Component release after exposure of Staphylococcus aureus cells to pulsed electric fields

The objective of this work was to get further insights on the mechanism of inactivation of bacterial cells by pulsed electric fields (PEF) through the study of the release of intracellular components after exposing Staphylococcus aureus cells in McIvlaine buffer (pH 7.0, 2 mS/cm) to PEF treatments of different intensity (18 and 25 kV/cm) and treatment times (from 20 to 400 mu s). Release of most compounds, except proteins, was almost immediate after the treatment, but the relative amount released depended on the molecule studied. A good correlation between the release of the smallest components studied (particularly ions) and membrane permeabilization (as measured by NaCl sensitization and PI entry) was observed. On the other hand, results obtained suggested that S. aureus inactivation by PEF would be related to the exit of cytoplasmic proteins of a molecular weight higher than 6 kDa. Results obtained in this work indicated that increasing PEF treatment time would reduce the capability of S. aureus cells to repair the electropores formed and suggested that this might be due to the formation of pores of a larger size, which S. aureus cells would be unable to reseal in a situation of homeostasis loss. Industrial relevance: Results reported here can help to design more effective treatments for microbial inactivation using PEF on food, and therefore facilitate its industrial implementation

Genotypic and phenotypic characterization of a Salmonella Typhimurium strain resistant to pulsed electric fields

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain

Protective effect of glutathione on Escherichia coli cells upon lethal heat stress

Heat treatments are widely used by the food industry to obtain safe and stable products, therefore a deeper knowledge of its mode of action on microorganisms would allow a better profit of this technology. Heat shows a multitarget mechanism of action on bacteria, affecting various cellular structures, and causing unbalances in several homeostatic parameters. The aim of this work was to study the effect of glutathione on bacterial survival against heat treatments, in order to acquire knowledge about the mode of action of heat on bacterial cells. Cells were treated in presence or absence of added glutathione and the level of inactivation, soluble protein concentration, enzymatic activities, intracellular ROS level and membrane damages were studied. Results showed that glutathione protected microorganisms against heat inactivation. Moreover, glutathione in the treatment medium preserved intracellular enzyme activity, membrane structure and reduced ROS detection. Besides, glutathione decreased sublethal injury in E. coli. Data presented in this work add new knowledge about bacterial inactivation and survival by heat

Experimental and computational analysis of microbial inactivation in a solid by ohmic heating using pulsed electric fields

Pulsed electric field technology (PEF) has traditionally been used as a technique to inactivate microorganisms in liquid foods at temperatures below those used in heat treatments; however, application of high-intensity PEF (E>1 kV/cm) at high frequencies (>10 Hz) can allow rapid and volumetric solid food electrical heating in order to replace traditional convection/conduction heating that progresses from the heating medium to the inside of the product. This investigation is the first one to evaluate the inactivation of Salmonella Typhimurium 878 in a solid product (cylinder of technical agar used as reference solid) by applying PEF treatments (2.5 and 3.75 kV/cm, and up to 9000 microseconds) at 50 Hz. The evolution of temperature in different locations of the agar cylinder was measured by observing the variability of heating rates depending on location and PEF intensity. Microbial inactivation was determined and compared with isothermal heat treatments that predicted similar inactivation values, but did not detect additional inactivation. Computational analysis enabled us to predict temperature and microbial inactivation for any spatial and temporal distribution of the cylinder agar by detecting the coldest point in the transition zone between the high-voltage electrode, the agar, and the plastic container of the treatment chamber. In order to evaluate the variability of the temperature, computational predictions were done each 0.5-mm. The difference between the coldest and the hottest point (e.g. at the center of the cylinder) resulted in around 10 °C and 10 second variation in temperature and processing time, respectively. In any case, it was possible to obtain 5-log10-reductions after 60 s of PEF treatments when using 2.5 kV/cm and 50% reduction for 3.75 kV/cm. These results suggested the potential of PEF technology as a rapid heating system based on ohmic heating for microbial inactivation in solid food products

Pulsed Electric Fields (PEF) applications in the inactivation of parasites in food

Background Parasites are concerning food-borne pathogens. Some of them are currently not being routinely controlled in food, probably because their burden on public health is underestimated and the relative importance of different transmission routes is not completely known. Parasitic incidences could be avoided if preventive technologies were applied during food processing. Effective inactivation treatments are currently based on heat or freezing, but their side effects collide head-on with current consumer trends and new culinary habits. Scope and approach This review describes the potential application of Pulsed Electric Field (PEF) technology in the control of food-borne parasites, with the aim of reducing the viability and infectivity of parasite transmission stages without affecting food quality. Results of published studies performed on different media are critically analyzed and factors affecting the outcomes are examined. Key findings and conclusions Recent studies on the topic demonstrate the feasibility of PEF as an alternative to traditional freezing processes for the inactivation of Anisakis in fish. The development of new PEF equipment is advancing at a rapid pace, allowing for food treatment at a scale that would have been unimaginable some years ago. A review of more basic-science studies carried out on buffer media would contribute to progress in addressing the underlying drawbacks that remain to be solved. Thoroughly different fields (parasitology, physics, food engineering, water sanitation, etc.) should converge to achieve the industrial implementation of PEF for the inactivation of food-borne parasites

Gaseous time projection chambers for rare event detection: Results from the T-REX project. II. Dark matter

As part of the T-REX project, a number of R&D and prototyping activities have been carried out during the last years to explore the applicability of Micromegas-read gaseous TPCs in rare event searches like double beta decay (DBD), axion research and low-mass WIMP searches. While in the companion paper we focus on DBD, in this paper we focus on the results regarding the search for dark matter candidates, both axions and WIMPs. Small ultra-low background Micromegas detectors are used to image the x-ray signal expected in axion helioscopes like CAST at CERN. Background levels as low as $0.8\times 10^{-6}$ c keV$^{-1}$cm$^{-2}$s$^{-1}$ have already been achieved in CAST while values down to $\sim10^{-7}$ c keV$^{-1}$cm$^{-2}$s$^{-1}$ have been obtained in a test bench placed underground in the Laboratorio Subterr\'aneo de Canfranc. Prospects to consolidate and further reduce these values down to $\sim10^{-8}$ c keV$^{-1}$cm$^{-2}$s$^{-1}$will be described. Such detectors, placed at the focal point of x-ray telescopes in the future IAXO experiment, would allow for 10$^5$ better signal-to-noise ratio than CAST, and search for solar axions with $g_{a\gamma}$ down to few 10$^{12}$ GeV$^{-1}$, well into unexplored axion parameter space. In addition, a scaled-up version of these TPCs, properly shielded and placed underground, can be competitive in the search for low-mass WIMPs. The TREX-DM prototype, with $\sim$0.300 kg of Ar at 10 bar, or alternatively $\sim$0.160 kg of Ne at 10 bar, and energy threshold well below 1 keV, has been built to test this concept. We will describe the main technical solutions developed, as well as the results from the commissioning phase on surface. The anticipated sensitivity of this technique might reach $\sim10^{-44}$ cm$^2$ for low mass ($<10$ GeV) WIMPs, well beyond current experimental limits in this mass range.Comment: Published in JCAP. New version with erratum incorporated (new figure 14

Radiopurity of Micromegas readout planes

Micromesh Gas Amplification Structures (Micromegas) are being used in an increasing number of Particle Physics applications since their conception fourteen years ago. More recently, they are being used or considered as readout of Time Projection Chambers (TPCs) in the field of Rare Event searches (dealing with dark matter, axions or double beta decay). In these experiments, the radiopurity of the detector components and surrounding materials is measured and finely controlled in order to keep the experimental background as low as possible. In the present paper, the first measurement of the radiopurity of Micromegas planes obtained by high purity germanium spectrometry in the low background facilities of the Canfranc Underground Laboratory (LSC) is presented. The obtained results prove that Micromegas readouts of the microbulk type are currently manufactured with radiopurity levels below 30 microBq/cm2 for Th and U chains and ~60 microBq/cm2 for 40K, already comparable to the cleanest detector components of the most stringent low background experiments at present. Taking into account that the studied readouts were manufactured without any specific control of the radiopurity, it should be possible to improve these levels after dedicated development.Comment: 15 pages, 2 figure

The X-ray Telescope of CAST

The Cern Axion Solar Telescope (CAST) is in operation and taking data since 2003. The main objective of the CAST experiment is to search for a hypothetical pseudoscalar boson, the axion, which might be produced in the core of the sun. The basic physics process CAST is based on is the time inverted Primakoff effect, by which an axion can be converted into a detectable photon in an external electromagnetic field. The resulting X-ray photons are expected to be thermally distributed between 1 and 7 keV. The most sensitive detector system of CAST is a pn-CCD detector combined with a Wolter I type X-ray mirror system. With the X-ray telescope of CAST a background reduction of more than 2 orders off magnitude is achieved, such that for the first time the axion photon coupling constant g_agg can be probed beyond the best astrophysical constraints g_agg < 1 x 10^-10 GeV^-1.Comment: 19 pages, 25 figures and images, replaced by the revised version accepted for publication in New Journal of Physic