Res Microbiol, 2003 Dec, 154(10), 659 - 67 The csgD promoter, a control unit for biofilm formation in Salmonella typhimurium; Gerstel U et al.; Expression of cellulose and curli fimbriae in Salmonella typhimurium is dependent on the transcriptional regulator CsgD . Transcription of csgD itself is influenced by a variety of regulatory stimuli . Complex nucleoprotein arrangements modulate the transcriptional activity of csgD and trigger the transition between the planktonic status and biofilm formation. J Trace Elem Med Biol, 2003, 17 Suppl 1, 37 - 43 Relationship between mineral content of domestic hot water and microbial contamination; Borella P et al.; The relationship between Legionella and Pseudomonas spp contamination and mineral content of domestic hot waters was investigated in a cross-sectional Italian survey . Pseudomonas spp contamination was associated with elevated Ca, but lower Fe and free chlorine content in the water . Waters with Cu concentrations > 50 microg/L prevented Legionella spp colonisation (OR = 0.14, 95% CI = 0.02-1.13), whereas the risk of legionellae occurrence increased in waters with Mn levels > 3 microg/L (OR = 2.37, 95% CI = 1.06-5.30) . Furthermore, Mn was positively associated with the risk of colonisation by eterotrophic bacteria growing at either 22 and 36 degrees C . Legionella species and serogroups were differently distributed according to Cu and Mn concentrations, suggesting that Legionella strains may have a different sensibility/resistance to trace elements . A specific action of Cu as decontamination factor is suggested and the consideration of Mn as a risk indicator for bacterial colonisation and biofilm presence is proposed. Biofouling, 2003 Oct, 19(5), 307 - 13 Bacterial biofilm in seawater: cell surface properties of early-attached marine bacteria; Grasland B et al.; The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria . The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized . Although these bacteria adhered to a hydrophilic support immersed for 3 and 6 h, they presented various physico-chemical properties . Eleven strains possessed a hydrophilic surface and five a hydrophobic surface . Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support . On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater . Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored . The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface. Biofouling, 2003 Oct, 19(5), 287 - 95 Effect of different concentrations of ortho-phthalaldehyde on biofilms formed by Pseudomonas fluorescens under different flow conditions; Simoes M et al.; The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity . The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities . The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide . The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application . Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions . However, OPA did not promote the detachment of biofilms from the surface . The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix . The biocide was more effective on suspended cells than on cells grown in biofilms . This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA. Biofouling, 2003 Oct, 19(5), 279 - 85 Viscoelastic properties of a mixed culture biofilm from rheometer creep analysis; Towler BW et al.; The mechanical properties of mixed culture biofilms were determined by creep analysis using an AR1000 rotating disk rheometer . The biofilms were grown directly on the rheometer disks which were rotated in a chemostat for 12 d . The resulting biofilms were heterogeneous and ranged from 35 microns to 50 microns in thickness . The creep curves were all viscoelastic in nature . The close agreement between stress and strain ratio of a sample tested at 0.1 and 0.5 Pa suggested that the biofilms were tested in the linear viscoelastic range and supported the use of linear viscoelastic theory in the development of a constitutive law . The experimental data was fit to a 4-element Burger spring and dashpot model . The shear modulus (G) ranged from 0.2 to 24 Pa and the viscous coefficient (eta) from 10 to 3000 Pa . These values were in the same range as those previously estimated from fluid shear deformation of biofilms in flow cells . A viscoelastic biofilm model will help to predict shear related biofilm phenomena such as elevated pressure drop, detachment, and the flow of biofilms over solid surfaces. Environ Microbiol, 2003 Dec, 5(12), 1350 - 69 Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics; Arevalo-Ferro C et al.; The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis . The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development . In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE) . This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains . We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs . These protein spots were characterized by MALDI-TOF peptide mapping . Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors . For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated . Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound . Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system . Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P . aeruginosa . In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type . These results add haemoglobin utilization to the list of phenotypes controlled through QS in P . aeruginosa . The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms . To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem. Expert Opin Biol Ther, 2003 Dec, 3(8), 1201 - 7 Bacterial biofilms of importance to medicine and bioterrorism: proteomic techniques to identify novel vaccine components and drug targets; Hassett DJ et al.; Biofilms are highly ordered microbial communities enmeshed in a carefully sculpted matrix designed for survival of organisms either in multi- or mono-genus/species in a specific microniche . In human disease, biofilm infections are some of the most recalcitrant to treat . Even with rigorous antibiotic regimens, some biofilms, such as those within the thick airway mucus of cystic fibrosis (CF) patients, persist throughout the course of the disease process . In this editorial, discussion will cover the utility of using advanced proteomic techniques to help identify potential weaknesses in the already impressive defensive armamentarium of biofilm bacteria . Two biofilm systems will be discussed herein, one of which is that of Pseudomonas aeruginosa biofilms within CF airway biofilms . The other is referred to as persistent 'bioterrorist agent biofilms' in which Francisella tularensis can grow on surfaces where environmental amoeba can phagocytose them, allowing for growth of F . tularensis within the amoebae. Microbiol Immunol, 2003, 47(11), 877 - 81 Biofilm formation by a fimbriae-deficient mutant of Actinobacillus actinomycetemcomitans; Inoue T et al.; Actinobacillus actinomycetemcomitans strain 310-TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth . The fimbriae-deficient mutant 310-DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel . Scanning electron microscopy revealed that on glass coverslips, 310-TR formed tight and spherical microcolonies, while 310-DF produced looser ones . These findings suggest that fimbriae are not essential for the surface-adherent growth but are required for enhancing cell-to-surface and cell-to-cell interactions to stabilize the biofilm . Treatment of the 310-DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation. Infect Immun, 2003 Dec, 71(12), 7188 - 92 Role of urease enzymes in stability of a 10-species oral biofilm consortium cultivated in a constant-depth film fermenter; Shu M et al.; Using a 10-species oral biofilm consortium and defined mutants, we show that high-level capacity to generate ammonia from a common salivary substrate is needed to maintain community diversity . This model appears to be suitable for the study of the effects of individual genetic determinants on the ecology of oral biofilms. Infect Immun, 2003 Dec, 71(12), 7154 - 8 Comparative analysis of Salmonella enterica serovar Typhimurium biofilm formation on gallstones and on glass; Prouty AM et al.; In this study, the roles of global regulators, motility, lipopolysaccharide, and exopolysaccharides were further characterized with respect to biofilm formation on both gallstones and glass surfaces . These studies show the complex nature of biofilms and demonstrate that characteristics observed for each biofilm are unique to the particular culture condition. Antimicrob Agents Chemother, 2003 Dec, 47(12), 3967 - 9 In vitro activity of tigecycline against Staphylococcus epidermidis growing in an adherent-cell biofilm model; Labthavikul P et al.; The activity of tigecycline against Staphylococcus epidermidis growing in an in vitro adherent-cell biofilm model was determined . Tigecycline minimum bactericidal concentrations (MBCs) ranged from 1 to 8 microg/ml for S . epidermidis growing in a biofilm of adherent cells, compared to MBCs of 0.12 to >32 microg/ml for freely growing cells . The killing activity of tigecycline against the adherent bacteria was at least fourfold better than that of vancomycin and daptomycin. FEMS Microbiol Lett, 2003 Nov 21, 228(2), 203 - 10 A three-tiered approach to differentiate Listeria monocytogenes biofilm-forming abilities; Marsh EJ et al.; The purpose of this research was to develop a system for cultivating and evaluating Listeria monocytogenes biofilms that produces consistent and reliable results . A three-tiered approach was used to evaluate biofilm-forming abilities of three L . monocytogenes strains that were originally associated with listeriosis outbreaks . A L . monocytogenes 'honeycomb' biofilm structure was described . L . monocytogenes strains Scott A and V7 were comparable in developing biofilm network structures and F2365 was less effective in forming biofilm . This three-tiered method can be very useful for further biofilm studies. Int J Dermatol, 2003 Dec, 42(12), 925 - 7 Microbiology's principle of biofilms as a major factor in the pathogenesis of acne vulgaris; Burkhart CN et al.; Propionibacterium acnes reside within the pilosebaceous unit in a biofilm . As such, they live in a community of bacteria that encase themselves within an extracellular polysaccharide lining, which the organisms secrete after adherence to the surface . This gylcocalyx polymer acts as a protective exoskeleton and serves as a physical barrier, limiting effective antimicrobial concentrations within the biofilm microenvironment . The gylcocalyx polymer secreted by P . acnes as a biofilm may explain the immunogenicity of the organism as well as the clinical course of the disease . The P . acnes' biofilm model explains many aspects of acne pathogenesis and therapy, including why prolonged antibiotic treatment is needed, why antibiotic resistance is not a reliable assessment of treatment outcome, why accutane offers long-lasting effectiveness, and why benzoyl peroxide radicals are beneficial . This microbiologic principle of biofilms as applied to acne leads to numerous new pathways of assessment and exploration. Eur J Oral Sci, 2003 Dec, 111(6), 465 - 71 Formation of Candida albicans biofilms on non-shedding oral surfaces; Lamfon H et al.; The aim of this investigation was to determine the ability of Candida albicans to form biofilms on enamel, dentine and denture acrylic of various surface roughnesses . Biofilms of C . albicans were grown on various materials in a constant depth film fermenter (CDFF) and maintained with artificial saliva . Enamel, dentine and denture acrylic were prepared to the same surface roughness using silicon carbide grit . In a separate experiment, denture acrylic was prepared to different surface roughnesses . At various intervals, the materials were removed from the CDFF, the biofilms disrupted and the number of yeast and hyphae present determined . The total number of yeast cells present on enamel was higher than either dentine or acrylic . The highest numbers of yeast cells were found on the roughest surfaces at 6 h; however, higher numbers of hyphae were found on enamel at 6 h and 24 h . Confocal laser scanning microscopy images revealed that the maturation of biofilms on denture acrylic may be dependent upon hyphal cells . The results of the present study show that both the type of surface and their roughness affect the initial formation and subsequent structure of developing of C . albicans biofilms. Curr Opin Otolaryngol Head Neck Surg, 2003 Dec, 11(6), 416 - 23 Otitis media: concepts and controversies; Darrow DH et al.; PURPOSE OF REVIEW: Otitis media is the most common bacterial infection among children, accounting for as many as 30 million office visits annually . Proper treatment has become critical as offending pathogens become increasingly resistant to antibiotics and the cost of managing the disorder has exceeded 3 billion dollars per year . However, data suggest that many practitioners still struggle with the diagnosis of otitis media and often recommend medical and surgical intervention inappropriately . This article presents recent advances in the otitis media literature and an evidence-based approach to its management . RECENT FINDINGS: Recent investigations have resulted in the following findings: (1) bacterial biofilms may account for the persistence of middle ear disease; (2) there is increasing evidence that heredity and reflux are risk factors for otitis media; (3) primary care providers may be receiving poor otitis media training, leading to inadequate diagnostic skills; (4) medical and surgical therapy are of limited utility in the management of acute and recurrent acute otitis media; (5) antibiotics and steroids are of limited value in the treatment of chronic middle ear effusion; (6) delayed management of effusion may not adversely affect development in children; (7) vaccination for pneumococcus may alter the serotypes responsible for otitis media; and (8) vaccine candidates for other middle ear pathogens are under investigation . SUMMARY: Management of otitis media is constantly evolving, based on research from a variety of medical subspecialties . It is incumbent on the otolaryngologist and primary care providers treating otitis media to keep pace with and synthesize these findings into a rational approach to treatment. Water Res, 2004 Jan, 38(1), 225 - 35 Distribution of bacteria in a domestic hot water system in a Danish apartment building; Bagh LK et al.; Bacterial growth in hot water systems seems to cause problems such as bad odor of the water, skin allergies and increased heat transfer resistance in heating coils . In order to establish a basis for long-term suppression of bacterial growth, we studied the distribution of bacteria in a Danish domestic hot water system . Heterotrophic plate counts (HPC) were measured in both water and biofilm samples from various sampling sites in the system . In hot water samples, where the temperature was 55-60 degrees C, the HPC were 10(3)-10(4)CFU/mL at incubation temperatures of 25 degrees C or 37 degrees C and 10(5)CFU/mL at 55 degrees C or 65 degrees C . In the cold water (10 degrees C) supplying the hot water system, the HPC at 25 degrees C or 37 degrees C was lower than in the hot water, and no bacteria were found after incubation at 55 degrees C or 65 degrees C . HPC constituted from 38% to 84% of the AODC results in hot water but only 2% in cold water, which showed a high ratio of culturable bacteria in hot water . Biofilm samples from the hot water tank and the inner surface of the pipes in the cold and hot water distribution system were collected by specially designed sampling devices, which were exposed in the system for 42 days . The quasi-steady-state number of bacteria in the biofilm, measured as the geometric mean of the HPC obtained between 21 and 42 days, was five-fold higher in the hot water pipe (13x10(5)CFU/cm(2) at 55 degrees C) than in the cold water pipe (2.8x10(5)CFU/cm(2) at 25 degrees C) . There was no significant difference between the number of bacteria in the biofilm samples from the top, middle and bottom of the hot water tank, and the number of bacteria in the biofilm counted at 55 degrees C ranged from 0.6x10(4) to 1.7x10(4)CFU/cm(2) . The surfaces of the sacrificial aluminum anodes and the heating coils in the hot water tank also contained high bacterial numbers . The measured number of bacteria in water and biofilm samples was related to the dimensions of the hot water system, and calculations showed that the majority of bacteria (72%) were located in the biofilm especially in the distribution system, which accounts for the greatest surface area . Free-living bacteria accounted for 26% and only a minor part of the bacteria were in the sludge in the hot water tank (2%). Respirology, 2003 Dec, 8(4), 522 - 4 Examination of expandable metallic stent removed at autopsy; Hosokawa Y et al.; OBJECTIVE: We evaluated the damage to expandable metallic stents (EMS) based upon analysis of EMS removed at autopsy . METHODOLOGY: Seventeen EMS were obtained during autopsy from the main bronchi of nine patients with lung cancer . Each EMS was categorized into one of the following three groups, according to the degree of EMS damage: no damage at all (ND), damage to part of the EMS (PD), and marked damage (MD) that caused loss of function of the airway stent . The removed EMS were examined using a scanning electron microscope . RESULTS: Of the 17 stents, ND occurred in 13 (7/9 primary, 6/8 secondary), PD in four (all secondary), and MD in none . None of the stents had completely lost the ability to function as an airway stent . Bacterial biofilm formation (BBF) was detected on EMS from four patients . CONCLUSIONS: Bacterial biofilm forms on EMS implanted into the airway . Some physical damage was present in four of 17 EMS, but all remained functional. Semin Dial, 2003 Nov-Dec, 16(6), 428 - 37 Continuous peritoneal dialysis-associated peritonitis: a review and current concepts; Troidle L et al.; The percentage of end-stage renal disease (ESRD) patients in the United States maintained on continuous peritoneal dialysis (CPD) therapy is decreasing . Complications from CPD therapy, including peritonitis, may be the reason for the decline . Improvements in CPD technology and a better understanding of the risk factors that predispose patients to the development of peritonitis have been responsible for a decline in the rate of peritonitis . Yet peritonitis remains a significant cause of patient morbidity and mortality and the overall outcome of peritonitis is not acceptable . Factors that have limited our ability to lessen the impact of peritonitis include a lack of data on dosing antibiotics in patients on continuous cycling peritoneal dialysis (CCPD) therapy, a lack of knowledge concerning the biology of bacterial biofilm, and the development of resistance to the current prophylactic antibiotic protocols . Further studies are needed concerning the optimal management of the peritoneal catheter and whether it is feasible to resume CPD therapy after catheter removal. Nature, 2003 Nov 20, 426(6964), 306 - 10 A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance; Mah TF et al.; Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts . One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells . We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa . We have identified a mutant of P . aeruginosa that, while still capable of forming biofilms with the characteristic P . aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics . The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans . Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm . Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents. Int J Hyg Environ Health, 2003 Oct, 206(6), 563 - 73 Contamination of drinking water by coliforms from biofilms grown on rubber-coated valves; Kilb B et al.; In water samples from drinking water distribution systems, coliform bacteria (predominantly Citrobacter species) were repeatedly detected . Disinfection and flushing of the systems did not erase the problem . The pattern of the coliform occurrences indicated contamination originating from biofilms . After inspection of internal surfaces of the systems, no significant biofilm growth was observed on pipe surfaces, but in a number of cases, visible biofilms were detected on rubber-coated valves which harboured the same coliform species as those found in the drinking water samples . In these cases, the rubber-coated valves seemed to act as point sources for the contamination of water. Biofouling, 2003 Aug, 19(4), 247 - 56 The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata; Kavouras JH et al.; This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes . Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year . Biofilms were developed over 1, 3, 8, and 14 d . Controls were clean glass and polystyrene Petri dishes . Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d . Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached . The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces . Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA) . There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA) . These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels. Biofouling, 2003 Aug, 19(4), 215 - 22 The use of cyclic voltammetry to detect biofilms formed by Pseudomonas fluorescens on platinum electrodes; Vieira MJ et al.; The development of an electrochemical detector to monitor the in situ formation of biofilms is described . The detector consisted of an electrochemical cell containing three electrodes, whose response to the application of a potential profile to the working electrode was sensitive to the amount of biofilm present on the surface . The electrochemical technique used was repetitive cyclic voltammetry . Differences between the response of the uncolonised electrode and after Pseudomonas fluorescens biofilms of different ages were grown on its surface were determined . The results show that cyclic voltammetry applied to platinum electrodes can be used to detect young biofilms . The development of the shape of the voltammogram as the potential is cycled may constitute a means of providing information on the coverage of the surface . Observation of the platinum electrodes before and after the electrochemical measurements showed that even after 30 min of recycling, most of the cells were still adhered to the surface, although some may have lost viability. Proteomics, 2003 Oct, 3(10), 2052 - 64 Carbon starvation survival of Listeria monocytogenes in planktonic state and in biofilm: a proteomic study; Helloin E et al.; The proteomes of Listeria monocytogenes expressed in suspension and biofilm state, in the presence and absence of a carbon source, were analysed by two-dimensional electrophoresis with the help of computer software . The up-regulated proteins in each case were identified by peptide sequencing using electrospray ionisation tandem mass spectrometry and a database search against the Listeria genome was performed . Relevant functions could be attributed to a number of the induced proteins which contribute to the understanding of the mechanisms of starvation survival of L . monocytogenes in planktonic state and in biofilm. Shock, 2003 Dec, 20(6), 503 - 10 Post-traumatic osteomyelitis: analysis of inflammatory cells recruited into the site of infection; Wagner C et al.; Device-associated infections after implants or endoprostheses inflict local inflammation and ultimately osteolysis, a clinical entity referred to as posttraumatic osteomyelitis . The underlying molecular mechanisms are not yet known; formation of bacterial biofilms on the implant is presumed, conferring resistance to antibiotics and to host defense mechanisms as well . To gain insight into the pathogenesis of post-traumatic osteomyelitis, the infected site was analyzed for the presence of immunocompetent cells . In 18 patients, the infected site was rinsed intraoperatively . This so-called lavage contained 1-2 x 107 leukocytes, predominantly highly activated polymorphonuclear neutrophils (PMNs), as characterized by low expression of CD62L (selectin), and high expression of the adhesion protein CD18, of the high-affinity immunoglobulin (IgG) receptor CD64, and of the LPS-receptor CD14 . CD16, the low-affinity IgG receptor, was affected in some patients only . Because the majority of infections were caused by staphylococci species, the effect of bacteria-derived lipoteichoic acid on PMN of healthy donors was tested in vitro . A similar activation pattern was found: rapid down-regulation of CD62L, a slower loss of CD16, and upregulation of CD18, CD64, and CD14 . Lipoteichoic acid signaling required p38 mitogen-activated protein kinase and resulted in induction of CD14-specific mRNA and de novo protein synthesis . We conclude that PMNs infiltrate the infected site, but despite local activation they are unable to clear the bacteria, presumably because of biofilm formation . Our data are consistent with the hypothesis that during the ineffective "frustrated" attempt to phagocytose, PMNs release cytotoxic and proteolytic entities that in turn contribute to the progression of tissue injury and ultimately to osteolysis. Microbes Infect, 2003 Nov, 5(13), 1213 - 9 Antimicrobial resistance of Pseudomonas aeruginosa biofilms; Drenkard E; Resistance to antimicrobial agents is the most important feature of biofilm infections . As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate . Although several mechanisms have been postulated to explain reduced susceptibility to antimicrobials in bacterial biofilms, it is becoming evident that biofilm resistance is multifactorial . The contribution of each of the different mechanisms involved in biofilm resistance is now beginning to emerge. Microbes Infect, 2003 Nov, 5(13), 1177 - 87 In vitro identification of two adherence factors required for in vivo virulence of Pseudomonas fluorescens; de Lima Pimenta A et al.; By enriching a random transposon insertion bank of Pseudomonas fluorescens for mutants affected in their adherence to the human extracellular matrix protein fibronectin, we isolated 23 adherence minus mutants . Mutants showed a defect in their ability to develop a biofilm on an abiotic surface and were impaired for virulence when tested in an in vivo virulence model in the fruit fly, Drosophila melanogaster . Molecular characterisation of these mutants showed that the transposon insertions localised to two distinct chromosomal locations, which were subsequently cloned and characterised from two mutants . A search in the databanks identified two loci in the Pseudomonas aeruginosa PAO1 genome with significant homology to the genes interrupted by the transposon insertions . Mutant IVC6 shows homology to gmd, coding for the enzyme GDP-mannose dehydratase, involved in the synthesis of A-band- O-antigen-containing lipopolysaccharide (LPS) . Mutant IVG7 is significantly similar to a probable outer membrane protein of strain PAO1, with no specific function attributed thus far, yet with significant homology to Escherichia coli FadL, involved in long-chain fatty acid transport . We propose that this protein, together with LPS, is involved in the first steps of P . fluorescens adherence leading to host colonisation . Results presented here also demonstrate the pathogenic potential of P . fluorescens, assessed in an in vivo Drosophila model system, correlated with its ability to adhere to the human extracellular matrix protein, fibronectin . Correlation between the mutant phenotypes with identified virulence factors and their actual role in the virulence of P . fluorescens is discussed. Mycoses, 2003, 46(9-10), 370 - 4 Changes in Candida albicans colonization and morphology under influence of voriconazole; Bernhardt H et al.; The aim was the investigation of fungal colonization and morphological alterations under the influence of voriconazole in an in vitro system . Voriconazole stopped growth and colonization of Candida albicans (wild type SC5314) on cover slips in microtiter plates dependent on drug concentration, the time of Candida growth before the input of voriconazole and oxygen concentration . The direct microscopy by fluorescence staining with the optical brightener Blancophor showed short bizarrely deformed mycelia looking swollen . The colonization on cover glass was diminished . Microcolonies or starting of biofilm formation as in the control was not observed . The metabolic activity was demonstrated by vital staining with FUN 1 resulting in red fluorescent structures in the yeast forms and mycelia in the controls . Under voriconazole influence the remaining cells only showed a green or pale yellow fluorescence . Most of the cells lost their metabolic activity. Mol Cell Biochem, 2003 Nov, 253(1-2), 167 - 77 The bacterial adaptive response gene, barA, encodes a novel conserved histidine kinase regulatory switch for adaptation and modulation of metabolism in Escherichia coli; Sahu SN et al.; Histidine kinases are important prokaryotic determinants of cellular adaptation to environmental conditions, particularly stress . The highly conserved histidine kinase, BarA, encoded by the bacterial adaptive response gene, barA, is a member of the family of tripartite histidine kinases, and is involved in stress adaptation . BarA has been implicated to play a role during infection of epithelial cells . Homologues and orthologues of BarA have been found in pathogenic yeast, fungi, mould and in plants . The primary aim of this review is to assimilate evidence present in the current literature linking the role of BarA in stress response, and to support it with preliminary experimental evidence indicating that, it is indeed a global response regulator . In particular, the review focuses on the unusual domain structure of the BarA protein, its role in oxidative, weak acid, and osmotic stress responses and its role in biofilm formation . A preliminary genomic approach to identify downstream genes regulated by the BarA signaling pathway, using DNA microarray, is reported . The results demonstrate that BarA plays a global response regulatory role in cell division, carbon metabolism, iron metabolism and pili formation . The evolutionary significance of these types of histidine kinase sensors is reviewed in light of their roles in pathogenesis. Biofouling, 2003 Jun, 19(3), 159 - 68 "Escherichia coli-milk" biofilm removal from stainless steel surfaces: synergism between ultrasonic waves and enzymes; Oulahal-Lagsir N et al.; Three different methods to standardize biofilm removal for in situ sanitary control of closed surfaces in the food industry have been developed and compared, i.e . sonication, enzymatic treatment and a combined treatment which involved the application of ultrasound to enzyme preparations . The biofilm studied was an Escherichia coli model biofilm, made with milk on stainless steel sheets . Plate counting and epifluorescence microscopy were used to assess the efficiency of each treatment . The results are expressed in percentages, 100% denoting total removal, obtained with a flat ultrasonic transducer (T1) developed and presented in a previous study . The application of ultrasound by a patented curved transducer, T2 (10 s, 40 kHz), specifically devised for closed surfaces, was not sufficient to completely remove the biofilm (30 +/- 7%) . This biofilm was dislodged by two proteolytic enzyme preparations tested by immersion, viz . a 15-min application of protease (84 +/- 1%) and a 30-min trypsin application (95 +/- 8%) . Using a combined treatment, the results showed a synergism between ultrasonic waves and proteolytic or glycolytic enzyme preparations, with removal of a significant amount of biofilm, i.e . 61-96% depending on the conditions tested, i.e . two to three times greater compared to sonication alone (30%) . This application was in agreement with an industrial control, i.e . a good reproducible recovery of the biofilm in 10 s compared with 30 or 15 min with the enzyme alone. Biofouling, 2003 Jun, 19(3), 151 - 7 Studies on the behaviour of Pseudomonas fluorescens biofilms after Ortho-phthalaldehyde treatment; Simoes M et al.; A relatively novel biocide, ortho-phthalaldehyde (OPA), was tested to control biofilms formed by Pseudomonas fluorescens on stainless steel surfaces . The toxic action of OPA was assessed in terms of inactivation and removal of the biofilm by means of, respectively, the determination of the respiratory activity and the variation in the dry weight of the biofilms . For comparison, the activity of OPA against suspended bacteria was also evaluated . The results showed that higher concentrations of OPA and longer exposure times are needed to inactivate P . fluorescens biofilms than planktonic populations, thus denoting that sessile bacteria have a reduced susceptibility to OPA . This appears to be associated with the reaction with the proteins of the matrix, as demonstrated by the reduction of the antimicrobial action of OPA in the presence of a protein (bovine serum albumin) . The application of OPA appeared to cause little effect in the removal of biofilms from the metal slides since the mass of biofilm that remained on the surfaces, after biocide treatment, was within the same range as those observed in the control tests . These results suggest that, with OPA application, biofilms can be inactivated but stay attached to the surfaces, decreasing thereby the success of the chemical treatment. Biofouling, 2003 Apr, 19 Suppl, 171 - 80 Chemical control of bacterial epibiosis and larval settlement of Hydroides elegans in the red sponge Mycale adherens; Lee OO et al.; The sponge, Mycale adherens, usually occurs within the fouling community of Hong Kong waters, yet its body surface is rarely fouled by other macro-organisms . In this study, sponge-associated bacteria were isolated using enrichment culture techniques and compared with indigenous bacterial isolates from an inanimate reference site in the close vicinity . Bacterial isolates were identified phylogenetically by 16S rRNA gene sequence analysis . The comparison between culturable bacterial communities from the sponge and indigenous benthic bacteria revealed differences both in the total number of isolates and their phylogenetic affiliation . Laboratory bioassays utilizing monospecies bacterial films revealed that a significant portion of sponge-associated bacteria had either an inhibitory or neutral effect on larval settlement of the fouling polychaete, Hydroides elegans . In contrast to natural biofilms, which harbor ca 65% of bacteria with at least some sort of inductive effect on H . elegans, statistical analysis showed that only 25% of sponge-associated bacteria were classified as "inductive" strains while the remaining 75% were classified as "non-inductive" strains . Waterborne metabolites of sponges affected the larvae of H . elegans in a concentration-dependent manner by either exerting a toxic or an anti-settlement effect . Organic solvent extracts of sponge tissue weakly inhibited growth of bacterial strains isolated from marine biofilms . A potential antifouling mechanism in the sponge M . adherens is discussed. Biofouling, 2003 Apr, 19 Suppl, 139 - 49 Mn cycling in marine biofilms: effect on the rate of localized corrosion; Dexter SC et al.; Microelectrodes of the Au-Hg amalgam type have been used together with square wave voltammetry to measure profiles of oxygen, peroxide, Fe, Mn and sulfur chemical species through the thickness of natural assemblage marine biofilms grown on stainless steel alloy Nitronic 50 (UNS S20910) . The data show Mn+2 and peroxide together at locations where the dissolved oxygen concentration was low . Oxidized species of Fe were also found at some locations . Sulfur species (predominantly S-2) was often found at locations where the dissolved oxygen concentration was below the detectable limit . Confocal scanning laser microscopy was used to image the microbial assemblage at the locations of the chemical profile data . Organisms with a filamentous morphology were found in consortia with rod and coccoidal shaped microbes at locations where dissolved Mn and peroxide were measured . The filamentous forms were usually absent at locations where Mn was not detected . It is suggested that the filamentous organisms may be Mn metabolizers, and that peroxidatic Mn re-oxidation may be taking place within the biofilm. Biofouling, 2003 Apr, 19(2), 139 - 50 The biofilm matrix; Allison DG; The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population . The matrix is composed of cells, water and secreted/released extracellular macromolecules . In addition, a range of enzymic and regulatory activities can be found within the matrix . Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium . The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear . Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells. Biofouling, 2003 Apr, 19(2), 133 - 8 Laser impact on marine planktonic diatoms: an experimental study using a flow cytometry system; Nandakumar K et al.; A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum . Three flow speeds, i.e . 9, 18 and 27 ml min-1 and three laser fluences, i.e . 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study . The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls . Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species) . A maximum mortality of 0.77 log10 (about 83%) for C . gracilis and 0.68 log10 (about 78%) for S . costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence . The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C . gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S . costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2 . In general, mortality increased with an increase in the laser fluence . The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures . The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water. Biofouling, 2003 Apr, 19(2), 125 - 32 Mutual influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their adhesion to stainless steel; Medilanski E et al.; The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp . desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments . It was found that P . aeruginosa promoted the adhesion of D . desulfuricans under conditions of turbulence, but not under quiescent conditions . The enhancement involved the alignment of most D . desulfuricans along P . aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P . aeruginosa to aligning D . desulfuricans cells . A slightly positive effect of preadhered D . desulfuricans on the adhesion of P . aeruginosa was found . Under condition of laminar flow, substantially better adhesion of D . desulfuricans to confluent P . aeruginosa biofilms than to steel was observed . The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P . aeruginosa biofilms. Biofouling, 2003 Apr, 19(2), 109 - 14 Laser impact on bacterial ATP: insights into the mechanism of laser-bacteria interactions; Nandakumar K et al.; The mechanisms of laser action on bacteria are not adequately understood . Here, an attempt has been made to study the fluctuation in ATP (adenosine triphosphate) concentration following laser irradiation from a pulsed Nd:YAG laser on a marine biofilm-forming bacterium Pseudoalteromonas carrageenovora . A stationary phase bacterial suspension (density 10(7-8) ml-1) was exposed to pulsed laser irradiations at a fluence of 0.1 J cm-2 (pulse width 5 ns, repetition rate 10 Hz) for different durations, ranging from 2 s to 15 min . The total viable count (TVC) and ATP concentration of the irradiated samples were determined immediately after the laser irradiation . While the maximum reduction in the TVC observed with respect to the control was 59% immediately after 15 min irradiation, the ATP concentration showed a reduction of about 86% for the same duration . The ATP concentration showed an abrupt reduction from 3 min of laser irradiation and continued to reduce significantly with increasing duration of irradiation . Thus, 3 min irradiation at a fluence of 0.1 J cm-2 is considered as an approximate threshold for ATP production in this bacterium . As the decreased level of ATP production continued, bacterial mortality resulted . The reduction in ATP production could be due to damage caused by the laser irradiations on bacterial metabolic processes such as cellular respiration. Biofouling, 2003 Apr, 19(2), 77 - 85 The use of cellulase in inhibiting biofilm formation from organisms commonly found on medical implants; Loiselle M et al.; A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants . A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source . Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density . Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units ml-1 . In addition, a control study using deactivated cellulase was performed . The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P . aeruginosa on glass surfaces . The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7 . The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P . aeruginosa exopolysaccharides (EPS) . The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme . An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P . aeruginosa . While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness. Biofouling, 2003 Feb, 19(1), 9 - 18 Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics; Cutter LA et al.; The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp . strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces . The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface . D . tiedjei was found to detach readily from surfaces when it was not the primary colonizer . The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species . Under certain experimental conditions the test organisms formed close associations with each other on the surfaces . These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms. J Clin Invest, 2003 Nov, 112(10), 1466 - 77 The application of biofilm science to the study and control of chronic bacterial infections; Costerton W et al.; Unequivocal direct observations have established that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms . The diagnostic and therapeutic strategies that have served us so well in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases . We discuss the potential benefits of the application of the new methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases. Clin Microbiol Infect, 2003 Sep, 9(9), 955 - 8 Influence of the incubation atmosphere on the production of biofilm by staphylococci; Stepanovic S et al.; Biofilm formation by Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus sciuri and Staphylococcus aureus in aerobic, anaerobic and CO2 incubation atmospheres was quantified by the modified microtiter plate test . The S . epidermidis and S . aureus strains showed significantly lower biofilm production when grown in a CO2-rich environment compared to that exhibited in aerobic incubation . The amount of biofilm produced by these strains under anaerobic conditions did not differ significantly from the biofilm formation detected in the aerobic incubation . The incubation atmosphere did not affect S . sciuri biofilm formation . Biofilm production by S . hemolyticus isolates was very low regardless of the experimental conditions used. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14357 - 62 Epub 2003 Nov 12. The Vibrio cholerae O139 O-antigen polysaccharide is essential for Ca2+-dependent biofilm development in sea water; Kierek K et al.; Vibrio cholerae is both an inhabitant of estuarine environments and the etiologic agent of the diarrheal disease cholera . Previous work has demonstrated that V . cholerae forms both an exopolysaccharide-dependent biofilm and a Ca2+-dependent biofilm . In this work, we demonstrate a role for the O-antigen polysaccharide of V . cholerae in Ca2+-dependent biofilm development in model and true sea water . Interestingly, V . cholerae biofilms, as well as the biofilms of several other Vibrio species, disintegrate when Ca2+ is removed from the bathing medium, suggesting that Ca2+ is interacting directly with the O-antigen polysaccharide . In the Bay of Bengal, cholera incidence has been correlated with increased sea surface height . Because of the low altitude of this region, increases in sea surface height are likely to lead to transport of sea water, marine particulates, and marine biofilms into fresh water environments . Because fresh water is Ca2+-poor, our results suggest that one potential outcome of an increase is sea surface height is the dispersal of marine biofilms with an attendant increase in planktonic marine bacteria such as V . cholerae . Such a phenomenon may contribute to the correlation of increased sea surface height with cholera. Bull Math Biol, 2003 Nov, 65(6), 1053 - 79 The dependence of quorum sensing on the depth of a growing biofilm; Chopp DL et al.; In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly . In this paper, a one-dimensional model of a growing Pseudomonas aeruginosa biofilm is examined . Quorum sensing has been included in the model through equations describing the production, degradation, and diffusion of the signaling molecules, acyl-homoserine lactones, in the biofilm . From this model, we are able to make some important observations about quorum sensing . First, in order for quorum sensing to initiate near the substratum, in accordance with experimental observations, the model suggests that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound . Second, the induction of quorum sensing is related to a critical biofilm depth; once the biofilm grows to the critical depth, quorum sensing is induced . Third, the critical biofilm depth varies with the pH of the surrounding fluid . Of particular interest is the prediction of a critical pH threshold, above which quorum sensing is not possible at any depth . These results highlight the importance of careful study of the relationship among metabolic activity of the bacterium, signal synthesis, and the chemistry of the surrounding environment. J Clin Microbiol, 2003 Nov, 41(11), 4961 - 5 Molecular epidemiology of Proteus mirabilis infections of the catheterized urinary tract; Sabbuba NA et al.; Proteus mirabilis compromises the care of many patients undergoing long-term indwelling bladder catheterization . It forms crystalline bacterial biofilms in catheters which block the flow of urine, causing either incontinence due to leakage or painful distention of the bladder due to urinary retention . If it is not dealt with, catheter blockage can lead to pyelonephritis and septicemia . We have examined the epidemiology of catheter-associated P . mirabilis infections by use of pulsed-field gel electrophoresis (PFGE) of NotI restriction enzyme digests of bacterial DNA . This technique was shown to be more discriminatory than the classical phenotypic Dienes typing technique . We demonstrated that each of 42 isolates from diverse environmental sources and 10 of 12 isolates from blood, wound swabs, and mid-stream urine samples of hospitalized patients had distinct genotypes . Examination of a set of 55 isolates of P . mirabilis, each from a different clinical or environmental source, identified 49 distinct genotypes and 43 Dienes types . The index of discrimination was 0.993 for the PFGE method and 0.988 for the Dienes method . Applying the PFGE method to isolates from catheter-associated urinary tract infections confirmed that the strains present in the crystalline catheter biofilms were identical to those isolated from the same patient's urine . An analysis of samples taken during a prospective study of infections in catheterized nursing home patients revealed that a single genotype of P . mirabilis can persist in the urinary tract despite many changes of catheter, periods of noncatheterization, and antibiotic therapy. Laryngoscope, 2003 Nov, 113(11), 1977 - 82 The resistance of maxillofacial reconstruction plates to biofilm formation in vitro; Emery BE et al.; OBJECTIVES/HYPOTHESIS: Bacterial biofilms, bacteria surrounded by a protective glycocalyx, have been demonstrated on bioimplants placed within and outside of the head and neck region . The presence of the biofilm often makes decontamination of an infected implant impossible, requiring removal of the implant . Infections attributable to biofilm formation within the facial skeleton after reconstruction with implants may result in delayed union, fibrous union, malunion, nonunion, and malocclusion . These complications often require removal of the implant and secondary surgery . Although the incidence of infections necessitating implant removal is relatively low, the increased numbers of implants being placed make this a growing problem . Previous work in the authors laboratory has demonstrated a resistance to biofilm formation on different types of pressure-equalizing tubes . The hypothesis evaluated in the study is that such resistance to biofilm formation is due to the inability of bacteria to adhere to the tubes because of the material's smoothness or surface charge . STUDY DESIGN: A controlled observational study . METHODS: Scanning electron microscopy was used to evaluate the formation of biofilms in vitro for a common strain of Staphylococcus aureus on four implantable materials . The implantable materials included titanium and polylactide resorbable plates . RESULTS: Consistent with the authors' prior findings, they were able to produce bacterial biofilm reliably on a silicone pressure equalizing tube but were unable to demonstrate biofilm formation on the titanium or resorbable implants . CONCLUSION: The absence of biofilm formation on these implants can best be explained by the surface charge or polarity properties of these materials . These findings are consistent with the relatively low incidence of infections among patients receiving these implants in maxillofacial applications. Appl Environ Microbiol, 2003 Nov, 69(11), 6946 - 8 Mycobacterium xenopi and drinking water biofilms; Dailloux M et al.; The ability of Mycobacterium xenopi to colonize an experimental drinking water distribution system (a Propella reactor) was investigated . M . xenopi was present in the biofilm within an hour following its introduction . After 9 weeks, it was always present in the outlet water (1 to 10 CFU 100 ml(-1)) and inside the biofilm (10(2) to 10(3) CFU cm(-2)) . Biofilms may be considered reservoirs for the survival of M . xenopi. Appl Environ Microbiol, 2003 Nov, 69(11), 6899 - 907 Long-term succession of structure and diversity of a biofilm formed in a model drinking water distribution system; Martiny AC et al.; In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old . Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 micro m in thickness to a final looser structure with an average thickness of 14.1 micro m and covering 76% of the surface . An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed . A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase . This step was followed by the growth of a bacterium which was related to Nitrospira, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness . After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including Nitrospira, Planctomyces, Acidobacterium, and Pseudomonas . The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system. Appl Environ Microbiol, 2003 Nov, 69(11), 6475 - 80 Effect of the environment on genotypic diversity of Actinomyces naeslundii and Streptococcus oralis in the oral biofilm; Paddick JS et al.; The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR . The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A . naeslundii organisms than the plaque sampled from the caries-free subjects . These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values . We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable . We found that the diversity of A . naeslundii strains did not change (chi2 = 0.68; P = 0.41) although the proportional representation of A . naeslundii was significantly reduced (P < 0.05) . Conversely, the diversity of the S . oralis strains increased (chi2 = 11.71; P = 0.0006) and the proportional representation of S . oralis did not change . We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S . oralis increased, resulting in the increased genotypic diversity of this species . Apparently, A . naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation . These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community. Microbiology, 2003 Nov, 149(Pt 11), 3221 - 9 A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface; Joshua GW et al.; To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied . Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C . elegans; one strain, lacking the haemin storage (hms) locus, caused no effect . Similarly, 15 strains of Yersinia enterocolitica caused no effect . Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity . The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9.5 % an intermediate infection, and 9.5 % a severe infection . There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C . elegans and those able to form a biofilm on an abiotic surface . Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm . Seven transposon mutants of Y . pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis . A screen of 15 defined C . elegans mutants identified four where mutation caused (complete) resistance to infection by Y . pseudotuberculosis YPIII pIB1 . These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface . This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms. J Electron Microsc (Tokyo), 2003, 52(4), 429 - 33 Comparison of hexamethyldisilazane and critical point drying treatments for SEM analysis of anaerobic biofilms and granular sludge; Araujo JC et al.; We present a fast procedure for scanning electron microscopy (SEM) analysis in which hexamethyldisilazane (HMDS) solvent, instead of the critical point drying, is used to remove liquids from a microbiological specimen . The results indicate that the HMDS solvent is suitable for drying samples of anaerobic cells for examination by SEM and does not cause cell structure disruption. Clin Oral Investig, 2003 Dec, 7(4), 181 - 8 Epub 2003 Nov 04. Oral microbial biofilms and plaque-related diseases: microbial communities and their role in the shift from oral health to disease; Sbordone L et al.; Many oral pathologies, such as dental caries, periodontal disease and peri-implantitis are plaque-related . Dental plaque is a microbial biofilm formed by organisms tightly bound to a solid substrate and each other by means of an exopolymer matrix . Bacteria exhibit different properties when contained within a biofilm . Knowing the mechanisms controlling the formation and development of biofilms can help to understand the emergence and progression of such pathologies and to plan effective treatment . Most periodontal pathogens are common saprophytes of the oral cavity, expressing their virulence only in a susceptible host or when some changes come about in the oral environment . Physical, metabolic and physiological interactions may cause positive or negative effects among the various microbiota present . Such mechanisms of antagonism/synergy select the bacterial population and alterations of its composition affect the balance with the host and may lead to pathology . The effectiveness of antimicrobial agents, as measured through in vitro tests, is dramatically reduced in vivo due to the properties of the microbial community: mature, intact biofilms are less sensitive to such agents, as the exopolymer matrix, bacterial enzymes and slow growth rate hinder the action of chemotherapeutic agents . The present literature review aims to examine the most representative studies, focusing on the characteristics of bacterial communities and the crucial shift from oral health to plaque-related diseases. J Clin Invest, 2003 Nov, 112(9), 1291 - 9 Interspecies communication in bacteria; Federle MJ et al.; Until recently, bacteria were considered to live rather asocial, reclusive lives . New research shows that, in fact, bacteria have elaborate chemical signaling systems that enable them to communicate within and between species . One signal, termed AI-2, appears to be universal and facilitates interspecies communication . Many processes, including virulence factor production, biofilm formation, and motility, are controlled by AI-2 . Strategies that interfere with communication in bacteria are being explored in the biotechnology industry with the aim of developing novel antimicrobials . AI-2 is a particularly attractive candidate for such studies because of its widespread use in the microbial kingdom. J Clin Invest, 2003 Nov, 112(9), 1288 - 90 Bacterial communication and group behavior; Greenberg EP; The existence of species-specific and interspecies bacterial cell-cell communication and group organization was only recently accepted . Researchers are now realizing that the ability of these microbial teams to communicate and form structures, known as biofilms, at key times during the establishment of infection significantly increases their ability to evade both host defenses and antibiotics . This Perspective series discusses the known signaling mechanisms, the roles they play in both chronic Gram-positive and Gram-negative infections, and promising therapeutic avenues of investigation. Res Microbiol, 2003 Nov, 154(9), 623 - 9 Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system; Hu JY et al.; Bacterial biofilm is a common phenomenon in both natural and engineered systems which often becomes a source of contamination and microbially influenced corrosion . It is thought that formation of biofilm in the monoculture of several bacterial species is regulated by acylhomoserine lactone (AHL) quorum-sensing signals . In this study, we investigated the microbial diversity and existence of AHL-producing and AHL-degrading bacterial species in the biofilm samples from a water reclamation system located in a tropical environment . 16S ribosomal DNA sequencing analysis indicated the presence of at least 11 bacterial species, including the frequently encountered bacterial pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae, and several rare pathogens . We showed that only two groups of isolates, belonging to P . aeruginosa and Enterobacter agglomerans, produced AHL signals . We also found that three bacterial isolates, i.e., Agrobacterium tumefaciens XJ01, Bacillus cereus XJ08, and Ralstonia sp . XJ12, expressed AHL degradation enzymes . Furthermore, we showed that P . aeruginosa isolate HL43 was virulent against animal model Caenorhabditis elegans and released 2-6-fold more pyocyanin cytotoxin than P . aeruginosa strains PA01 and PA14, the two commonly used laboratory strains . These data indicate the complexity and importance of biofilm research in water reclamation. Gastrointest Endosc, 2003 Nov, 58(5), 777 - 84 In vivo evaluation of a new bioabsorbable self-expanding biliary stent; Ginsberg G et al.; BACKGROUND: Bioabsorbable stents may offer advantages for the treatment of benign and malignant biliary strictures, including large stent diameter, decreased biofilm accumulation and proliferative changes, elimination of the need for stent removal and imaging artifacts, and prospects for drug impregnation . However, suboptimal expansion has hampered prior iterations . A new bioabsorbable biliary stent (BioStent) was evaluated in a porcine model . METHODS: BioStents were placed in 8 animals for long-term follow-up . The following were evaluated: accuracy and ease of delivery and deployment, radial expansion, and radiologic visualization . Stent function and biotolerance were assessed by cholangiography, serum bilirubin, and necropsy for histopathology performed in pairs at 2, 4, 6, and 12 months . RESULTS: Stents were delivered without sphincterotomy and were deployed easily, accurately, and with good immediate stent expansion and radiographic visualization . On follow-up, all stents were fully expanded and serum bilirubin levels remained within the normal range . Although there was no clinical evidence of biliary obstruction, filling defects were common at cholangiography . On histopathologic evaluation, there was neither bile duct integration or proliferative change . CONCLUSIONS: The BioStent bioabsorbable biliary stent, modified with axial runners, can be effectively deployed endoscopically, is self-expanding, is visualized radiographically, and remains patent up to 6 months . There was no bile duct integration or proliferative change, which are potential advantages . Stent occlusion and migration remain concerns. FEMS Microbiol Lett, 2003 Oct 24, 227(2), 287 - 93 Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans; Welin J et al.; Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries . Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions . In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR . Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types . For this, cells were pulse labeled with {(14)C}-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis . A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis . While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR . Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment . On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux. FEMS Microbiol Lett, 2003 Oct 24, 227(2), 171 - 4 Isoniazid resistance of exponentially growing Mycobacterium smegmatis biofilm culture; Teng R et al.; Biofilm growth of Mycobacterium smegmatis was found to be unaffected at an isoniazid concentration that inhibited growth of planktonic bacilli (i.e . at isoniazid minimum inhibitory concentration=10 microg ml(-1)) . Significant growth (50% of drug-free control) of biofilms was observed at up to 40 microg ml(-1) and the MIC for biofilm growth showed an increase to up to 80 microg ml(-1) isoniazid . Thus, the biofilm growth modus appears to be a strategy for replicating bacilli to evade the onslaught of antibacterials. J Dent Educ, 2003 Oct, 67(10), 1130 - 9 Caries vaccines for the twenty-first century; Smith DJ; Can infection with the dental caries pathogen, Streptococcus mutans, be intercepted or modified immunologically? Resolving this question requires answers to many questions: What are the pathways by which this cariogenic streptococcus enters and accumulates in the dental biofilm? Can bacterial components associated with virulence induce immune responses? What is the level of maturity of immune pathways in the oral cavity of the young child at the time of infection? Can immune strategies deal effectively with chronic S . mutans infections? Are these vaccines safe? Many such questions have been answered . For example, preclinical application of modern methods of mucosal vaccine design and delivery has routinely resulted in protection from dental caries caused by S . mutans infection, using antigens involved in the sucrose-independent or sucrose-dependent mechanisms of infection by these cariogenic streptococci . Passive administration of antibody to functional epitopes of S . mutans virulence antigens has also provided a degree of protection in preclinical studies and small-scale human investigations . The caries-protective capacity of active immunization with dental caries vaccines now awaits proof of principle in pediatric clinical trials. Microb Ecol . 2003 Oct 31; {Epub ahead of print} Comparison of the Phenotypes and Genotypes of Biofilm and Solitary Epiphytic Bacterial Populations on Broad-Leaved Endive; Boureau T et al.; The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety . Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor . However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies . Broad-leaved endive ( Cichorium endivia var . latifolia) harbors biofilms containing fluorescent pseudomonads . These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads . To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive . Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation . The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains . However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms . We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states . This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate. Biol Bull, 2003 Oct, 205(2), 121 - 32 Larval development and metamorphosis in Pleurobranchaea maculata, with a review of development in the notaspidea (Opisthobranchia); Gibson GD; Pleurobranchaea maculata is a carnivorous notaspidean that is common in New Zealand . This species produces small eggs (diameter 100 microm) and planktotrophic veligers that hatch in 8 d and are planktonic for 3 weeks before settling on biofilmed surfaces (14 degrees C) . Larval development is known in detail for only two other notaspidean species, P . japonica and Berthellina citrina . In all three species of pleurobranchids, mantle and shell growth show striking differences from veligers of other opisthobranch taxa . In young veligers of pleurobranchids, the shell is overgrown by the mantle, new shell is added by cells other than those of the mantle fold, and an operculum does not form . Thus some "adult" traits (e.g., notum differentiation, mechanism of shell growth, lack of operculum) are expressed early in larval development . This suggests that apomorphies characteristic of adult pleurobranchids evolved through heterochrony, with expression in larvae of traits typical of adults of other clades . The protoconch is dissolved post-settlement and not cast off as occurs in other opisthobranch orders, indicating that shell loss is apomorphic . P . maculata veligers are atypical of opisthobranchs in having a field of highly folded cells on the lower velar surface, a mouth that is posterior to the metatroch, and a richly glandular, possibly chemodefensive mantle . These data indicate that notaspidean larvae are highly derived in terms of the novel traits and the timing of morphogenic events . Phylogenetic analysis must consider embryological origins before assuming homology, as morphological similarities (e.g., shell loss) may have developed through distinct mechanisms. Int J Food Microbiol, 2003 Dec 15, 89(1), 1 - 10 Planktonic or biofilm growth affects survival, hydrophobicity and protein expression patterns of a pathogenic Campylobacter jejuni strain; Dykes GA et al.; The effect of planktonic or biofilm modes of growth on survival, hydrophobicity and cellular protein expression patterns of a pathogenic Campylobacter jejuni strain were determined . This was achieved by growing the strain in brain heart infusion broth (with 1% yeast extract), or attached to glass beads in the same medium, at 37 degrees C for 48 h under microaerophilic conditions . Cells from the broth or the bead surfaces were stored at different temperatures (4, 10, 25 and 37 degrees C) for 28 days in phosphate buffered saline (PBS) and monitored at appropriate time intervals for culturable numbers and hydrophobicity by standard methods . In addition, cells were inoculated onto the surface of two processed meat products (a bologna and a summer sausage) vacuum packaged and stored at 4 degrees C for 28 days . Numbers of culturable cells were monitored at appropriate time intervals by standard methods . Cells from the broth or the bead surfaces were also examined for protein expression using two-dimensional protein electrophoresis . Results indicated that numbers of culturable cells in phosphate buffered saline decreased from approximately 6 log colony forming units (cfu) g(-1) to undetectable levels within 14-day storage in a temperature dependent manner . Hydrophobicity of broth grown cells decreased from 15% to 0% adherence to xylene over the same time in a temperature independent manner . Cells grown in a biofilm mode initially displayed a <0.3% adherence to xylene which was maintained during storage . Furthermore, cells grown in the biofilm mode decreased in number more rapidly on storage in buffer than their counterparts grown in broth . Numbers of culturable cells on meat decreased from approximately 5 log cfu g(-1) to undetectable levels within 14-day storage in a product dependent manner, with the most rapid decrease observed for the more acidic summer sausage . Cells grown in a biofilm mode decreased in number more rapidly on storage than broth grown cells . The protein expression patterns differed between planktonic and biofilm cells with seven unique and 12 up-regulated protein spots expressed in a growth mode specific manner . A number of the differentially expressed spots were tentatively identified, by comparison to existing literature, as surface- and stress-associated proteins . Despite the elicitation of some putative stress proteins, this study importantly indicates that biofilm cells of C . jejuni are less resistant to stress than their planktonic counterparts and may lack a sophisticated adaptive stress-resistance response . These findings have implication in determining the risks of infection associated with C . jejuni contamination on food. Mol Cell Probes, 2003 Oct, 17(5), 237 - 43 Direct estimation of biofilm density on different pipe material coupons using a specific DNA-probe; Chang YC et al.; A variety of approaches to quantify biomass in biofilms without disruption due to detachment have been developed over the years . One basic approach is the combination of advanced microscopy with molecular staining . However, many stains (e.g . 4',6-diamino-2-phenylindole, acridine orange or live-dead stains) can be non-specific when corrosion products, precipitates, and pipe material are present . In addition, some pipe materials cause high background when using epifluorescent microscopy . The new refinement discussed in this presentation used fluorescence spectroscopy to obtain the spectra from four common distribution system pipe materials: PVC, 'concrete' lined cast iron, cast iron, and galvanized steel . The emission maximum for all four materials was between 500 and 550 nm, but emissions radically decreased around 575-600 nm . A molecular probe, BO-PRO-3 (Molecular Probes, Inc., Eugene, OR, USA) was identified which has an emission intensity maximum at 599 nm (red), with emission intensity 200 times greater when it is bound to DNA . The BO-PRO-3 has greatly reduced non-specific staining and background problems . In the preliminary experiment, using diluted waste water, a significant exponential relationship was found between stained surface area/total area ratio and fixed biofilm inventory measurements from scraping heterotrophic plate counts (SHPC) on R2A medium . In addition, the biofilm inventory on different pipe material coupons from pilot distribution systems was also correlated to the stained surface area fraction and SHPC. BMC Microbiol . 2003 Oct 27;3(1):22. Functional profiling of mercuric reductase (mer A) genes in biofilm communities of a technical scale biocatalyzer; Felske AD et al.; BACKGROUND: Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up . The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase . We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation . RESULTS: Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol . The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences . The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community . One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa . The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing . CONCLUSIONS: The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation . In such a way, a predominant mercury reducing Ps . aeruginosa strain was identified by its unique mercuric reductase gene. Antimicrob Agents Chemother, 2003 Nov, 47(11), 3657 - 9 Antifungal combinations against Candida albicans biofilms in vitro; Bachmann SP et al.; Candida biofilms display increased resistance to most antifungal agents . We have evaluated the efficacy of combinations of fluconazole (FLC), amphotericin B, and caspofungin (CSP) against Candida albicans biofilms in vitro . Indifference was observed for all the combinations of paired antifungal agents when a checkerboard titration method was used . Time-kill experiments revealed an antagonistic effect of high FLC doses with CSP. Antimicrob Agents Chemother, 2003 Nov, 47(11), 3580 - 5 In vitro and ex vivo activities of minocycline and EDTA against microorganisms embedded in biofilm on catheter surfaces; Raad I et al.; Minocycline-EDTA (M-EDTA) flush solution has been shown to prevent catheter-related infection and colonization in a rabbit model and in hemodialysis patients . We undertook this study in order to determine the activities of M-EDTA against organisms embedded in fresh biofilm (in vitro) and mature biofilm (ex vivo) . For the experiment with the in vitro model, a modified Robbin's device (MRD) was used whereby 25 catheter segments were flushed for 18 h with 10(6) CFU of biofilm-producing Staphylococcus epidermidis, Staphylococcus aureus, and Candida albicans per ml . Subsequently, each of the catheter segments was incubated in one of the following solutions: (i) streptokinase, (ii) heparin, (iii) broth alone, (iv) vancomycin, (v) vancomycin-heparin, (vi) EDTA, (vii) minocycline (high-dose alternating with low-dose), or (viii) M-EDTA (low-dose minocycline alternating with high-dose minocycline were used to study the additive and synergistic activities of M-EDTA) . All segments were cultured quantitatively by scrape sonication . For the experiment with the ex vivo model, 54 catheter tip segments removed from patients and colonized with bacterial organisms by roll plate were longitudinally cut into two equal segments and exposed to either saline, heparin, EDTA, or M-EDTA (with high-dose minocycline) . Subsequently, all segments were examined by confocal laser electron microscopy . In the in vitro MRD model, M-EDTA (with a low concentration of minocycline) was significantly more effective than any other agent in reducing colonization of S . epidermidis, S . aureus, and C . albicans (P < 0.01) . M-EDTA (with a high concentration of minocycline) eradicated all staphylococcal and C . albicans organisms embedded in the biofilm . In the ex vivo model, M-EDTA (with a high concentration of minocycline) reduced bacterial colonization more frequently than EDTA or heparin (P < 0.01) . We concluded that M-EDTA is highly active in eradicating microorganisms embedded in fresh and mature biofilm adhering to catheter surfaces. Antimicrob Agents Chemother, 2003 Nov, 47(11), 3407 - 14 Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces; Wu JA et al.; Staphylococci often form biofilms, sessile communities of microcolonies encased in an extracellular matrix that adhere to biomedical implants or damaged tissue . Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts . This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices . Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan . Lysostaphin kills Staphylococcus aureus within minutes (MIC at which 90% of the strains are inhibited {MIC(90)}, 0.001 to 0.064 microg/ml) and is also effective against Staphylococcus epidermidis at higher concentrations (MIC(90), 12.5 to 64 microg/ml) . The activity of lysostaphin against staphylococci present in biofilms compared to those of other antibiotics was, however, never explored . Surprisingly, lysostaphin not only killed S . aureus in biofilms but also disrupted the extracellular matrix of S . aureus biofilms in vitro on plastic and glass surfaces at concentrations as low as 1 microg/ml . Scanning electron microscopy confirmed that lysostaphin eradicated both the sessile cells and the extracellular matrix of the biofilm . This disruption of S . aureus biofilms was specific for lysostaphin-sensitive S . aureus, as biofilms of lysostaphin-resistant S . aureus were not affected . High concentrations of oxacillin (400 microg/ml), vancomycin (800 microg/ml), and clindamycin (800 microg/ml) had no effect on the established S . aureus biofilms in this system, even after 24 h . Higher concentrations of lysostaphin also disrupted S . epidermidis biofilms. Bioresour Technol, 2003 Dec, 90(3), 323 - 8 Reductive dechlorination of 2-chlorophenol in a hydrogenotrophic, gas-permeable, silicone membrane bioreactor; Chang CC et al.; A gas-permeable silicone membrane bioreactor was used to cultivate the biofilm under hydrogenotrophic condition for reductive dechlorination of 2-chlorophenol (2-CP) . The anaerobic sludge obtained from a swine wastewater treatment plant was immobilized by polyvinyl alcohol (PVA) so as to form a biofilm on the surface of the silicone tube . After acclimating for about 4 months, the bioreactor showed a high dechlorinating performance . Under the condition of continuous feeding with 2-CP at 25 mg/l and the hydraulic retention time of 15 h, the 2-CP removal efficiency reached 92.8% (2-CP decay rate: 0.67 g/m(2)d of surface area of silicone tube) . H(2) was used as electron donor for dechlorinating 2-CP, and produced the dechlorinating intermediate, phenol . Both nitrate and sulfate played important roles in inhibiting 2-CP dechlorination through different biological mechanisms . Nitrate can be easily utilized as an electron acceptor by the biofilm, while sulfate cannot . Results of this study demonstrated that nitrate competed with 2-CP as the electron acceptor, while sulfate retarded the activity of hydrogen-dechlorinating bacteria and thus inhibited the 2-CP dechlorination. Biotechnol Bioeng, 2003 Nov 20, 84(4), 424 - 32 Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI); Manz B et al.; The characterization of substrate transport in the bulk phase and in the biofilm matrix is one of the problems which has to be solved for the verification of biofilm models . Additionally, the surface structure of biofilms has to be described with appropriate parameters . Magnetic resonance imaging (MRI) is one of the promising methods for the investigation of transport phenomena and structure in biofilm systems . The MRI technique allows the noninvasive determination of flow velocities and biofilm structures with a high resolution on the sub-millimeter scale . The presented investigations were carried out for defined heterotrophic biofilms which were cultivated in a tube reactor at a Reynolds number of 2000 and 8000 and a substrate load of 6 and 4 g/m2d glucose . Magnetic resonance imaging provides both structure data of the biofilm surface and flow velocities in the bulk phase and at the bulk/biofilm interface . It is shown that the surface roughness of the biofilms can be determined in one experiment for the complete cross section of the test tubes both under flow and stagnant conditions . Furthermore, the local shear stress was calculated from the measured velocity profiles . In the investigated biofilm systems the local shear stress at the biofilm surface was up to 3 times higher compared to the mean wall shear stress calculated on the base of the mean flow velocity . Urol Res, 2003 Oct, 31(5), 306 - 11 Epub 2003 Jul 11. Why are Foley catheters so vulnerable to encrustation and blockage by crystalline bacterial biofilm? Stickler D, Young R, Jones G, Sabbuba N, Morris N. Many patients undergoing long-term bladder catheterisation experience blockage and encrustation of their catheters . The problem stems from infection by urease producing bacteria, particularly Proteus mirabilis . Bacterial biofilms colonise the catheters, the activity of urease raises the pH and induces the deposition of calcium and magnesium phosphate crystals . In this study, a laboratory model of the catheterised bladder has been used to examine the early stages in the formation of the crystalline biofilms . The results show that initial cell adhesion is to the irregular surfaces surrounding the catheter eye-holes . Microcolonies form in depressions in these surfaces and spread to cover the entire surface of the rims around the eye-holes . Crystals then form around the bacterial populations and the biofilm starts to move down the lumenal surfaces of the catheters . The encrustation develops most extensively and generally blocks the catheter at or just below the eye-hole . There is a need to improve catheter design and manufacturing procedures for the eye-holes if the problems associated with the current devices are to be reduced. Microb Ecol, 2004 Apr, 47(3), 266 - 70 Epub 2003 Oct 28. An aufwuchs chamber slide for high-resolution confocal laser scanning microscopy and stereo imaging of microbial communities in natural biofilms; Eaglesham BS et al.; Aufwuchs chamber slides were constructed by attaching a silicone rubber gasket to a glass slide with epoxy cement . For biofilm growth, the slides were suspended in Cayuga Lake near Ithaca, NY, for 27 days . Biofilms in the chamber were stained with 0.05% acridine orange . After rinsing, the chamber was filled with molten 1% agarose to stabilize filaments and delicate polymer structures at the biofilm surface . Areas of biofilm approximately 0.5 mm thick on the inner face of the wall of the chamber were selected for side-on optical sectioning in a confocal laser scanning microscope (CLSM) . Stacks of high-resolution optical images captured by the CLSM z-sectioning software, were used to create left-right stereo image pairs . At low magnification the stereo pairs showed 3-D details of the microbial landscape in the mature biofilms . Channels, pores, and other structural features of the biofilm matrix were observed in peripheral regions . Higher magnification images revealed the 3-D distribution of specific biofilm components such as filaments of sheathed bacteria projecting outward into the liquid milieu, and organic coatings, including bacterial cells on the surfaces of mineral particles. Infect Immun, 2003 Nov, 71(11), 6607 - 9 Role of actin filament network in Burkholderia multivorans invasion in well-differentiated human airway epithelia; Schwab UE et al.; The role of the actin-based cytoskeleton in the internalization process of Burkholderia multivorans by well-differentiated human airway epithelia was investigated by immunohistology and confocal microscopy . Our data suggest that an intact actin cytoskeleton is required for biofilm formation but not single cell entry or paracytosis. Biotechnol Lett, 2003 Sep, 25(18), 1521 - 4 Polymerization of cardanol using soybean peroxidase and its potential application as anti-biofilm coating material; Kim YH et al.; Soybean peroxidase (20 mg) catalyzed the oxidative polymerization of cardanol in 2-propanol/phospate buffer solution (25 ml, 1:1 v/v) and yielded 62% polycardanol over 6 h . Cobalt naphthenate (0.5% w/w) catalyzed the crosslinking of polycardanol and the final hardness of crosslinked polycardanol film exceeded 9 H scale as pencil scratch hardness, which shows a high potential as a commercial coating material . In addition, it showed an excellent anti-biofouling activity to Pseudomonas fluorescens compared to other polymeric materials such as polypropylene. Can J Microbiol, 2003 Jul, 49(7), 443 - 9 Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria; Martino PD et al.; We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1 . In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E . coli and other indole-producing bacteria . Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium . Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E . coli 3714 (S17-1 tnaA::Tn5) in LB medium . Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected . At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E . coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains . In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species . Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors. Pathol Biol (Paris), 2003 Oct, 51(8-9), 490 - 5 {Ecology and transmission of Mycobacterium ulcerans}; Marsollier L et al.; Mycobacterium ulcerans is an environmental pathogen concerning mainly the tropical countries; it is the causative agent of Buruli ulcer, which has become the third most important mycobacterial disease . In spite of water-linked epidemiological studies to identify the sources of M . ulcerans, the reservoir and the mode of transmission of this organism remain elusive . To determine the ecology and the mode of transmission of M . ulcerans we have set up an experimental model . This experimental model demonstrated that water bugs were able to transmit M . ulcerans by bites . In insects, the bacilli were localized exclusively within salivary glands, where it could both multiply contrary to other mycobacteria species . In another experimental study, we report that the crude extracts from aquatic plants stimulate in vitro the growth of M . ulcerans as much as the biofilm formation by M . ulcerans has been observed on aquatic plants . Given that the water bugs are essentially carnivorous, it is difficult to imagine a direct contact in the contamination of aquatic bugs and plants . It seems very likely that an intermediate host exists . In an endemic area of Daloa in Cote d'Ivoire, our observations were confirmed. FEMS Microbiol Lett, 2003 Oct 10, 227(1), 113 - 9 Identification of genes involved in the switch between the smooth and rugose phenotypes of Vibrio cholerae; Rashid MH et al.; Vibrio cholerae can switch to a 'rugose' phenotype characterized by an exopolysaccharide (EPS) matrix, wrinkled colony morphology, increased biofilm formation and increased survival under specific conditions . The vps gene cluster responsible for the biosynthesis of the rugose EPS (rEPS) is positively regulated by VpsR . We recently identified media (APW#3) promoting EPS production and the rugose phenotype and found epidemic strains switch at a higher frequency than non-pathogenic strains, suggesting this switch and the rugose phenotype are important in cholera epidemiology . In this study, transposon mutagenesis on a smooth V . cholerae strain was used to identify mutants that were unable to shift to the rugose phenotype under inducing conditions to better understand the molecular basis of the switch . We identified vpsR, galE and vps previously associated with the rugose phenotype, and also identified genes not previously associated with the phenotype, including rfbD and rfbE having roles in LPS (lipopolysaccharide) synthesis and aroB and aroK with roles in aromatic amino acid synthesis . Additionally, a mutation in amiB encoding N-acetylmuramoyl-L-alanine amidase caused defects in the switch, motility and cell morphology . We also found that a gene encoding a novel regulatory protein we termed RocS (regulation of cell signaling) containing a GGDEF and EAL domains and associated with c-di-GMP levels is important for the rugose phenotype, EPS, biofilm formation and motility . We propose that modulation of cyclic dinucleotide (e.g . c-di-GMP) levels might have application in regulating various phenotypes of prokaryotes . Our study shows the molecular complexity of the switch between the smooth and rugose phenotypes of V . cholerae and may be relevant to similar phenotypes in other species. Pathophysiology, 2003 May, 9(3), 161 - 178 Pathogenesis of various forms of infection in artificial hearts; Dobsak P et al.; Implanted biomaterials are often inevitably attacked by the bacterial infection . So far this problem has not been sufficiently explained and solved . It represents an 'evergreen' in the artificial heart research . Infection of biomaterials is a completely new clinical entity that profoundly differs from the common clinical course of various kinds of infections and their treatment . These infections are persistent; they resist host defense mechanisms and antibiotic therapy because the nature of these microorganisms has changed due to their protection by the biofilm of some bacteria on the surfaces of implanted biomaterials . In our 66 long-term experiments with total artificial heart (TAH) in 25 animals, the infection and sepsis were the main causes of death . The different organs, attacked by the bacterial and septic compli