Antibiotics and probiotics-induced effects on the total fatty acid composition of feces in a rat model.

Fatty acids (FAs) play important roles as membrane components and signal transduction molecules. Changes in short chain FA (SCFA) composition are associated with gut microbiota modifications. However, the effect of bacteria-driven changes on the detailed FA spectrum has not been explored yet. We investigated the effect of antibiotics (ABx) and/or probiotics, in four treatment groups on rat stool FA composition. Principal component analysis indicated that the chromatogram profiles of the treatment groups differ, which was also observed at different time points. Linear mixed effects models showed that in the parameters compared (sampling times, treatments. and their interactions), both the weight percentage and the concentration of FAs were affected by ABx and probiotic administration. This study found that the gut microbiome defines trans and branched saturated FAs, most saturated FAs, and unsaturated FAs with less carbon atoms. These results are among the first ones to demonstrate the restoring effects of a probiotic mixture on a substantial part of the altered total FA spectrum, and also revealed a previously unknown relationship between gut bacteria and a larger group of FAs. These findings suggest that intestinal bacteria produce not only SCFAs but also other FAs that may affect the host's physiological processes.

Evaluation of the effect of ozone disinfection on forensic identification of blood, saliva, and semen stains.

Good laboratory practice minimizes the biological hazard posed by potentially infectious casework samples. In certain scenarios, when the casework sample is contaminated with highly contagious pathogens, additional safety procedures such as disinfection might be advised. It was previously proven that ozone gas treatment does not hamper STR analysis, but there is no data on how the disinfection affects other steps of the forensic analysis. In this study, we aimed to assess the interference of ozone disinfection with forensic tests used to identify biological stains. A dilution series of blood, saliva, and semen samples were pipetted onto cotton fabric and let completely dry. Half of the samples were subjected to ozone treatment, while the rest served as controls. All the samples were tested with specific lateral flow immunochromatographic assays and for specific RNA markers with quantitative real-time PCR. Additionally, luminol test was carried out on blood spots, Phadebas® Amylase Test on saliva stains, and semen stains were examined with STK Lab kit and light microscope following Christmas Tree or Hematoxylin-Eosin staining. Ozone treatment had no detrimental effect on the microscopic identification of sperm cells. Undiluted blood samples were detected with luminol and immunoassay, but at higher dilution, the sensitivity of the test decreased after disinfection. The same decrease in sensitivity was observed in the detection of semen stains using STK Lab kit from STK® Sperm Tracker, and in the case of the immunoassay specific for prostate-specific antigen (PSA). Ozone treatment almost completely inhibited the enzymatic activity of amylase. The sensitivity of antibody-based detection of amylase was also greatly reduced. RNA markers showed degradation but remained detectable in blood and semen samples after incubation in the presence of ozone. In saliva, the higher Ct values of the mRNA markers were close to the detection limit, even before ozone treatment.

Highly Selective MIF Ketonase Inhibitor KRP-6 Diminishes M1 Macrophage Polarization and Metabolic Reprogramming.

Macrophage polarization is highly involved in autoimmunity. M1 polarized macrophages drive inflammation and undergo metabolic reprogramming, involving downregulation of mitochondrial energy production and acceleration of glycolysis. Macrophage migration inhibitory factor (MIF), an enigmatic tautomerase (ketonase and enolase), was discovered to regulate M1 polarization. Here, we reveal that KRP-6, a potent and highly selective MIF ketonase inhibitor, reduces MIF-induced human blood eosinophil and neutrophil migration similarly to ISO-1, the most investigated tautomerase inhibitor. We equally discovered that KRP-6 prevents M1 macrophage polarization and reduces ROS production in IFN-γ-treated cells. During metabolic reprogramming, KRP-6 improved mitochondrial bioenergetics by ameliorating basal respiration, ATP production, coupling efficiency and maximal respiration in LPS+IFN-γ-treated cells. KRP-6 also reduced glycolytic flux in M1 macrophages. Moreover, the selective MIF ketonase inhibitor attenuated LPS+IFN-γ-induced downregulation of PARP-1 and PARP-2 mRNA expression. We conclude that KRP-6 represents a promising novel therapeutic compound for autoimmune diseases, which strongly involves M1 macrophage polarization.

Microfluidic Analysis for the Determination of Protein Content in Different Types of Plant-Based Drinks.

The widespread consumption of plant-based drinks, driven by health and dietary reasons (including cow's milk allergy, lactose intolerance, milk protein intolerance, following a vegetarian or vegan diet) necessitates automated and accurate test methods. Our study demonstrates the simultaneous determination of protein components and total protein concentrations in plant-based milk alternatives using a rapid and reproducible microchip gel electrophoretic method. As expected, the electrophoretic profiles of each plant-based drink differed. Based on our analyses and statistical evaluation, it can be determined that the protein profiles of different plant-based beverages do not differ significantly between different manufacturers or different expiry dates. The measured total protein content was compared with the nominal values, i.e., the values stated on the beverage labels. As the number of consumers of functional and specialized plant-based milk alternatives continues to rise, it is important to prioritize methods that provide qualitative and quantitative information on protein composition and other nutrients.

Efficacy of lyophilised bacteria-rich faecal sediment and supernatant with reduced bacterial count for treating patients with Clostridioides difficile Infection - A novel method for capsule faecal microbiota transfer.

Background and aims: Faecal microbiota transfer (FMT) has managed to earn its place in the Clostridioides difficile infection (CDI) guidelines by having comparable efficacy and recurrence rate of fidaxomicin. After more than 100 successful FMT administration through nasogastric tube, we started using hard gelatine capsules filled with lyophilised faecal sediment and supernatant. Our main question was whether uncoated capsules (containing faecal sediment or supernatant) are comparable to the widely used nasogastric tubes in CDI. We also investigated the effect of storage and time on the survival rate of bacteria in the samples. Methods: We compared the efficacy of our capsules to other treatment options of CDI at the Department of Infectology at the University of Pécs (Hungary). For our study, stool was collected from a single donor. We treated 10 patients with relapsing CDI, 5 of them received supernatant, 5 received sediment. Donor samples were stored on 4 different temperatures and tested to determine the survival rates of bacteria. As pilot projects, we also assessed the changes of bacterial taxa, protein- and lipid compositions. Moreover, we selected 4 patients to compare their samples prior and after FMT by using microbiome (16S amplicon sequencing), protein, and lipid analyses. Results: 4 out of the 5 patients who received supernatant became symptomless within 2 days after FMT. In the sediment group 3 out of 5 patients were cured from CDI. Comparing the supernatant to the sediment, we found significantly lower number of colony-forming units in the supernatant. We found that -80°C is the most suitable temperature to store the samples. The stool lipid profiles of recipients showed a more diverse composition after FMT, and changes in the stool protein profiles were observed as well. In the microbiome analysis, we observed an increase in the alpha diversity after FMT. Conclusions: Our study of 10 patients showed good efficacy of lyophilised faecal supernatant using capsules. The single donor approach proved to be effective in our investigation. A significantly lower CFU number was sufficient for the effect, the separation can be achieved by widely available instruments. For storage temperature, -20°C was sufficient in our clinical practice.

The Effect of Mutation in Lipopolysaccharide Biosynthesis on Bacterial Fitness.

This paper presents the genome sequence of a Shigella sonnei mutant strain (S. sonnei 4351) and the effect of mutation in lipopolysaccharide biosynthesis on bacterial fitness. Lipopolysaccharides are the major component of the outer leaflet of the Gram-negative outer membrane. We report here a frameshift mutation of the gene gmhD in the genome of S. sonnei 4351. The mutation results in a lack of epimerization of the core heptose while we also found increased thermosensitivity, abnormal cell division, and increased susceptibility to erythromycin and cefalexin compared to the S. sonnei 4303. Comparative genomic analysis supplemented with structural data helps us to understand the effect of specific mutations on the virulence of the bacteria and may provide an opportunity to study the effect of short lipopolysaccharides.

Effect of Culture Conditions on Fatty Acid Profiles of Bacteria and Lipopolysaccharides of the Genus Pseudomonas-GC-MS Analysis on Ionic Liquid-Based Column.

The profiling of bacterial fatty acids is a well-established technique in identifying and classifying bacteria. Cultivation conditions may affect the biosynthesis, thereby, changing the fatty acid profile in bacteria. The effect of the culture conditions on the fatty acid components of Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa polyresistant and Pseudomonas putida all are aligned to the genus Pseudomonas. The fatty acids in the lipopolysaccharides of Pseudomonas aeruginosa PAO1 were also examined. The effects of the cultivation conditions were followed by using agar and blood agar media at the characteristic temperatures, 25 °C, 37 °C and 42 °C, respectively, and an analysis was made during the 1st, 3rd and 5th day following inoculation. In addition to quantitative differences, we also experienced qualitative differences in the fatty acid profiles which detect newly appearing fatty acids, due to changes in environmental factors. The application of ionic liquid-based column unveils new possibilities for the analyses of fatty acids in GC-MS experiments for bacterial fatty acid profiling. The validation results (response linearity, limit of detection, limit of quantification, system suitability, intraday and interday repeatability and accuracy) show the high separation efficiency of the ionic liquid-based column in the analyses.

The Effects of Rapamycin on the Intestinal Graft in a Rat Model of Cold Ischemia Perfusion and Preservation.

Attenuating the rheological and structural consequences of intestinal ischemia-reperfusion-injury (IRI) is important in transplant proceedings. Preconditioning is an often-proposed remedy. This technique uses physical or pharmacological methods to manipulate key ischemia pathways, such as oxidation, inflammation, and autophagy, prior to ischemia. This study determined the time-dependent effects of Rapamycin preconditioning on small-bowel grafts undergoing cold ischemia perfusion and preservation. Our main parameters were mucosa and cell injury and autophagy. A total of 30 male Wistar rats were divided into 5 groups: sham, preservation-control, and 3 treated groups (Rapamycin administered either 0, 30, or 60 min prior to perfusion). After perfusion, the intestines were placed in chilled IGL-1 solution for 12 h. Thereafter, they were reperfused. Histology and bioanalysis (LDH and lactate) were used to ascertain intestinal injury while immunohistochemistry was used for measuring changes in autophagy markers (Beclin-1, LC3B, and p62 proteins). The results show no significant difference amongst the groups after vascular perfusion. However, intestinal injury findings and autophagy changes demonstrate that administering Rapamycin 30 min or 60 min prior was protective against adverse cold ischemia and reperfusion of the intestinal graft. These findings show that Rapamycin is protective against cold ischemia of the small intestine, especially when administered 30 min before the onset.

Celebrities and Breast Cancer: A Multidimensional Quali-Quantitative Analysis of News Stories Shared on Social Media.

In 2020, breast cancer was the most frequent type of cancer in 158 countries. To advise the population about risk factors and the importance of preventive measures, celebrities can be of great help, acting as spokespersons for reliable scientific information. The goal of this study is to analyse the content of news stories about breast cancer shared on different social media, examining how stories with celebrity presence are constructed. We performed a quali-quantitative multidimensional analysis of news stories in English that addressed breast cancer on the following social media platforms: Facebook, Pinterest, Twitter and Reddit. We compared news stories with and without the presence of celebrities. Our sample consists of 1594 news stories that had at least 1000 total shares across all analysed social media; 262 news stories mention a celebrity (16.44%), while 1332 stories do not (83.56%). Nevertheless, the ones that feature celebrities are, as a rule, more shared. The percentage of stories with celebrities addressing breast cancer prevention is quite low (1.9%). The same can be said for mentions of scientific papers/specialist quotes (3.4%). This research may help outline some possible paths that healthcare organizations and communication professionals can take to improve breast cancer content available online.

The first nationwide surveillance of carbapenem-resistant Enterobacterales in the United Arab Emirates - increased association of Klebsiella pneumoniae CC14 clone with Emirati patients.

OBJECTIVES: To assess the current prevalence, distribution, and main clonal types of carbapenem-resistant Enterobacterales (CRE) in the United Arab Emirates. METHODS: A total of 504 CRE collected over a 9-month period in 15 hospitals were studied. Antibiotic susceptibility and the presence of common carbapenemase, 16S methylase, and mobile colistin resistance genes were assessed. Selected strains forming larger clusters by pulsed field gel electrophoresis were subjected to whole genome sequencing to identify their sequence types and core genome MLST. RESULTS: Strains expressing OXA and NDM type carbapenemases and 16S methylases were present in all major hospitals. Considerable interhospital differences were noticed, suggesting the role of specific clones. A total of three major Klebsiella pneumoniae clones (CC14, ST231, and CC147) were identified, accounting for 48.6% of all CRE. All clones were significantly more resistant than sporadic isolates. CC14 strains exhibited a significant association with Emirati patients. CONCLUSIONS: Nearly half of CRE infections in the country are due to a limited number of clones. The data indicate the possibility of interhospital transmission, combined in some hospitals with inadequate stewardship practices. The study also revealed an association of the largest, most resistant clone (CC14) with Emirati patients. The specific reasons for it should be clarified by further investigations.

Misinformation on social networks during the novel coronavirus pandemic: a quali-quantitative case study of Brazil.

BACKGROUND: One of the challenges posed by the novel coronavirus pandemic is the infodemic risk, that is, a huge amount of information being published on the topic, along with misinformation and rumours; with social media, this phenomenon is amplified, and it goes faster and further. Around 100 million people in Brazil (50% of the inhabitants) are users of social media networks - almost half of the country's population. Most of the information on the Internet is unregulated, and its quality remains questionable. METHODS: In this study, we examine the main characteristics of misinformation published on the topic. We analysed 232 pieces of misinformation published by the Brazilian fact-checking service "Agência Lupa". The following aspects of each news item were analysed: a) In what social media has it circulated?; b) What is the content classification, sentiment and type of misinformation?; d) Are there recurrent themes in the sample studied? RESULTS: Most were published on Facebook (76%), followed by WhatsApp, with 10% of total cases. Half of the stories (47%) are classified as "real-life", that is, the focus is on everyday situations, or circumstances involving people. Regarding the type of misinformation, there is a preponderance of fabricated content, with 53% of total, followed by false context (34%) and misleading content (13%). Wrong information was mostly published in text format (47%). We found that 92.9% of misinformation classified as "fabricated content" are "health tips", and 88.9% of "virtual scams" are also fabricated. CONCLUSION: Brazilian media and science communicators must understand the main characteristics of misinformation in social media about COVID-19, so that they can develop attractive, up-to-date and evidence-based content that helps to increase health literacy and counteract the spread of false information.

Breast cancer on social media: a quali-quantitative study on the credibility and content type of the most shared news stories.

BACKGROUND: Female breast cancer was the most diagnosed cancer in 2020, with more than two million new cases worldwide. Access to scientifically correct information can assist patients in early detection or prevention of the disease. However, misinformation on social networking sites (SNSs) about breast cancer can be propagated rapidly, posing a threat to health communication efforts. The aim of this study is to analyse the characteristics of the most shared news stories referencing the disease that circulated on SNSs, including the credibility of this content. METHODS: This is an exploratory quali-quantitative study. Data collection was conducted between June 2019 and June 2020. We performed statistical and content analysis of the stories that had at least 1,000 total shares. Each story was classified in accordance to the following aspects: credibility; type of rumour; source; content type; mentions prevention or early detection/screening exams. RESULTS: The abundance of news stories in our sample (n = 1,594) were not classified according to their credibility, as they do not address science, risk factors, prevention, treatment, or other aspects which can be assessed for scientific accuracy. However, content classified as "rumours" are 3.29 times more shared than those considered scientifically correct. Regarding content type, most stories are classified as 'real-life story' or 'solidarity' (67.69%). In our sample, 5.08% of the total comment on prevention and 19.7% reference early detection. CONCLUSION: We consider it can be a good strategy, in SNSs, to combine content of greater popularity, such as real-life stories, with subjects that can make a difference in a patient's life, such as early detection, breast cancer symptoms and disease prevention strategies. Doctors, scientists and health journalists can expand the dialogue with the lay public regarding breast cancer, helping to counteract online misinformation.

Forest Bathing Always Makes Sense: Blood Pressure-Lowering and Immune System-Balancing Effects in Late Spring and Winter in Central Europe.

Various formats of forest bathing have been receiving increasing attention owing to their perspectives in health promotion and the treatment of chronic lifestyle diseases. The majority of field studies are still being conducted in the Far Eastern region, and they often make psychological assessments mainly in the green season. In our pretest-posttest field experiment, twelve healthy, working-age volunteers participated in a 2-h leisurely forest walking program, first in the green season (May) and then in the winter season (January), in the Mecsek Hills, next to Pécs, Hungary. Systolic blood pressure decreased after the trips both in late spring and in the winter. Based on changes in the expressions of CD69, an early activation marker, NKG2D, a major recognition receptor, perforin, granzyme B, and TIM-3, an inhibitory immune checkpoint molecule, on CD8+ cytotoxic T, NK, NKdim, NKbright, and NKT cells, we detected the stimulation of NKbright cells and activation of all examined immune cell subsets in the green season. In the winter, a slight activating and an interesting balancing effect regarding TIM-3 could be observed considering our finding that basal (pretest) TIM-3 expression by NK cells was significantly lower in the winter. Our work expands the knowledge on and potentials of forest medicine.

Capillary zone electrophoresis of proteins applying ionic liquids for dynamic coating and as background electrolyte component.

The use of ionic liquids in capillary electrophoresis, either as coating material or as components of the background electrolyte needs systematic standardization to set up optimal conditions. Excellent separation of the proteins was achieved using 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF4 ]) or 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4 ]) ionic liquids using the properly made ionic-liquid-water binary mixtures for the experiments. The binary mixture has a distinctly stable and well perceptible low pH, which depends on the concentration of the ionic liquid, and on the preparation time of the mixture. Optimal conditions for the electrophoretic separation were obtained upon a multivariate analysis of the experimental parameters (applied voltage, migration time, concentration, and type of the ionic liquid). The standardized condition provides a low electroendosmotic flow toward the anode, which, however, did not hinder the proteins to migrate toward the cathode. The migration of cytochrome c, lysozyme, myoglobin, trypsin, and apo-transferrin at a pH around 2, far below the isoelectric points of the proteins, showed RSD values of the migration times less than 7.5% and less than 6.5% when using [emim][BF4 ] or [bmim][BF4 ], respectively, either in run-to-run or day-to-day experiments. The determination of the extent of the EOF is not possible with the commonly used EOF markers, due to interaction with the ionic-liquid constituents. The interaction of the ionic liquids with the proteins influences the migration order in zone electrophoresis. This method has been applied successfully for the analyses of real biological samples such as proteins from egg whites and human tears.

Microchip gel electrophoretic analysis of perchloric acid-soluble serum proteins in systemic inflammatory disorders.

Perchloric acid (PCA) precipitation is a well-known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA-soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods. After PCA treatment of the serum, the soluble proteins in the supernatant were analyzed by SDS-PAGE and by microchip gel electrophoresis (MGE). Characteristic changes of the electrophoretic patterns of the PCA-soluble fractions were observed. Four characteristic bands (at ∼11, ∼65, ∼85, and ∼120 kDa) with varying intensity were detected by MGE. The proportion of the ∼65, ∼85, and ∼120 kDa bands were significantly higher in systemic inflammatory conditions than in healthy individuals (p < 0.001), and characteristic patterns were observed in patients with acute inflammation. The marked differences in the acid-soluble protein patterns, which were observed in patients with ongoing systemic inflammation, might be a good indicator of inflammation. The MGE analysis is a fast screening and quantification method for the detection of characteristic changes among acid-soluble serum proteins.

Fast determination of lactic, succinic, malic, tartaric, shikimic, and citric acids in red Vranec wines by CZE-ESI-QTOF-MS.

A fast and simple method with CZE coupled to ESI/QTOF-MS was optimized and validated for quantitative determination of organic acids (lactic acid, succinic acid, malic acid, tartaric acid, shikimic acid, and citric acid) in red wines. The BGE was ammonium acetate and the separation of the analytes was performed in a polybrene-coated capillary in the presence of EOF. The sample preparation included dilution and filtration of the wine. The method showed satisfactory performance characteristics: good linearity for each organic acid, with correlation coefficients ranging from r2  = 0.9902 (shikimic acid) to r2  = 0.9990 (tartaric acid). The limit of quantification was between 0.0034 mM (for shikimic acid) and 0.107 mM (for citric acid), and the recovery data fell between 95.8% (malic acid) and 102.7% (lactic acid); the total run time was less than 4 min. The RSD values for the interday repeatability and intraday reproducibility were between 3.44 and 9.50%, and between 1.75 and 8.29%, respectively. Seventeen Macedonian red Vranec wines were studied demonstrating a wide variation in the organic acids' concentration, which should be most probably due to the variation of the climate conditions in the vine areas.

Capillary Electrophoresis Chips for Fingerprinting Endotoxin Chemotypes and Subclasses.

Endotoxins (lipopolysaccharides, LPS; lipooligosaccharides, LOS) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans, such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among the LPS from different bacterial serotypes and their mutants include variations mainly within the composition and length or missing of their O-polysaccharide chains. Microchip electrophoretic methodology enables the structural characterization of LPS molecules from several bacteria and the quantitative evaluation of components of endotoxin extracts. The improved microchip electrophoretic method is based on the direct labeling of endotoxins by covalent binding of a fluorescent dye. The classification of the S-type LPSs can be done according to their electrophoretic profiles, which are characteristics of the respective bacterial strains. According to the number, distribution, and the relative amounts of components in an endotoxin extract, it is possible to differentiate between the S-type endotoxins from different Gram-negative bacterial strains. The microchip electrophoresis affords high-resolution separation of pure and partially purified (e.g., obtained from whole-cell lysate) S and R endotoxins. This microchip technique provides a new, standardizable, fast, and sensitive method for the detection of endotoxins and for the quantitative evaluation of components of an endotoxin extract.

Effects of clary sage oil and its main components, linalool and linalyl acetate, on the plasma membrane of Candida albicans: an in vivo EPR study.

The effects of clary sage (Salvia sclarea L.) oil (CS-oil), and its two main components, linalool (Lol) and linalyl acetate (LA), on cells of the eukaryotic human pathogen yeast Candida albicans were studied. Dynamic and thermodynamic properties of the plasma membrane were investigated by electron paramagnetic resonance (EPR) spectroscopy, with 5-doxylstearic acid (5-SASL) and 16-SASL as spin labels. The monitoring of the head group regions with 5-SASL revealed break-point frequency decrease in a temperature dependent manner of the plasma membrane between 9.55 and 13.15 °C in untreated, in CS-oil-, Lol- and LA-treated membranes. The results suggest a significant increase in fluidity of the treated plasma membranes close to the head groups. Comparison of the results observed with the two spin labels demonstrated that CS-oil and LA induced an increased level of fluidization at both depths of the plasma membrane. Whereas Lol treatment induced a less (1 %) ordered bilayer organization in the superficial regions and an increased (10 %) order of the membrane leaflet in deeper layers. Acute toxicity tests and EPR results indicated that both the apoptotic and the effects exerted on the plasma membrane fluidity depended on the composition and chemical structure of the examined materials. In comparison with the control, treatment with CS-oil, Lol or LA induced 13.0, 12.3 and 26.4 % loss respectively, of the metabolites absorbing at 260 nm, as a biological consequence of the plasma membrane fluidizing effects. Our results confirmed that clary sage oil causes plasma membrane perturbations which leads to cell apoptosis process.

Antimicrobial and Virulence-Modulating Effects of Clove Essential Oil on the Foodborne Pathogen Campylobacter jejuni.

Our study investigated the antimicrobial action of clove (Syzygium aromaticum) essential oil (EO) on the zoonotic pathogen Campylobacter jejuni After confirming the clove essential oil's general antibacterial effect, we analyzed the reference strain Campylobacter jejuni NCTC 11168. Phenotypic, proteomic, and transcriptomic methods were used to reveal changes in cell morphology and functions when exposed to sublethal concentrations of clove EO. The normally curved cells showed markedly straightened and shrunken morphology on the scanning electron micrographs as a result of stress. Although, oxidative stress, as a generally accepted response to essential oils, was also present, the dominance of a general stress response was demonstrated by reverse transcription-PCR (RT-PCR). The results of RT-PCR and two-dimensional (2D) PAGE revealed that clove oil perturbs the expression of virulence-associated genes taking part in the synthesis of flagella, PEB1, PEB4, lipopolysaccharide (LPS), and serine protease. Loss of motility was also detected by a phenotypic test. Bioautographic analysis revealed that besides its major component, eugenol, at least four other spots of clove EO possessed bactericidal activity against C. jejuni Our findings show that clove EO has a marked antibacterial and potential virulence-modulating effect on C. jejuni IMPORTANCE: This study demonstrates that the components of clove essential oil influence not only the expression of general stress genes but also the expression of virulence-associated genes. Based on this finding, alternative strategies can be worked on to control this important foodborne pathogen.

Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution.

We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.