Bioengineering Lantibiotics for Therapeutic Success

peer-reviewedSeveral examples of highly modified antimicrobial peptides have been described. While many such peptides are non-ribosomally synthesized, ribosomally synthesized equivalents are being discovered with increased frequency. Of the latter group, the lantibiotics continue to attract most attention. In the present review, we discuss the implementation of in vivo and in vitro engineering systems to alter, and even enhance, the antimicrobial activity, antibacterial spectrum and physico-chemical properties, including heat stability, solubility, diffusion and protease resistance, of these compounds. Additionally, we discuss the potential applications of these lantibiotics for use as therapeutics.DF,CH,PC,RR are supported by the Irish Government under the National Development Plan, through a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA14/TIDA/2286) to DF, a SFI Investigator awards to CH and RR (10/IN.1/B3027),SFI-PIfunding(11/PI/1137) to PDC and the Alimentary Pharmabiotic Centre under Grant Number SFI/12/RC/2273

Antimicrobial antagonists against food pathogens; a bacteriocin perspective

peer-reviewedEfforts are continuing to find novel bacteriocins with enhanced specificity and potency. Traditional plating techniques are still being used for bacteriocin screening studies, however, the availability of ever more bacterial genome sequences and the use of in silico gene mining tools have revealed novel bacteriocin gene clusters that would otherwise have been overlooked. Furthermore, synthetic biology and bioengineering-based approaches are allowing scientists to harness existing and novel bacteriocin gene clusters through expression in different hosts and by enhancing functionalities. The same principles apply to bacteriocin producing probiotic cultures and their application to control pathogens in the gut. We can expect that the recent developments on bacteriocins from Lactic Acid Bacteria (LAB) described here will contribute greatly to increased commercialisation of bacteriocins in food systems.This work was funded by the Alimentary Pharmabiotic Centre, a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI (grant no. 12/RC/2273

Fermented beverages with health-promoting potential: Past and future perspectives

peer-reviewedFermentation is an ancient form of food preservation, which also improves the nutritional content of foods. In many regions of the world, fermented beverages have become known for their health-promoting attributes. In addition to harnessing traditional beverages for commercial use, there have recently been innovative efforts to develop non-dairy probiotic fermented beverages from a variety of substrates, including soy milk, whey, cereals and vegetable and fruit juices. On the basis of recent developments, it is anticipated that fermented beverages will continue to be a significant component within the functional food market

The Square Root Depth Wave Equations

We introduce a set of coupled equations for multilayer water waves that removes the ill-posedness of the multilayer Green-Naghdi (MGN) equations in the presence of shear. The new well-posed equations are Hamiltonian and in the absence of imposed background shear they retain the same travelling wave solutions as MGN. We call the new model the Square Root Depth equations, from the modified form of their kinetic energy of vertical motion. Our numerical results show how the Square Root Depth equations model the effects of multilayer wave propagation and interaction, with and without shear.Comment: 10 pages, 5 figure

Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources

peer-reviewedKefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.The Alimentary Pharmabiotic Centre is a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI CSET grant APC CSET 2 grant 07/CE/B1368

A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and ß-lactam resistance genes in the gut microbiota

peer-reviewedBackground: The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes. Results: The strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and β-lactam (including bla OXA, bla TEM, bla SHV and bla CTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes. Conclusions: This study has demonstrated that the gut can be a significant source of aminoglycoside and β-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes.Fiona Fouhy is in receipt of an Irish Research Council EMBARK scholarship and is a Teagasc Walsh fellow. Research in the PDC laboratory is also supported by the Irish Government under the National Development Plan through the Science Foundation Ireland Investigator award 11/PI/113

The Prevalence and Control of Bacillus and Related Spore-Forming Bacteria in the Dairy Industry

peer-reviewedMilk produced in udder cells is sterile but due to its high nutrient content, it can be a good growth substrate for contaminating bacteria. The quality of milk is monitored via somatic cell counts and total bacterial counts, with prescribed regulatory limits to ensure quality and safety. Bacterial contaminants can cause disease, or spoilage of milk and its secondary products. Aerobic spore-forming bacteria, such as those from the genera Sporosarcina, Paenisporosarcina, Brevibacillus, Paenibacillus, Geobacillus and Bacillus, are a particular concern in this regard as they are able to survive industrial pasteurization and form biofilms within pipes and stainless steel equipment. These single or multiple-species biofilms become a reservoir of spoilage microorganisms and a cycle of contamination can be initiated. Indeed, previous studies have highlighted that these microorganisms are highly prevalent in dead ends, corners, cracks, crevices, gaskets, valves and the joints of stainless steel equipment used in the dairy manufacturing plants. Hence, adequate monitoring and control measures are essential to prevent spoilage and ensure consumer safety. Common controlling approaches include specific cleaning-in-place processes, chemical and biological biocides and other novel methods. In this review, we highlight the problems caused by these microorganisms, and discuss issues relating to their prevalence, monitoring thereof and control with respect to the dairy industry.NG is funded by the Teagasc Walsh Fellowship Scheme and through the Irish Dairy Levy funded project ‘Thermodur-Out.

Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?

peer-reviewedBacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings.KE, DF, CH, PC, MR, RR are supported by the Irish Government under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries and Food, Ireland (DAFM 13/F/462) to PC and MR, a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA 14/TIDA/2286) to DF, SFI-PI funding (11/PI/1137) to PDC and the APC Microbiome Insitute under Grant Number SFI/12/RC/2273

Continuous and discrete Clebsch variational principles

The Clebsch method provides a unifying approach for deriving variational principles for continuous and discrete dynamical systems where elements of a vector space are used to control dynamics on the cotangent bundle of a Lie group \emph{via} a velocity map. This paper proves a reduction theorem which states that the canonical variables on the Lie group can be eliminated, if and only if the velocity map is a Lie algebra action, thereby producing the Euler-Poincar\'e (EP) equation for the vector space variables. In this case, the map from the canonical variables on the Lie group to the vector space is the standard momentum map defined using the diamond operator. We apply the Clebsch method in examples of the rotating rigid body and the incompressible Euler equations. Along the way, we explain how singular solutions of the EP equation for the diffeomorphism group (EPDiff) arise as momentum maps in the Clebsch approach. In the case of finite dimensional Lie groups, the Clebsch variational principle is discretised to produce a variational integrator for the dynamical system. We obtain a discrete map from which the variables on the cotangent bundle of a Lie group may be eliminated to produce a discrete EP equation for elements of the vector space. We give an integrator for the rotating rigid body as an example. We also briefly discuss how to discretise infinite-dimensional Clebsch systems, so as to produce conservative numerical methods for fluid dynamics

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

peer-reviewedThe bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers