Novel oxidising feed additives reduce in vitro methane emissions using the rumen simulation technique.

Enteric methane (CH4) produced by ruminant livestock is a potent greenhouse gas and represents significant energy loss for the animal. The novel application of oxidising compounds as antimethanogenic agents with future potential to be included in ruminant feeds, was assessed across two separate experiments in this study. Low concentrations of oxidising agents, namely urea hydrogen peroxide (UHP) with and without potassium iodide (KI), and magnesium peroxide (MgO2), were investigated for their effects on CH4 production, total gas production (TGP), volatile fatty acid (VFA) profiles, and nutrient disappearance in vitro using the rumen simulation technique. In both experiments, the in vitro diet consisted of 50:50 grass silage:concentrate on a dry matter basis. Treatment concentrations were based on the amount of oxygen delivered and expressed in terms of fold concentration. In Experiment 1, four treatments were tested (Control, 1× UHP + KI, 1× UHP, and 0.5× UHP + KI), and six treatments were assessed in Experiment 2 (Control, 0.5× UHP + KI, 0.5× UHP, 0.25× UHP + KI, 0.25× UHP, and 0.12× MgO2). All treatments in this study had a reducing effect on CH4 parameters. A dose-dependent reduction of TGP and CH4 parameters was observed, where treatments delivering higher levels of oxygen resulted in greater CH4 suppression. 1× UHP + KI reduced TGP by 28 % (p = 0.611), CH4% by 64 % (p = 0.075) and CH4 mmol/g digestible organic matter by 71 % (p = 0.037). 0.12× MgO2 reduced CH4 volume by 25 % (p > 0.05) without affecting any other parameters. Acetate-to-propionate ratios were reduced by treatments in both experiments (p < 0.01). Molar proportions of acetate and butyrate were reduced, while propionate and valerate were increased in UHP treatments. High concentrations of UHP affected the degradation of neutral detergent fibre in the forage substrate. Future in vitro work should investigate alternative slow-release oxygen sources aimed at prolonging CH4 suppression.

Antimicrobials offered from nature: Peroxidase-catalyzed systems and their mimics.

The control of antimicrobial resistance requires the development of novel antimicrobial alternatives and naturally occurring peroxidase-catalyzed systems may be of great value in this era of emerging antimicrobial resistance. In the peroxidase system, a peroxidase enzyme catalyzes the oxidation of a halide/pseudohalide, at the expense of hydrogen peroxide, to generate reactive products with broad antimicrobial properties. The appropriate use of peroxidase systems needs a better understanding of the identities and properties of the generated antimicrobial oxidants, specific targets in bacterial cells, their mode of action and the factors favoring or limiting their activity. Here, the ABCs (antibacterial activity, bacterial "backtalk" and cytotoxicity) of these systems and their mimics are discussed. Particular attention is paid to the concomitant use of thiocyanate and iodide dual substrates in peroxidase/peroxidase-free systems with implications on their antimicrobial activity. This review also provides a summary of actual applications of peroxidase systems as bio-preservatives in oral healthcare, milk industry, food/feed specialties and related products, mastitis and wound treatment; lastly, this review points to opportunities for further research and potential applications.

Mutation rate and efflux response of bacteria exposed to a novel antimicrobial iodo-thiocyanate complex.

OBJECTIVES: Antimicrobials, at sub-lethal concentrations, can act as selectors and promoters of resistance by increasing mutation rates. We measured the rate of Escherichia coli mutation from levofloxacin (LVX) sensitivity to resistance when it was grown under the near-lethal challenge of the novel biocidal iodo-thiocyanate complex (ITC). Another relevant factor affecting the emergence of antimicrobial resistance is the role of efflux pumps. Consequently, we evaluated whether ITC could potentially be a substrate for efflux pumps, and thus that efflux-mediated resistance could arise towards ITC. METHODS: The mutation rate was measured by fluctuation analysis, when multiple parallel E. coli cultures were grown in the absence and presence of ITC. Then the mutational events, which occurred independently in each culture, were scored by plating the fraction of the culture in LVX-selective solid media and compared with the total cell number. To detect if ITC is a substrate for efflux pumps, minimum inhibitory concentrations (MICs) were determined against Pseudomonas aeruginosa in the absence and presence of the efflux pump inhibitor (EPI). RESULTS: We have found that the E. coli exposed to the near-lethal level of ITC had a slight, but not significant, increase in mutation rate compared with unexposed cultures. Furthermore, the presence of EPI decreased the MIC of ITC by a modest 2-fold, showing that ITC was not a target for efflux pumps. CONCLUSIONS: ITC usage most likely will not promote resistance development via increased mutation rates, and efflux-mediated resistance emergence to it is less likely than for some other antimicrobials.

Continuous culture of Escherichia coli, under selective pressure by a novel antimicrobial complex, does not result in development of resistance.

We attempted to generate de novo resistance to a newly described biocidal complex, ITC (iodo-thiocyanate complex), and to levofloxacin (LVX) in Escherichia coli ATCC 25922, by means of selective chemostat culture. We measured resistance by determining the minimum inhibitory concentrations (MICs) for these agents. E. coli underwent 20-day parallel adaptive evolution routes under no antimicrobial selection, and gradually increasing ITC and LVX selection pressure. Long-term exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based comparisons of ITC-challenged and unchallenged bacterial populations. Moreover, the exposed E. coli population could not survive a 2 × MIC challenge of ITC. By contrast, resistance to LVX was rapidly induced (on day 1 the MIC had increased 16-fold), selected for (by day 14 the MIC had increased 64-fold) and enriched with a highly characteristic genome mutational pattern. WGS of this evolving population revealed that the majority of mutations appeared in the genes of LVX target proteins (GyrA, ParC, ParE) and drug influx (OmpF). This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.

In vitro comparative cytotoxicity study of a novel biocidal iodo-thiocyanate complex.

Novel biocides, which avoid the induction of cross-resistance to antibiotics, are an urgent societal requirement. Here, we compared the cytotoxic and bactericidal effects of a new antimicrobial agent, the iodo-thiocyanate complex (ITC), with those of the common antiseptics, hydrogen peroxide (H2O2), povidone iodine (PVP-I) and Lugol's iodine (Lugol). The antimicrobials were co-incubated for 10 min with HeLa and Escherichia coli cells in the presence and absence of organic matter (Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum). The cytotoxic concentrations of ITC were equivalent to its bactericidal concentrations (7.8 µg ml-1). By contrast, cytotoxic effects of H2O2, PVP-I and Lugol were apparent at concentrations lower than their bactericidal concentrations (250, 250 and 125 µg ml-1, respectively). The cellular effects of ITC were not quenched by organic matter, unlike the other antiseptics. ITC, PVP-I and Lugol had hemolytic effect on horse erythrocytes at high concentrations, while H2O2 showed no hemolysis. ITC, at 30 or 300 µg ml-1, did not cause DNA breakage in HeLa cells as assessed by an in vitro comet assay in the absence of S9 metabolic activation, whereas H2O2 caused extensive single-strand DNA breaks. The pronounced antimicrobial potency of ITC and its favorable cytotoxicity profile suggests that ITC should be considered for antiseptic applications.

Antibacterial Potential of an Antimicrobial Agent Inspired by Peroxidase-Catalyzed Systems.

Antibiotic resistance is an increasingly serious threat to global health. Consequently, the development of non-antibiotic based therapies and disinfectants, which avoid induction of resistance, or cross-resistance, is of high priority. We report the synthesis of a biocidal complex, which is produced by the reaction between ionic oxidizable salts-iodide and thiocyanate-in the presence of hydrogen peroxide as an oxidation source. The reaction generates bactericidal reactive oxygen and iodine species. In this study, we report that the iodo-thiocyanate complex (ITC) is an effective bactericidal agent with activity against planktonic and biofilm cells of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and methicillin-resistant S. aureus) bacteria. The minimum bactericidal concentrations and the minimum biofilm eradication concentrations of the biocidal composite were in the range of 7.8-31.3 and 31.3-250 µg ml-1, respectively. As a result, the complex was capable to cause a rapid cell death of planktonic test cultures at between 0.5 and 2 h, and complete eradication of dual and mono-species biofilms between 30 s and 10 min. Furthermore, the test bacteria, including a MRSA strain, exposed to the cocktail failed to develop resistance after serial passages. The antimicrobial activity of the ITC appears to derive from the combinational effect of the powerful species capable of oxidizing the essential biomolecules of bacteria. The use of this composition may provide an effective and efficient method for killing potential pathogens, as well as for disinfecting and removing biofilm contamination.

Embryonic stem cell technology: applications and uses in functional genomic studies.

In this postgenomic era, the role of functional genomics is becoming increasingly important and playing a key role in this field are embryonic stem cells. These cells are capable of proliferating indefinitely in a pluripotent state and have the potential to differentiate into all somatic cell types. Through a combination of their ease of genetic manipulation and directed in vitro differentiation they have proved themselves to be an extremely valuable tool in functional genomics. Here, some of their applications in functional genomic studies are discussed.

Embryonic stem cells: understanding their history, cell biology and signalling.

Embryonic stem cells offer enormous potential as a source of a variety of differentiated cells for cell therapy, drug discovery and toxicology screening. With the creation of human embryonic stem cell lines we now have a resource with the potential to differentiate into every tissue of the body. To fully harness this resource it is necessary to understand their biology. Here we give a background to their history, describe interesting elements of their cell biology and introduce the underlying signalling mechanisms that control their ability to self-renew and differentiate.