High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Cytoprotective and genoprotective effects of taxifolin against oxidative damage in HTR-8/SVneo human trophoblast cells.

An increase of reactive oxygen species in the placenta and oxidative disbalance has been recognized as a significant factor contributing to pregnancy complications. Dietary intake of food rich in antioxidants during pregnancy could exert a protective role in the prevention of adverse outcomes such as preeclampsia, miscarriage, and others. Flavonoid taxifolin has shown numerous health-promoting effects in a large number of studies conducted on animals, as well as various human cell types in vitro. However, its effects on human placental cells-trophoblasts-have yet to be determined. Therefore, cytoprotective and genoprotective effects of taxifolin on trophoblast cell line HTR-8/SVneo under induced oxidative stress were explored in this study. Cytotoxicity of a range of taxifolin concentrations (1-150 µM) was evaluated using the MTT and crystal violet assays. A model of oxidative stress was achieved by exposing HTR-8/SVneo cells to H2O2. To determine cytoprotective and antigenotoxic effects, the cells were pre-incubated with three concentrations of taxifolin (10, 50, and 100 µM) and then exposed to H2O2. Taxifolin in concentrations of 1, 5, 10, 25, 50, and 100 µM showed no cytotoxic effects on HTR-8/SVneo cells, but 150 µM of taxifolin caused a significant decrease in adherent cell number, as detected by crystal violet assay. Pretreatment with the chosen concentrations of taxifolin showed a significant cytoprotective effect on H2O2-induced cytotoxicity, as determined by the MTT assay. Furthermore, taxifolin showed a significant reduction in H2O2-induced DNA damage, measured by comet assay. This study showed protective effects of taxifolin on human trophoblast cells exposed to oxidative damage. Further studies are needed to explore the underlying mechanisms.

Inhibition of proliferation, migration, and adhesion of skin fibroblasts by enzymatic poly(gallic acid) grafted with L-Arginine, migration, and adhesion of skin fibroblasts by enzymatic poly(gallic acid) grafted with L-Arginine.

Psoriasis and atopic dermatitis (AD) are characterized by enhanced skin inflammation, which results in hyperproliferation and the recruitment of immune cells into the skin. For that reason, it is needed a chemical capable to reduce cell proliferation and the recruitment of cells. The search for new molecules for therapeutic skin treatment mainly focuses on the antioxidant and anti-inflammatory properties, highlighting the rheological properties of polymeric polypeptides. We studied L-arginine (L-Arg) grafted (-g-) to enzymatic poly(gallic acid) (PGAL). The latter is a multiradical antioxidant with greater properties and thermal stability. The derivative was enzymatically polymerized in an innocuous procedure. The poly(gallic acid)-g-L-Arg molecule (PGAL-g-L-Arg) inhibits bacterial strains which also have been involved in the progression of psoriasis and AD. However, it is important to analyze their biological effect on skin cells. The cell viability was analyzed by calcein/ethidium homodimer assays and crystal violet. The proliferation and cell attachment were determined by a curve of time and quantitation of the optical density of crystal violet. To analyze the cell migration a wound-healing assay was performed. This synthesis demonstrates that it is not cytotoxic at high concentrations (250 µg/mL). We observed a decrease in the proliferation, migration, and adhesion of dermal fibroblasts in vitro but the compound could not avoid the increase of reactive oxygen species in the cell. Based on our findings, PGAL-g-L-Arg is a promising candidate for treating skin diseases such as psoriasis and AD where decreasing the proliferation and cell migration could help to avoid inflammation.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Virulence and biofilm inhibition of 3-methoxycinnamic acid against Agrobacterium tumefaciens.

AIMS: In the current study the anti-virulence and anti-biofilm activities of the cinnamic acid derivative, 3-methoxycinnamic acid, was investigated against Agrobacterium tumefaciens. METHODS AND RESULTS: Based on the disc diffusion test and ß-galactosidase activity assay, 3-methoxycinnamic acid was shown to interfere with the quorum sensing (QS) system of A. tumefaciens. Crystal violet staining assay, phenol-sulfuric acid method, Bradford protein assay and confocal laser scanning microscopy (CLSM) revealed that the biofilm formation of A. tumefaciens was inhibited after the treatment of 3-methoxycinnamic acid. Employing high-performance liquid chromatography (HPLC) analysis of culture supernatant revealed that the production of 3-oxo-octanoylhomoserine lactone (3-oxo-C8-HSL) decreased concentration-dependently after treatment with 3-methoxycinnamic acid. Swimming and chemotaxis assays also indicated that 3-methoxycinnamic acid had a good effect on reducing the motility and chemotaxis of A. tumefaciens. In addition, the RT-qPCR, molecular docking and simulations further demonstrated that 3-methoxycinnamic acid could competitively inhibit the binding of 3-oxo-C8-HSL to TraR and down-regulate virulence-related genes. CONCLUSIONS: 3-Methoxycinnamic acid is proved to have good anti-virulence and anti-biofilm activities against A. tumefaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that investigates the anti-virulence and anti-biofilm activities of 3-methoxycinnamic acid against A. tumefaciens. With its potential QS-related virulence and biofilm inhibitory activities, 3-methoxycinnamic acid is expected to be developed as a potent pesticide or adjuvant for the prevention and treatment of crown gall caused by A. tumefaciens.

Antibiofilm mechanism of a novel milk-derived antimicrobial peptide against Staphylococcus aureus by downregulating agr quorum sensing system.

AIMS: Staphylococcus aureus has emerged as a serious threat to food safety owing to biofilm formation. The study aimed to examine the antibiofilm mechanism of a novel milk-derived antimicrobial peptide BCp12 against it. METHODS AND RESULTS: Antibiofilm activity of BCp12 was studied by crystal violet staining, MTT assay, motility, SEM and CLSM. TMT proteome, real-time PCR and molecular docking in silico were conducted to evaluate the mechanism of BCp12 against S. aureus biofilm. The results showed that BCp12 had significant antibiofilm activity at 1 × MIC and sub-MIC. BCp12 induced the dispersion of structure of S. aureus biofilm BCp12 inhibited the movement of S. aureus. A total of 703 proteins were downregulated and 334 proteins were upregulated after BCp12 treatment. The proteins (agrA, agrB, agrC and psmß) of the QS systems were downregulated. Additionally, the expression of the agr-related genes, agrA, agrB, agrC and psmß, was downregulated. BCp12 was bound to the receptor proteins agrA and agrC through hydrogen bonds and π-π bonds. CONCLUSIONS: The results showed the antibiofilm activity of BCp12 and it inhibits the biofilm formation by interfering agr QS system. SIGNIFICANCE AND IMPACT OF STUDY: BCp12 has the potential to be a novel antibiofilm agent against S. aureus biofilm and used in the food industry.

Asian Sand Dust Particles Enhance the Development of Aspergillus fumigatus Biofilm on Nasal Epithelial Cells.

BACKGROUND: Asian sand dust (ASD) and Aspergillus fumigatus are known risk factors for airway mucosal inflammatory diseases. Bacterial and fungal biofilms commonly coexist in chronic rhinosinusitis and fungus balls. We evaluated the effects of ASD on the development of A. fumigatus biofilm formation on nasal epithelial cells. METHODS: Primary nasal epithelial cells were cultured with A. fumigatus conidia with or without ASD for 72 h. The production of interleukin (IL)-6, IL-8, and transforming growth factor (TGF)-ß1 from nasal epithelial cells was determined by the enzyme-linked immunosorbent assay. The effects of ASD on A. fumigatus biofilm formation were determined using crystal violet, concanavalin A, safranin staining, and confocal scanning laser microscopy. RESULTS: ASD and A. fumigatus significantly enhanced the production of IL-6 and IL-8 from nasal epithelial cells. By coculturing A. fumigatus with ASD, the dry weight and safranin staining of the fungal biofilms significantly increased in a time-dependent manner. However, the increased level of crystal violet and concanavalin A stain decreased after 72 h of incubation. CONCLUSIONS: ASD and A. fumigatus induced the production of inflammatory chemical mediators from nasal epithelial cells. The exposure of A. fumigatus to ASD enhanced the formation of biofilms. The coexistence of ASD and A. fumigatus may increase the development of fungal biofilms and fungal inflammatory diseases in the sinonasal mucosa.

Efficacy of linked colour imaging in magnifying chromoendoscopy with crystal violet staining: a pilot study.

PURPOSE: Diagnosis of the depth of invasion is crucial in the endoscopic management of early colorectal cancer. Image-enhanced endoscopy (IEE) is a method for easily evaluating the depth of invasion. Linked colour imaging (LCI) is an IEE method that enables clearer identification of neoplastic lesions and mucosal inflammation. The aim of this experimental study was to explore the efficacy of LCI in vessel and pit pattern recognition when used in magnifying chromoendoscopy with crystal violet staining for superficial colorectal neoplasms. METHODS: This was an experimental study. Colour difference (CD) values between the surrounding mucosa and vessels and pits were measured on white light (WLI), blue laser (BLI), and LCI images. The CD values of 10 neoplastic lesions were calculated and compared between WLI and the other techniques. RESULTS: The CD value was 9.8 (interquartile range, 7.3-12.4) for WLI, 9.7 (6.7-13.4) for LCI, and 6.8 (5.1-9.3) for BLI. The CD value was statistically different between WLI and BLI but not between WLI and LCI. With regard to vessel description, the CD value was 7.5 (4.0-11.0) for WLI, 15.6 (11.6-23.9) for LCI, and 23.3 (15.8-30.4) for BLI. CONCLUSIONS: LCI provides more diagnostic information than other light modes. Further, it is superior to the other techniques in terms of vessel visibility and is comparable to them in terms of pit recognition. These unique features of LCI may lead to its use as an alternative to WLI and BLI for pit and vessel pattern evaluation in the future.

Synthesis and DNA binding profile of monomeric, dimeric, and trimeric derivatives of crystal violet.

Monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a-1f) have been synthesized for the purpose of evaluating their affinity and sequence selectivity for duplex DNA. Competitive ethidum displacement assays indicate that 1a-1f have apparent association constants for CT DNA in the range of 1.80-16.2 × 107 M-1 and binding site sizes of 10-14 bp. Viscosity experiments performed on ligand 1f confirmed that these dyes associate with duplex DNA by a non-intercalative mode of binding. Circular dichroism and competition binding studies of the tightest binding ligand 1e with known major and minor groove binding molecules suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers, suggesting a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove.

Biosorption of crystal violet dye using inactive biomass of the fungus Diaporthe schini.

An inactive biomass of a new fungus recently discovered, Diaporthe schini, was evaluated for the biosorption of crystal violet (CV) in simulated textile effluents. The characterization assays were performed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and N2 adsorption/desorption isotherms. The influences of pH and biosorbent dosage on the biosorption capacity were evaluated. Kinetics, equilibrium and thermodynamic studies were also carried out. Characterization techniques showed an amorphous biosorbent, with a rough surface containing irregular particles and surface area of 6.5 m2 g-1. The most adequate values of pH and biosorbent dosage were 7.5 and 0.4 g L-1, respectively. The Elovich kinetic model and the Sips equilibrium model were suitable to fit the experimental data. The biosorption capacity increased with temperature, reaching a maximum biosorption capacity of 642.3 mg g-1 at 328 K. The biosorption was a spontaneous and endothermic process. Diaporthe schini inactive biomass was an interesting biosorbent to treat colored effluents, presenting efficiency of 87% in the decolorization of a simulated dye house effluent.

Clinical impact of Candida spp. biofilm production in a cohort of patients with candidemia.

Persistent candidemia refers to the continued isolation of the same Candida species in the blood of a candidemic patient despite appropriate therapy. Despite the clinical importance of persistent candidemia, studies have superficially addressed the biological conditions behind this phenomenon. The aim of this study was to evaluate the correlation between the biofilm-forming ability by Candida bloodstream isolates and the persistence of infection. A total of 55 isolates of Candida were tested and characterized in two groups: (i) group I, which included seven patients with persistent candidemia, and (ii) group II, which included 18 patients with nonpersistent candidemia. Microorganisms were identified at the species level by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Biofilm quantification was evaluated by the crystal violet staining method and confocal scanning laser microscopy (CSLM). Molecular tests confirmed the identification of Candida albicans (92% group I and 94% group II) and Candida dubliniensis isolates (8% group I and 6% group II). All 55 isolates were able to form biofilms, but a higher biofilm mass was produced by C. albicans/C. dubliniensis strains cultured from the persistent group (P < .05). Our data suggest that Candida sp. biofilm production should be considered a relevant biologic variable in explaining patients who fail to clear a bloodstream infection despite adequate antifungal treatment with triazoles.

Planktonic growth and biofilm formation profiles in Candida haemulonii species complex.

Candida haemulonii species complex have emerged as multidrug-resistant yeasts able to cause fungemia worldwide. However, very little is known regarding their physiology and virulence factors. In this context, planktonic growth and biofilm formation of Brazilian clinical isolates of Candida haemulonii (n = 5), Candida duobushaemulonii (n = 4), and Candida haemulonii var. vulnera (n = 3) were reported. Overall, the fungal planktonic growth curves in Sabouraud dextrose broth reached the exponential phase in 48 h at 37°C. All the clinical isolates formed biofilm on polystyrene in a time-dependent event, as judged by the parameters evaluated: biomass (crystal violet staining), metabolic activity (XTT reduction), and extracellular matrix (safranin incorporation). No statistically significant differences were observed when the average measurements among the three Candida species were compared regarding both planktonic and biofilm lifestyles; however, typical isolate-specific differences were clearly noticed in fungal growth kinetics.

Fabrication of hybrid biosorbent nanoscale zero-valent iron-Sargassum swartzii biocomposite for the removal of crystal violet from aqueous solution.

A novel nanoscale zero-valent iron-Sargassum swartzii (nZVI-SS) biocomposite was synthesized and evaluated for its ability to adsorb crystal violet (CV) from aqueous solutions. Involvement of various functional groups of the biosorbent in preferential adsorption of cationic dye was observed using Fourier transform infrared (FTIR) spectroscopy. Morphological changes occurring on the biocomposite materials were characterized using scanning electron microscopy (SEM). Significant increase (∼90%) in the biosorption of cationic dye was observed with gradual increase in pH of the medium from 3 to 12. The effect of biosorbent concentration, initial pH, temperature, agitation rate, adsorption time, and initial dye concentration was studied for the biosorption of CV using nZVI biocomposite. During the optimization study, maximum biosorption capacity was observed at pH of 8. At various initial CV concentrations (20-100 mg/L), attainment of batch sorption equilibrium was observed within 120 min of reaction time. The Langmuir isotherm model expressed high coefficient of determination (R2 = 0.999). The maximum dye uptake of 200 mg/g was reported at pH 8. Kinetics and temperature profiles were evaluated and reported. Desorption study was carried out with 0.1 M HCl. Investigations proved that nZVI-SS is an excellent biosorbent for the sequestration of CV in aqueous media.

Exposure to Crystal Violet, Its Toxic, Genotoxic and Carcinogenic Effects on Environment and Its Degradation and Detoxification for Environmental Safety.

Crystal Violet (CV), a triphenylmethane dye, has been extensively used in human and veterinary medicine as a biological stain, as a textile dye in textile processing industries and also used to provide a deep violet color to paints and printing ink. CV is also used as a mutagenic and bacteriostatic agent in medical solutions and antimicrobial agent to prevent the fungal growth in poultry feed. Inspite of its many uses, CV has been reported as a recalcitrant dye molecule that persists in environment for a long period and pose toxic effects in environment. It acts as a mitotic poison, potent carcinogen and a potent clastogene promoting tumor growth in some species of fish. Thus, CV is regarded as a biohazard substance. Although, there are several physico-chemical methods such as adsorption, coagulation and ion-pair extraction reported for the removal of CV, but these methods are insufficient for the complete removal of CV from industrial wastewaters and also produce large quantity of sludge containing secondary pollutants. However, biological methods are regarded as cost-effective and eco-friendly for the treatment of industrial wastewaters, but these methods also have certain limitations. Therefore, there is an urgent need to develop such eco-friendly and cost-effective biological treatment methods, which can effectively remove the dye from industrial wastewaters for the safety of environment, as well as human and animal health.

Microwave treated Salvadora oleoides as an eco-friendly biosorbent for the removal of toxic methyl violet dye from aqueous solution--A green approach.

In the present study, microwave treated Salvadora oleoides (MW-SO) has been investigated as a potential biosorbent for the removal of toxic methyl violet dye. A batch adsorption method was experimented for biosorptive removal of toxic methyl violet dye from the aqueous solution. The effect of various operating variables, viz., adsorbent dosage, pH, contact time and temperature on the removal of the dye was studied and it was found that nearly 99% removal of the dye was possible under optimum conditions. Kinetic study revealed that a pseudo-second-order mechanism was predominant and the overall process of the dye adsorption involved more than one step. Hence, in order to investigate the rate determining step, intra-particle diffusion model was applied. Adsorption equilibrium study was made by analyzing Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) adsorption isotherm models and the biosorption data was found to be best represented by the Langmuir model. The biosorption efficiency of MW-SO was also compared with unmodified material, Salvadora oleoides (SO). It was found that the sorption capacity (qmax) increased from 58.5 mg/g to 219.7 mg/g on MW treatment. Determination of thermodynamic parameters such as free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) confirmed the spontaneous, endothermic and feasible nature of the adsorption process. The preparation of MW-SO did not require any additional chemical treatment and a high percentage removal of methyl violet dye was obtained in much lesser time. Thus, it is in agreement with the principles of green chemistry. The results of the present research work suggest that MW-SO can be used as an environmentally friendly and economical alternative biosorbent for the removal of methyl violet dye from aqueous solutions.

Purification and characterization of extracellular laccase produced by Ceriporiopsis subvermispora and decolorization of triphenylmethane dyes.

Laccases of white-rot fungi provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. The aim of this study was production, characterization, and application of laccases from the white-rot fungus Ceriporiopsis subvermispora ATCC 90467 for decolorization of triphenylmethane dyes that could remain persistent in wastewater. Laccase was purified from a C. subvermispora culture by a four-step method resulting high specific activity of 2,571 U g-1 , 88-fold higher than crude laccase. Purified laccase (molecular weight 45 kDa) had the optimum activity at pH 2.0 and the optimum temperature 50 °C using ABTS as chromogenic substrate. Laccases efficiently decolorized triphenylmethane dyes such as Malachite Green (87.8%), Bromocresol Purple (71.6%), and Methyl Violet (68.1%) without redox mediator. However, decolorization percentage of hardly degradable triphenylmethane dyes such as Phenol Red, Bromophenol Blue, and Brilliant Blue R-250 was increased the presence of some low-molecular weight compounds (natural or synthetic redox mediators). Purified laccases were resistant to Mg2+ , Ca2+ , Ba2+ , Mn2+ , Fe2+ , Cu2+ , Zn2+ , and Sn2+ (10 mmol L-1 ). These findings suggest that laccases from C. subvermispora are able to decolorize triphenylmethane dyes without the negative influence of metal ions that can be found in wastewater.

Biological treatment of colored wastewater by Streptomyces fulvissimus CKS 7.

This study aims to investigate the biological processes related to the biodegradable potential of growing microbial cells for contaminated water treatment. Thus, the use of the Streptomyces fulvissimus CKS 7 (CKS7) has been evaluated for decolorizing efficiency of a solution containing a cationic triphenylmethane dye, crystal violet. The color reduction was monitored by UV-Vis spectroscopic analysis, through changes in their absorption spectrum and comparing the results with those of the respective controls. It was found that the CKS7 performed well and reached up to 100% effectiveness. The required process parameters have been apparently mild and include the reaction temperature of 27-30 °C, 10% inoculum size, under shaking conditions, whereas the time course of decolorization had been concentration dependent. A possible mechanism for removing dye from the working medium was accomplished in two steps: the binding of the dye on the bacterial cell surface, in addition to the dye biodegradation by the bacterial intracellular enzymes. After one cycle of the complete dye removal, the adapted culture was successfully reused for the same purpose. The phytotoxicity analysis revealed that non-toxic compounds were present in decolorized medium, indicating that the CKS7 bacteria seem to be a promising application for contaminated water treatment.

In vitro antifungal activity and in vivo antibiofilm activity of cerium nitrate against Candida species.

OBJECTIVES: The objective of this study was to clarify the antifungal properties of cerium, a lanthanide member, against Candida species. A comprehensive study with planktonic and sessile cells was performed. The ability of cerium nitrate (CN) to impair in vitro and in vivo biofilm formation was evaluated and its potential use in biofilm treatment was also evaluated. METHODS: Forty-eight clinical isolates of different Candida species and the type strain ATCC 90028 were tested according to the protocol M27-A3. The MICs and minimum lethal concentrations were determined. A time-kill assay was performed and a cytometric kinetic study was performed using live/dead markers. Biofilm inhibition and biofilm susceptibility in the presence of cerium was evaluated by quantification of the biofilm metabolic activity and total biomass with XTT and crystal violet assays, respectively. CN in vivo efficacy as a coating for medical indwelling devices was evaluated for the first time for Candida parapsilosis, using a mouse subcutaneous foreign body model using polyurethane catheter segments. Scanning electron microscopy was used to assess biofilm architecture after CN treatment. RESULTS: The MICs for planktonic cells correlated with severe cellular metabolic activity impairment and membrane damage after 3 h of incubation. Moreover, CN efficiently prevented biofilm formation both in vitro and in vivo in segments of polyurethane catheters. At higher concentrations, it was also able to disorganize and almost eradicate preformed biofilms. CONCLUSIONS: Our results strongly suggest that CN application in the clinical setting might be effective in preventing the formation of biofilm-associated infections, namely through catheter coating and ultimately as an antimicrobial lock therapy.

Adhesion of smooth and rough phenotypes of Flavobacterium psychrophilum to polystyrene surfaces.

Phenotypic smooth cells of the fish pathogenic bacterium Flavobacterium psychrophilum have previously been reported to be more adhesive to polystyrene surfaces than corresponding rough cells. In this study, the adhesion ability of smooth and rough cells of F. psychrophilum to polystyrene surfaces was investigated in detail with a crystal violet staining method. By treating both polystyrene surfaces with fish mucus and carbohydrates and the bacterial cells with carbohydrates, the involvement of lectins in the adhesion process was investigated. Smooth cells showed significantly higher adhesion ability to untreated polystyrene surfaces compared with corresponding rough cells and increasing water hardness had an inhibitory effect on the adhesion. Treatment of polystyrene surfaces with D-glucose, D-galactose and fish mucus increased the adhesion ability of smooth cells to polystyrene. Furthermore, treatment of the smooth cells with D-glucose, D-galactose and sialic acid decreased the adhesion ability of the cells, indicating that the adhesion is likely mediated by complementary lectins on the surface of the cells. Sodium (meta)periodate treatment of smooth cells also decreased the adhesion ability to polystyrene, suggesting that the lectins, such as the dominating sialic acid-binding lectin, are probably localized in the extracellular polysaccharides surrounding the cells.

Genetic characterization of plasmid-associated triphenylmethane reductase in Listeria monocytogenes.

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes.