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Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50–100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

Bacterial biohybrids, composed of self-propelling bacteria carrying micro/nanoscale materials, can deliver their payload to specific regions under magnetic control, enabling additional frontiers in minimally invasive medicine. However, current bacterial biohybrid designs lack high-throughput and facile construction with favorable cargoes, thus underperforming in terms of propulsion, payload efficiency, tissue penetration, and spatiotemporal operation. Here, we report magnetically controlled bacterial biohybrids for targeted localization and multistimuliresponsive drug release in three-dimensional (3D) biological matrices. Magnetic nanoparticles and nanoliposomes loaded with photothermal agents and chemotherapeutic molecules were integrated onto Escherichia coil with similar to 90% efficiency. Bacterial biohybrids, outperforming previously reported E. coli-based microrobots, retained their original motility and were able to navigate through biological matrices and colonize tumor spheroids under magnetic fields for on-demand release of the drug molecules by near-infrared stimulus. Our work thus provides a multifunctional microrobotic platform for guided locomotion in 3D biological networks and stimuli-responsive delivery of therapeutics for diverse medical applications., Max Planck Society, This study was funded by the Max Planck Society.

Background: Pathogen genomics is increasingly being translated from the research setting into the activities of public health professionals operating at different levels. This survey aims to appraise the literacy level and gather the opinions of public health experts and allied professionals working in the field of infectious diseases in Belgium concerning the implementation of next-generation sequencing (NGS) in public health practice. Methods: In May 2019, Belgian public health and healthcare professionals were invited to complete an online survey containing eight main topics including background questions, general attitude towards pathogen genomics for public health practice and main concerns, genomic literacy, current and planned NGS activities, place of NGS in diagnostic microbiology pathways, data sharing obstacles, end-user requirements, and key drivers for the implementation of NGS. Descriptive statistics were used to report on the frequency distribution of multiple choice responses whereas thematic analysis was used to analyze free text responses. A multivariable logistic regression model was constructed to identify important predictors for a positive attitude towards the implementation of pathogen genomics in public health practice. Results: 146 out of the 753 invited public health professionals completed the survey. 63% of respondents indicated that public health agencies should be using genomics to understand and control infectious diseases. Having a high level of expertise in the field of pathogen genomics was the strongest predictor of a positive attitude (OR = 4.04, 95% CI = 1.11 - 17.23). A significantly higher proportion of data providers indicated to have followed training in the field of pathogen genomics compared to data end-users (p < 0.001). Overall, 79% of participants expressed interest in receiving further training. Main concerns were related to the cost of sequencing technologies, data sharing, data integration, interdisciplinary working, and bioinformatics expertise. Conclusions: Belgian health professionals expressed favorable views about implementation of pathogen genomics in their work activities related to infectious disease surveillance and control. They expressed the need for suitable training initiatives to strengthen their competences in the field. Their perception of the utility and feasibility of pathogen genomics for public health purposes will be a key driver for its further implementation., SCOPUS: ar.j, info:eu-repo/semantics/published

The increasing emergence of drug-resistant fungal infections has necessitated a search for new compounds capable of combating fungal pathogens of plants, animals, and humans. Microorganisms represent the main source of antibiotics with applicability in agriculture and in the clinic, but many aspects of their metabolic potential remain to be explored. This report describes the discovery and characterization of a new antifungal compound, solanimycin, produced by a hybrid polyketide/nonribosomal peptide (PKS/NRPS) system in Dickeya solani, the enterobacterial pathogen of potato. Solanimycin was active against a broad range of plant-pathogenic fungi of global economic concern and the human pathogen Candida albicans. The genomic cluster responsible for solanimycin production was defined and analyzed to identify the corresponding biosynthetic proteins, which include four multimodular PKS/NRPS proteins and several tailoring enzymes. Antifungal production in D. solani was enhanced in response to experimental conditions found in infected potato tubers and high-density fungal cultures. Solanimycin biosynthesis was cell density dependent in D. solani and was controlled by both the ExpIR acyl-homoserine lactone and Vfm quorum-sensing systems of the bacterial phytopathogen. The expression of the solanimycin cluster was also regulated at the post-transcriptional level, with the regulator RsmA playing a major role. The solanimycin biosynthetic cluster was conserved across phylogenetically distant bacterial genera, and multiple pieces of evidence support that the corresponding gene clusters were acquired by horizontal gene transfer. Given its potent broad-range antifungal properties, this study suggests that solanimycin and related molecules may have potential utility for agricultural and clinical exploitation. IMPORTANCE Fungal infections represent a major clinical, agricultural, and food security threat worldwide, which is accentuated due to the difficult treatment of these infections. Microorganisms represent a prolific source of antibiotics, and current data support that this enormous biosynthetic potential has been scarcely explored. To improve the performance in the discovery of novel antimicrobials, there is a need to diversify the isolation niches for new antibiotic-producing microorganisms as well as to scrutinize novel phylogenetic positions. With the identification of the antifungal antibiotic solanimycin in a broad diversity of phytopathogenic Dickeya spp., we provide further support for the potential of plant-associated bacteria for the biosynthesis of novel antimicrobials. The complex regulatory networks involved in solanimycin production reflect the high metabolic cost of bacterial secondary metabolism. This metabolic regulatory control makes many antibiotics cryptic under standard laboratory conditions, and mimicking environmental conditions, as shown here, is a strategy to activate cryptic antibiotic clusters., The work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; UK) through award BB/N008081/1 to G.P.C.S., F.L., and J.M. M.A.M. was supported by an EU Marie-Curie Intra-European Fellowship for Career Development (FP7-PEOPLE-2011-IEF) grant number 298003. Work in the Matilla laboratory was supported by grant from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-103972GA-I00). Work with plant pathogens was carried out under DEFRA license no. 50864/197900/1.

The inhibition of Escherichia coli ATCC 25922 (E. coli), Staphylococcus aureus ATCC6538 (S. aureus), Salmonella Enteritidis ATCC 13076 (S. Enteritidis), and Listeria monocytogenes DSM12464 (L. monocytogenes) is one of the main aims of the food industry. This study was the first in which the use of ultrasound and microwave power were applied to optimize the values of the bioactive components, amino acids, and mineral compositions of tangerine juice and to inhibit Escherichia coli, Staphylococcus aureus, Salmonella Enteritidis, and Listeria monocytogenes. The response surface methodology (RSM) was used to describe the inactivation kinetics, and the effects of ultrasound treatment time (X1: 12–20 min), ultrasound amplitude (X2:60–100%), microwave treatment time (X3: 30–40 s), and microwave power (X4:200–700 W). The optimum parameters applied to a 5-log reduction in E. coli were determined as ultrasound (12 min, 60%) and microwave (34 s, 700 W). The optimum condition ultrasound–microwave treatment was highly effective in tangerine juice, achieving up to 5.27, 5.12, and 7.19 log reductions for S. aureus, S. Enteritidis, and L. monocytogenes, respectively. Ultrasound–microwave treatment increased the total phenolic compounds and total amino acids. While Cu, K, Mg, and Na contents were increased, Fe and Ca contents were lower in the UM-TJ (ultrasound–microwave-treated tangerine juice) sample. In this case, significant differences were detected in the color values of ultrasound–microwave-treated tangerine juice (UM-TJ) (p < 0.05). The results of this study showed that ultrasound–microwave treatment is a potential alternative processing and preservation technique for tangerine juice, resulting in no significant quality depreciation.

The detection and quantification of sublethal injury (SI) of pathogenic microorganisms has become a common procedure when assessing the efficiency of microbial inactivation treatments. However, while a plethora of studies investigates SI in function of time, no suitable modelling procedure for SI data has been proposed thus far. In this study, a new SI model structure was developed that relies on existing microbial inactivation models. This model is based on the description of inactivation kinetics between the subpopulations of healthy, sublethally injured and dead cells. The model was validated by means of case studies on previously published results, modelled by different inactivation models, i.e., (i) log-linear inactivation, (ii) biphasic inactivation, and (iii) log-linear inactivation with tailing. Results were compared to those obtained by the traditional method that relies on calculating SI from independent inactivation models on non-selective and selective media. The log-linear inactivation case study demonstrated that the SI model is equivalent to the use of independent models when there can be no mistake in calculating SI. The biphasic inactivation case study illustrated how the SI model avoids unrealistic calculations of SI that would otherwise occur. The final case study on log-linear inactivation with tailing clarified that the SI model provides a more mechanistic description than the independent models, in this case allowing the reduction of the number of model parameters. As such, this paper provides a comprehensive overview of the potential and applications for the newly presented SI model.

Bacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific ‘universal stress responders’, that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex)., Nature Communications, 12 (1), ISSN:2041-1723

Novel processing methods such as cold atmospheric plasma (CAP) and natural antimicrobials like nisin, are of interest to replace traditional food decontamination approaches as, due to their mild nature, they can maintain desirable food characteristics, i.e., taste, texture, and nutritional content. However, the microbial growth characteristics (planktonic growth/surface colonies) and/or the food structure itself (liquid/solid surface) can impact the inactivation efficacy of these novel processing methods. More specifically, cells grown as colonies on a solid(like) surface experience a completely different growth environment to cells grown planktonically in liquid, and thus could display a different response to novel processing treatments through stress adaptation and/or cross protection mechanisms. The order in which combined treatments are applied could also impact their efficacy, especially if the mechanisms of action are complementary. This work presents a fundamental study on the efficacy of CAP and nisin, alone and combined, as affected by food system structure. More specifically, Listeria innocua was grown planktonically (liquid broth) or on a viscoelastic Xanthan gum gel system (1.5% w/v) and treated with CAP, nisin, or a combination of the two. Both the inactivation system, i.e., liquid versus solid(like) surface and the growth characteristics, i.e., planktonic versus colony growth, were shown to impact the treatment efficacy. The combination of nisin and CAP was more effective than individual treatments, but only when nisin was applied before the CAP treatment. This study provides insight into the environmental stress response/adaptation of L. innocua grown on structured systems in response to natural antimicrobials and novel processing technologies, and is a step towards the faster delivery of these food decontamination methods from the bench to the food industry. ispartof: INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY vol:337 ispartof: location:Netherlands status: published

Do islands harbour less diverse disease communities than mainland? The island biogeography theory predicts more diverse communities on mainland than on islands due to more niches, more diverse habitats and availability of greater range of hosts. We compared bacteria prevalences of Campylobacter, Chlamydia and Salmonella in cloacal samples of a small shorebird, the Kentish plover (Charadrius alexandrinus) between two island populations of Macaronesia and two mainland locations in the Iberian Peninsula. Bacteria were found in all populations but, contrary to the expectations, prevalences did not differ between islands and mainland. Females had higher prevalences than males for Salmonella and when three bacteria genera were pooled together. Bacteria infection was unrelated to bird’s body condition but females from mainland were heavier than males and birds from mainland were heavier than those from islands. Abiotic variables consistent throughout breeding sites, like high salinity that is known to inhibit bacteria growth, could explain the lack of differences in the bacteria prevalence between areas. We argue about the possible drivers and implications of sex differences in bacteria prevalence in Kentish plovers

Molecules 25(8), 1940 - (2020). doi:10.3390/molecules25081940, NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a ‘sink’ for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains., Published by MDPI7969, Basel

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, fifth in the series, evaluates the safety of NFCs containing linalool and/or other characteristic mono- and sesquiterpenoid tertiary alcohols and esters using the safety evaluation procedure published by the FEMA Expert Panel in 2005 and updated in 2018. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of the chemical constituents of each NFC into well-defined congeneric groups. The safety of each NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of both the constituent congeneric groups and the NFCs. Sixteen NFCs, derived from the Lavandula, Aniba, Elettaria, Daucus, Salvia, Coriandrum, Ribes, Guaiacum/Bulnesia, Citrus, Pogostemon, Melaleuca and Michelia genera, were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

Bacteria commonly form dense biofilms encased in extracellular polymeric substances (EPS). Biofilms are often extremely tolerant to antimicrobials but their reliance on shared EPS may also be a weakness as social evolution theory predicts that inhibiting shared traits can select against resistance. Here we show that EPS of Salmonella biofilms is a cooperative trait whose benefit is shared among cells, and that EPS inhibition reduces both cell attachment and antimicrobial tolerance. We then compare an EPS inhibitor to conventional antimicrobials in an evolutionary experiment. While resistance against conventional antimicrobials rapidly evolves, we see no evolution of resistance to EPS inhibition. We further show that a resistant strain is outcompeted by a susceptible strain under EPS inhibitor treatment, explaining why resistance does not evolve. Our work suggests that targeting cooperative traits is a viable solution to the problem of antimicrobial resistance. ispartof: Nature Communications vol:11 issue:1 ispartof: location:England nrpages: 11 status: published

Bacteria-host interactions are characterized by the delivery of bacterial virulence factors, i.e., effectors, into host cells where they counteract host immunity and exploit host responses allowing bacterial survival and spreading. These effectors are translocated into host cells by means of dedicated secretion systems such as the type 3 secretion system (T3SS). A comprehensive understanding of effector translocation in a spatio-temporal manner is of critical importance to gain insights into an effector’s mode of action. Various approaches have been developed to understand timing and order of effector translocation, quantities of translocated effectors and their subcellular localization upon translocation into host cells. Recently, the existing toolset has been expanded by newly developed state-of-the art methods to monitor bacterial effector translocation and dynamics. In this review, we elaborate on reported methods and discuss recent advances and shortcomings in this area of tracking bacterial effector translocation.

Traditionally, predictive growth models for food pathogens are developed based on experiments in broth media, resulting in models which do not incorporate the influence of food microstructure. The use of model systems with various microstructures is a promising concept to get more insight into the influence of food microstructure on microbial dynamics. By means of minimal variation of compositional and physicochemical factors, these model systems can be used to study the isolated effect of certain microstructural aspects on microbial growth, survival and inactivation. In this study, the isolated effect on microbial growth dynamics of Listeria monocytogenes of two food microstructural aspects and one aspect influenced by food microstructure were investigated, i.e., the nature of the food matrix, the presence of fat droplets, and microorganism growth morphology, respectively. To this extent, fish-based model systems with various microstructures were used, i.e., a liquid, a second more viscous liquid system containing xanthan gum, an emulsion, an aqueous gel, and a gelled emulsion. Growth experiments were conducted at 4 and 10 °C, both using homogeneous and surface inoculation (only for the gelled systems). Results regarding the influence of the growth morphology indicated that the lag phase of planktonic cells in the liquid system was similar to the lag phase of submerged colonies in the xanthan system. The lag phase of submerged colonies in each gelled system was considerably longer than the lag phase of surface colonies on these respective systems. The maximum specific growth rate of planktonic cells in the liquid system was significantly lower than for submerged colonies in the xanthan system at 10 °C, while no significant differences were observed at 4 °C. The maximum cell density was higher for submerged colonies than for surface colonies. The nature of the food matrix only exerted an influence on the maximum specific growth rate, which was significantly higher in the viscous systems than in the gelled systems. The presence of a small amount of fat droplets improved the growth of L. monocytogenes at 4 °C, resulting in a shorter lag phase and a higher maximum specific growth rate. The obtained results could be useful in the determination of a set of suitable microstructural parameters for future predictive models that incorporate the influence of food microstructure on microbial dynamics. ispartof: International Journal Of Food Microbiology vol:283 pages:7-13 ispartof: location:SPAIN, Cordoba status: published

The second plague pandemic, caused by Yersinia pestis, devastated Europe and the nearby regions between the 14th and 18th centuries AD. Here we analyse human remains from ten European archaeological sites spanning this period and reconstruct 34 ancient Y. pestis genomes. Our data support an initial entry of the bacterium through eastern Europe, the absence of genetic diversity during the Black Death, and low within-outbreak diversity thereafter. Analysis of post-Black Death genomes shows the diversification of a Y. pestis lineage into multiple genetically distinct clades that may have given rise to more than one disease reservoir in, or close to, Europe. In addition, we show the loss of a genomic region that includes virulence-related genes in strains associated with late stages of the pandemic. The deletion was also identified in genomes connected with the first plague pandemic (541–750 AD), suggesting a comparable evolutionary trajectory of Y. pestis during both events., The second plague pandemic, caused by Yersinia pestis, started in the 14th century and recurred in Europe until the 18th century. Here, the authors reconstruct ancient Y. pestis genomes from human remains spanning this period, shedding light into the phylogeography and population structure of the European strains.

Bacteria are confronted with a multitude of stressors when occupying niches within the host. These stressors originate from host defense mechanisms, other bacteria during niche competition or result from physiological challenges such as nutrient limitation. To counteract these stressors, bacteria have developed a stress-induced network to mount the adaptations required for survival. These stress-induced adaptations include the release of membrane vesicles from the bacterial envelope. Membrane vesicles can provide bacteria with a plethora of immediate and ultimate benefits for coping with environmental stressors. This review addresses how membrane vesicles aid Gram-negative bacteria to cope with host-associated stress factors, focusing on vesicle biogenesis and the physiological functions. As many of the pathways, that drive vesicle biogenesis, confer we propose that shedding of membrane vesicles by Gram-negative bacteria entails an integrated part of general stress responses.

The aim of the study was to develop marination liquids (MLs) enriched with probiotics (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus or their combination) to improve the safety and sensory quality of meat during marination. The total acidity, total phenolic content, antioxidant and antimicrobial activities of MLs were in the range of 0.70-0.92 g tartaric acid/100 mL, 331.00-513.80 mg GAE/L, 71.10%-93.37% and 6.50-10.00 mm, respectively. At the end of the marination, the numbers of Escherichia coli O157: H7, Listeria monocytogenes and Salmonella Typhimurium on meat samples ( approximately equal to 6 log CFU/g) were decreased in the range of 0.7-2.7, 2.1-3.3 and 0.8-2.0 log CFU/g, respectively, depending on the type of ML and the treatment time used. Additionally, meat sample marinated with MLs containing L. casei was the most preferred sample in terms of appearance, color, flavor and general acceptability. These results showed that the existing effects of koruk juice were increased by probiotics.

The uptake of nickel [Ni(II)] by Paenibacillus sp., Methylobacterium sp., Paraburkholderia sp., and Pseudomonas sp. strains isolated from a boreal bog was studied using batch experiments. All strains removed Ni(II) from the solution and the uptake efficiency was affected by the nutrient source, incubation temperature, time, and pH. As highest Ni uptake (with a maximum Kd of 1890 L/kg DW) was recorded for the Pseudomonas sp. strains, these bacteria were used in the following protein expression (SDS-PAGE and MALDI-TOFF), transmission electron microscopy (TEM) and EDS experiments. In addition, Freundlich and Langmuir sorption isotherms were determined. In the Ni(II) treated cells, dense crystalline intra-cellular accumulations were observed in TEM examinations, which were identified as Ni accumulations using EDS. SDS-PAGE and MALDI-TOFF spectra of Ni(II) treated cells showed several changes in the protein profiles, which can indicate active accumulation of Ni in these bacteria. Concurrently, we observed Ni(II) uptake to follow Freundlich and Langmuir isotherms, suggesting straight cellular biosorption in addition to the intra-cellular accumulation. The role of cellular (cell membrane and cell wall) functional groups involved in Ni(II) binding were therefore studied using Fourier transformation infrared spectroscopy. These analyses supported the potential role of the alcoholic hydroxyl, carboxyl and amine groups in Ni(II) binding in these bacteria, therefore suggesting two different Ni(II) uptake mechanisms; (i) intra-cellular accumulation [possibly connected to detoxification of Ni(II)], and (ii) straight biosorption on cell membrane/wall functional groups.

Acoustic wave based sensors have a major impact in the detection of low concentration of biological and chemical agents. Magnetoelastic resonator platforms are particularly interesting due to their low cost and wireless detection process. However, efforts in improving their performance are still focused only in the reduction of the size. In the present study, a new way to increase the sensitivity of magnetoelastic sensor platforms is proposed. This work demonstrates, both theoretically and experimentally, that the mass load sensitivity can be improved by tailoring sensor geometry and mass load position. Triangular and arched triangular shapes have been tested and compared with the traditional rectangular ones. It has been observed an increase in the mass sensitivity of more than 400% in Metglas (R) 2826MB magnetoelastic sensor by using new sensor platform geometries. Even higher sensitivities have been obtained by doing partial coatings on the sensor edge/tip instead of the traditional complete uniform coating. New geometries present an increase up to 6400% in partial coatings. The obtained results clearly show the key role of magnetoelastic resonator platforms geometries in the increase of mass load sensitivity and their importance in the draw of future labor-free and wireless sensors for low mass detection systems. This work was financially supported by the "Ministerio de Economia, Industria y Competitividad" (MAT2016-76739R(AEI/FEDER, UE)) and by the "Gobierno Vasco", under projects IT-630-13 (Basque University Research System Groups), ACTIMAT KK-2018/00099 and muF4 KK-2017/00089 (Elkartek Program). A.C. Lopes thanks to MSCA-IF-2015 (Marie Sklodowska Curie Actions) of the European Union's Horizon 2020 Programme for the received funds under grant agreement no [701852].

Objectives Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. Methods Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. Results The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. Conclusions Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria.

No abstract available.

Antibiotic resistance is posing a continuous threat to global public health and represents a huge burden for society as a whole. In the past decade, the interference with bacterial quorum sensing (QS) (i.e., cell cell communication) mechanisms has extensively been investigated as a valid therapeutic approach in the pursuit of a next generation of antimicrobials. (S)-4,5-Dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule that modulates QS in both Gram-negative and Gram-positive bacteria, is phosphorylated by LsrK, and the resulting phospho-DPD activates QS. We designed and prepared a small library of DPD derivatives, characterized by five different scaffolds, and evaluated their LsrK inhibition in the context of QS interference. SAR studies highlighted the pyrazole moiety as an essential structural element for LsrK inhibition. Particularly, four compounds were found to be micromolar LsrK inhibitors (IC50 ranging between 100 mu M and 500 mu M) encouraging further exploration of novel analogues as potential new antimicrobials.

Resistance to antibiotics is an increasingly serious threat to global public health and its management translates to significant health care costs. The validation of new Gram-negative antibacterial targets as sources for potential new antibiotics remains a challenge for all the scientists working in this field. The interference with bacterial Quorum Sensing (QS) mechanisms represents a potentially interesting approach to control bacterial growth and pursue the next generation of antimicrobials. In this context, our research is focused on the discovery of novel compounds structurally related to (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule able to modulate bacterial QS in both Gram-negative and Gram-positive bacteria. In this study, a practical and versatile synthesis of racemic DPD is presented. Compared to previously reported syntheses, the proposed strategy is short and robust: it requires only one purification step and avoids the use of expensive or hazardous starting materials as well as the use of specific equipment. It is therefore well suited to the synthesis of derivatives for pharmaceutical research, as demonstrated by four series of novel DPD-related compounds described herein.

Systemic immunization with soluble flagellin (sFliC) from Salmonella Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103(+) CD11b(+) cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4(+) T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and CD11c-cre.Irf4(fl/fl) mice (which have reduced numbers of cDC2, particularly intestinal CD103(+) CD11b(+) cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103(+) CD11b(+) cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103(+) CD11b(+) cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103(+) CD11b(+) cDC2 in the induction of Th2-associated responses. CD11c-cre.Irf4(fl/fl) mice showed a reduced primary FliC-specific Th2-associated IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103(+) CD11b(+) cDC2.

The main parameters of magnetoelastic resonators in the detection of chemical (i.e., salts, gases, etc.) or biological (i.e., bacteria, phages, etc.) agents are the sensitivity S (or external agent change magnitude per Hz change in the resonance frequency) and the quality factor Q of the resonance. We present an extensive study on the experimental determination of the Q factor in such magnetoelastic resonant platforms, using three different strategies: (a) analyzing the real and imaginary components of the susceptibility at resonance; (b) numerical fitting of the modulus of the susceptibility; (c) using an exact mathematical expression for the real part of the susceptibility. Q values obtained by the three methods are analyzed and discussed, aiming to establish the most adequate one to accurately determine the quality factor of the magnetoelastic resonance. Ana Catarina Lopes thanks MSCA-IF-2015 (Marie Sklodowska Curie Actions) of the European Union's Horizon 2020 Programme for the received funds under grant agreement no. [701852]. Ariane Sagasti wishes to thank BCMaterials Centre for financial support. Jon Gutierrez, Andoni Lasheras, and Jose Manuel Barandiaran would like to acknowledge the financial support from the Basque Government under the ACTIMAT project (Etortek 2018 program) and Research Groups IT711-13 project. Dimitris Kouzoudis is thankful for financial support under Erasmus + Mobility Agreement between the University of Patras and the University of the Basque Country (UPV/EHU). Technical and human support provided by SGIker (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged.

Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications.

Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications.

Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named "Jerseyvirinae" and consisting of three genera, "Jerseylikevirus", "Sp3unalikevirus" and "K1glikevirus". To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phages Jersey, vB_SenS_AG11, vB_SenS-Ent1, vB_SenS-Ent2, vB_SenS-Ent3, FSL SP-101, SETP3, SETP7, SETP13, SE2, SS3e and wksl3 form the proposed genus "Jerseylikevirus". The proposed genus "K1glikevirus" consists of Escherichia phages K1G, K1H, K1ind1, K1ind2 and K1ind3. The proposed genus "Sp3unalikevirus" contains one member so far. Jersey-like phages appear to be widely distributed, as the above phages were isolated in the UK, Canada, the USA and South Korea between 1970 and the present day. The distinguishing features of this subfamily include a distinct siphovirus morphotype, genomes of 40.7-43.6 kb (49.6-51.4 mol % G +C), a syntenic genome organisation, and a high degree of nucleotide sequence identity and shared proteins. All known members of the proposed subfamily are strictly lytic.

In food safety management, sampling is an important tool for verifying control. Sampling by nature is a stochastic process. However, uncertainty regarding results is made even greater by the uneven distribution of microorganisms in a batch of food. This article reviews statistical aspects of sampling and describes the impact of distributions on the sampling results. Five different batch contamination scenarios are illustrated: a homogeneous batch, a heterogeneous batch with high- or low-level contamination, and a batch with localized high- or low-level contamination. These batch contamination scenarios showed that sampling results have to be interpreted carefully, especially when heterogeneous and localized contamination in food products is expected.

Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses.

Bacteria are confronted with a multitude of stressors when occupying niches within the host. These stressors originate from host defense mechanisms, other bacteria during niche competition or result from physiological challenges such as nutrient limitation. To counteract these stressors, bacteria have developed a stress-induced network to mount the adaptations required for survival. These stress-induced adaptations include the release of membrane vesicles from the bacterial envelope. Membrane vesicles can provide bacteria with a plethora of immediate and ultimate benefits for coping with environmental stressors. This review addresses how membrane vesicles aid Gram-negative bacteria to cope with host-associated stress factors, focusing on vesicle biogenesis and the physiological functions. As many of the pathways, that drive vesicle biogenesis, confer we propose that shedding of membrane vesicles by Gram-negative bacteria entails an integrated part of general stress responses.

Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccp Ax). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000–3000 μm and a diameter of 10–20 μm, which were remarkably larger than the fibres of BC typically produced by G. hansenii. Electronic supplementary material The online version of this article (10.1007/s00449-017-1864-1) contains supplementary material, which is available to authorized users.

There are no funders and sponsors indicated explicitly in the document. This deposit is composed by the main article plus the supplementary materials of the publication. Escherichia coli is both a harmless commensal in the intestines of many mammals, as well as a dangerous pathogen. The evolutionary paths taken by strains of this species in the commensal-to-pathogen transition are complex and can involve changes both in the core genome, as well in the pan-genome. One way to understand the likely paths that a commensal strain of E. coli takes when evolving pathogenicity is through experimentally evolving the strain under the selective pressures that it will have to withstand as a pathogen. Here, we report that a commensal strain, under continuous pressure from macrophages, recurrently acquired a transposable element insertion, which resulted in two key phenotypic changes: increased intracellular survival, through the delay of phagosome maturation and increased ability to escape macrophages. We further show that the acquisition of the pathoadaptive traits was accompanied by small but significant changes in the transcriptome of macrophages upon infection. These results show that under constant pressures from a key component of the host immune system, namely macrophage phagocytosis, commensal E. coli rapidly acquires pathoadaptive mutations that cause transcriptome changes associated to the host-microbe duet. There are no funders and sponsors indicated explicitly in the document. info:eu-repo/semantics/publishedVersion

peer-reviewed Bovine lactoferrin (LF) has been shown to prevent adhesion to and invasion of mammalian cell lines by pathogenic bacteria, with evidence for direct bacterial binding by the milk glycoprotein. However, the glycosylation pattern of LF changes over the lactation cycle. In this study, we aim to investigate the effect that this variation has on the milk glycoprotein’s ability to interact with pathogens. Surface plasmon resonance technology was employed to compare the binding of LF from colostrum (early lactation) and mature milk (late lactation) to a panel of pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Cronobacter sakazakii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella typhimurium). Novel interactions with LF were identified for C. sakazakii, S. pneumoniae and P. aeruginosa with the highest binding ability observed for mature milk LF in all cases, with the exception of S. typhimurium. The difference in bacterial binding observed may be as a result of the varying glycosylation profiles. This work demonstrates the potential of LF as a functional food ingredient to prevent bacterial infection

Burkholderia pseudomallei is a Gram-negative intracellular pathogen and the causative agent of melioidosis, a severe disease of both humans and animals. Melioidosis is an emerging disease which is predicted to be vastly under-reported. Type III Secretion Systems (T3SSs) are critical virulence factors in Gram negative pathogens of plants and animals. The genome of B. pseudomallei encodes three T3SSs. T3SS-1 and -2, of which little is known, are homologous to Hrp2 secretion systems of the plant pathogens Ralstonia and Xanthomonas. T3SS-3 is better characterized and is homologous to the Inv/Mxi-Spa secretion systems of Salmonella spp. and Shigella flexneri,respectively. Upon entry into the host cell, B. pseudomallei requires T3SS-3 for efficient escape from the endosome. T3SS-3 is also required for full virulence in both hamster and murine models of infection. The regulatory cascade which controls T3SS-3 expression and the secretome of T3SS-3 have been described, as well as the effect of mutations of some of the structural proteins. Yet only a few effector proteins have been functionally characterized to date and very little work has been carried out to understand the hierarchy of assembly, secretion and temporal regulation of T3SS-3. This review aims to frame current knowledge of B. pseudomallei T3SSs in the context of other well characterized model T3SSs, particularly those of Salmonella and Shigella.

How the flagellum knows when to stop The bacterial flagellum is important in bacterial pathogenesis and biofilm formation. It is a rotary nanomotor that allows bacteria to propel themselves through liquids and across surfaces. Researchers interested in nanoscale robotics use the bacterial flagellum as a model for a machine that self-assembles on the nanoscale. Cohen et al. examined exactly how the flagellum precisely measures its shaft so that it spans, but does not extend beyond the edge of, the periplasm. The growing flagellum uses a mechanism by which it “senses” when it hits the outer membrane and stops growing. Changing the width of the periplasmic space by remodeling a particular lipid changed the length of the flagellar shaft. Science , this issue p. 197

Thomas et al. show that a novel protein, Eros, controls the abundance of components of the phagocyte NADPH oxidase, making it essential for the phagocyte respiratory burst and defense against common infections., The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox. Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

International audience; Thiram (tetramethylthiuram disulfide) is a representative dithiocarbamate (DTC) pesticide used in both the field and as a seed protectant. The widespread use of Thiram and other DTC pesticides has raised concerns for health, because these compounds can exert neuropathic, endocrine disruptive, and carcinogenic effects. These toxic effects are thought to rely, at least in part, on the reaction of Thiram (and certain of its metabolites) with cellular protein thiols with subsequent loss of protein function. So far, a limited number of molecular targets of Thiram have been reported, including few enzymes such as dopamine beta-hydroxylase, 11 beta-hydroxysteroid dehydrogenase, and brain glycogen phosphorylase. We provide evidence that Thiram is an inhibitor (K-I = 23 mu M; k(inact) = 0.085 second(-1); k(inact)/K-I = 3691 M-1.s(-1)) of human arylamine N-acetyltransferase 1 (NAT1), a phase II xenobiotic-metabolizing enzyme that plays a key role in the biotransformation of aromatic amine xenobiotics. Thiram was found to act as an irreversible inhibitor through the modification of NAT1 catalytic cysteine residue as also reported for other enzymes targeted by this pesticide. We also showed using purified NAT1 and human keratinocytes that Thiram impaired the N-acetylation of 3,4-dichloroaniline (3,4-DCA), a major toxic metabolite of aromatic amine pesticides (such as Diuron or Propanil). As coexposure to different classes of pesticides is common, our data suggest that pharmacokinetic drug-drug interactions between DTC pesticides such as Thiram and aromatic amine pesticides may occur through alteration of NAT1 enzymes functions.

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer's patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle- associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed.

Semipermeable membranes of cells frequently pose an obstacle in metabolic engineering by limiting uptake of substrates, intermediates, or xenobiotics. Previous attempts to overcome this barrier relied on the promiscuous nature of peptide transport systems, but often suffered from low versatility or chemical instability. Here, we present an alternative strategy to transport cargo molecules across the inner membrane of Escherichia coli based on chemical synthesis of a stable cargo-peptide vector construct, transport through the peptide import system, and efficient intracellular release of the cargo by the promiscuous enzyme γ-glutamyl transferase (GGT). Retaining the otherwise periplasmic GGT in the cytoplasm was critical for the functionality of the system, as was fine-tuning its expression in order to minimize toxic effects associated to cytoplasmic GGT expression. Given the established protocols of peptide synthesis and the flexibility of peptide transport and GGT, the system is expected to be suitable for a broad range of cargoes. publisher: Elsevier articletitle: Overcoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors journaltitle: Metabolic Engineering articlelink: http://dx.doi.org/10.1016/j.ymben.2016.10.016 content_type: article copyright: © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. ispartof: Metabolic Engineering vol:39 pages:60-70 ispartof: location:Belgium status: published

In this study, simulated solar disinfection of secondary effluent was followed by dark storage at different temperatures or different receiving water matrices. Escherichia coil illumination was followed by 3-day monitoring of the bacterial population and its adaptation in different temperature conditions in the dark. The subsequent survival was linked to the dose received during exposure to light, and results were obtained on the environmentally induced prolongation of survival, maintenance of population or excessive growth, at 4 degrees C, 20 degrees C and 37 degrees C, respectively. An additional set of experiments at 20 degrees C was subjected to dilution in E. coli-free synthetic wastewater, water from Lake Leman, (synthetic) seawater and Mili-Q water. Post-irradiation monitoring was also conducted, studying 50%, 10% and 1% dilution rates, and the results were attributed to the two parameters of dilution medium and dilution ratio. However, different responses were found based on the acquired dose during pre-treatment. This indicates the importance of the illumination prior to storage, and the preference of bacteria in some matrices over the others. Survival was linked to initial population, dose, dilution and medium; shorter illumination times are to be considered according to the receiving water matrix. (C) 2014 Elsevier B.V. All rights reserved.

Studying macrophage biology in the context of a whole living organism provides unique possibilities to understand the contribution of this extremely dynamic cell subset in the reaction to infections, and has revealed the relevance of cellular and molecular processes that are fundamental to the cell-mediated innate immune response. In particular, various recently established zebrafish infectious disease models are contributing substantially to our understanding of the mechanisms by which different pathogens interact with macrophages and evade host innate immunity. Transgenic zebrafish lines with fluorescently labeled macrophages and other leukocyte populations enable non-invasive imaging at the optically transparent early life stages. Furthermore, there is a continuously expanding availability of vital reporters for subcellular compartments and for probing activation of immune defense mechanisms. These are powerful tools to visualize the activity of phagocytic cells in real time and shed light on the intriguing paradoxical roles of these cells in both limiting infection and supporting the dissemination of intracellular pathogens. This Review will discuss how several bacterial and fungal infection models in zebrafish embryos have led to new insights into the dynamic molecular and cellular mechanisms at play when pathogens encounter host macrophages. We also describe how these insights are inspiring novel therapeutic strategies for infectious disease treatment.

Pulsed electrical field (PEF) technology can be used for the inactivation of micro-organisms and therefore for preservation of food products. It is a mild technology compared to thermal pasteurization because a lower temperature is used during processing, leading to a better retention of the quality. In this study, pathogenic and spoilage micro-organisms relevant in refrigerated fruit juices were studied to determine the impact of process parameters and juice composition on the effectiveness of the PEF process to inactivate the micro-organisms. Experiments were performed using a continuous-flow PEF system at an electrical field strength of 20 kV/cm with variable frequencies to evaluate the inactivation of Salmonella Panama, Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae in apple, orange and watermelon juices. Kinetic data showed that under the same conditions, S. cerevisiae was the most sensitive micro-organism, followed by S. Panama and E. coli, which displayed comparable inactivation kinetics. L. monocytogenes was the most resistant micro-organism towards the treatment conditions tested. A synergistic effect between temperature and electric pulses was observed at inlet temperatures above 35 °C, hence less energy for inactivation was required at higher temperatures. Different juice matrices resulted in a different degree of inactivation, predominantly determined by pH. The survival curves were nonlinear and could satisfactorily be modeled with the Weibull model.

The metabolic fate of a compound is determined by numerous factors including its chemical structure. Although the metabolic options for a variety of functional groups are well understood and can often provide a rationale for the comparison of toxicity based on structural analogy, at times quite minor structural variations may have major consequences for metabolic outcomes and toxicity. In this perspective, the effects of structural variations on metabolic outcomes is detailed for a group of related hydroxy- and alkoxy-substituted allyl- and propenylbenzenes. These classes of compounds are naturally occurring constituents of a variety of botanical-based food items. The classes vary from one another by the presence or absence of alkylation of their para-hydroxyl substituents and/or the position of the double bond in the alkyl side chain. We provide an overview of how these subtle structural variations alter the metabolism of these important food-borne compounds, ultimately influencing their toxicity, particularly their DNA reactivity and carcinogenic potential. The data reveal that detailed knowledge of the consequences of subtle structural variations for metabolism is essential for adequate comparison of structurally related chemicals. Taken together, it is concluded that predictions in toxicological risk assessment should not be performed on the basis of structural analogy only but should include an analogy of metabolic pathways across compounds and species.

Transfer of Listeria monocytogenes was investigated from surface-inoculated cooked ham to commercial slicing machine surfaces, from spot-inoculated ham to the slicing machine blade, and vise-versa from a contaminated slicer to clean ham. With balances the proportion transfer from the source to the various destinations were investigated as well as the kinetics of transfer during successive slicing, using a difference equation. For inoculated ham, the transfer ratio to the machine was highest to the table (0.06), followed by the handle board (0.01) and plate, guard, and front and back of the blade (

International audience; Bartonella henselae is a zoonotic pathogen that possesses a flea-cat-flea transmission cycle and causes cat scratch disease in humans via cat scratches and bites. In order to establish infection, B. henselae must overcome oxidative stress damage produced by the mammalian host and arthropod vector. B. henselae encodes for putative Fe2+ and Mn2+ transporter SitABCD. In B. henselae, Sitknockdown increases sensitivity to hydrogen peroxide. We consistently show that Sitknockdown decreases the ability of B. henselae to survive in both human endothelial cells and cat fleas, thus demonstrating that the SitABCD transporter plays an important role during the B. henselae infection cycle.

Processive DNA synthesis by the alpha epsilon theta core of the Escherichia coli Pol III replicase requires it to be bound to the beta(2) clamp via a site in the a polymerase subunit. How the epsilon proofreading exonuclease subunit influences DNA synthesis by alpha was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of epsilon. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel beta-binding site in e that, in conjunction with the site in a, maintains a closed state of the alpha epsilon theta-beta(2) replicase in the polymerization mode of DNA synthesis. The epsilon-beta interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.