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

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

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

Analysis of educational leadership at rural early-childhood and primary schools: a case study in Teruel (Aragon, Spain)

Rural schools are a distinctive feature in the autonomous community of Aragon, as state-run rural schools are the main cultural institution in small towns. This research was conducted in four grouped rural schools (hereafter referred to by their Spanish acronym, CRAs) in the province of Teruel in 2017. Our objectives included analising the views of management team members and teachers on educational leadership with a special emphasis on the rural school’s differentiating elements. Four CRAs were chosen in the province of Teruel. Three teachers and six members of management teams participated and the study data were collected using a qualitative interview. The content analysis of the interview was conducted using the NVivo program and was organised into two fundamental areas: views of educational leadership and the skills this leadership includes. The general perception of management teams and teachers is positive; they value the effort put into performing the work and understand that both management and CRAs are complex. They also appreciate the management teams’ organisation of training and its contents. This analysis suggests a set of conditions that provides a qualitative insight into management possibilities and limitations in the CRA model

Functional Expression and Characterization of the Highly Promiscuous Lanthipeptide Synthetase SyncM, Enabling the Production of Lanthipeptides with a Broad Range of Ring Topologies

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies

Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reduce ATP production and inhibit the activity of efflux pumps), impairs the respiration chain, promote the production of reactive oxygen species (ROS) in bacterial cells and induce intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa

Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens

The development and dissemination of antibiotic-resistant bacterial pathogens is a growing global threat to public health. Novel compounds and/or therapeutic strategies are required to face the challenge posed, in particular, by Gram-negative bacteria. Here we assess the combined effect of potent cell-wall synthesis inhibitors with either natural or synthetic peptides that can act on the outer-membrane. Thus, several linear peptides, either alone or combined with vancomycin or nisin, were tested against selected Gram-negative pathogens, and the best one was improved by further engineering. Finally, peptide D-11 and vancomycin displayed a potent antimicrobial activity at low μM concentrations against a panel of relevant Gram-negative pathogens. This combination was highly active in biological fluids like blood, but was non-hemolytic and non-toxic against cell lines. We conclude that vancomycin and D- 11 are safe at >50-fold their MICs. Based on the results obtained, and as a proof of concept for the newly observed synergy, a Pseudomonas aeruginosa mouse infection model experiment was also performed, showing a 4 log10 reduction of the pathogen after treatment with the combination. This approach offers a potent alternative strategy to fight (drug-resistant) Gram-negative pathogens in humans and mammals.China Scholarship Council 201306770012European Union (EU)NWO-NACTAR progra

Light yield determination in large sodium iodide detectors applied in the search for dark matter

Application of NaI(Tl) detectors in the search for galactic dark matter particles through their elastic scattering off the target nuclei is well motivated because of the long standing DAMA/LIBRA highly significant positive result on annual modulation, still requiring confirmation. For such a goal, it is mandatory to reach very low threshold in energy (at or below the keV level), very low radioactive background (at a few counts/keV/kg/day), and high detection mass (at or above the 100 kg scale). One of the most relevant technical issues is the optimization of the crystal intrinsic scintillation light yield and the efficiency of the light collecting system for large mass crystals. In the frame of the ANAIS (Annual modulation with NaI Scintillators) dark matter search project large NaI(Tl) crystals from different providers coupled to two photomultiplier tubes (PMTs) have been tested at the Canfranc Underground Laboratory. In this paper we present the estimates of the NaI(Tl) scintillation light collected using full-absorption peaks at very low energy from external and internal sources emitting gammas/electrons, and single-photoelectron events populations selected by using very low energy pulses tails. Outstanding scintillation light collection at the level of 15~photoelectrons/keV can be reported for the final design and provider chosen for ANAIS detectors. Taking into account the Quantum Efficiency of the PMT units used, the intrinsic scintillation light yield in these NaI(Tl) crystals is above 40~photoelectrons/keV for energy depositions in the range from 3 up to 25~keV. This very high light output of ANAIS crystals allows triggering below 1~keV, which is very important in order to increase the sensitivity in the direct detection of dark matter