, that preadsorption to clay did not prevent viruses from being inactivated by chlorine . Moreover, with no clay added, a greater amount of viruses was recovered from the biofilm than from the water flow (by a factor of 2 or 10 in the absence or presence of chlorine, respectively), indicating a tendency for virus accumulation within biofilms.

APMIS Suppl, 2003, (116), 1 - 47
Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection . Mechanism of antibiotic resistance and target of the humoral immune response; Ciofu O; The intensive antibiotic treatment of cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa has improved the survival rate and the clinical condition of Danish patients . Acquirement of resistance to anti-pseudomonal antibiotics is one of the main drawbacks of this therapeutic strategy and our results showed the development of resistance of P . aeruginosa to several antibiotics during 25 years of intensive antibiotic treatment . Our studies have been concentrating on the development of resistance to beta-lactam antibiotics . We have shown an association between the development of resistance to beta-lactam antibiotics and the occurrence of high beta-lactamase producing strains and between the MIC of the beta-lactams and the levels of beta-lactamase expression . Partially derepressed mutants, characterized by high basal levels of beta-lactamase with the possibility of induction to even higher levels during treatment with beta-lactam antibiotics, were the most frequent phenotype found among resistant Danish P . aeruginosa CF isolates . We have also shown that the high alginate producing P . aeruginosa isolates, that characterize the chronic lung infection in CF patients, are more susceptible to antibiotics and produce less beta-lactamase than the non-mucoid paired isolates . We propose that the non-mucoid isolates are exposed to a relatively higher antibiotic pressure than the mucoid isolates and therefore, they become easily antibiotic resistant and in consequence produce high levels of beta-lactamase . The beta-lactamase produced by the non-mucoid isolates might play a protective role in the biofilm, defending the mucoid isolates from the action of beta-lactam antibiotics and helping them to maintain their antibiotic susceptibility . We have also shown that beta-lactamase, which is a periplasmic enzyme, can be secreted extracellulary packed in membrane vesicles liberated by high beta-lactamase-producing P . aeruginosa . The continuos presence in the CF lungs of bacteria producing high basal levels of beta-lactamase (partial derepressed) induces a humoral immune response to beta-lactamase . We have shown that antibodies against the chromosomally encoded beta-lactamase (a beta ab) might be considered a marker of the development of resistance to beta-lactam antibiotics . We investigated the humoral immune response to beta-lactamase by quantifying a beta ab specific IgG and IgG subclass antibodies, by investigating the influence of the allotypes on the IgG subclass response and by measuring the avidity of the IgG a beta ab . We found that CF patients with good lung function had in the early stages of the chronic lung infection higher titers of a beta ab of good avidity than patients with poor lung function . Therefore, we raised the hypothesis that some of the a beta ab might have beta-lactamase neutralizing effect, playing a beta-lactamase inhibitor role and improving the effect of the treatment with beta-lactam antibiotics . Finally, we tested our hypothesis in the rat model of chronic lung infection by assessing the effect of a beta ab raised by vaccination with purified chromosomal beta-lactamase on the outcome of the treatment with ceftazidime of bacteria resistant to beta-lactam antibiotics . Our results showed that significantly lower bacterial load and better lung pathology were found in rats with neutralizing antibodies compared to non-immunized rats or rats without neutralizing antibodies . Our findings might be of potential importance for the improvement of the treatment with beta-lactam antibiotics of resistant P . aeruginosa hyperproducing chromosomal beta-lactamase that represent a threat especially for patients with CF and chronic lung infection.

Infect Immun, 2004 Jan, 72(1), 133 - 44
Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity; Head NE et al.; Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF) . In this study, a group of genomically diverse P . aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation . In an in vitro biofilm assay system, P . aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters . Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation . Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model . Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm . While P . aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1 . To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization . Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402) . Together, P . aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF . P . aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.

Infect Immun, 2004 Jan, 72(1), 106 - 13
Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae; Swords WE et al.; Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis . The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community . Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi . Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions . In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo . LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment . Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems . The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system . These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

J Antimicrob Chemother, 2004 Feb, 53(2), 127 - 9 Epub 2003 Dec 19.
Trends in the treatment of orthopaedic prosthetic infections; Bernard L et al.; The most commonly used therapy for prosthetic joint infection is a two-stage prosthetic exchange separated by 6 weeks of intravenous antibiotic therapy . This often results in long periods of hospitalization, morbidity, severe functional impairment and sometimes increased mortality . Therefore novel and challenging therapeutic approaches have been attempted, particularly in hip prosthetic infection . This includes, whenever possible, according to the type of microorganism, antibacterial susceptibility and clinical presentation (including age and comorbidities): (i) less aggressive surgical techniques (debridement and prosthesis retention, or re-implantation with a single-stage exchange arthroplasty); and (ii) antibiotic combinations active against biofilm-associated bacteria, including rifampicin (particularly with quinolones) with excellent bio-availability which allow prolonged and efficient oral therapy.

Clin Microbiol Infect, 2003 Dec, 9(12), 1179 - 86
Epidemiology of methicillin-resistant staphylococci in Europe; Stefani S et al.; Methicillin-resistant staphylococci are mostly resistant not only to all beta-lactams but also to a wide range of other antibiotics, and have emerged as major nosocomial pathogens during the past two decades . Considerable variations in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) exist between institutions and between geographic areas . In Europe, in general, a north-south gradient is observed, MRSA strains being rare in Scandinavian hospitals (<2%) and far more prevalent in Mediterranean hospitals (>40%) . Whether low or high, the rates of MRSA prevalence in European countries have remained approximately the same during the last decade . Recent findings suggest that MRSA might also be emerging as a community-acquired pathogen . The first stage in the emergence of MRSA is its acquisition by methicillin-susceptible S . aureus, and the integration into its chromosome, of the mecA gene, which, together with the other mec genes, is carried on a mobile genetic element, the staphylococcal chromosomal cassette mec (SCCmec) . The origin of SCCmec elements as well as the mechanisms of their acquisition remain unknown . Molecular epidemiology studies using different techniques clearly indicate that the massive geographic spread of MRSA results from the dissemination of relatively few highly epidemic clones . Five major lineages (the so-called Iberian, Brazilian, Hungarian, New York/Japan and pediatric pandemic MRSA clones) have been defined . In Europe, the Iberian clone has been reported in several countries; the Brazilian, pediatric and Hungarian clones have also been detected, but less frequently . A unique Italian clone is predominant in Italy . As with S . aureus, coagulase-negative staphylococci (CNS) represent a serious concern in hospital-acquired infections . Despite marked geographic variations, in some areas of Europe high proportions (60-70%) of CNS are methicillin resistant . The formation of biofilm is a key virulence factor of S . epidermidis, the prominent CNS pathogen, which is the most common cause of bacteremia in device-related infections . Another emerging nosocomial pathogen, S . hemolyticus, is characterized by a tendency to develop multiple antibiotic resistances, with a unique predisposition to glycopeptide resistance.

MMWR Recomm Rep, 2003 Dec 19, 52(RR-17), 1 - 61
Guidelines for infection control in dental health-care settings--2003; Kohn WG et al.; This report consolidates previous recommendations and adds new ones for infection control in dental settings . Recommendations are provided regarding 1) educating and protecting dental health-care personnel; 2) preventing transmission of bloodborne pathogens; 3) hand hygiene; 4) personal protective equipment; 5) contact dermatitis and latex hypersensitivity; 6) sterilization and disinfection of patient-care items; 7) environmental infection control; 8) dental unit waterlines, biofilm, and water quality; and 9) special considerations (e.g., dental handpieces and other devices, radiology, parenteral medications, oral surgical procedures, and dental laboratories) . These recommendations were developed in collaboration with and after review by authorities on infection control from CDC and other public agencies, academia, and private and professional organizations.

Caries Res, 2004, 38 Suppl 1, 9 - 15
Dental plaque as a biofilm: a pilot study of the effects of nutrients on plaque pH and dentin demineralization; Zaura E et al.; Direct or in situ methods (e.g., confocal microscopy, microsensors) are used to study non-oral biofilms for almost two decades, and they have recently been introduced in the research of dental plaque . We combined a pH microsensor technique and transversal microradiography (TMR) in a pilot study where the effects of nutrient availability on plaque acidogenicity and on the development of caries-like lesions were assessed . One volunteer accumulated dental plaque for 7 days in 0.2-mm-wide and 0.8-mm-deep dentin grooves at four conditions: (1) saliva only, (2) 8 x 5 min/day dipping in 10% sucrose solution, (3) 7 x 5 min/day consumption of sweet cookies plus one meal/day, and (4) subject's regular diet . Plaque pH versus time and depth profiles in the grooves were recorded ex vivo before and after sugar challenge . 'Saliva' plaque responded to sugar with slow pH decrease--minimum pH 5.6-5.8 was reached after 30 min, while sugar dipping resulted in metabolically active plaque (minimum pH 5.3-5.5 within 4-8 min) . TMR analysis revealed no demineralization after these two periods . Metabolically active plaque leading to distinct lesions resulted from frequent plaque exposure to diets rich in starch and carbohydrates (groups 3 and 4) . These findings strengthen the view that the plaque acidogenicity does not necessarily reflect cariogenicity, and that retention of food components may account for increased cariogenicity at plaque retention sites in the mouth .

Caries Res, 2004 Jan-Feb, 38(1), 54 - 61
Demineralization of dentin by Streptococcus mutans biofilms grown in the constant depth film fermentor; Deng DM et al.; To develop a bacterial demineralization model, we grew Streptococcus mutans biofilms in a constant depth film fermentor (CDFF) and studied the effects of sucrose pulsing frequency (SPF) in time on dentin demineralization . S . mutans biofilms were grown in dentin specimens with grooves and on dentin surface specimens for 20 days . During the experiments, 2% sucrose was pulsed either 4 or 8 times per day for periods of 30 min . Diluted brain-heart infusion medium containing 25 mM PIPES buffer and 1.5 mM CaCl2 was pulsed as the alternative growth medium . Specimens with intact biofilms were taken out on days 5, 12 and 20 . The model was assessed by viable counts of the biofilm, mineral loss and lesion depth in the dentin specimens (by transversal microradiography) and pH measurements in the groove (by pH microelectrode) . The results showed that biofilms formed on the dentin surface specimens were constant in viable counts for the low SPF, while this parameter tended to increase with time under the high SPF . Lesions with intact surfaces were formed and the lesion size increased significantly over time and increased significantly with increasing SPF . Typical Stephan curves were found after sucrose pulsing . The pH inside the groove returned to neutral under low SPF, but remained below 6.5 under high SPF . With the CDFF S . mutans biofilm model, lesions can be created in dentin within reasonable experimental time periods, as a result of the presence of a biofilm and in response to carbohydrate challenges .

Plant Physiol, 2004 Jan, 134(1), 307 - 19 Epub 2003 Dec 18.
Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production; Bais HP et al.; Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants . Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil . Using this infection model, we demonstrated the biocontrol ability of a wild-type B . subtilis strain 6051 against P . syringae . Arabidopsis root surfaces treated with B . subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B . subtilis biofilm . It is known that formation of biofilms by B . subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent . To determine the role of surfactin in biocontrol by B . subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production . B . subtilis M1 was ineffective as a biocontrol agent against P . syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces . The antibacterial activity of surfactin against P . syringae was determined in both broth and agar cultures and also by live-dead staining methods . Although the minimum inhibitory concentrations determined were relatively high (25 microg mL(-1)), the levels of the lipopeptide in roots colonized by B . subtilis are likely to be sufficient to kill P . syringae . Our results collectively indicate that upon root colonization, B . subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.

J Periodontol, 2003 Nov, 74(11), 1647 - 51
Effects of chlorhexidine, minocycline, and metronidazole on Porphyromonas gingivalis strain 381 in biofilms; Noiri Y et al.; BACKGROUND: Porphyromonas gingivalis is found in subgingival biofilm and is associated with periodontal disease . Bacteria in biofilms are able to resist higher antimicrobial concentrations than in suspension . Little is known about the susceptibility of P . gingivalis in biofilms to antimicrobial agents . The effects of chlorhexidine gluconate, minocycline hydrochloride, and metronidazole on P . gingivalis biofilms were examined in vitro . METHODS: P . gingivalis strain 381 biofilms were prepared on 32 hydroxyapatite disks . At 0, 24, 72, and 144 hours after perfusion of the three antimicrobial agents, two disks from each device were used to assess the antimicrobial effects by adenosine triphosphate (ATP) bioluminescence, and for morphological investigation by scanning electron microscopy (SEM) . RESULTS: Close relationships were found between the results of the ATP analyses and the SEM observations in all groups examined . A significant decrease (P < 0.001) in ATP content was found between the chlorhexidine-treated and control groups . The extracellular matrix structure and P . gingivalis cells were altered in the presence of chlorhexidine . Minocycline hydrochloride also caused a decrease (P < 0.05) in the ATP content and morphological change on P . gingivalis biofilms . Metronidazole showed no significant efficacy against P . gingivalis biofilms . CONCLUSION: Chlorhexidine gluconate was effective at reducing the viability of P . gingivalis 381 cells in biofilms, while minocycline hydrochloride and metronidazole exhibited weaker effects.

J Periodontol, 2003 Nov, 74(11), 1595 - 609
Formation and decontamination of biofilms in dental unit waterlines; Wirthlin MR et al.; BACKGROUND: Biofilms are a natural occurrence in aquatic environments, including community drinking water systems . The interior of small-diameter tubings in dental unit waterlines (DUWL) are also sites of biofilm formation . In the lumen of the tubings, the flow is minimal, and the water becomes stagnant when the units are not in use . Molecules precipitate from the water onto the interior wall and promote the adherence of planktonic microorganisms from the water . Once they become sessile, the microorganisms change their phenotype . After adherence, there is a so-called surface-associated lag time, and the organisms then enter a growth phase and produce exopolysaccharides that coat the organisms in a slime layer . Within the biofilm, the microorganisms can signal one another, transfer nutrients, and exchange genetic material . The insoluble exopolysaccharides shield the microorganisms from displacement and from penetration by predator organisms, antibiotics, and disinfectants . The external surface layer of microorganisms is faster growing and may detach as "swarmer" cells . Detachment of microorganisms from dental unit biofilm flushed into the oral cavity could theoretically infect the patient . Splatter and aerosols from dental procedures may possibly infect health care personnel . METHODS: This study compared three DUWL cleaners (an alkaline peroxide product, a freshly mixed chlorine dioxide product, and a buffer-stabilized chlorine dioxide product) in 16 dental units with self-contained water systems, 6 months after installation in a periodontal teaching clinic . One unit treated by flushing and drying served as a control . Units were sampled daily for 10 days with heterotrophic plate count (HPC) sampler plates . The plates were incubated for 7 days at room temperature, and colonies were counted at 10.5x magnification . Samples of internal water tubing before and after the use of waterline cleaners were processed and examined by scanning electron microscopy . RESULTS: The estimated mean HPC was derived from original and replicate independent counts of two investigators of undiluted and diluted samples, reported as colony forming units (CFU)/ml . Shock treatments with the alkaline peroxide product (n = 5) reduced the HPC from baseline, but in the ratio of daily counts to control, there was a large variance and a trend to return of high counts as days passed . The mean daily HPC was significantly better than the control for only 3 of the 9 days of treatment and exceeded the goal of 200 on 3 days . Freshly mixed chlorine dioxide (n = 4) and the buffer-stabilized chlorine dioxide (n = 5) both reduced HPC to near 0 on all days . Their ratios of daily estimated means to that of the control were significantly (P < 0.001) better at all times . In comparing treatments, the freshly mixed chlorine dioxide was better (P < 0.001) than the alkaline peroxide on 8 of 9 days . The buffered chlorine dioxide treatment was better than the alkaline peroxide at all times . The two chlorine dioxide treatments each had so many HPC counts of 0 that a meaningful statistical difference between them was not calculated . Scanning electron microscopy of plastic waterline tubing samples taken before and after treatments showed reductions in biofilm coverage, but the differences were not statistically significant . CONCLUSIONS: Chlorine dioxide waterline cleaners are effective in decontaminating DUWL biofilm . Chlorine dioxide has advantages over other chlorine products . Controlling DUWL biofilm may have beneficial effects on nosocomial infections.

Water Sci Technol, 2003, 48(8), 79 - 87
Microbial activity of biofilm during start-up period of anaerobic hybrid reactor at low and high upflow feeding velocity; Suraruksa B et al.; With an aim to shorten start-up time of an Anaerobic Hybrid Reactor (AHR), initial biofilm development was studied, particularly at different upflow feeding velocities . At a low (0.01 m x h(-1)) upflow velocity, initial biofilm was found to develop via the attachment of suspended biomass in the packed zone, while microbial growth on the film was insignificant . Contrarily, with higher (1.0 m x h(-1)) upflow velocity, initial biofilm development was from both microbial attachment and growth on supporting media . Biofilm thickness was determined using confocal laser scanning microscopy (CLSM), which indicated that the biofilm developed faster with the higher velocity, due to the contribution of the microbial growth on supporting media . When operated beyond the initial biofilm development with the lower velocity, both the activity of acetogens and the methanogens increased, although there was a lower amount of attached biomass on the supporting media . Whereas, both groups were found to decrease with higher upflow velocity, but acidogenic activity increased . It can be concluded that higher upflow velocity positively affected the initial stage of biofilm development and has the potential to accelerate attached biomass on supporting media during the initial phase . Subsequently, the upflow velocity should be reduced to the normal rate to enhance the methanogenic activity.

Microb Ecol, 2004 May, 47(4), 316 - 28
Biofilm structure and function and possible implications for riverine DOC dynamics; Romani AM et al.; Biofilms are major sites of carbon cycling in streams and rivers . Here we elucidate the relationship between biofilm structure and function and river DOC dynamics . Metabolism (extracellular enzymatic activity) and structure (algae, bacteria, C/N content) of light-grown (in an open channel) and dark-grown (in a dark pipe) biofilms were studied over a year, and variations in dissolved organic carbon (DOC) and biodegradable DOC (BDOC) were also recorded . A laboratory experiment on 14C-glucose uptake and DOC dynamics was also performed by incubating natural biofilms in microcosms . On the basis of our field (annual DOC budget) and laboratory results, we conclude that light-grown biofilm is, on annual average, a net DOC consumer . This biofilm showed a high monthly variability in DOC uptake/release rates, but, on average, the annual uptake rate was greater than that of the dark-grown biofilm . The higher algal biomass and greater structure of the light-grown biofilm may enhance the development of the bacterial community (bacterial biomass and activity) and microbial heterotrophic activity . In addition, the light-grown biofilm may promote abiotic adsorption because of the development of a polysaccharide matrix . In contrast, the dark-grown biofilm is highly dependent on the amount and quality of organic matter that enters the system and is more efficient in the uptake of labile molecules (higher 14C-glucose uptake rate per mgC) . The positive relationships between the extracellular enzymatic activity of biofilm and DOC and BDOC content in flowing water indicate that biofilm metabolism contributes to DOC dynamics in fluvial systems . Our results show that short-term fluvial DOC dynamics is mainly due to the use and recycling of the more labile molecules . At the river ecosystem level, the potential surface area for biofilm formation and the quantity and quality of available organic carbon might determine the effects of biofilm function on DOC dynamics.

J Bacteriol, 2004 Jan, 186(1), 226 - 34
Inactivation of ompX causes increased interactions of type 1 fimbriated Escherichia coli with abiotic surfaces; Otto K et al.; During the initial steps of biofilm formation, bacteria have to adapt to a major change in their environment . The adhesion-induced phenotypic changes in a type 1 fimbriated Escherichia coli strain included reductions in the levels of several outer membrane proteins, one of which was identified as OmpX . Here, the phenotypes of mutant strains that differ at the ompX locus were studied with regard to adhesion, cell surface properties, and resistance to stress and antimicrobial compounds . The kinetics of adhesion were measured online by an extended quartz crystal microbalance technique for wild-type and mutant strains with a fimbriated or nonfimbriated background . Deletion of ompX led to significantly increased cell-surface contact in fimbriated strains but to decreased cell-surface contact in a nonfimbriated strain . Phenotypic characterization of the ompX mutant demonstrated that ompX interferes with proper regulation of cell surface structures that play a key role in mediating firm contact of the cell with a surface (i.e., type 1 fimbriae, flagellae, and exopolysaccharides) . These phenotypic changes were accompanied by increased tolerance to several antibiotic compounds and sodium dodecyl sulfate . Based on these results, we propose that changes in the composition of outer membrane proteins during fimbria-mediated adhesion may be part of a coordinated adaptive response to the attached mode of growth.

Oral Microbiol Immunol, 2004 Feb, 19(1), 61 - 4
A comparison of the antibacterial efficacies of essential oils against oral pathogens; Takarada K et al.; Cariogenic bacteria and periodontopathic bacteria are present in dental plaque as biofilms . In this study, we investigated the antibacterial effects of essential oils on the following oral bacteria: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sobrinus . We tested manuka oil, tea tree oil, eucalyptus oil, lavandula oil, and romarinus oil and determined their minimum inhibitory concentration and minimum bactericidal concentration . The essential oils inhibited the growth of the bacteria tested, manuka oil being the most effective . Minimum bactericidal concentration values showed that lavandula oil acts bacteriostatically, and the remaining oils, bactericidally . Periodontopathic bacterial strains tested were killed completely by exposure for 30 s to 0.2% manuka oil, tea tree oil or eucalyptus oil . Tea tree oil and manuka oil showed significant adhesion-inhibiting activity against P . gingivalis . All the essential oils tested inhibited the adhesion of S . mutans . This study showed that, among the essential oils tested, manuka oil and tea tree oil in particular had strong antibacterial activity against periodontopathic and cariogenic bacteria . From the viewpoint of safety, we also examined the effects of these essential oils on cultured human umbilical vein endothelial cells and found that, at a concentration of 0.2%, they had little effect on cultured cells.

Oral Microbiol Immunol, 2004 Feb, 19(1), 31 - 8
Physiologic actions of zinc related to inhibition of acid and alkali production by oral streptococci in suspensions and biofilms; Phan TN et al.; Zinc is a known inhibitor of acid production by mutans streptococci . Our primary objective was to extend current knowledge of the physiologic bases for this inhibition and also for zinc inhibition of alkali production by Streptococcus rattus FA-1 and Streptococcus salivarius ATCC 13419 . Zinc at concentrations as low as 0.01-0.1 mm not only inhibited acid production by cells of Streptococcus mutans GS-5 in suspensions or in biofilms but also sensitized glycolysis by intact cells to acidification . Zinc reversibly inhibited the F-ATPase of permeabilized cells of S . mutans with a 50% inhibitory concentration of about 1 mm for cells in suspensions . Zinc reversibly inhibited the phosphoenolpyruvate: sugar phosphotransferase system with 50% inhibition at about 0.3 mm ZnSO4, or about half that concentration when the zinc-citrate chelate was used . The reversibility of these inhibitory actions of zinc correlates with findings that it is mainly bacteriostatic rather than bactericidal . Zinc inhibited alkali production from arginine or urea and was a potent enzyme inhibitor for arginine deiminase of S . rattus FA-1 and for urease of S . salivarius . In addition, zinc citrate at high levels of 10-20 mm was weakly bactericidal.

J Appl Microbiol, 2004, 96(1), 177 - 84
Biofilm formation and cell-to-cell signalling in Gram-negative bacteria isolated from a food processing environment; Van Houdt R et al.; AIMS: To investigate the biofilm-forming capacity and the production of quorum signals in Gram-negative bacteria isolated from a food production environment, and the possible correlation between both phenotypes . METHODS AND RESULTS: Sixty-eight Gram-negative bacteria were isolated from equipment and working surfaces in a raw vegetable processing line, and tested for biofilm-forming capacity using an in vitro microplate assay . All isolates showed significantly higher biofilm-forming capacity than Escherichia coli laboratory strain DH5alpha, which was included as a negative control, and differed up to 56-fold in relative biofilm-forming capacity . Various assays based on reporter bacteria were used to detect quorum signals produced by the isolates . Twenty-six isolates produced autoinducer-2, five isolates produced N-acyl-homoserine lactones (AHLs), and none produced the Pseudomonas quinolone signal . CONCLUSIONS: No correlation was found between in vitro biofilm-forming capacity and production of quorum signalling molecules among the 68 strains isolated from the raw vegetable processing line . SIGNIFICANCE AND IMPACT OF THE STUDY: Several recent studies have shown a role of AHL-based quorum sensing in biofilm formation of specific Gram-negative bacterial strains . The current work shows that production of AHL and other quorum signals is not widespread in Gram-negative isolates from a raw vegetable processing line, and is not a general requirement for biofilm formation, at least in vitro.

Ann Agric Environ Med, 2003, 10(2), 151 - 7
Biofilm and dental unit waterlines; Szymanska J; Aquatic biofilms, which are well-organized communities of microorganisms, are widespread in nature . They constitute a major problem in many environmental, industrial and medical settings . The use of advanced techniques has revealed biofilm structure, formation and ecology . Special attention was given to the build-up of biofilm in dental unit waterlines (DUWLs), which are small-bore flexible plastic tubing to bring water to different handpieces . They are coated with well-established biofilms . Active biofilm is a source of microbial contamination of DUWLs water . The safety of dental treatment requires a good quality of the water used . The knowledge of nature, formation and the ways to eliminate the biofilm is the first step towards reducing health risk, both for patients and dental personnel . The article reviews these issues.

Appl Microbiol Biotechnol, 2004 Mar, 64(1), 1 - 6 Epub 2003 Dec 12.
Knowledge of the Bacillus subtilis genome: impacts on fundamental science and biotechnology; Tosato V et al.; The advent of genomics has greatly influenced fundamental and applied microbiology . This has become paradigmatic in the case of Bacillus subtilis, a primary model bacterium for research and biotechnology . Indeed, mining its genome has provided more fruitful information than classical approaches would have yielded in a longer period of time . Through advanced analysis of its genome and transcriptome, fundamental discoveries dealing with the informational architecture of the B . subtilis chromosome, as well as with the elucidation of its pathway-level regulation of gene expression, have been achieved . The possibility of performing a complete metabolic manipulation of the secretory pathway of Bacillus is promising important biotechnological fallouts . Similar emphasis exists for the possibility of controlling the cell in the formation of biofilms with specific physical and chemical characteristics . At the theoretical level, the new concept of genetic superinformation has been formulated and its analytical approach implemented, while the understanding of the minimal genetic requirements for the existence of a reproducing bacterial cell is being tackled . In summary, the impact of the B . subtilis genome has philosophically revolutionised the way that basic knowledge is translated into applied microbiology and biotechnology, making this bacterium the workhorse of post-genomic microbiology.

Skinmed, 2003 Jul-Aug, 2(4), 222 - 8
Assessment of etiologic agents in acne pathogenesis; Burkhart CN et al.; Acne is a chronic inflammatory disease of the pilosebaceous units . Traditional etiologic factors include increased sebum production, ductal hyperkeratosis, abnormality of the microbial flora within the pilosebaceous unit, and mediators of inflammation . Recent developments do not refute these familial elements, but rather refine particular aspects . Interleukin-1a influences hypercornification of the infundibulum as well as the inflammatory response by inducing the production of vascular endothelial growth factor in dermal papilla cells and follicular keratinocytes of the pilosebaceous unit . New retinoids have been developed based on controlling cellular proliferation and differentiation in the pilosebaceous unit by their action on nuclear receptors of cells . Dermal inflammation is not due to presence of bacteria, but from biologically active mediators produced by Propionibacterium acnes . The environment within the pilosebaceous unit is probably more important than the absolute number of P . acnes organisms . Indeed, the major role of the sebaceous gland appears to be supplying P . acnes needed nutrients . Moreover, the microbiologic principle of biofilms appears to be applicable to P . acnes in acne.

J Food Prot, 2003 Dec, 66(12), 2258 - 66
Influence of extended acid stressing in fresh beef decontamination runoff fluids on sanitizer resistance of acid-adapted Escherichia coli O157:H7 in biofilms; Stopforth JD et al.; This study evaluated resistance to sanitizing solutions of Escherichia coli O157:H7 cells forming biofilms on stainless steel coupons exposed to inoculated meat decontamination runoff fluids (washings) . A previously acid-adapted culture of a rifampicin-resistant derivative of E . coli O157:H7 strain ATCC 43895 was inoculated in unsterilized or sterilized combined hot-water (85 degrees C) and cold-water (10 degrees C) (50/50 {vol/vol}) composite water (W) washings (pH 6.29 to 6.47) and in W washings mixed with 2% acetic acid (pH 4.60 to 4.71) or in 2% lactic acid W washings (pH 4.33 to 4.48) at a ratio of 1/99 (vol/vol) . Stainless steel coupons (2 by 5 by 0.08 cm) were submerged in the inoculated washings and stored for up to 14 days at 15 degrees C . Survival of E . coli O157:H7 was determined after exposure (0 to 60 s for cells in suspension and 0 to 300 s for attached cells) to two commercial sanitizers (150 ppm peroxyacetic acid and 200 ppm quaternary ammonium compound) at 2, 7, and 14 days . E . coli O157:H7 attached more rapidly to coupons submerged in washings containing the natural flora than to those without . The attached cells were more resistant to the effects of the sanitizers than were the cells in suspension, and survival was highest in the presence of the natural flora . Attached cells in the presence of dilute acid washings were more sensitive to subsequent sanitizer treatments than were cells generated in the presence of W washings . Under the conditions of this study, cells of E . coli O157:H7 in W washings were more sensitive to acidic (peroxyacetic acid) than to alkaline (quaternary ammonium) sanitizers during storage . These results suggest that meat processing plants that apply no decontamination or that use only water washings of meat should consider using acidic sanitizers to enhance biofilm removal . Plants that apply both water and acidic washings may create a sublethal acid-stressing environment in the runoff fluids, sensitizing biofilm cells to subsequent sanitizing treatments.

Biodegradation, 2003 Dec, 14(6), 437 - 52
Advances in biotreatment of acid mine drainage and biorecovery of metals: 2 . Membrane bioreactor system for sulfate reduction; Tabak HH et al.; Several biotreatmemt techniques for sulfate conversion by the sulfate reducing bacteria (SRB) have been proposed in the past, however few of them have been practically applied to treat sulfate containing acid mine drainage (AMD) . This research deals with development of an innovative polypropylene hollow fiber membrane bioreactor system for the treatment of acid mine water from the Berkeley Pit, Butte, MT, using hydrogen consuming SRB biofilms . The advantages of using the membrane bioreactor over the conventional tall liquid phase sparged gas bioreactor systems are: large microporous membrane surface to the liquid phase; formation of hydrogen sulfide outside the membrane, preventing the mixing with the pressurized hydrogen gas inside the membrane; no requirement of gas recycle compressor; membrane surface is suitable for immobilization of active SRB, resulting in the formation of biofilms, thus preventing washout problems associated with suspended culture reactors; and lower operating costs in membrane bioreactors, eliminating gas recompression and gas recycle costs . Information is provided on sulfate reduction rate studies and on biokinetic tests with suspended SRB in anaerobic digester sludge and sediment master culture reactors and with SRB biofilms in bench-scale SRB membrane bioreactors . Biokinetic parameters have been determined using biokinetic models for the master culture and membrane bioreactor systems . Data are presented on the effect of acid mine water sulfate loading at 25, 50, 75 and 100 ml/min in scale-up SRB membrane units, under varied temperatures (25, 35 and 40 degrees C) to determine and optimize sulfate conversions for an effective AMD biotreatment . Pilot-scale studies have generated data on the effect of flow rates of acid mine water (MGD) and varied inlet sulfate concentrations in the influents on the resultant outlet sulfate concentration in the effluents and on the number of SRB membrane modules needed for the desired sulfate conversion in those systems . The pilot-scale data indicate that the SRB membrane bioreactors systems can be applied toward field-scale biotreatment of AMD and for recovery of high purity metals and an agriculturally usable water.

Arch Inst Pasteur Tunis, 1998 Jul-Oct, 75(3-4), 205 - 9
Bacterial composition of the biofilm on the surface of course sediment of the Danube: with special reference to the clinically important bacteria; Zalmum AA et al.; On monthly intervals and over a period of 14 months (November, 1993-December, 1994) biofilm samples from sediments taken at the Szentendre Island on the Danube were culturel and the isolated organisms were examined macromorphologically and micromorphogically and tested for oxidase and catalase production and their ability to oxidise and ferment glucose . The majority (85%) of the strains isolated were catalase positive . 43% were oxidase positive, 38% were glucose oxidisers and only 19% fermented glucose . In the sediment upmost biofilm layers Gram-negatives dominate (10 samples out of 14) . A representative set strains has been subjected to complete identification using the Biolog automated identification system . The coryneform bacteria (e.g . Arthrobacter spp.) and members of the genus Rhodococcus dominated the Gram-positive bacteria while the dominant Gram-negative bacteria were facultative H2 autotrophs, methylotrophs (e.g . Xanthobacter flavus, Hydrogenophaga spp.) and different Aeromonas, Enterobacter and Pseudomonas species . Because the water supply of Budapest (the capital of Hungary) comes from bankwall filtred Danube water resources, the presence of clinically important microorganisms (i.e . Aeromonas, Enterobacter, Pseudomonas and Rhodococcus spp) in the Danube-sediments may pose a public health problem to the users (in particular the immunocompromised) of such water supplies.

Eukaryot Cell, 2003 Dec, 2(6), 1266 - 73
EAP1, a Candida albicans gene involved in binding human epithelial cells; Li F et al.; Candida albicans adhesion to host tissues contributes to its virulence and adhesion to medical devices permits biofilm formation, but we know relatively little about the molecular mechanisms governing C . albicans adhesion to materials or mammalian cells . Saccharomyces cerevisiae provides an attractive model system for studying adhesion in yeast because of its well-characterized genetics and gene expression systems and the conservation of signal transduction pathways among the yeasts . In this study, we used a parallel plate flow chamber to screen and characterize attachment of a flo8Delta S . cerevisiae strain expressing a C . albicans genomic library to a polystyrene surface . The gene EAP1 was isolated as a putative cell wall adhesin . Sequence analysis of EAP1 shows that it contains a signal peptide, a glycosylphosphatidylinositol anchor site, and possesses homology to many other yeast genes encoding cell wall proteins . In addition to increasing adhesion to polystyrene, heterologous expression of EAP1 in S . cerevisiae and autonomous expression of EAP1 in a C . albicans efg1 homozygous null mutant significantly enhanced attachment to HEK293 kidney epithelial cells . EAP1 expression also restored invasive growth to haploid flo8Delta and flo11Delta strains as well as filamentous growth to diploid flo8/flo8 and flo11/flo11 strains . Transcription of EAP1 in C . albicans is regulated by the transcription factor Efg1p, suggesting that EAP1 expression is activated by the cyclic AMP-dependent protein kinase pathway.

Microbiology, 2003 Dec, 149(Pt 12), 3473 - 84
Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system; Tomaras AP et al.; Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices . In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated . This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid-air interface of cultures incubated stagnantly . The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation . Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain . Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus . Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria . Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative . These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A . baumannii cells.

Curr Opin Microbiol, 2003 Dec, 6(6), 578 - 85
Bacterial biofilms: prokaryotic adventures in multicellularity; Webb JS et al.; The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms . Recent advances in the field provide new insight into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity.

J Biomater Sci Polym Ed, 2003, 14(10), 1005 - 28
Surface engineering of biomaterials with plasma techniques; Poncin-Epaillard F et al.; In this study, 3 types of plasma techniques, i.e . plasma modification, plasma deposition and plasma followed by grafting reaction, are used for the fabrication of tools, medical devices and biomaterials . Depending on purpose, bioadhesion of cells and biomolecules is either looked for or avoided . Since the mechanisms of bioadhesion depend on the characteristics of the surface (hydrophilic or hydrophobic), modifying the surface by a treatment will alter the bioadhesion . These treatments are developed for the anti-fouling process, the sterilisation and the improvement of the formation of biofilms . They have also proved useful for the synthesis of biomimicking devices.

J Clin Invest, 2003 Dec, 112(11), 1626 - 32
Quorum sensing and biofilm formation in Streptococcal infections; Cvitkovitch DG et al.; Members of the bacterial genus Streptococcus are responsible for causing a wide variety of infections in humans . Many Streptococci use quorum-sensing systems to regulate several physiological properties, including the ability to incorporate foreign DNA, tolerate acid, form biofilms, and become virulent . These quorum-sensing systems are primarily made of small soluble signal peptides that are detected by neighboring cells via a histidine kinase/response regulator pair.

J Clin Invest, 2003 Dec, 112(11), 1620 - 5
Quorum sensing in Staphylococcus infections; Yarwood JM et al.; Quorum sensing via the accessory gene regulator (agr) system has been assigned a central role in the pathogenesis of staphylococci, particularly Staphylococcus aureus . While the control of virulence gene expression in vitro by agr has been relatively straightforward to describe, regulation of both the quorum response itself and virulence genes in vivo is considerably more complex . The quorum response is highly dependent upon the environment in which the organism is grown and is strongly influenced by additional regulators that respond to signals other than cell density . There is increasing evidence that the agr phenotype may influence the behavior and pathogenesis of biofilm-associated S . aureus and S . epidermidis and may contribute to the chronic nature of some biofilm-associated infections.

Appl Environ Microbiol, 2003 Dec, 69(12), 7336 - 42
Variation in biofilm formation among strains of Listeria monocytogenes; Borucki MK et al.; Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces . Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products . Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L . monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L . monocytogenes . Eighty L . monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment . Statistically significant differences were detected between phylogenetic divisions . Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks . Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains . There were no significant differences detected among serotypes . Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains . Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm . These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.

Appl Environ Microbiol, 2003 Dec, 69(12), 7298 - 309
Metagenome survey of biofilms in drinking-water networks; Schmeisser C et al.; Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities . Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies . First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group . Our second strategy included a snapshot genome sequencing approach . Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups . Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig . The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community . Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.

Appl Environ Microbiol, 2003 Dec, 69(12), 7063 - 72
Linked redox precipitation of sulfur and selenium under anaerobic conditions by sulfate-reducing bacterial biofilms; Hockin SL et al.; A biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated from a naturally occurring mixed biofilm and identified by 16S rDNA analysis as a strain of Desulfomicrobium norvegicum, rapidly removed 200 micro M selenite from solution during growth on lactate and sulfate . Elemental selenium and elemental sulfur were precipitated outside SRB cells . Precipitation occurred by an abiotic reaction with bacterially generated sulfide . This appears to be a generalized ability among SRB, arising from dissimilatory sulfide biogenesis, and can take place under low redox conditions and in the dark . The reaction represents a new means for the deposition of elemental sulfur by SRB under such conditions . A combination of transmission electron microscopy, environmental scanning electron microscopy, and cryostage field emission scanning electron microscopy were used to reveal the hydrated nature of SRB biofilms and to investigate the location of deposited sulfur-selenium in relation to biofilm elements . When pregrown SRB biofilms were exposed to a selenite-containing medium, nanometer-sized selenium-sulfur granules were precipitated within the biofilm matrix . Selenite was therefore shown to pass through the biofilm matrix before reacting with bacterially generated sulfide . This constitutes an efficient method for the removal of toxic concentrations of selenite from solution . Implications for environmental cycling and the fate of sulfur and selenium are discussed, and a general model for the potential action of SRB in selenium transformations is presented.

Anal Bioanal Chem, 2004 Feb, 378(4), 1095 - 101 Epub 2003 Dec 05.
Biofilms as bio-indicator for polluted waters? Total reflection X-ray fluorescence analysis of biofilms of the Tisza river (Hungary); Mages M et al.; The aim of this work was to investigate the heavy metal accumulation by natural biofilms living in the catchment area of the Tisza river in Hungary, as well as in biofilms cultivated in vitro . Laboratory tests have demonstrated that metals can be adsorbed on biofilms, depending on their concentration and on the availability of free sorptive places . Biofilms were cultivated in vitro in natural freshwater from the Saale river, Germany . After reaching the plateau phase, Cu was added to reach a concentration of 100 micro g/L . An increase of its mass fraction in the biofilm was observed, which caused the decrease of the concentration in the water phase . Unfortunately, the reactor wall was also found to act as adsorbent for Cu . More detailed results of our in vitro experiments will be published in a forthcoming paper . Naturally grown biofilm samples from exposed as well as background places at the Hungarian rivers Szamos and Tisza were collected in 2000 and 2002 after the cyanide spill, and analysed using total reflection X-ray fluorescence analysis (TXRF) . Metal mass fraction differences as high as two orders of magnitude were found between polluted and unpolluted (background) sampling points . Extremely high concentration values, e.g . 5600 microg/g Zn in biofilm, were found at highly polluted sampling points . This means an enrichment factor of ca . 10,000 compared to the water phase.

J Orthop Res, 2004 Jan, 22(1), 39 - 43
Adhesion of Staphylococcus to orthopaedic metals, an in vivo study; Sheehan E et al.; This study describes a new model of biofilm study in rabbits . The primary focus of this study was to assess biofilm adhesion to orthopaedic metals in their first 48 h in a femoral intramedullary implantation model . Two previous inoculation methods i.e . that of pre- and direct inoculation were studied with two bacterial isolates namely Staphylococcus aureus and epidermidis, on titanium and stainless steel metallic implants . A method of sonication and log dilution/plating was used to assess biofilm bacteria adhering to implants . Silver coated metals were then compared with their respective control metals in the new model . The direct inoculation model gave larger and more reproducible biofilm adhesion to implanted metals . Staphylococcus epidermidis shows lower adhesion ability to metals, and biofilms adhere in greater numbers to stainless steel over titanium . Silver coated metals show no statistical difference over control metals when exposed to orthopaedic biofilms.

Lancet Infect Dis, 2003 Dec, 3(12), 794 - 803
Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental situations; Russell AD; Antibiotics are used as chemotherapeutic drugs, and biocides are used as antiseptics, disinfectants, and preservatives . Several factors affect biocidal activity, notably concentration, period of contact, pH, temperature, the presence of interfering material, and the types, numbers, location, and condition of microorganisms . Bacterial cells as part of natural or artificial (laboratory) biofilm communities are much less susceptible than planktonic cells to antibiotics and biocides . Assessment of biocidal activity by bactericidal testing is more relevant than by determination of minimum inhibitory concentrations . Biocides and antibiotics may show some similarities in their mechanisms of action and common mechanisms of bacterial insusceptibility may apply, but there are also major differences . In the laboratory, bacteria can become less susceptible to some biocides . Decreased resistance may be stable or unstable and may be accompanied by a low-level increase in antibiotic resistance . Laboratory studies are useful for examining stress responses and basic mechanisms of action and of bacterial insusceptibility to antibacterial agents . Translation of such findings to the clinical and environmental situations to provide evidence of a possible relation between biocide use and clinical antibiotic resistance is difficult and should be viewed with caution.

Mol Microbiol, 2003 Dec, 50(5), 1665 - 82
The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface; Ferrieres L et al.; Bacteria are often found associated with surfaces as sessile bacterial communities called biofilms, and the formation of a biofilm can be split up into different stages each requiring the expression of specific genes . The production of extracellular polysaccharides (EPS) is important for the maturation of biofilms and is controlled by the Rcs two-component pathway in Escherichia coli (and other Gram-negative bacteria) . In this study, we show, for the first time, that the RcsC sensor kinase is required for normal biofilm development in E . coli . Moreover, using a combination of DNA macroarray technology and transcriptional fusion analysis, we show that the expression of > 150 genes is controlled by RcsC in E . coli . In silico analyses of the RcsC regulon predicts that 50% of the genes encode proteins that are either localized to the envelope of E . coli or have activities that affect the structure/properties of the bacterial surface, e.g . the production of colanic acid . Moreover, we also show that RcsC is activated during growth on a solid surface . Therefore, we suggest that the RcsC sensor kinase may play an important role in the remodelling of the bacterial surface during growth on a solid surface and biofilm formation.

Mol Microbiol, 2004 Jan, 51(1), 283 - 96
Structure-function analysis of the self-recognizing Antigen 43 autotransporter protein from Escherichia coli; Klemm P et al.; Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains . Expression of Ag43 confers aggregation and fluffing of cells, promotes biofilm formation and is associated with enhanced resistance to antimicrobial agents . Ag43 is an autotransporter protein and consists of two moieties: a transporter, the beta-module, and a passenger domain, the alpha-module . Here we have employed various molecular approaches to probe structure/function aspects of Ag43 . An entire family of Ag43 variants was identified . The gene encoding Ag43 (flu) was cloned from a diverse range of E . coli subtypes and found to encode variant proteins with different properties . Several novel variants were identified and characterized that were unable to promote cell-cell aggregation . By employing a combination of linker insertion mutagenesis and domain swapping between clumping and non-clumping variants, we have pinpointed the region of the protein responsible for autoaggregation to be located within the N-terminal one-third of the passenger domain . Our data suggest that ionic interactions between charged residues residing in interacting pairs of Ag43alpha domains may be important for the self-recognition process . Based on its similarity to other related proteins, we predict the passenger, Ag43alpha, domain primarily to consist of an extended beta-helix structure in which numerous repeats or rungs are stacked in parallel orientation in an extended cylindrical formation . Finally, we found that in spite of their different aggregative pattern all Ag43 variants promoted biofilm formation to abiotic surfaces.

Mol Microbiol, 2004 Jan, 51(1), 97 - 113
Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms; Kuiper I et al.; Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons . PCL1445 produces biosurfactant activity at the end of the exponential growth phase . High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses . A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases . Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9 . The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II . We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC) . Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms . Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2003 Oct, 21(5), 389 - 91
{Evaluation of in vitro cario-static effect of Galla chinensis with biofilm model}; Li J et al.; OBJECTIVE: To evaluate the in vitro cario-static effect of Galla chinesis with biofilm model . METHODS: A four-organism bacterial consortium was cultured in a biofilm model on hydroxyapatite (HA) discs in a continuous culture system and exposed to repeated solution pulsing . There were three groups with different solution pulsed in the model: negative control group was pulsed with distilled water, positive control group was pulsed with 100 mmol/L sucrose solution and experimental group was pulsed with 100 mmol/L sucrose solution containing 4.0 g/L Galla chinensis . During the experiment, the dynamic changes of pH were recorded . After 6 pulses, surface structure of the biofilm was observed with a scanning electron microscope and the population on the biofilm was enumerated . RESULTS: Galla chinesis significantly inhibited the adherence of Actinomyces naelundii to HA disc compared with the control group and facilitated the removal of acid products . It was also found that the extra-cellular polysaccharide was reduced with the pulsing of Galla chinesis . CONCLUSION: Galla chinesis in the biofilm model can partially reduce the cario-genic response of sucrose solution.

Nature, 2003 Nov 27, 426(6965), 439 - 42
Contributions of microbial biofilms to ecosystem processes in stream mesocosms; Battin TJ et al.; In many aquatic ecosystems, most microbes live in matrix-enclosed biofilms and contribute substantially to energy flow and nutrient cycling . Little is known, however, about the coupling of structure and dynamics of these biofilms to ecosystem function . Here we show that microbial biofilms changed the physical and chemical microhabitat and contributed to ecosystem processes in 30-m-long stream mesocosms . Biofilm growth increased hydrodynamic transient storage-streamwater detained in quiescent zones, which is a major physical template for ecological processes in streams-by 300% and the retention of suspended particles by 120% . In addition, by enhancing the relative uptake of organic molecules of lower bioavailability, the interplay of biofilm microarchitecture and mass transfer changed their downstream linkage . As living zones of transient storage, biofilms bring hydrodynamic retention and biochemical processing into close spatial proximity and influence biogeochemical processes and patterns in streams . Thus, biofilms are highly efficient and successful ecological communities that may also contribute to the influence that headwater streams have on rivers, estuaries and even oceans through longitudinal linkages of local biogeochemical and hydrodynamic processes.

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