Antimicrobial photodynamic therapy fighting polymicrobial infections - a journey from in vitro to in vivo.

Rapidly evolving multidrug resistance renders conventional antimicrobial strategies increasingly inefficient. This urges the exploration of alternative strategies with a lower potential of resistance development to control microbial infections. A promising option is antimicrobial photodynamic therapy (aPDT), especially in the setting of wound infections. In this study its effectiveness was tested as a treatment option for polymicrobially infected wounds in both in vitro and in vivo models. First, aPDT was applied to wound-relevant Gram-positive and Gram-negative bacteria in planktonic culture as the standard in vitro test system and compared different media to show a possible dependency of the therapy on the surrounding environment. In a second step, aPDT was investigated in an in vitro model mimicking the wound bed conditions using fibrin-coated culture plates. Finally, we tested aPDT in vivo in a polymicrobial infected wound healing model in immunocompromised BALB/c mice. In vitro, it was shown that the bactericidal effectiveness of aPDT was strongly dependent on the surrounding environment of the phototoxic reaction. In vivo, the significant delay in wound healing induced by polymicrobial infection was drastically diminished by a two-times application of aPDT using 100 µM methylene blue (generally regarded as safe for topical application on human skin) and 24 J cm-2 pulsed red LED light. Our experiments suggest that aPDT is capable of significantly improving wound healing also in complicated polymicrobially infected wound situations.

In Vitro and In Vivo Effects of the Polymyxin-Vorinostat Combination Therapy Against Multidrug-Resistant Gram-Negative Pathogens.

With the stagnancy of antibiotics development, polymyxins have become the last defense for treatment of multidrug-resistant (MDR) Gram-negative bacteria, whereas the effect of polymyxin monotherapy is limited by resistance. The objective of this study was to evaluate the effects of polymyxin B (PMNB)-vorinostat (SAHA) combination therapy against Gram-negative pathogens in vitro and in vivo. The antibacterial activities of PMNB and SAHA were evaluated by susceptibility testing. The synergistic effect was assessed by checkerboard tests and time-killing kinetics experiments. Cellular morphology studies and reactive oxygen species (ROS) assay were conducted to explore potential mechanisms. Also, Galleria mellonella models were made to evaluate the antibacterial effects in vivo. PMNB-SAHA had the synergistic effect against all tested isolates, reducing >2 log10 colony-forming units (CFU)/mL at 40 minutes, and showed more powerful antibacterial effects than PMNB alone in the 24-hour window. Cellular morphology study showed the change of membrane and disruption of integrity. ROS assay showed more oxidative stress in combination than PMNB or SAHA monotherapy. In animal models, PMNB-SAHA showed a higher survival rate than that of monotherapy. This study is the first to report the synergistic antibacterial effect of PMNB-SAHA therapy against MDR Gram-negative bacteria. Further clinical research is needed to confirm the results.

Towards osteogenic and bactericidal nanopatterns?

Recent discoveries have shown that nanopatterns with feature sizes ≤100 nm could direct stem cell fate or kill bacteria. These effects could be used to develop orthopedic implants with improved osseointegration and decreased chance of implant-associated infections. The quest for osteogenic and bactericidal nanopatterns is ongoing but no controlled nanopatterns with dual osteogenic and bactericidal functionalities have been found yet. In this study, electron beam induced deposition (EBID) was used for accurate and reproducible decoration of silicon surfaces with four different types of nanopatterns. The features used in the first two nanopatterns (OST1 and OST2) were derived from osteogenic nanopatterns known to induce osteogenic differentiation of stem cells in the absence of osteogenic supplements. Two modifications of these nanopatterns were also included (OST2-SQ, OST2-H90) to study the effects of controlled disorder and lower nanopillar heights. An E. coli K-12 strain was used for probing the response of bacteria to the nanopatterns. Three nanopatterns (OST2, OST2-SQ, and OST2-H90) exhibited clear bactericidal behavior as evidenced by severely damaged cells and disrupted formation of extracellular polymeric substance. These findings indicate that controlled nanopatterns with features derived from osteogenic ones can have bactericidal activity and that EBID represents an enabling nanotechnology to achieve (multi)functional nanopatterns for bone implants.

Targeted inactivation of antibiotic-resistant Escherichia coli and Pseudomonas aeruginosa in a soil-lettuce system by combined polyvalent bacteriophage and biochar treatment.

High abundances of antibiotic-resistant pathogenic bacteria (ARPB) and antibiotic resistance genes (ARGs) in agricultural soil-plant systems have become serious threats to human health and environmental safety. Therefore, it is crucial to develop targeted technology to control existing antibiotic resistance (AR) contamination and potential dissemination in soil-plant systems. In this work, polyvalent bacteriophage (phage) therapy and biochar amendment were applied separately and in combination to stimulate ARPB/ARG dissipation in a soil-lettuce system. With combined application of biochar and polyvalent phage, the abundance of Escherichia coli K-12 (tetR) and Pseudomonas aeruginosa PAO1 (ampR + fosR) and their corresponding ARGs (tetM, tetQ, tetW, ampC, and fosA) significantly decreased in the soil after 63 days' incubation (p < 0.05). Similar results for endophytic K-12 and PAO1, and ARGs, were also obtained in lettuce tissues following combined treatment. Additionally, high throughput sequencing revealed that biochar and polyvalent phage synergetically improved the structural diversity and functional stability of the indigenous bacterial communities in soil and the endophytic ones in lettuce. Hence, this work proposes a novel biotechnology that combines biochar amendment and polyvalent phage therapy to achieve targeted inactivation of ARPB, which stimulates ARG dissipation in soil-lettuce systems.

Violet 405 nm light: A novel therapeutic agent against ß-lactam-resistant Escherichia coli.

BACKGROUND AND OBJECTIVE: Approximately 1.7 million patients are affected by hospital-acquired infections every year in the United States. The increasing prevalence of multidrug-resistant bacteria associated with these infections prompts the investigation of alternative sterilization and antibacterial therapies. One method currently under investigation is the antibacterial properties of visible light. This study examines the effect of a visible light therapy (VLT) on ß-lactam-resistant Escherichia coli, a common non-skin flora pathogen responsible for a large percentage of indwelling medical device-associated clinical infection. MATERIALS AND METHODS: 405 nm light-emitting diodes were used to treat varying concentrations of a common laboratory E. coli K-12 strain transformed with the pCIG mammalian expression vector. This conferred ampicillin resistance via expression of the ß-lactamase gene. Bacteria were grown on sterile polystyrene Petri dishes plated with Luria-Bertani broth. Images of bacterial growth colonies on plates were processed and analyzed using ImageJ. Irradiance levels between 2.89 ± 0.19 and 9.45 ± 0.63 mW cm(-2) and radiant exposure levels between 5.60 ± 0.39 and 136.91 ± 4.06 J cm(-2) were tested. RESULTS: VLT with variable irradiance and constant treatment time (120 minutes) demonstrated significant reduction (P < 0.001) in E. coli between an irradiance of 2.89 mW cm(-2) (81.70%) and 9.37 mW cm(-2) (100.00%). Similar results were found with variable treatment time with constant irradiance. Log10 reduction analysis produced between 1.98 ± 0.53 (60 minute treatment) and 6.27 ± 0.54 (250 minute treatment) log10 reduction in bacterial concentration (P < 0.001). CONCLUSIONS: We have successfully demonstrated a significant bacterial reduction using high intensity 405 nm light. Illustrating the efficacy of this technology against a ß-lactam-resistant E. coli is especially relevant to the need for novel methods of sterilization in healthcare settings. These results suggest that VLT using 405 nm light could be a suitable clinical option for eradication of ß-lactam-resistant E. coli. Visible light kills statistically significant concentrations of E. coli. Antibiotic-resistant Gram-negative bacteria exhibits sensitivity to 405 nm light. Greater than 6 log10 reduction in ß-lactam-resistant E. coli when treated with visible light therapy.

Influence of Plantaginaceae species on E. coli K12 growth in vitro: Possible relation to phytochemical properties.

CONTEXT: The data concerning the influence of Plantaginaceae water extracts on bacterial growth are contradictory. OBJECTIVE: This study investigates the influence of Plantago maxima Juss. ex Jacq., Plantago lanceolata L., Plantago major L., Veronica teucrium L., Veronica spicata L., and Veronica incana L. aqueous extracts on growth of Escherichia coli K12 culture and the relation to antioxidant, reducing, and iron-binding activities. MATERIALS AND METHODS: Aqueous extracts were prepared from the dried leaves with the final concentration of 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, and 1/40 (w/w). Comparative analysis of total flavonoids, iridoids, and tannins in Plantaginaceae species was performed. Iron-binding, antioxidant, and reducing activities of plant extracts were analyzed spectrophotometrically. The influence of plant extracts on E. coli K12 growth was studied in vitro by estimating the bacterial growth in the extract-containing medium. RESULTS: Total tannin content in plant leaves positively correlated with iron-binding activity (r = 0.641), whereas total flavonoids correlated with antioxidant activity (r = 0.687). In an in vitro model, it is estimated that water extracts of studied Plantaginaceae species stimulated bacterial growth. Prebiotic activity significantly of 1/20 and 1/40 plant extracts positively correlated with antioxidant (r = 0.589; r = 0.576, respectively) and reducing activity (r = 0.721; r = 0.620, respectively) of plant aqueous extracts at 6-24 h. Negative correlation was observed between iron-binding activity and bacterial growth (r = -0.503 and r = -0.534 for 1/20 and 1/40 extracts, respectively). CONCLUSION: Aqueous Plantaginaceae extracts possess prebiotic activity depending on the phytochemical content of plant leaves.

Light-induced surface graft polymerizations initiated by an anthraquinone dye on cotton fibers.

Anthraquinone and its derivatives could serve as photo-sensitizers and generate radicals and reactive oxygen species in polymers under exposure of UVA or day light. Such a property was utilized in development of novel light-induced surface radical graft polymerizations on cotton fibers that were dyed with an anthraquinone derivative, 2-ethylanthraquinone. Several functional monomers were directly grafted onto the dyed cotton fibers upon UVA exposure. The chemical and morphological structures and thermal properties of the grafted fibers were confirmed and characterized by Fourier transform infrared spectrometer (FTIR), scanning electron microscope (SEM) and thermal gravimetric analysis (TGA). Reaction conditions including concentrations of the photosensitizer, the amount of monomers, as well as UVA irradiation time could influence grafting efficiencies. More interestingly, the surface graft polymerization did not significantly change the light active functions of the agent, evidenced by the light-active antimicrobial functions of the grafted fibers.

Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human ß-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human ß-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

Ginkgolic acids and Ginkgo biloba extract inhibit Escherichia coli O157:H7 and Staphylococcus aureus biofilm formation.

Infection by enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem, and there is no effective therapy. Biofilm formation is closely related to EHEC infection and is also a mechanism of antimicrobial resistance. Antibiofilm screening of 560 purified phytochemicals against EHEC showed that ginkgolic acids C15:1 and C17:1 at 5µg/ml and Ginkgo biloba extract at 100µg/ml significantly inhibited EHEC biofilm formation on the surfaces of polystyrene and glass, and on nylon membranes. Importantly, at their working concentrations, ginkgolic acids and G. biloba extract did not affect bacterial growth. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, and these findings were in-line with reduced fimbriae production and biofilm reductions. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of a commensal E. coli K-12 strain. In addition, ginkgolic acids and G. biloba extract inhibited the biofilm formation of three Staphylococcus aureus strains. The findings of this study suggest that plant secondary metabolites represent an important resource for biofilm inhibitors.

Antioxidant supplementation enhances bacterial peritonitis in mice by inhibiting phagocytosis.

Antioxidants are known to exhibit numerous health benefits including anti-ageing, anti-apoptotic and immuno-stimulatory effects. However, we present the data showing counterproductive effects of therapeutically relevant antioxidants on bacterial clearance by the immune system in a murine peritonitic model. The antioxidants ascorbic acid, glutathione and N-acetylcysteine augmented morbidity and mortality in mice carrying Eshcerichia coli-induced acute bacterial peritonitis. Treatment of peritonitic mice with antioxidants significantly increased their bacterial load in the range of 0.3-2 logs. Antioxidant administration to peritonitic mice resulted in decreased numbers of macrophages, B-cells and dendritic cells at the primary site of infection and increased neutrophil infiltration. Serum TNF-α levels were also decreased in antioxidant-treated peritonitic mice. In vitro experiments showed that antioxidants reduced the phagocytic efficacy of peritoneal macrophages by ~60-75% and also decreased E. coli-induced oxidative burst in macrophages cells. Taken together, our data indicate that the antioxidants increased the severity of peritonitis by decreasing the phagocytic efficiency, oxidative burst, and TNF-α production, and increasing neutrophil infiltration. Based on these results, we propose that antioxidant supplementation during the course of bacterial infection is not recommended as it could be detrimental for the host. In addition, the present study underlines the importance of timing and context of antioxidant administration rather than indiscriminate usage to gain the best possible therapeutic advantage of these redox compounds.

Synthesis and characterization of nano-encapsulated black pepper oleoresin using hydroxypropyl beta-cyclodextrin for antioxidant and antimicrobial applications.

Previous studies have reported antimicrobial and antioxidant activity of black pepper oleoresin which is associated to its phenolic compounds and piperine. The ability of cyclodextrins to form an inclusion complex with a guest molecule could improve black pepper oleoresin application, bioavailability, and stability in foods. Hydroxypropyl beta-cyclodextrin (HPBCD) inclusion complex with black pepper olereosin were synthesized using the kneading method and characterized for its physico-chemical properties and its antioxidant and antimicrobial activities. Inclusion complex size was 103.9 ± 7.6 nm and indicated to be a polydisperse system. The entrapment efficiency was 78.3 ± 3.6%, which suggests that other constituents in black pepper oleoresin have higher affinities for HPBCD than piperine (major compound in black pepper oleoresin). Thermograms showed the disappearance of oxidation peaks of black pepper oleoresin, proving complex formation with HPBCD. Phase solubility results indicated 1:1 stoichiometric inclusion complex formation and an increase of black pepper oleoresin aqueous solubility with HPBCD concentration. Nano-encapsulation with HPBCD did not affect (P > 0.05) total phenolic content; however, it enhanced (P < 0.05) black pepper oleoresin antioxidant activity. Black pepper oleoresin and its inclusion complex were analyzed for their antimicrobial activity against Escherichia coli K12 and Salmonella enterica serovar Typhimurium LT2. Both free and encapsulated black pepper oleoresin effectively inhibited bacterial growth within the concentration range tested. Black pepper oleoresin encapsulated in HPBCD was able to inhibit Salmonella at lower (P < 0.05) concentrations than its corresponding free extract. Therefore, black pepper oleoresin-HPBCD nanocapsules could have important applications in the food industry as antimicrobial and antioxidant system.

Cytotoxic and genotoxic potential of tannery waste contaminated soils.

Soil samples from agricultural fields in the vicinity of industrial area of Jajmau, Kanpur (India) were collected and found to be heavily contaminated with various toxic heavy metals. GC-MS analysis revealed the presence of organic compounds mainly phthalates in contaminated soils. Samples were extracted using dichloromethane (DCM) and hexane solvents, and the extracts were assayed for genotoxic potential using three different bioassays namely Ames Salmonella/mammalian microsome test, DNA repair defective Escherichia coli K-12 mutants and Allium cepa chromosomal aberration assay. TA98 was found to be the most sensitive strain to all the soil extracts tested. The highest mutagenic potential was observed in DCM extracts of soil as compared with hexane extracts for each strain of Salmonella typhimurium. DCM extracts of the soil exhibited maximum damage to the cells at a dose of 40 µl of soil extracts/ml of culture after a 6-h treatment. The survival was 23% in polA, 40% in lexA and 53% in recA mutants when treated with DCM extract of site I. In A. cepa assay, all the test concentrations of soil extracts (5-100%) affected mitotic index in a dose-dependent manner and several types of abnormalities were observed at different mitotic stages with the treatments: C-mitosis, anaphase bridges, laggards, binucleated cells, stickiness, broken and unequal distributions of chromosomes at anaphase stage of cell division. The soil is accumulating a large number of pollutants as a result of wastewater irrigation and this practice of accumulation has an adverse impact on soil health.

Assessment of cytotoxic and genotoxic potential of refinery waste effluent using plant, animal and bacterial systems.

The work described here presents the toxic effect of Mathura refinery wastewater (MRWW) in plant (Allium cepa), bacterial (E. coli K12) and human (blood) system. The samples were collected from adjoining area of Mathura refinery, Dist. Mathura, U.P. (India). Chromosomal aberration test and micronucleus assay in (A. cepa) system, E. coli K12 survival assay as well as hemolysis assay in human blood were employed to assess the toxicity of MRWW. MRWW exposure resulted in the formation of micronuclei and bridges in chromosomes of A. cepa cells. A significant decline occurred in survival of DNA repair defective mutants of E. coli K12 exposed to MRWW. On incubation with MRWW, calf thymus DNA-EtBr fluorescence intensity decreased and percent hemolysis of human blood cells increased. An induction in the MDA levels of MRWW treated A. cepa roots indicated lipid peroxidation also. Collectively, the results demonstrate a significant genotoxic and cytotoxic potential of MRWW.

A new Transcriptional Effect Level Index (TELI) for toxicogenomics-based toxicity assessment.

This study proposes and demonstrates the potential application of a new Transcriptional Effect Level Index (TELI) to convert the information-rich toxicogenomic data into integrated and quantitative endpoints. A library of transcriptional fusions of green fluorescent protein (GFP) that includes different promoters for 91 stress-related genes in E. coli K12, MG1655 is employed to evaluate the gene expression alteration induced by exposure to four nanomaterials (NMs), nano silver (nAg), nano titanium dioxide anatase (nTiO2_a), nano titanium dioxide rutile (nTiO2_r), and fullerene soot. TELI is determined for each toxicogenomic assay, and it incorporates the number and identity of genes that had altered expression, the magnitude of alteration, and the temporal pattern of gene expression change in response to toxicant exposure. TELI values exhibit a characteristic "sigmoid" shaped toxicity dose-response curve, based on which TELI(MAX) (the maximal value of TELI), TELI50 (concentration that yields half of TELI(MAX)), NOTEL(TELI) (TELI-based no observed transcriptional effect level), and Slope(TELI) (the slope of TELI-dose response curve) are obtained. TELI-based endpoints are compared to currently used endpoints such as EC50 and no observed transcriptional effect level (NOTEL). The agreement of NOTEL(TELI) and NOTEL values validates the concept and application of TELI. Multiple endpoints derived from TELI can describe the dose response behavior and characteristics more completely and holistically than single points such as NOTEL alone. TELI values determined for genes in each stress response category (e.g., oxidative stress, DNA repair) indicate mode of action (MOA)-related comparative transcriptional level toxicity among compounds, and it reveals detailed information of toxic response pathways such as different DNA damage and repair mechanisms among the NMs. This study presents a methodology for converting the rich toxicogenomic information into a readily usable and transferable format that can be potentially linked to regulation endpoints and incorporated into a decision-making framework.

[Effect of phytocompositions on biological activity of probiotic strains of Bacillus subtilis].

Influence of phytocompositions containing silica and medical herbs on growth and biological activity of probiotic strains of Bacillus subtilis and some representatives of obligate microflorae of intestine has been studied. Biocompatibility of studied strains with both pure aerosil bacteria, and its phytocompositions has been shown in the article. It is supposed that phytocompositions can be considered as components for creation of complex probiotic preparations from bacilli for the purpose to increase their therapeutic efficiency.

Inhibition of infectious diseases by components from Aloe vera.

The ability to eliminate Escherichia coli K-12 from the peritoneal cavity in the early stage of infection (48 h) was improved by the pre-administration of an aloe sample to BALB/c mice. Our results suggest that the aloe sample could inhibit infectious diseases by stimulating the host defense mechanism, especially the phagocytic and killing activities of macrophages.

Comparative study on the antioxidant and biological activities of carvacrol, thymol, and eugenol derivatives.

Four derivatives of thymol, carvacrol, and eugenol were synthesized: 4-(hydroxymethyl)-5-isopropyl-2-methylphenol, 4,4'-methylenebis(5-isopropyl-2-methyl)phenol, 4-allyl-6-(hydroxymethyl)-2-methoxyphenol, and 4-(hydroxymethyl)-2-isopropyl-5-methylphenol. The obtained derivatives showed remarkably better antioxidative properties according to 1,1-diphenyl-2-picrylhydrazyl assay (50% inhibitory concentrations = 4-156 microg/mL) and Rancimat assay (protection factors = 1.55-5.84) when compared with parent compounds and values similar to or better than those of butylated hydroxytoluene and vitamin C. At concentrations of 10 mM carvacrol derivatives had no toxic effect on viability of Escherichia coli K-12 (determined by minimum inhibitory concentrations). Other phenol derivatives showed reduced cytotoxic effect on E. coli K-12 at concentrations of 2-5 mM on the basis of 50% lethal dose measurements. In comparison with the parent compounds, phenol derivatives showed reduced cytotoxic effect for Saccharomyces cerevisiae cells (determined by yeast colony reduction). On the other hand, the majority of synthesized compounds had dose-dependent antiproliferative effects on human uterine carcinoma cells (HeLa), which makes them potentially interesting for the adjuvant experimental cancer treatments. The 4,4'-methylenebis(5-isopropyl-2-methyl)phenol derivative of carvacrol showed lower inhibiting capacity also for the HeLa cells, which makes this particular derivative attractive as an efficient antioxidant with negligible cytotoxic effects.

Catalytic sterilization of Escherichia coli K 12 on Ag/Al2O3 surface.

Bactericidal action of Al(2)O(3), Ag/Al(2)O(3) and AgCl/Al(2)O(3) on pure culture of Escherichia coli K 12 was studied. Ag/Al(2)O(3) and AgCl/Al(2)O(3) demonstrated a stronger bactericidal activity than Al(2)O(3). The colony-forming ability of E. coli was completely lost in 0.5 min on both of Ag/Al(2)O(3) and AgCl/Al(2)O(3) at room temperature in air. The configuration of the bacteria on the catalyst surface was observed using scanning electron microscopy (SEM). Reactive oxygen species (ROS) play an important role in the expression of the bactericidal activity on the surface of catalysts by assay with O(2)/N(2) bubbling and scavenger for ROS. Furthermore, the formation of CO(2) as an oxidation product could be detected by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and be deduced by total carbon analysis. These results strongly support that the bactericidal process on the surface of Ag/Al(2)O(3) and AgCl/Al(2)O(3) was caused by the catalytic oxidation.

Optimization of ferrioxamine E concentration as effective supplementation for selective isolation of Salmonella enteritidis in egg white.

Utilization of ferrioxamine E (FE) as a sole source of iron distinguishes Salmonella from a number of related species, including Escherichia coli. FE is not able to serve as a source of iron for E. coli or the Proteus-Providencia-Morganella group. This confers a selective advantage on Salmonella Enteritidis in egg white supplemented with FE. The optimum concentration of FE that promoted a selective advantage for Salmonella in egg white was determined. Four supplementation concentrations were evaluated (25, 50, 200, and 500 microg/ml) in egg white artificially inoculated with proportionally mixed cultures of a rifampin-resistant strain of Salmonella Enteritidis (0.1 ml of 102 CFU/ml) and E. coli K-12 (0.1 ml of 10(1) through 10(8) CFU/ml). After a 24-h incubation at 37 degrees C, Salmonella and E. coli populations were enumerated. At higher concentrations of FE (>50 microg/ml), both Salmonella and E. coli were able to use the iron supplement (1 to 8.5 log CFU/ml and 1.8 to 8 log CFU/ml, respectively); however, lower FE concentrations (< or = 50 microg/ml) exclusively promoted Salmonella growth. Salmonella was unrecoverable without supplementation. This study indicates that optimum levels of FE supplementation in egg can improve the selective detection for Salmonella Enteritidis among other competitive organisms.

Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid.

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 microg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 microg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 microg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 microg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).