Minimum inhibitory and bacterial concentrations of theaflavin and synergistic combinations with epicatechin and quercetin against clinical isolates of Stenotrophomonas Maltophilia

Stenotrophomonas maltophilia is an important nosocomial pathogen with intrinsic resistance to multiple antibiotics. Previous investigations have shown flavanols from black tea to possess antibacterial activity. This study describes the determination of minimum inhibitory concentrations and minimum bactericidal concentration for theaflavin independently and in formulations with the polyphenols epicatechin and quercetin against nine clinical isolates of Stenotrophomonas maltophilia and the control isolate NCTC 130141 via the microtitre assay. The results demonstrate that theaflavin has strong antibacterial activity and also shows significant synergism with epicatechin and quercetin. The minimum inhibitory concentrations of the isolates range between 200-400 g/mL for theaflavin and 100-200 g/mL for both theaflavin:epicatechin and theaflavin:quercetin combinations. The minimum bactericidal concentrations were discovered to be a 2 fold increase on those of the minimum inhibitory concentrations. The research highlights the potential use of polyphenols for the clinical treatment of highly antibiotic resistant bacteria.

Use of in vitro critical inhibitory concentration, a novel approach to predict in vivo synergistic bactericidal effect of combined amikacin and piperacillin against pseudomonas aeruginosa in a systemic rat infection model

PURPOSE: This study was undertaken to explore the use of in vitro critical inhibitory concentration (CIC) as a surrogate marker relating the pharmacokinetic (PK) parameters to in vivo bactericidal synergistic effect [pharmacodynamic (PD)] of amikacin + piperacillin combination against Pseudomonas aeruginosa in a systemic rat infection model. METHODS: The in vitro antibacterial activities of amikacin and piperacillin, alone and in combinations at various ratios of the concentrations, were tested against a standard [5 x 10(5) colony-forming units (CFU)/ml] and a large (1.5 x 10(8) CFU/ml) inoculum of P. aeruginosa ATCC 9027 using a modified survival-time method. The CIC of each individual antibiotic for the different combinations was determined using a cup-plate method. In vivo studies were performed on Sprague-Dawley rats using a systemic model of infection with P. aeruginosa ATCC 9027. PK profiles and in vivo killing effects of the combination at different dosing ratios were studied. RESULTS: An inoculum effect was observed with the antibiotics studied. Synergy was seen against both the inocula at the following concentration ratios: 70% C(ami) + 30% C(pip) and 75% C(ami) + 25% C(pip), where C(ami) and C(pip) are the concentrations of amikacin and piperacillin to produce a 1000-fold decrease in bacterial population over 5 h, respectively. The CIC values determined corroborated with the order of in vitro bacterial killing observed for the antibiotic combinations. The dosing ratio of 12.6 mg/kg amikacin + 36 mg/kg piperacillin (a 70:30 ratio of the individual doses) exhibited the greatest killing in vivo when compared to the other ratios. The PK-PD relationships were described by simple, linear regression equations using the area under the in vivo killing curve as a PD marker and the AUCIC(ami)/CIC(ami) + AUCIC(pip)/CIC(pip), AUC(ami)/CIC(ami) + AUC(pip)/CIC(pip), C(max,ami)/CIC(ami) + C(max,pip)/CIC(pip), and AUCIC(ami)/MIC(ami) + AUCIC(pip)/MIC(pip) as PK markers for the amikacin + piperacillin combination. CONCLUSION: The combination of amikacin and piperacillin exhibited synergistic killing effect on P. aeruginosa that could be modeled using CIC as a surrogate marker relating the PK parameters to in vivo bactericidal effect.

Australian Enterococcal Sepsis Outcome Programme, 2011

From 1 January to 31 December 2011, 29 institutions around Australia participated in the Australian Enterococcal Sepsis Outcome Programme (AESOP). The aim of AESOP 2011 was to determine the proportion of enterococcal bacteraemia isolates in Australia that are antimicrobial resistant, with particular emphasis on susceptibility to ampicillin and the glycopeptides, and to characterise the molecular epidemiology of the Enterococcus faecalis and E. faecium isolates. Of the 1,079 unique episodes of bacteraemia investigated, 95.8% were caused by either E. faecalis (61.0%) or E. faecium (34.8%). Ampicillin resistance was detected in 90.4% of E. faecium but not detected in E. faecalis. Using Clinical and Laboratory Standards Institute breakpoints (CLSI), vancomycin non-susceptibility was reported in 0.6% and 31.4% of E. faecalis and E. faecium respectively and was predominately due to the acquisition of the vanB operon. Approximately 1 in 6 vanB E. faecium isolates however, had an minimum inhibitory concentration at or below the CLSI vancomycin susceptible breakpoint of ≤ 4 mg/L. Overall, 37% of E. faecium harboured vanA or vanB genes. Although molecular typing identified 126 E. faecalis pulsed-field gel electrophoresis (PFGE) pulsotypes, more than 50% belonged to 2 pulsotypes that were isolated across Australia. E. faecium consisted of 73 PFGE pulsotypes from which 43 multilocus sequence types were identified. Almost 90% of the E. faecium were identified as clonal complex 17 clones, of which approximately half were characterised as sequence type 203, which was isolated Australia-wide. In conclusion, the AESOP 2011 has shown that although polyclonal, enterococcal bacteraemias in Australia are frequently caused by ampicillin-resistant vanB E. faecium.

Novel 1,4-substituted-1,2,3-triazoles as antitubercular agents

Tuberculosis (TB) remains a pressing unmet medical need, particularly with the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. Here, a series of 1,4-substituted-1,2,3-triazoles have been synthesized and evaluated as potential antitubercular agents. These compounds were assembled via click chemistry in high crude purity and in moderate to high yield. Of the compounds tested, 12 compounds showed promising antitubercular activity with six possessing minimum inhibitory concentration (MIC) values <10 μg mL^-1, and total selectivity for Mycobacterium tuberculosis (Mtb) growth inhibition. A second set of 21 compounds bearing variations on ring C were synthesized and evaluated. This second library gave an additional six compounds displaying MIC values ≤10 μg mL^-1 and total selectivity for Mtb growth inhibition. These compounds serve as an excellent starting point for further development of antitubercular therapies.

Antifungal synergy of theaflavin and epicatechin combinations against Candida albicans

New antifungal agents are required to compensate for the increase in resistance to standard antifungal agents of Candida albicans, which is an important opportunistic fungal pathogen that causes minor infections in many individuals but very serious infections in those who are immune-compromised. In this study, combinations of theaflavin and epicatechin are investigated as potential antifungal agents and also to establish whether antifungal synergy exists between these two readily accessible and cost-effective polyphenols isolated from black and green tea. The results of disc diffusion assays showed stronger antibacterial activity of theaflavin:epicatechin combinations against C. albicans NCTC 3255 and NCTC 3179, than that of theaflavin alone. Minimum inhibitory concentrations (MICs) of 1,024 μg/ml with theaflavin and 128-256 μg/ml with theaflavin:epicatechin combinations were found. The fractional inhibitory concentration indexes were calculated, and the synergy between theaflavin and epicatechin against both isolates of C. albicans was confirmed. Theaflavin:epicatechin combinations show real potential for future use as a treatment for infections caused by C. albicans.

Method design and validation for the determination of uranium levels in human urine using high-resolution alpha spectrometry

Quantification of uranium in human urine is a valuable technique for assessing occupational and public exposure to uranium. A reliable method has been developed and validated in the ARPANSA Radiochemistry Laboratory by means of standard radiochemical separation and purification techniques and measurement using high-resolution alpha spectrometry. This method can be used to evaluate the levels of naturally occurring ²³⁴U, ²³⁵U and ²³⁸U in urine. Method design and validation is the process of defining an analytical requirement, and then confirming that the method under consideration has performance capabilities consistent with what the application requires. The method was designed to measure levels down to 2 mBq/day of total uranium, corresponding to approximately 1/100th of the annual committed effective dose of 20 mSv. Validation tests were developed to assess selectivity, accuracy, recovery and quantification of uncertainty. The radiochemical recovery of this method was measured using ²³²U tracer. The typical minimum detectable concentration for total uranium for 24-h urine samples is approximately 0.6 mBq/day or 0.019 μg/day.

Monitoring the early stage self-assembly of enzyme-assisted peptide hydrogels

The early stages of the self-assembly of peptide hydrogels largely determine their final material properties. Here we discuss experimental methodologies for monitoring the self-assembly kinetics which underpin peptide hydrogel formation. The early stage assembly of an enzyme-catalysed Fmoc-trileucine based self-assembled hydrogel was examined using spectroscopic techniques (circular dichroism, CD, and solution NMR) as well as chromatographic (HPLC) and mechanical (rheology) techniques. Optimal conditions for enzyme-assisted hydrogel formation were identified and the kinetics examined. A lag time associated with the formation and accumulation of the self-assembling peptide monomer was observed and a minimum hydrogelator concentration required for gelation was identified. Subsequent formation of well defined nano-and microscale structures lead to self-supporting hydrogels at a range of substrate and enzyme concentrations. 1H NMR monitoring of the early self-assembly process revealed trends that were well in agreement with those identified using traditional methods (i.e. HPLC, CD, rheology) demonstrating 1H NMR spectroscopy can be used to non-invasively monitor the self-assembly of peptide hydrogels without damaging or perturbing the system.

Exploration of the anticandidal mechanism of Cassia spectabilis in debilitating candidiasis

Candida albicans has become resistant to the commercially available, toxic, and expensive anti-Candida agents that are on the market. These factors force the search for new antifungal agents from natural resources. Cassia spectabilis had been traditionally employed by healers for many generations. The possible mechanisms of the C. spectabilis leaf extract were determined by potassium leakage study and the effect of the extract on the constituents of the cell wall and enzymes as well as the morphological changes on C. albicans cells were studied along with cytotoxicity assays. The cytotoxicity result indicated that the extract is nontoxic as was clearly substantiated by a half maximal inhibitory concentration (IC50) value of 59.10 μg/mL. The treated cells (C. spectabilis extract) demonstrated potassium leakage of 1039 parts per million (ppm) compared to Amphotericin B (AmpB)-treated cells with a released potassium value of 1115 ppm. The effects of the extract on the cell wall proteins illustrated that there were three major types of variations in the expression of treated cell wall proteins: the presence of new proteins, the absence of proteins, and the amount of expressed protein. The activities of two enzymes, α-glucosidase and proteinase, were determined to be significantly high, thereby not fully coinciding with the properties of the antifungal reaction triggered by C. spectabilis. The morphology of C. albicans cells treated with the C. spectabilis extract showed that the cells had abnormalities and were damaged or detached within the microcolonies. Our study verifies C. spectabilis leaf extract as an effective anti-C. albicans agent.

Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells

CONTEXT: Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments. OBJECTIVE: The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells. MATERIALS AND METHODS: Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00 µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation. RESULTS: Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC50) values was 25.26 µg/mL at 24 h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC50 value of 10.01 µg/mL at 24 h. DISCUSSION AND CONCLUSION: This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.

Probing synechocystis-arsenic interactions through extracellular nanowires.

Microbial nanowires (MNWs) can play an important role in the transformation and mobility of toxic metals/metalloids in environment. The potential role of MNWs in cell-arsenic (As) interactions has not been reported in microorganisms and thus we explored this interaction using Synechocystis PCC 6803 as a model system. The effect of half maximal inhibitory concentration (IC50) [~300 mM As (V) and ~4 mM As (III)] and non-inhibitory [4X lower than IC50, i.e., 75 mM As (V) and 1 mM As (III)] of As was studied on Synechocystis cells in relation to its effect on Chlorophyll (Chl) a, type IV pili (TFP)-As interaction and intracellular/extracellular presence of As. In silico analysis showed that subunit PilA1 of electrically conductive TFP, i.e., microbial nanowires of Synechocystis have putative binding sites for As. In agreement with in silico analysis, transmission electron microscopy analysis showed that As was deposited on Synechocystis nanowires at all tested concentrations. The potential of Synechocystis nanowires to immobilize As can be further enhanced and evaluated on a large scale and thus can be applied for bioremediation studies.

Quantifying minimum monolith size and solute dilution from multi-compartment percolation sampler data

Preferential flow affects solute transport in natural soils, leading to high spatiotemporal variation of concentration. A multicompartment solute sampler (MCS), yielding multiple breakthrough curves at a given depth, can monitor tracer movement in a heterogeneous soil. We present a technique to estimate from MCS data whether a soil monolith is sufficiently large to capture preferential flow, which is a necessity for tracer breakthrough curves to be representative. For several soils, we estimate that an MCS should be larger than 0.1 to 0.2 m². We also expand dilution theory to analyze the concentration variations of a tracer passing the control plane monitored by the MCS, in addition to the conventional plume spreading analysis. We characterize the set of locally observed breakthrough curves by the entropy-based dilution index. For given first and second-central moment, the spatially uniform log-normal breakthrough curve maximizes the dilution index. The ratio between observed and maximum dilution index is denoted reactor ratio. For a 300-compartment solute sampler, covering an area of 0.75 m², we compute a reactor ratio of 0.665, compared with 0.04 for stochastic-convective and 1 for convective-dispersive transport. With a single, large collector the reactor ratio would be 0.958, severely underestimating concentration variations. Large collector areas are clearly inadequate to estimate dilution. Values of the dilution index and the reactor ratio for individual sampling compartments indicate efficient longitudinal mixing in most but not all cases, and considerable spatial variation of the leaching process.

Effects of dietary astaxanthin concentration and feeding period on the skin pigmentation of Australian snapper Pagrus auratus (Bloch & Schneider, 1801)

A single-factor experiment was conducted to investigate the effects of dietary astaxanthin concentration on the skin colour of snapper. Snapper (mean weight=129 g) were held in white cages and fed one of seven dietary levels of unesterified astaxanthin (0, 13, 26, 39, 52, 65 or 78 mg astaxanthin kg⁻¹) for 63 days. Treatments comprised four replicate cages, each containing five fish. The skin colour of all fish was quantified using the CIE L^*, a^*, b^* colour scale after 21, 42 and 63 days. In addition, total carotenoid concentrations of the skin of two fish cage−1 were determined after 63 days. Supplementing diets with astaxanthin strongly affected redness (a^*) and yellowness (b^*) values of the skin at all sampling times. After 21 days, the a^* values increased linearly as the dietary astaxanthin concentration was increased before a plateau was attained between 39 and 78 mg kg⁻¹. The b^* values similarly increased above basal levels in all astaxanthin diets. By 42 days, a^* and b^* values increased in magnitude while a plateau remained between 39 and 78 mg kg⁻¹. After 63 days, there were no further increases in measured colour values, suggesting that maximum pigmentation was imparted in the skin of snapper fed diets >39 mg kg⁻¹ after 42 days. Similarly, there were no differences in total carotenoid concentrations of the skin of snapper fed diets >39 mg kg⁻¹ after 63 days. The plateaus that occurred in a^* and b^* values, while still increasing in magnitude between 21 and 42 days, indicate that the rate of astaxanthin deposition in snapper is limited and astaxanthin in diets containing >39 mg astaxanthin kg⁻¹ is not efficiently utilized. Astaxanthin retention after 63 days was greatest from the 13 mg kg⁻¹ diet; however, skin pigmentation was not adequate. An astaxanthin concentration of 39 mg kg⁻¹ provided the second greatest retention in the skin while obtaining maximum pigmentation. To efficiently maximize skin pigmentation, snapper growers should commence feeding diets containing a minimum of 39 mg unesterified astaxanthin kg⁻¹ at least 42 days before sale.

Amylase and glucosidase enzyme inhibitory activity of ginger (Zingiber officinale Roscoe) an in vitro study

The prevalence of type 2 diabetes has reached to an epidemic proportion in Sri Lanka. The need for achieving better control of blood glucose level has been evident in diabetes management. However it is not easy to achieve this goal in a large proportion of patients. This is partly due to limitations of currently available pharmacological agents which stimulate research on novel anti-diabetic agents with different mechanisms. Digestive enzymes have been targeted as potential avenues for modulation of blood glucose concentration through inhibition of the enzymatic breakdown of complex carbohydrates to meal derived glucose absorption. Acarbose is a widely used oral anti-diabetic drug which inhibits the α-glucosidase, enzyme responsible for breaking down of disaccharides and polysaccharides into glucose. Many herbal extracts have been found to posses similar inhibitory effects. Ginger (Zingiber officinale Roscoe) has developed a reputation in treatment of several diseases. In vitro enzymic inhibitory effect of ginger was investigated in this study. Enzymes α -amylase and α -glucosidase treated with either Acarbose or ginger extract were allowed to react with cooked rice and percentages of glucose content were measured. The glucosidase and amylase activities on the rice were inhibited by addition of ginger cause significant reduction in glucose percentages (36.86± 1.05 to 26.87± 2.17, P<0.05 and 49.04±0.65 to 35.35±2.22, P<0.05) which showed comparable results with Acarbose on glucosidase activity (36.86± 1.05 to, 27.8±1.32 P<0.05). Results of the study indicates ginger as a potential plant based amylase and glucosidase inhibitor in carbohydrate digestion but usage in glycaemic control in human has to be investigated further.

Physiology and biochemistry of budburst in Vitis vinifera

Both the physiological and biochemical control of budburst in the grapevine, Vitis Vinifera L. were investigated. It was found that the accuracy of a predictive model for grapevine budburst based on ambient temperature was limited under the experimental conditions. There was a significant correlation of 4.7 ± 0.3 days between the days of maximal xylem exudation and budburst over the 3 years of investigation. The co-relationships between daily xylem exudate volume and a range of environmental parameters were considered. It was found that soil temperature was highly correlated against daily xylem exudation. Ambient temperature and soil moisture were significantly correlated with xylem exudation, however the coefficients of correlation were much lower than that of soil temperature. Rainfall showed only a very limited correlation with daily xylem exudate flow. Seasonal variations in the pH and the carbohydrate and inorganic nutrient concentrations of xylem exudate were investigated. Exudate carbohydrate concentrations fell from 660 µM before the day of maximal xylem exudation to zero levels within 4 weeks. Xylem exudate pH was found to consistently fall to a minimum at the time of maximal exudate flow. Exudate concentrations of the metallic cofactors Ca, K, Mg, Mn and Zn varied directly with daily exudate flow, suggesting some sort of flow-dependent mobilisation of these nutrients. A growth promontory oligosaccharide fraction was prepared by partial acid hydrolysis of grapevine primary cell wall material. This fraction significantly increased control growth of the Lemna minor L. bioassay over a limited ‘window’ of bioactivity. A growth inhibitory oligosaccharide fraction, similar in activity to abscisic acid was isolated from grapevine xylem exudate prior to budburst. The exudate concentration or efficacy of this substance declined after budburst such that there was no apparent growth inhibition. A model is proposed for grapevine budburst whereby an oligosaccharide growth inhibitor is gradually removed from the xylematic stream under the effects of soil temperature, allowing the surge of metabolic activity and vegetative growth that constitute budburst.

The inhibitory effect of stevioside on bacillus cereus growth in milk: validation and its response surface optimization

Stevioside, a glycoside present in the leaves of Stevia rebaudiana Bertoni, offers therapeutic benefits such as anti-hyperglycemic, anti-hypertensive, antiinflammatory, anti-tumor, diuretic and immune influencing properties. In this work antimicrobial activity of stevioside against Bacillus cereus, a major source of milk contamination was investigated. The isolate was confirmed by various biochemical and 16S rRNA gene sequencing. The effect of temperature, incubation time and concentration of stevioside was optimized from a central composite response surface design. The standard plate count (SPC) of pasteurized milk was drastically reduced in comparison to toned and fresh milk. The optimal temperature, incubation time and stevioside concentration were observed to be 60.23°C, 21 h, and 275 μg/ mL respectively. The synergism of stevioside with the external factors (temperature and time) against B. cereus was observed. Our studies showed that addition of stevioside in fresh as well as pasteurised milk would control growth of B. cereus in milk.