Infect Immun, 2001 Dec, 69(12), 7933 - 6 Identification of the sigB operon in Staphylococcus epidermidis: construction and characterization of a sigB deletion mutant; Kies S et al.; The role of the alternative sigma factor sigma(B) in Staphylococcus epidermidis was investigated by the construction, complementation, and characterization of a sigB deletion mutant . Electrophoretic analyses confirmed a profound influence of sigma(B) on the expression of exoproteins and cytoplasmic proteins . Detailed investigation revealed reduced lipase and enhanced protease activity in the sigma(B) mutant . Furthermore, no significant influence of sigma(B) on heterologous biofilm formation or on the activity of the global regulator agr was detected. Infect Immun, 2001 Dec, 69(12), 7851 - 7 Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation; Bateman BT et al.; An inducible promoter system provides a powerful tool for studying the genetic basis for virulence . A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible P(BAD) promoter, but each of these systems has limitations in its application to Staphylococcus aureus . In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S . aureus in vitro and inside epithelial cells as well as in an animal model of infection . Using the xyl/tetO promoter::gfp(uvr) fusion carried on a shuttle plasmid, we demonstrated that dose-dependent tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low . To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter . Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells . Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB. Infect Immun, 2001 Dec, 69(12), 7625 - 34 Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS; Fong KP et al.; The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors . Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi . Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V . harveyi luxS and secretes an AI-2-like signal . Cell-free conditioned medium from A . actinomycetemcomitans or from a recombinant Escherichia coli strain (E . coli AIS) expressing A . actinomycetemcomitans luxS induced luminescence in V . harveyi BB170 >200-fold over controls . AI-2 levels peaked in mid-exponential-phase cultures of A . actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures . Incubation of early-log-phase A . actinomycetemcomitans cells with conditioned medium from A . actinomycetemcomitans or from E . coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide . In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E . coli AIS(-), a recombinant strain in which luxS was insertionally inactivated . A . actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A . actinomycetemcomitans . Finally, A . actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P . gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism . Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A . actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm. Pesqui Odontol Bras, 2001 Jul-Sep, 15(3), 215 - 22 {Contribution to the study of dental caries in 0-30-month-old infants}; de Barros SG et al.; This study evaluated the oral health conditions of 340 children, aged 0-30 months (21.3 +/- 5.6)--54.4% of girls and 45.6% of boys--from 20 public day nurseries of Salvador (Brazil), as to the presence of incipient carious lesions . The exam was carried out by a single examiner, who utilized a mirror, a probe and a penlight . The teeth were wiped with gauzes in order to remove the dental plaque . A questionnaire was answered by the children's parents or caretakers in order to assess information regarding knowledge on caries, caries risk factors, socioeconomic status of the family and utilization of fluoride . Two hundred and twenty-nine answered questionnaires (67.35%) were obtained . The observed lesions were classified in five stages, according to their severity (C0-C4; active/inactive) . The data were analyzed using the Epi-info 6.02 . The prevalence of caries was 55.3% when all stages were registered: 25% for subjects aged 0-12 months, 51.18% for subjects aged 13-24 months and 71.03% for those aged 25-30 months (chi 2 = 25.31, p < 0.01) . When only active white spots were considered, 49.7% of the children were affected; 17.6% of the children presented with cavitated lesions . Among the affected children, 90.96% had lesions only on anterior teeth: 80% of the lesions were white spots and 20%, cavities . No significant difference was observed between genders . The increased amount of biofilm was positively associated with dental decay in toddlers (chi 2 = 67.61, p < 0.01), and the number of affected children increased when the sleep-time nursing habit was present (chi 2 = 0.24, p = 0.62) . The prevalence of lesions increased with age and with the number of erupted teeth (chi 2 = 25.31, p < 0.01; chi 2 = 122.95, p < 0.01) . Early oral health attention, diagnosis of incipient lesions, as well as educative and preventive programs to change oral hygiene and dietary habits are suggested. Mol Microbiol, 2001 Oct, 42(2), 415 - 26 Expression of the Pho regulon negatively regulates biofilm formation by Pseudomonas aureofaciens PA147-2; Monds RD et al.; We report the isolation of insertional mutations to the pstC and pstA genes of the phosphate-specific transport (pst) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147-2 . Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P . aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (P(i)) transport and repression of Pho regulon expression . Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular P(i) . The two-component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to P(i)-limiting environments . By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P . aureofaciens in P(i)-limiting conditions . This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low-P(i) environments, and that phoR mutations uncouple PA147-2 from such regulatory constraints . Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non-limiting P(i) environments . Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147-2 . During phenotypic analysis of pst mutants, we also uncovered novelties in relation to P(i) assimilation and Pho regulon control in P . aureofaciens. Zhonghua Yi Xue Za Zhi, 2001 Aug 10, 81(15), 934 - 6 {Effect of macrolides on biofilm of mucoid Pseudomonas aeruginosa}; Chai D et al.; OBJECTIVE: To observe the effect of macrolides with different structures on biofilm of mucoid Pseudomonas aeruginosa so as to provide a theoretical basis for treatment of bacterial biofilm diseases . METHODS: Modified plate culture method was used to establish in vitro bacterial biofilm model, which was identified by silver nitrate staining . After the biofilm was treated by gatifloxacin and/or macrolides with different structures and of different concentrations, the number of viable bacteria was measured by MTT method and bacterial adherence was measured by crystal violet staining . RESULTS: Clarithromycin and azithromycin of 1/4 and 1/16 MIC combined with gatifloxacin significantly inhibited the adherence of mucoid Pseudomonas aeruginosa (P < 0.05) and reduced the number of viable mucoid Pseudomonas aeruginosa compared with gatifloxacin alone (P < 0.05), while such effects could not be seen for rovamycin . CONCLUSION: Inhibiting the adherence of mucoid Pseudomonas aeruginosa may be one of the mechanisms by which macrolides such as clarithromycin and azithromycin enhance the antibacterial activity of gatifloxacin against mucoid Pseudomonas aeruginosa. Arch Microbiol, 2001 Nov, 176(5), 347 - 54 Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings; Schabereiter-Gurtner C et al.; A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany . The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses . The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium . The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter . Rubrobacter-related 16S rDNA sequences were the most abundant . In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified . No Rubrobacter-related bacteria were detected in non-rosy biofilms . The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings. Biomaterials, 2001 Dec, 22(24), 3235 - 47 Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology; Willcox MD et al.; Contact lens wear is a successful form of vision correction . However, adverse responses can occur during wear . Many of these adverse responses are produced as a consequence of bacterial colonization of the lens . The present study demonstrated that during asymptomatic contact lens wear lenses are colonized by low levels of bacteria with gram-positive bacteria, such as coagulase negative staphylococci, predominating . Gram-negative bacteria are frequently the causative agents of adverse responses during contact lens wear . Measuring the adhesion of different strains and/or species of bacteria to different contact lens materials demonstrated considerable differences . In particular . Pseudormonas aeruginosa strains Paerl and 6294 and Aeromonas hydrophilia strain Ahyd003 adhered in larger numbers to the highly oxygen permeable contact lenses Balafilcon A compared to hydrogel lenses manufactured from either Etafilcon A or HEMA . Furthermore, after Balafilcon A lenses had been worn for 6 h during the day bacteria were able to adhere in greater numbers to the worn lenses compared to the unworn lenses with increases in adhesion ranging from 243% to 1393% . However, wearing Etafilcon A lenses usually resulted in a decrease in adhesion (22-48%) . Bacteria were able to grow after adhesion to lenses soaked in artificial tear fluid and formed biofilms, visualized by scanning confocal microscopy . Chemostat grown bacterial cultures were utilized to enable control of bacterial growth conditions and bacteria were shown to adhere in the greatest numbers if grown under low temperature (25 degrees C compared to 37 degrees C) . The changes in growth temperature was shown . using 2D gel electrophoresis, to change the experssion of cell-surface proteins and, using ID gel electrophoresis, to change the expression of surface lipopolysaccharide of P . aeruginosa Paerl . Thus, these surface changes would have been likely to have mediated the increased adhesion to Etafilcon A contact lenses. Microbiology, 2001 Nov, 147(Pt 11), 3121 - 6 Role of biofilms in the survival of Legionella pneumophila in a model potable-water system; Murga R et al.; Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa . Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media . The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell . This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis . The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons . The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp . The levels of L . pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H . vermiformis, and it was found that, although unable to replicate in the absence of H . vermiformis, L . pneumophila was able to persist. J Bacteriol, 2001 Dec, 183(23), 6875 - 84 Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms; Li YH et al.; Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates . The ability of S . mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries . Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S . mutans growing in high-cell-density biofilms . It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S . mutans . This study was initiated to examine the acid tolerance response (ATR) of S . mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation . S . mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates . The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation . Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h . Both biofilm and planktonic cells were removed to assay percentages of survival . The results showed that S . mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation . Cell density was found to modulate acid adaptation in S . mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density . The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S . mutans . Heat or proteinase treatment abolished the induction by the culture filtrate . The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR . Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR . This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S . mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication. J Bacteriol, 2001 Dec, 183(23), 6746 - 51 Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials; Spoering AL et al.; Biofilms are considered to be highly resistant to antimicrobial agents . Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells . Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance . It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells . A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study . Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms . Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state . Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic . The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters . The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin . At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture . Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms . In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined . We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted . We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells. Lett Appl Microbiol, 2001 Nov, 33(5), 344 - 8 A rapid, non-destructive method for the determination of Staphylococcus epidermidis adhesion to surfaces using quartz crystal resonant sensor technology; Pavey KD et al.; AIMS: To investigate the use of quartz crystal resonant sensor (QCRS) technology to determine the adhesion of Staphylococcus epidermidis to fibronectin-coated surfaces . METHODS AND RESULTS: QCRS sensors (14 MHz) with 4 mm gold electrodes were coated with fibronectin and exposed for 15 min to suspensions of Staph . epidermidis ranging in concentration from 1 x 10(2) to 1 x 10(6) cfu ml(-1) . Changes in resonant frequency were recorded and showed a linear relationship with the logarithm of cell concentration over the range tested . CONCLUSIONS: QCRS technology was shown to be a rapid, sensitive and non-destructive method for measuring the adhesion of bacteria to surfaces . SIGNIFICANCE AND IMPACT OF THE STUDY: This report demonstrates that QCRS technology has the potential to be used for a range of applications requiring measurement of bacteria on surfaces . In particular, it may be used for the real-time monitoring of bacterial biofilm formation. Eur J Oral Sci, 2001 Oct, 109(5), 316 - 24 Proteolytic degradation of oral biofilms in vitro and in vivo: potential of proteases originating from Euphausia superba for plaque control; Berg CH et al.; This paper deals with enzymatic removal of dental plaque, in vitro as well as in vivo, using proteases from the Antarctic krill shrimp (Euphausia superba), referred to as Krillase . Krillase exhibits both endo- and exopeptidase activity but has no microbicidal effect . In model systems with pure cultures of oral microorganisms . Krillase demonstrated inhibition of microbial adhesion to saliva-coated hydroxyapatite . Furthermore, a protocol for the growth of reproducible in vitro plaque films has been developed, and effects of Krillase on the plaque film were investigated by means of scanning electron microscopy (SEM) . The results showed that Krillase efficiently released microorganisms from plaque in vitro, the effect being dependent on the enzymatic activity . The surface energy of the substratum had a minor influence on the formation and removal of plaque in vitro . Ellipsometric studies on the formation and enzymatic removal of a salivary pellicle indicated that the enzymatic effect on plaque may partly depend on degradation of the salivary pellicle . Krillase was also able to remove plaque accumulated on dentures in vivo . Our results demonstrate the potential of Krillase for plaque control, and that these enzymes are worthy of further investigations including clinical studies and work to find a suitable vehicle. Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 182 - 5 Strains degrading polysaccharides produced by bacteria from paper machines; Ratto M et al.; Biofilm-degrading enzymes are potential agents for slime control in paper machines . In this work, extracellular polysaccharides were produced by bacteria isolated from paper machines and the isolated polysaccharides were used as substrates for the screening of polysaccharide-degrading microbes . Polysaccharide yields of 1.5-3.5 g/l were obtained by ethanol precipitation from cultures of strains of Klebsiella pneumoniae, Bacillus licheniformis and Pseudomonas fluorescens on sucrose medium . Two K . pneumoniae strains apparently produced an identical heteropolysaccharide containing galacturonic acid . Fructose-containing polysaccharides were the main products of B . licheniformis and P . fluorescens . Bacteria capable of hydrolyzing the fructose-containing polymers (levans) appeared to be relatively common among the strains selected for screening . None of the bacteria or mixed cultures screened were able to utilize the Klebsiella heteropolysaccharides. Biodegradation, 2001, 12(1), 1 - 10 Enhanced biodegradation of methylhydrazine and hydrazine contaminated NASA wastewater in fixed-film bioreactor; Nwankwoala AU et al.; The aerobic biodegradation of National Aeronautics and Space Administration (NASA) wastewater that contains mixtures of highly concentrated methylhydrazine/hydrazine, citric acid and their reaction product was studied on a laboratory-scale fixed film trickle-bed reactor . The degrading organisms, Achromobacter sp., Rhodococcus B30 and Rhodococcus J10, were immobilized on coarse sand grains used as support-media in the columns . Under continuous flow operation, Rhodococcus sp . degraded the methylhydrazine content of the wastewater from a concentration of 10 to 2.5 mg/mL within 12 days and the hydrazine from approximately 0.8 to 0.1 mg/mL in 7 days . The Achromobacter sp . was equally efficient in degrading the organics present in the wastewater, reducing the concentration of the methylhydrazine from 10 to approximately 5 mg/mL within 12 days and that of the hydrazine from approximately 0.8 to 0.2 mg/mL in 7 days . The pseudo first-order rate constants of 0.137 day(-1) and 0.232 day(-1) were obtained for the removal of methylhydrazine and hydrazine, respectively, in wastewater in the reactor column . In the batch cultures, rate constants for the degradation were 0.046 and 0.079 day(-1) for methylhydrazine and hydrazine respectively . These results demonstrate that the continuous flow bioreactor afford greater degradation efficiencies than those obtained when the wastewater was incubated with the microbes in growth-limited batch experiments . They also show that wastewater containing hydrazine is more amenable to microbial degradation than one that is predominant in methylhydrazine, in spite of the longer lag period observed for hydrazine containing wastewater . The influence of substrate concentration and recycle rate on the degradation efficiency is reported . The major advantages of the trickle-bed reactor over the batch system include very high substrate volumetric rate of turnover, higher rates of degradation and tolerance of the 100% concentrated NASA wastewater . The results of the present laboratory scale study will be of great importance in the design and operation of an industrial immobilized biofilm reactor for the treatment of methylhydrazine and hydrazine contaminated NASA wastewater. Clin Microbiol Infect, 2001, 7 Suppl 4, 1 - 7 Coagulase-negative staphylococci as a cause of infections related to intravascular prosthetic devices: limitations of present therapy; Schulin T et al.; Coagulase-negative staphylococci (CNS) are an important cause of catheter-related bloodstream infections . This review will shed light on the pathogenesis related to biofilm formation, and will discuss antimicrobial susceptibility of CNS to older and newer antibiotics, as well as therapeutic options. J Clin Periodontol, 2001 Nov, 28(11), 1074 - 8 Effect of an enamel matrix protein derivative (Emdogain) on ex vivo dental plaque vitality; Sculean A et al.; BACKGROUND: A common clinical observation following surgical periodontal therapy with an enamel matrix derivative (Emdogain) is the improved healing of the soft tissues and the limited inflammation of the operated areas . These clinical observations are empirical and difficult to explain . One of the factors influencing the early wound healing might be a potential antimicrobial effect of Emdogain . AIM: To investigate the effect of Emdogain on the vitality of ex vivo supragingival dental plaque and to compare this effect to that of a standard 0.2% chlorhexidine solution . MATERIALS AND METHODS: 24 patients suffering from adult periodontitis were included in the study . At the beginning of the experiment, all participants were given a professional tooth cleaning . For the following 4 days, they had to refrain from any kind of oral hygiene measures . At day 5, from each of the volunteers, a voluminous plaque biofilm sample was taken with a sterile curette from the vestibular surfaces of the 1st lower molars and divided into 5 equal parts . Each part was mounted with 5 microl of the following solutions: (1) NaCl, (2) enamel matrix derivative dissolved in water (EMD), (3) enamel matrix derivative dissolved in the vehicle (Emdogain), (4) vehicle (propylene glycol alginate, PGA), (5) 0.2% chlorhexidine digluconate (CHX) . After a reaction time of 2 min the test solutions were sucked off, and subsequently the biofilm was stained with a fluorescence dye . The vitality of the plaque flora after the treatments was evaluated under the fluorescence microscope (VF%) . RESULTS: Plaque samples treated with NaCl showed a mean vitality of 76.8+/-8% . The EMD, Emdogain, PGA and CHX showed VF values of 54.4+/-9.2, 21.4+/-10.6%, 19.6+/-11.6% and 32.3+/-11.8%, respectively . Emdogain, PGA and CHX showed statistically highly significant reductions (p<0.0001) in terms of bacteria vitality when compared to water (negative control) and EMD . Both Emdogain and PGA were found to be statistically significantly different compared to CHX (p<0.001) (positive control) . CONCLUSION: The results of this study indicate that Emdogain might have an antibacterial effect on the vitality of the ex vivo supragingival dental plaque flora. J Clin Periodontol, 2001 Nov, 28(11), 1045 - 9 Plaque removal characteristics of electric toothbrushes using an in vitro plaque model; Carter K et al.; BACKGROUND/AIM: The purpose of the study was to investigate and quantify the efficacy of plaque removal by commercially available electric toothbrushes using an in vitro system based on artificial plaque grown on glass slides . MATERIAL AND METHODS: Artificial plaque based on Streptococcus sanguis and Streptococcus mutans was cultured in a series of phosphate buffer solution, sucrose solution and brain heart infusion on sterile glass slides, for a period of 5 weeks . 7 different electric toothbrushes of current designs were operated in contact with the glass slides under loads of 1 or 2N for 10 s after which image analysis of the brushed slides was undertaken to calculate the absolute and relative areas of removal . RESULTS: The electric toothbrushes showed a larger area of plaque removal as loading force increased from 1 to 2N . The area of biofilm removed also depended on the head design and ranged from 258 mm(2) (1N, Interplak) and 314 mm(2) (2N, Interplak), to 30 mm(2) (1N, Braun 3D), and 148 mm(2) (2N, Blend-a-Dent) . The area of biofilm removal was significantly different among all electric toothbrushes at p<0.01 (ANOVA) . All electric toothbrushes removed more biofilm than the actual contacting tufted area of the head . CONCLUSION: The artificial plaque model system under different loading conditions showed differences in the absolute area of removal per electric toothbrush and also differences in the relative removal per unit contact bristle area . These differences on operating variables should be considered when evaluating new electric toothbrushes. Res Microbiol, 2001 Oct, 152(8), 753 - 60 Biofilms augment the number of free-living amoebae in dental unit waterlines; Barbeau J et al.; Freshwater amoebae are ubiquitous . Some species can cause infections in humans while others can ingest and protect opportunistic bacteria . Although the presence of free-living amoebae in various water sources has been reported, few studies have looked at their concentration, which may be clinically relevant, especially if they are present in healthcare devices . A simple technique was used to detect, observe, and evaluate the concentration of free-living amoebae in dental unit and tap water samples . Fifty-three water samples were collected from 35 dental units (air/water syringes) and 18 water taps . The technique was based on the ability of waterborne bacteria to create a biofilm and serve as substratum for the development of amoebae naturally present in the water samples . Laboratory-grown freshwater biofilms support the proliferation of a wide variety of free-living amoebae . All the dental unit water samples tested contained amoebae at concentrations up to 330/mL, or more than 300 times the concentration in tap water from the same source . Hartmanella, Vanella, and Vahlkampfia spp . were the most frequently encountered . Naegleria and Acanthamoeba spp . were also present in 40% of the samples . Four of the samples collected from dental units, but none from water taps, contained amoebae able to proliferate at 44 degrees C . Biofilms that form inside some dental instruments can considerably increase the concentration of free-living amoebae, some of which are potential human pathogens. Pharm Res, 2001 Sep, 18(9), 1247 - 54 Biofilm consortia on biomedical and biological surfaces: delivery and targeting strategies; Sihorkar V et al.; Microbial biofilms have been observed as congregates and attached communities on a diverse range of microecosystems of medicinal and industrial importance . Until recently, most investigations have been performed on planktonic (floating or fluid phase) microorganisms . After realization of the biofilm existence and their recalcitrance toward conventionally adopted preventive strategies and antimicrobial agents, research has been shifted toward novel therapeutics based drug delivery and targeting approaches . With the emergence of various biofilm models and methods to assess biofilm formation and physiology, it is pivotal to discuss various novel strategies that may become the therapeutic tools and clinically adaptable strategies of the future . This review explores various novel research strategies studied to date for their potential in effective biofilm eradication. Photochem Photobiol, 2001 Oct, 74(4), 570 - 8 Exposure of arctic field scientists to ultraviolet radiation evaluated using personal dosimeters; Cockell CS et al.; During July 2000 we used an electronic personal dosimeter (X-2000) and a biological dosimeter (Deutsches Zentrum fur Luft- und Raumfahrt: Biofilm) to characterize the UV radiation exposure of arctic field scientists involved in biological and geological fieldwork . These personnel were working at the Haughton impact structure on Devon Island (75 degrees N) in the Canadian High Arctic under a 24 h photoperiod . During a typical day of field activities under a clear sky, the total daily erythemally weighted exposure, as measured by electronic dosimetry, was up to 5.8 standard erythemal dose (SED) . Overcast skies (typically 7-8 okta of stratus) reduced exposures by a mean of 54% . We estimate that during a month of field activity in July a typical field scientist at this latitude could potentially receive approximately 80 SED to the face . Because of body movements the upper body was exposed to a UV regimen that often changed on second-to-second time-scales as assessed by electronic dosimetry . Over a typical 10 min period on vehicle traverse, we found that erythemal exposure could vary to up to 87% of the mean exposure . Time-integrated exposures showed that the type of outdoor field activities in the treeless expanse of the polar desert had little effect on the exposure received . Although absolute exposure changed in accordance with the time of day, the exposure ratio (dose received over horizontal dose) did not vary much over the day . Under clear skies the mean exposure ratio was 0.35 +/- 0.12 for individual activities at different times of the day assessed using electronic dosimetry . Biological dosimetry showed that the occupation was important in determining daily exposures . In our study, scientists in the field received an approximately two-fold higher dose than individuals, such as medics and computer scientists, who spent the majority of their time in tents. Nature, 2001 Oct 25, 413(6858), 860 - 4 Gene expression in Pseudomonas aeruginosa biofilms; Whiteley M et al.; Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment . Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections . To gain insights into the differences between free-living P . aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays . Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms . Some of the regulated genes are known to affect antibiotic sensitivity of free-living P . aeruginosa . Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes . We propose that this response is critical for the development of biofilm resistance to tobramycin . Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences . Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics. Postgrad Med, 2001 Oct, 110(4), 63 - 4, 69-70, 73-6 Coagulase-negative staphylococci . Pathogens have major role in nosocomial infections; von Eiff C et al.; Coagulase-negative staphylococci live naturally on the skin and mucous membranes of humans and are therefore often found in clinical specimens . Distinguishing clinically significant, pathogenic strains from contaminant strains is one of the major challenges facing clinical microbiology laboratories . S epidermidis and other novobiocin-susceptible coagulase-negative staphylococci have emerged as a major cause of nosocomial infections, particularly of nosocomial bacteremia in immunocompromised patients . S epidermidis also is common in injecting drug users, who are particularly susceptible to right-sided endocarditis, and--most important--in patients with such indwelling foreign bodies as intravenous catheters . Depending on the kind of device and its insertion site, different infection syndromes generate a variety of clinical presentations . In these patients, the host defense mechanisms often seem unable to handle the infection and, in particular, to eliminate the staphylococci from the infected device because of a biofilm on the foreign body surface . S saprophyticus, the most commonly isolated bacterium of the novobiocin-resistant coagulase-negative staphylococci, is a common pathogen of the urogenital tract; it generally infects immunocompetent patients, particularly young, sexually active men and women. J Bacteriol, 2001 Nov, 183(22), 6579 - 89 Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth; Sauer K et al.; The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface . A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria . In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns . Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated . N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism . cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of P . putida . Twenty-eight of these genes had known homologs . As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth . The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface . Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis . Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms . In contrast, PilA was observed in 12-h biofilms but not in planktonic culture . Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development . To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P . putida and pure C(12)-HSL were added to 6-h planktonic cultures of P . putida, and cell extracts were analyzed by 2-D gel profiles . Differential expression of 16 proteins was observed following addition of HSLs . One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition . The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth . The results presented here demonstrate that P . putida undergoes a global change in gene expression following initial attachment to a surface . Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes. J Calif Dent Assoc, 2001 Sep, 29(9), 679 - 84 Controlling biofilm and microbial contamination in dental unit waterlines; Lee TK et al.; Despite the fact that the ADA had set the goal of less than 200 colony-forming units per milliliter of unfiltered output water from dental unit waterlines to be achieved voluntarily by the year 2000, there is much confusion and resistance within the profession with regard to waterlines . Many in the profession are still wondering what the most effective means are to predictably achieve the goal . It is a well-established fact that bacterial biofilm can readily form within dental unit waterlines and degrade the microbial quality of the water in dental units regardless of the water source . These biofilms are primarily formed by various microcolonies of bacteria that attach to surfaces over time within the waterlines . An increasing number of medically compromised and immunocompromised patients being treated in dental offices and increased public awareness have brought about renewed interest in this issue . There are generally four categories of products that are available to address this issue: independent water systems, sterile water delivery systems, filtration, and chemical treatment protocols . A recent study at the University of California at Los Angeles demonstrates that the Ultra chemical treatment protocol can be an effective means of controlling biofilm in dental unit waterlines. J Am Dent Assoc, 2001 Sep, 132(9), 1287 - 91 Evaluation of a hydrogen peroxide disinfectant for dental unit waterlines; Linger JB et al.; BACKGROUND: The purpose of this study was to investigate the use of a hydrogen peroxide-based dental unit waterline, or DUWL, treatment to reduce the colonization and growth of heterotrophic bacteria . METHODS: Twenty-three dental units with self-contained water systems were randomly selected . Three of the units and tap water served as controls . Twenty-four water samples were taken at baseline and once a week for five weeks . They were serially diluted, spread-plated in duplicate onto R2A agar plates and incubated at 37 C for seven days . RESULTS: At baseline, the tap water control had a mean count of 0 colony-forming units/milliliter, or CFU/mL, the three control DUWLs had a median count of 8,440 CFU/mL and the 20 treated DUWLs had a median count of 9,760 CFU/mL . By week 1, 19 (95 percent) of the 20 treated DUWLs had counts of less than 200 CFU/mL, and by week 4, the median count for all of the treated DUWLs was 0 CFU/mL . The measurement at week 5 showed that the reduction to below 200 CFU/mL had been maintained . Scanning electron micrographs from processed DUWL tubing samples revealed a similar pattern of results, with biofilm accumulation more evident in the untreated control specimens . CONCLUSIONS: Following the parameters of this study, the authors used a hydrogen peroxide-based disinfectant to achieve the ADA goal of no more than 200 CFU of heterotrophic, mesophilic bacteria per milliliter of unfiltered output water . CLINICAL IMPLICATIONS: An easy-to-use hydrogen peroxide-based DUWL disinfectant demonstrated effectiveness in improving the quality of water used for intraoral procedures . Protocol compliance meets the ADA year 2000 goal. Environ Sci Technol, 2001 Oct 1, 35(19), 3863 - 8 Characterization of Fe plaque and associated metals on the roots of mine-waste impacted aquatic plants; Hansel CM et al.; Iron plaque on aquatic plant roots are ubiquitous and sequester metals in wetland soils; however, the mechanisms of metal sequestration are unresolved . Thus, characterizing the Fe plaque and associated metals will aid in understanding and predicting metal cycling in wetland ecosystems . Accordingly, microscopic and spectroscopic techniques were utilized to identify the spatial distributions, associations, and chemical environments of Fe, Mn, Pb, and Zn on the roots of a common, indigenous wetland plant (Phalaris arundinacea) . Iron forms a continuous precipitate on the root surface, which is composed dominantly of ferrihydrite (ca . 63%) with lesser amounts of goethite (32%) and minor levels of siderite (5%) . Although Pb is juxtaposed with Fe on the root surface, it is complexed to organic functional groups, consistent with those of bacterial biofilms . In contrast, Mn and Zn exist as discrete, isolated mixed-metal carbonate (rhodochrosite/hydrozincite) nodules on the root surface . Accordingly, the soil-root interface appears to be a complex biochemical environment, containing both reduced and oxidized mineral species, as well as bacterial-induced organic-metal complexes . As such, hydrated iron oxides, bacterial biofilms, and metal carbonates will influence the availability and mobility of metals within the rhizosphere of aquatic plants. Res Microbiol, 2001 Sep, 152(7), 613 - 9 Bacterial infections of free-living amoebae; Winiecka-Krusnell J et al.; Free-living amoebae are a diverse group of ubiquitous unicellular organisms, some of which cause severe central nervous system infections and keratitis . However, the focus of research has shifted from the direct pathogenic effects of free-living amoebae towards their role as carriers of pathogenic bacteria . Large outbreaks of legionellosis with numerous fatal cases, both in hospitals and in the community, appear to be the visible tip of the iceberg of complex relationships between amoebae and bacteria in biofilms . The recognition of amoebae as reservoirs and vehicles for bacterial spread leads us to public health issues such as the development of pathogenicity, antibiotic resistance, quality of public water supplies, housing standards, sanitation and decontamination measures . In this review we discuss bacterial infections of free-living amoebae from both a "biological" and general "infection control" point of view. Verh K Acad Geneeskd Belg, 2001, 63(4), 359 - 77 Legionellosis: a new disease of Western hemisphere? Goossens H. Legionnaires' disease is an acute bacterial pneumonia caused by an organism of the family of Legionellaceae . L . pneumophila serotype 1 is responsible for most infections . The incidence of Legionnaires' disease in Belgium is not known because the diagnosis cannot be made based on clinical or radiological grounds and because there is a microbiological underdiagnosis . Legionellae are widely distributed in nature, as free living organisms, in protozoa, and in biofilms . As a consequence, these bacteria are particularly resistant to disinfection and heating . We recently investigated an outbreak of legionnaires' disease among visitors to a fair in Kapellen, Belgium . The annual commercial fair at Kapellen was held between October 29 to November 7, 1999 . Ninety-three people, who met the case definition, were identified . Among these 93 patients, 41 could be considered as confirmed cases, 14 as presumptive, and 38 as possible cases . Five patients died in as a consequence of the epidemic . A case-control study showed a causal link between a visit to the fair and the occurrence of the infection . A contaminated aerosol from a whirlpool was probably responsible for the outbreak . This epidemic illustrates the need for preventive measures to control for the growth of Legionellae in aerosol producing instruments. Int Endod J, 2001 Oct, 34(7), 547 - 53 A new in vitro model for the study of microbial microleakage around dental restorations: a preliminary qualitative evaluation; Matharu S et al.; AIM: The aim of this study was to develop an in vitro model to replicate microbial microleakage at a tooth/ restoration interface using a constant depth film fermentor (CDFF) . METHODOLOGY: Amalgam restorations were placed in machined bovine dentine cylinders and sealed externally with varnish, leaving a 1-mm perimeter exposed around the tooth/restoration interface . The dentine cylinders were housed in a CDFF and 300-microm thick microcosm dental plaques were grown over their exposed surfaces . The biofilms were maintained with a mucin-containing artificial saliva for up to 8 weeks . Cylinders were aseptically removed from the CDFF (at 1, 2, 4, & 8 weeks) and surface-decontaminated with validated protocols prior to splitting and sampling of apposing amalgam and dentine surfaces . Scanning electron microscopy (SEM) was used to ascertain the position and structure of the bacterial aggregates . Bacterial viability was determined by vital staining of the bacteria in situ . RESULTS: At all sampling times, SEM showed cocci, rods and filaments on both amalgam and dentine surfaces; some originated as cascades from the surface biofilm and extended into the tooth/restoration microspace . Vital staining showed the majority of bacteria from both dentine and amalgam surfaces to be viable . CONCLUSION: This preliminary investigation showed that the CDFF may be a valuable tool for the in vitro study of the dynamics of microbial microleakage around dental restorations. Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 826 - 30 Investigations into the application of a process for the determination of microbial activity in biofilms; Holtmann D et al.; The formation of biofilms in a waste paper medium was studied in a pilot plant by analysing the redox potential in the biofilm . Miniaturised redox electrodes were applied at the reactor wall/biofilm phase boundary . With this measurement set-up, it was possible to demonstrate the effectiveness of biocides and thus to avoid under- and over-doses with these agents . The redox signals measured were correlated with reference methods, such as colony-forming units and dehydrogenase activity. Antimicrob Agents Chemother, 2001 Nov, 45(11), 3262 - 6 Eradication of biofilm-forming Staphylococcus epidermidis (RP62A) by a combination of sodium salicylate and vancomycin; Polonio RE et al.; Staphylococcus epidermidis is a major cause of infections associated with indwelling medical devices . Biofilm production is an important virulence attribute in the pathogenesis of device-related infections . Therefore, elimination of these biofilms is an ideal treatment . Salicylate (5 mM) combined with 1 microg of vancomycin per ml inhibited biofilm formation by S . epidermidis (RP62A) by >or=99.9% . When biofilm-coated polystyrene beads were exposed to 5 mM sodium salicylate and 4 microg of vancomycin per ml (one-half the minimum biofilm eradication concentration), there was a >99.9% reduction in viable count. Med Microbiol Immunol (Berl), 2001 Sep, 189(4), 217 - 23 Interaction of Staphylococcus epidermidis with endothelial cells in vitro; Merkel GJ et al.; Staphylococcus epidermidis is a leading cause of nosocomial bacteremia, yet virtually nothing is known about how this pathogen interacts with human endothelial cells . We present evidence here that two biofilm-producing strains of S . epidermidis adhere to two types of endothelial cell lines in vitro and that adherence is significantly increased after briefly heat-treating the bacteria at 40 degrees C in the presence of calcium . This mild heat treatment resulted in bacteria that were 5 to more than 20 times more adherent than untreated controls . While the adherence of bacteria in all phases of growth was increased after heat treatment, heat-treated late stationary phase cells were generally the most adherent . Electron microscopy demonstrated that S . epidermidis was internalized and appeared to exist free in the cytoplasm . Adherence to endothelium, should it occur in vivo during bacteremia, may be a virulence factor associated with this bacterium's pathogenesis. Infect Immun, 2001 Nov, 69(11), 7046 - 56 Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation; Rogers JD et al.; Interactions between bacteria and salivary components are thought to be important in the establishment and ecology of the oral microflora . alpha-Amylase, the predominant salivary enzyme in humans, binds to Streptococcus gordonii, a primary colonizer of the tooth . Previous studies have implicated this interaction in adhesion of the bacteria to salivary pellicles, catabolism of dietary starches, and biofilm formation . Amylase binding is mediated at least in part by the amylase-binding protein A (AbpA) . To study the function of this protein, an erythromycin resistance determinant {erm(AM)} was inserted within the abpA gene of S . gordonii strains Challis and FAS4 by allelic exchange, resulting in abpA mutant strains Challis-E1 and FAS4-E1 . Comparison of the wild-type and mutant strains did not reveal any significant differences in colony morphology, biochemical metabolic profiles, growth in complex or defined media, surface hydrophobicity, or coaggregation properties . Scatchard analysis of adhesion isotherms demonstrated that the wild-type strains adhered better to human parotid-saliva- and amylase-coated hydroxyapatite than did the AbpA mutants . In contrast, the mutant strains bound to whole-saliva-coated hydroxyapatite to a greater extent than did the wild-type strains . While the wild-type strains preincubated with purified salivary amylase grew well in defined medium with potato starch as the sole carbohydrate source, the AbpA mutants did not grow under the same conditions even after preincubation with amylase . In addition, the wild-type strain produced large microcolonies in a flow cell biofilm model, while the abpA mutant strains grew much more poorly and produced relatively small microcolonies . Taken together, these results suggest that AbpA of S . gordonii functions as an adhesin to amylase-coated hydroxyapatite, in salivary-amylase-mediated catabolism of dietary starches and in human saliva-supported biofilm formation by S . gordonii. Infect Immun, 2001 Nov, 69(11), 6931 - 41 Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates; Mattos-Graner RO et al.; Streptococcus mutans, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm . These include glucosyltransferases, their glucan products, and proteins that bind glucan . At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel . The gbpB gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus . Examination of gbpB genes from clinical isolates of S . mutans revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein . The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay. Peptides, 2001 Oct, 22(10), 1603 - 8 Staphylococcus aureus and Staphylococcus epidermidis peptide pheromones produced by the accessory gene regulator agr system; Otto M; The accessory gene regulator (agr) system of staphylococci regulates the expression of virulence factors in response to cell density . The extracellular signaling molecule encoded by this system is a thiolactone-containing pheromone peptide whose primary sequence varies among staphylococcal strains . A post-translational modification of the peptide is believed to be carried out by an enzyme with a novel function, AgrB . Staphylococcal pheromones show cross-inhibiting properties: Pheromones of self and pheromones of non-self induce and suppress the agr response, respectively, and have therefore been proposed as novel anti-staphylococcal drugs . As inhibition of agr leads to diminished expression of toxins, but to increased expression of colonization factors and biofilm formation, their therapeutic potential remains yet to be evaluated in depth. Peptides, 2001 Oct, 22(10), 1579 - 96 Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior; Kleerebezem M et al.; Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner . Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see {38}) . The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms . These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space . The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value . Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones . Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see {47,48}), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see {37,61}) . However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see {20}).This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria. Biodegradation, 2000, 11(6), 359 - 64 Acetate conversion in anaerobic biogas reactors: traditional and molecular tools for studying this important group of anaerobic microorganisms; Schmidt JE et al.; Different methods were applied to study the role of aceticlastic methanogens in biogas reactors treating solid waste and wastewater . We used traditional microbiological methods, immunological and 16S rRNA ribosomal probes for detection of the methanogens . Using this approach we identified the methanogenic spp . and their activity . In biofilm systems, such as the UASB reactors the presence of the two aceticlastic methanogens could be correlated to the difference in the kinetic properties of the two species . In biogas reactors treating solid wastes, such as manure or mixture of manure and organic industrial waste, only Methanosarcina spp . were identified . Methanosarcina spp . isolated from different plants had different kinetics depending on their origin . Relating the reactor performance data to measurement of the activity by conventional microbiological methods gave a good indication of the microbial status of specific trophic groups . 16S rRNA probing confirmed these observations and gave a more detailed picture of the microbial groups present. Scanning, 2001 Sep-Oct, 23(5), 346 - 50 Direct wet surface imaging of an anaerobic biofilm by environmental scanning electron microscopy: application to landfill clay liner barriers; Darkin MG et al.; To contain domestic waste and its associated pollution within a landfill, engineered mineral (clay) barriers are used and are designed to have a permeability of 1 x 10(-9) m/s (Westlake 1995) . The rate of permeability of various porous media has shown to be influenced by the clogging of flow paths (media pores) due to biofilm formation (Charckalis and Marshall 1990, Cunningham et al . 1991) . The term biofilm is given to describe the colonies of surface adherent microorganisms (Donlan et al . 1994) . In this study, permeability experiments were built and modified to act as microcosms to investigate the influence of biofilm formation on the permeability of clay barriers . Traditional scanning electron microscopy methods disrupt or destroy the biofilm and previous anaerobic studies have involved building closed cells (such as miniature continuous culture chambers) that utilise light microscopes (Robin Jones et al . 1997) . This paper examines the application of the environmental scanning electron microscope (ESEM) to the direct examination of the clay interface and biofilm formation in situ within the microcosm. FEMS Microbiol Lett, 2001 Sep 25, 203(2), 263 - 7 Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system; Bardouniotis E et al.; The importance of non-tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention . Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC) and to compare biocide susceptibility of planktonic and biofilm organisms . Scanning electron microscopy was also carried out to observe biofilm morphology . With the exception of Sporicidin and Virkon the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values . The MBEC assay system was seen to produce multiple and reproducible biofilms of M . phlei and to be a useful tool for susceptibility studies. FEMS Microbiol Lett, 2001 Sep 25, 203(2), 161 - 4 Inactivation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by Porphyromonas gingivalis; Grenier D et al.; In response to periodontal inflammation, host cells release matrix metalloproteinases (MMPs) that contribute to periodontal tissue breakdown unless the tissue inhibitors of metalloproteinases (TIMPs) neutralize their activity . In this study, the capacity of Porphyromonas gingivalis to inactivate TIMP-1 was investigated . Proteolytic digestion of TIMP-1 was monitored by SDS-PAGE and Western immunoblotting . Planktonic cells and biofilms of P . gingivalis degraded TIMP-1 with production of several lower molecular mass fragments . Incorporation of human serum in the assay mixture had no effect on the degradation of TIMP-1 by P . gingivalis, whereas a cysteine proteinase inhibitor caused a complete inhibition . Using a fluorogenic assay, it was found that TIMP-1 treated with P . gingivalis lost its capacity to inhibit MMP-9 activity . This study revealed the potential of P . gingivalis to inactivate TIMP-1 through proteolytic degradation . This phenomenon may contribute to increasing significantly the level of active MMPs in affected periodontal sites and subsequently favor tissue destruction. Acta Otolaryngol, 2001 Jul, 121(5), 643 - 6 Does metal coating improve the durability of silicone voice prostheses? Arweiler-Harbeck D, Sanders A, Held M, Jerman M, Ehrich H, Jahnke K. Voice prostheses, which are used for voice rehabilitation in cancer patients after laryngectomy, usually become colonized with a mixed biofilm of bacteria and Candida after 2-4 months and lose their efficiency . It is essential to ensure the stability and biocompatibility of these implants . With the aid of surface frame analysis we have shown that local antifungal treatment is inadequate for eliminating the deep infiltration and encapsulation of Candida colonies in silicone . A surface that prevents the adhesion of microorganisms is required . Because of its special properties there are few methods available for coating silicone . We employed, for the first time, a new method of surface modification using anodic vacuum arc coating . Using this method it was possible to obtain a solid film of gold or titanium metal with a layer thickness < 100 nm . Resistance against Candida colonization and destruction of coated prostheses were tested both in vitro and in vivo . A titanium coating seemed to provide the optimal solution to the problem, because surface adhesion and the smoothness of the material appeared to be superior to those of a gold coating. Infect Control Hosp Epidemiol, 2001 Jul, 22(7), 414 - 8 Pseudo-outbreak of Mycobacterium chelonae and Methylobacterium mesophilicum caused by contamination of an automated endoscopy washer; Kressel AB et al.; OBJECTIVE: To evaluate an unusual number of rapidly growing acid-fast bacilli, later identified as Mycobacterium chelonae, and pink bacteria, later identified as Methylobacterium mesophilicum, from fungal cultures obtained by bronchoscopy . DESIGN: Outbreak investigation . SETTING: An academic medical center performing approximately 500 bronchoscopies and 4,000 gastrointestinal endoscopies in 1998 . PATIENTS: Patients undergoing bronchoscopy July 21 to October 2, 1998 . METHODS: The infection control department reviewed patient charts and bronchoscopy logs; obtained cultures of source water, faucets, washers, unopened glutaraldehyde, glutaraldehyde from the washers, and endoscopes; observed endoscope and bronchoscope cleaning and disinfecting procedures; reviewed glutaraldehyde monitoring records; and sent M . chelonae isolates for DNA fingerprinting . RESULTS: M . chelonae, M . mesophilicum, gram-negative bacteria, and various molds grew from endoscopes, automated washers, and glutaraldehyde from the washers but not from unopened glutaraldehyde . The endoscopy unit regularly monitored the pH of glutaraldehyde, and the logs contained no deficiencies . The above sources remained positive for the same organisms after a glutaraldehyde cleaning cycle of the automated washers . DNA finger-printing of the M . chelonae revealed that they were clonally related . CONCLUSIONS: The automated washers were contaminated with a biofilm that rendered them resistant to decontamination . The washers then contaminated the endoscopes and bronchoscopes they were used to disinfect . Our institution purchased new endoscopes and a new paracetic acid sterilization system. J Antimicrob Chemother, 2001 Oct, 48(4), 573 - 7 A combination of roxithromycin and imipenem as an antimicrobial strategy against biofilms formed by Staphylococcus aureus; Yamasaki O et al.; We examined the effects of a combination of roxithromycin and imipenem on a biofilm model of Staphylococcus aureus . Treatment with roxithromycin alone and imipenem alone did not decrease the number of viable bacterial cells compared with the control . However, a combination treatment of roxithromycin and imipenem significantly decreased the number of viable bacterial cells on day 8 after inoculation in the in vivo model (P < 0.01) . On days 5 and 8 after inoculation, numerous polymorphonuclear leucocytes and macrophages invaded the bacterial clusters in the roxithromycin- and roxithromycin/imipenem-treated groups, but did not invade the control or imipenem-treated groups . The present study indicated that a combination of roxithromycin and imipenem is a potentially effective treatment for S . aureus biofilm-associated skin infections as it can induce the invasion of polymorphonuclear leucocytes into the biofilm. Front Biosci, 2001 Oct 01, 6, E93 - 103 The role of bacteria in gallstone pathogenesis; Swidsinski A et al.; Bacteria are often found in high concentrations in brown pigment and less so in cholesterol gallstones . Although it is intriguing to hypothesize that cholesterol stone formation is non-bacterial in nature and principally different from the pathogenesis of "infectious" brown pigment gallstones, it is more likely that significant overlap exists between the two processes . Most gallstones are composite in nature . Using molecular-genetic methods, bacteria can be found in most pure cholesterol gallstones (i.e . those whose structure consists of more than 90% cholesterol) . The natural history of the gallstones development is unknown . It is likely that brown pigment stones can evolve in their chemical composition after the termination of the infectious process that initiate their formation, and may further develop into either mixed or nearly pure cholesterol stones . In a similar fashion, cholesterol-poor or black pigment gallstones may act as foreign bodies to enhance the propensity of bacterial colonization in the presence of pre-existing gallstones or cholangitis, thereby activating pathways of bacterial lithogenesis and resulting in the encasement of cholesterol nuclei with pigment shells and/or in the internal remodeling of extant stones . It is often difficult, if not impossible, to ascertain whether bacterial infection of bile arose before stone formation or vice-versa . The development of gallstones (nucleation, assembly of microcalculi, growth, remodeling) includes the interaction of both bacterial and non-bacterial mechanisms, working discontinuously over years and decades and shaping the structural individuality of each stone . At cholecystectomy, the gallstone removed from the patient represents the end product of a long pathologic process . Although our understanding of the exact temporal contribution of bacteria in lithogenesis is incomplete, it is important for the clinician to realize that most gallstones are colonized by a bacterial biofilm, even though the bile may be culture-negative. J Periodontol, 2001 Sep, 72(9), 1210 - 20 Effects of smoking and treatment status on periodontal bacteria: evidence that smoking influences control of periodontal bacteria at the mucosal surface of the gingival crevice; Eggert FM et al.; BACKGROUND: We examined whether smoking status could influence growth of potentially pathogenic bacteria in the periodontal environment of treated and untreated periodontal patients . METHODS: We have previously reported effects of treatment status on marker bacteria in our patients . We established a history of any smoking during 6 months prior to microbiological sampling (F-ME, 16 smokers out of 64; MHM, 70 smokers out of 185) . We used a commercial immunoassay to quantitate Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in paper point samples from periodontal sites . RESULTS: Logistic regression showed that in smokers, neither P . gingivalis nor A . actinomycetemcomitans was quantitatively increased, while P intermedia was somewhat increased . Multiple regression demonstrated that smoking disrupts the positive relationship between increasing probing depth and increasing bacterial growth that is found in non-smokers . In smokers, growth of marker bacteria at shallow sites (< or =5 mm) was significantly increased to the levels found at deeper sites (>5 mm) in both smokers and non-smokers . Supragingival plaque biofilm was identified as a reservoir for marker bacteria; smokers and nonsmokers had equal ranges of oral cleanliness . CONCLUSIONS: Smoking-associated periodontitis is not simply a reflection of oral cleanliness . Smoking extends a favorable habitat for bacteria such as P . gingivalis, P . intermedia, and A . actinomycetemcomitans to shallow sites (< or =5 mm) . Molecular byproducts of smoking interfere with mechanisms that normally contain growth of damaging bacteria at the surface of the oral mucosa in gingival crevices . In this way, smoking can promote early development of periodontal lesions. J Periodontol, 2001 Sep, 72(9), 1201 - 9 Performance of a commercial immunoassay for detection and differentiation of periodontal marker bacteria: analysis of immunochemical performance with clinical samples; Eggert FM et al.; BACKGROUND: We employed a commercial immunoassay for simultaneous detection and differentiation of marker bacteria Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia and reassessed the immunochemical performance of the assay . METHODS: We compared the analytical performance of the immunoassay in our study of clinical samples from 249 periodontal patients in 2 private periodontal practices with the previously reported analytical performance of the same immunoassay . We also compared immunoassay measurements of the marker bacteria in clinical samples with values obtained in other studies by direct culture of the same organisms . RESULTS: The assay produced 3 times more high-end readings than reported previously . We also reassessed and revised previously published calibration curves for the immunoassay . The immunoassay provided measurements of the marker bacteria in clinical samples from our patients that were comparable to and consistent with measurements of the same bacteria by direct culture in other studies . CONCLUSIONS: We ascribe the increased sensitivity of the immunoassay in our study to: 1) a more standardized and vigorous sample dispersion that improves release of particulate and soluble antigens from dental plaque biofilm, and 2) better visualization of the reaction product of the enzyme-linked immunoassay . High-technology assays, such as diagnostic immunoassays, have a significant potential for future development in dental diagnosis, because they simplify detection and measurement of biologically important markers such as specific bacteria in clinical samples . Commercial assays also have an important potential for standardization of clinical measurements of biological markers. Clin Infect Dis, 2001 Nov 1, 33(9), 1567 - 72 Epub 2001 Sep 26. Device-associated infections: a macroproblem that starts with microadherence; Darouiche RO; Medical devices are responsible for a large portion of nosocomial infections, particularly in critically ill patients . Device-associated infections can cause major medical and economic sequelae . Bacterial colonization of the indwelling device can be a prelude to both infection and malfunction of the device . The pathogenesis of device-associated infection centers around the multifaceted interaction among the bacteria, the device, and the host . Bacterial factors are probably the most important in pathogenesis of infection, whereas device factors are the most amenable to modification with the objective of preventing infection . Some, but not all, of the studied bacterial receptors satisfy the proposed "adherence/infection" version of Koch's postulates . Traditional surface-modifying preventive approaches have largely focused on antimicrobial coating of devices and resulted in variable clinical success in preventing device-associated infections . The potential protective role of newer innovative approaches, such as biofilm modification and bacterial interference, ought to be further investigated. Microbiology, 2001 Oct, 147(Pt 10), 2841 - 8 Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate; Li Y et al.; Streptococcus mutans produces a number of extracellular sucrose-metabolizing enzymes that contribute to the ability of the organism to cause dental caries, including three glucosyltransferases, the products of the gtfB, gtfC and gtfD genes, and a fructosyltransferase, encoded by the ftf gene . To better understand the regulation of the expression of these genes under environmental conditions that more closely mimic those in dental plaque, two strains of S . mutans harbouring fusions of the gtfBC (SMS102) and ftf (SMS101) promoters to a chloramphenicol acetyltransferase (CAT) gene were examined in biofilms formed in vitro . The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium . CAT specific activity in biofilm cells was measured at quasi-steady state or following additions of 25 mM sucrose or glucose, with or without pH control . After approximately 10 generations of biofilm growth, the ftf and gtfBC genes of S . mutans were found to be expressed at levels different from those reported for planktonic cells growing under otherwise similar conditions . The expression of these genes was induced by the addition of sucrose to the quasi-steady-state cultures . Expression of the gtfBC genes was influenced by environmental pH, since CAT specific activities in quasi-steady-state biofilms of strain SMS102 grown without pH control were twice those produced by cells grown with pH control . Moreover, addition of glucose to quasi-steady-state biofilms resulted in increased expression of the gtfBC-cat fusion, although the magnitude of the induction was less than that seen with sucrose . The effect of pH on ftf expression was negligible . A modest and transient induction of ftf was observed in biofilms pulsed with excess glucose and the kinetics and level of induction of ftf by excess carbohydrate were dependent on the pH of the biofilms . This study demonstrates that the type and amount of carbohydrate and the environmental pH have a major influence on transcription of the gtfBC and ftf genes when the organisms are growing in biofilms, and provides evidence for previously undisclosed regulatory circuits for exopolysaccharide gene expression in S . mutans. J Microbiol Methods, 2001 Nov, 47(2), 169 - 80 Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images; Xavier JB et al.; The determination of volumes and interface areas from confocal laser scanning microscopy (CLSM) images requires the identification of component objects by segmentation . An automated method for the determination of segmentation thresholds for CLSM imaging of biofilms was developed . The procedure, named objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold selection (RATS) method . OTS is based on the statistical properties of local gray-values and gradients in the image . By characterizing the dependence between a volumetric feature and the intensity threshold used for image segmentation, the former can be determined with an arbitrary confidence level, with no need for user intervention . The identification of an objective segmentation procedure renders the possibility for the full automation of volume and interfacial area measurement . Images from two distinct biofilm systems, acquired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area . The reliability of measurements for each case was analyzed to identify optimal procedure for image acquisition . The automated OTS method was shown to reproduce values obtained manually by an experienced operator. J Appl Microbiol, 2001 Oct, 91(4), 725 - 34 Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions; Kalmokoff ML et al.; AIMS: To determine whether isolates of Listeria monocytogenes differ in their ability to adsorb and form biofilms on a food-grade stainless steel surface . METHODS AND RESULTS: Strains were assessed for their ability to adsorb to a test surface over a short time period . Although some differences in numbers of bound cells were found among the strains, there were no correlations between the degree of adsorption and either the serotype or source of the strain . The ability of each strain to form a biofilm when grown with the test surface was also assessed . With the exception of a single strain, all strains adhered as single cells and did not form biofilms . Significant differences in adherence levels were found among strains . Strains demonstrating enhanced attachment produced extracellular fibrils, whereas those which adhered poorly did not . A single strain formed a biofilm consisting of adhered single cells and aggregates of cells . CONCLUSIONS: Significant differences were found in the ability of various L . monocytogenes strains to attach to a test surface . In monoculture, the majority of strains did not form biofilms . SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in attachment and biofilm formation among strains provide a basis to study these characteristics in L . monocytogenes. J Appl Microbiol, 2001 Oct, 91(4), 646 - 51 Biofilms and microbially influenced cuprosolvency in domestic copper plumbing systems; Critchley MM et al.; AIMS: To survey biofilm accumulation within domestic copper plumbing pipes in South Australian drinking water distribution systems and examine its role in copper solvation (cuprosolvency) . METHODS AND RESULTS: Cold water copper pipes were sampled from two different plumbing systems receiving filtered and unfiltered potable water respectively . Biomass was quantified by total organic carbon measurements and viable cell counts and microbial activity by respirometry . Biofilm accumulation was related to water chemistry within the systems, particularly nutrients, alkalinity and conductivity, as well as water turbulence . Laboratory coupon experiments were used to determine the effect of extracted biofilm on copper solvation . Biofilms were shown to be capable of both increasing and decreasing aqueous copper concentrations in comparison to sterile controls . CONCLUSIONS: The results suggest that water quality may influence the accumulation of biofilms in copper plumbing systems, as well as potential cuprosolvency activity . SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of biofilms in copper plumbing systems and their ability to influence aqueous copper concentrations has implications for both public health and the management of distribution systems. Water Sci Technol, 2001, 44(4), 197 - 204 Influence of porosity and composition of supports on the methanogenic biofilm characteristics developed in a fixed bed anaerobic reactor; Picanco AP et al.; This paper reports on the influence of the material porosity on the anaerobic biomass adhesion on four different inert matrices: polyurethane foam, PVC, refractory brick and special ceramic . The biofilm development was performed in a fixed-bed anaerobic reactor containing all the support materials and fed with a synthetic wastewater containing protein, lipids and carbohydrates . The data obtained from microscopic analysis and kinetic assays indicated that the material porosity has a crucial importance in the retention of the anaerobic biomass . The polyurethane foam particles and the special ceramic were found to present better retentive properties than the PVC and the refractory brick . The large specific surface area, directly related to material porosity, is fundamental to provide a large amount of attached biomass . However, different supports can provide specific conditions for the adherence of distinct microorganism types . The microbiological exams revealed a distinction in the support colonization . A predominance of methanogenic archaeas resembling Methanosaeta was observed both in the refractory brick and the special ceramic . Methanosarcina-like microorganisms were predominant in the PVC and the polyurethane foam matrices. J Dairy Sci, 2001 Sep, 84(9), 1926 - 36 Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment; Somers EB et al.; Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB) . The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied . Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001) . During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers . Biofilms of L . curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese . The starter culture also survived the cleaning process . Additionally, L . curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening . On the other hand, biofilms of L . fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening . Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese . Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11621 - 6 Fruiting body formation by Bacillus subtilis; Branda SS et al.; Spore formation by the bacterium Bacillus subtilis has long been studied as a model for cellular differentiation, but predominantly as a single cell . When analyzed within the context of highly structured, surface-associated communities (biofilms), spore formation was discovered to have heretofore unsuspected spatial organization . Initially, motile cells differentiated into aligned chains of attached cells that eventually produced aerial structures, or fruiting bodies, that served as preferential sites for sporulation . Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for exopolysaccharide and surfactin production . The formation of aerial structures was robust in natural isolates but not in laboratory strains, an indication that multicellularity has been lost during domestication of B . subtilis . Other microbial differentiation processes long thought to involve only single cells could display the spatial organization characteristic of multicellular organisms when studied with recent natural isolates. Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11897 - 902 Epub 2001 Sep 25. Pb(II) distributions at biofilm-metal oxide interfaces; Templeton AS et al.; The distribution of aqueous Pb(II) sorbed at the interface between Burkholderia cepacia biofilms and hematite (alpha-Fe(2)O(3)) or corundum (alpha-Al(2)O(3)) surfaces has been probed by using an application of the long-period x-ray standing wave technique . Attached bacteria and adsorbed organic matter may interfere with sorption processes on metal oxide surfaces by changing the characteristics of the electrical double layer at the solid-solution interface, blocking surface sites, or providing a variety of new sites for metal binding . In this work, Pb L(alpha) fluorescence yield profiles for samples equilibrated with 10(-7) to 10(-3.8) M Pb(II) were measured and modeled to determine quantitatively the partitioning of Pb(II) at the biofilm-metal oxide interface . Our data show that the reactive sites on the metal oxide surfaces were not passivated by the formation of a monolayer biofilm . Instead, high-energy surface sites on the metal oxides form the dominant sink for Pb(II) at submicromolar concentrations, following the trend alpha-Fe(2)O(3) (0001) > alpha-Al(2)O(3) (1102) > alpha-Al(2)O(3) (0001), despite the greater site density within the overlying biofilms . At {Pb} > 10(-6) M, significant Pb uptake by the biofilms was observed. Appl Environ Microbiol, 2001 Oct, 67(10), 4717 - 25 Transmission to eels, portals of entry, and putative reservoirs of Vibrio vulnificus serovar E (biotype 2); Marco-Noales E et al.; Vibrio vulnificus serovar E (formerly biotype 2) is the etiologic agent that is responsible for the main infectious disease affecting farmed eels . Although the pathogen can theoretically use water as a vehicle for disease transmission, it has not been isolated from tank water during epizootics to date . In this work, the mode of transmission of the disease to healthy eels, the portals of entry of the pathogen into fish, and their putative reservoirs have been investigated by means of laboratory and field experiments . Results of the experiments of direct and indirect host-to-host transmission, patch contact challenges, and oral-anal intubations suggest that water is the prime vehicle for disease transmission and that gills are the main portals of entry into the eel body . The pathogen mixed with food can also come into the fish through the gastrointestinal tract and develop the disease . These conclusions were supported by field data obtained during a natural outbreak in which we were able to isolate this microorganism from tank water for the first time . The examination of some survivors from experimental infections by indirect immunofluorescence and scanning electron microscopy showed that V . vulnificus serovar E formed a biofilm-like structure on the eel skin surface . In vitro assays demonstrated that the ability of the pathogen to colonize both hydrophilic and hydrophobic surfaces was inhibited by glucose . The capacity to form biofilms on eel surface could constitute a strategy for surviving between epizootics or outbreaks, and coated survivors could act as reservoirs for the disease. Huan Jing Ke Xue, 2001 Jul, 22(4), 86 - 90 {Effect of anaerobic acidification treatment on nitrogen removal and toxicity reduction of coke plant wastewater}; Li Y et al.; Coke plant wastewater was treated by anaerobic acidification-anoxic-aerobic (A1-A2-O) biofilm process and anoxic-aerobic (A/O) biofilm process respectively . Toxicity reduction effects of the two processes were compared . The toxicity of coke plant wastewater was equal to that of mercury chloride with concentration of 0.19 mg/L . Its toxicity reduction was related to the removal of organic nitrogen . Anaerobic acidification unit acted an important role on removal of both organic nitrogen and reduction of toxicity of the wastewater . After treatment by A1-A2-O process, its toxicity can be decreased greatly . When HRT was 37.9 hours, The relative luminosity of the treated effluent to photobacterium can attain 96.8%, that is equal to the toxicity of mercury chloride with concentration of 0.023 mg/L. J Hosp Infect, 2001 Oct, 49(2), 117 - 21 Non-touch fittings in hospitals: a possible source of Pseudomonas aeruginosa and Legionella spp; Halabi M et al.; Non-touch fittings are gradually becoming very common in the bathrooms and toilets of public facilities and restaurants . Hospitals and other healthcare facilities have recently started to install these types of water taps to lower water consumption, thus saving costs, and to prevent healthcare workers from touching the tap, thus promoting hygiene . This study analysed the bacteriological water quality of 38 non-touch water taps in different settings in a 450-bed secondary-care hospital in Upper Austria . Two different tap types were installed: 23 taps were without temperature selection and 15 were with temperature selection (cold and warm) . A membrane filtration method was used, and the authors screened for both indicator organisms and Pseudomonas aeruginosa in 100 ml water samples . In 10 non-touch taps without temperature selection, the authors also screened for Legionella spp . in 500 ml water samples . Seventy four percent of the taps without temperature selection and 7% of the taps with temperature selection showed contamination with P . aeruginosa (P<0.001) . None of the taps showed contamination with indicator organisms . Detailed analysis of the source of contamination revealed that the magnetic valve and the outlet itself were heavily contaminated, whereas the junction from the central pipe system was free of contamination.All 10 analysed taps showed contamination with Legionella spp . It was concluded that the local contamination of non-touch fittings is a result of the low amount of water that flows through the outlet, the low water pressure and the column of water, which is 'still-standing' and has a temperature of about 35 degrees C, thus providing nearly ideal growth conditions for P . aeruginosa . Additionally, the presence of materials such as rubber, PVC, etc . in the fittings enhances the adhesion of P . aeruginosa and thus the production of biofilms . In conclusion, the authors wish to encourage infection control teams to evaluate the use of non-touch fittings in hospitals, especially when they are installed in risk areas . J Bacteriol, 2001 Oct, 183(20), 6074 - 84 Genetic and physiologic analysis of the groE operon and role of the HrcA repressor in stress gene regulation and acid tolerance in Streptococcus mutans; Lemos JA et al.; Our working hypothesis is that the major molecular chaperones DnaK and GroE play central roles in the ability of oral bacteria to cope with the rapid and frequent stresses encountered in oral biofilms, such as acidification and nutrient limitation . Previously, our laboratory partially characterized the dnaK operon of Streptococcus mutans (hrcA-grpE-dnaK) and demonstrated that dnaK is up-regulated in response to acid shock and sustained acidification (G . C . Jayaraman, J . E . Penders, and R . A . Burne, Mol . Microbiol . 25:329-341, 1997) . Here, we show that the groESL genes of S . mutans constitute an operon that is expressed from a stress-inducible sigma(A)-type promoter located immediately upstream of a CIRCE element . GroEL protein and mRNA levels were elevated in cells exposed to a variety of stresses, including acid shock . A nonpolar insertion into hrcA was created and used to demonstrate that HrcA negatively regulates the expression of the groEL and dnaK operons . The SM11 mutant, which had constitutively high levels of GroESL and roughly 50% of the DnaK protein found in the wild-type strain, was more sensitive to acid killing and could not lower the pH as effectively as the parent . The acid-sensitive phenotype of SM11 was, at least in part, attributable to lower F(1)F(0)-ATPase activity . A minimum of 10 proteins, in addition to GroES-EL, were found to be up-regulated in SM11 . The data clearly indicate that HrcA plays a key role in the regulation of chaperone expression in S . mutans and that changes in the levels of the chaperones profoundly influence acid tolerance. J Bacteriol, 2001 Oct, 183(20), 5927 - 36 Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase; Bhattacharjee MK et al.; Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces . The bacteria produce long fibrils of bundled pili, which are required for adherence . Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence . We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili . The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins . In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes . Here we report studies on the function of TadA . The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus . The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A . actinomycetemcomitans tadA mutants . Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A . actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane . TadA-T7 was purified and found to hydrolyze ATP in vitro . The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration . TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases . Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding . Both mutants were found to be defective in their ability to complement tadA mutants . We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A . actinomycetemcomitans. J Bacteriol, 2001 Oct, 183(20), 5848 - 54 Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation; Mireles JR 2nd et al.; Swarming motility plays an important role in surface colonization by several flagellated bacteria . Swarmer cells are specially adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement of slime . The external slime provides the milieu for motility and likely harbors swarming signals . We recently reported the isolation of swarming-defective transposon mutants of Salmonella enterica serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis . Here, we have examined the biofilm-forming abilities of the swarming mutants using a microtiter plate assay . A whole spectrum of efficiencies were observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation . Since we have postulated that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm formation . We report that surfactin inhibits biofilm formation of wild-type S . enterica grown either in polyvinyl chloride microtiter wells or in urethral catheters . Other bio- and chemical surfactants tested had similar effects. J Microbiol Methods, 2001 Oct, 47(1), 1 - 10 Growing reproducible biofilms with respect to structure and viable cell counts; Jackson G et al.; We have developed a new method of growing 4-day-old biofilms that are reproducible, with respect to viable cell number and biofilm structure . To demonstrate the utility of the method, we grew biofilms composed of Pseudomonas aeruginosa (ATCC#700829), P . fluorescens (ATCC#700830) and Klebsiella pneumoniae (ATCC#700831), 18 times in flat-plate reactors under well-defined conditions of: flow rate, nutrient concentration, temperature, inoculum and growth rate . The resulting 4-day-old biofilms were approximately 200-300 microm thick and exhibited a high degree of reproducibility . The number of viable cells that accumulated per unit surface area and the biofilm areal porosity were reproduced within 10% error . We have also quantified other parameters characterizing biofilm structure using biofilm-imaging techniques: fractal dimension, textural entropy and diffusion distance as auxiliary parameters characterizing the reproducibility of biofilm accumulation . As a result of analysis, we have introduced a new parameter to better quantify and characterize the number of viable cells in biofilms, "specific number of viable cells" (SNVC) . This parameter is the viable cell number normalized with respect to the surface area covered by the biofilm and with respect to the biomass of the biofilm . This new descriptor represents the dynamics of biofilm accumulation better than the traditionally used colony-forming unit (CFU) per surface area covered by the biofilm because it accounts not only for the surface coverage but also for the biofilm thickness. Clin Infect Dis, 2001 Oct 15, 33(8), 1387 - 92 Epub 2001 Sep 20. Biofilm formation: a clinically relevant microbiological process; Donlan RM; Microorganisms universally attach to surfaces and produce extracellular polysaccharides, resulting in the formation of a biofilm . Biofilms pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices . An appreciation of the role of biofilms in infection should enhance the clinical decision-making process. J Food Prot, 2001 Sep, 64(9), 1369 - 76 Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp; Bremer PJ et al.; Contaminated surfaces of food processing equipment are believed to be a significant source of Listeria monocytogenes to foods . However, very little is known about the survival of Listeria in processing environments . In a mixed bacterial biofilm of L . monocytogenes and Flavobacterium spp., the number of L . monocytogenes cells attaching to stainless steel increased significantly compared to when L . monocytogenes was in a pure culture . The L . monocytogenes cells in the mixed biofilms were also recoverable for significantly longer exposure periods . On colonized coupons held at 15 degrees C and 75% humidity, decimal reduction times were 1.2 and 18.7 days for L . monocytogenes in pure and mixed biofilms, respectively . With increasing exposure time, the proportion of cells that were sublethally injured (defined as an inability to grow on selective agar) increased from 8.1% of the recoverable cell population at day 0 to 91.4% after 40 days' exposure . At 4 and -20 degrees C, decimal reduction times for L . monocytogenes in pure culture were 2.8 and 1.4 days, respectively, and in mixed culture, 10.5 and 14.4 days, respectively . The enhanced colonization and survival of L . monocytogenes on "unclean" surfaces increase the persistence of this pathogen in food processing environments, while the increase in the percentage of sublethally injured cells in the population with time may decrease the ability of enrichment regimes to detect it. J Orthop Res, 2001 Sep, 19(5), 820 - 6 A simple infection model using pre-colonized implants to reproduce rat chronic Staphylococcus aureus osteomyelitis and study antibiotic treatment; Monzon M et al.; Staphylococcus aureus biofilms formed on medical implants represent a serious problem, being difficult to eradicate with antibiotic therapy and leading to chronic infections . Simplified in vivo and in vitro antibiotic susceptibility assays using biofilm bacteria are needed . In this work, a novel chronic osteomyelitis infection model was developed in rats in the absence of bacterial suspension, requiring the use of only 10(6) bacteria in biofilms at the site of surgery, with a full success in reproducing infection . Stainless-steel implants pre-colonized for 12 h with a highly adherent S . aureaus isolate were introduced into the rat tibiae . In animals not submitted to antibiotic treatment, infection was found in the implants and spread to bone in all cases, indicating the high efficacy of the model to reproduce osteomyelitis . The effect of a 21-day treatment with cefuroxime, vancomycin, tobramycin or ciprofloxacin on infection was studied in this model 42 days after surgery . Bone colonization was inhibited by vancomycin and cefuroxime . Cefuroxime (the most efficient antibiotic, able to sterilize 1 out of 8 implants) reduced the number of bacteria in biofilms adhered to implants at a higher extent than vancomycin, trobramycin and ciprofloxacin . Analogous observations were made in this work in vivo and in vitro on the relative antibiotic efficacy against S . aureus biofilm bacteria . suggesting the usefulness of both tests as a potential tool to study antibiotic suceptibility, and the need for new antimicrobials against these bacteria. Eur J Clin Microbiol Infect Dis, 2001 Jul, 20(7), 502 - 4 Influence of dynamic conditions on biofilm formation by staphylococci; Stepanovic S et al.; The modified microtiter plate test was used to investigate biofilm formation by staphylococci under both static and dynamic conditions . The quantity of biofilm produced under static conditions was used as a reference . Dynamic conditions, which were achieved by incubating microtiter plates on a horizontal shaker with and without the presence of glass beads in wells, either reduced biofilm formation or left it unchanged . Dynamic conditions particularly affected the capacity of certain species to produce biofilm: these species included the causative agents of infections associated with a foreign body (Staphylococcus epidermidis, Staphylococcus aureus) . On the basis of these results, dynamic conditions should be included as a parameter for evaluating biofilm formation by staphylococci in vitro. Am J Trop Med Hyg, 2001 Sep, 65(3), 177 - 9 A cluster of melioidosis cases from an endemic region is clonal and is linked to the water supply using molecular typing of Burkholderia pseudomallei isolates; Currie BJ et al.; Nine cases of melioidosis with four deaths occurred over a 28-month period in members of a small remote Aboriginal community in the top end of the Northern Territory of Australia . Typing by pulsed-field gel electrophoresis showed isolates of Burkholderia pseudomallei from six of the cases to be clonal and also identical to an isolate from the community water supply, but not to soil isolates . The clonality of the isolates found in this cluster contrasts with the marked genetic diversity of human and environmental isolates found in this region which is hyperendemic for B . pseudomallei . It is possible that the clonal bacteria persisted and were propagated in biofilm in the water supply system . While the exact mode of transmission to humans and the reasons for cessation of the outbreak remain uncertain, contamination of the unchlorinated community water supply is a likely explanation. Lett Appl Microbiol, 2001 Oct, 33(4), 320 - 4 The differential adherence capabilities of two Listeria monocytogenes strains in monoculture and multispecies biofilms as a function of temperature; Norwood DE et al.; AIMS: To determine the differential adherence capabilities at three different temperatures of Listeria monocytogenes Scott A, a clinical food pathogen, and L . monocytogenes FM876, a persistent strain from a milk-processing environment, to stainless steel . METHODS AND RESULTS: Differential adherence was investigated by submerging stainless steel coupons in both 48-h Listeria monocultures and mixed cultures additionally containing Staphylococcus xylosus DP5H and Pseudomonas fragi ATCC 4973 . Immunofluorescent microscopy and image analysis techniques were utilized to identify and quantify the L . monocytogenes cells adhering to the steel at 4 degrees C, 18 degrees C and 30 degrees C . The monoculture biofilms consistently contained greater L . monocytogenes numbers than the multispecies biofilms, with the persistent strain FM876 showing significantly greater adherence than strain Scott A . Optimum adherence occurred at 18 degrees C in monoculture biofilms . CONCLUSION: L . monocytogenes strains exhibit differential, temperature-dependent, adherence to stainless steel . SIGNIFICANCE AND IMPACT OF THE STUDY: These results demo