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.

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.

Functional analysis of the genes encoding diaminopropionate ammonia lyase in Escherichia coli and Salmonella enterica serovar Typhimurium.

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on dl-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of dl-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

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.

The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli.

Siderophore-interacting proteins (SIPs), such as YqjH from Escherichia coli, are widespread among bacteria and commonly associated with iron-dependent induction and siderophore utilization. In this study, we show by detailed biochemical and genetic analyses the reaction mechanism by which the YqjH protein is able to catalyze the release of iron from a variety of iron chelators, including ferric triscatecholates and ferric dicitrate, displaying the highest efficiency for the hydrolyzed ferric enterobactin complex ferric (2,3-dihydroxybenzoylserine)(3). Site-directed mutagenesis revealed that residues K55 and R130 of YqjH are crucial for both substrate binding and reductase activity. The NADPH-dependent iron reduction was found to proceed via single-electron transfer in a double-displacement-type reaction through formation of a transient flavosemiquinone. The capacity to reduce substrates with extremely negative redox potentials, though at low catalytic rates, was studied by displacing the native FAD cofactor with 5-deaza-5-carba-FAD, which is restricted to a two-electron transfer. In the presence of the reconstituted noncatalytic protein, the ferric enterobactin midpoint potential increased remarkably and partially overlapped with the effective E(1) redox range. Concurrently, the observed molar ratios of generated Fe(II) versus NADPH were found to be ~1.5-fold higher for hydrolyzed ferric triscatecholates and ferric dicitrate than for ferric enterobactin. Further, combination of a chromosomal yqjH deletion with entC single- and entC fes double-deletion backgrounds showed the impact of yqjH on growth during supplementation with ferric siderophore substrates. Thus, YqjH enhances siderophore utilization in different iron acquisition pathways, including assimilation of low-potential ferric substrates that are not reduced by common cellular cofactors.

[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.

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.

In vitro effect of Reiki treatment on bacterial cultures: Role of experimental context and practitioner well-being.

OBJECTIVE: To measure effects of Reiki treatments on growth of heat-shocked bacteria, and to determine the influence of healing context and practitioner well-being. METHODS: Overnight cultures of Escherichia coli K12 in fresh medium were used. Culture samples were paired with controls to minimize any ordering effects. Samples were heat-shocked prior to Reiki treatment, which was performed by Reiki practitioners for up to 15 minutes, with untreated controls. Plate-count assay using an automated colony counter determined the number of viable bacteria. Fourteen Reiki practitioners each completed 3 runs (n = 42 runs) without healing context, and another 2 runs (n = 28 runs) in which they first treated a pain patient for 30 minutes (healing context). Well-being questionnaires were administered to practitioners pre-post all sessions. RESULTS: No overall difference was found between the Reiki and control plates in the nonhealing context. In the healing context, the Reiki treated cultures overall exhibited significantly more bacteria than controls (p < 0.05). Practitioner social (p < 0.013) and emotional well-being (p < 0.021) correlated with Reiki treatment outcome on bacterial cultures in the nonhealing context. Practitioner social (p < 0.031), physical (p < 0.030), and emotional (p < 0.026) well-being correlated with Reiki treatment outcome on the bacterial cultures in the healing context. For practitioners starting with diminished well-being, control counts were likely to be higher than Reiki-treated bacterial counts. For practitioners starting with a higher level of well-being, Reiki counts were likely to be higher than control counts. CONCLUSIONS: Reiki improved growth of heat-shocked bacterial cultures in a healing context. The initial level of well-being of the Reiki practitioners correlates with the outcome of Reiki on bacterial culture growth and is key to the results obtained.

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).

Carica papaya seed macerate as inhibitor of conjugative R plasmid transfer from Salmonella typhimurium to Escherichia coli in vitro and in the digestive tract of gnotobiotic mice.

In this study, the effect of Carica papaya seed macerate on conjugal R plasmid transfer from Salmonella typhimurium to Escherichia coli was investigated in vitro and in the digestive tract of gnotobiotic mice. Twenty-five micrograms per milliliter and 430 mg (administered intragastrically twice a day) of papaya seed macerate concentrations were used during conjugation for in vitro and in vivo assays, respectively. High frequency of conjugation inhibition by macerate was observed for both in vitro and in vivo experiments, independently of bacterial growth and mating conditions. Papaya seed macerate caused a reduction of the transconjugant population ranging from 71% to about 100%. There was no lethal effect of the seed macerate on donor or recipient cells in the concentrations used. Once the mechanisms and magnitude of resistance gene transfer are clearly understood, strategies to reduce or minimize the dissemination of these genes could be relevant. The data here obtained show a clinical potential use of papaya seed macerate on this transfer.

The expression profile of Escherichia coli K-12 in response to minimal, optimal and excess copper concentrations.

The gene expression profile of Escherichia coli K-12 MG1655 grown in minimal medium supplemented with elevated copper concentrations (as copper-glycine) has been analysed using whole-genome oligonucleotide microarrays. At 750 muM copper-glycine, the expression of both the cue and cus copper-export systems is evident. At near-lethal copper concentrations (2 mM copper-glycine), the expression of these two regulons increases significantly. Other regulons with increased transcription in response to elevated concentrations of copper-glycine include those for the superoxide stress response, iron homeostasis, and envelope stress. Furthermore, a variety of ORFs with decreased expression in response to increased copper-glycine has been identified, including the zinc ABC transporter and genes involved in the chemotactic response.