Resistome analysis of Escherichia coli isolates from layers in Hungary.

The authors aimed to investigate eight strains of Escherichia coli (E. coli) strains from Hungarian layer flocks for antimicrobial resistance genes (ARG), using metagenomic methods. The strains were isolated from cloacal swabs of healthy adult layers. This study employed shotgun sequencing-based genetic and bioinformatic analysis along with determining phenotypic minimum inhibitory concentrations. A total of 59 ARGs were identified in the eight E. coli isolates, carrying ARGs against 15 groups of antibiotics. Among these, 28 ARGs were identified as transferable. Specifically, four ARGs were plasmid-derived, 18 ARGs were phage-derived and an additional six ARGs were predicted to be mobile, contributing to their mobility and potential spread between bacteria.

Antiviral Drug Candidate Repositioning for Streptococcus suis Infection in Non-Tumorigenic Cell Models.

The increasing prevalence of antimicrobial resistance against zoonotic bacteria, including Streptococcus (S.) suis, highlights the need for new therapeutical strategies, including the repositioning of drugs. In this study, susceptibilities of bacterial isolates were tested toward ten different 3-amidinophenyalanine (Phe(3-Am)) derivatives via determination of minimum inhibitory concentration (MIC) values. Some of these protease inhibitors, like compounds MI-432, MI-471, and MI-476, showed excellent antibacterial effects against S. suis. Their drug interaction potential was investigated using human liver microsomal cytochrome P450 (CYP450) measurements. In our work, non-tumorigenic IPEC-J2 cells and primary porcine hepatocytes were infected with S. suis, and the putative beneficial impact of these inhibitors was investigated on cell viability (Neutral red assay), on interleukin (IL)-6 levels (ELISA technique), and on redox balance (Amplex red method). The antibacterial inhibitors prevented S. suis-induced cell death (except MI-432) and decreased proinflammatory IL-6 levels. It was also found that MI-432 and MI-476 had antioxidant effects in an intestinal cell model upon S. suis infection. Concentration-dependent suppression of CYP3A4 function was found via application of all three inhibitors. In conclusion, our study suggests that the potential antiviral Phe(3-Am) derivatives with 2',4' dichloro-biphenyl moieties can be considered as effective drug candidates against S. suis infection due to their antibacterial effects.

In Vitro Microevolution and Co-Selection Assessment of Amoxicillin and Cefotaxime Impact on Escherichia coli Resistance Development.

The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.

Determining the In Vivo Efficacy of Plant-Based and Probiotic-Based Antibiotic Alternatives against Mixed Infection with Salmonella enterica and Escherichia coli in Domestic Chickens.

Restrictions on the use of antimicrobial compounds have led to a surge of interest in alternative solutions, such as natural, plant-based compounds. In our study, we investigated the efficacy of three feed supplements containing different additives, namely, probiotics (Lactobacillus spp., "Test substance A"), turmeric (Curcuma longa L., "Test substance B"), and fenugreek (Trigonella foenum graecum, "Test substance C"). In the experiment, we tested 180 birds of the Bábolna Tetra-SL laying hybrid breed that were infected with Salmonella enteritidis strains. The birds were randomly divided into six groups: three groups treated with the different additives, a negative control group, a positive control group, and an antibiotic-treated group using enrofloxacin. We examined the maturation and the time course of shedding of Salmonella; at the end of rearing, pathological and histopathological examinations were performed. When Salmonella was isolated from the cloacal swab samples, the enrofloxacin-treated group had a high number of animals shedding Salmonella by day 9, which was like the group treated with test material C. The greatest reduction in Salmonella shedding was observed in the groups treated with test materials A and B. In terms of pathological parameters, villus length and crypt depth were significantly better in the group treated with test material C compared to the positive and negative controls, and when comparing the body weight of the tested animals, the group treated with test material B had a significantly larger absorption surface area compared to the positive control group. Overall, the supplement with test material C proved to be the most effective. In the future, it is worthwhile to investigate the combination of the tested active substances for their possible synergistic effects and to perform a dose-response study to select the optimal dosage.

In Vitro Microevolution and Co-Selection Assessment of Florfenicol Impact on Escherichia coli Resistance Development.

The issue of antimicrobial resistance is becoming an increasingly serious challenge in both human and veterinary medicine. Prudent antimicrobial use in veterinary medicine is warranted and supported by international guidelines, with the Antimicrobial Advice Ad Hoc Expert Group (AMEG) placing particular emphasis on the critically important group B antimicrobials. These antimicrobials are commonly employed, especially in the poultry and swine industry. The impact of florfenicol, a veterinary antibiotic, was studied on the resistance development of Escherichia coli. The aim of the study was to investigate the effect of the use of florfenicol on the development of phenotypic and genomic resistances, not only to the drug itself but also to other drugs. The minimum inhibitory concentrations (MICs) of the antibiotics were investigated at 1×, 10×, 100× and 1000× concentrations using the adapted Microbial Evolution and Growth Arena (MEGA-plate) method. The results demonstrate that florfenicol can select for resistance to fluoroquinolone antibiotics (167× MIC value increase) and cephalosporins (67× MIC value increase). A total of 44 antimicrobial resistance genes were identified, the majority of which were consistent across the samples. Chromosomal point mutations, including alterations in resistance-associated and regulatory genes (acrB, acrR, emrR and robA), are thought to trigger multiple drug efflux pump activations, leading to phenotypically increased resistance. The study underscores the impact of florfenicol and its role in the development of antimicrobial resistance, particularly concerning fluoroquinolone antibiotics and cephalosporins. This study is the first to report florfenicol's dose-dependent enhancement of other antibiotics' MICs, linked to mutations in SOS-box genes (mdtABC-tolC, emrAB-tolC and acrAB-tolC) and increased multidrug efflux pump genes. Mutations in the regulatory genes acrR, emrR and rpbA support the possibility of increased gene expression. The results are crucial for understanding antimicrobial resistance and its development, highlighting the promising potential of in vitro evolutionary and coselection studies for future research.

Immune response to zinc oxide inhalation in metal fume fever, and the possible role of IL-17f.

Metal fume fever (MFF) is a work-related disease caused by the inhalation of metal particles, including zinc oxide. Chronic asthma may develop as a long-term consequence of exposure, particularly for welders and metal workers who are most at risk. In this study, we investigated the effects of ZnO fume inhalation on multiple inflammation-related cytokine- and cytokine receptor genes in mice from lung and lymph node samples, to explore the role of these in the pathogenesis of MFF. In our experiments, the animals were treated with a sub-toxic amount of ZnO fume for 4 h a day for 3 consecutive days. Sampling occurred 3 and 12 h post-treatment. We are the first to demonstrate that ZnO inhalation causes extremely increased levels of IL-17f gene expression at both sampling time points, in addition to increased gene expression rates of several other interleukins and cytokines, such as IL-4, IL-13, CXCL5, CSF-3, and IFN-γ. Our animal experiment provides new insights into the immunological processes of early metal fume fever development. IL-17f plays a crucial role in connecting immunological and oxidative stress events. The increased levels of IL-4 and IL-13 cytokines may explain the development of long-term allergic asthma after exposure to ZnO nanoparticles, which is well-known among welders, smelters, and metal workers.

Deep Sequencing of Porcine Reproductive and Respiratory Syndrome Virus ORF7: A Promising Tool for Diagnostics and Epidemiologic Surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major concern worldwide. Control of PRRSV is a challenging task due to various factors, including the viral diversity and variability. In this study, we evaluated an amplicon library preparation protocol targeting the ORF7 region of both PRRSV species, Betaarterivirus suid 1 and Betaarterivirus suid 2. We designed tailed primers for a two-step PCR procedure that generates ORF7-specific amplicon libraries suitable for use on Illumina sequencers. We tested the method with serum samples containing common laboratory strains and with pooled serum samples (n = 15) collected from different pig farms during 2019-2021 in Hungary. Testing spiked serum samples showed that the newly designed method is highly sensitive and detects the viral RNA even at low copy numbers (corresponding to approx. Ct 35). The ORF7 sequences were easily assembled even from clinical samples. Two different sequence variants were identified in five samples, and the Porcilis MLV vaccine strain was identified as the minor variant in four samples. An in-depth analysis of the deep sequencing results revealed numerous polymorphic sites along the ORF7 gene in a total of eight samples, and some sites (positions 12, 165, 219, 225, 315, 345, and 351) were found to be common in several clinical specimens. We conclude that amplicon deep sequencing of a highly conserved region of the PRRSV genome could support both laboratory diagnosis and epidemiologic surveillance of the disease.

Effects of Lactobacillus rhamnosus DSM7133 on Intestinal Porcine Epithelial Cells.

Antimicrobial resistance is one of the biggest health challenges nowadays. Probiotics are promising candidates as feed additives contributing to the health of the gastrointestinal tract. The beneficial effect of probiotics is species/strain specific; the potential benefits need to be individually assessed for each probiotic strain or species. We established a co-culture model, in which gastrointestinal infection was modeled using Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium). Using intestinal porcine epithelial cells (IPEC-J2), the effects of pre-, co-, and post-treatment with Lactobacillus (L.) rhamnosus on the barrier function, intracellular (IC) reactive oxygen species (ROS) production, proinflammatory cytokine (IL-6 and IL-8) response, and adhesion inhibition were tested. E. coli- and S. Typhimurium-induced barrier impairment and increased ROS production could be counteracted using L. rhamnosus (p < 0.01). S. Typhimurium-induced IL-6 production was reduced via pre-treatment (p < 0.05) and post-treatment (p < 0.01); increased IL-8 secretion was decreased via pre-, co-, and post-treatment (p < 0.01) with L. rhamnosus. L. rhamnosus demonstrated significant inhibition of adhesion for both S. Typhimurium (p < 0.001) and E. coli (p < 0.001 in both pre-treatment and post-treatment; p < 0.05 in co-treatment). This study makes a substantial contribution to the understanding of the specific benefits of L. rhamnosus. Our findings can serve as a basis for further in vivo studies carried out in pigs and humans.

Susceptibility of Actinobacillus pleuropneumoniae, Pasteurella multocida and Streptococcus suis Isolated from Pigs in Hungary between 2018 and 2021.

Porcine respiratory disease complex (PRDC) has been a major animal health, welfare, and economic problem in Hungary; therefore, great emphasis should be put on both the prevention and control of this complex disease. As antibacterial agents are effective tools for control, antibiotic susceptibility testing is indispensable for the proper implementation of antibacterial therapy and to prevent the spread of resistance. The best method for this is to determine the minimum inhibitory concentration (MIC) by the broth microdilution method. In our study, we measured the MIC values of 164 Actinobacillus pleuropneumoniae, 65 Pasteurella multocida, and 118 Streptococcus suis isolates isolated from clinical cases against the following antibacterial agents: amoxicillin, ceftiofur, cefquinome, oxytetracycline, doxycycline, tylosin, tilmicosin, tylvalosin, tulathromycin, lincomycin, tiamulin, florfenicol, colistin, enrofloxacin, and sulfamethoxazole-trimethoprim. Outstanding efficacy against A. pleuropneumoniae isolates was observed with ceftiofur (100%) and tulathromycin (100%), while high levels of resistance were observed against cefquinome (92.7%) and sulfamethoxazole-trimethoprim (90.8%). Ceftiofur (98.4%), enrofloxacin (100%), florfenicol (100%), and tulathromycin (100%) were found to be highly effective against P. multocida isolates, while 100% resistance was detected against the sulfamethoxazole-trimethoprim combination. For the S. suis isolates, only ceftiofur (100%) was not found to be resistant, while the highest rate of resistance was observed against the sulfamethoxazole-trimethoprim combination (94.3%). An increasing number of studies report multi-resistant strains of all three pathogens, making their monitoring a high priority for animal and public health.

Effect of hydroxylated and methylated flavonoids on cytochrome P450 activity in porcine intestinal epithelial cells.

Cytochrome P450 (CYP) oxidases are among the main metabolizing enzymes that are responsible for the transformation of xenobiotics, including clinically important drugs. Their activity can be influenced by several compounds leading to decreased efficacy or increased toxicity of co-administered medicines. Flavonoids exert various beneficial effects on human and animal health; therefore they are used as food and feed supplements. However, they are also well-known for their CYP modulating potential. Since the amount of CYP enzymes is highest in the liver, interaction studies are mainly conducted in hepatocytes, however, CYP activity in the gastrointestinal tract is also remarkable. In this study, effects of apigenin (API), quercetin (QUE) and their methylated derivatives trimethylapigenin (TM-API), 3-O-methylquercetin (3M-QUE) and 3',7-di-O-methylquercetin (3'7DM-QUE) on the CYP enzyme activity was examined in IPEC-J2 porcine intestinal epithelial cells. Potential food-drug interactions were studied using flavonoid treatment in combination with inducer and inhibitor compounds. API, TM-API, QUE and 3M-QUE significantly inhibited the CYP3A29 enzyme, while 3'7DM-QUE did not alter its activity. Enzyme inhibition has also been observed in case of some food-drug combinations. Our results support previous findings about CYP modulating effects of flavonoids and highlights the possibility of interactions when flavonoid-containing supplements are consumed during drug treatments.

Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model.

The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte-non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H2O2 and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.

Pharmacokinetics and Pharmacodynamics of Florfenicol in Plasma and Synovial Fluid of Pigs at a Dose of 30 mg/kgbw Following Intramuscular Administration.

A major problem of our time is the ever-increasing resistance to antimicrobial agents in bacterial populations. One of the most effective ways to prevent these problems is to target antibacterial therapies for specific diseases. In this study, we investigated the in vitro effectiveness of florfenicol against S. suis, which can cause severe arthritis and septicemia in swine herds. The pharmacokinetic and pharmacodynamic properties of florfenicol in porcine plasma and synovial fluid were determined. After a single intramuscular administration of florfenicol at 30 mg/kgbw, the AUC0-∞ was 164.45 ± 34.18 µg/mL × h and the maximum plasma concentration was 8.15 ± 3.11 µg/mL, which was reached in 1.40 ± 0.66 h, whereas, in the synovial fluid, these values were 64.57 ± 30.37 µg/mL × h, 4.51 ± 1.16 µg/mL and 1.75 ± 1.16 h, respectively. Based on the MIC values of the 73 S. suis isolates tested, the MIC50 and MIC90 values were 2 µg/mL and 8 µg/mL, respectively. We successfully implemented a killing-time curve in pig synovial fluid as a matrix. Based on our findings, the PK/PD breakpoints of the bacteriostatic (E = 0), bactericidal (E = -3) and eradication (E = -4) effects of florfenicol were determined and MIC thresholds were calculated, which are the guiding indicators for the treatment of these diseases. The AUC24h/MIC values for bacteriostatic, bactericidal and eradication effects were 22.22 h, 76.88 h and 141.74 h, respectively, in synovial fluid, and 22.42 h, 86.49 h and 161.76 h, respectively, in plasma. The critical MIC values of florfenicol against S. suis regarding bacteriostatic, bactericidal and eradication effects in pig synovial fluid were 2.91 ± 1.37 µg/mL, 0.84 ± 0.39 µg/mL and 0.46 ± 0.21 µg/mL, respectively. These values provide a basis for further studies on the use of florfenicol. Furthermore, our research highlights the importance of investigating the pharmacokinetic properties of antibacterial agents at the site of infection and the pharmacodynamic properties of these agents against different bacteria in different media.

In Vitro Pharmacokinetic Behavior of Antiviral 3-Amidinophenylalanine Derivatives in Rat, Dog and Monkey Hepatocytes.

Type II transmembrane serine proteases represent pharmacological targets for blocking entry and spread of influenza or coronaviruses. In this study, the depletion rates of the 3-amidinophenylalanine (3-APhA)-derived matriptase/TMPRSS2 inhibitors MI-463, MI-472, MI-485 or MI-1900 were determined by LC-MS/MS measurements over a period of 300 min using suspensions of rat, dog and cynomolgus monkey primary hepatocytes. From these in vitro pharmacokinetic (PK) experiments, intrinsic clearance values (Clint) were evaluated, and in vivo pharmacokinetic parameters (hepatic clearance, hepatic extraction ratio and bioavailability) were predicted. It was found that rat hepatocytes were the most active in the metabolism of 3-APhA derivatives (Clint 31.9-37.8 mL/min/kg), whereas dog and monkey cells displayed somewhat lower clearance of these compounds (Clint 6.6-26.7 mL/min/kg). These data support elucidation of important PK properties of anti-TMPRSS2/anti-matriptase 3-APhAs using mammalian hepatocyte models and thus contribute to the optimization of lead compounds.

Epidemiology and Clinical Manifestation of West Nile Virus Infections of Equines in Hungary, 2007-2020.

West Nile virus (WNV) is an emerging pathogen in Hungary, causing severe outbreaks in equines and humans since 2007. The aim of our study was to provide a comprehensive report on the clinical signs of West Nile neuroinvasive disease (WNND) in horses in Hungary. Clinical details of 124 confirmed equine WNND cases were collected between 2007 and 2019. Data about the seasonal and geographical presentation, demographic data, clinical signs, treatment protocols, and disease progression were evaluated. Starting from an initial case originating from the area of possible virus introduction by migratory birds, the whole country became endemic with WNV over the subsequent 12 years. The transmission season did not expand significantly during the data collection period, but vaccination protocols should be always reviewed according to the recent observations. There was not any considerable relationship between the occurrence of WNND and age, breed, or gender. Ataxia was by far the most common neurologic sign related to the disease, but weakness, behavioral changes, and muscle fasciculation appeared frequently. Apart from recumbency combined with inappetence, no other clinical sign or treatment regime correlated with survival. The survival rate showed a moderate increase throughout the years, possibly due to the increased awareness of practitioners.

Luteolin and Chrysin Could Prevent E. coli Lipopolysaccharide-Ochratoxin A Combination-Caused Inflammation and Oxidative Stress in In Vitro Porcine Intestinal Model.

Ochratoxin A (OTA) and lipopolysaccharide (LPS) intake can cause gastrointestinal disorders. Polyphenolic chrysin (CHR) and luteolin (LUT) display anti-inflammatory and antioxidant properties. Porcine intestinal epithelial (jejunal) IPEC-J2 cells were treated with OTA (1 µM, 5 µM and 20 µM), E. coli LPS (10 µg/mL), CHR (1 µM) and LUT (8.7 µM) alone and in their combinations. Cell viabilities (MTS assay) and extracellular (EC) hydrogen-peroxide (H2O2) production (Amplex red method) were evaluated. Intracellular (IC) reactive oxygen species (ROS) were assessed using a 2'-7'dichlorodihydrofluorescein diacetate (DCFH-DA) procedure. ELISA assay was used to evaluate IL-6 and IL-8 secretion. OTA decreased cell viabilities (p < 0.001) which could not be alleviated by LUT or CHR (p > 0.05); however, EC H2O2 production was successfully suppressed by LUT in IPEC-J2 cells (p < 0.001). OTA with LPS elevated the IC ROS which was counteracted by CHR and LUT (p < 0.001). IL-6 and IL-8 secretion was elevated by LPS + OTA (p < 0.001) which could be inhibited by LUT (p < 0.01 for IL-6; p < 0.001 for IL-8). Based on our results, CHR and LUT exerted beneficial effects on IC ROS levels and on cytokine secretion (LUT) in vitro; thus, they might be used as dietary and feed supplements to avoid OTA- and LPS-related health risks.

Photoreactive Coating Material as an Effective and Durable Antimicrobial Composite in Reducing Bacterial Load on Surfaces in Livestock.

Titanium dioxide (TiO2) is a well-known photocatalytic compound that can be used to effectively reduce the presence of pathogens in human and animal hospitals via ROS release. The aim of this study was to investigate the efficacy of a polymer-based composite layer containing TiO2 and zinc oxide (ZnO) against Escherichia coli (E. coli) of animal origin. We showed that the photocatalyst coating caused a significant (p < 0.001) reduction in pathogen numbers compared to the control with an average reduction of 94% over 30 min. We used six light sources of different wattages (4 W, 7 W, 9 W, 12 W, 18 W, 36 W) at six distances (35 cm, 100 cm, 150 cm, 200 cm, 250 cm, 300 cm). Samples (n = 2160) were taken in the 36 settings and showed no significant difference in efficacy between light intensity and distance. We also investigated the influence of organic contaminant that resulted in lower activity as well as the effect of a water jet and a high-pressure device on the antibacterial activity. We found that the latter completely removed the coating from the surface, which significantly (p < 0.0001) reduced its antibacterial potential. As a conclusion, light intensity and distance does not reduce the efficacy of the polymer, but the presence of organic contaminants does.

The Impact of Quercetin and Its Methylated Derivatives 3-o-Methylquercetin and Rhamnazin in Lipopolysaccharide-Induced Inflammation in Porcine Intestinal Cells.

Oxidative stress in the small intestine can lead to inflammation and barrier malfunction. The present study describes the effect of quercetin (Q), 3-o-methylquercetin (QM), and rhamnazin (R) on cell viability, paracellular permeability, production of intracellular reactive oxygen species (ROS), extracellular hydrogen peroxide (H2O2), and interleukin-6 (IL-6) after challenging jejunal cells (IPEC-J2) with different types (Salmonella enterica ser. Typhimurium, Escherichia coli O111:B4, and E. coli O127:B8) of lipopolysaccharides (LPS) applied in 10 µg/mL concentration. The intracellular ROS level increased after all LPS treatments, which could be decreased by all tested flavonoid compounds in 50 µM concentration. Extracellular H2O2 production significantly increased after Q and R treatment (50 µM). S. Typhimurium LPS could significantly increase IL-6 production of enterocytes, which could be alleviated by Q, QM, and R (50 µM) as well. Using fluorescein isothiocyanate dextran (FD4) tracer dye, we could demonstrate that S. Typhimurium LPS significantly increased the permeability of the cell layer. The simultaneous treatments of S. Typhimurium LPS and the flavonoid compounds showed no alteration in FD4 penetration compared to untreated cells. These results highlight that Q, QM, and R are promising substances that can be used to protect intestinal epithelial cells from the deteriorating effects of oxidative stress.

Interspecies Comparisons of the Effects of Potential Antiviral 3-Amidinophenylalanine Derivatives on Cytochrome P450 1A2 Isoenzyme.

In vitro models of animals vulnerable to SARS-CoV-2 infection can support the characterization of effective antiviral drugs, such as synthetic inhibitors of the transmembrane protease serine 2 (TMPRSS2). Changes in cytochrome P450 (CYP) 1A2 activities in the presence of the potential TMPRSS2/matriptase inhibitors (MI) were measured using fluorometric and luminescent assays. Furthermore, the cytotoxicity of these inhibitors was evaluated using the MTS method. In addition, 60 min-long microsomal stability assays were performed using an UPLC-MS/MS procedure to elucidate depletion rates of the inhibitors. CYP1A2 was influenced significantly by MI-463 and MI-1900 in rat microsomes, by MI-432 and MI-482 in beagle microsomes, and by MI-432, MI-463, MI-482, and MI-1900 in cynomolgus monkey microsomes. The IC50 values in monkey microsomes were 1.30 ± 0.14 µM, 2.4 ± 1.4 µM, 0.21 ± 0.09 µM, and 1.1 ± 0.8 µM for inhibitors MI-432, MI-463, MI-482, and MI-1900, respectively. The depletion rates of the parent compounds were lower than 50%, independently of the investigated animal species. The host cell factor TMPRSS2 is of key importance for the cross-species spread of SARS-CoV-2. Studies of the in vitro biotransformation of TMPRSS2 inhibitors provide additional information for the development of new antiviral drugs.

Preliminary study to investigate the distribution and effects of certain metals after inhalation of welding fumes in mice.

The most important welding processes used are the gas metal arc (GMA) welding, the tungsten inert gas (TIG) welding, and the manual metal arc (MMA) welding processes. The goal of our investigation was to monitor the distribution of iron (Fe), manganese (Mn), calcium (Ca), and magnesium (Mg) in the lung, spleen, liver, and kidney of mice after inhalation exposure of different welding methods using different steel base materials. The treatment groups were the following: MMA-mild steel, MMA-molybdenum-manganese (MoMn) alloy, TIG-mild steel, and TIG-stainless steel. The samples were taken 24 and 96 h after the treatments. Most importantly, it was found that the Mn concentration in the lung' samples of the MMA-mild steel and the MMA-MoMn groups was increased extremely at both sampling times and in the spleen' samples also. In the TIG groups, the rise of the Mn concentration was only considerable in the lungs and spleens at 24 h, and emerged concentration was found in the liver in 96 h samples. Histopathology demonstrated emerged siderin content in the spleens of the treated animals and in siderin filled macrophages in the lungs mostly in all treated groups. Traces of high-level glycogen retention was found in the MMA groups at both sampling times. Similar glycogen retention in TIG-Ms and TIG stainless group's liver samples and emerged number of vacuoles, especially in the hepatocytes of the TIG-stainless steel 96 h group were also found. The mentioned results raise the consequence that there is a considerable difference in the kinetics of the Mn distribution between the MMA- and the TIG-fume-treated groups. Hence, the result suggests that manganese has a particle-size-dependent toxico-kinetics property. The anomaly of the glycogen metabolism indicates the systemic effect of the welding fumes. Also, the numerous vacuoles mentioned above show a possible liver-specific adverse effect of some components of the TIG-stainless steel welding fumes.

Synovial and Systemic Pharmacokinetics of Florfenicol and PK/PD Integration against Streptococcus suis in Pigs.

Florfenicol is a member of the phenicol group, a broad-spectrum antibacterial agent. It has been used for a long time in veterinary medicine, but there are some factors regarding its pharmacokinetic characteristics that have yet to be elucidated. The aim of our study was to describe the pharmacokinetic profile of florfenicol in synovial fluid and plasma of swine after intramuscular (i.m.) administration. In addition, the dosage regimen of treatment of arthritis caused by S. suis was computed for florfenicol using pharmacokinetic/pharmacodynamic (PK/PD) indices. As the first part of our investigation, the pharmacokinetic (PK) parameters of florfenicol were determined in the plasma and synovial fluid of six pigs. Following drug administration (15 mg/kgbw, intramuscularly), blood was drawn at the following times: 10, 20, 30, 40, 50 and 60 min, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48 and 72 h; synovial fluid samples were taken after 1, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h. The concentration of florfenicol was determined by a validated liquid chromatography-mass spectrometry (LC-MS/MS) method via multiple reaction monitoring (MRM) modes. As the second part of our research, minimum inhibitory concentration (MIC) values of florfenicol were determined in 45 S. suis strains isolated from clinical samples collected in Hungary. Furthermore, a strain of S. suis serotype 2 (SS3) was selected, and killing-time curves of different florfenicol concentrations (0.5 µg/mL, 1 µg/mL and 2 µg/mL) were determined against this strain. Peak concentration of the florfenicol was 3.58 ± 1.51 µg/mL in plasma after 1.64 ± 1.74 h, while it was 2.73 ± 1.2 µg/mL in synovial fluid 3.4 ± 1.67 h after administration. The half-life in plasma was found to be 17.24 ± 9.35 h, while in synovial fluid it was 21.01 ± 13.19 h. The area under the curve (AUC24h) value was 54.66 ± 23.34 µg/mL·h for 24 h in plasma and 31.24 ± 6.82 µg/mL·h for 24 h in synovial fluid. The drug clearance scaled by bioavailability (Cl/F) in plasma and synovial fluid was 0.19 ± 0.08 L/h/kg and 0.29 ± 0.08 L/h/kg, respectively. The mean residence time (MRT) in plasma and synovial fluid was 24.0 ± 13.59 h and 27.39 ± 17.16 h, respectively. The steady-state volume of distribution (Vss) in plasma was calculated from Cl/F of 0.19 ± 0.08 L/h/kg, multiplied by MRT of 24.0 ± 13.59 h. For the PK/PD integration, average plasma and synovial fluid concentration of florfenicol was used in a steady-state condition. The obtained MIC50 value of the strains was 2.0 µg/mL, and MIC90 proved to be 16.0 µg/mL. PK/PD integration was performed considering AUC24h/MIC breakpoints that have already been described. This study is the first presentation of the pharmacokinetic behavior of florfenicol in swine synovia as well as a recommendation of extrapolated critical MICs of S. suis for therapeutic success in the treatment of S. suis arthritis in swine, but it should be noted that this requires a different dosage regimen to that used in authorized florfenicol formulations.