Biofilms

Adv Perit Dial, 1997, 13, 214 - 7
The effects of macrolide and quinolone antibiotics in methicillin-resistant Staphylococcus aureus biofilm growth; Dasgupta MK et al.; Recent studies demonstrate that 14-membered macrolides increase permeability and destruction of Pseudomonas biofilms . The effect of a macrolide antibiotic, erythromycin, on methicillin-resistant Staphylococcus aureus (MRSA) biofilm on Silastic catheter materials in comparison with two different quinolone antibiotics, sparfloxacin (SPFX) and a new quinolone, SYN 1193, was examined . Two different MRSA strains were grown in biofilm, using Mueller-Hinton broth with and without the addition of 10% pooled normal human serum (PNHS), in a modified Robbins device, at 37 degrees C for 24, 48, and 72 hours . Two different clinical MRSA strains were used and minimum bactericidal concentration (MBC) were determined at the time intervals mentioned . Three different dosages of each antibiotic were tested: 5.0, 20.0, and 50.0 micrograms/mL . In addition, a constant dosage of SPFX and SYN 1193, in combination with varying dosages of erythromycin, was tested under similar experimental conditions . SYN 1193 demonstrated the highest MBC in comparison to SPFX; addition of PNHS did not alter the effect of SYN 1193 . However, erythromycin alone and in combination with SPFX and SYN 1193 had no effect on MBC . We conclude that (1) macrolide antibiotic erythromycin has poor MRSA biofilm permeability and killing in comparison to SPFX and SYN 1193, and (2) SYN 1193 had the highest MBC to MRSA biofilm.

Sci Total Environ, 1997 Oct 7, 205(1), 25 - 49
Nutrient dynamics in a lowland stream impacted by sewage effluent: Great Ouse, England; House WA et al.; Nutrient and ancillary chemical changes in a stretch of the Great Ouse river near Brackley in Northamptonshire, UK, were measured on a seasonal basis over one year with an initial pilot study in the spring of 1994 . River bed-sediments were characterized by their physical, adsorptive and chemical properties and a batch returned to the laboratory for experiments in a fluvarium channel . These experiments involved studies of the release and uptake of soluble reactive phosphorus to or from the overlying water in oxic conditions and release when the dissolved oxygen concentration was reduced to near zero . There was no impact of the point-sources on the concentration of nitrate in the river, a slight effect on the concentration of silicon during low-flow conditions in the summer and net uptake in the spring caused by the growth of diatoms . However there was a substantial impact on phosphorus concentrations, particularly during the summer sampling when the river was in low flow . The results for the winter showed little impact of point discharges because of the high dilution of the treated effluent . The bed-sediments at this time were found to be close to equilibrium with respect to the concentration of soluble reactive phosphorus in the overlying water . Both the fluvarium channel and field measurements obtained in the autumn are consistent with a lower net uptake of phosphorus and degradation of vegetation in the river . In the spring and summer visits, the phosphorus concentrations increased immediately downstream of the main point input and then decreased in concentration at the next downstream site . This effect was particularly noticeable in the summer and was consistent with a large uptake of phosphorus to the bed-sediment and associated vegetation . The contribution of the bed-sediment was estimated using a chemical model describing the uptake kinetics by the Elovich equation and also a parabolic equation . The stability of the waters with respect to calcite and calcium phosphate minerals was assessed in detail . Seasonal changes in the sediment composition were consistent with the deposition of calcite and coprecipitation of inorganic phosphate in the lattice of calcite, either abiotically, or in association with algal biofilms in the sediment . Good correlation between the total phosphorus and calcium contents of the sediments were evident, particularly at the sites furthest from the main sewage input . Measurements of the equilibrium phosphate concentrations of the surface sediments showed that they did not respond quickly to the higher concentrations of dissolved phosphorus found in the summer . It is also evident that the use of the equilibrium phosphate concentration to predict the magnitude of the release of soluble reactive phosphorus becomes less reliable as the solution concentration approaches the equilibrium phosphate concentration . Perturbations may arise because of changes in the surface micro-layer caused by a number of processes such as particle-size fractionation, biological activity or changes in the local redox conditions . However bearing these constraints in mind, with an equilibrium phosphate concentration of the sediments generally below 5 mumol dm-3, the release of phosphorus to the overlying water is not expected until the concentration is below this value . The results also show that phosphorus is not accumulating in the surface sediment and that much of the phosphorus in the sediment is not easily desorbed.

J Comp Pathol, 1997 Aug, 117(2), 165 - 70
Adaptation of a fluorocarbon-based non-aqueous fixation regime for the ultrastructural study of the teleost epithelial mucous coat; Sanchez JG et al.; Studies on the microanatomy of the mucus-rich biofilm surface of normal or damaged teleost skin tissue have been limited because conventional fixation regimes do not effectively retain mucus during tissue preparation . A non-aqueous fixation method, based on a technique devised to retain airway mucous for ultrastructural study, and consisting of the use of an inert perfluorocarbon solvent with osmium teroxide 1%, was successfully used to prepare skin tissues of healthy juvenile rainbow trout . The skin's mucous coat was examined by transmission electron microscopy and the results were compared with those obtained with tissues prepared by a conventional glutaraldehyde-based method . In samples fixed with glutaraldehyde, the cell-surface structures retained were limited to microridges and a poorly discernible glycocalyx layer . In contrast, those fixed by the non-aqueous method had a more clearly demonstrated glycocalyx layer, and a second fibrillar layer, resembling mucus, which was separated from the glycocalyx layer by an electron-lucent zone.

Appl Environ Microbiol, 1997 Oct, 63(10), 4107 - 10
Magnification of tributyl tin toxicity to oyster larvae by bioconcentration in biofilms of Shewanella colwelliana; Labare ML et al.; The toxic effects of dissolved versus bioconcentrated tributyl tin (TBT) on oyster larvae were compared . Water column TBT levels, which had no effect in solution, inhibited natural attachment and metamorphosis of oyster larvae on bottom surfaces due to bioconcentration by biofilms . This mechanism should be considered when evaluating heavy metal toxicity in the environment.

J Antimicrob Chemother, 1997 Sep, 40(3), 419 - 21
Antibacterial efficacy of tobramycin against anaerobic Escherichia coli cultures in the presence of electron acceptors; Tresse O et al.; The antimicrobial activity of tobramycin against anaerobic cultures of Escherichia coli was tested in the presence of various electron carriers . The presence of 2,6-dichlorophenol 4-indophenol (DCIP) significantly enhanced the killing efficacy of tobramycin . Only 0.003% of the initial cell population (i.e . 10(6) cfu/mL) remained viable after exposure for 10 h to the mixture of antibiotic (20 x MIC, i.e . 40 mg/L) and electron acceptor (10(-3) M), as compared with 9% of surviving organisms in the presence of tobramycin alone . Less synergy was obtained with p-benzoquinone and 1,2-naphthoquinone . Fumarate did not affect the efficiency of the antibiotic . The mixture of tobramycin and DCIP was ineffective against agar-entrapped bacteria which, like biofilm organisms, are subject to oxygen limitation.

J Antimicrob Chemother, 1997 Sep, 40(3), 329 - 34
The development of a small-scale biofilm model suitable for studying the effects of antibiotics on biofilms of gram-negative bacteria; Gander S et al.; A method for the study of membrane-associated biofilm populations of Escherichia coli ATCC 8739 using modified Swinnex filter units was developed . Biofilms were established under carbon limitation in a chemically defined simple salts medium . Cells, pressure filtered on to cellulose nitrate membranes in situ, were perfused from the sterile side . Steady-state conditions were attained at which the growth rate of the sessile cells could be demonstrated to be proportional to the flow rate of medium . The antibiotic susceptibility of these biofilms was examined by including ciprofloxacin within the perfusing medium . Susceptibility of the biofilms to ciprofloxacin was found to be affected not only by its concentration, but also by the growth rate of the bacteria.

Clin Microbiol Rev, 1997 Oct, 10(4), 597 - 610
Uses of inorganic hypochlorite (bleach) in health-care facilities; Rutala WA et al.; Hypochlorite has been used as a disinfectant for more than 100 years . It has many of the properties of an ideal disinfectant, including a broad antimicrobial activity, rapid bactericidal action, reasonable persistence in treated potable water, ease of use, solubility in water, relative stability, relative nontoxicity at use concentrations, no poisonous residuals, no color, no staining, and low cost . The active species is undissociated hypochlorous acid (HOCl) . Hypochlorites are lethal to most microbes, although viruses and vegetative bacteria are more susceptible than endospore-forming bacteria, fungi, and protozoa . Activity is reduced by the presence of heavy metal ions, a biofilm, organic material, low temperature, low pH, or UV radiation . Clinical uses in health-care facilities include hyperchlorination of potable water to prevent Legionella colonization, chlorination of water distribution systems used in hemodialysis centers, cleaning of environmental surfaces, disinfection of laundry, local use to decontaminate blood spills, disinfection of equipment, decontamination of medical waste prior to disposal, and dental therapy . Despite the increasing availability of other disinfectants, hypochlorites continue to find wide use in hospitals.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1997 Sep, 84(3), 310 - 4
Coronal leakage of mixed anaerobic bacteria after obturation and post space preparation; Barrieshi KM et al.; OBJECTIVES: The purpose of this study was to assess bacterial leakage of a mixed anaerobic community of organisms in obturated canals after post space preparation . STUDY DESIGN: A mixed microbial community of strict anaerobic organisms (F . nucleatum, P . micros and C . rectus) was developed . With the use of an in vitro model system, coronal leakage was assessed in 40 anterior teeth after obturation and post space preparation . The specific leakage time in days for each organism to penetrate through the obturating material was determined . In addition, colonization of the apical canal space was assessed by scanning electron microscope after longitudinal splitting of randomly selected specimens . RESULTS: Eighty percent of the teeth demonstrated coronal leakage of F . nucleatum and C . rectus by the 90 day interval . Bacterial penetration occurred from 48 days to 84 days . Scanning electron microscope examination showed a heterogeneous biofilm of coccal and bacillary species colonizing the apical portion of the canal wall . CONCLUSIONS: This study demonstrated that coronal leakage phenomena do occur after loss of coronal seals . The model system developed using mixed, anaerobic bacterial cultures is more clinically relevant and may be used to assess bacterial penetration through gutta percha obturation.

FEMS Microbiol Lett, 1997 Sep 15, 154(2), 259 - 63
Evidence of autoinducer activity in naturally occurring biofilms; McLean RJ et al.; N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria . Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens . In nature, bacteria often grow as surface-adherent biofilm communities . As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology . However, proof of AHL production within biofilms has heretofore been lacking . In this study we have employed a cross-feeding assay, using A, tumefaciens A136 (traI::lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones . AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm . This represents the first report of AHL activity in naturally occurring biofilms.

Int J Food Microbiol, 1997 Jul 22, 37(2-3), 145 - 53
Adherence to stainless steel by foodborne microorganisms during growth in model food systems; Hood SK et al.; Biofilm formation on stainless steel by Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Pseudomonas fragi and Pseudomonas fluorescens during growth in model food systems was studied . Test growth media included tryptic soy broth (TSB), diluted TSB (dTSB), 1% reconstituted skim milk (RSM) and diluted meat juice (DMJ) . Adherent cells were stained with acridine orange and enumerated using epifluorescent microscopy and computerized image analysis . Cells were observed on the stainless steel surface after 1 h in all of the media . However, the increases in the number of adherent cells over time was seen only with S . typhimurium in DMJ, E . coli O157:H7 in TSB, dTSB and DMJ, P . fragi in RSM and P . fluorescens in RSM . The medium which produced the highest observed level of adherent cells was different for each microorganism.

Chemotherapy, 1997 Sep-Oct, 43(5), 340 - 5
Permeation of antimicrobial agents through Pseudomonas aeruginosa biofilms: a simple method; Shigeta M et al.; In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method . Bacteria used were a leucine-requiring mucoid mutant . Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days . At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured . PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25% . Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM . This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.

Curr Microbiol, 1997 Oct, 35(4), 249 - 54
Enzyme-linked lectinsorbent assay measures N-acetyl-D-glucosamine in matrix of biofilm produced by Staphylococcus epidermidis; Thomas VL et al.; An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification of in situ biofilm produced by Staphylococcus epidermidis in polystyrene 96-well tissue culture plates with phosphatase-labeled wheat germ agglutinin (WGA) as a specific probe for the GlcNAcbeta-1,4n component of exocellular matrix material (EMM) that is responsible for intercellular adhesion and accumulation . The ELLA and the modified Christensen dye assay were used to test 13 laboratory strains of coagulase-negative staphylococci and 10 clinical isolates of S . epidermidis . Four biofilm-positive laboratory strains of S . epidermidis were positive by both tests, and six biofilm-negative strains were negative by both . One strain of S . hemolyticus was positive by the ELLA only . Two of the 10 clinical isolates of S . epidermidis were positive by both assays, two were negative by both, and the remaining were positive by the ELLA only . The ELLA was objective, reproducible, specific, sensitive, and useful for screening strains for their capacity to adhere to plastic, produce EMM, and form biofilm.

Behring Inst Mitt, 1997 Feb, (98), 350 - 60
Rationale for development of immunotherapies that target mucoid Pseudomonas aeruginosa infection in cystic fibrosis patients; Pier GB; Despite a complex sputum bacteriology, the progressive decline in pulmonary function that is the hallmark of the genetic disease cystic fibrosis (CF) is attributable to a single infecting pathogen, mucoid Pseudomonas aeruginosa . Therefore, active and passive immunotherapies that target this particular variant of the bacterium should be of value in attenuating infection and interfering with the decline in pulmonary function . The major surface antigen of mucoid P . aeruginosa is referred to as either mucoid exopolysaccharide (MEP) or alginate, a random polymer of D-mannuronic and L-guluronic acid residues linked beta 1-4 . During chronic infection CF patients make antibodies to MEP that fail to mediate opsonic killing of bacteria in vitro . These antibodies can be elicited by vaccination in 35-40% of plasma donors given a preparation of MEP comprised of only the highest molecular-weight polymers; inclusion in human vaccines of smaller polymers normally produced by the bacterium fails to elicit opsonic antibodies, just like in infected CF patients . Opsonic, but not non-opsonic, antibodies to MEP protect animals against chronic endobronchial infection . CF patients do produce opsonic antibodies to mucoid P . aeruginosa that are in a planktonic or suspended state, but these antibodies are not directed at the MEP antigen and they fail to kill P . aeruginosa growing in a biofilm . This is the state that the bacteria grow in the lung . Therefore immunoglobulin G preparations with opsonic antibodies to MEP could provide CF patients with antibodies that they normally do not produce during chronic lung infection and may improve their clinical course.

Can J Microbiol, 1997 Aug, 43(8), 751 - 8
Influence of trophic conditions on exopolysaccharide production: bacterial biofilm susceptibility to chlorine and monochloramine; Samrakandi MM et al.; This study examines the controversial efficacy of chlorine and monochloramine against biofilms that differ in their extracellular polysaccharide (EPS) content . The results point out a net variability of bacterial biofilm susceptibility according to the nutrients present . Chlorine and monochloramine showed an equal biocidal activity on lactose medium-grown E . coli ATCC 10536 and glycerol-ammonium nitrate medium-grown nonmucoid Pseudomonas aeruginosa biofilms . In contrast, the effect of monochloramine is greater compared with that of chlorine on E . coli and mucoid P . aeruginosa biofilms grown in sucrose and glycerol-ammonium nitrate media, respectively . In these culture conditions, treatment with 25 mg monochloramine/L for 2 h reduced culturable cells by 4.5 logs (99.997%) for E . coli and about 3 logs (99.87%) for mucoid P . aeruginosa while the similar treatment with chlorine reduced culturable cells in these biofilms by 2.2 logs (99.4%) and 1 log (10%), respectively . The decrease of chlorine disinfection efficacy on sucrose and glycerol-ammonium nitrate medium-grown biofilms is postulated to be linked to the higher polysaccharide production observed in these media . It seems likely that monochloramine produces a high leakage of material absorbing at 260 nm from sucrose medium-grown E . coli biofilm, which could indicate its better penetration into biofilms.

Antimicrob Agents Chemother, 1997 Sep, 41(9), 1876 - 9
Influence of electric fields and pH on biofilm structure as related to the bioelectric effect; Stoodley P et al.; Mixed species biofilms of Klebsiella pneumoniae, Pseudomonas fluorescens, and Pseudomonas aeruginosa were grown in a flow cell fitted with two platinum wire electrodes . The biofilm growing on the wires reached a thickness of approximately 50 microm after 3 days . When a voltage was applied with oscillating polarity, the biofilm attached to the wire expanded and contracted . The biofilm expanded by approximately 4% when the wire was cathodic but was reduced to 74% of the original thickness when the wire was anodic . The phenomenon was reproduced by alternately flushing the flow cell with media adjusted to pH 3 and pH 10 with no electric current . At pH 10 the biofilm was unaltered, but it became compacted to 69% of the original thickness at pH 3 . We explained these phenomena in terms of the molecular interactions between charged acidic groups in the biofilm slime and the bacterial cell walls . Contraction of the biofilm under acidic conditions may be caused by (i) the elimination of electrostatic repulsion from neutralization of negatively charged carboxylate groups through protonation and (ii) subsequent hydrogen bonding between the carboxylic acids and oxygen atoms in the sugars . Electrostatic interactions between negatively charged groups in the biofilm and the charged wire may also be expected to cause biofilm expansion when the wire was cathodic and contraction when the wire was anodic . The consequences of the explanation of the increased susceptibility of biofilm cells to antibiotics in an electric field, the "bioelectric effect," are discussed.

FEMS Microbiol Rev, 1997 Jul, 20(3-4), 573 - 90
Microbial studies in the Canadian nuclear fuel waste management program; Stroes-Gascoyne S et al.; Atomic Energy of Canada Limited (AECL) has developed a concept for permanent geological disposal of used nuclear fuel in Canada . This concept, based on a multibarrier system, would involve disposal of nuclear fuel waste in titanium or copper containers, surrounded by compacted clay-based buffer and backfill materials, in a vault 500-1000 m deep in granitic rock of the Canadian Shield . Subsurface environments will not be sterile and an experimental program was initiated in 1991 by AECL to address and quantify the potential effects of microbial action on the integrity of the disposal vault . This microbial program focuses on answering specific questions in areas such as the survival of bacteria in compacted clay-based buffer materials under relevant radiation, temperature and desiccation conditions; mobility of microbes in compacted buffer materials; the potential for microbially influenced corrosion of containers; microbial gas production in backfill material; introduction of nutrients as a result of vault excavation and operation; the presence and activity of microbes in deep granitic groundwaters; and the effects of biofilms on radionuclide migration in the geosphere . This paper summarizes the results to date from the research activities at AECL.

FEMS Microbiol Rev, 1997 Jul, 20(3-4), 291 - 303
Interactions between biofilms and the environment; Beveridge TJ et al.; The surfaces of bacteria are highly interactive with their environment . Whether the bacterium is Gram-negative or Gram-positive, most surfaces are charged at neutral pH because of the ionization of the reactive chemical groups which stud them . Since prokaryotes have a high surface area-to-volume ratio, this can have surprising ramifications . For example, many bacteria can concentrate dilute environmental metals on their surfaces and initiate the development of fine-grained minerals . In natural environments, it is not unusual to find such bacteria closely associated with the minerals which they have helped develop . Bacteria can be free-living (planktonic), but in most natural ecosystems they prefer to grow on interfaces as biofilms; supposedly to take advantage of the nutrient concentrative effect of the interface, although there must also be gained some protective value against predators and toxic agents . Using a Pseudomonas aeruginosa model system, we have determined that lipopolysaccharide is important in the initial attachment of this Gram-negative bacterium to interfaces and that this surface moiety subtly changes during biofilm formation . Using this same model system, we have also discovered that there is a natural tendency for Gram-negative bacteria to concentrate and package periplasmic components into membrane vesicles which bleb-off the surface . Since some of these components (e.g., peptidoglycan hydrolases) can degrade other surrounding cells, the vesicles could be predatory; i.e., a natural system by which neighboring bacteria are targeted and lysed, thereby liberating additional nutrients to the microbial community . This obviously would be of benefit to vesicle-producing bacteria living in biofilms containing mixed microbial populations.

J Am Dent Assoc, 1997 Sep, 128(9), 1235 - 43
Comparison of dental water quality management procedures; Murdoch-Kinch CA et al.; The effectiveness of ADA-recommended approaches for reducing dental unit waterline, or DUWL, contamination was investigated using newly installed dental units . Over a 2-month period, the authors studied independent water reservoirs, a sodium hypochlorite disinfection regimen, daily draining and purging of DUWLs and point-of-use filters by assessing microbial contamination and biofilm development using scanning electron microscopy . The findings demonstrate that DUWL contamination can be controlled when dental personnel use available technologies and adhere to recommended maintenance protocols.

Arch Oral Biol, 1997 Jul, 42(7), 513 - 20
Colonization of Candida albicans on cleaned human dental hard tissues; Sen BH et al.; Candida albicans is a fungus that commonly infects oral mucosal surfaces . Limited data exist on biofilm formation by C . albicans on dental surfaces . Human premolar teeth were infected with C . albicans for 10 days and hard-tissue surfaces were examined with a scanning electron microscope . Enamel, cementum and dentine, in the absence or presence of a smear layer, were readily colonized by this micro-organism . Hyphae penetrated into cracks, followed the ridges of the cavities and migrated into dentinal tubules . Blastospores and hyphae were embedded in an extracellular material . These findings suggest that dental hard tissues may be invaded by C . albicans and thus can potentially present a reservoir for disseminating candidal infections.

Appl Environ Microbiol, 1997 Sep, 63(9), 3724 - 8
Enzymatic removal and disinfection of bacterial biofilms; Johansen C et al.; Model biofilms of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and Pseudomonas aeruginosa were made on steel and polypropylene substrata . Plaque-resembling biofilms of Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum were made on saliva-coated hydroxyapatite . The activity of enzymes against bacterial cells in biofilm was measured by fluorescence microscopy and an indirect conductance test in which evolution of carbon dioxide was measured . Glucose oxidase combined with lactoperoxidase was bactericidal against biofilm bacteria but did not remove the biofilm from the substrata . A complex mixture of polysaccharide-hydrolyzing enzymes was able to remove bacterial biofilm from steel and polypropylene substrata but did not have a significant bactericidal activity . Combining oxidoreductases with polysaccharide-hydrolyzing enzymes resulted in bactericidal activity as well as removal of the biofilm.

Appl Environ Microbiol, 1997 Sep, 63(9), 3352 - 8
Substratum topography influences susceptibility of Salmonella enteritidis biofilms to trisodium phosphate; Korber DR et al.; Established (48- and 72-h) Salmonella enteritidis biofilms grown in glass flow cells with or without artificial crevices (0.5-, 0.3-, and 0.15-mm widths) were subjected to a 10% trisodium phosphate (TSP) solution under different flow regimens (0.3, 0.6, 1.2, and 1.8 cm s-1) . The abundance of biofilm remaining after TSP treatment, the biocidal efficacy of TSP, and the factors which contributed to bacterial survival were then evaluated by using confocal laser microscopy and a fluorescent viability probe . Biofilm age affected the amount of biofilm which remained following a 15-s exposure to TSP . After TSP treatment of 48-h biofilms, 29% of the original biofilm remained at the biofilm-liquid interface, whereas 75% of the biofilm remained at the base (the attachment surface) . Following TSP treatment of 72-h biofilms, 27% of the biofilm material remained at the biofilm-liquid interface, 73% remained at the 5-micron depth, and 91% remained at the biofilm base . Results obtained using the BacLight viability probe indicated that TSP exposure killed all the cells in 48-h biofilms, whereas in the thicker 72-h biofilms, surviving bacteria (approximately 2% of the total) were found near the 5- and 0-micron depths . In the presence of artificially constructed crevices, an inverse relationship was shown to exist between bacterial survival (ranging from approximately 13 to 83% of total biofilm material) and crevice width . This relationship was further influenced by the velocity of TSP flow; high TSP flow velocities (1.8 cm s-1) resulted in the lowest number of surviving bacteria at the base of crevices (approximately 42% survival) . Extended time courses demonstrated that after TSP stress was relieved, biofilms continued to grow within crevices but not in systems without crevices . It is suggested that advective TSP flux into crevices and through the biofilm matrix was enhanced under conditions of high flow . These results suggest that the inherent roughness of the substratum on which the biofilm was grown and the timing of TSP application are important factors controlling the efficacy of TSP treatment.

J Oral Rehabil, 1997 Aug, 24(8), 594 - 604
Effects of salivary or serum pellicles on the Candida albicans growth and biofilm formation on soft lining materials in vitro; Nikawa H et al.; The effects of salivary or serum pellicle on Candida albicans growth, biofilm formation and cavitation on the soft lining materials were examined . Both saliva and serum pellicles reduced the antifungal effects of soft liners . The fungal biofilm formation on these materials varied depending upon both the materials tested and protein-coats, and the pellicles which significantly enhanced the biofilm formation . Similarly, the pellicles enhanced the firm colonization and hyphal invasion of the yeasts on the specimens, although the cavitation appeared to be regulated by the plasticizer used . These results suggest that the interactions between proteinaceous pellicle, tissue conditioners and fungi are complex . They also suggest that denture pellicles facilitate fungal plaque formation onto soft lining materials through several mechanisms such as reduction of the antifungal effects of soft liners, facilitation of biofilm formation, firm colonization and hyphal invasion . In addition, the composition of the materials is also involved in the susceptibility to the fungi.

Curr Microbiol, 1997 Sep, 35(3), 169 - 74
Biofouling of groundwater systems by Thiothrix spp; Brigmon RL et al.; Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida . Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem . The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida . Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp . The detection of Thiothrix spp . was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures . It was estimated through immunocytochemical procedures that Thiothrix spp . comprised 18% of the biofilm in the municipal water storage tanks . These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.

Spinal Cord, 1997 Aug, 35(8), 521 - 5
Urinary catheter blockage depends on urine pH, calcium and rate of flow; Burr RG et al.; Urinary catheters tend to block when biofilm from urease-producing organisms build up on the catheter surface . This is a locally-occurring process that influences and influenced by the composition of the urine . In this work we relate urine pH and calcium to catheter blockage and suggest how to reduce the rate of encrustation . Sixty patients with indwelling urinary catheters were studied, 26 of them being troubled by frequent blockage of their catheters, 34 of them not . A series of small urine samples were collected during a 24 h period . Urinary pH and calcium concentration were combined into discriminant functions designed to separate Blockers from Non-blockers and achieved a 95% correct classification . The results indicate that a high and uniform rate of fluid intake is mandatory for the patient with a tendency for catheter blockage . Excessive total fluid intake may be avoided by attention to uniformity . Other avoidable risk factors include: excess dietary calcium from certain protein supplements and antacids; excess dietary magnesium from certain beverages and antacids; alkali from effervescent tablets; excess dietary citrate from some fruit juices and cordials; intermittent dehydration from alcohol ingestion . Less tractable risk factors include infection of the urinary tract with urease-positive organisms, hypercalciuria of immobilisation, hyperhydrosis and postural oliguria . The processes involved in catheter encrustation and blockage provide a model for the formation of calculi in spinal cord injured patients . Therefore the above considerations may also be relevant to the management of stone disease in paraplegic and tetraplegic patients.

Curr Eye Res, 1997 Aug, 16(8), 776 - 81
Protein-lipid interaction on the surface of a hydrophilic contact lens in vitro; Bontempo AR et al.; PURPOSE: To investigate the mechanism of protein-lipid interactions responsible for biofilm formation on the surface of hydrophilic contact lenses in vitro . METHODS: New, never-worn hydrophilic contact lenses were individually incubated in a protein-only, lipid-only, or combination protein-lipid artificial tear solution for 24 hours at 37 degrees C with constant stirring . Deposited lipids were removed with a methanol based extraction procedure, separated using high-performance, thin layer chromatography and quantitatively analyzed densitometrically . Deposited proteins were extracted with 4M urea, separated using gel electrophoresis and quantitatively analyzed densitometrically . RESULTS: The presence of protein in the artificial tear solution has a profound effect on the nature of lipid deposition for each group of hydrophilic lens, whereas the presence of lipid has a significant effect on the nature of protein deposition for only a group IV lens . In addition, the presence of lipid deposits on a group IV lens decreases the adsorption of lysozyme, while the presence of protein deposits reduces the amount of total lipid adhering to a group II lens . CONCLUSIONS: Protein adsorption on a group IV lens renders the lens surface less hydrophilic and, thereby, more susceptible to lipid deposition, which in turn increases surface hydrophobicity and inhibits additional protein deposition . For a group II lens, positively charged protein competes with and replaces some of the polar lipids attached to the lens . Thus, the interaction of protein and lipid on a lens surface most prone to a particular contaminant apparently makes it less likely for that contaminant to bind.

Appl Microbiol Biotechnol, 1997 Jul, 48(1), 11 - 7
Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion; Jayaraman A et al.; Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escherichia coli DH5 alpha has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 degrees C . In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was comparable to that observed in anaerobic sterile media . To corroborate this result, a continuous reactor and electrochemical impedance spectroscopy were used to show that both P . fragi K and E . coli DH5 alpha decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion found with sterile medium in the absence of oxygen and with E . coli DH5 alpha grown anaerobically . In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion rate that was observed upon killing the P . fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion . Hence, pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface.

J Periodontal Res, 1997 Jul, 32(5), 473 - 7
Effects of metronidazole on Porphyromonas gingivalis biofilms; Wright TL et al.; Subgingival bacteria exist within a biofilm consisting of cells and extracellular matrix which may afford organisms protection from both antibiotics and components of the host immune system . MIC values for planktonic Porphyromonas gingivalis treated with metronidazole were compared with those obtained for the same strain in biofilms associated with hydroxyapatite (HA) surfaces . The treated biofilms were examined for growth and studied by scanning electron microscopy . A broth assay resulted in an MIC of 0.125 microgram/ml for metronidazole against P . gingivalis, P . gingivalis biofilms exhibited growth after treatment with 20 micrograms/ml metronidazole, which was 160 times the MIC for planktonic organisms . The results of this study indicate that biofilm-associated P . gingivalis may be resistant to metronidazole at concentrations which are usually attained by systemic administration.

Vet Res, 1997 Jul-Aug, 28(4), 353 - 63
Resistance of Escherichia coli growing as biofilms to disinfectants; Ntsama-Essomba C et al.; The bactericidal activity of various disinfectants (cationic or amphoteric surfactants, oxidizing agents, phenolic derivatives) was determined against Escherichia coli CIP 54127 obtained by culture on tryptic soy agar (in-suspension or on-germ-carrier test) or in the form of biofilms produced in a continuous culture system . The bacteria tested on germ-carriers or included in biofilms were more resistant than the same strain in suspension . The extent of the reduction in activity depended on the nature of the disinfectant . In the two cases, the greatest reduction was observed with benzalkonium chloride and hexadecyl trimethylammonium bromide, the agents with the lowest hydrophile-lipophile balance . The activity of the oxidizing agents (sodium hypochlorite, peracetic acid/H2O2) and alkyl trimethylammonium derivatives (C12 and C14) was somewhat reduced, while that of the phenolic derivatives (o-cresol, phenol) was either slightly attenuated or unaffected . The reduction in sensitivity was attributed to a reduced accessibility of the bacterial cells to the disinfectants, due to the fact that the former adhered to a support . Furthermore, the interfering action of the substances in contact with the bacteria (milk in the germ-carrier test and exopolymers in the biofilms) could play a role . The reduced sensitivity of the bacteria in the biofilms was not due to any alteration in the metabolic state of the bacteria (mostly in a quiescent state) since this resistance was lost after the mechanical resuspension of the cells before the contact with the disinfectants.

Microbiology, 1997 Jul, 143 ( Pt 7), 2407 - 13
The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus; Williams I et al.; Sensitivity of Staphylococcus aureus to the antibiotics tetracycline, benzylpenicillin and vancomycin was found to decrease by 2-10 fold when cells were grown adherent to silicone catheter surfaces . Sensitivity to rifampicin and fusidic acid was not significantly altered in adherent cells . Susceptibility further decreased with increased adherence time prior to antibiotic challenge . The resistance observed was not genotypic, or due to the presence of a specialized subpopulation of bacteria, as it disappeared when the bacteria were removed from the catheter, subcultured and retested . Also, adherent bacteria were found to grow more slowly than bacteria growing planktonically . It is concluded that the decrease in antibiotic susceptibility of adherent bacteria is a function of the physiological status of the individual cells rather than a function of biofilm formation or slime production . The decrease in growth rate of the adherent bacteria is a result of the adherence process rather than a result of nutrient depletion . The decrease in growth rate is implicated, but is not the sole factor, in the decreased antibiotic susceptibility of adherent bacteria.

Br J Urol, 1997 Jul, 80(1), 58 - 63
Which indwelling urethral catheters resist encrustation by Proteus mirabilis biofilms?
Morris NS, Stickler DJ, Winters C.
OBJECTIVE: To test the resistance of currently available types of indwelling urethral catheters to blockage by encrustation with mineralized Proteus mirabilis biofilms . MATERIALS AND METHODS: Encrustation was studied in a simple laboratory model of the catheterized bladder . Artificial urine was supplied to the bladder chamber at 0.5 mL/min . The bladder urine was inoculated with a clinical strain of P . mirabilis that had been isolated from an encrusted catheter . The models were operated until the catheters blocked and atomic absorption spectrometry was used to assess the amounts of calcium and magnesium deposited on the catheters . Scanning electron microscopy was also used to locate and assess the degree of encrustation . RESULTS: The mean times to blockage ranged from 21 h for the Bard hydrogel/silver-coated latex catheter to 56 h for the Eschmann Folatex S all-silicone catheter . The calcium and magnesium salts were mainly deposited on the 10 cm below the eye-holes of the catheters, complete blockage generally occurring in the 2 cm immediately below the eye-hole . CONCLUSION: None of the 18 types of catheter tested, including those coated with hydrogel or silver, were capable of resisting encrustation by P . mirabilis biofilm.

Appl Environ Microbiol, 1997 Jul, 63(7), 2533 - 42
Optimization of L-(+)-lactic acid production by ring and disc plastic composite supports through repeated-batch biofilm fermentation; Ho KL et al.; Four customized bioreactors, three with plastic composite supports (PCS) and one with suspended cells (control), were operated as repeated-batch fermentors for 66 days at pH 5 and 37 degrees C . The working volume of each customized reactor was 600 ml, and each reactor's medium was changed every 2 to 5 days for 17 batches . The performance of PCS bioreactors in long-term biofilm repeated-batch fermentation was compared with that of suspended-cell bioreactors in this research . PCS could stimulate biofilm formation, supply nutrients to attached and free suspended cells, and reduce medium channelling for lactic acid production . Compared with conventional repeated-batch fermentation, PCS bioreactors shortened the lag time by threefold (control, 11 h; PCS, 3.5 h) and sixfold (control, 9 h; PCS, 1.5 h) at yeast extract concentrations of 0.4 and 0.8% (wt/vol), respectively . They also increased the lactic acid productivity of Lactobacillus casei subsp . rhamnosus (ATCC 11443) by 40 to 70% and shortened the total fermentation time by 28 to 61% at all yeast extract concentrations . The fastest productivity of the PCS bioreactors (4.26 g/liter/h) was at a starting glucose concentration of 10% (wt/vol), whereas that of the control (2.78 g/liter/h) was at 8% (wt/vol) . PCS biofilm lactic acid fermentation can drastically improve the fermentation rate with reduced complex-nutrient addition.

Appl Environ Microbiol, 1997 Jul, 63(7), 2516 - 23
Ingredient selection for plastic composite supports for L-(+)-lactic acid biofilm fermentation by Lactobacillus casei subsp . rhamnosus; Ho KL et al.; Plastic composite supports containing 50% agricultural products (oat hulls, soybean hulls, yeast extract, soybean flour, dried bovine erythrocytes, bovine albumin, and/or mineral salts) and 50% (wt/wt) polypropylene were produced by high-temperature twin-screw extrusion . The research employed two half sets of a five-factorial fractional design (2(5 - 1)) to evaluate the effects of different agricultural components on the properties of the plastic composite supports and to select the best plastic composite support formulation for lactic acid fermentation . The biofilm population was affected by the contact angle and relative hydrophobicity of the supports (r = 0.79 to 0.82) . Lactic acid was produced by the suspended cells (r = 0.96) and the biofilm on the plastic composite support discs (r = 0.85) . Incorporation of yeast extract into plastic composite supports enhanced growth of free and attached cells in minimal medium (P < 0.0001) . The presence of soybean hulls, yeast extract, or mineral salts in plastic composite supports produced less hydrophobic supports (P < 0.0001) and enhanced cell attachment (P < 0.03) . Under all conditions, suspended-cell and polypropylene disc controls gave negligible lactic acid production and cell density . Plastic composite supports containing soybean hulls, yeast extract, soybean flour, bovine albumin, and mineral salts gave the highest biofilm population (2.3 x 10(9) CFU/g of support), cell density (absorbance of 1.8 at 620 nm), and lactic acid concentration (7.6 g/liter) in minimal medium.

Arch Biochem Biophys, 1997 Jun 15, 342(2), 389 - 95
The major 20-kDa polysaccharide of Staphylococcus epidermidis extracellular slime and its antibodies as powerful agents for detecting antibodies in blood serum and differentiating among slime-positive and -negative S . epidermidis and other staphylococci species; Karamanos NK et al.; Staphylococcus epidermidis has been recognized as an important pathogen in immunocompromised hosts and patients with prosthetic or implanted medical devices . A highly adhesive extracellular material (slime or biofilm) produced by certain strains is associated with bacterial adherence to and growth on biomaterials contributing to pathogenesis of bacteremia . We have recently reported on the isolation and characterization of a sulfated 20-kDa acidic polysaccharide which constitutes slime's major component . Immunization of rabbits with crude slime and 20-kDa polysaccharide gave rise to readily reactive sera without manipulation of the 20-kDa polysaccharide structure . Immunological studies using purified polyclonal antibodies to 20-kDa polysaccharide by direct and competitive ELISA showed that they exhibit a high degree of reactivity and specificity with the homologous antigen . A significant proportion of the reactivity of antibodies to crude slime was also shown to be attributed to the 20-kDa polysaccharide . This polysaccharide is immunogenic in humans since blood sera derived from patients 10-15 days after confirmation of slime-producing S . epidermidis bacteremia gave approximately 16 times higher reactivity than that of healthy individuals . Antibodies to 20-kDa polysaccharide were able to recognize and react specifically with slime-positive S . epidermidis strains compared to slime-negative ones (2 to 5 times higher reactivity) . Moreover, these antibodies exhibited statistically significant (P < 0.05) differences in the degree of reactivity among S . epidermidis and other staphylococci species . These results open a new area in the diagnosis of S . epidermidis infection by direct analysis in blood sera, in differentiating among slime-positive and slime-negative strains as well as in distinguishing slime-producing S . epidermidis from other staphylococci species by simple laboratory tests.

Endoscopy, 1997 Jun, 29(5), 372 - 9
Automatic washer disinfector for flexible endoscopes: a new evaluation process; Pineau L et al.; BACKGROUND AND STUDY AIMS: Many automatic washer disinfectors for flexible endoscopes have been marketed and offered as an alternative method of preventing infections, but they are frequently unsatisfactory . There is therefore clearly a need to test prototypes prior to marketing, following an evaluation process that is sufficiently reliable and rigorous to guarantee the efficacy of the decontamination processes . MATERIALS AND METHODS: The present study describes an experimental method based on the follow-up of the decontamination of a Tygon tube, the internal surface of which was contaminated by a bacterial biofilm . This method is proposed as a preliminary test for evaluating washer disinfectors . RESULTS: An analysis of the results obtained after technical modifications of the first prototype of the Fibro-Cleaner showed that complementary activities of each successive cycle phase allow a reduction in the number of adherent bacteria of more than 8 log per cm2 of support . With the three different biofilms tested (Escherichia coli, Pseudomonas aeruginosa mucoid and Staphylococcus aureus), no microorganisms were recovered from the support at the end of the decontamination process . CONCLUSIONS: The experimental protocol suggested here seems to be well suited for assessing washer disinfectors during the development phase of the prototype, as well as for comparative studies.

J Ind Microbiol Biotechnol, 1997 Jun, 18(6), 396 - 401
Importance of biofilm formation for corrosion inhibition of SAE 1018 steel by axenic aerobic biofilms; Jayaraman A et al.; To investigate if corrosion inhibition by aerobic biofilms is a general phenomenon, carbon steel (SAE 1018) coupons were exposed to a complex liquid medium (Luria-Bertani) and seawater-mimicking medium (VNSS) containing fifteen different pure-culture bacterial suspensions representing seven genera . Compared to sterile controls, the mass loss in the presence of these bacteria (which are capable of developing a biofilm to various degrees) decreased by 2- to 15-fold . The extent of corrosion inhibition in LB medium depended on the nature of the biofilm: an increased proportion of live cells, observed with confocal scanning laser microscopy (CSLM) and image analysis, decreased corrosion . Corrosion inhibition in LB medium was greatest with Pseudomonas putida (good biofilm formation), while metal coupons exposed to Streptomyces lividans in LB medium (poor biofilm formation) corroded in a manner similar to the sterile controls . Pseudomonas mendocina KR1 reduced corrosion the most in VNSS . It appears that only a small layer of active, respiring cells is required to inhibit corrosion, and the corrosion inhibition observed is due to the attached biofilm.

J Ind Microbiol Biotechnol, 1997 Jun, 18(6), 364 - 9
Fiber-reinforced polymeric composites are susceptible to microbial degradation; Gu JD et al.; A mixed culture of fungi, enriched from degraded polymeric materials, formed biofilms on coupons of fiber-reinforced polymeric composites (FRPCs) . They grew actively in aqueous extracts of the composites under ambient conditions . The data indicate that the fungi utilized the resins or fiber chemical sizing as carbon and energy sources . A progressive decline in impedance from above 10(7) Ohms to below 10(8) Ohms was detected in the inoculated FRPC panels by electrochemical impedance spectroscopy (EIS) after 179 days of incubation, but not on the sterile controls . The degradation proceeds through an initial ingress of water into the resins, followed by degradation of bonding between fiber surfaces and resins and finally separation of fibers from the resins . At the end of EIS study, the extent of disbonding in the inoculated composite was greater than the control observed by scanning electron microscopy . These results suggest that the composite materials are susceptible to microbial attack by providing nutrients for growth.

Nippon Hinyokika Gakkai Zasshi, 1997 Jun, 88(6), 596 - 604
{A clinical study on combination therapy of antimicrobial agents for complicated urinary tract infection--with special reference to combination with clarithromycin}; Sano M et al.; PURPOSE: To confirm the clinical efficacy of the combined therapy to complicated urinary tract infection (UTI), we conducted a comparative clinical study of the combined therapy with ciprofloxacin (CPFX) and clarithromycin (CAM) acting an biofilm elimination or CPFX alone in patients with complicated UTI . PATIENTS AND METHODS: The study was carried out in patients with complicated UTI having WBCs with 5/hpf or more in urinary sediment and bacteriuria at least 10(4) CFU/ml . The combined therapy was CPFX and CAM, each 600 mg/day, for 14 days, and the single therapy group CPFX, 600 mg/day, for 14 days . On Day 7 and 14, the eradication rate and efficacy rate (according to the criteria of the Japanese UTI committee) were determined . In the patients with indwelling catheter, the surface of the catheter tip was observed under a scanning electron microscope (SEM) on Day 14 . RESULTS: In both cases with and without catheters, clinical efficacy was higher in the combined therapy group than in the single therapy group . In particular, the efficacy rates at 14 Day were significantly higher in the former group . Furthermore, we investigated the therapeutic effect in the below MIC breakpoint of CPFX in complicated UTI . The combined therapy group showed a higher clinical efficacy in both cases with and without indwelling catheter than the single therapy group, although there was not statistically significant . Biofilm on the surface of the catheter tip was eliminated in 75% of the combined therapy group . However, none of the biofilm was eliminated in the single therapy group . CONCLUSION: From the above results, we surmise that the combined use of CPFX and CAM will show some degree of efficacy in eliminating both the causative organism and its biofilm in the complicated UTI.

Can J Microbiol, 1997 Jun, 43(6), 534 - 40
Recombinant plasmid mobilization between E . coli strains in seven sterile microcosms; Lebaron P et al.; Transfer by mobilization of a pBR derivative recombinant plasmid lacking transfer functions (oriT+, tra-, mob-) from one E . coli K12 strain to another was investigated in seven sterile microcosms corresponding to different environments . These microcosms were chosen as representative of environments that genetically engineered microorganisms (GEMOs) encounter after accidental release, namely attached biomass in aquatic environments (biofilm), soil, seawater, freshwater, wastewater, mouse gut, and mussel gut, GEMOs survived in the same way as the host strains in all microcosms . Recombinant DNA mobilization occurred in the mouse gut, in sterile soil, and in biofilm . The plasmid transfer rates principally reflected the environmental conditions encountered in each microcosm.

J Antimicrob Chemother, 1997 Jun, 39(6), 817 - 20
Factors affecting development of rifampicin resistance in biofilm-producing Staphylococcus epidermidis; Svensson E et al.; Selection and regrowth of variants resistant to 0.016-32 mg/L of rifampicin, which were present at a frequency of 10(-7) in the initial inoculum, were seen when large inocula (> 10(5) cfu/mL) of Staphylococcus epidermidis ATCC 35984 were incubated with the drug . Conventional MIC determinations using approximately 10(5) cfu/mL did not detect the resistant variants . Larger inocula increased the MIC by > 8000-fold . Population analysis showed that rifampicin concentrations above the MIC (measured at an inoculum of approximately 10(5) cfu/mL) select highly resistant variants (MIC > 256 mg/L) when large inocula (> or = 10(5) cfu/mL) were incubated with rifampicin . The resistant variants were stable through ten passages . It was not possible to prevent regrowth of the resistant variants by increasing the rifampicin concentration further . At subinhibitory concentrations there was no development of rifampicin resistance.

Mol Microbiol, 1997 Jun, 24(5), 1013 - 24
Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface; Heilmann C et al.; Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign-body-associated infections . We have characterized two distinct classes of biofilm-negative Tn917 mutants in S . epidermidis affected in initial attachment (class A) or intercellular adhesion (class B) . mut1 (class A mutant) lacks five surface-associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild-type DNA fragment . The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1 . Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment . Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface . DNA-sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148kDa . The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE . By analogy with the S . aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing . The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal- and propeptide . The 45 and 38kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively . Additionally, AtlE was found to exhibit vitronectin-binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during later stages of adherence . Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.

Curr Opin Biotechnol, 1997 Jun, 8(3), 328 - 34
Understanding and advancing wastewater treatment; Stams AJ et al.; In bioreactors used for the purification of wastewater, microorganisms are active in biofilms or aggregates . Insight into the factors that determine the structure and function of aggregated biomass is increasing steadily . Besides conventional techniques, modem molecular techniques are used increasingly to get a better understanding of the complex microbial communities in wastewater treatment systems . In recent years, the combined use of these techniques has led to a good insight into the population dynamics of different types of microbes in bioreactors.

Antimicrob Agents Chemother, 1997 Jun, 41(6), 1352 - 8
Comparison of recalcitrance to ciprofloxacin and levofloxacin exhibited by Pseudomonas aeruginosa bofilms displaying rapid-transport characteristics; Vrany JD et al.; Attenuated total reflection Fourier transform infrared spectroscopy was used to measure transport of the fluoroquinolones (FQs) ciprofloxacin and levofloxacin into Pseudomonas aeruginosa biofilms . Biofilms were exposed to each FQ at dose levels of 100, 250, and 500 microg/ml for 30 min . A mathematical transport model was used to extract the diffusion coefficient, binding site density, and adsorption and desorption rates for each experiment . Recalcitrance of the biofilms toward each FQ was evaluated by comparison of efficacies with planktonic bacteria . By this criterion, biofilms were found to exhibit more recalcitrance toward levofloxacin than ciprofloxacin under the experimental conditions . These results cannot be explained by the more hindered transport of levofloxacin, implicating the domination of physiological factors.

Biochim Biophys Acta, 1997 May 22, 1326(1), 37 - 46
Reactive liposomes encapsulating a glucose oxidase-peroxidase system with antibacterial activity; Hill KJ et al.; Liposomes were prepared from phospholipid mixtures of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylinositol (PI), encapsulating the enzymes glucose oxidase (GO) and GO in combination with horse radish peroxidase (HRP) by both extrusion (VET) and reverse-phase evaporation (REV) . The optimum level of PI in DPPC/PI liposomes for targeting to biofilms of the oral bacterium Streptococcus gordonii has been established . The liposomes were characterised in terms of the content and activity of the encapsulated enzymes . The antibacterial activity of these 'reactive' liposomes arising from hydrogen peroxide and oxyacids in the presence of the substrates glucose and iodide ions, after targeting to the biofilms, were measured both as a function of liposome-biofilm incubation time and incubation time with the substrates . Bacterial inhibition increases with both liposome-biofilm and substrate-biofilm incubation time and with the extent of enzyme encapsulation . The reactive liposomes also display antibacterial activity in the presence of saliva . The reactive liposomes have potential value in the context of oral hygiene.

J Antimicrob Chemother, 1997 May, 39(5), 643 - 6
Influence of N-acetylcysteine on the formation of biofilm by Staphylococcus epidermidis; Perez-Giraldo C et al.; The influence of various concentrations (0.003-8 mg/mL) of N-acetylcysteine on the formation of biofilms by 15 strains of Staphylococcus epidermidis has been studied . A dose-related decrease in biofilm formation was observed, except with the lowest concentrations . The 'slime' index relative to the control was 63%, 55%, 46%, 34%, 26% and 26% in the presence of 0.25, 0.5, 1, 2, 4, and 8 mg/mL of N-acetylcysteine, respectively . These data are statistically significant . The inhibitory effect of 2 mg/mL of N-acetylcysteine on slime formation was also verified by electron microscopy.

J Appl Microbiol, 1997 May, 82(5), 663 - 8
Synergism in biofilm formation between Salmonella enteritidis and a nitrogen-fixing strain of Klebsiella pneumoniae; Jones K et al.; A laboratory reactor, which simulates biofilm formation in water pipes, was used to study interactions in biofilm formation between a nitrogen-fixing strain of Klebsiella pneumoniae and Salmonella enteritidis . The level of attachment of Salm . enteritidis was higher in the binar biofilm than in the single species biofilm . In the initial colonization phase the binary biofilm contained a much higher proportion of metabolically active cells than in single species biofilms formed by either Salm . enteritidis or Kl . pneumoniae . When a pulse of Salm . enteritidis was passed over an already established biofilm of Kl . pneumoniae it rapidly became integrated into the biofilm, from where it was subsequently released into the water column, along with Kl . pneumoniae . Klebsiella pneumoniae fixed nitrogen in the presence of Salm . enteritidis in both types of biofilm.

J Dairy Res, 1997 May, 64(2), 253 - 60
Binding of Staphylococcus aureus to milk fat globules increases resistance to penicillin-G; Ali-Vehmas T et al.; The susceptibility to penicillin-G of Staphylococcus aureus strains that cause mastitis was tested in milk and in Iso-sensitest broth (ISB): The minimal inhibitory concentrations (MIC) of beta-lactamase-positive strains in milk were 10-100-fold those in ISB, whereas the MIC of beta-lactamase-negative strains in milk were some 10-fold those in ISB; beta-lactamase production was induced by milk in beta-lactamase-positive strains . Much of the increase in resistance to penicillin-G caused by milk can be attributed to milk fat globules; the increase in resistance was related to the binding capacity of the bacteria to milk fat globules as well as to capsule formation by the bacteria . It appears that the binding of the staphylococci to the fat globules and bacterial capsule formation resulted in a biofilm type of growth . In this case, the staphylococci behaved differently from the planktonic type of growth in artificial broth medium in which antibiotic susceptibility testing is usually carried out.

J Med Microbiol, 1997 May, 46(5), 425 - 9
Susceptibilities of Actinobacillus actinomycetemcomitans biofilms to oral antiseptics; Thrower Y et al.; The susceptibilities of Actinobacillus actinomycetemcomitans cultures, grown as 1- or 3-day-old biofilms or as planktonic suspensions, to concentrations of chlorhexidine digluconate, cetylpyridinium chloride or triclosan used in commercial mouthwashes were compared . Three-day biofilms were the most resistant form of the organism and chlorhexidine was the most active antiseptic . Comparison of solutions of the pure antibacterial agent with commercial products containing the same concentration of antiseptic showed little difference in in-vitro activities . The results emphasise that the testing of antimicrobial mouthwashes should be performed on bacteria grown as biofilms.

J Appl Microbiol, 1997 Apr, 82(4), 519 - 26
Glutaraldehyde-resistant Mycobacterium chelonae from endoscope washer disinfectors; Griffiths PA et al.; Glutaraldehyde is used to disinfect flexible and other heat-sensitive endoscopes often with the aid of automated systems . Mycobacterium chelonae is being isolated with increasing frequency from these washer disinfectors and processed endoscopes . This has, on occasions, led to misdiagnosis and iatrogenic infections . Recent reports suggest that disinfecting machines, on a sessional or regular basis, with 2% glutaraldehyde may have selected and therefore encouraged the growth of strains of Myco . chelonae, possibly in biofilm, with decreasing susceptibility to glutaraldehyde . In view of this, the resistance of three strains of Myco . chelonae var . chelonae (the type strain NCTC 946 and two machine isolates) was tested against 2% glutaraldehyde and a wide range of alternative disinfectants . Disinfectants tested were a chlorine releasing agent, sodium dichloroisocyanurate at 1000 ppm and 10,000 ppm av Cl, 0.35% peracetic acid (NuCidex, Johnson & Johnson), 70% industrial methylated spirit (IMS), 1% peroxygen compound ('Virkon', Antec International) and 10% succine dialdehyde ('Gigasept', Sanofi Winthrop) . Suspension and carrier tests were carried out in the presence and absence of an organic load . Results showed the type strain, which had not been exposed to the selective pressure of disinfectant usage, to be very sensitive to most disinfectants with the exception of 1% Virkon . The washer disinfector isolates, on the other hand, were extremely resistant to 2% glutaraldehyde and showed greater resistance to 1% Virkon and 1000 ppm NaDCC . Purchasing machines in which the entire fluid pathways, including those for delivering rinse water, are disinfected with an appropriate agent during each cycle are preferred . If this is not possible then sessional cleaning and disinfection at the start of each day and regular maintenance should prevent biofilm formation and contamination with disinfectant-resistant strains of mycobacteria . In addition to machine disinfection, the use of sterile or bacteria-free (filtered < 0.45 microm) water is essential for bronchoscopes and all invasive endoscopes . If there is doubt that the effectiveness of the machine disinfection procedure or water quality, the channels and surfaces of endoscopes may be rinsed with 70% IMS after automated processing.

Nutrition, 1997 Apr, 13(4 Suppl), 5S - 9S
Microbial interactions with catheter material; Lewis WJ et al.; The use of central venous catheters to deliver parenteral nutrition therapy is often complicated by infection . The original source of these infections has been debated but it appears that organisms colonizing the skin or those contaminating the catheter hub are most often responsible . Before forming a biofilm, an organism must first successfully attach to a surface . To do this, microbes have evolved strategies that allow them to adhere to surfaces and evade forces that would favor their detachment . Once a biofilm is formed on a catheter, the organisms are relatively safe from a host immune response and antibiotics . In this review, what is known about these interactions is discussed.

Kansenshogaku Zasshi, 1997 Apr, 71(4), 337 - 41
{Efficacy of erythromycin inhalation in chronic respiratory infection caused by Pseudomonas aeruginosa}; Yanagihara K et al.; The prognosis of chronic respiratory tract infection with Pseudomonas aeruginosad considered to be poor . However, low-dose and long-term 14 membered macrolide, such as erythromycin or clarithromycin, treatment has been reported as effective clinically in chronic lower respiratory tract disease . There was no report to investigate the effect of macrolide on chronic biofilm related Pseudomonas aeruginosa respiratory tract infection in vivo . In a newly established murine model of chronic Pseudomonas aeruginosa respiratory infection mimicking diffuse panbronchiolitis (DPB), we investigated the effect of erythromcin inhalation on viable bacteria in the lungs . Infection was produced by placement of a plastic tube in the bronchus with inoculating . Pseudomonas aeruginosa suspended in saline was also inoculated in bronchus after intubation of the tube . Viable bacteria were constantly isolated at 10(4) 10(6) cfu-specimen from the lungs for more than 30 days . Treatment with erythromycin inhalation for four days reduced the number of viable bacteria in the lungs with statistically significant . Our result indicated that our new model of chronic respiratory tract infection is simple and provides a useful tool to study the pathogenic process and treatment of such infection . Our results also suggest that the erythromycin inhalation is effective on chronic Pseudomons aeruginosa respiratory infection.

Appl Microbiol Biotechnol, 1997 Apr, 47(4), 352 - 7
Immobilisation of whole bacterial cells for anaerobic biotransformations; Raihan S et al.; Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction . A flow-rate/activity plot was constructed for each flow-through reactor and RA1/2 values (residence time corresponding to 50% nitrite removal) calculated for each reactor type . Cells immobilised in flat and hollow-fibre membranes were the most effective (RA1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50% nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively . Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors . Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively) . Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively).

Pharm Res, 1997 Apr, 14(4), 503 - 7
The effect of intestinal bacteria adherence on drug diffusion through solid films under stationary conditions; Rubinstein A et al.; PURPOSE: To study the in vitro and in vivo the role of surface bacterial adhesion on the diffusion of model drugs at stationary conditions . METHODS: Salicylic acid (SA) diffusion through ethyl cellulose (EC) films was measured in vitro in side-by-side diffusion cells with and without E . coli of intestinal origin . Insulin (I) release from paper strips coated or uncoated with pectin films, with or without antibiotic treatment, was measured in vivo in conscious rats after cecal implantation by comparing blood glucose levels at Tmax of the pharmacodynamic effect . RESULTS: During five hours of diffusion studies which were performed immediately following incubation of EC films with bacteria, the diffusion rate of SA throughout the films was 2.72-fold lower in the presence of bacteria compared with the diffusion rate in the control studies conducted without bacteria . The mean blood glucose levels dropped in the rat to 40.6 +/- 21.6% of glucose basal levels within 2.4 +/- 1.4 h when uncoated I solid carriers were used . Glucose levels did not change for pectin-coated dosage forms . After antibiotic treatment which prevented the formation of bacterial biofilm on the surface of the I solid dosage forms, blood glucose levels dropped to 22.0 +/- 4.7% and 50.9 +/- 20.5% of glucose basal levels within 7.4 +/- 2.6 h and 1.8 +/- 0.9 h for pectin uncoated or coated dosage forms, respectively . Maximum bacterial adherence occurred at stationary conditions (RPM = 0), while at maximum agitation (200 RPM), almost no adherence occurred . CONCLUSIONS: (a) Bacterial adherence shows down the diffusion rate of SA through EC films; (b) Under stationary conditions bacterial adherence may also interfere with drug release from biodegradable (pectin) films; (c) Successful functioning of biodegradable colon-specific delivery systems depends on agitation and surface friction in the lumen of the colon.

Int J Food Microbiol, 1997 Mar 3, 34(3), 197 - 207
Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage; Hibma AM et al.; Phage breeding was employed to produce a bacteriophage (Listeria monocytogenes phage ATCC 23074-B1) which was specific for L-forms of L . monocytogenes . The bred phage was compared to its unbred parent for lytic activity and specificity . It was also tested for its ability to prevent L-form biofilm formation on stainless steel and compared with an organic acid (lactic) at L-form biofilm inactivation on stainless steel . The bred phage lysed only L-forms of L . monocytogenes in broth culture and only plaqued on L-form lawns . Likewise, the unbred phage performed similarly with classical cell-walled culture and lawns . The bred phage successfully inhibited L-form biofilm formation on stainless steel and was as successful as lactic acid (130 ppm) at inactivating pre-formed L-form biofilms . Both reduced viable cell numbers by 3-long cycles over a 6 h period . It appears that phage breeding technology may be an attractive alternative to chemical sanitizers which lack specificity and can be toxic.

Chemotherapy, 1997 Mar-Apr, 43(2), 137 - 41
Effect of the growth rate of Pseudomonas aeruginosa biofilms on the susceptibility to antimicrobial agents; Shigeta M et al.; The growth rate of biofilm cells of a leucine-requiring mutant Pseudomonas aeruginosa HU1 was regulated by the leucine concentration in the chemically defined medium, and the effect of the growth rate of biofilm cells on the antimicrobial activities of the antimicrobial agents piperacillin (PIPC), imipenem (IPM) and ofloxacin (OFLX) were evaluated . PIPC showed little effect on the biofilm bacteria regardless of the leucine concentration in the medium . IPM showed weak bactericidal activity to biofilm cells; activity was greater in younger biofilm cells growing in high concentrations of leucine . On the other hand, OFLX revealed strong bactericidal activity to biofilm bacteria regardless of the growth rate . Our data suggest that the bactericidal action of antimicrobial agents to biofilm bacteria is different from that to planktonic bacteria . The bioassay using mutants with regulated growth is useful for the evaluation of the efficacy of antimicrobial agents against biofilm bacteria.

Lett Appl Microbiol, 1997 Mar, 24(3), 177 - 9
Elimination of adhering bacteria from surfaces by pulsed laser beams; Sadoudi AK et al.; As an alternative to the use of chemicals for cleaning and disinfecting surfaces of equipment in food industry, the efficacy of pulsed laser beams for removal and killing of adherent bacteria from stainless steel surfaces was assessed . Escherichia coli biofilms were produced under dynamic conditions in diluted nutritive medium incubated at 37 degrees C for 24 h . Influence of energy density and number of shots were tested at three wavelengths (1064, 532 and 355 nm) . With one 20 ns pulse, results range from 3.5 decimal reductions of the microbial load with < or = 50 MW cm-2 without visible alteration of the surface, to more than 6 decimal reductions with < or = 600 MW cm-2 . The measured effect was largely attributed to removal of the micro-organisms and transfer to the surrounding air . The treatment could therefore be improved with respect to the numbers remaining associated with the surface by venting.

Infect Immun, 1997 Mar, 65(3), 890 - 6
Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates; Ziebuhr W et al.; Staphylococcus epidermidis is a common cause of catheter-associated infections and septicemia in immunocompromised patients . To answer the question whether S . epidermidis skin isolates differ from isolates causing septicemic diseases, 51 strains obtained from blood cultures, 1 strain from shunt-associated meningitis, and 36 saprophytic isolates were characterized . The study demonstrates that most of the blood culture strains formed a multilayered biofilm on plastic material, whereas skin and mucosal isolates did not . Moreover, biofilm-producing strains were found to generate large bacterial autoaggregates in liquid culture . Autoaggregation and biofilm formation on polymer surfaces was associated with the presence of a DNA sequence encoding an intercellular adhesion gene cluster (ica) that mediates the production of a polysaccharide intercellular adhesin . The presence of the intercellular adhesion genes in blood culture isolates was also found to be correlated with the exhibition of black colonies on Congo red agar, whereas the adhesin-negative strains formed red colonies . Upon subcultivation on Congo red agar, the black colony forms of the blood culture strains exhibited red colony variants which were biofilm and autoaggregation negative and occurred at a frequency of 10(-5) . The DNA analysis of these S . epidermidis variants by pulsed-field gel electrophoresis and Southern hybridization with an ica-specific gene probe revealed no detectable difference between the black and red colony types . Moreover, after repeated passage, the phenotype of the parent strain could be restored . Therefore, these colony forms were regarded as phase variants . This phenotypic change was observed exclusively in adhesin-positive clinical isolates and not in adhesin-negative saprophytic strains of S . epidermidis.

Community Dent Oral Epidemiol, 1997 Feb, 25(1), 126 - 34
Promoting changes in clinical practice . Treatment time and outcome studies in a Danish public child dental health clinic; Thylstrup A et al.; This paper examines the characteristics of the process of innovation as applied to provision of dental health to children . The data are from a demonstration project conducted in a Danish public child dental health clinic to evaluate the appropriateness and effectiveness of an individualized and alternative, mainly non-operative caries treatment strategy that was implemented in 1987 . The aim of the strategy was to avoid operative intervention and sealants, and was based on three principles: caries is a localized disease; intensive training in home-based treatment, and individualized risk assessments and controls in conjunction with professional plaque removal . The major influence on the innovation process appeared to be personal education of the personnel, active participation in research, and feedback from clinical observations . In 1992 an additional feedback measure was introduced addressing the question: how much time does the individual child spend on treatment during one year? Mean total treatment time varied from 12 min 1- to 4-yr-olds to 44-58 min for 7- to 17-yr-olds . About 60% of the total treatment time was used on non-operative caries treatment (diagnosis, risk assessment, plaque removal, toothbrushing training) and about 25% on operative caries treatment for 3- to 17-yr-olds . The 1-2-yr-olds required only non-operative treatment . The new treatment strategy reduced operative procedures by 60% . The paper introduces an easily applicable measure of treatment outcome in terms of teeth that have not been restored or sealed as a result of regular professional treatment or care . The main conclusion drawn is that the dominant moving forces in the innovation process were professional responsibility and self-adjustment by a constant learning process involving clinical feedback measures in conjunction with modern understanding of caries as a localized disease caused by local evolution of microbial cariogenic biofilms.

Community Dent Oral Epidemiol, 1997 Feb, 25(1), 76 - 81
Does assessment of microbial composition of plaque/saliva allow for diagnosis of disease activity of individuals?
Bowden GH.
Microbiological tests are limited in their applicability in the assessment of caries activity and in caries prediction . They can be effective in group of persons with high or low caries experience . The reasons for the limitation of microbiological tests rests with unique characteristics of the microflora and local environments of the oral cavity, which will modify the cariogenicity of plaque in an individual . Thus, high numbers of S . mutans may be associated with the development of a lesion at a site, while a second susceptible site with high levels of this organism in the same subject will remain caries free . This paper identifies some aspects of oral bacteria which can contribute to the unique nature of the microflora associated with plaque in an individual . Firstly, the range of bacteria potentially involved in caries has widened and now includes, for example, 'low pH' non-mutans streptococci . The presence of such organisms in plaque in an individual may influence early enamel demineralization . Most significantly, Streptococcus mutans, Streptococcus mitis and Actinomyces naeslundii have been shown to be comprise many distinct clones, with different distribution among subjects . Little is known of the impact of clonal diversity on caries activity but in some bacterial diseases particular clones are associated with virulence . Therefore, possession of a particular clone or clones by an individual could be related to caries activity . Also, the extent of clonal diversity may reflect the nature of the oral environment . Recent studies suggest that cells are released from biofilms, during adherence and growth, i.e . the early phases of development . Thus, determination of the numbers of a given species in non-stimulated saliva may indicate whether it is actively growing in plaque . Microbiological tests on the oral flora should perhaps be used to monitor the status of the oral cavity, after establishing a norm for the individual patient . Research on species and clonal diversity of oral bacteria among human populations; diversity and its role in the caries process; and the liberation of biofilm cells could provide data to allow better appreciation and evaluation of the results of microbiological testing.

Infect Immun, 1997 Feb, 65(2), 519 - 24
A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces; Hussain M et al.; Two distinct pathogenic mechanisms, adhesion to polymer surfaces and subsequent accumulation of sessile bacterial cells, are considered important pathogenic steps in foreign body infections caused by Staphylococcus epidermidis . By using mitomycin mutagenesis, we have recently generated a mutant, strain M7, from S . epidermidis RP62A which is unaffected in adhesion but deficient in accumulation on glass or polystyrene surfaces and lacks a 115-kDa extracellular protein (designated the 140-kDa antigen; F . Schumacher-Perdreau, C . Heilmann, G . Peters, F . Gotz, and G . Pulverer, FEMS Microbiol . Lett . 117:71-78, 1994) . To evaluate the role of this protein in accumulation, we harvested extracellular proteins from S . epidermidis RP62A grown on dialysis membranes placed over chemically defined medium, purified the protein by using ion-exchange chromatography, determined its N-terminal amino acid sequence, and raised antiserum in rabbits . The antibody recognized only a single band in a Western immunoblot of the crude extracellular extract . With the microtiter biofilm test, antiserum at a dilution of < or =1:1,000 blocked accumulation of RP62A up to 98% whereas preimmune serum did not . The 140-kDa antigen was found only in extracellular products from bacteria grown under sessile conditions . Of 58 coagulase-negative clinical isolates, 32 strains were 140-kDa antigen positive and produced significantly larger amounts of biofilm than the 26 strains that were 140-kDa antigen negative . The 140-kDa protein appears to be biochemically and functionally unrelated to any previously described factors associated with biofilm formation . Thus, the 140-kDa antigen, referred to as accumulation-associated protein, may be a factor essential in S . epidermidis accumulation and, due to its immunogenicity, may allow the development of novel immunotherapeutic strategies for prevention of foreign body infection.

Eur Arch Otorhinolaryngol, 1997, 254(6), 261 - 3
A new method for in vivo evaluation of biofilms on surface-modified silicone rubber voice prostheses; Everaert EP et al.; A new method is presented that permits a rapid and accurate in vivo evaluation of biofilm formation on surface-modified silicone rubber voice prostheses . The method is based on partial modification of a Groningen button voice prosthesis by exposing half of the prosthesis to an argon plasma . This results in one side of the prosthesis becoming hydrophilic while leaving the unmodified side hydrophobic as a control . Modified prostheses were placed in patients for an evaluation period of approximately 4 weeks . Despite making the silicone rubber surface hydrophilic, biofilm formation was stimulated when compared to unmodified, hydrophobic silicone rubber . Findings show that biofilm formation on voice prostheses is influenced by hydrophobicity of a silicone rubber surface . The method of partial surface modification used was seen to be suitable for demonstrating such influences regardless of nutrition and other variations in the patient's lifestyle . Microbiological analysis of the biofilms on both sides of the prosthesis valve did not show any changes in microbial composition, with Candida albicans, streptococci and staphylococci being the most commonly isolated strains.

Ann Pharm Fr, 1997, 55(2), 49 - 54
{Bacterial biofilms and resistance to disinfectants}; Vidal DR et al.; When bacteria grow in close association with solid surface, they constitute a microbial community tight included in the exopolymer glycocalyx . Many laboratory studies have shown that these bacteria are 10 to 100 folds more resistant to disinfectants than the bacteria of the same strain in suspension . Several factors are responsible for this resistance: the glycocalyx which limits the diffusion and reacts with the disinfectant, the more or less dense repartition of the bacteria inside the biofilm, their physiologic state with reduced metabolism, and the surface on which is the biofilm . The activity assessment of disinfectant agents is achieved with standardized methods . They must take into account not only the conditions in which the disinfectants are employed, but also the micro-organism state . Experimental results showing the resistance of biofilm bacteria must lead to elaborate methods allowing the assessment of bactericide activity of disinfectants against biofilm bacteria.

Caries Res, 1997, 31(3), 194 - 200
A method for the quantitative site-specific study of the biochemistry within dental plaque biofilms formed in vivo; Robinson C et al.; The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ . A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro . Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources . Analytical data can be related to plaque volume rather than weight . Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.

Int Orthop, 1997, 21(1), 46 - 51
Adherence of Staphylococcus aureus slime-producing strain variants to biomaterials used in orthopaedic surgery; Gracia E et al.; The adherence of Staphylococcus aureus to biomaterials used in orthopaedic surgery (polymethylmethacrylate, fresh bone, steel and titanium alloys) and to glass was studied in vitro at 1, 2, 6, 24 and 48 h of incubation . Nonslime-producing strains (72, 80 and 510) and slime-producing variants of these strains were used . An automated and fast method of ATP-bioluminiscence was applied to determine bacterial viability . The lowest adherence corresponded to polymethylmethacrylate and bone, and the highest to metals . Significant adherence was detected in all cases after 6 h and was strain dependent, being lowest for strain 72 . In most cases, adherence of nonslime-producing variants was not significant compared with controls, and slime-producing were more adherent than nonslime-producing variants . These differences were maximal at 6 h or 48 h, depending on the strain and the material . The findings suggest that the appearance of slime-producing cells within a given nonslime-producing bacterial population may jeopardise postoperative immune systems and antibiotic efficacy as a consequence of biofilm formation on implants and prostheses.

Ann Biomed Eng, 1997 Jan-Feb, 25(1), 69 - 76
The effect of ultrasonic frequency upon enhanced killing of P . aeruginosa biofilms; Qian Z et al.; It is widely recognized that the bacteria sequestered in a biofilm on a medical implant are much more resistant to antibiotics than their planktonic counterparts . Recent studies have shown that application of antibiotic along with low power ultrasound significantly increases the killing of planktonic bacteria by the antibiotic . Herein is reported a similar application of antibiotic and ultrasound to sessile bacteria in biofilms of Pseudomonas aeruginosa on a polyethylene substrate . Biofilm viability was measured after exposure to 12 micrograms/ml gentamicin sulfate and 10 mW/cm2 ultrasound at frequencies of 70 kHz, 500 kHz, 2.25 MHz, and 10 MHz . The results indicate that a significantly greater fraction of the bacteria was killed by gentamicin when they were subjected to ultrasound . However, ultrasound by itself did not have any deleterious effect on the biofilm viability . In addition, lower-frequency insonation is significantly more effective than higher frequency in reducing bacterial viability within the biofilm . The possible mechanisms of synergistic action are discussed.

ASAIO J, 1997 Jan-Feb, 43(1), 8 - 12
In vitro and in vivo effects of rifampin on Staphylococcus epidermidis graft infections; Garrison JR Jr et al.; Rifampin, bound in high concentrations to prosthetic grafts, has been proposed for the treatment of vascular graft infections . The optimum antibiotic concentration and duration of treatment for infected grafts is not known . This study compared the in vitro and in vivo efficacy of varying concentrations of rifampin against three different strains of slime producing Staphylococcus epidermidis (RP62A, KC2, and KB1) bound to the knitted Dacron at high and low concentrations at 10(4) and 10(7) CFU/cm2 of prosthetic . Time kill experiments were performed at 4, 18, and 42 hr, in which each Dacron bound bacterial strain was exposed in vitro to 4X, 64X, 100X, and 1,000X minimum inhibitory concentration (MIC) of rifampin . In vitro, the Dacron bound laboratory strain RP62A was implanted subcutaneously in the backs of male Swiss-Webster mice and exposed to 4X, 100X, and 1,000X the MIC of rifampin for similar time periods . In addition, systemic vancomycin (10 mg/kg) was assessed for synergy and prevention of rifampin resistance . In vitro, all concentrations of rifampin showed near total killing (< 1 log) of all bacterial strains at low initial concentrations (10(4) CFU/cm2) but not high (10(7) CFU/cm2) to 42 hr . Importantly, resistance was shown to develop in all three strains of S . epidermidis with high initial bacterial biofilm concentrations . In vivo, rifampin concentrations between 4X MIC and 100X MIC achieved a balance between optimal killing and prevention of resistance . Systemic vancomycin slightly improved bacterial clearance but did not alter the development of rifampin resistance at high local concentrations . Caution is advised with the use of antibiotic bonded grafts because resistance may develop even with the addition of systemic antibiotics.

Ophthalmologica, 1997, 211 Suppl 1, 9 - 14
A new perspective in ocular infection and the role of antibiotics; Miyanaga Y; A new concept of ocular infectious diseases includes two modes of pathogenesis, one being an acute inflammation caused by the attachment of free-floating bacteria, and another being intractable biofilm-related ocular infections . 15-20% of Staphylococcus epidermidis strains isolated from human conjunctiva exhibit slime formation and can be pathogens causing inflammation . Topical application, in the form of, for example, eye drops, of a combination of two antimicrobial agents is usually applied to treat such infections . Any antimicrobial agent having a post-antibiotic effect is generally administered in a twice-a-day regimen.

Appl Microbiol Biotechnol, 1997 Jan, 47(1), 18 - 22
Electrochemical disinfection of drinking water using an activated-carbon-fiber reactor capable of monitoring its microbial fouling; Okochi M et al.; An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water . The application of an alternating potential of 1.0 V and -0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h . Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately . The bacterial cell density in treated water was always been less than 20 cells/ml . It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling.

Biomaterials, 1997 Jan, 18(1), 87 - 91
Adhesion of Lactobacillus species in urine and phosphate buffer to silicone rubber and glass under flow; Millsap KW et al.; Coating uroepithelial cells or catheter materials with lactobacilli has been shown to retard the development of a uropathogenic biofilm, with biosurfactant production and strong adhesion being two prerequisite properties of the Lactobacillus strains to be employed . In this paper, adhesion of six selected Lactobacillus strains to silicone rubber and glass in urine and in a phosphate buffer was studied using a parallel plate flow chamber . In addition, adhesive cell surface properties of the lactobacilli, i.e . the pH dependences of their zeta potentials and their hydrophobicities by water contact angles, were determined . L . acidophilus ATCC 4356 and L . fermentum B54 were the only strains showing significant adhesion to both hydrophobic silicone rubber and hydrophilic glass, possibly by virtue of their high cell surface hydrophobicities (water contact angles of 68 and 75 degrees, respectively) and small zeta potentials (-10.0 and -8.1 mV in buffer, respectively) . Both hydrophobic Lactobacillus strains adhered less well in urine than in buffer . The remaining Lactobacillus strains studied were hydrophilic, with water contact angles between 25 and 36 degrees, and had highly negative zeta potentials, reaching -37.7 mV in buffer . Adhesion of these highly negatively charged, hydrophilic strains in buffer was essentially absent, while for some of these strains minor adhesion in urine was observed . This study demonstrates that the adhesion of lactobacilli to substrata differs with strain hydrophobicity and charge, and that urinary components can affect the ability of hydrophilic Lactobacillus strains to adhere to substrata.

Biomaterials, 1997 Jan, 18(1), 53 - 7
Corrosion of the intra-oral magnets by multi-species biofilms in the presence and absence of sucrose; Wilson M et al.; The purpose of this study was to determine the corrosive effects of multi-species biofilms on intra-oral magnets in the presence and absence of sucrose . Using pooled human saliva as an inoculum, biofilms were grown on the surfaces of 90 neodymium-iron-boron (Nd2Fe14B) magnets in a constant depth film fermentor under aerobic conditions at 37 degrees C . The fluid phase was a mucin-containing artificial saliva (delivered at a rate of 0.72/litres day-1), and, after 15 days, 100 ml of 10% (w/v) sucrose was added (as three pulses of 33.3 ml) each day for a further 15 days . Six magnets with attached biofilms were removed periodically . On each sampling occasion the numbers of aerobes, anaerobes, streptococci, veillonellae and actinomyces in each biofilm, the pH of the fermentor effluent and the dry mass of the magnets were determined . Addition of sucrose to the fermentor resulted in a fall in pH (from a mean of 6.94 to a mean of 4.96), an increase in the proportion of streptococci and a decrease in the proportion of veillonellae comprising the biofilms . The decrease in mass of the magnets was 28-fold greater in the presence of sucrose than in its absence . The results of this study have shown that the presence of sucrose affects the microbial composition of multi-species biofilms growing on Nd2Fe14B magnets and results in a marked increase in corrosion of the magnets.

Appl Environ Microbiol, 1997 Jan, 63(1), 270 - 6
Succession and convergence of biofilm communities in fixed-film reactors treating aromatic hydrocarbons in groundwater; Massol-Deya A et al.; Community composition, succession, and performance were compared in three fluidized bed reactors (FBR) operated to test preemptive colonization and the influence of toluene compared with a mixture of benzene, toluene, and p-xylene (BTX) as feeds . One reactor was inoculated with toluene-degrading strains Pseudomonas putida PaW1, Burkholderia cepacia G4, and B . pickettii PKO1 . PaW1 outcompeted the other two strains . When groundwater strains were allowed to challenge the steady-state biofilm developed by inoculated strains, they readily displaced the inoculated strains and further reduced the toluene effluent concentration from 0.140 to 0.063 mg/liter for 98% removal . Amplified ribosomal DNA restriction analysis (ARDRA) of reactor community DNA showed a succession of populations to a pattern that was stable for at least 4 months of operation . Parallel reactors fed toluene and BTX but inoculated directly from groundwater had the same treatment performance and the same ARDRA profiles as each other and as the seeded reactor once the groundwater community took over . Convergence and stability of populations were confirmed by genotype analysis of 120 isolates taken from all reactors and at several times . Ninety percent of the isolates were of 4 of the 12 genotypes found, and their ARDRA patterns accounted for most of the community ARDRA patterns . Estimates of the maximum specific growth rates (mu max), half-saturation constants (K(m)), and maximum substrate utilization rates (Vmax) of the 12 genotypes isolated revealed a rather high diversity of toluene use kinetics even though the toluene in the feed was constant . The climax populations, however, generally showed kinetic parameters indicative of greater competitiveness than the inocula . rRNA sequence analysis of three codominant strains showed them to be members of the alpha, beta, and gamma subdivisions of the Proteobacteria . Two were similar to Comamonas and Pseudomonas putida, but the member of the alpha group was somewhat distant from any organism in the rRNA database . The convergence of communities to the same composition from three different starting conditions and their constancy over several months suggests that a rather stable community was selected.

J Gastroenterol, 1996 Dec, 31(6), 836 - 43
Bacterial adhesion on hydrophilic heparinized catheters, with compared with adhesion on silicone catheters, in patients with malignant obstructive jaundice; Homma H et al.; To study the inhibitory effects on bacterial adhesion of a newly devised, hydrophilic heparinized catheter to be used in patients with malignant obstructive jaundice, a randomized controlled study of indwelling endoprostheses was performed, using implantable port-connected heparinized catheters (n = 25) and silicone catheters (n = 21) . Catheters withdrawn from patients were cultured for bacteria and examined by electron microscopy for the presence of adherent organisms . In vitro examination of the two type of catheters exposed to suspensions of Eschericia coli and Staphylococcus aureus was performed using electron microscopy and a luminometer . The formation of a biofilm coated with glycocalyces was found in silicone catheters, but not in the heparinized catheters . In vitro experiments demonstrated little bacterial adhesion to the heparinized surface, but significant formation of biofilm on the silicone surface . Anionically charged heparinized catheters have inhibitory effects on bacterial adhesion, and the surface charge of the catheter may be a factor in inhibiting this adhesion.

J Antimicrob Chemother, 1996 Dec, 38(6), 987 - 1000
The efficacy of antibiotics enhanced by electrical currents against Pseudomonas aeruginosa biofilms; Jass J et al.; Low electrical currents are reported to enhance the activity of tobramycin and biocides against biofilm bacteria . We report that the activity of those antibiotics to which the bacterium is susceptible in its planktonic state may be enhanced by a low electrical current against the more resistant biofilm cells . Pseudomonas aeruginosa biofilms were formed on a dialysis membrane suspended between two parallel electrode plates in an electrical colonisation cell . Ciprofloxacin, polymyxin B and piperacillin were tested on biofilms at ten times their MIC for 12 h in the presence of 0 (control) and 9 mA/cm2 current density . At these concentrations the antibiotics alone reduced the biofilm population, but in the presence of an electrical current the population was further reduced by ciprofloxacin and polymyxin B though not by piperacillin . Exposure of the planktonic bacteria to the same concentrations of antibiotics demonstrated that ciprofloxacin and polymyxin B reduced the population to below 1% after 12 h and below 0.1% after 24 h, while piperacillin was bacteriostatic . The results suggest that the electrical current can enhance the activity against biofilms of only those antibiotics that are effective against planktonic cells.

Int J Food Microbiol, 1996 Dec, 33(2-3), 157 - 67
In vivo bioluminescence to detect the attachment of L-forms of Listeria monocytogenes to food and clinical contact surfaces; Hibma AM et al.; A bioluminescent phenotype of Listeria monocytogenes was employed to study the adhesion and biofilm formation of both classical and L-forms to relevant food and clinical contact surfaces . Attachment of both parental (cell-walled) and L-forms to intravenous tubing and stainless steel was compared using viable counts and bioluminescence . Both cell types attached themselves to intravenous tubing and stainless steel . Parental cell numbers attached to intravenous tubing were 1 log higher than L-form numbers, and on stainless steel, attached L-form numbers were 1 log higher . Bioluminescence measurements yielded a pattern similar to viable count measurements . There was a clear relationship between light output of attached bacteria and their viability and, thus, a bioluminescent phenotype provides a rapid monitor of both microbial viability and biofilm formation by L-forms.

J Colloid Interface Sci, 1996 Nov 10, 183(2), 498 - 505
The Use of Microelectrodes to Study the Precipitation of Calcite upon Algal Biofilms
Hartley AM, House WA, Leadbeater BSC, Callow ME.
Calcium and pH ion-selective microelectrodes are used for the first time to measure chemical activity profiles above laboratory-grown algal biofilms (green alga, Chlorococcum sp) . The results are used to calculate the solution composition changes above an actively photosynthesizing biofilm . These changes include the total calcium concentration, degree of supersaturation of the solution with respect to calcite, and the partial pressure of carbon dioxide . Measurements are made in a flow-cell, which was designed to hold the templates on which the biofilms are grown . Measurements show pronounced gradients of pH and calcium concentration, within a distance of approximately 500 μm above the biofilm . The high degree of supersaturation caused by the low concentrations of carbon dioxide near the biofilm surface leads to the precipitation of calcite on the biofilm . SEM micrographs illustrate the close association of calcite crystals with the algal cells . Preliminary results are presented of the precipitation rates estimated from the diffusion flux of calcium at the biofilm surface . The results are in broad agreement with the rates expected from calcite seed growth experiments in similar solution conditions.

Braz J Med Biol Res, 1996 Nov, 29(11), 1455 - 9
Biliary and pancreatic stent blockage by bacterial biofilm: presentation of two cases; Brant CQ et al.; Biliary and pancreatic stents are effective tools in the management of obstructive jaundice (both malignant and benign), pancreatic pseudocyst drainage, and as treatment for biliary and pancreatic fistulae . Unfortunately, stents may become blocked and require replacement in a number of patients . In the present study a blocked stent from a patient with transpapillary drainage of pancreatic pseudocyst and another from a patient with obstructive jaundice resulting from cancer of the head of the pancreas associated with Mirizzi syndrome were characterized by electron microscopy . Stent blockage was diagnosed by a pressure test and stent cultures were performed . Electron microscopy of the blocked stents revealed the sludge to consist of microcolonies of bacteria mixed with amorphous material, and cultures of both stents were positive for Klebsiella sp and E . coli.

J Antimicrob Chemother, 1996 Nov, 38(5), 757 - 69
Mathematical model of beta-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released beta-lactamases; Dibdin GH et al.; We present a mathematical model that describes penetration of an antibacterial agent into a bacterial biofilm and, in particular, the penetration of a beta-lactam compound into a biofilm of Pseudomonas aeruginosa . As well as dealing with this penetration, and the consequent bacterial lysis, the model considered diffusion of the released beta-lactamases in the extracellular space and the consequent inactivation there of further incoming antibiotic; it also allowed for any chosen fraction of the total beta-lactamase to be permanently accessible to exogenous substrate . The modelling scheme was validated against analytical solutions under appropriately simplified conditions . Using published experimental data we show here that lysis of cells in the surface layers of a film could have an important protective effect on the viability of underlying bacteria, especially in thick biofilms.

Curr Eye Res, 1996 Nov, 15(11), 1092 - 100
The effect of eye closure on protein and complement deposition on Group IV hydrogel contact lenses: relationship to tear flow dynamics; Sack RA et al.; PURPOSE: This study was designed to determine the effect of overnight eye closure on the rate and composition of protein deposition on high water content ionic matrix soft contact lenses (Group IV SCLs) and to extrapolate from this data information on the probable change in the rate of reflex-type tear secretion associated with eye closure . METHODS: Group IV SCLs were temporally sampled after equivalent periods of wear under closed eye (C) or open eye (O) conditions . Lenses were rinsed in saline and the majority of the tightly bound protein extracted at 90 degrees C in 40% urea, containing 1% SDS, 1 mM DTT, 100 mM Tris-HCl (pH 8.00) . Residual protein was determined by Coomassie staining of the extracted lenses and densitometric analysis . Extracted protein was quantitated and separated by SDS-PAGE . Gels were either stained with Coomassie blue or reversibly stained with imidazole-zinc and blotted . Blots were PAS stained, or lectin and antibody probed for glycoproteins, secretory IgA (sIgA), IgG, lysozyme and complement C3 . Laboratory simulated deposition studies were carried out on unworn lenses exposed to HPLC purified lysozyme . RESULTS: The protein in the saline rinse, to a large degree mirrored the composition of tear fluid in which the lens had been residing (O or C) . This would suggest that the saline wash consists of residual tear fluid and loosely adherent protein . In contrast, the urea extracts were highly homogeneous consisting primarily of lysozyme and to lesser extent lysozyme dimer . This supports the contention that the Group IV SCL functions in the eye much as cationic exchange resin selectively absorbing lysozyme . C extracts also proved relatively enriched in trace amounts of sIgA, IgG and complement C3 and its breakdown products . High levels of C3 and C3 breakdown products were specifically recovered only in the C worn lens extracts from a subject experiencing unilateral contact lens associated corneal infiltrates from the affected eye . In all subjects, markedly less protein (lysozyme) was recovered in urea extracts of lenses exposed to 7-8 h of closed eye as compared to open eye wear (0.20 +/- .08 versus 0.79 +/- .15 mg/lens (n = 6)) . Temporal studies further revealed that deposition was linearly related to duration of wear during the initial phase of conditioning film formation giving rise to rate constants for lysozyme deposition of 2.2 +/- 0.29 (n = 5) and 0.20 +/- 0.06 microgram/min (n = 4) under open and closed eye conditions respectively . With further wear, deposition eventually reached a steady state . Under laboratory conditions, lysozyme was much rapidly and quantitatively removed from solution in a manner following a hyperbolic plot . This suggests that during the initial phase of deposition the rate of deposition is limited by the capacity of the tear fluid to deliver lysozyme to the lens surface under these two extremes of conditions . CONCLUSIONS: Eye closure profoundly affects the rate of lysozyme deposition on Group IV hydrogels and the composition of minor biofilm constituents in a manner that could affect biocompatibility . Findings support the contention that eye closure results in a > 90% reduction in the rate of reflex-type tear secretion.

Antimicrob Agents Chemother, 1996 Nov, 40(11), 2517 - 22
Theoretical aspects of antibiotic diffusion into microbial biofilms; Stewart PS; Antibiotic penetration into microbial biofilm was investigated theoretically by the solution of mathematical equations describing various combinations of the processes of diffusion, sorption, and reaction . Unsteady material balances on the antibiotic and on a reactive or sorptive biomass constituent, along with associated boundary and initial conditions, constitute the mathematical formulations . Five cases were examined: diffusion of a noninteracting solute; diffusion of a reversibly sorbing, nonreacting solute; diffusion of an irreversibly sorbing, nonreacting solute; diffusion of a stoichiometrically reacting solute; and diffusion of a catalytically reacting solute . A noninteracting solute was predicted to penetrate biofilms of up to 1 mm in thickness relatively quickly, within a matter of seconds or minutes . In the case of a solute that does not sorb or react in the biofilm, therefore, the diffusion barrier is not nearly large enough to account for the reduced susceptibility of biofilms to antibiotics . Reversible and irreversible sorption retards antibiotic penetration . On the basis of data available in the literature at this point, the extent of retardation of antibiotic diffusion due to sorption does not appear to be sufficient to account for reduced biofilm susceptibility . A catalytic (e.g., enzymatic) reaction, provided it is sufficiently rapid, can lead to severe antibiotic penetration failure . For example, calculation of beta-lactam penetration indicated that the reaction-diffusion mechanism may be a viable explanation for failure of certain of these agents to control biofilm infections . The theory presented in this study provides a framework for the design and analysis of experiments to test these mechanisms of reduced biofilm susceptibility to antibiotics.

Clin Orthop, 1996 Nov, (332), 184 - 9
Efficacy of antibiotics alone for orthopaedic device related infections; Isiklar ZU et al.; Treatment of orthopaedic device related infections with antibiotics alone generally has been thought to be inadequate . A rabbit model was used to compare the efficacy of 4 different antibiotic regimens for treating orthopaedic device related infection caused by slime producing Staphylococcus epidermidis . After bacterial inoculation of a hole drilled through the intercondylar notch, a stainless steel screw was placed into the femur . Two weeks later, rabbits were randomized to receive a 2-week course of antibiotics: (1) 9 rabbits received vancomycin alone; (2) 10 rabbits received minocycline alone; (3) 10 rabbits received vancomycin plus rifampin; and (4) 10 rabbits received minocycline plus rifampin . Quantitative bone cultures were performed, and antibiotic levels in serum, bone, and biofilm were determined . Despite high levels of vancomycin in biofilm, infection was never cured by vancomycin alone and was eradicated in only 20% of rabbits that received minocycline alone . The highest cure rate (90%) was achieved with the combination of vancomycin and rifampin, whereas the combination of minocycline and rifampin yielded a cure rate of 70% . These results encourage the clinical evaluation of the combination of vancomycin and rifampin in patients in whom infected orthopaedic device cannot be removed.

Appl Environ Microbiol, 1996 Nov, 62(11), 4014 - 8
Effect of growth conditions and substratum composition on the persistence of coliforms in mixed-population biofilms; Camper AK et al.; Laboratory reactors operated under oligotrophic conditions were used to evaluate the importance of initial growth rate and substratum composition on the long-term persistence of coliforms in mixed-population biofilms . The inoculum growth rate had a dramatic effect on the ability of coliforms to remain on surfaces . The most slowly grown coliforms (mu = 0.05/h) survived at the highest cell concentration . Antibody staining revealed that Klebsiella pneumoniae existed primarily as discrete microcolonies on the surface . Both coliforms and heterotrophic plate count bacteria were supported in larger numbers on a reactive substratum, mild steel, than on polycarbonate.

Appl Environ Microbiol, 1996 Nov, 62(11), 3954 - 9
Multiparametric analysis of waterline contamination in dental units; Barbeau J et al.; Microbial contamination of dental unit waterlines is thought to be the result of biofilm formation within the small-bore tubing used for these conduits . Systematic sampling of 121 dental units located at the dental school of Universite de Montreal showed that none of the waterlines was spared from bacterial contamination . Multilevel statistical analyses showed significant differences between samples taken at the beginning of the day and samples taken after a 2-min purge . Differences were also found between water from the turbine and the air/water syringe . Random variation occurred mainly between measurements (80%) and to a lesser extent between dental units (20%) . In other analyses, it was observed to take less than 5 days before initial bacterial counts reached a plateau of 2 x 10(5) CFU/ml in newly installed waterlines . Sphyngomonas paucimobilis, Acinetobacter calcoaceticus, Methylobacterium mesophilicum, and Pseudomonas aeruginosa were the predominant isolates . P . aeruginosa showed a nonrandom distribution in dental unit waterlines, since 89.5% of the all the isolates were located in only three of the nine clinics tested . Dental units contaminated by P . aeruginosa showed significantly higher total bacterial counts than the others . By comparison, P . aeruginosa was never isolated in tap water remote from or near the contaminated dental unit waterlines . In conclusion, dental unit waterlines should be considered an aquatic ecosystem in which opportunistic pathogens successfully colonize synthetic surfaces, increasing the concentration of the pathogens in water to potentially dangerous levels . The clinical significance of these findings in relation to routine dental procedures is discussed.

Appl Environ Microbiol, 1996 Nov, 62(11), 3939 - 47
Bacterial plasmolysis as a physical indicator of viability; Korber DR et al.; Bacterial plasmolytic response to osmotic stress was evaluated as a physical indicator of membrane integrity and hence cellular viability . Digital image analysis and either low-magnification dark-field, high-magnification phase-contrast, or confocal laser microscopy, in conjunction with pulse application of a 1.5 M NaCl solution, were used as a rapid, growth-independent method for quantifying the viability of attached biofilm bacteria . Bacteria were considered viable if they were capable of plasmolysis, as quantified by changes in cell area or light scattering . When viable Salmonella enteritidis biofilm cells were exposed to 1.5 M NaCl, an approximately 50% reduction in cell protoplast area (as determined by high-magnification phase-contrast microscopy) was observed . In contrast, heat- and formalin-killed S . enteritidis cells were unresponsive to NaCl treatment . Furthermore, the mean dark-field cell area of a viable, sessile population of Pseudomonas fluorescens cells (approximately 1,100 cells) increased by 50% as a result of salt stress, from 1,035 +/- 162 to 1,588 +/- 284 microns2, because of increased light scattering of the condensed, plasmolyzed cell protoplast . Light scattering of ethanol-killed control biofilm cells underwent little change following salt stress . When the results obtained with scanning confocal laser microscopy and a fluorescent viability probe were compared with the accuracy of plasmolysis as a viability indicator, it was found that the two methods were in close agreement . Used alone or in conjunction with fluorochemical probes, physical indicators of membrane integrity provided a rapid, direct, growth-independent method for determining the viability of biofilm bacteria known to undergo plasmolysis, and this method may have value during efficacy testing of biocides and other antimicrobial agents when nondestructive time course analyses are required.

J Dent Res, 1996 Oct, 75(10), 1779 - 88
Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci; Schupbach P et al.; The protective effects of milk and milk products against dental caries have been demonstrated in many animal studies . We have shown that this effect was mediated by micellar casein or caseinopeptide derivatives . A reduction in the Streptococcus sobrinus population in the oral microbiota of animals fed diets supplemented with these milk components was consistently observed . A possible explanation for these findings is that milk components are incorporated into the salivary pellicle, thereby reducing the adherence of S . sobrinus . This hypothesis was tested in vitro by the incubation of bovine enamel discs with unstimulated saliva . The resulting pellicle was washed and incubated with caseinoglycomacropeptide (CGMP) and/or caseinophosphopeptide (CPP) labeled with 17- and 12-nm gold particles . All samples were prepared for electron microscopy by high-pressure freezing followed by freeze-substitution . It was demonstrated by high-resolution scanning electron microscopy with back-scattered electron imaging, as well as by transmission electron microscopy, that both peptides were incorporated into the pellicle in exchange for albumin, confirming previous findings . This protein was identified with a mouse anti-human serum albumin followed by goat anti-mouse IgG labeled with 25-nm gold particles . Incorporation of CGMP and/or CPP into salivary pellicles reduced the adherence of both S . sobrinus and S . mutans significantly . It is suggested that the calcium and phosphate-rich micellar casein or caseinopeptides are incorporated into the pellicle . The resulting ecological shifts, together with the increased remineralization potential of this biofilm, may explain its modified cariogenic potential.

Br J Urol, 1996 Oct, 78(4), 579 - 88
An assessment of the ability of a silver-releasing device to prevent bacterial contamination of urethral catheter drainage systems; Stickler DJ et al.; OBJECTIVE: To investigate the ability of a silver-releasing device to protect the catheterized bladder from infection by blocking the ascending migration of bacteria from contaminated urine-drainage bags . MATERIALS AND METHODS: A simple physical model of the catheterized bladder and drainage system was used with the device located in the drainage-tube just below the sampling port . Urine was supplied to the model at 1.0 mL/min and the drainage bag was contaminated with Pseudomonas aeruginosa, Escherichia coli and Proteus mirabilis . Over 10 days, urine from the bag, catheter sampling-port and bladder were examined for contamination . RESULTS: Bacteriological analysis showed that the mean time for test organisms to reach the sampling ports of four different control bag systems ranged from 5.7 to 7.7 days . Urine from the sampling ports of the test systems incorporating the silver device remained sterile for 10 days . The device also prevented the growth of the large populations of bacteria (10(8) colony-forming units per mL) that occurred in the control bags . While scanning electron microscopy showed the formation of bacterial biofilm throughout the control drainage systems, no bacterial colonization was visible on the surfaces of the test systems . Chemical analysis established that the devices consistently generated concentrations of silver in urine ranging from 1.0 to 2.0 micrograms/mL . CONCLUSION: The incorporation of the silver-releasing device into the drainage systems produced an antibacterial barrier which protected the catheterized bladder from intraluminal contamination for at least 10 days . These in vitro tests suggest a useful role for the device in controlling infection in patients undergoing short-term indwelling bladder catheterization.

Clin Podiatr Med Surg, 1996 Oct, 13(4), 767 - 91
Infections following implant arthroplasties of the forefoot; Shapiro SE et al.; This article discusses the causes, pathogenesis, diagnosis, prevention, and treatment of infections associated with implant arthroplasties of the forefoot . Topics covered include isolated organisms, routes of infection, bacterial glycocalyx (that is, biofilm or slime layer) production, preoperative evaluation, air filtration systems, surgical technique, use of prophylactic antibiotics, inflammatory reactions (arthritic detritus, metallosis), differential diagnosis, and treatment protocols.

Clin Orthop, 1996 Oct, (331), 146 - 50
Salvage of infected total knee components; McLaren AC et al.; Debridement and retention of components for the treatment of infected total knee replacements has not been effective, with a failure rate of more than 70% . In cases where there are solidly fixed components, particularly with long stems, a method of successfully retaining the components is desirable . A protocol of radical debridement has been developed to eradicate the biofilm related bacteria associated with these infections . Assessment of the interfaces and elimination of all spaces and unsealed interfaces that will allow penetration by bacteria is essential . Four cases of infected total knee replacements are reported without recurrence after a minimum of 18 months . Refinement of the protocol and longer followup on more cases is planned to validate the early results.

Biomaterials, 1996 Oct, 17(20), 1975 - 80
Effect of low-intensity ultrasound upon biofilm structure from confocal scanning laser microscopy observation; Qian Z et al.; Ultrasonic irradiation at 500 kHz and 10 mW cm-2 of a 24 h old biofilm of P . aeruginosa enhanced the killing of bacteria by gentamicin . To determine whether this bioacoustic effect was caused by ultrasonic-induced changes in the biofilm morphology (biofilm breakup or disruption), the biofilms were examined by confocal scanning laser microscopy (CSLM) . Such disruption would be undesirable in the possible ultrasonic treatment of implant infections . The CSLM results showed that the biofilm is a partial monolayer of cells with occasional aggregates of cells, non-cellular materials and extracellular spaces . The aggregates contained large amounts of exopolysaccharide . The structure of biofilm was not changed when the biofilm was exposed to continuous ultrasound at 500 kHz and 10 mW cm-2, the same irradiation parameters that increased cell killing by nearly two orders of magnitude . The observation that low-intensity ultrasound does not disrupt biofilm or disperse the bacteria has significance in the possible use of ultrasound to enhance the action of antibiotics against biofilms.

Epidemiol Infect, 1996 Oct, 117(2), 267 - 80
Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime; Deighton MA et al.; The ability to produce large quantities of biofilm on solid surfaces in vitro is believed to distinguish potentially pathogenic strains of Staphylococcus epidermidis from commensals . Biofilm consists of staphylococcal cells encased in a matrix of extracellular polysaccharide (also referred to as slime), firmly adherent to each other and to the underlying surface structure . The association of slime with colonization of catheter surfaces in vivo has been examined extensively . Less attention has been paid to the contribution of slime to infections that occur in the absence of an inserted device . In a mouse model of subcutaneous infection without an implanted device 10 S . epidermidis strains (5 slime-positive, 5 slime-negative) produced abscesses; thus a foreign body is not essential for the expression of virulence by S . epidermidis . Biofilm-positive strains produced significantly more abscesses, that persisted longer than biofilm-negative strains . In these chronic infections, large numbers of staphylococci were associated with macrophages and viable staphylococci were cultured from specimens of pus collected at autopsy . Thus slime or components of slime appear to delay the clearance of S . epidermidis from host tissues, possibly by interfering with intracellular killing mechanisms . However, differences in the capacity to produce abscesses, within both the slime-positive and slime-negative groups, indicate that other factors also contribute to the virulence of S . epidermidis.

Int J Syst Bacteriol, 1996 Oct, 46(4), 871 - 5
Anaerofilum pentosovorans gen . nov., sp . nov., and Anaerofilum agile sp . nov., two new, strictly anaerobic, mesophilic, acidogenic bacteria from anaerobic bioreactors; Zellner G et al.; Strictly anaerobic, gram-positive, nonsporing, thin rod-shaped organisms whose cells were 0.2 to 0.6 by 3 to 6 microns were isolated from a Hoechst Biohochreaktor (strain FaeT {T = type strain}) and from the biofilm population of a fixed-film reactor treating sour whey (strain FT) . Strain FT was vigorously motile during early logarithmic growth by means of peritrichously inserted flagella, while strain FaeT was seldom motile and usually possessed no flagella . During the stationary growth phase both strains formed spheroplasts . The temperature optimum was close to 37 degrees C (temperature range for growth, > or = 17 to < 45 degrees C) and the pH optimum was 7.0 to 7.4 (pH range, 6.5 to 8.0) for both strains . The two organisms grew chemoorganotrophically on a number of mono- and disaccharides, including glucose and xylose; yeast extract was required for growth . The principal fermentation products from glucose included lactate, acetate, ethanol, formate, and CO2 . Hydrogen was not generated . The G + C contents of the DNAs of strains FaeT and FT were 55 and 54.5 mol%, respectively . The cell wall architecture was typical of gram-positive bacteria; the cells had an extraordinarily thin type A3 alpha' peptidoglycan layer containing muramic acid . Analysis of 16S ribosomal DNA sequences of the two new isolates demonstrated that they represent members of a new genus of bacteria in Clostridium cluster IV of the domain Bacteria and that the misclassified organism Fusobacterium prausnitzii and Clostridium leptum are among their closest relatives . The names Anaerofilum pentosovorans gen . nov., sp . nov . (type strain, strain Fae {= DSM 7168}) and Anaerofilum agile sp . nov . (type strain, strain F {= DSM 4272}) are proposed.

J Infect Dis, 1996 Oct, 174(4), 881 - 4
Association of biofilm production of coagulase-negative staphylococci with expression of a specific polysaccharide intercellular adhesin; Mack D et al.; An association between adherent biofilm production on tissue culture plates and expression of a specific polysaccharide intercellular adhesion (PIA), which is functionally involved in cell clustering, was investigated for 179 Staphylococcus epidermidis isolates . Of the S . epidermidis strains, 50.8% were biofilm producers (A570 of > 0.1) . There was a significant positive association between biofilm production and PIA expression: 86.8% of biofilm-producing S . epidermidis strains produced PIA as detected with a specific coagglutination assay . In contrast, 88.6% of the biofilm-negative isolates did not express PIA (P < .001) . A linear association between the amount of PIA produced as detected by inhibition ELISA and the amount of biofilm produced was established for 49 S . epidermidis strains, representing a continuum from biofilm-negative to strongly biofilm-producing (r = .81, P < .001) . Apparently, PIA is important for biofilm accumulation in the majority of clinical S . epidermidis isolates.

J Am Vet Med Assoc, 1996 Oct 1, 209(7), 1262 - 4
Interlocking intramedullary nail stabilization of a femoral fracture in a dog with osteomyelitis; Muir P et al.; Complications developed in a dog that underwent intramedullary pin and cerclage wire fixation of a comminuted femoral fracture . The fracture was unstable, the intramedullary pin protruded through the skin over the hip, and Staphylococcus sp was isolated from the fracture site . The loose pin was removed, and the fracture was restabilized with a 6.0-mm interlocking intramedullary nail and 3 interlocking screws . Cephalexin was given orally for 6 weeks . Eight weeks after the second surgery, the fracture was healed radiographically . Seventeen months later, the dog had a persistent mild lameness that was associated with low-grade osteomyelitis . The lameness resolved after the implants were removed, and antibiotics were administered . In human beings, interlocking intramedullary nail stabilization is an accepted treatment for open and contaminated fractures . Chronic infection of bone may be attributable to bacteria in biofilm surrounding implants, necessitating implant removal for resolution.

Biochim Biophys Acta, 1996 Sep 4, 1283(2), 207 - 14
The interaction of cationic liposomes with the skin-associated bacterium Staphylococcus epidermidis: effects of ionic strength and temperature; Sanderson NM et al.; Cationic liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol) and stearylamine (SA) . These phospholipid vesicles were exposed to adsorbed biofilms of the skin-associated bacteria Staphylococcus epidermidis, to which they showed a strong affinity . The interaction (as assessed by the apparent monolayer coverage of the biofilms by liposomes) was described in terms of a Langmuir adsorption isotherm which enabled determination of the maximum theoretical coverage of the bacterial surface and association/dissociation constants . The interaction was shown to be dependent on the ionic strength of the surrounding medium; on increasing the ionic strength the biofilm-vesicle dissociation constant decreased . This suggested that the adsorption was mediated by electrostatic effects . The adsorption of the vesicles was examined at various temperatures, enabling determination of thermodynamic parameters for the interaction . The adsorbed state of the liposomes was energetically favoured and the interaction was enthalpy driven . The Gibbs energies of adsorption were in a range from -15 to -19 kJ mol-1 and the enthalpies of adsorption from -26 to -22 kJ mol-1 . Studies using cell populations of different hydrophobicity showed that the hydrophobic character of the bacterial cells also had an effect on the adsorption of the vesicles to the biofilm.

ASAIO J, 1996 Sep-Oct, 42(5), M655 - 60
Effects of plasma on adhesion of biofilm forming Pseudomonas aeruginosa and Staphylococcus epidermidis to fibrin substrate; Benson DE et al.; Bacterial adhesion has been identified as the critical initial step in the pathogenesis of foreign body related infection . Recent investigations have shown microbial binding to implanted polymeric materials using specific adhesion of bacteria to immobilized plasma proteins, such as fibrin . These proteins are though to function as bridging molecules to facilitate bacterial colonization of the surface . The authors' results indicated a significant reduction in adhesion of biofilm forming Pseudomonas aeruginosa and coagulase negative Staphylococcus epidermidis to immobilized fibrin strands in the presence of platelet poor plasma (PPP) as compared to studies performed with phosphate buffered saline and Hank's balanced salt solution . A 10-fold decrease in the number of adherent bacteria was noted for samples exposed to PPP as compared to control samples . The effective range of PPP concentrations capable of producing the marked decrease in binding to fibrin strands was determined to be 1-100% for P . aeruginosa and 4-100% for S . epidermidis.

Microbiologia, 1996 Sep, 12(3), 347 - 58
Are bacterial biofilms constrained to Darwin's concept of evolution through natural selection?
Caldwell DE, Costerton JW.
Numerous antimicrobial agents have been developed which act at the molecular, cellular, and organismal levels . However, few have been developed which act at the community-level . This results largely from the failure of Darwinian selection theory to envision communities as units of proliferation and evolution . It is thus difficult to conceive of microbial communities as causative agents and to develop antimicrobials which are effective against them . Consequently, we find it necessary to consider a more comprehensive biological paradigm which envisions biofilm communities and other microbial associations (e.g . mixed infections, food spoilage, tooth decay) as units of existence, activity, ecology, proliferation, survival, and evolution . These communities exist in the same sense that organisms exist as units of ecological activity . This is a simpler, more comprehensive, and more unifying theory of ecology . It is simpler in that it no longer requires convoluted explanations of altruistic behavior in terms of individual selection . It is more comprehensive by not constraining evolution to the selection of any single level of biological organization (genes, races, lineages, or groups) . It unifies in that it bridges the boundaries between microbial ecology, evolutionary ecology and ecosystem ecology . The basis for this theory lies in recognizing that life consists of various forms of information (order) which evolve not only through genetic recombination and mutation, but also through the recombination of organisms within communities (as well as other mechanisms, some of which are considered beyond the realm of biology) . It also involves setting aside the concept of evolution through selection and competition, in favor of evolution through proliferation and association.

Antimicrob Agents Chemother, 1996 Sep, 40(9), 2012 - 4
Bacterial biofilms and the bioelectric effect; Wellman N et al.; Bacterial biofilms are acknowledged to be a major factor in problems of ineffective sterilization often encountered in clinics, hospitals, and industrial processes . There have been indications that the addition of a relatively small direct current electric field with the sterilant used to combat the biofilm greatly increases the efficacy of the sterilization process . The results of the experiments reported in this paper support the concept of the "bioelectric effect" as reported by J.W . Costerton, B . Ellis, K . Lam, F . Johnson, and A.E . Khoury (Antimicrob . Agents Chemother, 38:2803-2809, 1994) . With a current of 1 mA flowing through the chamber containing the biofilm, an increase in the killing of the bacteria of about 8 log orders was observed at the end of 24 h (compared with the control with the same amount of antibacterial agent but no current) . We also confirmed that the current alone does not affect the biofilm and that there appear to be optimum levels of both the current and the sterilant that are needed to obtain the maximum effect.

Chemotherapy, 1996 Sep-Oct, 42(5), 363 - 73
Role of bacterial biofilms in the chemotherapy of cholangitis with brown pigment stones; Uetera Y et al.; The purpose of the present study was to clarify the role of biofilms in the chemotherapy of cholangitis . In 1 case of recurrent cholangitis with intrahepatic stones, the patient underwent right hepatectomy after chemotherapy was performed for 42 weeks . In the other case, chemotherapy including ciprofloxacin was performed during an attack of recurrent cholangitis . Repeated culture of bile specimens yielded negative results, whereas culture of the choledochal stone yielded Pseudomonas aeruginosa . We observed an acute transient IgM response to a component of the biofilm of P . aeruginosa (alginate) in this case . In both cases, electron microscopy revealed viable bacteria covered with biofilm in the component of brown pigment stones . It was concluded that biofilm is a factor of drug resistance.

J Am Dent Assoc, 1996 Aug, 127(8), 1188 - 93
Molecular techniques reveal high prevalence of Legionella in dental units; Williams HN et al.; Legionella bacteria are ubiquitous in freshwater aquatic systems, and humans are infected by them primarily through inhalation of contaminated aerosols . This study analyzed a total of 47 water samples from dental lines in private dental offices and university and hospital dental clinics for Legionella using the polymerase chain reaction, direct fluorescent antibody staining and culture techniques . The typical temperature of dental waterlines (23 C) combined with Legionella's ability to form biofilms, stagnation of the water in the lines and a low chlorine residual all potentially create a unique niche for this microorganism.

Clin Orthop, 1996 Aug, (329), 255 - 62
Disinfecting agents for removing adherent bacteria from orthopaedic hardware; Moussa FW et al.; This investigation seeks to determine whether surfactants or detergents can be used to clean and disinfect orthopaedic wounds with implanted hardware . Thus, a stepwise investigation of biocompatible surfactants and detergents was performed to identify an irrigation agent for disinfecting orthopaedic wounds . Bacterial adhesions assays, irrigation studies, and bactericidal assays determined that benzalkonium chloride showed the greatest efficacy . Testing involved stainless steel screws colonized with a preformed biofilm of Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa, which were immersed in benzalkonium chloride solutions for various time intervals under static conditions . After 10 minutes, benzalkonium chloride achieved a minimum 4 log kill (10,000-fold) for all 3 strains of bacteria . Additional studies demonstrated that the high mechanical energy of jet irrigation improved the disinfecting properties of this agent . With jet lavage, both 1:1000 and 1:5000 concentrations of benzalkonium chloride achieved a minimum 2 log kill (100-fold) for all 3 bacteria . The results or this study suggest that at tissue compatible concentrations, benzalkonium chloride has significant disinfection properties for in vitro colonized orthopaedic devices, and these properties may be enhanced via jet lavage.

J Appl Bacteriol, 1996 Aug, 81(2), 120 - 5
The effect of chlorhexidine on defined, mixed culture oral biofilms grown in a novel model system; Kinniment SL et al.; In order to develop an improved method to evaluate antimicrobial agents for use in clinical dentistry, a constant-depth film fermenter (CDFF) has been used to generate biofilms of fixed depth comprising nine species of bacteria commonly found in dental plaque in health and disease . These bacteria were grown together initially in a conventional chemostat which was used to inoculate the CDFF over an 8 h period . Medium was then supplied directly to the CDFF and biofilms allowed to develop . The biofilms were then challenged with eight short pulses of two concentrations of chlorhexidine (0.0125 and 0.125% w/v) . The lower concentration had a limited effect on the composition of the biofilms while a differential and substantial inhibition was obtained with a higher concentration . Actinomyces naeslundii was lost from the biofilm, and the viable counts of streptococci, Fusobacterium nucleatum and Porphyromonas gingivalis were inhibited by over three orders of magnitude by 0.125% chlorhexidine, whereas Veillonella dispar was only transiently affected . The findings were consistent with those from clinical studies of dental plaque, suggesting that this model would have a predictive value when evaluating novel antiplaque or antimicrobial inhibitors.

Appl Environ Microbiol, 1996 Aug, 62(8), 2994 - 8
Natural transformation in river epilithon; Williams HG et al.; Natural transformation was demonstrated in unenclosed experiments incubated in river epilithon . Strains of Acinetobacter calcoaceticus were transformed to prototrophy by either free DNA (lysates) or live donor cells . The sources of transforming DNA and recipient culture were immobilized on filters, secured to stones, and incubated midstream in the river . The transfer frequency generally increased with temperature . No transfer was detected in the river Taff below 10 degrees C . The age of the recipient culture affected the transformation frequencies in situ but did not significantly affect the transfer frequency on laboratory media . Transformation of recipient cultures which had been incorporated into the natural epilithic biofilm and transformation of the plasmid pQM17 in situ were also demonstrated . This study provides the first direct evidence of natural transformation in situ of bacteria incorporated into an indigenous community.

Appl Environ Microbiol, 1996 Aug, 62(8), 2783 - 8
Purification and characterization of a novel antibacterial protein from the marine bacterium D2; James SG et al.; A biofilm-forming marine bacterium, D2, isolated from the surface of the tunicate Ciona intestinalis, was found to produce a novel, 190-kDa protein with antibacterial activity . The protein contained at least two subunits of 60 and 80 kDa, joined together by noncovalent bonds, and was shown to be released by D2 cells into the surrounding medium during stationary phase . N-terminal sequence analysis revealed no close similarity of this protein to any other proteins within the Swiss Prot database . Bacteriocidal activity against a wide variety of marine and medical bacterial isolates was observed, 77% of the strains tested being sensitive to the protein . Bacterial strains varied in their resistance to the D2 protein, with D2 itself being among the most sensitive with an MBC in liquid suspension of 4 micrograms/ml . An apparent increased resistance of D2 to the protein as the cells progressed further into stationary phase was observed and seen as a possible explanation for its survival despite the production of an autoinhibitory factor . The ability of the D2 bacterium to produce an antibacterial factor in addition to its inhibitory effects on marine invertebrates and algae (S . Egan et al., unpublished data) indicates that D2 has the potential to greatly affect the survival of a range of colonizers of the marine surface environment.

Curr Opin Ophthalmol, 1996 Aug, 7(4), 65 - 70
Infectious diseases of the conjunctiva and cornea; van Bijsterveld OP et al.; The modern local antibiotics, such as the aminoglycosides and quinolones, are very successful in treating infectious conjunctivitis and keratitis . More notably in some Third World countries, however, suppurative keratitis is found in more than half of the infectious disease cases caused by Fusarium species . Here, of course, treatment should be antifungal . The emergence of some problematic microorganisms is related to contact lens wear . Pseudomonas, for example, have the ability to adhere to contact lenses and thus form microcolonies, which are protected by biofilm that predisposes to infection . Acanthamoeba infections of the cornea are a direct consequence of inappropriate or inadequate disinfection of contact lens systems . Occasionally the diagnosis of herpes simplex manifestations of the outer eye can be very difficult . Even more confusing is the delayed appearance of zoster manifestations, such as pseudodendrites, particularly in cases of zoster sine herpete eruptione . The polymerase chain reaction is of particular value in demonstrating the presence of varicella zoster DNA . Although infectious disease of the outer eye remains common, the incidence and complications have increased because of frequent use of antimicrobial agents . In the under-developed areas of the world, however, infections are still very common, are frequently caused by fungi, and are the cause of serious ocular complications . In the Western World infectious eye disease does not seem to be a major diagnostic or therapeutic point at present . Some organisms that have been in the environment all along, however, have emerged in the past half century as a major problem . Thus, in the past years a number of new techniques in diagnosis as well as new insights in pathophysiology and new developments in treatment have emerged that are of interest.

J Ind Microbiol, 1996 Jul, 17(1), 6 - 10
Variation in sessile microflora during biofilm formation on AISI-304 stainless steel coupons; de Franca FP et al.; Coupons of stainless steel type AISI-304 were exposed to the industrial cooling system of a petrochemical plant fed by seawater from the Guanabara Bay, Rio de Janeiro, Brazil, in order to study the in situ formation of biofilms . Bacteria, microalgae and fungi were detected on the coupons as soon as 48 h after exposure . Their respective numbers were determined at times 48, 96 and 192 h and over the following 8 weeks . Aerobic, anaerobic and sulfate-reducing bacteria were quantified according to the technique of the most probable number, and fungi by the pour plate technique . The number of microorganisms present in the forming biofilm varied over the experimental period, reaching maximal levels of 14 x 10(11) cells cm-2, 30 x 10(13) cells cm-2, 38 x 10(11) cells cm-2 and 63 x 10(5) cells cm-2, respectively, for aerobic bacteria, anaerobic bacteria, sulfate-reducing bacteria and fungi, and the dynamics of this variation depended on the group of microorganisms . Bacillus sp, Escherichia coli, Serratia sp and Pseudomonas putrefaciens were identified among the aerobic bacteria isolated . Additionally, microalgae and bacteria of the genus Gallionella were also detected . Nonetheless, no evidence of corrosion was found on the stainless steel type AISI-304 coupons over the experimental period.

J Photochem Photobiol B, 1996 Jul, 34(2-3), 123 - 8
Phototoxicity of argon laser irradiation on biofilms of Porphyromonas and Prevotella species; Henry CA et al.; Species of Prevotella (Pr.) and Porphyromonas (Po.) and other microorganisms were cultivated as biofilms on agar medium and examined for their susceptibility to argon laser irradiation (continuous mode; wavelengths, 488-514 nm; fluences, 20-200 J cm(-2)) . Fluences of 35 to 80 J cm(-2) inhibited biofilm growth in Po . endodontalis, Po . gingivalis, Pr . denticola, Pr . intermedia, Pr . melaninogenica and Pr . nigrescens . A fluence of 70 J cm(-2) did not affect biofilm growth in species of Bacillus, Candida, Enterobacter, Proteus, Pseudomonas, Staphylococcus and Streptococcus . The phototoxic effects of argon laser irradiation against Prevotella and Porphyromonas species were: (1) caused by the radiation alone; (2) modified by biofilm age; (3) dependent on the presence of atmospheric oxygen; (4) influenced by medium supplements of hemin, hemoglobin and blood; (5) greater when compared with other microbial species; (6) demonstrated without augmentation with an exogenous photosensitizer; and (7) apparently unrelated to the protoporphyrin content of the cells . Overall, these in vitro findings suggest that low doses of argon laser radiation may be effective in the treatment and/or prevention of clinical infections caused by biofilm-associated species of Prevotella or Porphyromonas.

Am J Gastroenterol, 1996 Jul, 91(7), 1301 - 8
Prevention of biliary stent clogging: a clinical review; Libby ED et al.; Endoscopic stenting is a well established treatment for obstructive jaundice . The major complication of the technique is late stent blockage, which results from bacterial biofilm and sludge deposition . Numerous approaches to overcoming this problem have been proposed . Large diameter stents can provide longer patency, but they do not prevent blockage indefinitely . Although many plastics have been investigated for resistance to biofilm adherence, there is no convincing evidence that any material prevents clogging in vivo . Changes in stent design and placement techniques to prevent bacterial colonization may provide more lasting effects . Long term antibiotic prophylaxis offers an intriguing possibility for prolonging stent patency . However, its efficacy remains uncertain, and more studies are required to assess timing, dosage, and the optimal spectrum of antibacterial coverage . Metal stent designs now permit delivery of larger diameters; these must be improved to prevent tumor ingrowth and to allow subsequent stent removal . In summary, our understanding of the behavior of bacterial biofilm and its role in stent blockage has improved, but we are still searching for methods to maintain stent function indefinitely.

Acta Stomatol Belg, 1996 Jun, 93(2), 73 - 8
Antibacterial effects of amalgams on mutans streptococci in an in vitro biofilm test procedure; Netuschil L et al.; Conflicting data continue to be presented in the literature regarding the antibacterial potential of various amalgam alloy compositions . The aim of the present study was to compare the antibacterial effects of 4 different amalgam samples on mutans streptococci using two in vitro test procedures . Glass and bovine enamel served as negative controls . The first test, one commonly used, consisted of immersing freshly prepared disks of the six materials in culture broth inoculated with mutans streptococci . Optical density measurements of the broth served to evaluate bacterial growth . This was followed by a biofilm technique which provided more intimate contact between the bacteria and specimen surfaces . With the exception of one high-copper lathe-cut amalgam, the first test revealed no antibacterial potential of the samples whereas the second test with the biofilm design elucidated significant differences in antibacterial potential between the amalgam alloy compositions and the controls . The biofilm technique, used in an in vitro test procedure, reflects the conditions of the oral environment more accurately than conventional test designs.

Compend Contin Educ Dent, 1996 Jun, 17(6), 538 - 40, 542 passim; quiz 558
Microbial contamination of dental unit waterlines: origins and characteristics; Williams JF et al.; Microbial adherence to the internal surface of dental tubing and the formation of a highly protective biofilm layer is predictable, given the ideal growth conditions in the tubing . The slime layer is the most common cause of dental water contamination . Various environmental and human-derived potential pathogens have been reported worldwide . Dental equipment such as retracting shut-off valves, antiretracting valves that tend to fail, or waterlines that are inaccessible contribute to a situation in which virtually every standard dental unit contains contaminated water . While exposure to Pseudomonas, Moraxella, Staphylococcus, and Legionella has been linked to dental water, the medical risk of dental unit waterline contamination is most significant to immune-deficient individuals . Regulations and technological devices are emerging to manage dental water quality.

J Ind Microbiol, 1996 Jun, 16(6), 331 - 41
Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes; Doolittle MM et al.; Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage-infected host cells of Escherichia coli and Pseudomonas aeruginosa . Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized . Biofilms of each bacterium were infected by labeled phage . SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms . The spread of the recombinant T4 phage, YZA1 (containing an rII-LacZ fusion), within a lac E . coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose . Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded beta-galactosidase activity . Fluorescent antibodies were used to detect non-labeled progeny phage . Phage T4 infected both surface-attached and surface-associated E . coli while phage E79 adsorbed to P . aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted . Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.

Oral Microbiol Immunol, 1996 Jun, 11(3), 188 - 92
Susceptibility of biofilms of Streptococcus sanguis to chlorhexidine gluconate and cetylpyridinium chloride; Wilson M et al.; Biofilms of Streptococcus sanguis and planktonic cells of the organism were exposed to chlorhexidine gluconate and cetylpyridinium chloride, at concentrations used clinically, and survivors enumerated . S . sanguis exhibited a lower susceptibility to both antiseptics when it comprised a biofilm than when the organism was in the planktonic form . No viable bacteria were detectable after 5 min of exposure of planktonic cells to 0.2% (w/v) chlorhexidine gluconate or 0.05% (w/v) cetylpyridinium chloride, whereas viable bacterial survived in biofilms of S . sanguis even after exposure to these agents for 4 h . Older biofilms (7 days old) had similar susceptibilities to the antiseptics as younger biofilms (4 days old) . Chlorhexidine achieved kills corresponding to approximately a 2 log10 reduction in the viable count of biofilms, containing approximately 10(7) colony-forming units after 5 min of incubation, whereas the corresponding kills achieved by cetylpyridinium chloride amounted to approximately a 1 log10 reduction . However, on a molar basis, cetylpyridinium chloride was the more effective of the two antiseptics . Minimum inhibitory concentration determinations showed chlorhexidine gluconate to be more effective against S . sanguis than cetylpyridinium chloride . The results of this study have revealed that the minimum inhibitory concentration is not a reliable predictor of the relative effectiveness of antimicrobial agents against S . sanguis biofilms.

Mol Microbiol, 1996 Jun, 20(5), 1083 - 91
Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis; Heilmann C et al.; The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum . PIA was shown to be a linear beta-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyranosyl residues of which 80-85% are N-acetylated, the rest being non-N-acetylated and positively charged . A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production . The mutant could be complemented by transformation with the icaABC-carrying plasmid pCN27 . Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression . The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped icaA promoter . IcaA contains four potential transmembrane helices, indicative of a membrane location . The deduced IcaA sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity) . This similarity suggests that IcaA has N-acetylglucosaminyltransferase activity in the formation of the beta-1, 6-linked N-acetyl-D-glucosaminyl polymer . IcaB is secreted into the medium and contains a typical signal peptide . IcaC is hydrophobic and contains six predicted transmembrane helices distributed over its entire length, typical for an integral membrane protein . Neither IcaB nor IcaC shares similarity with known proteins, and their function is unknown . Inactivation of icaA, icaB, or icaC in pCN27 led to the complete loss of the intercellular adhesion phenotype in S . carnosus, suggesting that all three genes are involved in intercellular adhesion, PIA expression, and translocation.

J Am Soc Nephrol, 1996 Jun, 7(6), 877 - 82
Biofilm causes decreased production of interferon-gamma; Dasgupta MK; Interferon-gamma, a cytokine, is produced by lymphocytes when they are stimulated by cytokines from activated macrophages, and is essential for macrophage-mediated bactericidal operations . To investigate whether a strain of bacteria can activate macrophages and lymphocytes, the interferon-gamma levels may thus be measured . Current literature maintains that peritoneal dialysis patients with recurrent peritonitis have "unhealthy" macrophages and lymphocytes unable to produce interferon-gamma, but that the administration of interferon improves the rates of peritonitis . In an in vitro experiment, Staphylococcus epidermidis, in both its planktonic and biofilm forms, was added to a suspension of peritoneal dialysis effluents, macrophages, and healthy peripheral blood lymphocytes, which were incubated at 37 degrees C for 18 h and then centrifuged . Subsequent levels of interferon-gamma were measured in the supernatants . Three such experiments were done with peritoneal macrophages and dialysis effluents collected from each of the three different patients involved in the study . It was found that little or no interferon-gamma (0.42 +/- 0.17 U/mL) was produced when biofilm bacteria were tested, but significant amounts of interferon-gamma (9.25 +/- 4.63 U/mL) resulted in conjunction with the planktonic form of the same bacteria . To eliminate experimental errors, all conditions were left identical, appropriate control groups were added, and each of the three experiments was duplicated . These in vitro data therefore provide new insight in the role of biofilm in the pathogenesis of recurrent peritonitis in peritoneal dialysis patients . Further clinical studies are required.

J Can Dent Assoc, 1996 Jun, 62(6), 492 - 5
Dental unit water contamination; Peters E et al.; Bacteria-laden biofilms located on the lumen surface of dental unit water lines have resulted in the persistent and widespread microbial contamination of dental unit water supplies . These biofilms are resistant to chemical disinfection . As such, they act as reservoirs that facilitate the continuous re-contamination of dental unit water . The microbial populations of the biofilms found in dental unit water lines include opportunistic pathogens of unknown significance . Strategies to control the contamination are discussed.

Hinyokika Kiyo, 1996 Jun, 42(6), 433 - 8
{Clinical study of Silver Lubricath Foley catheter}; Nakada J et al.; We evaluated the Silver Lubricath Foley Catheter (silver catheter) coated with silver and hydrogel developed to prevent urinary infection, in comparison with the silicone-coated catheter (silicone catheter) . Twelve patients ranging from 71 to 95 years of age (median age, 82 years) were catheterized and the 16 of 18F catheter was replaced every 2 weeks . They answered a questionnaire which included inquiry about the treatment with urinary catheter . Because of less leakage and discomfort to the urethra, the silver catheter had advantages over the silicone catheter . The risk of bacteriuria after 14 days of catheterization was not significantly different between the two types of catheter . Scanning electron microscopic analysis revealed that bacterial biofilm developed on the inner surface of both catheters after 14 days of catheterization . On the other hand, the amount of bacterial biofilm on the outer surface of a silver catheter was less than that on the outer surface of a silicone catheter.

Dent Mater, 1996 May, 12(3), 208 - 14
Biochemical and physiological considerations for modeling biofilms in the oral cavity: a review; Glantz PO et al.; This review describes some of the biochemical and physiological factors that should be considered in designing in vitro models of the oral cavity . The significance of the non-Newtonian properties and internal structures of saliva are reported as well as the ability of saliva to form biological films on all types of solid surfaces . The presence of compositional biochemical variations between absorbed salivary films is discussed for solid surfaces having different surface chemistries . The significance of the presence of small amounts of organic material in enamel and dentin is also stressed . Finally, by selecting other, more easily available secretional products than saliva, more relevant model systems for materials testing in vitro may be established than if only simple aqueous salt solutions are used.

Chemotherapy, 1996 May-Jun, 42(3), 186 - 91
The influence of azithromycin on the biofilm formation of Pseudomonas aeruginosa in vitro; Ichimiya T et al.; The influence of azithromycin on biofilm formation by Pseudomonas aeruginosa, a cause of refractory chronic respiratory tract infection, was investigated . Alginic acid produced by a mucoid strain of P . aeruginosa was quantified by high-performance liquid chromatography from colonies growing on an agar medium . Polysaccharides in the biofilm formed on silicon chips by a nonmucoid strain were determined by a tryptophan reaction . The effect of azithromycin was examined at concentrations below the minimum inhibitory concentration (sub-MIC) for each strain . Azithromycin significantly inhibited the production of alginic acid from the mucoid strain at > or = 1/256 MIC, and the production of exopolysaccharides from the nonmucoid strain at > or = 1/16 MIC . The inhibition of biofilm formation by azithromycin was also observed by scanning electron microscopy . These findings suggest that azithromycin inhibits biofilm formation by P . aeruginosa at concentrations well below the MIC.

Appl Microbiol Biotechnol, 1996 May, 45(4), 562 - 8
Aerobic dechlorination of low-chlorinated biphenyls by bacterial biofilms in packed-bed batch bioreactors; Fava F et al.; Cells of an aerobic three-membered bacterial co-culture, designated as ECO3, capable of cometabolizing and aerobically dechlorinating low-chlorinated biphenyls in the presence of biphenyl, were immobilized on Manville silica beads, on frosted-glass beads and on polyurethane foam cubes in packed-bed bioreactors continuously fed with a biphenyl-saturated air stream . The ECO3 biofilm reactors were found to be capable of extensively mineralizing several pure dichlorobiphenyls (75 mg/l) and Aroclor 1221 (75 mg/l) in batch mode . Immobilized ECO3 cells could aerobically degrade and dechlorinate the dichlorobiphenyls tested more extensively than suspended ECO3 cells . Among the three biofilm reactors, the glass bead bioreactor and the polyurethane bioreactor exhibited the highest capability of mineralizing both dichlorobiphenyls and Aroclor 1221; the polychlorinated biphenyl availability in the bioreactors more than the biomass availability, both depending on the nature of the support employed, significantly governed the efficiency of the treatment . These results are of interest for the possible development of a bioreactor system for continuous treatment of polychlorinated-biphenyl-contaminated wastewaters.

Pathol Biol (Paris), 1996 May, 44(5), 397 - 404
{In vivo and in vitro analysis of the ability of urinary catheter to microbial colonization}; Brisset L et al.; Bacterial adhesion to biomaterials is a complex phenomenon involving numerous factors . The ability to reduce urinary catheters infections simply by general hygiene and asepsis is low: an ascending colonization cannot be avoided . This will lead to a clinical infection only if several factors favour the bacterial adhesion or the bacterial coaggregation and the feeding of the bacterial biofilm . Among the many factors involved in bacterial adhesion, we focused in this paper on the physical parameters of surface hydrophobicity of the urinary catheters (Van der Waals and acido-basic forces) and the surface hydrophobicity of the bacteria (BATH and zeta potential) . We also compared scanning electron microscopy (SEM) of in vivo and in vitro infected urinary catheters . We provided evidence that the more hydrophobic the bacteria, the more they are able to colonize hydrophobic materials, whereas hydrophilic cells are able to colonize hydrophilic materials more easily . Some biomaterials were found to display an irregular texture of hydrophobic and hydrophilic areas: they favour both types of adhesion . Moreover the divalent cations (MgII) drastically increased the bacterial coaggregation and favour bacterial growth within the biofilm . Finally, an increase in urinary pH and ionic strength increases the colonization risk . Consequently, choice of urinary catheter biomaterials is essential as patient hygiene and diet in order to avoid clinical infections.

Cornea, 1996 May, 15(3), 301 - 4
Biofilm formation in infectious crystalline keratopathy due to Candida albicans; Elder MJ et al.; Infectious crystalline keratopathy (ICK) is an uncommon, indolent corneal infection in which the slow clinical course contrasts with the rapid laboratory growth and microbiological sensitivities of the infecting organism . This prospective study aimed to determine whether biofilm production was the cause of this disparity . A case of failed medical management of ICK in a patient with Stevens-Johnson syndrome is presented . A penetrating keratoplasty yielded corneal tissue that was freshly fixed for electron microscopy using 0.05% ruthenium red and 2.5% gluteraldehyde . Candida albicans was grown from 3/3 broths, and fungi with morphology consistent with Candida were seen on histological examination . Electron microscopy revealed microorganisms morphologically typical of Candida surrounded by a polysaccharide-rich glycocalyx consistent with a biofilm . We concluded that Candida albicans is capable of producing a biofilm and is a known cause of ICK . This case is supportive evidence that biofilm production is associated with cases of ICK and may explain the chronic, pauciinflammatory features of ICK and its relative resistance to antibiotic treatment.

Biotechnol Prog, 1996 May-Jun, 12(3), 316 - 21
Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm; Wentland EJ et al.; The use of acridine orange to visualize and quantify spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm was investigated . Bacterial colonies supported on polycarbonate filter membranes were grown on R2A agar plates . Some colonies were sampled for cell enumeration, while others were cryoembedded, sectioned, and stained with the fluorescent nucleic acid stain acridine orange . Spatial patterns of fluorescent color and intensity with depth in the colony were quantified using confocal microscopy and image analysis of stained cross sections . Colonies sampled in the midexponential phase were thin (20 microns), had high average specific growth rates (> 1 h-1), and had all the cells stained bright orange . Colonies sampled after more than 24 h of growth were thick (> 200 microns) and were growing slowly (mu < 0.15 h-1) . These older colonies were characterized by distinct bands of orange at the colony edges and a dark green center . Stained biofilm cross sections displayed a similar orange band at the biofilm-bulk fluid interface and a green interior . Colony-average specific growth rates, determined by calculating the local slope of the cell accumulation versus time data, were correlated with colony-average fluorescence intensities . There was no correlation between average specific growth rate and orange or green intensity individually, but growth rate did correlate with the orange:green intensity ratio (r2 = 0.57) . The resulting regression was used to predict specific growth rate profiles within colonies . These profiles indicated that bacteria were growing rapidly near the air and agar interfaces and more slowly in the center of the colonies when thicker than about 30 microns . The dimension of the orange bands ranged from 10 to 30 microns, which may indicate the thickness of growing regions . The inherent variability associated with this technique suggests that it is best applied in single species systems and that the results should be regarded as qualitative in nature.

Curr Microbiol, 1996 Apr, 32(4), 176 - 8
Effect of Staphylococcus epidermidis on hydrogel contact lens retention on the rabbit eye; Gabriel MM et al.; A slime-producing isolate of Staphylococcus epidermidis attached to FDA Group II hydrogel contact lenses persisted on rabbit eyes for up to 14 days, but except for minor redness of the eye no other effect was observed . Eye flora of eight representative New Zealand White rabbits included four different species of Staphylococcus including S . epidermidis and one species of Micrococcus, none of which produced overtly obvious biofilms . The slime-producing strain of S . epidermidis adhered more effectively to lenses than a non-slime-producing strain, and lenses challenged with the slime-producing strain remained on the rabbit eye for longer time periods than those with a non-slime-producing strain . Bacteria associated with the contact lens may affect the retention of the lens on the rabbit cornea during experimental studies.

Ecotoxicol Environ Saf, 1996 Apr, 33(3), 271 - 80
Functional effects of the bacterial insecticide Bacillus thuringiensis var . kurstaki on aquatic microbial communities; Kreutzweiser DP et al.; Epilithic microbial communities were colonized on leaf disks and exposed to commercial preparations of Bacillus thuringiensis var . kurstaki (Btk) in aquatic microcosms . Responses in terms of microbial respiration, bacterial cell density, protozoan density, and microbial decomposition activity were measured . Test concentrations for treatments with Dipel 64AF and Dipel 8AF in microcosms were the expected environmental concentration (EEC) of 20 IU/ml, 100x the EEC, and 1000x the EEC . Bacterial cell density in the biofilm of leaf disks was significantly increased at concentrations as low as the EEC . There were no concomitant alterations in protozoan density . Microbial respiration was significantly increased, and decomposition activity was significantly decreased, but only at the artificially high concentration of 1000x the EEC . This effect was attributed to the spore-crystal component rather than formulation ingredients . Microbial decomposition of leaf material was also determined in outdoor stream channels treated at concentrations ranging from the EEC to 100x the EEC . Although there tended to be reduced decomposition activity in treated channels, there were no significant differences in mass loss of leaf material between treated and control channels . Various regression, classification, and ordination procedures were applied to the experimental data, and none indicated significant treatment effects . These results from laboratory and controlled field experiments indicate that contamination of watercourses with Btk is unlikely to result in significant adverse effects on microbial community function in terms of detrital decomposition.

Scand J Gastroenterol, 1996 Apr, 31(4), 398 - 403
Bacterial adherence and biofilm formation on latex and silicone T-tubes in relation to bacterial contamination of bile; Koivusalo A et al.; BACKGROUND: T-tube-related bacteriobilia causes infectious complications and obstruction . To prevent these, the choice of T-tube material may be of importance . METHODS: Transected common bile ducts (CBDs) of 17 piglets were sutured over latex or silicone T-tubes, or without a T-tube . RESULTS: After 6 weeks bacteriobilia was found in all of 12 CBDs with and in 1 of 5 CBDs without a T-tube (p < 0.05) . By scanning electron microscopy (SEM) four of five latex and none of five silicone T-tubes had bacterial biofilms (p < 0.05) . All tubes remained patent . Segments of T-tubes were incubated with five different bacterial strains . Sonication and SEM showed that 0.1-1.1% of 10(7) colony-forming units of inoculum adhered to T-tubes . Two to six times more bacteria adhered to latex than to silicone (p < 0.05) . CONCLUSIONS: Silicone offers better long-term patency than latex . Less infectious complications occur if T-tubes are omitted.

J Ind Microbiol, 1996 Apr, 16(4), 249 - 56
Continuous ethanol production by Zymomonas mobilis and Saccharomyces cerevisiae in biofilm reactors; Kunduru MR et al.; Continuous ethanol fermentations were performed in duplicate for 60 days with Zymomonas mobilis ATCC 331821 or Saccharomyces cerevisiae ATCC 24859 in packed-bed reactors with polypropylene or plastic composite-supports . The plastic composite-supports used contained polypropylene (75%) with ground soybean-hulls (20%) and zein (5%) for Z . mobilis, or with ground soybean-hulls (20%) and soybean flour (5%) for S . cerevisiae . Maximum ethanol productivities of 536 g L-1 h-1 (39% yield) and 499 g L-1 h-1 (37% yield) were obtained with Z . mobilis on polypropylene and plastic composite-supports of soybean hull-zein, respectively . For Z . mobilis, an optimal yield of 50% was observed at a 1.92 h-1 dilution rate for soybean hull-zein plastic composite-supports with a productivity of 96 g L-1 h-1, whereas with polypropylene-supports the yield was 32% and the productivity was 60 g L-1 h-1 . With a S . cerevisiae fermentation, the ethanol production was less, with a maximum productivity of 76 g L-1 h-1 on the plastic composite-support at a 2.88 h-1 dilution rate with a 45% yield . Polypropylene-support bioreactors were discontinued due to reactor plugging by the cell mass accumulation . Support shape (3-mm chips) was responsible for bioreactor plugging due to extensive biofilm development on the plastic composite-supports . With suspension-culture continuous fermentations in continuously-stirred benchtop fermentors, maximum productivities of 5 g L-1 h-1 were obtained with a yield of 24 and 26% with S . cerevisiae and Z . mobilis, respectively . Cell washout in suspension-culture continuous fermentations was observed at a 1.0 h-1 dilution rate . Therefore, for continuous ethanol fermentations, biofilm reactors out-performed suspension-culture reactors, with 15 to 100-fold higher productivities (g L-1 h-1) and with higher percentage yields for S . cerevisiae and Z . mobilis, respectively . Further research is needed with these novel supports to evaluate different support shapes and medium compositions that will permit medium flow, stimulate biofilm formation, reduce fermentation costs, and produce maximum yields and productivities.

APMIS, 1996 Apr, 104(4), 280 - 4
Resistance of Streptococcus sanguis biofilms to antimicrobial agents; Larsen T et al.; Bacteria living in biofilms as dental plaque on tooth surfaces are generally more resistant to antimicrobial agents than bacteria in batch culture normally used for in vitro susceptibility testing . In order to compare the resistance of free-living and surface-grown oral bacteria, the MIC of Streptococcus sanguis 804 and ATCC 10556 to amoxicillin, doxycycline and chlorhexidine was determined by a broth dilution method . Subsequently, S . sanguis biofilms established in an in vitro flow model were perfused with the antimicrobial agents for 48 h at concentrations equal to and up to 500 times the MIC, and biofilm cell number was determined during this period . The antibiotics at the MIC did not affect the cell number of S . sanguis biofilms compared to the starting point, and only after 48 h at 500 times the MIC were the biofilm bacteria eliminated . At intermediate concentrations biofilm cell number gradually decreased . Chlorhexidine also gradually reduced biofilm cell number, but was inhibitory at concentrations closer to the MIC than was the case for the antibiotics . Thus S . sanguis in biofilms survived up to 500 times the MIC found in batch culture for up to 48 h.

Microbiology, 1996 Mar, 142 ( Pt 3), 631 - 8
Development of a steady-state oral microbial biofilm community using the constant-depth film fermenter; Kinniment SL et al.; The complexity of biofilm communities like dental plaque suggests that laboratory model biofilm growth systems may help to understand their structure and function . This study describes the use of a constant-depth film fermenter (CDFF) to investigate biofilm formation by a nine-membered community of oral bacteria . The community was grown to steady state in a chemostat incubated anaerobically . The chemostat output was fed into the CDFF incubated aerobically . Viable counts for each species from the chemostat and the CDFF at steady state showed major differences; however, all nine organisms were present under both conditions . There was evidence of succession during biofilm formation with obligately anaerobic species only establishing after several days . A steady-state biofilm community was achieved which remained stable over time . Electron microscopy showed evidence of spatial differentiation with what appeared to be Neisseria subflava dominant near the upper surface and Fusobacterium nucleatum largely confined to the middle portion.

Microbiology, 1996 Mar, 142 ( Pt 3), 623 - 9
Effect of oxygen, inoculum composition and flow rate on development of mixed-culture oral biofilms; Bradshaw DJ et al.; The effect of aeration on the development of a defined biofilm consortium of oral bacteria was investigated in a two-stage chemostat system . An inoculum comprising 10 species, including both facultatively anaerobic and obligately anaerobic bacteria, and species associated with oral health and disease, was inoculated into an anaerobic first-stage chemostat vessel . The effluent from this chemostat was linked to an aerated {200 ml CO2/air (5:95, v/v) min-1} second-stage vessel, in which removable hydroxyapatite discs were inserted to allow biofilm formation . Comparisons were made of planktonic and biofilm communities in the aerated second-stage vessel by means of viable counts . Both planktonic and early biofilm communities were dominated by Neisseria subflava, comprising > 40% of total c.f.u . in the fluid phase, and > 80% of c.f.u . in 2 h biofilms . Obligate anaerobes persisted in this mixed culture, and succession in biofilms led them to predominate only after 7 d . Despite the continuous addition of air, the dissolved oxygen tension (dO2) within the culture remained low (< 5% of air saturation), and the redox potential (Eh) was -275 mV . In order to assess the significance of the presence of N . subflava in community development, a subsequent experiment omitted this aerobe from the inoculum, to produce a nine-species culture . The planktonic phase was predominated by three streptococcal species, Prevotella nigrescens and Fusobacterium nucleatum . Biofilms again underwent successional changes, with anaerobes increasing in proportion with time . In contrast to the culture including N . subflava, dO2 was 50-60% of air saturation, and the Eh was +50 mV . In the final experiment, the rate of addition of first-stage culture was reduced to 1/10 of that in the previous experiment, in order to determine whether anaerobes were growing, rather than merely persisting in the aerated culture . The data for the planktonic phase indicated that the anaerobes were growing in aerated (dO2 40-50%, Eh +100 mV) conditions . Once again, anaerobes increased in proportion in older biofilms . The study indicates that mixed cultures can protect obligate anaerobes from the toxic effects of oxygen, both in the biofilm and planktonic modes of growth.

J Dermatol Sci, 1996 Mar, 11(3), 234 - 8
Staphylococcus aureus infection on cut wounds in the mouse skin: experimental staphylococcal botryomycosis; Akiyama H et al.; Staphylococcus aureus cells were inoculated on the cut wounds in the skin of cyclophosphamide-treated mice . Biopsy specimens were taken from three mice at 1, 3, 6, 12, 24, 36, 48 and 60 h after the inoculation and were examined by light and electron microscopies . One hour after the inoculation Staphylococcus aureus cells were seen around the cut wound and deeper into the subcutaneous tissue . By 6 h after the inoculation, Staphylococcus aureus cells formed clusters of bacterial colonies . By 36 h after the inoculation inflammatory cells, mainly polymorphonuclear leukocytes and macrophages, were seen around the clusters . Electron microscopic examination revealed fibril-like structures around the Staphylococcus aureus cells at 1 h . The Staphylococcus aureus cells were enclosed in membrane-like structures at 3 h . The membrane-like structures and the fibril-like structures were positive for Ruthenium red . By 12 h after the inoculation, the membrane-like structures increased in thickness and in electron density . Inflammatory cells were seen around but outside of the membrane-like structures at 24, 36 and 48 h . At 60 h the tissues around the membrane-like structures were degenerated and almost necrotic . These results suggest that Staphylococcus aureus cells may form biofilm in dermal or subcutaneous tissues in a neutropenic condition.

Eur J Clin Microbiol Infect Dis, 1996 Mar, 15(3), 233 - 7
Microbiological contamination of drinking water in a commercial household water filter system; Daschner FD et al.; The microbiological quality of filtered water in a commercial water filter system (Brita) was tested in households and in two laboratories . In 24 of 34 filters used in households, bacterial counts increased in the filtered water up to 6,000 cfu/ml . In 4 of 6 filters tested in the laboratory, bacterial counts in the fresh filtrate were higher than in tap water after approximately one week of use both at room temperature and at 4 degrees C, suggesting growth or biofilm formation in the filter material . In some cases colony counts in the filtered water were 10,000 times those in tap water . The filter material of 5 of 13 new commercial filters was contaminated with bacteria or moulds . National or international regulatory agencies should ensure that water filters marketed for domestic use do not allow deterioration in the microbiological quality of drinking water.

J Laryngol Otol, 1996 Mar, 110(3), 240 - 2
Candida'the cancer of silastic'; Ell SR; Candida species have the propensity to colonize and erode into the surface of silastic causing a loss of function . Colonization of silastic tracheo-oesophageal speaking-valves is an important cause of valve failure post-laryngectomy: the exact nature of the colonization is unknown and light microscopic studies have not been reported previously . Microbiological examination of scrapings taken from the colonized oesophageal surface of failed speaking valves was arranged before sectioning for light microscopy . Conventional ultrathin sectioning of silastic was difficult, therefore 15 micrometre sections were cut after cooling the material to -30 degrees C . These sections were then stained with toluidine blue; Candida species taking up the stain . Microbiological examination of the colonies confirmed the predominance of Candida species in the biofilm . Light microscopy of the 15 micrometer sections revealed that the Candida colonies had invaded the silastic . This work demonstrates that the colonization of the silastic valve prosthesis in the oesophageal area is due predominantly to Candida species which actively invade the structure of the silastic . This is an important cause of speaking-valve failure post-laryngectomy.

Infect Immun, 1996 Mar, 64(3), 1035 - 8
Detachment of Streptococcus mutans biofilm cells by an endogenous enzymatic activity; Lee SF et al.; Previous studies have shown that Streptococcus mutans NG8 possesses an endogenous surface protein-releasing enzyme (SPRE) activity that liberates its own surface proteins (S . F . Lee, Infect . Immun . 60:4032-4039, 1992) . The present study was initiated to investigate the possible role of the release of surface proteins by SPRE in the detachment of biofilm cells in vitro . Initially, the characteristics of surface protein release by the strain (S . mutans BM71) used in this study were shown to be the same as those previously described for S . mutans NG8 . BM71 displayed characteristics identical to those of NG8 in terms of pH optima and inhibitor sensitivity for protein release . Monolayer biofilms of S . mutans BM71 were formed on hydroxylapatite rods in a modified chemostat . Detachment of the biofilm cells was measured by viable cell counts of bacteria liberated after incubation of the biofilms in buffers . Results showed that biofilm cells were detached in a pH- dependent manner with a maximum rate of pH 5 (P = 0.016) to 6 (P = 0.002), a range similar to that for optimal surface protein release . The detachment of the biofilm cells was found to be inhibited by ZnCl2 (P = 0.002 to 0.023), which also inhibited surface protein release . Detachment was not inhibited significantly by CaCl2 (P = 0.525 to 0.784), precluding an ionic effect on inhibition by ZnCl2 . The extent of detachment could be increased (P = 0.046) by the addition of an SPRE preparation from S . mutans but not heat-inactivated SPRE (P = 0.665) or SPRE in the presence of ZnCl2 (P = 0.199) . Detachment was also studied by using biofilms of resting (viable but not dividing) cells . Results similar to those for biofilms formed from growing cells were obtained, indicating that cells detached from biofilms were not daughter cells . The results presented above show that monolayer biofilm cells of S . mutans under conditions of minimal shear force have the ability to detach from a surface and suggest that this detachment was mediated by an endogenous SPRE activity.

Biotechnol Appl Biochem, 1996 Feb, 23 ( Pt 1), 7 - 12
Pattern of growth and respiratory activity of Saccharomyces cerevisiae (baker's yeast) cells growing entrapped in an insolubilized gelatin gel; Parascandola P et al.; Yeast cells immobilized by entrapment in an insolubilized gelatin gel have been investigated with regard to their pattern of growth and respiratory activity . Mass-transfer resistance offered by the matrix and growth of the entrapped cells determine a gradient of nutrients throughout the gel which is responsible for both a lower specific growth rate of immobilized cells with respect to that of free ones, and a heterogeneous biomass distribution, with progressively increased cellular density from the inside to the outside of the matrix . The spatial organization of the growing cells leads to the formation of a biofilm perfectly adherent to the surface of the matrix . Measurements of the oxygen-uptake rate of the immobilized cells, after having been released from the matrix by trypsin digestion, and confocal microscopy of cells stained with Rhodamine 123 demonstrate the occurrence of a gradient of respiratory activity throughout the immobilized culture.

Scand J Urol Nephrol, 1996 Feb, 30(1), 19 - 24
Bacterial adhesion and biofilm formation on various double-J stents in vivo and in vitro; Cormio L et al.; The aim of this study was to determine whether bacterial adhesion and biofilm formation occur on various ureteric stent materials in vivo and in vitro and whether there is any correlation between biofilm formation and urinary tract infection . Using an open surgical procedure, 23 double-J stents of 8 different types were inserted in the ureters of 13 piglets . After 6 weeks intubation, 2 (9%) of the 23 stented renal units yielded positive urine culture . Scanning Electron Microscopy (SEM) showed sporadic bacteria on 7 (30%) of the 23 indwelling ureteric stents and bacterial biofilms on only 2 (9%), those indwelling the renal units which yielded positive urine cultures . Precut segments of the 8 double-J stent brands were incubated with 5 different bacterial strains . Microbiological and SEM analysis of the stents revealed that only 0.1-0.01% of the bacterial inoculum adhered to the stents . There was no statistically significant difference in bacterial adherence to the various stent materials . Interestingly, the two Escherichia coli strains with P-fimbriae adhered significantly more than the two without P-fimbriae . Sporadic bacterial adhesion to ureteric stents seemed to have little clinical significance whereas biofilm formation seemed to result in urinary tract infection . None of the tested stents proved to be superior to the others in this respect, and adhesion appeared to depend on the virulence of the bacteria rather than the properties of the biomaterials.

J Antimicrob Chemother, 1996 Feb, 37(2), 377 - 81
Killing of Streptococcus sanguis in biofilms using a light-activated antimicrobial agent; Wilson M et al.; The aim of this study was to determine whether Streptococcus sanguis, when in a biofilm, could be killed using a light-activated antimicrobial agent . Biofilms were grown on hydroxyapatite, irradiated with up to 12.2 J of light from a gallium aluminium arsenide laser in the presence of aluminium disulphonated phthalocyanine (AlPcS2) and survivors enumerated . No significant decrease in the viable count was found when either the AlPcS2 or the laser light was used alone . There was a light dose related decrease in the viable counts of irradiated AlPcS2-treated biofilms . No viable streptococci were detectable following irradiation with 12.2 J of laser light.

J Am Dent Assoc, 1996 Feb, 127(2), 181 - 9
Biofilm and the dental office; Shearer BG; The author provides a brief overview of the basic processes involved in biofilm formation and explores the implications these biofilms have for health care facilities such as hospitals and dental offices . Included with this article are suggestions dentists may consider for improving water quality and a white paper on waterlines adopted by the ADA.

J Hosp Infect, 1996 Feb, 32(2), 105 - 15
Examination of biofilm formation and risk of infection associated with the use of urinary catheters with leg bags; Rogers J et al.; Urinary catheters and legs bags were simultaneously colonized by Escherichia coli and Proteus vulgaris using a model urinary drainage system . the system was continuously supplied with filter-sterilized artificial urine using a diurnal flow pattern . The extent of colonization was determined by assessment of both planktonic and biofilm formation over time . Contamination of the catheters resulted in rapid colonization of the whole system within a 24 h period . Contamination of the leg bags resulted in an ascending biofilm formation over a four-day period . Results indicated that infection risk could be minimized by changing the catheter and leg bags at least once a week . The design of the leg bags was not found to influence the rate or extent of biofilm formation.

Scand J Gastroenterol, 1996 Feb, 31(2), 175 - 81
A bacteriologic and scanning electron microscope study after implantation of foreign bodies in the biliary tract in rats; Yu JL et al.; BACKGROUND: Bacterial adherence to the stent surfaces, concomitant colonization, and possible stent blockage are the main complications after the use of biliary stents . The present study was assigned to investigate bacteriologic and morphologic changes in the biliary tract after the implantation of biliary drain materials . METHODS: Rubber and silicone pieces with a surface area of 1 cm2 were implanted into the biliary tract in rats after temporary obstruction of the common bile duct by the use of a mini-occluder . The animals were killed at 4, 8 and 14 weeks, respectively, after implantation, and the implants were retrieved, cultured, and examined by scanning electron microscopy (SEM) . Bacterial culture and SEM were also performed on tissue samples obtained from the mucosal surface of the biliary tract . RESULTS: Bacterial colonization and biofilm formation were found on the surfaces of the implanted materials and on the mucosal surface of the biliary tract in animals with implants but not on the biliary tract mucosa in rats without implants . CONCLUSION: Foreign bodies implanted in the biliary tract facilitate bacterial adherence not only to the surface of the implants but also to the mucosal surface in the biliary tract.

J Med Microbiol, 1996 Feb, 44(2), 79 - 87
Susceptibility of oral bacterial biofilms to antimicrobial agents; Wilson M; There is great interest in the use of antimicrobial agents for the prevention and treatment of plaque-related oral diseases and many publications have reported the results of studies in which the minimum inhibitory concentrations of agents for cariogenic and periodontopathogenic bacteria have been determined . However, such data are relevant only to situations where the organisms of interest are in aqueous suspension, whereas in caries and the inflammatory periodontal diseases the target organisms are in the form of biofilms . On the basis of studies with medically important bacteria, it has been established that bacteria in biofilms are invariably less susceptible to antimicrobial agents than their planktonic counterparts . Therefore, in the laboratory assessment of agents which may be suitable for treating plaque-related diseases, the target organisms should be in the form of biofilms . While laboratory evaluation of chemical agents for the prevention of plaque formation has usually employed biofilm-based models, the search for antimicrobial agents effective in the treatment of plaque-related diseases has not . Therefore, there are few data available regarding those characteristics of antimicrobial agents (e.g., their biofilm eliminating concentrations or biofilm killing concentrations) that could be used to judge their suitability for treating plaque-related diseases . In this review the limited information available concerning the antimicrobial susceptibility of oral bacteria in biofilms is presented.

J Appl Bacteriol, 1996 Feb, 80(2), 225 - 32
Comparison of chemical dehydration and critical point drying for the stabilization and visualization of aging biofilm present on interior surfaces of PVC distribution pipe; Carr JH et al.; In this study, fixation of attached glycocalyx on the interior surfaces of polyvinyl chloride distribution pipe remnants was compared with and without ruthenium red/osmium tetroxide and, in the final preparatory phase, with chemical dehydration and critical point drying . SEM examination of interior surface of the polyvinyl chloride pipe showed varying concentrations of adherent bacteria, depending on the preparatory technique used . It was concluded that using a combination of ruthenium red/osmium tetroxide and critical point drying is the optimum method for visually demonstrating aging biofilm on the interior surface of contaminated polyvinyl chloride pipe.

J Appl Bacteriol, 1996 Feb, 80(2), 124 - 30
A modified chemostat system to study the ecology of oral biofilms; Bradshaw DJ et al.; Previously, we developed a chemostat system to study the behaviour and properties of a community of up to 10 species of oral bacteria . The present study describes modification of this system to incorporate removable and replaceable hydroxyapatite (the major mineral in human dental enamel) disks on which biofilms could develop . Hydroxyapatite disks were immersed in the chemostat for known time periods, and the bacterial content of biofilms determined by viable counting . Initial deposition rates were rapid, with all 10 species detected after 1 h, and the numbers of bacteria in biofilms continued to increase for 21 d . The species composition of biofilms reflected that of the surrounding fluid phase, and showed only limited signs of the type of 'species succession' which is observed in developing dental plaque in vivo, although anaerobic species increased in proportion in older biofilms . Four-day biofilms showed the least variability and were chosen as the 'standard biofilm' for more detailed study . Variability in the bacterial composition of 4-d biofilms was comparable both within a single chemostat run and between independent chemostat runs . Glucose pulsing in the absence of pH control resulted in the selection of cariogenic species; the disruption of the biofilm community was less marked than that of the equivalent planktonic culture . The model system has considerable potential in studying the effects of a variety of factors on biofilm development, as well as in comparing the efficacy of antimicrobial systems against biofilms.

J Photochem Photobiol B, 1996 Feb, 32(3), 189 - 96
Biological dosimetry of solar radiation for different simulated ozone column thicknesses; Horneck G et al.; During the Spacelab mission D-2, in the experiment RD-UVRAD, precalibrated biofilms consisting of dry monolayers of immobilised spores of Bacillus subtilis (strain Marburg) were exposed, for defined intervals, to extraterrestrial solar radiation filtered through an optical filtering system, to simulate different ozone column thicknesses . After the mission, the biofilms were processed and optical densities indicative of any biological activity were determined for each exposure condition by image analysis . For the different simulated ozone column thicknesses, biologically effective irradiances were experimentally determined from the biofilm data and compared with calculated data using a radiative transfer model and the known biofilm action spectrum . The data show a strong increase in biologically effective solar UV irradiance with decreasing (simulated) ozone concentrations . The full spectrum of extraterrestrial solar radiation leads to an increment of the biologically effective irradiance by nearly three orders of magnitude compared with the solar spectrum at the surface of the Earth for average total ozone columns.

J Calif Dent Assoc, 1996 Jan, 24(1), 47 - 52
Microbes in dental unit water; Miller CH; Microbes are present in dental unit water and exist in the biofilm that lines the inside walls of water tubing . Improving the microbial quality of dental unit water as means become available is a natural part of maintaining the high quality of patient care and staff protection for which dentistry is well-noted.

Microbiol Immunol, 1996, 40(12), 931 - 9
Effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on biomaterial-associated staphylococcal infection in mice; Rozalska B et al.; Staphylococcal infections are a major complication in the usage of biomaterials . Different modifications of polymers have been made to reduce the incidence of such infections . We studied the effects of modifying heparinized polyethylene (H-PE) with mouse recombinant granulocyte-macrophage stimulating factor (rGM-CSF) . The elimination of staphylococci (Staphylococcus aureus, S . epidermidis) from the peritoneum of mice implanted with rGM-CSF-coated H-PE was slightly more effective than the elimination of the bacteria from the peritoneum of animals implanted with uncoated H-PE . Most interestingly, the number of staphylococci present in the biofilms covering rGM-CSF-coated implants were significantly lower than the number of bacteria detected on the surface of H-PE not coated with rGM-CSF . In vitro, rGM-CSF restored the anti-bacterial potency of the phagocytes, which had been reduced by surface contact with H-PE . The results suggest that modification of biomaterials with rGM-CSF could be one way of preventing staphylococcal infections; especially in neutropenic disorders, which constitute the highest risk factor for foreign body-associated infections.

J Drug Target, 1996, 4(3), 181 - 9
Encapsulation of vancomycin and gentamicin within cationic liposomes for inhibition of growth of Staphylococcus epidermidis; Sanderson NM et al.; Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol) and dimethyldioctadecylammonium bromide (DDAB) . The cationic vesicles adsorb to biofilms of the skin-associated bacteria Staphylococcus epidermidis, which have a negative charge . Encapsulation of the antibacterial drug vancomycin into such liposomes enhanced its activity relative to the free agent . The effectiveness of the preparation was dependent on the fluidity of the liposomal membrane and on the level of drug entrapment within the aqueous core of the vesicles . The aminoglycoside antibiotic gentamicin was also encapsulated within similar liposomes but was less effective, possibly due to its slow passage through the membrane . The liposomal vancomycin preparation has potential medical use in treating bacterial infections of foreign body biomedical devices (e.g . catheters), with either topical or intravenous administration.

Acta Otolaryngol Suppl, 1996, 525, 61 - 3
Inhibitory effect of roxithromycin on biofilm formation of Pseudomonas aeruginosa; Ozeki M et al.; Biofilm formed by bacteria has recently has been shown to be involved in making infectious diseases intractable . We indirectly measured and evaluated the inhibitory effect of roxithromycin (RXM) on biofilm formation on the inside of a plastic test tube by determining the number of living bacteria in the biofilm . As a result, RXM was found to inhibit biofilm formation even though it does not have antimicrobial activity against Pseudomonas aeruginosa . In addition, this inhibitory effect on biofilm formation was observed at in vivo concentrations which are possible in clinical use of RXM . Thus, there is a possibility that RXM is effective against infectious otolaryngological diseases when biofilm formation is likely to be pathologically involved, even if the detected bacteria are not sensitive to the drug.

Acta Otolaryngol Suppl, 1996, 525, 56 - 60
Inhibitory activity of clarithromycin on biofilm synthesis with Pseudomonas aeruginosa; Kondoh K et al.; Inhibitory activity of clarithromycin (CAM) on biofilm synthesis with Pseudomonas aeruginosa was observed both qualitatively and quantitatively . Using scanning electron microscopy it was seen that biofilms on a Teflon sheet with CAM decreased markedly in a dose-dependent manner as compared with a control Teflon sheet without CAM . The quantity of hexose, a key biofilm ingredient, decreased with increasing concentrations of CAM, even though CAM has no direct bactericidal activity against P . aeruginosa . The results indicated that CAM inhibits the synthesis of biofilm via inhibition of polysaccharides synthesis . This inhibition of CAM was confirmed at 10 micrograms/ml or higher, levels attainable in human tissue at clinical dosage . We concluded that CAM has a considerable potential in the treatment of intractable infection caused by biofilm.

Microbios, 1996, 86(347), 105 - 16
Biofilms associated with poultry processing equipment; Lindsay D et al.; Aerobic and Gram-negative bacteria were enumerated on non-metallic surfaces and stainless steel test pieces attached to equipment surfaces by swabbing and a mechanical dislodging procedure, respectively, in a South African grade B poultry processing plant . Changes in bacterial numbers were also monitored over time on metal test pieces . The highest bacterial counts were obtained from non-metallic surfaces such as rubber fingered pluckers and plastic defeathering curtains which exceeded the highest counts found on the metal surfaces by at least 1 log CFU cm-2 . Gram-negative bacterial counts on all non-metallic surface types were at least 2 log CFU cm-2 lower than corresponding aerobic plate counts . On metal surfaces, the highest microbial numbers were obtained after 14 days exposure, with aerobic plate counts ranging from 3.57 log CFU cm-2 to 5.13 log CFU cm-2, and Gram-negative counts from 0.70 log CFU cm-2 to 3.31 log CFU cm-2 . Scanning electron microscopy confirmed the presence of bacterial cells on non-metallic and metallic surfaces associated with poultry processing . Rubber 'fingers', plastic curtains, conveyor belt material and stainless steel test surfaces placed on the scald tank overflow and several chutes revealed extensive and often confluent bacterial biofilms . Extracellular polymeric substances, but few bacterial cells were visible on test pieces placed on evisceration equipment, spinchiller blades and the spinchiller outlet.

Arch Oral Biol, 1996 Jan, 41(1), 27 - 34
Inhibition by ethanol of the growth of biofilm and dispersed microcosm dental plaques; Sissons CH et al.; Inhibition of microcosm plaque biofilm growth by periodic application of ethanol was compared with the minimum inhibitory concentration (MIC) and bactericidal effects of ethanol on liquid cultures of dispersed plaque bacteria . Microcosm plaques were cultured from saliva in a multiplaque 'artificial mouth' and their growth in wet weight measured daily . Nutrient conditions included: a continuous supply of a medium containing 0.25 percent mucin, and 8-hourly 5 percent (w/v) sucrose (1.5 ml over 6 min) . Plaque biofilm growth was strongly inhibited by exposure to 40 percent (v/v) ethanol applied in volumes of 3.75 ml over 15 min, six times daily . Application of 1.5 ml over 6 min inhibited much less or not at all . Ethanol concentrations lower than 40 percent caused less inhibition, with 10 percent having almost no effect . The pH response to sucrose was unchanged by prior application of 40 percent ethanol for 30 min . Some evidence was obtained for either bacterial adaptation to ethanol or selection of ethanol-resistant bacteria . The MIC and bactericidal effects of ethanol were assessed by growth of dispersed plaque in liquid culture; the bactericidal effect was measured as the induced delay in growth . The aerobic and anaerobic MIC of ethanol for growth was 10 percent and 8 percent; 50 percent inhibition of growth rate occurred at 3.7 percent and 2.8 percent . Ethanol (40 percent) was bactericidal within 1-2 min, but 10 percent had almost no effect . It was concluded that, despite the well-known high ethanol sensitivity of dispersed plaque bacteria, prolonged application of ethanol concentrations in the order of 40 percent are necessary to inhibit growth of plaque biofilms.

Microsc Res Tech, 1996 Jan 1, 33(1), 73 - 86
Applications of laser scanning microscopy for analysis of aquatic microhabitats; Ghiorse WC et al.; Laser scanning microscopy was used 1) to analyze Mn oxide-encrusted biofilms and particles in marine Mn-oxidizing enrichment cultures, 2) to optimize fluorescence in situ hybridization protocols of 16S ribosomal RNA-targeted oligonucleotide signature probes for single bacterial cell identification in particles from a wetland, and 3) to develop a combined immunofluorescence-microautoradiography procedure for analysis of the distribution of 14C-labeled organic compounds and 14C-mineralizing bacteria in groundwater seep sediments . The results demonstrated the wide applicability and benefits of using laser scanning microscopy for analysis of complex microbial assemblages.

J Ind Microbiol, 1996 Jan, 16(1), 48 - 56
A dual fluorescence technique for visualization of Staphylococcus epidermidis biofilm using scanning confocal laser microscopy; Sanford BA et al.; A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced by Staphylococcus epidermidis . Chemostat cultures of RP62A (a well-characterized slime-producing strain of S . epidermidis) were used to produce mature biofilm on polyvinylchloride (PVC) disks immobilized in a modified Robbins device using a 'seed' and 'feed' model system . Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained on in situ biofilm using the dual fluorescence procedure . Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser . Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker . Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies . Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm . The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture of S . epidermidis biofilm formed in in vivo and in vitro on medical grade virgin or modified inert polymer surfaces.

Microbios, 1996, 86(346), 49 - 58
Biofilm formation by Acinetobacter baumannii; Vidal R et al.; Acinetobacter baumannii, an important nosocomial pathogen is usually found on various surfaces in the hospital environment . In this work, the ability to form biofilms on the surface of sterile coverslips by one clinical isolate of A . baumannii was studied . Sessile cells which adhered to coverslips after being immersed in a nutrient-deficient mineral medium were observed by epifluorescence and scanning electron microscopy at various times of incubation . A rapid increase in the number of sessile cells in young biofilms, followed by a slower increase of such cells was found . At 48 h biofilms were clearly visible and an amorphous material similar to the exopolysaccharide described in some other bacteria covered sessile cells was evident . Biofilm formation by A . baumannii probably favours its maintenance on solid surfaces in the hospital environment and protects the micro-organism against some antibacterial factors.

Microbios, 1996, 85(345), 223 - 30
Efficacy of disinfectants against biofilm cells of methicillin-resistant Staphylococcus aureus; Oie S et al.; The effect of disinfectants on biofilm cells of methicillin-resistant Staphylococcus aureus (MRSA) was evaluated . Eradication of biofilm cells of MRSA by chlorhexidine gluconate, benzalkonium chloride, alkyldiaminoethyl glycine and sodium hypochlorite required a much longer time than that of planktonic cells in suspensions . Benzalkonium chloride (0.1%) and alkyldiaminoethyl glycine (0.1%) were ineffective for eradication of biofilm cells even after 1 h, but were effective for eradication of planktonic cells within 20 s . Sodium hypochlorite (0.01%) was also ineffective for eradication of biofilm cells even after 30 min, but was lethal to planktonic cells within 20 s.

Eur J Clin Microbiol Infect Dis, 1996 Jan, 15(1), 88 - 92
Bacteria involved in the blockage of biliary stents and their susceptibility to antibacterial agents; Molinari G et al.; Endoscopically inserted stents are used for the palliation of obstructive jaundice, but infections and blockage of these stents by biliary sludge and bacterial biofilm may develop, presenting major complications . To analyze which bacteria are involved in this process, 25 biliary stents were examined . Eighty-one microorganisms were isolated: 59 gram-negative bacteria (54 Enterobacteriaceae and 5 Pseudomonas aeruginosa), 19 gram-positive bacteria (all Enterococcus spp.), and 3 Candida albicans . The Enterobacteriaceae were sensitive to netilmicin (100%), imipenem (98%), ciprofloxacin (96%), cefotaxime (69%), and piperacillin (57%), whereas Enterococcus spp . were sensitive to imipenem (79%), piperacillin (75%), ciprofloxacin (63%), and ampicillin (58%) . The unpredictable aetiology and high rates of antibiotic resistance suggest that bacteriological monitoring is mandatory to avoid treatment failures in these patients.

Infect Immun, 1996 Jan, 64(1), 277 - 82
Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation; Heilmann C et al.; Biofilm formation is thought to result from the concerted action of primary attachment to a specific surface and accumulation in multilayered cell clusters . Here we describe the isolation and characterization of transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay . Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolated . Each mutant carried one copy of Tn917 . The mutants were divided into two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a) . Mutants of phenotypic class A lacked four cell surface proteins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters . They were able to form a biofilm on a glass surface, a trait that was even more pronounced than in the wild-type stain O-47 . Loss of several surface proteins might have led to the reduced surface hydrophilic structures, thus favoring primary attachment to a glass surface and leading to subsequent biofilm formation . Mutants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a glass surface, mut1 and mut2 could be complemented by wild-type DNA fragments containing the Tn917 insertion sites of mut1 and mut2, respectively . The complemented biofilm-positive clone mut1 (pRC20) produced a 60-kDa protein which is postulated to function as the adhesin for binding to plastic . The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically distinct.

J Bacteriol, 1996 Jan, 178(1), 175 - 83
The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis; Mack D et al.; The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to biofilm production of Staphylococcus epidermidis, which is thought to contribute to virulence in biomaterial-related infections . We purified a specific polysaccharide antigen of biofilm-producing S . epidermidis 1457 and RP62A, which was recently shown to have a function in the accumulative phase of biofilm production by mediating intercellular adhesion (D . Mack, M . Nedelmann, A . Krokotsch, A . Schwarzkopf, J . Heesemann, and R . Laufs, Infect . Immun . 62:3244-3253, 1994) . Following Sephadex G-200 gel filtration, this antigen was separated by Q-Sepharose chromatography into a major polysaccharide, polysaccharide I (> 80%), which did not bind to Q-Sepharose, and a minor polysaccharide, polysaccharide II (< 20%), which was moderately anionic . As shown by chemical analyses and nuclear magnetic resonance spectroscopy, polysaccharide I is a linear homoglycan of at least 130 beta-1,6-linked 2-deoxy-2-amino-D-glucopyranosyl residues . On average, 80 to 85% of them are N acetylated; the rest are non-N-acetylating and positively charged . Chain cleavage by deamination with HNO2 revealed a more or less random distribution of the non-N-acetylated glucosaminyl residues, with some prevalence of glucosaminyl-rich sequences . Cation-exchange chromatography separated molecular species whose content of non-N-acetylated glucosaminyl residues varied between 2 and 26% . Polysaccharide II is structurally related to polysaccharide I but has a lower content of non-N-acetylated D-glucosaminyl residues and contains phosphate and ester-linked succinate, rendering it anionic . Enzyme-linked immunosorbent assay inhibition with various monosaccharides revealed the beta-anomeric form and the acetylated amino group of the D-glucosaminyl residues as important for reactivity with the specific antiserum . The unbranched polysaccharide structure favors long-range contacts and interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular adhesion and biofilm accumulation . The structure of the polysaccharide is, so far, considered to be unique and, according to its function, is referred to as S . epidermidis polysaccharide intercellular adhesin (PIA).

Philos Trans R Soc Lond B Biol Sci, 1995 Dec 29, 350(1334), 325 - 43
Diffusion and binding measurements within oral biofilms using fluorescence photobleaching recovery methods; Birmingham JJ et al.; Numerous studies have postulated that bacteria which reside in a biofilm differ from planktonic bacteria . These differences are thought to affect biofilm permeability and, indirectly, the susceptibility of biofilm bacteria to antibacterial agents . In this study fluorescence recovery after photobleaching (FRAP) was used to monitor the diffusion and binding characteristics of a set of size fractionated fluorescein isothiocyanate (FTTC)-conjugated dextrans over small areas (ca . 10 micron) in bacterial biofilms . From these measurements it was straightforward to calculate apparent diffusion rates . Initial studies on the concentration dependence of dextran interaction with planktonic bacteria showed that no irreversible interaction was occurring, however, anomalous faster than free solution diffusion rates were obtained . This phenomenon was modelled using novel analytical and numerical methods which incorporate reversible binding with associated fluorescence changes . Apparent diffusion rates measured in biofilms were highly dependent on biofilm preparation . Sucrose starved biofilms produced an apparent slow-down of two- to fivefold depending on dextran molecular mass and location within the biofilm, indicating that diffusion within the biofilm is hindered . Sucrose supplemented biofilms produced apparent diffusion rates close to those in free solution, suggesting less hindered diffusion . Ex vivo plaque showed diffusion and binding similar to the sucrose supplemented biofilms . The FRAP approach provides a fast and convenient method for determining diffusion rates over small areas within bacterial biofilms . This study reinforces the importance of considering the influence of reversible binding and associated fluorescence changes, as these may have a marked effect on the measured apparent diffusion rate.

Am J Med, 1995 Dec 29, 99(6A), 26S - 30S
Biofilm disease: its clinical manifestation and therapeutic possibilities of macrolides; Kobayashi H; In the investigation of the disease manifestation of DPB from the viewpoint of biofilm disease, the important factors are an excess antigen-antibody reaction in which alginate acts as the antigen and the resulting formation of immune complex . The effects of 14- and 15-membered macrolides are inhibition of immunoreaction induced by alginate and their inhibitory effect on alginate production serving as an antigen at the GMD level . Further, the specificity of macrolides on these actions was also evidenced from the standpoint of structural activity . In my opinion, based on the similarity in pathogenic process between patients with infected cystic fibrosis and patients with Pseudomonas biofilm disease, long-term administration of 14- or 15-membered ring macrolides can be tried in patients with infected cystic fibrosis.

Enferm Infecc Microbiol Clin, 1995 Dec, 13(10), 581 - 6
{Antimicrobial activity in Staphylococcus epidermidis biofilms on polyvinyl chloride catheters}; Ramirez de Arellano E et al.; OBJECTIVES: We have evaluated the effect of polyvinylchloride (PVC) catheters on the activity of eight antimicrobials . Secondly the activity of these antimicrobials against Staphylococcus epidermidis biofilms on PVC catheters was also studied . METHOD: The MIC and MBC values of amikacine, clindamycine, cloxacilline, ciprofloxacine, vancomycine, teicoplanine, daptomycine and rifampicine was evaluated by a microdilution assay in the presence of 0.5 cm length segments of PVC catheters . Catheter segments with bacteria attached for different periods of time were used as inocula for the studies of antimicrobial activity against bacterial biofilms . Both slime producing and non-producing strains were evaluated . RESULTS: The presence of PVC did not affect the activity of the antimicrobials evaluated against S . epidermidis . For the non-slime producing strains MBC values against sessile bacteria were significantly higher than those against planktonic bacteria . For the slime-producing strain MBCs of ciprofloxacine, amikacine and teicoplanine against both bacterial populations were similar . At high concentrations (16 x MBC), clindamycine was the only antimicrobial that sterilized non-slime producing S . epidermidis biofilms on PVC . Against the slime producing strains ciprofloxacine yielded the highest reduction on bacterial viability (99.9%) . CONCLUSIONS: S . epidermidis biofilms on PVC were more resistant to antimicrobials than planktonic bacteria . Slime production is not the only factor involved in this phenomenon.

Clin Otolaryngol, 1995 Dec, 20(6), 555 - 6
Microbial colonization of the Groningen speaking valve and its relationship to valve failure; Ell SR et al.; Speaking valve failure may be related to biofilm development . An inter-observer validated method of assessing microbial overgrowth in defective prostheses was developed . Two independent observers recorded colonisation of the valves on a 100 mm linear analogue scale in several defined areas . Inter-observer agreement was high in all areas . In vitro opening pressure and derived forward resistances were measured . The relationship between colonization of the valves and the in vitro measurements was investigated . Significant correlations for a two-tailed Spearman's test were recorded between the resistance and microbial colonization of the hinge and oesophageal areas (rs 0.22: P < 0.05, rs 0.25: P < 0.05, n = 80) . There was no significant association for resistance and colonization of the tracheal surface, and no significant association between the opening pressure and the colonization . This work suggests that microbial colonization of the prosthesis in the oesophageal and hinge areas is associated with valve failure.

Anal Chem, 1995 Dec 1, 67(23), 4349 - 53
Tandem mass spectrometry of herbicide residues in lipid-rich tissue; Headley JV et al.; A tandem mass spectrometry procedure, originally developed for bacterial biofilms was adapted for the identification of herbicide residues in lipid-rich tissue of amphipods collected from microcosms in a prairie wetland . For this application, the amounts of tissue employed (less than 1 mg wet weight), and detection of target analytes at picogram levels, were similar to the values reported for bacterial biofilms . Described is an application of the technique for the identification of residues of the herbicide S-2,3,3-trichloroallyl diisopropyl thiocarbamate (triallate; trade name Avadex-BW) . For amphipods collected from microcosms exposed to the herbicide 2-{4-(2,4-dichlorophenoxy)phenoxy}propionic acid methyl ester (diclofop-methyl, trade name Hoe Grass), there were detectable levels of only the hydrolysis product, diclofop acid, in the lipid-rich tissue . Other transformation products reported for bacterial biofilms were not observed in the amphipods.

Oral Microbiol Immunol, 1995 Dec, 10(6), 334 - 41
The effect of sodium hypochlorite on potential pathogenic traits of Candida albicans and other Candida species; Webb BC et al.; Strains of Candida albicans, Candida krusei, Candida kefyr, Candida tropicalis, Candida parapsilosis and Candida guilliermondii were grown in the presence or absence of concentrations of sodium hypochlorite below the minimal inhibitory concentration and tested for a range of characteristics that may be associated with pathogenicity . Sodium hypochlorite is used routinely in hospitals in Australia for disinfection procedures, and these experiments were designed to assess the efficacy of hypochlorite as a sterilizing agent for acrylic dentures . Candida showed varying abilities to adhere to surfaces that may be present in the oral cavity . Sodium hypochlorite reduced the adhesion of all C . albicans strains and most other Candida species to both polystyrene and buccal epithelial cells . A biofilm of Streptococcus gordonii reduced the adhesion of most C . albicans strains and most other Candida species to polystyrene . However, Candida species were able to coaggregate with S . gordonii in suspension, with one strain of C . albicans, GDH 2346, showing greater coaggregating ability than the other strains or species . Sodium hypochlorite increased coaggregation of all C . albicans strains and most other Candida species . Examination of cell wall proteins from strains of C . albicans and a strain of C . parapsilosis showed that growth in hypochlorite increased the amount of protein in some existing bands and, in one strain of C . albicans, increased the number of detectable protein bands ranging from 56 to 26 kDa . Only 4 strains of C . albicans were able to produce hyphae, and 3 of these same strains and C . parapsilosis were able to produce proteinase . Hypochlorite increased the rate of blastospore to hyphal transition but had no effect on proteinase production or activity . It is concluded that hypochlorite at a concentration below the minimal inhibitory concentration may function as an anti-adhesin for Candida species but may not affect their more pathogenic characteristics.

J Infect Dis, 1995 Dec, 172(6), 1509 - 18
Characterization of the hemagglutinin of Staphylococcus epidermidis; Rupp ME et al.; Adherence to biomaterials and production of biofilm is thought to be pivotal in the pathogenesis of prosthetic device infection by Staphylococcus epidermidis . In this study a strong association (P < .001) of hemagglutination with adherence and biofilm production was observed . Hemagglutination was not associated with cell surface hydrophobicity (P = .906) . Hemagglutination inhibition studies revealed that hemagglutination was not affected by heat, pH, cation concentration, proteolytic enzymes, biologic detergent, serum proteins, or subinhibitory antibiotics . Hemagglutination was abolished by periodate oxidation and digestion with glycosidases . It was markedly inhibited by beta-lactose and its monosaccharide constituents in a concentration-dependent fashion . Hemagglutinin expression depended on the presence of glucose . Chemical analysis of a partially purified hemagglutinin preparation and cell-free hemagglutinating supernatants revealed little or no protein and small quantities of reducing sugars, pentose, ketose, hexosamine, uronic acid, and phosphate . Hemagglutinin of S . epidermidis appears to be a polysaccharide distinct from other known adhesins of S . epidermidis.

Arch Surg, 1995 Dec, 130(12), 1345 - 50
Immunosuppression augments growth of graft-adherent Staphylococcus epidermidis; Bergamini TM et al.; OBJECTIVE: To determine if systemic suppression of host defenses during graft implantation alters the initial adherence and subsequent growth of Staphylococcus epidermidis on vascular prostheses . DESIGN: Dacron grafts 1 cm2 were implanted in the back subcutaneous tissue of Swiss-Webster mice (n = 247), followed by topical inoculation with 2 x 10(7), 2 x 10(5), 2 x 10(3), or 2 x 10(1) colony-forming units of S epidermidis . Half of the mice were immunosuppressed with cyclophosphamide (150 mg/kg intraperitoneally), to achieve a consistent, significant decrease in the white blood cell count and major histocompatibility complex class II (Ia) expression . Control mice received an equal volume of saline solution . Graft bacterial biofilm concentrations were determined at 1 day for adherence and within 2 weeks for bacterial growth, by using sonication and quantitative agar culture . RESULTS: Immunosuppression did not significantly alter the initial adherence of bacteria to vascular grafts . Immunosuppressed animals that were inoculated with 2 x 10(7) and 2 x 10(5) colony-forming units of S epidermidis had significantly higher bacterial biofilm concentrations as compared with those in control animals . Graft infection persisted at 14 days in all animals, with and without immunosuppression . CONCLUSIONS: Suppression of immune function during graft implantation augmented growth of adherent bacteria . The effect of short-term perioperative immunosuppression on late-appearing S epidermidis graft infection needs further study.

Adv Dent Res, 1995 Nov, 9(3), 244 - 54; discussion 255-69
The role of microbiology in models of dental caries; Marsh PD; Models of dental caries (laboratory, animal, and human in situ models) vary markedly in their microbiological complexity . Laboratory models range from mono-cultures of cariogenic species providing an acidic challenge to enamel, to the development of diverse mixed cultures growing on a habitat-simulating medium in an artificial mouth or chemostat . The latter systems are of value in determining either mechanisms of action or cause-and-effect relationships--e.g., between dietary components or antimicrobial agents and the microflora . Laboratory models have also shown that the sensitivity of oral bacteria to inhibitors is markedly reduced when growing in biofilms such as dental plaque . Animal models have proved unequivocally that caries is an infectious and transmissible disease . Their use has enabled comparisons to be made of (a) the cariogenic potential of different bacterial species, (b) the role of the diet, and (c) the effects of potential anti-caries agents . It has been claimed that no caries-protective agent currently in use has failed a rodent test . In situ human models have been designed to permit the development of "natural" plaque on standardized enamel surfaces freely exposed to the human oral environment . The microflora that develops on unadulterated surfaces is similar in composition to that found at comparable sites on vital teeth . Demineralization can be accelerated by the inoculation of additional cariogenic bacteria coupled with either intra- or extra-oral sucrose rinses . The increased realism associated with the transition from laboratory to human in situ models is countered by a reduced ability to control or manipulate the system for experimental purposes . Thus a hierarchy of tests is needed for the study of anti-caries agents, each requiring a varying degree of microbiological complexity.

Int J Prosthodont, 1995 Nov-Dec, 8(6), 527 - 34
Cleansing efficacy of commercial denture cleansers: ability to reduce Candida albicans biofilm activity; Nikawa H et al.; A simple method to measure Candidal biofilm activity using pH change of Stomastat was developed and used to evaluate the efficacy of 11 commercial denture cleansers on Candida albicans biofilm . The initial number of yeasts inoculated correlated with the pH value of Stomastat after both 24- and 30-hour incubation periods (r=0.992; r=0.988, respectively; P<.01), which supported the method's validity . The ability of the cleansing agents to decrease fungal biofilm activity varied depending upon the components of the agents . In general, peroxide denture cleansers, a disinfectant, and one enzyme cleanser were more efficacious than the other types tested in this study.

Biomaterials, 1995 Nov, 16(17), 1339 - 43
Adsorption of human salivary proteins to titanium powder . I . Adsorption of human salivary albumin; Steinberg D et al.; Titanium (Ti) is among the most widely used implant materials in dentistry today . The success of Ti implants is associated with their interactions with the surrounding tissues and biological fluids . In the present study, the adsorption of salivary proteins to Ti and the effect of calcium (Ca) on this process were investigated . Untreated and Ca-treated Ti powders were suspended in human clarified whole saliva . After incubation, the supernatant fluid was collected for protein analysis . The powders were then washed and resuspended in EDTA to desorb proteins from Ti surfaces . Sodium dodecylsulphate polyacrylamide gel electrophoresis and Bradford protein assay were conducted to determine the concentration and type of proteins that adsorbed onto Ti surfaces . The presence of Ca ions enhanced the adsorption of salivary proteins to Ti . A 66 kDa protein, identified by immunoblotting as albumin, was found as the main adsorbed salivary protein . Adsorption of albumin to Ti pretreated with Ca was significantly greater than to native Ti . The Ca-dependent adsorption process was reversed by EDTA . The data suggest that salivary albumin is one of the main constituents of a salivary biofilm formed on Ti dental implants and its adsorption to Ti surfaces is Ca-dependent . The presence of albumin on Ti dental implants may affect plaque accumulation on the implants and the biocompatibility of Ti implants.

J Appl Bacteriol, 1995 Nov, 79(5), 558 - 68
Assessment of biological activity and fate of organic compounds in a reactor for the measurement of biodegradable organic carbon in water; Ribas F et al.; A new, rapid method for the determination of biodegradable dissolved organic carbon (BDOC), especially suited to water industry needs, was recently proposed by the authors . This dynamic method measured the BDOC of circulating water continuously pumped over a biofilm attached to a special support (sinterized porous glass) that fills a system of two glass columns . The BDOC value corresponds to the difference in dissolved organic carbon (DOC) between inflow and outflow water samples . The analytical results are not significantly different from those of other bioassays that use indigenous bacteria, and the total duration of the analysis is less than 3 h . However, a problem common to all the BDOC methods based on attached bacteria is the extent to which the decrease in DOC during the BDOC analysis is due to true biodegradation or to adsorption of organic matter to the reactor . In the present study, a reasonable support is provided for the hypothesis that this decrease, at least in the dynamic method, is predominantly due to microbiological activity . After comparing the support (sinterized porous glass) with a good physical adsorbent (granular activated carbon), the influence of temperature, residual chlorine and sodium azide on the reactor performance was tested, and a sensitivity only attributable to biological activity was observed . Another set of experiments were performed to assess the fate and specific elimination of different organic substances, explicable assuming that biodegradation processes were involved.

J Med Microbiol, 1995 Nov, 43(5), 368 - 76
Efficacy of sustained release ciprofloxacin microspheres against device-associated Pseudomonas aeruginosa biofilm infection in a rabbit peritoneal model; Owusu-Ababio G et al.; The relative effectiveness of a poly(L-lactic acid) ciprofloxacin hydrochloride (CIP) microsphere formulation (250-425 microns) against peritoneal implanted biofilm of Pseudomonas aeruginosa was investigated in a rabbit model . Correlations between in-vivo CIP pharmacokinetics in peritoneal dialysate and serum after intraperitoneal administration, in-vivo cell counts and rabbit survival rate were obtained . Dialysate and serum concentrations after 12 h (C12h) were greater than those obtained with free drug whereas maximum serum concentrations (Cmax) were lower and the time to reach Cmax(tmax) was longer . A silastic implant device pre-colonised with P . aeruginosa for 2 days was implanted in the rabbit peritoneum, and dialysate with or without drug or microspheres was administered via a catheter . Rabbits receiving no antibiotic and those receiving free drug (10 mg in dialysate) died of peritonitis and septicaemia, whereas all rabbits given CIP microspheres recovered completely from infection . The viable count of P . aeruginosa was markedly reduced or eliminated from the catheter, the device and the peritoneal wall in CIP microsphere-treated rabbits but not in rabbits treated with free drug, as determined from histological and scanning electronmicroscopic evidence . These results demonstrate that sustained release of antibiotics at biofilm eradication concentrations (BEC) is required to treat biofilm infections associated with peritoneal implanted devices.

Antimicrob Agents Chemother, 1995 Oct, 39(10), 2262 - 8
Modulation of biofilms of Pseudomonas aeruginosa by quinolones; Yassien M et al.; The interaction between four fluoroquinolones (ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin) and biofilms of Pseudomonas aeruginosa in wells of microtiter plates and on segments of vascular catheters were studied in an in vitro model of vascular catheter colonization . Subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of the fluoroquinolones reduced the adherence of P . aeruginosa to 30 to 33, 44 to 47, and 61 to 67% of that of controls, respectively . The addition of high concentrations of the fluoroquinolones (12.5 and 400 micrograms/ml) to preformed biofilms (grown for 48 h at 37 degrees C) decreased the adherence of P . aeruginosa to 69 to 77 and 39 to 60% of that of controls, respectively . In an in vitro model of vascular catheter colonization, subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of fluoroquinolones reduced the number of adherent bacteria to 21 to 23, 40 to 46, and 55 to 70% of that of the controls, respectively . Scanning electron microscopy demonstrated a significant reduction in glycocalyx formation and adherent bacteria in the presence of pefloxacin at one-half to one-eight of the MIC . Vascular catheter segments precolonized with P . aeruginosa for 24 h and exposed to the fluoroquinolones at 4 to 25 times the MIC (50 micrograms/ml) for 2 h showed <5% growth of adherent cells compared with controls . No adherent organisms were cultured in the presence of 8 to 50 times the MIC (100 micrograms/ml) . Scanning electron microscopy studies of preformed biofilms exposed to pefloxacin verified the results obtained by culture . These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adherence of P . aeruginosa to plastic surfaces and vascular catheters . Clinically achievable concentrations of fluoroquinolones (50 to 100 micrograms/ml) were able to eradicate preformed biofilms on vascular catheters.

J Ind Microbiol, 1995 Oct, 15(4), 391 - 6
Prevention of biofilm formation by polymer modification; Jansen B et al.; Bacterial biofilm formation on synthetic polymers plays an important role in industry and in modern medicine, leading, for example, to difficult-to-treat infections caused by colonized foreign bodies . Prevention of biofilm formation is a necessary step in the successful prophylaxis of such infections . One approach is to inhibit bacterial adherence by polymer surface modification . We have investigated polymer modification by glow discharge treatment in order to study the influence of the modified surface on bacterial adherence . Surface roughness, surface charge density and contact angles of the modified polymers were determined and related to the adherence of Staphylococcus epidermidis KH6 . Although no influence of surface roughness and charge density on bacterial adherence was noticed, a correlation between the free enthalpy of adhesion (estimated from contact angle measurements) and adherence was observed . There seems to exist a certain minimum bacterial adherence, independent of the nature of the polymer surface . Modified polymers with negative surface charge allow for bacterial adherence close to the adherence minimum . These polymers could be improved further by the ionic bonding of silver ions to the surface . Such antimicrobial polymers are able to prevent bacterial colonization, which is a prerequisite for biofilm formation . It is suggested that modification of polymers and subsequent surface coupling of antimicrobials might be an effective approach for the prevention of bacterial biofilm formation.

J Ind Microbiol, 1995 Oct, 15(4), 384 - 90
Control of Legionella pneumophila in a hospital water system by chlorine dioxide; Walker JT et al.; Immuno-compromised patients are particularly susceptible to Legionnaires' Disease . After three cases of the disease occurred in a hospital, a continuous dosing regime using chlorine dioxide was initiated to replace chlorination of the water system . This study identified a number of factors which may have resulted in conditions that would encourage the growth of the water-borne pathogen Legionella pneumophila . The residual chlorination was inadequate for microbial control at the taps furthest from the four storage tanks, of which two were found to be in excess for demand . The temperature of the water in the storage tanks was also found to be above 20 degrees C; a temperature that would encourage microbial growth . A back-up calorifier was present and was found to contain L . pneumophila, and linseed oil-based sealants that provide nutrients for microbial growth were also prevalent as jointing compounds in the water circuit . Although the shower heads were routinely disinfected, a requirement was identified to also disinfect the shower hoses . No L . pneumophila were recovered from the water system after the chlorine reduced dioxide disinfection trial . Biofilm was also dramatically reduced after disinfection; however, small microcolonies were identified and proved to be metabolically active when tested with a metabolic indicator . Using light and fluorescence microscopy, the pipe samples removed from the water system were rapidly analysed for biofouling, complementing existing microbiological methods.

J Ind Microbiol, 1995 Oct, 15(4), 377 - 83
A paint incorporating silver to control mixed biofilms containing Legionella pneumophila; Rogers J et al.; A three-stage chemostat containing a mixed consortium of microorganisms, including Legionella pneumophila, was used to determine the suitability of a silver-containing paint to control biofouling in water systems . The paint was efficient in controlling total surface colonisation by heterotrophic microorganisms and growth of the pathogen over a 2-week period . Biodiversity was limited in the presence of the silver paint and this was thought to help control L . pneumophila numbers . Glass control tiles suspended alongside the silver painted tiles also had reduced colonisation for the 2-week period, suggesting that low levels of silver leached from the paint surface . This loss of silver was confirmed since the inhibition of biofouling and inclusion of the pathogen was not maintained after the 2-week period . Although this paint was unsuitable for controlling biofouling over extended time periods, the data suggest that a reformulated paint or electrochemical method of introducing silver ions may be successful.

J Ind Microbiol, 1995 Oct, 15(4), 361 - 71
Regulation of expression of group IA capsular polysaccharides in Escherichia coli and related extracellular polysaccharides in other bacteria; Whitfield C et al.; Bacterial surface polysaccharides fulfill a number of important roles in cell-cell interactions, survival in natural environments, and formation of biofilms . Consequently, the mechanisms involved in regulation of extracellular polysaccharides are predicted to have a significant impact on microbial adaptation . Strains of Escherichia coli, Klebsiella spp, and Erwinia spp produce extracellular polysaccharides which share structural features . There are also similarities in the organization of genes required for synthesis of these cell surface polymers and, in some cases, the mechanism of synthesis may be related . Despite the diverse habitats of these bacteria, the systems which regulate expression of their extracellular polysaccharides appear to share components and mechanisms . Understanding these regulatory processes may lead to novel therapeutic approaches for pathogens, or for control of unwanted biofilm formation in industrial settings.

J Ind Microbiol, 1995 Oct, 15(4), 311 - 7
Modification by surface association of antimicrobial susceptibility of bacterial populations; Allison DG et al.; In the majority of natural situations in which bacteria are found, they are associated with and attached to surfaces . In the presence of moisture and nutrients, they grow to form extensive bacterial films which are often enveloped within copius exopolymeric matrices . Biofilms are ubiquitous to many different situations in industry, the environment and medicine . Their presence can be either beneficial or more commonly detrimental to such systems . In this respect, biofilm populations possess physiological properties distinct from those of unattached, planktonic bacteria . Moreover, it is generally accepted that bacteria growing within a biofilm are more resistant to antimicrobial agents than their planktonic counterparts . However, although the consequences of attachment to antimicrobial resistance have been known for many years, the mechanistic bases for such effects have still to be fully elucidated . In this article the nature of different resistance mechanisms, including those of the exopolymeric matrix, environmental modulation, attachment-specific physiologies and quorum sensing are reviewed.

CLAO J, 1995 Oct, 21(4), 242 - 6
Resistance of adhered bacteria to rigid gas permeable contact lens solutions; May LL et al.; We examined eight conditioning or disinfecting solutions recommended for use with rigid gas permeable (RGP) or hard contact lenses for their efficacy against planktonic and attached cells of bacteria and Candida albicans . Most solutions, particularly those containing phenylmercuric nitrate and polyaminopropyl biguanide, gave marked inhibition (99.99% reduction within 4 hours) of planktonic cells of bacteria . Planktonic cells of Serratia marcescens and C . albicans survived in certain solutions containing chlorhexidine and benzalkonium chloride . In contrast, cells of all test microorganisms adhered to wells of polyethylene contact lens cases showed various degrees of survival after 4, 6, and 12 hours of exposure to most contact lens solutions . Drying of the lens case with adhered cells for 10 hours prior to addition of the lens solution usually reduced the incidence of recovery . Studies demonstrated that biofilms developed in cases with solutions of low and high efficacy . The enhanced resistance of adhered cells requires the periodic disinfection or replacement of lens cases used with RGP lenses.

Chest, 1995 Oct, 108(4), 1049 - 52
Structural features of tracheal tube biofilm formed during prolonged mechanical ventilation; Inglis TJ et al.; The dissemination of tracheal tube biofilm into the mechanically ventilated lung has been proposed as a contributory factor in the pathogenesis of ventilator-associated pneumonia . In the present study, conventional light microscopy, confocal laser scanning microscopy, and scanning electron microscopy were used to examine luminal tracheal tube biofilm in tubes from ten consecutive medical intensive care patients . Biofilms also were cultured . No tube contained a predominantly microbial aggregate . Microorganisms were either dispersed throughout the biofilm or restricted to the most superficial layer . Neutrophil polymorphonuclear cells were present in all biofilms in a pattern suggesting that a layering or stratification had taken place . The distribution of neutrophils and microorganisms was consistent with a progressive accretion of respiratory secretions, rather than formation of a predominantly microbial biofilm.

J Antimicrob Chemother, 1995 Sep, 36(3), 521 - 6
The role of oxygen limitation in the resistance of agar-entrapped, sessile-like Escherichia coli to aminoglycoside and beta-lactam antibiotics; Tresse O et al.; Viable cells of Escherichia coli were entrapped in agar gel layers to form artificial biofilm-like structures . Killing assays of immobilized organisms by latamoxef and tobramycin were performed under different oxygenation conditions of the culture medium and compared with free-cell experiments . Under moderate aeration, agar-entrapped bacteria displayed higher resistance to the two antibiotics than suspended cells . Slow-growing free-cell cultures were resistant to latamoxef but not to tobramycin . In anaerobic incubation conditions, suspended organisms were highly resistant to the two antibiotics . Sustained oxygenation enhanced tobramycin efficacy against free and immobilized cells . These results show that oxygen deficiency in the gel layer contributes to the enhanced antibiotic resistance of sessile-like cells.

Int J Prosthodont, 1995 Sep-Oct, 8(5), 434 - 44
Commercial denture cleansers--cleansing efficacy against Candida albicans biofilm and compatibility wit soft denture-lining materials; Nikawa H et al.; Chemical cleansing has been suggested as one of the most effective methods for plaque control, particularly for resilient denture-lining materials . These materials are known to be easily colonized and infected by Candida albicans . Maintaining the cleanliness of such materials is difficult because of porosity and incompatibility with some types of denture cleansers . Therefore, denture cleansers used for soft liners must address both microbiologic and physical requirements . This study evaluated the antifungal efficacy of 11 commercial denture cleansers on C albicans biofilm, studied the compatibility of tissue conditioners with denture cleansers, and makes suggestions for the chemical cleansing of dentures lined with tissue conditioners.

Antimicrob Agents Chemother, 1995 Sep, 39(9), 2128 - 31
Resistance of Candida albicans biofilms to antifungal agents in vitro; Hawser SP et al.; Biofilms formed by Candida albicans on small discs of catheter material were resistant to the action of five clinically important antifungal agents as determined by {3H}leucine incorporation and tetrazolium reduction assays . Fluconazole showed the greatest activity, and amphotericin B showed the least activity against biofilm cells . These findings were confirmed by scanning electron microscopy of the biofilms.

J Ind Microbiol, 1995 Sep, 15(3), 248 - 53
Influence of lactobacilli on the adhesion of Staphylococcus aureus and Candida albicans to fibers and epithelial cells; Reid G et al.; The ability of organisms to adhere to and form biofilms on fibrous materials is believed to be an important initiating step in the induction of several diseases, such as toxic shock syndrome . Using an in vitro assay, a moderately hydrophobic strain of Staphylococcus aureus (water contact angle 35 degrees) and a hydrophilic Candida albicans (shown by a hexadecane test) were highly adherent to commercial diaper fibers . The lumen side of the diaper was porous and the fibers were very hydrophobic (> 140 degrees), but the internal section was very hydrophilic (0 degrees), presumably for lus strains was present . Surfaces precoated with lactobacilli inhibited staphylococcal adhesion by 26-97%, and candida by 0-67% . When the lactobacilli were used to challenge adherent pathogens, there was 99% displacement of the S . aureus and up to 91% displacement of C . albicans . Hydrophobic L . acidophilus 76 (54 degrees) and T-13 (80 degrees) were the most effective of five Lactobacillus isolates tested at interference by precoating . The moderately hydrophilic L . casei var rhamnosus GR-1 (33 degrees) was the most effective at displacing the yeast . Experiments with uroepithelial cells also showed that the lactobacilli could significantly interfere with the adhesion of both pathogens to the cells . The results demonstrate the rapidity with which two pathogens adhered to fibers and epithelial cells, and raised the possibility that members of the normal female urogenital flora might interfere with infections caused by these organisms.

J Ind Microbiol, 1995 Sep, 15(3), 234 - 42
The effect of electrical currents and tobramycin on Pseudomonas aeruginosa biofilms; Jass J et al.; The combined use of antibiotics with low levels of electrical current has been reported to be more effective in controlling biofilms (the bioelectric effect) than antibiotics alone . An electrical colonisation cell was designed to study the effect of antibiotics on biofilms formed on a dialysis membrane away from the electrode surface . To avoid the electrochemical generation of toxic products, Pseudomonas aeruginosa biofilms were formed in minimal salts medium that excluded chloride-containing compounds . Under these conditions, electrical currents of up to 20 mA cm-2 did not prevent biofilm formation or have any detrimental effect on an established biofilm . Tobramycin alone at concentrations of 10 micrograms ml-1 did not affect the biofilm, but were significantly enhanced by 9 mA cm-2 . The effect of tobramycin concentrations of 25 micrograms ml-1 were enhanced by a 15 mA cm-2 electrical current . In both cases higher levels of electrical current, up to 20 mA cm-2, did not further enhance the effect of the antibiotic . The possible mechanisms of action of the bioelectric effect have been reported to involve electrophoresis, iontophoresis and electroporesis, thus overcoming the biofilm biomass and cell wall barriers . Our results suggest that other factors may also be important, such as the metabolic activity and growth rate of the bacteria . Such factors may be critical in maximising antibiotic efficacy.

J Ind Microbiol, 1995 Sep, 15(3), 214 - 26
Colonization factors of diarrheagenic E . coli and their intestinal receptors; Cassels FJ et al.; While Escherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains of E . coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion . After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness . Enterotoxigenic E . coli (ETEC) is the most extensively studied of the five categories of E . coli that cause diarrheal disease, and has the greatest impact on health worldwide . ETEC can be isolated from domestic animals and humans . The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described . Another major category, enteropathogenic E . coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC . An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response . Three additional categories of E . coli diarrheal disease, their colonization factors and their host cell receptors, are discussed . It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and that E . coli is part of these biofilms as both commensals and pathogens.

J Ind Microbiol, 1995 Sep, 15(3), 208 - 13
Interactions of Candida albicans with bacteria and salivary molecules in oral biofilms; Holmes AR et al.; The yeast Candida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells . C . albicans and Candida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind to Streptococcus gordonii NCTC 7869 while two other Candida species (Candida krusei and Candida kefyr) do not . Adherence of C . albicans was greatest when the yeast had been grown at 30 degrees C to mid-exponential growth phase . For 21 strains of C . albicans there was a positive correlation between the ability to adhere to S . gordonii and adherence to experimental salivary pellicle . Whole saliva either stimulated or slightly inhibited adherence of C . albicans to S . gordonii depending on the streptococcal growth conditions . The results suggest that the major salivary adhesins and coaggregation adhesins of C . albicans are co-expressed.

J Ind Microbiol, 1995 Sep, 15(3), 198 - 207
Oxygen metabolism, oxidative stress and acid-base physiology of dental plaque biofilms; Marquis RE; Dental plaque is a natural biofilm which has been a focus of attention for many years because of its known roles in caries and periodontal diseases . Acid production by plaque bacteria leads to the erosion of tooth mineral in caries, and the cariogenicity of plaque is related to population levels of acid-tolerant organisms such as mutans streptococci . However, the biofilm character of plaque allows for survival of a diverse flora, including less acid-tolerant organisms, some of which can produce ammonia from arginine or urea to counter acidification . Plaque is often considered to be relatively anaerobic . However, evidence is presented here that both supragingival and subgingival plaque have active oxygen metabolism and that plaque bacteria, including anaerobes, have developed defenses against oxidative stress . Even in subgingival plaque associated with periodontitis, measured residual oxygen levels are sufficient to allow for oxygen metabolism by organisms considered to be extremely anaerobic such as Treponema denticola, which metabolizes oxygen by means of NADH oxidases and produces the protective enzymes superoxide dismutase and NADH peroxidase . The finding that plaque bacteria produce a variety of protective enzymes is a good indicator that oxidative stress is a part of their everyday life . The biofilm character of plaque allows for population diversity and coexistence of aerobes, anaerobes and microaerophiles . Overall, agents that affect oxidative metabolism offer possibilities for reducing the pathogenic activities of plaque.

J Ind Microbiol, 1995 Sep, 15(3), 193 - 7
Isolation and characterization of coaggregation-defective (Cog-) mutants of Streptococcus gordonii DL1 (Challis); Clemans DL et al.; Streptococcus gordonii DL1 (Challis) bears coaggregation-mediating surface adhesins which recognize galactoside-containing surface polysaccharides on Streptococcus oralis 34, Streptococcus oralis C104, and Streptococcus SM PK509 . Fifty-nine spontaneously-occurring coaggregation-defective (Cog-) mutants of S . gordonii DL1 unable to coaggregate with partner streptococci were isolated . Six representative Cog- mutants were characterized by their coaggregation properties with four Actinomyces naeslundii strains (T14V, PK947, PK606, PK984), Veillonella atypica PK1910, and Propionibacterium acnes PK93 . The six representative Cog- mutants showed altered coaggregation with their streptococcal partners, A . naeslundii PK947, and P . acnes PK93 . Based on the coaggregation phenotypes of these mutants, a model for the lactose-inhibitable coaggregation between S . gordonii DL1 and its partner bacteria is proposed . The potential use of these mutants in studies of oral biofilms is discussed.

J Ind Microbiol, 1995 Sep, 15(3), 186 - 92
Genetic analysis of adherence by oral streptococci; Jenkinson HF; Streptococci are one of the most successful bacterial colonizers of the human body and are major components of oral biofilms . The bacterial cells express multiple cell-surface adhesins that are responsible for the ability of streptococci to adhere to a wide range of substrates which include salivary and serous proteins, epithelial cells and other bacterial cells . Analysis of adherence-defective mutants has indicated the importance of high molecular mass wall-associated polypeptides and of enzymes catalyzing extracellular glucan polysaccharide synthesis to the adherence and accumulation of oral streptococci . The analysis of isogenic mutants of streptococci, generated through insertional inactivation (or allelic exchange), has confirmed the essential roles of specific surface polypeptides both to adhesive processes and to correct assembly of the cell wall layers.

J Ind Microbiol, 1995 Sep, 15(3), 169 - 75
Dental plaque as a biofilm; Marsh PD et al.; Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin . Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface . Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle) . To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface . Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions . These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession . As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogenous environment . Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases) . Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.

J Ind Microbiol, 1995 Sep, 15(3), 162 - 8
Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide; Boyd A et al.; Pseudomonas aeruginosa synthesizes an exopolysaccharide called alginate in response to environmental conditions . Alginate serves to protect the bacteria from adversity in its surroundings and also enhances adhesion to solid surfaces . Transcription of the alginate biosynthetic genes is induced upon attachment to the substratum and this leads to increased alginate production . As a result, biofilms develop which are advantageous to the survival and growth of the bacteria . In certain circumstances, P . aeruginosa produces an alginate lyase enzyme which cleaves the polymer into short oligosaccharides . This negates the anchoring properties of the alginate and results in increased detachment of the bacteria away from the surface, allowing them to spread and colonize new sites . Thus, both alginate biosynthetic and degradative enzymes are important for the development, maintenance and spread of P . aeruginosa biofilms.

J Ind Microbiol, 1995 Sep, 15(3), 156 - 61
Lectin-biotin assay for slime present in in situ biofilm produced by Staphylococcus epidermidis using transmission electron microscopy (TEM); Sanford BA et al.; A lectin-biotin assay was developed for use in the specific detection of slime produced by Staphylococcus epidermidis RP62A and M187sp11 grown in a chemically defined medium . Mature biofilm was formed on polyvinylchloride (PVC) disks using a combined chemostat-modified Robbins device (MRD) model system . Specimens fixed in situ were: 1) stained with ruthenium red; 2) reacted overnight with biotin-labeled lectins (WGA, succinyl-WGA, Con A, or APA) followed by treatment with gold-labeled extravidin; or 3) reacted with antibodies against S . epidermidis RP62A capsular polysaccharide/adhesin (PS/A) using an immunogold procedure . WGA and succinyl-WGA (S-WGA), which specifically bind N-acetylglucosamine, were shown by TEM to react only with slime, both cell-associated and exocellular . In contrast, Con A, APA and anti-PS/A reacted with the bacterial cell surface but did not react with slime . These results indicate the usefulness of WGA lectin as a specific marker for detection of the presence and distribution of slime matrix material in S . epidermidis biofilm.

J Ind Microbiol, 1995 Sep, 15(3), 152 - 5
Bacterial biofilm and clogging of biliary stents; Sung JJ; Endoscopic biliary stenting has become a standard palliative treatment for obstructive jaundice due to malignancies of the pancreas and the hepatobiliary system . Despite the high initial success rate in achieving biliary drainage, durable endoscopic stenting has been limited by the clogging of biliary stents, usually after 4-5 months, due to formation of an adherent bacterial biofilm . Various methods have been investigated for the prevention of bacterial adhesion and prolongation of stent patency . These include: 1) prophylactic use of antimicrobial agents and bile salts; 2) testing of new stent material and new designs for these biliary stents; and 3) the recent introduction of self-expandable metal stents . Each method has its own merits as well as specific problems . This article reviews the pathogenesis of biofilm formation on the biliary stents and the latest status of research in avoiding the problem of stent occlusion.

J Ind Microbiol, 1995 Sep, 15(3), 148 - 51
Tolerance of Staphylococcus epidermidis grown from indwelling vascular catheters to antimicrobial agents; Khardori N et al.; During a prospective study of indwelling vascular catheter-related infections, 134 isolates of Staphylococcus epidermidis were grown from 700 catheter tips . In vitro antimicrobial susceptibility testing of these isolates to oxacillin, vancomycin and ofloxacin was performed using the standard broth microdilution technique . These results were compared to those for the same organisms grown in biofilm before the addition of antimicrobial agents . In 96-well flat bottom microtiter plates, 10(4)-10(5) colony forming units of S . epidermidis in 0.1 ml broth were grown for 18 h at 37 degrees C, at which time a biofilm was observed for all isolates . Different concentrations of antimicrobial agents (0.1 ml) were then added to the plates . The plates were incubated for 18 h at 37 degrees C . Since MICs could not be estimated in these plates, all the wells were subcultured after mixing the biofilm with the broth . Minimum bactericidal concentrations (MBCs) were defined as 99.9% reduction in colony forming units . For organisms grown in suspension, 100% of the isolates were susceptible to vancomycin, 81% to ofloxacin and 40% to oxacillin . MBCs of susceptible isolates were within four-fold differences for vancomycin (53%), oxacillin (50%), and ofloxacin (51%) . When grown as a biofilm, 78%, 93% and 71% of isolates had MBCs of > or = 2048 micrograms ml-1 of oxacillin, vancomycin and ofloxacin respectively . These data demonstrate the reduced bactericidal activity of antimicrobial agents against S . epidermidis in a biofilm and a simple method for its detection in the microbiology laboratory.

J Ind Microbiol, 1995 Sep, 15(3), 141 - 7
Biofilms in device-related infections; Khardori N et al.; The use of various medical devices including indwelling vascular catheters, cardiac pacemakers, prosthetic heart valves, chronic ambulatory peritoneal dialysis catheters and prosthetic joints has greatly facilitated the management of serious medical and surgical illness . However, the successful development of synthetic materials and introduction of these artificial devices into various body systems has been accompanied by the ability of microorganism to adhere to these devices in the environment of biofilms that protect them from the activity of antimicrobial agents and from host defense mechanisms . A number of host, biomaterial and microbial factors are unique to the initiation, persistence and treatment failures of device-related infections . Intravascular catheters are the most common devices used in clinical practice and interactions associated with these devices are the leading cause of nosocomial bacteremias . The infections associated with these devices include insertion site infection, septic thrombophlebitis, septicemia, endocarditis and metastatic abscesses . Other important device-related infections include infections of vascular prostheses, intracardiac prostheses, total artificial hearts, indwelling urinary catheters, orthopedic prostheses, endotracheal tubes and extended wear lenses . The diagnosis and management of biofilm-associated infections remain difficult but critical issues . Appropriate antimicrobial therapy is often not effective in eradicating these infections and the removal of the device becomes necessary . Several improved diagnostic and therapeutic modalities have been reported in recent experimental studies . The clinical usefulness of these strategies remains to be determined.

J Ind Microbiol, 1995 Sep, 15(3), 137 - 40
Overview of microbial biofilms; Costerton JW; As the success of this two-issue special section of the Journal of Industrial Microbiology attests, the study of microbial biofilms is truly burgeoning as the uniqueness and the importance of this mode of growth is increasingly recognized . Because of its universality the biofilm concept impacts virtually all of the subdivisions of Microbiology (including Medical, Dental, Agricultural, Industrial and Environmental) and these two issues incorporate contributions from authors in all of these disciplines . Some time ago we reasoned that bacteria cannot possibly be aware (sic) of their precise location, in terms of this spectrum of anthrocentric subspecialties, and that their behavior must be dictated by a standard set of phenotypic responses to environmental conditions in what must seem to them (sic) to be a continuum of very similar aquatic ecosystems . In this overview I will, therefore, stress the common features of microbial biofilms that we should bear in mind as we use this simple universal concept to seek to understand bacterial behavior in literally hundreds of aquatic ecosystems traditionally studied by dozens of subspecies of microbiologists reared in sharply different scientific and academic conventions.

J Am Dent Assoc, 1995 Sep, 126(9), 1255 - 60
Contribution of biofilm bacteria to the contamination of the dental unit water supply; Williams HN et al.; The authors studied the contribution of bacterial biofilm to the contamination of the dental unit water supply, as well as the effects of flushing and sodium hypochlorite treatment on reducing the number of contaminants . This study demonstrated that biofilm in the dental tubing was the primary source of contaminated water delivered by dental units.

Nippon Hinyokika Gakkai Zasshi, 1995 Sep, 86(9), 1440 - 9
{A semi-quantitative measurement of glycocalyx and an ATP bioluminescent assay for the analysis of Pseudomonas aeruginosa biofilm}; Ono N; BACKGROUND: In order to analyze each element of a bacterial biofilm, a semi-quantitative measurement of glycocalyx using toluidine blue and an improved ATP bioluminescence assay were developed . RESULTS: In the toluidine blue assay, a linear correlation was obtained between optical density and the amount of gellan gum, which is an exopolysaccharide derived from Pseudomonas elodea . By using the improved ATP assay, ATP at concentration of 1 x 10(-4) mol/l or floating bacterial cells of 10(3) cfu could be detected . The applicability of these two methods was examined in evaluating a total amount of glycocalyx and ATP content of Pseudomonas aeruginosa biofilms, which were developed or treated with antimicrobial agents in a modified Robbins device . CONCLUSION: The changes in optical density and ATP luminescence were reasonable, and well correlated with those obtained by scanning electron microscopy . These results indicate that the toluidine blue assay and ATP assay are useful in analyzing bacterial biofilms.

Biochim Biophys Acta, 1995 Aug 17, 1245(1), 85 - 93
Differentiation of viable and non-viable bacterial biofilms using electrorotation; Zhou XF et al.; A new technique for studying the properties of biofilms has been developed, based on the phenomenon of electrorotation . Biofilms of Klebsiella rubiacearum were formed on the surfaces of 6 microns diameter polystyrene beads, and the presence of such films was found to alter their electrorotation spectra . The effects of adding a biocide (polyhexanide) to the surrounding aqueous medium was also investigated . The dielectric properties of the beads with biofilms, before and after biocide treatment, were interpreted from the electrorotation spectra using modelling methods that have been well tested for other heterogeneous biological systems . The technique is of value in understanding the physico-chemical properties of biofilms and can be adapted for monitoring the presence of toxic chemicals and for testing the activity of biocides against biofilms.

J Immunol, 1995 Aug 15, 155(4), 2029 - 38
Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients; Meluleni GJ et al.; Serum opsonophagocytic-killing titers often indicate the level of immune resistance to bacterial pathogens, yet in almost all cystic fibrosis (CF) patients that have chronic lung infections with mucoid Pseudomonas aeruginosa, high titers of opsonic-killing Abs can be measured and the infectious pathology still progresses through pulmonary failure and death . This anomalous finding may be due to the use of suspended cells of P . aeruginosa to evaluate phagocytic killing, whereas in the lungs of CF patients the organisms grow in a microcolony or biofilm, encased in mucoid exopolysaccharide (MEP, also called alginate) . To determine whether the microcolony mode of growth contributes to bacterial resistance to host defenses, we evaluated opsonophagocytic killing of mucoid P . aeruginosa growing in a biofilm . Abs from infected CF patients were poorly able to mediate opsonic killing of biofilm, but not suspended, mucoid P . aeruginosa cells . Bacterial resistance to killing could be overcome by disruption of the biofilm layer with an enzyme that degrades MEP . Chronically infected CF patients also fail to produce opsonic-killing Abs specific to MEP, and when these Abs were evaluated in sera of older, noninfected CF patients and humans vaccinated with MEP, comparable killing of P . aeruginosa in biofilms and suspensions was obtained . In this case, C3 was deposited onto the MEP layer and could be visualized by fluorescence microscopy deposited throughout the biofilm . We conclude that opsonic Abs made by CF patients in response to chronic infection are ineffective at mediating phagocytic killing and elimination of bacterial cells growing as microcolonies in their lungs.

J Antimicrob Chemother, 1995 Aug, 36(2), 425 - 30
Activity of sparfloxacin on Staphylococcus epidermidis attached to plastic catheters; Pascual A et al.; The activity of sparfloxacin on Staphylococcus epidermidis biofilms on different plastic catheters was evaluated . Sparfloxacin showed high bactericidal activity against S . epidermidis biofilms on Vialon and polyvinylchloride catheters . The combination of sparfloxacin with amikacin or rifampicin significantly increased its activity against bacterial biofilms on polyurethane and Teflon catheters.

Kansenshogaku Zasshi, 1995 Aug, 69(8), 913 - 8
{Formation of experimental rat bladder calculus and adherence of Pseudomonas aeruginosa to the calculus}; Takahata M et al.; The formation of experimental bladder calculus was studied . The calculus was formed by the uptake of ethylene glycolwater (1%) and retaining the silk thread in rat bladder with high frequency . The components of the calculus were calcium oxalate and calcium phosphate from the results of the electron prove micro analysis (EPMA) and ion chromatography . On the 7th day after the beginning of experiment, Pseudomonas aeruginosa was inoculated to the rat bladder via the urethra . Seven days after the infection, P . aeruginosa adhered to the surface of the calculus such as an aspect of a biofilm . It was considered that this experimental model was useful to study the adherence of bacteria, biofilm formation and its chemotherapy by antibacterial agents.

J Dairy Res, 1995 Aug, 62(3), 509 - 19
Development of bacterial biofilms in dairy processing lines; Austin JW et al.; Adherence of bacteria to various milk contact sites was examined by scanning electron microscopy and transmission electron microscopy . New gaskets, endcaps, vacuum breaker plugs and pipeline inserts were installed in different areas in lines carrying either raw or pasteurized milk, and a routine schedule of cleaning-in-place and sanitizing was followed . Removed cleaned and sanitized gaskets were processed for scanning or transmission electron microscopy . Adherent bacteria were observed on the sides of gaskets removed from both pasteurized and raw milk lines . Some areas of Buna-n gaskets were colonized with a confluent layer of bacterial cells surrounded by an extensive amorphous matrix, while other areas of Buna-n gaskets showed a diffuse adherence over large areas of the surface . Most of the bacteria attached to polytetrafluoroethylene (PTFE or Teflon) gaskets were found in crevices created by insertion of the gasket into the pipeline . Examination of stainless steel endcaps, pipeline inserts, and PTFE vacuum breaker plugs did not reveal the presence of adherent bacteria . The results of this study indicate that biofilms developed on the sides of gaskets in spite of cleaning-in-place procedures . These biofilms may be a source of post-pasteurization contamination.

Tuber Lung Dis, 1995 Aug, 76(4), 318 - 23
Dental units: an environmental study of sources of potentially pathogenic mycobacteria; Schulze-Robbecke R et al.; SETTING: Infections caused by non-tuberculous mycobacteria (NTM) are generally thought to be acquired from environmental sources . However, little is known about the situations in which transmission occurs . OBJECTIVE: In an attempt to identify situations of relevant contact with NTM we investigated the water to which patients are exposed during dental treatment . DESIGN: The concentration and species of NTM were determined in 43 cooling and spray water samples from 21 dental units in ten offices . In addition, mycobacterial colonization of 16 biofilm samples from the waterlines of two dental units was investigated . RESULTS: The mean NTM concentration in the water samples was 365 colony-forming units (cfu) per mL, exceeding the mean drinking water concentration by a factor of almost 400 . In the biofilm samples the mean NTM density amounted to 1165 cfu/cm2 . The species identified included Mycobacterium gordonae, M . flavescens, M . chelonae, 'M . chelonae-like organism' and M . simiae . CONCLUSION: High numbers of NTM may be swallowed, inhaled or inoculated into oral wounds during dental treatment, possibly resulting in colonization, sensitization or infection . Mycobacterial proliferation in biofilms forming within dental units may explain the extent of NTM contamination of dental spray and cooling water.

Ecotoxicol Environ Saf, 1995 Aug, 31(3), 211 - 7
Xenobiotic biodegradation test using attached bacteria in synthetic seawater; Osswald P et al.; The aerobic biodegradability of aniline, used as reference chemical, has been performed in synthetic seawater with attached biomass in a continuously fed reactor (biofilm chemostat reactor, BCR) . Marine bacteria inocula came from local marine fish aquarium filters to limit the geographic and seasonal variations in quality . A pretreatment of these inocula combining 5-microns filtration and centrifugation was used to concentrate bacteria and remove organic carbon contamination of the test . The performances of the BCR were tested in comparison with simple shake flask tests . Among the different variables tested, the ratio S0/X0 (initial concentration of xenobiotic to initial density of the inoculum), the presence of dissolved oxygen, and the hydraulic residence time appear to be the key parameters controlling the length of the biodegradation process . On the other hand, the addition of a cosubstrate (easily biodegradable compound) does not provide advantages . Thus, marine biofilm chemostat reactors with a high density of attached bacteria (around 10(7) cells cm-2) and fed with synthetic seawater plus nitrogen provide good tools for screening biodegradability of chemicals in the marine environment.

ASAIO J, 1995 Jul-Sep, 41(3), M365 - 8
Inhibition of bacterial adhesion and biofilm formation by a heparinized hydrophilic polymer; Nagaoka S et al.; The adhesion and growth of two pathogenic bacteria (Escherichia coli and Staphylococcus aureus) on the surface of a heparinized hydrophilic polymer were studied . Heparinized hydrophilic polymer is composed of poly(vinyl chloride) grafted with poly(ethylene glycol) monomethacrylate, diethylaminoethyl methacrylate, and ionically bound heparin . Poly(vinyl chloride) was used as a control . Plasma protein pre coated polymers were also prepared to evaluate the effect of proteins on bacterial adhesion . Polymer films were stored in bacterial suspensions under gentle shaking at 37 degrees C for 24 hr . The amount of adherent bacterial cells was measured by the bioluminescent assay of bacterial adenosine triphosphate . Their structure was observed by use of a scanning electron microscope . These evaluations demonstrated that a large amount of bacterial adhesion and biofilm formation was found on the surface of poly(vinyl chloride), whereas significant reductions in bacterial adhesion and no biofilm formation were observed on heparinized hydrophilic polymer . Bacterial adhesion onto plasma protein pre coat polymer films were also investigated, and it was clear that the bacterial adhesion on these surfaces was dependent upon the amount and species of absorbed proteins.

J Antimicrob Chemother, 1995 Jul, 36(1), 7 - 21
Speculations on the influence of infecting phenotype on virulence and antibiotic susceptibility of Legionella pneumophila; Barker J et al.; It is not clear how Legionella pneumophila, which is a ubiquitous aquatic organism not possessing a mammalian reservoir, evolved the ability to cause human disease . The unusual ecology of the organism may play an important role in the transmission and virulence of legionella infections . L . pneumophila can infect and kill specific species of free-living amoebae as well as multiplying as an intracellular parasite in human phagocytic cells . In nature L . pneumophila can survive and possibly replicate in free suspension, and grow in biofilms and in protozoa thus leading to diverse phenotypes, potentially with diverse virulence and susceptibility properties . Indeed, recent evidence shows that intra-amoeba growth induces a phenotype that is dramatically different physiologically to that obtained in vitro, with altered virulence and susceptibility properties . Growth in macrophages also has profound effect on the physiological properties of L . pneumophila . Many different stress proteins are expressed by the organism as a result of intra-macrophage growth . A heat shock protein is abundantly synthesised and may be presented on the surface of infected macrophages, which allows them to be targeted by T-lymphocytes for destruction . The difficulties in successfully treating Legionnaires' disease are probably influenced by the intracellular location of L . pneumophila . Retrospective clinical studies show that it is only drugs such as erythromycin, ciprofloxacin and rifampicin, which are capable of accumulating in phagocytic cells, that are efficacious in the treatment of legionnaires' disease . Despite the use of such drugs treatment failures occur, but these do not appear to be associated with the emergence of resistant strains . Studies have shown that although erythromycin and rifampicin can inhibit the multiplication of L . pneumophila in macrophages the organism is not killed and can resume multiplying when the drugs are removed . Thus a competent cell mediated immune response is important in recovery from legionella infections . There is an urgent need for greater understanding of how the changes induced by intracellular growth affect sensitivity to antibiotics and of how the changes induced by intracellular growth affect sensitivity to antibiotics and host defences . Immunocompromised patients, who have the highest mortality rates, are likely to gain the most from progress in the treatment of L . pneumophila infections.

Can J Microbiol, 1995 Jul, 41(7), 647 - 54
Bacterial succession within a biofilm in water supply lines of dental air-water syringes; Tall BD et al.; Biofilms have been implicated as reservoirs for bacterial contamination of water delivered by dental air-water syringes . A 6-month study was done of bacterial colonization and biofilm formation in plastic water supply lines connected to dental air-water syringes . Changes in biofilm flora were observed by both scanning electron microscopy and bacteriologic culture . By day 7, many rod- and spiral-shaped bacteria had colonized the ridged surface of the luminal wall of the tubing, as revealed by scanning electron microscopy . By day 30, individual microcolonies were embedded in extracellular polymeric material . By day 120, these microcolonies had begun to coalesce, and by day 180 the biofilm had developed into a multilayered, heterogeneous mixture of microcolonies . The mean aerobic plate counts of colony-forming units of planktonic and biofilm populations were, in log10 values, 5.9 +/- 0.54/mL and 4.2 +/- 0.82/cm2, respectively . Early colonizers were predominantly Pseudomonas spp., but included Pasteurella, Moraxella, Ochrobactrum, and Aeromonas spp . Flavobacterium and Acinetobacter spp . were observed later . Many of these organisms are opportunistic pathogens . These results demonstrate the longitudinal dynamics of biofilm formation.

Int J Food Microbiol, 1995 Jul, 26(2), 147 - 64
Influence of culture conditions on biofilm formation by Escherichia coli O157:H7; Dewanti R et al.; Biofilms of Escherichia coli O157:H7 were developed on stainless steel chips in trypticase soy broth (TSB), 1/5 dilution of TSB, 0.1% Bacto peptone (BP) and a minimal salts medium (MSM) supplemented with 0.04% of one of the following carbon sources: glucose, glycerol, lactose, mannose, succinic acid, sodium pyruvate or lactic acid . It was found that biofilms developed faster and a higher number of adherent cells (ca . 10(6) CFU/cm2) were recovered when the organisms were grown in the low nutrient media . Regardless of the carbon source, biofilms developed in MSM consisted of shorter bacterial cells and thicker extracellular matrix (ECM), with glucose as the best substrate for stable biofilm formation . Fewer bacteria in initial attachment, non-hydrophobicity of bacterial cells, lack of ECM formation and easy detachment of the biofilm bacteria may contribute to poor biofilm formation in TSB . ECM is probably important for the stability of biofilms; however, at 10 degrees C and under anaerobic conditions, ECM seems to be unnecessary.

J Obstet Gynecol Neonatal Nurs, 1995 Jul-Aug, 24(6), 525 - 31
The impact of recent advances in microbiology and immunology on perinatal and women's health care; Munro CL; Microbiology and immunology have offered important advances in the care of women and newborns . Understanding of the interplay between individuals and the microorganisms associated with them has provided the framework for important changes in practice . An excellent historical example of this idea, well known to nurses, involves puerperal fever . A recent example is knowledge of the relationship of breastfeeding and human immunodeficiency virus . Recent advances in microbiology and immunology that have improved women's health include improved diagnostic tests and a more complete understanding of the vaginal biofilm.

Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6195 - 9
Three-dimensional tracking of motile bacteria near a solid planar surface; Frymier PD et al.; Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers . In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface . Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria . The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al . {Ramia, M., Tullock, D . L . & Phan-Tien, N . (1993) Biophys J . 65,755-778}, which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate . The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces . The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.

FEMS Microbiol Lett, 1995 Jun 15, 129(2-3), 183 - 8
Reporter genes and fluorescent probes for studying the colonisation of biofilms in a drinking water supply line by enteric bacteria; Robinson PJ et al.; Biofilms containing diverse microflora were developed on bitumen-painted steel and glass tiles suspended in a chemostat model of a water distribution system . Escherichia coli, taken from a naturally occurring biofilm, was transformed with a plasmid containing the anaerobically induced nirB promoter fused to the lacZ reporter gene . The resulting transformant, PRB1, was introduced into the chemostat . After 7 and 13 days, an E . coli strain with an anaerobically induced Lac+ phenotype was present in the biofilm . Development of an episcopic differential interference contrast technique combined with UV fluorescence microscopy enabled the simultaneous visualization of E . coli in the biofilm using a fluorescent probe to detect expression of the gusA reporter gene and a lacZ fluorescent probe to monitor anaerobic expression of beta-galactosidase from pnirB.

Appl Environ Microbiol, 1995 Jun, 61(6), 2252 - 6
Nonuniform spatial patterns of respiratory activity within biofilms during disinfection; Huang CT et al.; Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine . Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h . Relatively little biofilm removal occurred as evidenced by total cell direct counts . Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter . The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms . Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment . These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis . Cells near the biofilm-bulk fluid interface lost respiratory activity first . After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.

Br J Ophthalmol, 1995 Jun, 79(6), 601 - 5
Acanthamoeba, bacterial, and fungal contamination of contact lens storage cases; Gray TB et al.; BACKGROUND--Microbial corneal infection is the most serious complication of contact lens wear . Contact lens cases are a recognised potential source of pathogens associated with corneal ulcers . METHODS--This survey established the incidence of protozoal, bacterial, and fungal contact lens case contamination in 101 asymptomatic daily wear cosmetic contact lens wearers from a domiciliary contact lens practice . RESULTS--Eighty two (81%) contact lens cases were found to be contaminated, with 19 (19%) sterile . Of all contact lens cases, 78 (77%) grew bacteria, 24 (24%) fungi, and 20 (20%) protozoa . Acanthamoeba spp were isolated from eight (8%) contact lens cases . Fifty six (55%) contact lens cases yielded mixed bacterial contamination . This is the first contact lens case survey in which hydrogen peroxide disinfection was the major method of contact lens disinfection (75% of subjects) and no home made saline was used . All the contaminating organisms were shown to possess the enzyme catalase that breaks down hydrogen peroxide to oxygen and water . The polymicrobial nature of the biofilms found in many contact lens cases is illustrated electron micrographically . CONCLUSION--Based on data from this and previous studies, the authors conclude with recommendations for contact lens wearers: (1) regular scrubbing of contact lens case interior to disrupt biofilms; (2) exposure of contact lens case to very hot water (> or = 70 degrees C) will kill Acanthamoeba contaminants; (3) allow contact lens case to air dry between uses; (4) if hydrogen peroxide disinfection is preferred, use a two step system; (5) replace contact lens case regularly.

Kansenshogaku Zasshi, 1995 Jun, 69(6), 738 - 46
{Studies on characteristics of bacteria which cause infectious stone}; Hayashi T; Culture of stones and electrical microscopic observation were performed with stones collected from forty-eight patients who underwent surgery for urolithiasis . Thirty-six strains of bacteria in the stones obtained form stone culture were classified as nineteen strains of bacteria deriving from primary infection stones (Group A-I) and seventeen strains of bacteria deriving from metabolic stones (Group A-M) . As for their ability to produce urease and glycocalyx, they were studied in comparison with forty-nine strains of stone surface-adhering bacteria (Group B) . Glycocalyx producing ability was examined by the provisional quantitative toluidine blue assay and safranine straining method . As for the electrical microscopic observation, formation of biofilm bacterium was observed in all twenty cases of primary infection stones and in thirteen cases (46.4%) of twenty-eight cases of metabolic stones . Urease producing ability per stone was 6/14 (43.9%) in Group A-I, 4/13 (30.8%) in Group A-M and, 4/32 (12.5%) in Group B . There was a significant difference (p < 0.05) between Group A-I and Group B . Similarly for the glycocalyx producing ratio (toluidine blue assay), the values were 9/14 (64.3%) in Group A-I, 11/3 (84.6%) in Group A-M and 10/32 (31.3%) in Group B . As for the glycocalyx producing ration (safranine method), the values were 10/14 (71.4%) in Group A-I, 9/13 (69.2%) in Group A-M and 9/32 (28.1%) in Group B . As for the glycocalyx producing ability, Group A-I and Group A-M both showed significantly higher production ratios compared to Group B for both toluidine blue method and safranine method.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomaterials, 1995 Jun, 16(9), 721 - 5
Corrosion of intra-oral magnets in the presence and absence of biofilms of Streptococcus sanguis; Wilson M et al.; Intra-oral magnets are used in dentistry for a variety of purposes, and their susceptibility to corrosion is of great clinical importance . Although a number of in vitro models have been developed to study corrosion of intra-oral magnets, none have attempted to determine the possible contribution to corrosion made by oral bacteria, which are known to form a biofilm on intra-oral appliances . We have exposed demagnetized neodymium/iron/boron (Nd2Fe14B) magnets to an artificial saliva in the presence and absence of a biofilm of Streptococcus sanguils, one of the predominant organisms in the oral cavity . Over a 21-d period, a 3.2% decrease in the mass of the magnets was observed when S . sanguis was present, while in the absence of the organism the decrease in mass was 1.4% . We also examined the ability of poly(para-xylylene), a commonly-used coating, to protect the magnets against corrosion . No decrease in the mass of magnets coated with poly(para-xylylene) was detected over a 21-d period in the presence of S . sanguis . This study has shown that biofilms of S . sanguis cause appreciable corrosion of Nd2Fe14B magnets which is greater than that occurring in the absence of the organism, and that a coating of poly(para-xylylene) provides protection against such corrosion.

Antimicrob Agents Chemother, 1995 Jun, 39(6), 1281 - 6
Effects of ciprofloxacin and protamine sulfate combinations against catheter-associated Pseudomonas aeruginosa biofilms; Soboh F et al.; Infection is a common complication associated with the use of transcutaneous and implanted medical devices . These infections are generally difficult to treat and frequently require removal of the biomaterial before the infection can be completely eradicated . The presence of a bacterial biofilm recalcitrant to treatment often mediates these infections . We studied the influence of a polycationic protein, protamine sulfate, on the efficacy of the fluoroquinolone ciprofloxacin against a clinical isolate of Pseudomonas aeruginosa . A P . aeruginosa biofilm was developed on 1-cm sections of red rubber catheter material and then treated with various combinations of protamine sulfate and ciprofloxacin . The present work demonstrated that ciprofloxacin in combination with protamine was more effective against biofilms than was ciprofloxacin alone . Protamine sulfate at 50 micrograms/ml combined with antibiotic at 0.5 microgram/ml reduced the number of viable cells by an average of 98.97%, while protamine sulfate at 50 micrograms/ml alone resulted in an average 107.8% increase and antibiotic alone resulted in an average 58.6% reduction after 24 h . Furthermore, protamine sulfate, in combination with ciprofloxacin, inhibited P . aeruginosa in a dose-dependent fashion . It was further observed that treatment with the combination of protamine sulfate and ciprofloxacin had a more drastic effect on planktonic organisms as compared with the P . aeruginosa biofilms; the MBC was reduced to < 0.05 microgram/ml in the presence of 25 micrograms of protamine sulfate per ml . These findings were substantiated by ultrastructure studies of treated cells using scanning and transmission electron microscopy . The synergism between ciprofloxacin and protamine sulfate significantly enhanced the efficacy of ciprofloxacin against planktonic and biofilm P . aeruginosa.

Oral Microbiol Immunol, 1995 Jun, 10(3), 160 - 7
Patterns and rates of growth of microcosm dental plaque biofilms; Sissons CH et al.; Rates of growth in wet weight and changes in them over time were established for microcosm dental plaques cultured from the mixed salivary bacteria in an artificial mouth . Standardized conditions included a continuous supply of medium containing 0.25% mucin and 1.5 ml of 5% w/v sucrose in 6 min every 8 h . Plaques were weighed daily . Plaque wet weight and total protein were highly correlated . Plaque doubling times were 3-7 h over day 1 and 9-21 h over day 2, which is similar to in vivo plaques . Subsequently, growth curves were either linear or between a linear and exponential increase . Evidence was obtained for plaque blooms . Methyl paraben (0.2%) applied for 15 min (3.75 ml) 6 times daily inhibited growth but only for 3 days, after which the rate was similar to control plaques, indicating that selection for resistance had occurred . It was concluded that the regulation of plaque growth rates is complex and does not conform to simple growth pattern models . Detailed studies of plaque growth and the effects of antiplaque agents can be carried out using this experimental system.

J Hosp Infect, 1995 Jun, 30 Suppl, 54 - 63
The prevention of orthopaedic implant and vascular graft infections; Strachan CJ; The infection rate for any surgical prosthesis insertion should be less than 1% in the first postoperative year . If infection occurs the patients will lose their new found mobility, lose their independence, be hospitalized with sepsis, both local and systemic, and perhaps die . Preoperative and intraoperative measures to prevent infection are well established in orthopaedic surgery but less scientifically applied in peripheral vascular surgery . In both specialties the problem of late infection has promoted research on the protection of the peri-prosthetic environment against both bacteria and biofilm . In orthopaedics, the incorporation of various antibiotics into bone cement is well accepted in revision surgery, but still debated for the primary operation . On-going research on bioresorbable ceramics and the incorporation of antibiotics more effective against coagulase-negative staphylococci should eventually counter late infections . As HIV-positive patients increasingly present with sepsis around implanted prostheses this need will increase . In vascular surgery as the risk factors for biomaterial infection are better understood, new generations of protein-sealed grafts are permitting ionically compatible antibiotic coatings . Large well-designed clinical trials have begun and are needed to confirm the forecast of improved long-term clinical outcomes.

J Hosp Infect, 1995 Jun, 30 Suppl, 409 - 13
New insights into the pathogenesis of ventilator-associated pneumonia; Inglis TJ; Colonization of the lower respiratory tract of critically ill patients with Gram-negative bacilli is an early stage in the pathogenesis of ventilator-associated pneumonia . Recent studies have emphasized the endogenous source of these bacteria and have pointed to the upper gastrointestinal tract as a major source . Gastric bacterial overgrowth is known to be promoted by raised intragastric pH . Further evidence suggests that duodenal reflux is just as important in gastric bacterial overgrowth and subsequent colonization of the mechanically ventilated lung . Work on the biomaterials lining the inside of the tracheal tube in critically ill patients has shown that this biofilm can be scattered many centimetres as a result of ventilator gasflow . In vitro and in vivo evidence has been found to support a fluid dynamic process that results in dissemination of tracheal tube biofilm, which also contains neutrophils and their contents . The dissemination of combined bacteria, effete neutrophils and their breakdown products into already compromised lungs may explain the complexity of the clinical condition known as ventilator-associated pneumonia . It may thus be more correct to refer to the condition as 'ventilator-induced pneumonitis'.

Dig Dis Sci, 1995 Jun, 40(6), 1167 - 73
Endoscopic stenting for palliation of malignant biliary obstruction . A review of progress in the last 15 years; Sung JJ et al.; Since the late 1970s, endoscopic biliary stenting has become a standard palliative treatment for obstructive jaundice due to malignancies of the pancreas and the hepatobiliary system . Despite the high initial success rate in achieving biliary drainage, endoscopic stenting therapy has been limited by the clogging of biliary stents, usually after four to five months, due to formation of adherent bacterial biofilm and accumulation of biliary sludge . Various methods for the prevention of bacterial adhesion and prolongation of stent patency have been investigated, including prophylactic antimicrobial agents and bile salts, new stent materials, and new stent designs . Recently, the introduction of self-expandable metal stents has significantly improved the duration of stent patency but the cost is considerably higher . Each method has its own merits as well as specific problems . This article reviews the pathogenesis of biofilm formation on the biliary stents and the latest status of research in avoiding stent occlusion.

FEMS Microbiol Lett, 1995 May 15, 128(3), 229 - 34
Initial microbial adhesion is a determinant for the strength of biofilm adhesion; Busscher HJ et al.; This paper presents a hypothesis on the importance of initial microbial adhesion in the overall process of biofilm formation . The hypothesis is based on the realization that dynamic shear conditions exist in many environments, such as in the oral cavity, or on rocks and ship hulls . Recognizing that an entire biofilm is detached during high shear once the bond between the initially adhering organisms and a surface (often constituted through a so-called 'conditioning film') is broken, it becomes clear that research should focus on detachment rather than adhesion . Experiments were done in a parallel plate flow chamber in which attempts were made to detach adhering oral streptococci from glass by applying a high shear caused by the passage of a bubble, giving an air-liquid interface . Detachment of streptococci from bare glass and from an initially adhering actinomycete strain appeared not to occur . However, substantial detachment of adhering streptococci occurred when adhesion was mediated through a salivary conditioning film, presumably because of cohesive failure in the conditioning film.

Ophthalmology, 1995 May, 102(5), 831 - 6
A novel antibiofilm technology for contact lens solutions; Farber BF et al.; PURPOSE: Nonsteroidal anti-inflammatory drugs, including sodium salicylate, inhibit extracellular bacterial biofilm production . The authors studied the effect of the addition of sodium salicylate on bacterial adherence and biofilm formation on contact lenses and cases and commonly used medical polymers . METHODS: The study was done in vitro with bacterial adherence and biofilm measured on lenses and cases that were exposed to saline contaminated with Staphylococcus epidermidis and Pseudomonas aeruginosa with and without 1 and 3 mm sodium salicylate . Bacterial adherence to contact lenses was quantitated by a vortex assay and by scanning electron microscopy . Biofilm formation on contact lens cases and other polymers was measured by an optical density assay and a radiolabeling assay . RESULTS: Inhibition of biofilm formation was demonstrated on plastic contact lens cases in a dose-related manner with 1 and 3 mm sodium salicylate . A dose-related decrease in bacterial adherence also was noted . Assays with contact lenses also demonstrated less adherence in the presence of sodium salicylate . Electron micrographs of the contact lens showed less biofilm, most noticeable with 3 mm salicylate . Other studies demonstrated decreased adherence of S . epidermidis to polyethylene and polystyrene . Sodium salicylate also decreased biofilm on plastic tissue culture wells, but sorbic acid paradoxically increased deposition . CONCLUSION: The authors found that the addition of low-dose sodium salicylate to saline decreased the adherence of S . epidermidis and P . aeruginosa to contact lenses and lens cases . Biofilm production also was decreased on the lens cases and on medical polymers used to make plastic cases . These studies suggest that sodium salicylate deserves additional study to determine its use in contact lens solutions.

Crit Care Med, 1995 May, 23(5), 894 - 900
Decreased bacterial adherence and biofilm formation on chlorhexidine and silver sulfadiazine-impregnated central venous catheters implanted in swine; Greenfeld JI et al.; OBJECTIVE: To determine if antiseptic central venous catheters impregnated with silver sulfadiazine and chlorhexidine (antiseptic) reduce bacterial adherence and biofilm formation without producing local or systemic toxicity . DESIGN: Prospective, randomized, controlled trial . SETTING: Experimental laboratory in a university teaching hospital . SUBJECTS: Ten outbred New Hampshire pigs . INTERVENTIONS: Nonimpregnated (control) and antiseptic-impregnated catheters were inserted intravascularly into swine for 7 days . After explantation, the catheters were assessed for bacterial adherence and biofilm formation, and the surrounding tissue was assessed for signs of toxicity . Before retrieval, systemic concentrations of antimicrobials were determined . MEASUREMENTS AND MAIN RESULTS: Sequential roll plate and centrifuging were used to detect moderately and tightly adherent bacteria on the outer and luminal surfaces of the catheter . The presence of biofilm was detected by scanning electron microscopy . Tissues surrounding the catheters were examined histopathologically; systemic concentrations of chlorhexidine, sulfadiazine, and silver were determined by atomic absorption and high-performance liquid chromatography . As compared with the controls, antiseptic catheters had significantly (p < .01) fewer moderately and tightly adherent bacteria on outer and luminal surfaces, and fewer adherent bacteria when outer surfaces alone were examined (p < .01) . Scanning electron microscopy showed bacterial biofilm and adherence on the control catheters but not on the antiseptic catheters . There were no abnormal histopathologic changes associated with the test catheter, and serum concentrations of the antibacterial agents were shown to be within nontoxic ranges . CONCLUSION: The antiseptic-impregnated catheters prevented bacterial adherence and biofilm formation and produced no local or systemic toxicity.

J Prosthet Dent, 1995 May, 73(5), 492 - 4
Cross contamination control and the dental handpiece; Clappison RA; The issue of heat sterilization of handpieces for every patient every time evokes visions of higher cost, damage to handpiece sheaths and turbines, and increased staff involvement . The introduction of fast turn-around steam autoclaves reduces the number of handpieces required and helps lower costs . The damage can be contained by using high-quality handpieces and by maintaining them properly . Staff involvement time can be reduced through the use of handpiece scrubbers, handpiece flush systems, and simple, adequate maintenance standards . Because of the discovery that biofilms contribute to the microbial contamination of dental unit water lines, the need for a clean water system is apparent because it will further reduce the potential danger of cross contamination.

APMIS, 1995 May, 103(5), 339 - 44
Development of a flow method for susceptibility testing of oral biofilms in vitro; Larsen T et al.; Bacteria in biofilms are known to be more resistant than bacteria in batch cultures to antimicrobial agents . The purpose of the present study was to develop a flow method for formation of oral biofilms permitting susceptibility testing of plaque bacteria . A brain heart infusion (BHI) Streptococcus sanguis 804 culture was pumped through a modified Robbins Device (MRD) with 25 exchangeable silicone disks at 40 ml/h . After 24-48 h disks were removed and biofilm cells dispersed by vortex mixing and low-output ultrasonication . Colony forming units (cfu)/cm2 were determined after aerobic incubation on blood agar plates . Optimal biofilm formation was found after growth for 48 h at 37 degrees C in BHI + 1% sucrose, using saliva-coated silicone disks in inverted MRDs, yielding on average 4.4 x 10(5) cfu/cm2 . Similar results were obtained for S . sanguis ATCC 10556 and five clinical isolates . Testing the susceptibility of S . sanguis to chlorhexidine gluconate showed increased resistance of biofilms compared to batch culture . Thus an appropriate biofilm model for susceptibility testing of oral microorganisms has been established.

Appl Environ Microbiol, 1995 May, 61(5), 1946 - 52
Construction of a rhizosphere pseudomonad with potential to degrade polychlorinated biphenyls and detection of bph gene expression in the rhizosphere; Brazil GM et al.; The genetically engineered transposon TnPCB, contains genes (bph) encoding the biphenyl degradative pathway . TnPCB was stably inserted into the chromosome of two different rhizosphere pseudomonads . One genetically modified strain, Pseudomonas fluorescens F113pcb, was characterized in detail and found to be unaltered in important parameters such as growth rate and production of secondary metabolites . The expression of the heterologous bph genes in F113pcb was confirmed by the ability of the genetically modified microorganism to utilize biphenyl as a sole carbon source . The introduced trait remained stable in laboratory experiments, and no bph-negative isolates were found after extensive subculture in nonselective media . The bph trait was also stable in nonselective rhizosphere microcosms . Rhizosphere competence of the modified F113pcb was assessed in colonization experiments in nonsterile soil microcosms on sugar beet seedling roots . F113pcb was able to colonize as efficiently as a marked wild-type strain, and no decrease in competitiveness was observed . In situ expression of the bph genes in F113pcb was found when F113pcb bearing a bph'lacZ reporter fusion was inoculated onto sugar beet seeds . This indicates that the bph gene products may also be present under in situ conditions . These experiments demonstrated that rhizosphere-adapted microbes can be genetically manipulated to metabolize novel compounds without affecting their ecological competence . Expression of the introduced genes can be detected in the rhizosphere, indicating considerable potential for the manipulation of the rhizosphere as a self-sustaining biofilm for the bioremediation of pollutants in soil . Rhizosphere bacteria such as fluorescent Pseudomonas spp . are ecologically adapted to colonize and compete in the rhizosphere environment . Expanding the metabolic functions of such pseudomonads to degrade pollutants may prove to be a useful strategy for bioremediation.

Jpn J Antibiot, 1995 May, 48(5), 595 - 601
{Synergism of ciprofloxacin and roxithromycin to Pseudomonas aeruginosa biofilm}; Gotoh N et al.; Synergism of ciprofloxacin (CPFX) and roxithromycin (RXM) to bacterial biofilms formed by Pseudomonas aeruginosa was investigated . Measurement of antibacterial activities and morphological observation with a scanning electron-microscope suggested that RXM eradicated the biofilms by unknown mechanism and thereby enhanced the bactericidal activity of CPFX to P . aeruginosa in biofilms.

Antimicrob Agents Chemother, 1995 May, 39(5), 1054 - 8
Mathematical corrections for bacterial loss in pharmacodynamic in vitro dilution models; Keil S et al.; In vitro dilution models are used to simulate in vivo drug concentration-time profiles and thus to study the effects of various antibiotic concentrations on the bacteria investigated . The major disadvantage of these models is permanent dilution of the bacterial culture, which falsifies the resulting kill curves . Known equations, which usually correct bacterial loss by simple first-order kinetics, do not take into account special test conditions, such as variable elimination rate constants, exceptionally long periods of investigation, or formation of biofilms . In the present investigation, we examined the validity of these equations with regard to the test conditions mentioned . We simulated the concentration-time curves resulting from continuous infusion of 1,000 mg of meropenem with steady-state levels of 2.5, 5.0, and 7.5 micrograms/ml in an in vitro dilution model . The resulting kill curves were compared with the kill curves obtained from incubation of bacteria in an undiluted system with meropenem at constant concentrations corresponding to the above-mentioned steady-state levels . Comparison of the matching kill curves showed that the common corrections, which do not consider the formation of biofilms in the compartments, partly overestimated the effect of bacterial dilution . We defined a factor, f, as an extension to the known equations which compensates for the effect of biofilms . Another extension was developed to allow the investigation of variable elimination rate constants . With the help of these extended mathematical corrections, we were able to fit the kill curves resulting from the in vitro dilution model exactly to the kill curves given by an undiluted system.

Antimicrob Agents Chemother, 1995 May, 39(5), 1038 - 44
Combination effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa growing in a biofilm; Kumon H et al.; We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa in biofilms by using an in vitro experimental system with a modified Robbins device . Sessile cells in a mature or immature biofilm, developed on a silicon disk, were used, and an ATP bioluminescence assay was employed to assess antibacterial effects . A synergistic effect of fosfomycin and ofloxacin was clearly detected when concentrations at which each drug independently produced no detectable decrease in the bioactivity of sessile cells were used . Exposure of the cells in a mature biofilm to fosfomycin at concentrations of one-eighth of the MIC to 10 times the MIC (6.25 to 500 micrograms/ml) and ofloxacin at three or 10 times the MIC (18.75 or 62.5 micrograms/ml) resulted in reduction of the bioactivity to 1.5 to 4.5% after 72 h . Young sessile cells in an immature biofilm were more susceptible to this combination therapy . With a combination of fosfomycin at three times the MIC and ofloxacin at three times the MIC, complete eradication was confirmed by both ATP assay and scanning electron microscopy.

Kansenshogaku Zasshi, 1995 May, 69(5), 572 - 81
{Experimental pneumonia with Pseudomonas aeruginosa in immunosuppressed guinea pigs as a model for biofilm-associated infection}; Ishida Y; To establish a model where the role of bacterial biofilms in chronic pneumonia with Pseudomonas aeruginosa could be investigated, hydrocortisone-treated guinea pigs were given P . aeruginosa, strain 2126 by inhalation which were used throughout this study, in planktonic form . In these animals, the bacteria were recovered only from the lungs more than 4 weeks after infection . The persistence in bacterial colonization in the lungs coincided with the formation of glanulomatous lesions that surrounded spherical grains consisting of outer shell and inner bacterial colonies . The outer shell of grain was stained with ruthenium red and was presumed to be polyanionic and therefore to be a biofilm-like material . In normal animals without hydrocortisone-treatment, the number of neutrophils recovered from bronchoalveolar lavage fluid increased significantly from 3 hours after infection and subsequently the inhaled bacteria were eliminated from the lungs by day 3 of infection . This early influx of neutrophils into the lungs tended to be suppressed by treatment with hydrocortisone . The formation of grains did not take place in the lungs of normal animals, indicating the significant role of grain-formation in the initiation and the prolongation of bacterial colonization in the lungs . P . aeruginosa, strain 2126, incubated in saline formed thick biofilms on the surface of teflon piece . Levofloxacin (LVFX), a quinolone antibacterial, exhibited killing activity against the bacteria in in vitro-forming biofilms at MIC . In contrast, gentamicin (GM), an aminoglycosid antibiotic, and ceftazidime (CAZ), a beta-lactams antibiotic, showed no such killing activity at MIC . Treatment of this model with oral LVFX achieved complete eradication of the bacteria, whereas subcutaneous injection of GM or CAZ was hardly effective . The pharmacokinetic study on these antibacterials revealed that the doses used in this study were sufficient to obtain the pulmonary levels of these drugs far above MIC even in GM and CAZ . These data indicate that the outer shell of grains, a characteristic finding in the pulmonary lesions of this model, may be one of the forms of pseudomonal biofilms and that this model represents the significant role of biofilm mode of growth of P . aeruginosa in persistence in pulmonary colonization.

Quintessence Int, 1995 May, 26(5), 331 - 7
Bacterial contamination of the water supply in newly installed dental units; Williams HN et al.; Bacterial contamination of the water supply of newly installed dental units was investigated . Water samples were collected from water supply lines to the dental operatories prior to connection of the dental units . Within hours following connection, and continuing for up to the 6 months of the study, water samples were obtained from the air-water syringe of the units . The samples were serially diluted 10-fold and plated on culture media for quantitative analysis . The formation of bacterial biofilm in the dental water supply tubing was monitored by scanning electron microscopy . The results of these studies revealed that the building's water supply to the dental units was contaminated prior to connection to the units . The water supply from the air-water syringe was therefore contaminated as well . The number of contaminant bacteria in the dental unit water supply increased for several weeks and then stabilized . The lumen of the dental tubing became progressively contaminated with bacterial biofilm, which subsequently became the primary reservoir for maintenance of the contamination of the dental unit water supply.

J Dent, 1995 Apr, 23(2), 113 - 5
Effectiveness of ethylene oxide for sterilization of dental handpieces; Parker HH 4th et al.; Ethylene oxide gas has been utilized as an alternative method for sterilization of dental handpieces, as it is less corrosive than steam . However, its effectiveness for sterilization of the internal components of dental handpieces has not been established . The objective of this study was to compare the effectiveness of ethylene oxide and steam for sterilization of dental handpieces . Unused handpieces and handpieces which had been exposed to clinical dental procedures ('clinical') were contaminated with Streptococcus mutans, exposed to steam or ethylene oxide, and flushed with sterile saline . Washings were plated on mitis-salivarius agar, and colonies identified and counted . No viable colonies could be established from washings from 'clinical' or 'unused' handpieces exposed to steam . However, viable colonies could be established from 'clinical' handpieces exposed to ethylene oxide . This data suggests that a substance entrapped within 'clinical' handpieces (possibly the biofilm) may protect bacteria from ethylene oxide gas, preventing adequate sterilization.

Urology, 1995 Apr, 45(4), 720 - 4
Synergy between protamine and vancomycin in the treatment of Staphylococcus epidermidis biofilms; Lee CK et al.; OBJECTIVES . To test for synergy between protamine and vancomycin by analyzing their bactericidal activities against slime-producing Staphylococcus epidermidis ATCC 35983 under planktonic and biofilm conditions . METHODS . We evaluated the activity of vancomycin and protamine separately against planktonic S epidermidis in broth by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each agent according to the standard macrobroth dilution method . To assess the possibility of synergy between these two agents, planktonic S epidermidis was exposed to vancomycin and protamine together in varying concentrations . Biofilms containing S epidermidis were then prepared and subjected to incubation with vancomycin and protamine separately as well as combined in varying concentrations . The bacterial viability of S epidermidis in the planktonic and biofilm phases after exposure to these two agents was assessed by qualitative culture and determination of viable colony blots . RESULTS . Standard antibacterial susceptibility tests revealed that the MICs of protamine and vancomycin were 1 and 2 micrograms/mL, respectively, and their MBCs were 4 micrograms/mL . The MICs were unchanged when protamine and vancomycin were combined in varying concentrations . Neither agent exhibited significant bactericidal activity against S epidermidis in the biofilm phase at concentrations < or = 32 micrograms/mL . However, a combination of both agents, each at 32 micrograms/mL, resulted in a 7-log decrease in viable bacterial counts . CONCLUSIONS . Protamine alone exhibited significant antibacterial activity against planktonic S epidermidis . No synergy was noted between protamine and vancomycin against S epidermidis in the planktonic phase . However, synergy was demonstrated when a combination of protamine and vancomycin was used on S epidermidis in the biofilm phase . Thus, protamine shows promise as an adjunctive agent to vancomycin in the treatment of S epidermidis in biofilms.

Pathol Biol (Paris), 1995 Apr, 43(4), 371 - 9
{Prevention of cariogenic dental plaque . Study of the structures implicated in the adhesion and coaggregation in Streptococcus mutans and Streptococcus sobrinus}; Jacquelin LF et al.; Cariogenic dental plaque may be assimilated to a biofilm resulting from the adhesion of S . mutans, then from the coaggregation of other streptococci, or other genus . We used a static monospecific biofilm model . Supports or bacteria were treated with inhibitors before adhesion in order to clarify the nature of adhesins responsible for the primary adhesion of S . mutans and S . sobrinus on Tygon . To determine the bindings of coaggregation, inhibitors were applied on one-day-old biofilms . Analysis of effects were performed by automatic inoculator Spiral (Interscience) for microbiological methods, and by SEM JEOL 5400 LV for microscopic methods . In the aim of preventing adhesion and coaggregation, different traps were assayed:sugars, chemical inhibitors such as F- and EDTA salts . Of these, only the latter showed efficiency . This confirmed the role of bivalent mineral ions and electrostatic attraction forces in the adhesion and coaggregation of streptococci.

Appl Environ Microbiol, 1995 Mar, 61(3), 920 - 5
Activity of synchronized cells of a steady-state biofilm recirculated reactor during xenobiotic biodegradation; Ascon-Cabrera MA et al.; The maintenance of a steady-state biofilm in a continuous-flow fixed-bed reactor, as a consequence of the reproduction-detachment of cells (an interfacial cell physiology phenomenon of steady-state biofilm) during the biodegradation of 2,4,6-trichlorophenol by Pseudomonas cells, was determined . After cell adhesion on an open-pore glass support, the biofilm was formed in a packed-bed recirculated reactor . After the steady-state biofilm was reached, the mechanisms of the interfacial cell detachment (at the biofilm-liquid interface) were determined . It was established that (i) the hydrophobicity of immobilized sessile cells (parent cells) increased (from 50 to 80%) as the dilution rate increased, while the hydrophobicity of detached suspended cells (daughter cells) remained constant (about 45%); and (ii) the immediately detached suspended cells showed a synchronized growth in about three generations . These results indicate that (i) the immobilized sessile and suspended detached cells grew synchronically at the end and at the beginning of the cell cycle, respectively; and (ii) the hydrophobicity difference of immobilized sessile and suspended detached cells permitted the cells detachment . Therefore, it is probable that independent of shear stress (due to recirculated flow), the synchronized growth and hydrophobicity of cells (which vary during the cell cycle) are the main factors permitting the maintenance of a steady-state xenobiotic-degrading biofilm reactor (in which the overall accumulation of biofilm is determined by the average growth rate of the biofilm cells minus the rate of detachment of cells from the biofilm).

Appl Environ Microbiol, 1995 Mar, 61(3), 860 - 7
Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture; Davies DG et al.; Reporter gene technology was used to observe the regulation of the alginate biosynthesis gene, algC in a mucoid strain of Pseudomonas aeruginosa in developing and mature biofilms in continuous culture on Teflon and glass substrata . The plasmid pNZ63, carrying an algC-lacZ transcriptional fusion, was shown to not be diluted in continuous culture over a period of 25 days in the absence of selection pressure . Biofilm cells under bulk phase steady-state conditions demonstrated fluctuations in algC expression over a 16-day period, but no trend of increased or decreased expression over the time interval was indicated . In vivo detection of algC up-expression in developing biofilms was performed with a fluorogenic substrate for the plasmid-borne lacZ gene product (beta-galactosidase) by using microscopy coupled with image analysis . By this technique, cells were tracked over time and analyzed for algC activity . During the initial stages of biofilm development, cells already attached to a glass surface for at least 15 min exhibited up-expression of algC, detectable as the development of whole-cell fluorescence . However, initial cell attachment to the substratum appeared to be independent of algC promoter activity . Furthermore, cells not exhibiting algC up-expression were shown to be less capable of remaining at a glass surface under flowing conditions than were cells in which algC up-expression was detected.

Eur J Clin Microbiol Infect Dis, 1995 Feb, 14(2), 134 - 7
Role of catheter colonization and infrequent hematogenous seeding in catheter-related infections; Anaissie E et al.; Adult cancer patients were prospectively studied to determine the relationship between ultrastructural and microbiologic catheter colonization and clinical catheter-related infections . Participants included 38 patients whose central venous catheters were removed because of suspected catheter infection (16 patients) or other noninfectious causes (22 controls) . The presence of clinical infection was determined . Catheters were examined by microbiologic methods (sonication and roll-plate culture) and by scanning and transmission electron microscopy . Ultrastructural microbial colonization and biofilm formation were universal and occurred as early as one day after catheter insertion . The extent of biofilm formation was unrelated to the clinical status of patient or the catheter microbiological findings . Secondary seeding of catheters was rarely seen . Catheter microbial biofilm formation occurs early, is universal and does not necessarily represent an infectious condition.

Appl Environ Microbiol, 1995 Feb, 61(2), 769 - 77
Channel structures in aerobic biofilms of fixed-film reactors treating contaminated groundwater; Massol-Deya AA et al.; Scanning electron microscopy, confocal scanning laser microscopy, and fatty acid methyl ester profiles were used to study the development, organization, and structure of aerobic multispecies biofilm communities in granular activated-carbon (GAC) fluidized-bed reactors treating petroleum-contaminated groundwaters . The sequential development of biofilm structure was studied in a laboratory reactor fed toluene-amended groundwater and colonized by the indigenous aquifer populations . During the early stages of colonization, microcolonies were observed primarily in crevices and other regions sheltered from hydraulic shear forces . Eventually, these microcolonies grew over the entire surface of the GAC . This growth led to the development of discrete discontinuous multilayer biofilm structures . Cell-free channel-like structures of variable sizes were observed to interconnect the surface film with the deep inner layers . These interconnections appeared to increase the biological surface area per unit volume ratio, which may facilitate transport of substrates into and waste products out of deep regions of the biofilm at rates greater than possible by diffusion alone . These architectural features were also observed in biofilms from four field-scale GAC reactors that were in commercial operation treating petroleum-contaminated groundwaters . These shared features suggest that formation of cell-free channel structures and their maintenance may be a general microbial strategy to deal with the problem of limiting diffusive transport in thick biofilms typical of fluidized-bed reactors.

Cytobios, 1995, 84(338-339), 141 - 6
Effect of iron limitation on the amount of slime produced by strains of Staphylococcus epidermidis; Elci S et al.; To investigate the effect of iron limitation on the amount of slime production by most strains of Staphylococcus epidermidis, ethylenediamine-di-o-hydroxyphenol acetic acid (EDDA), was added to the growth medium and twenty clinically relevant strains were assayed for slime production during growth in standard tryptic soy broth (TSB) and iron-limited TSB . Findings revealed that the density of the biofilm which formed on the base of microtitre plates was dependent on the degree of iron limitation, characteristics of the test strains and the stage of the growth cycle.

Ophthalmologica, 1995, 209(6), 315 - 8
Ocular infections: antibiotics and bacterial adhesion on biomaterials used in ocular surgery; Marone P et al.; The in vitro antibacterial activity of ofloxacin, sagamycin and other antibiotics was evaluated against 85 bacterial isolates {coagulase-negative staphylococci (CNS), n = 37, Staphylococcus aureus, n = 28, and Pseudomonas aeruginosa, n = 20} obtained from patients with ocular infections . The antistaphylococcal activity of ofloxacin was quite elevated with a 90% minimal inhibitory concentration (MIC90) of 1.56 mg/l against CNS and S . aureus . Rokitamycin and erythromycin showed a good activity against methicillin-sensitive staphylococci, but were less active than ofloxacin and sagamycin against methicillin-resistant strains (MIC90 > 100 mg/l) . Sagamycin was highly effective against staphylococci (MIC90 0.78 mg/l) and appeared to be the most active compound against P . aeruginosa (MIC90 6.25 mg/l), followed by ofloxacin, tobramycin and gentamicin . In a successive part of the study, the adhesive properties of slime-producing staphylococci were tested on biomaterials used in ocular surgery . Intraocular lenses, Silastic sheetings, circling bands and grooved strips showed a high affinity for slime-producing strains, while round silicone sponges were not covered by bacterial biofilm . In the last part of our study, we demonstrated how subMIC levels of ofloxacin increased the adhesion of slime-producing staphylococci . Our data confirmed the excellent activity of ofloxacin and sagamycin against ocular pathogens and the key role of adhesion in promoting colonization and infections of biomaterials.

Annu Rev Microbiol, 1995, 49, 711 - 45
Microbial biofilms; Costerton JW et al.; Direct observations have clearly shown that biofilm bacteria predominate, numerically and metabolically, in virtually all nutrient-sufficient ecosystems . Therefore, these sessile organisms predominate in most of the environmental, industrial, and medical problems and processes of interest to microbiologists . If biofilm bacteria were simply planktonic cells that had adhered to a surface, this revelation would be unimportant, but they are demonstrably and profoundly different . We first noted that biofilm cells are at least 500 times more resistant to antibacterial agents . Now we have discovered that adhesion triggers the expression of a sigma factor that derepresses a large number of genes so that biofilm cells are clearly phenotypically distinct from their planktonic counterparts . Each biofilm bacterium lives in a customized microniche in a complex microbial community that has primitive homeostasis, a primitive circulatory system, and metabolic cooperativity, and each of these sessile cells reacts to its special environment so that it differs fundamentally from a planktonic cell of the same species.

Appl Environ Microbiol, 1995 Jan, 61(1), 187 - 93
Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms; Brown ML et al.; Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria . Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent . This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853) . Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates . The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples . Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples . Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction . However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples . Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment . The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

J Surg Res, 1995 Jan, 58(1), 105 - 10
Antistaphylococcal activity of rifampin-bonded gelatin-impregnated Dacron grafts; Gahtan V et al.; The affinity of rifampin to bond to Dacron permits implantation of a vascular prosthesis with anti-staphylococcal bioactivity . The performance of rifampin-bonded Dacron grafts was evaluated in vitro and in vivo to develop a bonding technique with optimal graft bioactivity which was then used for in situ reconstruction of a biofilm graft infection . In vitro bioactivity was measured at 24-hr intervals for three types of Dacron prostheses (plain, gelatin, and collagen-impregnated) exposed to rifampin at varied concentrations and immersion times . Gelatin-impregnated grafts demonstrated superior bioactivity (P < 0.05) . Rifampin concentration and graft type had a greater effect on bioactivity than immersion time (P < 0.01) . Gelatin-impregnated grafts immersed in a 60 mg/ml rifampin solution for 15 min produced optimum bioactivity . Six grafts prepared in this fashion were used to replace the canine aorta . The level and duration of in vivo antistaphylococcal activity to Staphylococcus aureus and Staphylococcus epidermidis were less (P < 0.05) than those measured in vitro, but rifampin levels exceeded the study strain maximum inhibitory concentration for up to 48 hr . In a canine model, the rifampin-bonded gelatin-impregnated (N = 14) or nonbonded control (N = 10) grafts were used as in situ replacement for an established aortic graft infection caused by S . epidermidis . Replacement with a rifampin-bonded graft resulted in successful anatomic healing of perigraft and anastomotic tissue . Persistent biofilm colonization was confirmed in 8 of 10 controls versus 4 of 14 rifampin-bonded grafts (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Microbios, 1995, 82(330), 49 - 67
Effects of ciprofloxacin and vancomycin on physicochemical surface properties of Staphylococcus epidermidis, Escherichia coli, Lactobacillus casei and Lactobacillus acidophilus; Cuperus PL et al.; Under clinical conditions, during antibiotic treatment, micro-organisms often grow at sub-inhibitory concentrations . This may lead to altered adhesive cell surface properties and to a disruption of the indigenous microflora, in addition to the creation of a more pathogenic biofilm . The effects of growing Staphylococcus epidermidis, Escherichia coli and lactobacilli in the presence of sub-inhibitory concentrations of ciprofloxacin and vancomycin were determined . Growing the cells under antibiotic burden sometimes led to altered cell surface hydrophobicity (by adhesion to hexadecane), changes in the pH-dependence of zeta potentials, and elemental surface compositions or in different SDS-PAGE protein profiles . For several isolates only one of the surface properties was altered by the presence of an antibiotic in the growth medium and no systematic effects were observed for all isolates representing a certain species or even strain . The important conclusion to be drawn from the results of this study is that the effects of growing cells under antibiotic burden on their adhesive cell surface properties can only be established when using a variety of techniques.

Diagn Microbiol Infect Dis, 1995 Jan, 21(1), 9 - 14
Variations in DNA subtype, antifungal susceptibility, and slime production among clinical isolates of Candida parapsilosis; Pfaller MA et al.; Candida parapsilosis is an important nosocomial pathogen that can proliferate in high concentrations of glucose and form biofilms on prosthetic materials . We investigated the genotypic diversity, slime production, and antifungal susceptibility among 60 isolates of C . parapsilosis from 44 patients and 10 patient care providers from five different medical centers . Molecular typing was performed using macrorestriction digest profiles with BssHII followed by pulsed-field gel electrophoresis (REAG) and by electrophoretic karyotyping (EK) . Slime production was evaluated by growing the organisms in Sabouraud broth with 8% glucose and examining the walls of the tubes for the presence of an adherent slime layer . Antifungal susceptibility to amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole was determined using National Committee for Clinical Laboratory Standards proposed standard methods . Overall 28 different DNA types were identified by REAG and EK methods . MIC90 values ranged from 0.12 microgram/ml for itraconazole to 1.0 microgram/ml for fluconazole and amphotericin B . Sixty-five percent of the isolates produced slime: 37% were moderately to strongly positive, 28% were weakly positive, and 35% were negative . Overall, 83% of blood and catheter isolates were slime positive versus 53% of isolates from all other sites (P < 0.05) . These data underscore the genetic diversity and susceptibility of C . parapsilosis to antifungal agents . Slime production may be important in enabling C . parapsilosis to cause catheter-related bloodstream infections.

Bioprocess Technol, 1995, 20, 187 - 231
Microbial biofilms and biofilm reactors; Peyton BM et al.; Scientists and engineers have realized the industrial and environmental significance of biofilm accumulation and activity . The ability to predict and control biofilm formation has led to less fouling and corrosion in industrial systems and a better understanding of biofilm importance in natural aquatic systems . Understanding the fundamental processes contributing to biofilm formation is beneficial to anyone involved with natural or industrial systems where biofilms may play a significant role in determining variables such as bulk water quality, toxic compound biodegradation, or product quality.

Antonie Van Leeuwenhoek, 1995, 67(1), 47 - 77
Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia; Field JA et al.; Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems . Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora . Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria . These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further . The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack . Sequencing anaerobic and and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants . The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g . biofilm, soil aggregate, etc.) . Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers . The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm . Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments . Consequently an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances . The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation . Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups . The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.

Urol Res, 1995, 22(6), 383 - 7
Effect of prednisolone on ascending renal infection due to biofilm disease and lower urinary tract obstruction in rats; Haraoka M et al.; A model of renal infection due to lower urinary tract obstruction and biofilm disease was constructed for the study of renal scarring by inserting glass beads coated with bacterial biofilm into the bladder of rats and then clamping the urethra . We previously reported the effect of antimicrobial therapy used in combination with the anti-inflammatory agent prednisolone to prevent renal scarring . In this study we investigated the effect of prednisolone on renal scar formation using our new model . Renal scarring could not be prevented in the group in which prednisolone was administered in the period during which the urethra was regularly being clamped . In contrast, scarring was prevented in the group that began to receive prednisolone after the period of clamping had ended . Therefore, in cases of lower urinary tract obstruction prednisolone should only be administered for the prevention of renal scarring after the obstruction has been resolved.

Can J Microbiol, 1995 Jan, 41(1), 12 - 8
Lytic infection of Escherichia coli biofilms by bacteriophage T4; Doolittle MM et al.; Escherichia coli 3000 XIII formed biofilms on the surface of polyvinylchloride coupons in a modified Robbins device . Bacteriophage T4D+ infected cells in the biofilm and replicated . It is commonly held that bacteriophage cannot infect surface-attached bacteria (biofilms) because such bacteria are protected by an exopolymeric matrix that binds macromolecules and prevents their diffusion into the biofilm . To our knowledge this is the first observation that a bacteriophage can infect and multiply within cells growing as a biofilm.

Eye, 1995, 9 ( Pt 1), 102 - 9
Biofilm-related infections in ophthalmology; Elder MJ et al.; A biofilm is a functional consortium of microorganisms organised within an extensive exopolymer matrix . Organisms within a biofilm are difficult to eradicate by conventional antimicrobial therapy and can cause indolent infections . This paper reviews the pathophysiology of biofilms and their application of ophthalmology . Under certain environmental conditions such as nutrient limitation, some bacteria may secrete and reside in an exopolysaccharide glycocalyx polymer . This confers relative protection from humoral and cellular immunity, antibiotics and surfactants . Biofilms occur in natural aquatic ecosystems, on ship hulls, in pipelines and on the surface of biomaterials . They cause clinical infections of prosthetic hip joints, heart valves and catheters . Biofilm formation may occur rapidly on contact lenses and their cases and hence contribute to the pathogenesis of keratitis . Formation of biofilms is also implicated in delayed post-operative endophthalmitis and crystalline keratopathy . Bacteria within biofilms are 20-1000 times less sensitive to antibiotic than free-living planktonic organisms . Existing experimental methods for modifying biofilm include the use of macrolide antibiotics that specifically impair biofilm production, and the use of enzymes to digest it . These may have clinical applications, as potential adjunctive therapies to antibiotic treatment, for these resistant infections . In conclusion, biofilm is an important cause of infections associated with biomaterials . Novel strategies are needed to deal with these.

J Clin Periodontol, 1995 Jan, 22(1), 1 - 14
The influence of surface roughness and surface-free energy on supra- and subgingival plaque formation in man . A review of the literature; Quirynen M et al.; In the oral cavity, an open growth system, bacterial adhesion to the non-shedding surfaces is for most bacteria the only way to survive . This adhesion occurs in 4 phases: the transport of the bacterium to the surface, the initial adhesion with a reversible and irreversible stage, the attachment by specific interactions, and finally the colonization in order to form a biofilm . Different hard surfaces are available in the oral cavity (teeth, filling materials, dental implants, or prostheses), all with different surface characteristics . In a healthy situation, a dynamic equilibrium exists on these surfaces between the forces of retention and those of removal . However, an increased bacterial accumulation often results in a shift toward disease . 2 mechanisms favour the retention of dental plaque: adhesion and stagnation . The aim of this review is to examine the influence of the surface roughness and the surface free energy in the adhesion process . Both in vitro and in vivo studies underline the importance of both variables in supragingival plaque formation . Rough surfaces will promote plaque formation and maturation, and high-energy surfaces are known to collect more plaque, to bind the plaque more strongly and to select specific bacteria . Although both variables interact with each other, the influence of surface roughness overrules that of the surface free energy . For the subgingival environment, with more facilities for microorganisms to survive, the importance of surface characteristics dramatically decreases . However, the influence of surface roughness and surface-free energy on supragingival plaque justifies the demand for smooth surfaces with a low surface-free energy in order to minimise plaque formation, thereby reducing the occurrence of caries and periodontitis.

Acta Microbiol Immunol Hung, 1995, 42(2), 221 - 7
Biofilms produced by Pseudomonas aeruginosa and by Staphylococcus aureus on model medical devices; Ketyi I; Polyethylene, teflon, tygon, polypropylene, silicon rubber, and rubber tubes or rings contaminated with Pseudomonas aeruginosa or Staphylococcus aureus were implanted subcutaneously into mice . After 5 days the colony forming units developing on, and attaching to them were determined . The highest numbers of bacteria were observed on rubber and silicon rubber, polyethylene was next in order, while significantly lower values were obtained on teflon and on tygon and polypropylene . Rubber devices were better colonized after heat than after UV sterilization . The number of bacteria rose further, if the already used rubber implant was resterilized, recontaminated and reimplanted . The model seems suitable to test the development of bacterial biofilms on different materials pretreated in different manners.

Acta Microbiol Immunol Hung, 1995, 42(2), 215 - 9
A model for testing drug susceptibility of Pseudomonas aeruginosa and Staphylococcus aureus grown in biofilms on medical devices; Ketyi I; UV-sterilized polyethylene rings infected with Pseudomonas aeruginosa or with Staphylococcus aureus were implanted into artificial wounds of mice . During 5 days incubation biofilm was formed on the plastic surfaces . To determine the Minimal Bactericidal Dose on sessile bacteria, rings with biofilm were removed and incubated in Luria broth containing serial dilutions of different antibiotics . Parallel, the sensitivities of planktonic phase organisms were also determined using cells grown in broth . The biofilm mode of growth strongly reduced the sensitivity of the strains against most of the antibiotics used, especially against polymyxin B . On the other hand, beta-lactam type antibiotics were equally effective against bacteria both in the sessile and planktonic phase of growth.

Kansenshogaku Zasshi, 1995 Jan, 69(1), 114 - 22
{Observation of Pseudomonas aeruginosa biofilm with confocal laser scanning microscope}; Watanabe T; Confocal laser scanning microscope (CLSM) is a new type of microscope, which permits non-invasive optical sectioning of biomaterials by reducing out-of-focus haze . A biofilm of Pseudomonas aeruginosa was developed on the silicon disks using a modified Robbins device and used to visualize hydrated living biofilm with CLSM . In order to examine optimum staining agents and conditions, acridine orange, fluorescein isothiocyanate (FITC), FITC-conjugated concanavalin A (FITC-ConA), Evans blue, safranine and rhodamine-conjugated concanavalin A were used in three different medium (pH 4.5, pH 7.5, pH 9.5) . Only extracellular matrix was stained as a net-like structure for FITC-ConA and only bacteria for acridine orange and safranine . Although the staining patterns with FITC and Evans blue were affected markedly by the medium pH, those with other staining agents were not affected significantly . It was considered that the staining characteristics specific for each agent and changes of staining pattern by pH were probably due to relative differences among matrix, bacteria and staining agents in their electrostatic charges . In addition to sagittal sectioning images of P . aeruginosa biofilm, clear doubles staining images, which differentiates bacteria from matrix in the identical material, could be obtained with the combination of safranine and FITC-ConA.

Microbiology, 1995 Jan, 141 ( Pt 1), 29 - 39
In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy; Manz W et al.; Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria . Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae . Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp . and 72 non-legionellae reference strains . Using optimized conditions both probes hybridized to all tested strains of L . pneumophila . Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively . With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule . In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates . L . pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u . ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization . Even after incubation for 1 year a proportion of the released cells was still detectable . In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms . A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L . pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis . Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

Appl Environ Microbiol, 1994 Dec, 60(12), 4339 - 44
Direct measurement of chlorine penetration into biofilms during disinfection; De Beer D et al.; Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation . The electrode had a tip diameter of ca . 10 microm and was sensitive to chlorine in the micromolar range . The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae . Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid . Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h . The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics . The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix . Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface . These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.

J Chemother, 1994 Dec, 6(6), 388 - 91
Susceptibility of agar-entrapped Escherichia coli cultures to cefotaxime as assessed by the potentiometric measurement of lipoic acid reduction; Tresse O et al.; We used an original potentiometric procedure to monitor the metabolic activity of biofilm-like structures and to assess their susceptibility to betalactam antibiotics . The reduction of exogeneous alpha-lipoic acid to dihydrolipoic acid by planktonic and agar-entrapped bacteria, metabolizing glucose, was followed by potential-time measurements using gold versus reference electrodes . Immobilized cultures displayed a lower susceptibility to cefotaxime than their free counterparts . The redox potential-time courses showed that the gel matrix was more rapidly oxygen depleted than the free-cell culture medium . The depletion of both glucose and dissolved O2 inside the gel induced a breakdown in the reducing activity of agar-entrapped organisms.

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