In vitro assessment of antimicrobial and genotoxic effect of metallosurfactant based nickel hydroxide nanoparticles against Escherichia coli and its genomic DNA.

In the present study, we have synthesized nickel hydroxide nanosuspensions (Ns) using microemulsion technique. This approach is eco-friendly and makes use of Tween 80 (a non-ionic biocompatible surfactant) and newly synthesized metallosurfactants for the formation of uniform nanoparticles in the form of nanosuspensions (Ns). The nickel hydroxide Ns's were derived from three different metallosurfactants i.e. NiCTAC (Bishexadecyltrimethylammonium nickel tetrachloride), NiDDA (Bisdodecylamine nickel dichloride) and NiHEXA (bishexadecylamine nickel dichloride). Three different nickel-based metallosurfactants were synthesized and characterized using various methods such as CHN, 1HNMR, and FTIR. Fabrication of nanosuspension was confirmed using different characterization methods such as Transmission electron microscopy (TEM), Field Emission Scanning Electron Microscope (FESEM), Energy Dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), pH and Zeta potential. These particles were further investigated for their genotoxic and cytotoxic effects on gram-negative bacteria, Escherichia coli (E. coli). Effect of nanosuspensions on E. coli was confirmed using colony forming unit count, agar well diffusion, and gram staining method. Through colony forming unit count method, nanosuspensions influence on the colony-forming capacity of E. coli cells was confirmed. Agar well diffusion method provides the estimation of antimicrobial activity, and the largest inhibition zone was observed for NiCTAC Ns and smallest for NiHEXA Ns which is related to maximum and minimum bactericidal properties of Ns, respectively. The interaction behavior of bacterial DNA with Ni nanosuspension was analyzed using agarose gel electrophoresis and circular dichroism. Along with, the role of different chemical scavengers was also evaluated in DNA damage using gel electrophoresis. Furthermore, the antioxidant activity of Ni nanosuspension was also confirmed using DPPH assay.

Mutagenicity, anticancer activity and blood brain barrier: similarity and dissimilarity of molecular alerts.

The aim of the present work is an attempt to define computable measure of similarity between different endpoints. The similarity of structural alerts of different biochemical endpoints can be used to solve tasks of medicinal chemistry. Optimal descriptors are a tool to build up models for different endpoints. The optimal descriptor is calculated with simplified molecular input-line entry system (SMILES). A group of elements (single symbol or pair of symbols) can represent any SMILES. Each element of SMILES can be represented by so-called correlation weight i.e. coefficient that should be used to calculate descriptor. Numerical data on the correlation weights are calculated by the Monte Carlo method, i.e. by optimization procedure, which gives maximal correlation coefficient between the optimal descriptor and endpoint for the training set. Statistically stable correlation weights observed in several runs of the optimization can be examined as structural alerts, which are promoters of the increase or the decrease of a biochemical activity of a substance. Having data on several runs of the optimization correlation weights, one can extract list of promoters of increase and list of promoters of decrease for an endpoint. The study of similarity and dissimilarity of the above lists has been carried out for the following pairs of endpoints: (i) mutagenicity and anticancer activity; (ii) mutagenicity and blood brain barrier; and (iii) blood brain barrier and anticancer activity. The computational experiment confirms that similarity and dissimilarity for pairs of endpoints can be measured.

Mortality, intensive care treatment, and cost evaluation: Role of a polymerase chain reaction assay in patients with sepsis.

Objective We examined whether patients with a positive SeptiFast (SF) assay (LightCycler SeptiFast; Roche Diagnostics, Basel, Switzerland) developed higher long-term mortality, a more difficult course of treatment, and a higher antimicrobial treatment cost than patients with a negative SF assay. Methods We performed a post-hoc analysis of data collected in a 1-year prospective interventional study of adults with severe sepsis and septic shock. In addition to the standard treatment, an additional 5 ml of blood was obtained for an SF assay, and the antimicrobial treatment was changed according to the SF results. Results We included 57 patients, and the SF assay was positive (SF+) in 10 (17.5%) and negative (SF-) in 47 (82.5%) patients. A trend toward a higher 6-month, 1-year, and 2-year mortality rate was observed in the SF+ group. In the SF+ group, we observed a significantly greater need for second-line vasopressor therapy, a higher initial procalcitonin concentration, and higher maximum C-reactive protein and lactate concentrations. We found no significant differences in cost of antimicrobial treatment between the SF+ and SF- groups. Conclusions We observed a trend toward higher long-term mortality and a more difficult course of treatment but no difference in the cost of antimicrobial treatment.

Safety assessment of a traditionally used extract from leaves of Boldoa purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: Boldoa purpurascens Cav. (Nyctaginaceae) is a plant species used in traditional medicine in Cuba as a diuretic. AIM OF THE STUDY: The aim of the present investigation was to evaluate the safety profile of a hydroalcoholic extract from leaves of Boldoa purpurascens. MATERIALS AND METHODS: First, an experimental study to assess the oral acute toxicity at a dose of 2000mg/kg body weight of the extract was carried out. Potential genotoxicity of the extract was evaluated using the Ames test and the micronucleus induction assay in mouse bone marrow. In the Ames test a concentration range of 50, 100, 150, 300 and 500µg/plate was tested. In the micronucleus induction assay, doses of 500, 1000 and 2000mg/kg of body weight were tested. For completeness, since the extract contains saponins, the evaluation of the hemolytic activity, ocular and skin irritation were included. RESULTS: No signs or symptoms of toxicity were observed in the oral acute toxicity test (body weight at baseline, seven days and end of the experiment of 236.41±20.07, 256.81±30.44 and 240.02±26.16 respectively for the treated group). The hydroalcoholic extract from the leaves was not mutagenic in the Ames test, and no genotoxicity was observed in the micronucleus assay. A hemolysis test at concentration of 1mg/mL confirmed hemolytic activity, which is not a safety concern since saponins are not absorbed after oral administration. In order to evaluate the percentage of protein denaturation, the ocular irritability index was calculated. The extract was found to be irritating. Finally, skin irritability was evaluated and the irritation index was equal to zero. CONCLUSIONS: Based on the toxicological evaluation of a traditionally used hydroalcoholic extract from the leaves of Boldoa purpurascens we can confirm the safety of its oral use.

Application of comet assay for the assessment of DNA damage caused by chemical genotoxins in the dairy yeast Kluyveromyces lactis.

Kluyveromyces lactis, also known as dairy yeast, has numerous applications in scientific research and practice. It has been approved as a GRAS (Generally Recognized As Safe) organism, a probiotic, a biotechnological producer of important enzymes at industrial scale and a bioremediator of waste water from the dairy industry. Despite these important practical applications the sensitivity of this organism to genotoxic substances has not yet been assessed. In order to evaluate the response of K. lactis cells to genotoxic agents we have applied several compounds with well-known cyto- and genotoxic activity. The method of comet assay (CA) widely used for the assessment of DNA damages is presented here with new special modifications appropriate for K. lactis cells. The comparison of the response of K. lactis to genotoxins with that of Saccharomyces cerevisiae showed that both yeasts, although considered close relatives, exhibit species-specific sensitivity toward the genotoxins examined.

Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay.

BACKGROUND: Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. RESULTS: Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 microg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 microg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 microg/ml but scarce after 10 microg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. CONCLUSION: This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

Genetic toxicity assessment: employing the best science for human safety evaluation part III: the comet assay as an alternative to in vitro clastogenicity tests for early drug candidate selection.

Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell-based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates.

Genetic toxicity assessment: employing the best science for human safety evaluation. Part I: Early screening for potential human mutagens.

Results of genetic toxicology tests are used by FDA's Center for Drug Evaluation and Research as a surrogate for carcinogenicity data during the drug development process. Mammalian in vitro assays have a high frequency of positive results which can impede or derail the drug development process. To reduce the risk of such delays, most pharmaceutical companies conduct early non-GLP (good laboratory practices) studies to eliminate drug candidate with mutagenic or clastogenic activity. Early screens include in silico structure activity assessments and various iterations of the ultimate regulatory mandated GLP studies.

Dissemination of vancomycin-resistant enterococci harboring vanA through disposal of waste derived from industrial production of vancomycin.

We demonstrated the occurrence of vancomycin-resistant enterococci (VRE) in waste derived from the industrial production of vancomycin and their dissemination through disposal of such waste into a sewage treatment plant. Bacteriological counts on a medium selective for enterococci (Slanetz-Bartley agar) revealed the presence of high numbers of presumptive VRE (approximately 10(6) CFU/ml) in the waste originating from the fermentation biomass used for vancomycin production. The waste was also found to contain active residues of vancomycin (64-1,024 microg/ml) by bioassays using a vancomycin-susceptible enterococcal strain. Randomly amplified polymorphic DNA (RAPD) analysis of 65 presumptive VRE isolates from the waste allowed distinction of four genotypes, two of which (A and D) belonged to the genus Enterococcus, most likely E. faecium, and harbored the vanA gene conferring high-level vancomycin resistance. The same VRE strains found in the waste occurred also in the biological tanks and the final effluent of the sewage treatment plant receiving the waste, as demonstrated by the detection of undistinguishable pulsed-field gel electrophoresis (PFGE) patterns in VRE isolated from these sources. These results indicate the need to assess the possible dissemination of VRE and other antibiotic-resistant bacteria through disposal of waste derived from antibiotic production.

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants.

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.

Rapid flow cytometric assessment of mecillinam and ampicillin bacterial susceptibility.

The effects of antibiotics on Escherichia coli during exponential growth in liquid medium were monitored by flow cytometry measuring cellular DNA content and cell size of individual cells in large numbers as well as cell counts. A statistically significant increase in cellular DNA as well as size was recorded after 20 and 40 min of incubation with the MIC of ampicillin and mecillinam, respectively. The optical density (OD600nm) of treated cultures continued to increase for at least 80 minutes, showing that the increase in cellular DNA and size reflected continued growth and elongation of cells coupled with inhibition of cell division, the latter being confirmed by the cell counting. Since fixation, staining and flow-cytometric analysis can be carried out within a few minutes, the present results suggest that flow cytometry may have potential as a rapid and quantitative technique that can be automated for clinical and experimental susceptibility testing of antibacterial drugs.