Preliminary investigation into long-term stress by isolated captivity-related changes of reproduction hormones in Cynomolgus monkey.

BACKGROUND: Stress profoundly affects physical and emotional well-being, extending its physiological influence to the female menstrual cycle, impeding the hypothalamus-pituitary-gonadal (HPG) axis, and affecting fertility by suppressing sex-stimulating hormones. METHODS: In this study, we meticulously analyzed menstrual cycles and corresponding hormonal fluctuations in three female Cynomolgus monkeys. RESULTS: The preliminary findings indicated lower-than-normal levels of cortisol, follicle-stimulating hormone (FSH), and estradiol. Anovulatory bleeding occurred in one monkey, which could be linked to stress. In contrast to cortisol, alkaline phosphatase (ALP), which is correlated to cortisol levels, was consistently elevated in menstruating monkeys, suggesting its potential as a stress indicator. The non-menstruating group exhibited stress-related weight loss, emphasizing the observed ALP trends. CONCLUSIONS: Non-menstruating monkeys may experience more stress than menstruating monkeys. The implications of this study extend beyond the confines of primate studies and offer a valuable method for enhancing the welfare of female Cynomolgus monkeys.

Sappanone A Prevents Left Ventricular Dysfunction in a Rat Myocardial Ischemia Reperfusion Injury Model.

The incidence of myocardial infarction, among the causes of cardiovascular morbidity and mortality, is increasing globally. In this study, left ventricular (LV) dysfunction, including LV systolic and diastolic function, was investigated in a rat myocardial ischemia/reperfusion injury model with echocardiography. The homoisoflavanone sappanone A is known for its anti-inflammatory effects. Using echocardiography, we found that sappanone A administration significantly improved LV systolic and diastolic function in a rat myocardial ischemia/reperfusion injury model, especially in the early phase development of myocardial infarction. Based on myocardial infarct size, serum cardiac marker assay, and histopathological evaluation, sappanone A showed higher efficacy at the doses used in our experiments than curcumin and was evaluated for its potential to improve LV function.

Preventive effects of a novel herbal mixture on atopic dermatitis-like skin lesions in BALB/C mice.

BACKGROUND: A combination of parts of Cornus officinalis, Rosa multiflora, Lespedeza bicolor, Platycladus orientalis, and Castanea crenata is commonly used for alleviating inflammatory skin disorders. Therefore, this study was carried out to evaluate the in vitro and in vivo preventive effects of a novel herbal formula made from the five plants (C2RLP) against atopic dermatitis in BALB/C mice. METHODS: Mice were allocated into five groups (n = 8) including, control (Normal, petrolatum, and betamethasone treated) and treatment groups (treated with 2.5 and 5% C2RLP ointment). Atopic lesion was induced by applying 1-Chloro-2, 4-dinitrobenzene to the dorsal thoracic area of mice. Macroscopical and histological evaluations were performed to determine the effects of treatment on the progress of the skin lesions. The effects of treatment on the production and release of interleukins, interferon -ϒ, nitrite, prostaglandin E2, thymus and activation-receptor chemokine, and ß-hexosaminidase were evaluated and comparisons were made between groups. In addition, the chemical compounds present in C2RLP were identified by Liquid Chromatography-Mass Spectrometry. RESULTS: Topical application of C2RLP reduced the dermatitis score and suppressed histopathological changes in mice. Treatment significantly reduced (P < 0.05) plasma IL-4 level, the production of nitrite, prostaglandin E2, and thymus and activation-receptor chemokine production. The lipopolysaccharide-induced iNOS-mRNA expression in RAW 264.7 cells was also suppressed by high concentrations of C2RLP. In addition, C2RLP showed an inhibitory effect against DPPH free radical (IC50 = 147.5 µg/ml) and ß-hexosaminidase release (IC50 = 179.5 µg/ml). Liquid Chromatography-Mass Spectrometry analysis revealed the presence of various compounds, including loganin, ellagic acid, and kaempferol 3-glucoside. CONCLUSION: Down-regulation of T- helper 2 cellular responses and suppression of inflammatory mediators contributed to the protective effects of C2RLP from atopic dermatitis in BALB/C mice.

Inhibition of Salmonella Typhimurium adhesion, invasion, and intracellular survival via treatment with methyl gallate alone and in combination with marbofloxacin.

Salmonella enterica serovar Typhimurium infects intestinal epithelia and macrophages, which is prevented by inhibiting adhesion and cell invasion. This study aimed to investigate the role of methyl gallate (MG) in adhesion, invasion, and intracellular survival of Salmonella Typhimurium in Caco-2 and RAW 264.7 cells via a gentamicin protection assay, confocal microscopy, and quantitative reverse-transcription polymerase chain reaction. MG (30 µg/mL) inhibited adhesion and invasion of Salmonella Typhimurium by 54.01% and 60.5% in RAW 264.7 cells, respectively. The combination of MG with sub-minimum inhibitory concentration (MIC) of marbofloxacin (MRB) inhibited the adhesion, invasion, and intracellular survival by 70.49%, 67.36%, and 74%, respectively. Confocal microscopy further revealed reductions in bacterial count in Caco-2 cells treated with MG alone or with sub-MIC of MRB. Furthermore, MG alone or in combination with sub-MIC of MRB decreased the motility of Salmonella Typhimurium. Quorum sensing genes including sdiA, srgE, and rck were downregulated by 52.8%, 61.7%, and 22.2%, respectively. Moreover, rac-1 was downregulated by 56.9% and 71.9% for MG alone and combined with sub-MIC of MRB, respectively, in mammalian cells. Furthermore, MG downregulated virulence genes of Salmonella Typhimurium including cheY, ompD, sipB, lexA, and ompF by 59.6%, 60.2%, 20.5%, 31.4%, and 16.2%, respectively. Together, the present results indicate that MG alone or in combination with a sub-MIC of MRB effectively inhibited the adhesion, invasion, and intracellular survival of Salmonella Typhimurium in vitro by downregulating quorum sensing and virulence genes.

Using In Vitro Dynamic Models To Evaluate Fluoroquinolone Activity against Emergence of Resistant Salmonella enterica Serovar Typhimurium.

The objectives of this study were to determine pharmacokinetic/pharmacodynamic (PK/PD) indices of fluoroquinolones that minimize the emergence of resistant Salmonella enterica serovar Typhimurium (S Typhimurium) using in vitro dynamic models and to establish mechanisms of resistance. Three fluoroquinolones, difloxacin (DIF), enrofloxacin (ENR), and marbofloxacin (MAR), at five dose levels and 3 days of treatment were simulated. Bacterial killing-regrowth kinetics and emergence of resistant bacteria after antibacterial drug exposure were quantified. PK/PD indices associated with different levels of antibacterial activity were computed. Mechanisms of fluoroquinolone resistance were determined by analyzing target mutations in the quinolone resistance-determining regions (QRDRs) and by analyzing overexpression of efflux pumps. Maximum losses in susceptibility of fluoroquinolone-exposed S Typhimurium occurred at a simulated AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) of 47 to 71. Target mutations in gyrA (S83F) and overexpression of acrAB-tolC contributed to decreased susceptibility in fluoroquinolone-exposed S Typhimurium. The current data suggest AUC/MIC (AUC/mutant prevention concentration [MPC])-dependent selection of resistant mutants of S Typhimurium, with AUC/MPC ratios of 69 (DIF), 62 (ENR), and 39 (MAR) being protective against selection of resistant mutants. These values could not be achieved in veterinary clinical areas under the current recommended therapeutic doses of the fluoroquinolones, suggesting the need to reassess the current dosing regimen to include both clinical efficacy and minimization of emergence of resistant bacteria.

Elimination of A-234 from the environment: Effect of different decontaminants.

With the fact that there are Novichoks in the list of toxic chemicals by the Chemical Weapons Convention parties, it is necessary to develop methods of effective neutralization of the agents as well as for other organophosphorus toxic substances. However, experimental studies on their persistence in the environment and effective decontamination measures remain scarce. Therefore, here, we investigated the persistence behavior and decontamination methods of A-234 (ethyl N-[1-(diethylamino)ethylidene]phosphoramidofluoridate), a Novichok series, A-type nerve agent to assess its potential risk to the environment. Different analytical methods were implemented, including 31P solid-state magic angle spinning nuclear magnetic resonance (NMR), liquid 31P NMR, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry, and vapor-emission screening using a microchamber/thermal extractor with GC-MS. Our results showed that A-234 is extremely stable in sand and poses a long-lasting risk to the environment even when released in trace quantities. Moreover, the agent is not easily decomposed by water, dichloroisocyanuric acid sodium salt, sodium persulfate, and chlorine-based water-soluble decontaminants. However, it is efficiently decontaminated by Oxone® monopersulfate, calcium hypochlorite, KOH, NaOH, and HCl within 30 min. Our findings provide valuable insights for eliminating the highly dangerous Novichok agents from the environment.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats.

BACKGROUND: Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats. METHODS: OVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 µg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman's correlation coefficients were defined and analyzed. Additionally, gas chromatography-mass spectrometry analysis was conducted to uncover additional physiologically active ingredients. RESULTS: TPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. CONCLUSIONS: TPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.

Serum biomarker-based osteoporosis risk prediction and the systemic effects of Trifolium pratense ethanolic extract in a postmenopausal model.

BACKGROUND: Recent years, a soaring number of marketed Trifolium pratense (red clover) extract products have denoted that a rising number of consumers are turning to natural alternatives to manage postmenopausal symptoms. T. pratense ethanolic extract (TPEE) showed immense potential for their uses in the treatment of menopause complications including osteoporosis and hormone dependent diseases. Early diagnosis of osteoporosis can increase the chance of efficient treatment and reduce fracture risks. Currently, the most common diagnosis of osteoporosis is performed by using dual-energy x-ray absorptiometry (DXA). However, the major limitation of DXA is that it is inaccessible and expensive in rural areas to be used for primary care inspection. Hence, serum biomarkers can serve as a meaningful and accessible data for osteoporosis diagnosis. METHODS: The present study systematically elucidated the anti-osteoporosis and estrogenic activities of TPEE in ovariectomized (OVX) rats by evaluating the bone microstructure, uterus index, serum and bone biomarkers, and osteoblastic and osteoclastic gene expression. Leverage on a pool of serum biomarkers obtained from this study, recursive feature elimination with a cross-validation method (RFECV) was used to select useful biomarkers for osteoporosis prediction. Then, using the key features extracted, we employed five classification algorithms: extreme gradient boosting (XGBoost), random forest, support vector machine, artificial neural network, and decision tree to predict the bone quality in terms of T-score. RESULTS: TPEE treatments down-regulated nuclear factor kappa-B ligand, alkaline phosphatase, and up-regulated estrogen receptor ß gene expression. Additionally, reduced serum C-terminal telopeptides of type 1 collagen level and improvement in the estrogen dependent characteristics of the uterus on the lining of the lumen were observed in the TPEE intervention group. Among the tested classifiers, XGBoost stood out as the best performing classification model with the highest F1-score and lowest standard deviation. CONCLUSIONS: The present study demonstrates that TPEE treatment showed therapeutic benefits in the prevention of osteoporosis at the transcriptional level and maintained the estrogen dependent characteristics of the uterus. Our study revealed that, in the case of limited number of features, RFECV paired with XGBoost model could serve as a powerful tool to readily evaluate and diagnose postmenopausal osteoporosis.

Colistin Induces Resistance through Biofilm Formation, via Increased phoQ Expression, in Avian Pathogenic Escherichia coli.

This study aimed to optimize the colistin-based antibacterial therapy to prevent antimicrobial resistance related to biofilm formation in avian pathogenic Escherichia coli (APEC) in chicken. Of all the bacterial isolates (n = 136), 69 were identified as APEC by polymerase chain reaction (PCR). Through a series of antibiotic susceptibility tests, susceptibility to colistin (<2 µg/mL) was confirmed in all isolates. Hence, a mutant selection window (MSW) was determined to obtain colistin-induced resistant bacteria. The minimum inhibitory concentration (MIC) of colistin against the colistin-induced resistant APEC strains ranged from 8 to 16 µg/mL. To identify the inhibitory activity of colistin against the resistant strains, the mutant prevention concentration (MPC) was investigated for 72 h, and the single and multi-dose colistin activities were determined through the time-kill curve against APEC strains. Bacterial regrowth occurred after 12 h at a double MIC50 concentration (1.00 µg/mL), and regrowth was not inhibited even during multiple exposures. However, upon exposure to 8 µg/mL-a concentration that was close to the MPC-the growth of APEC was inhibited, including in the resistant strains. Additionally, colistin-induced resistant strains showed a slower growth compared with the susceptible ones. Colistin-induced resistant APEC strains did not show colistin resistance gene (mcr-1). However, the expression of higher mgrB and phoQ levels was observed in the resistant strains. Furthermore, these strains showed increased formation of biofilm. Hence, the present study indicated that colistin could induce resistance through the increased formation of biofilm in APEC strains by enhancing the expression of phoQ.

Ethanol-Induced Hepatotoxicity and Alcohol Metabolism Regulation by GABA-Enriched Fermented Smilax china Root Extract in Rats.

Chronic alcohol consumption can cause hepatic injury and alcohol-induced toxicities. Extracts from Smilax china root have been widely used in traditional medicine and for their potential pharmacological benefits. We aimed to determine if fermented Smilax china extract (FSC) regulates alcoholic fatty liver and liver injury using two in vivo experiments. Sprague-Dawley rats were administered ethanol (3 g/kg b.w.; po) with or without FSC pretreatment to induce an acute hangover. In another experiment, rats were fed either a normal or Lieber-DeCarli ethanol (6.7%) diet with or without FSC pretreatment (125, 250, and 500 mg/kg b.w.; po) for 28 days. Serum biomarkers, liver histopathology, and the mRNA levels of anti-inflammatory, antioxidant, lipogenic, and lipolytic genes were analyzed. FSC pretreatment significantly reduced blood alcohol and acetaldehyde concentrations, upregulated the mRNA expression of alcohol dehydrogenase, aldehyde dehydrogenase, and superoxide dismutase, and decreased the activities of liver enzymes in a dose-dependent manner. It also downregulated SERBP-1c and upregulated PPAR-α and reduced the gene expression of the anti-inflammatory cytokine IL-6 in the liver. The final extract after fermentation had increased GABA content. Furthermore, FSC was found to be safe with no acute oral toxicity in female rats. Thus, FSC increases alcohol metabolism and exhibits antioxidant and anti-inflammatory effects to induce hepatoprotection against alcohol-induced damage. It may be used as a functional food ingredient after excess alcohol consumption.

Impact of sphingosine and acetylsphingosines on the aggregation and toxicity of metal-free and metal-treated amyloid-ß.

Pathophysiological shifts in the cerebral levels of sphingolipids in Alzheimer's disease (AD) patients suggest a link between sphingolipid metabolism and the disease pathology. Sphingosine (SP), a structural backbone of sphingolipids, is an amphiphilic molecule that is able to undergo aggregation into micelles and micellar aggregates. Considering its structural properties and cellular localization, we hypothesized that SP potentially interacts with amyloid-ß (Aß) and metal ions that are found as pathological components in AD-affected brains, with manifesting its reactivity towards metal-free Aß and metal-bound Aß (metal-Aß). Herein, we report, for the first time, that SP is capable of interacting with both Aß and metal ions and consequently affects the aggregation of metal-free Aß and metal-Aß. Moreover, incubation of SP with Aß in the absence and presence of metal ions results in the aggravation of toxicity induced by metal-free Aß and metal-Aß in living cells. As the simplest acyl derivatives of SP, N-acetylsphingosine and 3-O-acetylsphingosine also influence metal-free Aß and metal-Aß aggregation to different degrees, compared to SP. Such slight structural modifications of SP neutralize its ability to exacerbate the cytotoxicity triggered by metal-free Aß and metal-Aß. Notably, the reactivity of SP and the acetylsphingosines towards metal-free Aß and metal-Aß is determined to be dependent on their formation of micelles and micellar aggregates. Our overall studies demonstrate that SP and its derivatives could directly interact with pathological factors in AD and modify their pathogenic properties at concentrations below and above critical aggregation concentrations.

Hepatoprotective effects of gamma-aminobutyric acid-enriched fermented Hovenia dulcis extract on ethanol-induced liver injury in mice.

BACKGROUND: Various extracts of Hovenia dulcis have been commonly used in Asia for cases of alcohol-related disorders. Fermentation is reported to enhance the level and biological activities of various bio-constituents of plant extracts. Therefore, this study was undertaken to evaluate the effects of fermented H. dulcis extract (FHDE) on ethanol-induced liver injury in mice. METHODS: FHDE was prepared using Bacillus subtilis and Lactobacillus plantarum. The effects of FHDE on ethanol-induced liver injury were evaluated in C57BL/6 N CrSlc mice. A mixed feed preparation containing the fermented extract with and without ethanol was given to mice for 29 days, according to its group. At the end of the experiment, blood and liver samples were collected from all mice in the group. Plasma biochemical analysis and histopathological investigation were performed to evaluate the impacts of treatment on the biomarkers of hepatic damage and inflammatory changes. Besides, the expression of genes that regulate the activities of enzymes associated with alcohol metabolism, antioxidant activity, and fatty acid oxidation was assessed using a quantitative real-time polymerase chain reaction. Moreover, the amino acid contents and the active ingredients of the extract were evaluated before and after fermentation. RESULTS: Fermentation resulted in a marked increase and decrease in the amount of Gamma-Amino-n-butyric acid (GABA) and glutamic acid, respectively. FHDE enhanced the body weight gain of mice compared to ethanol. Besides, plasma levels of triglyceride, low-density lipoprotein, the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly (P < 0.05) reduced in the FHDE-treated groups relative to the ethanol-treated control. FHDE upregulated the expression of genes associated with enzymes involved in alcohol dehydrogenation (Adh1 and Aldh2), antioxidant activity (SOD and CAT), and fatty acid oxidation (PPAR-α and PGC-1α). However, the expressions of Cytochrome peroxidase Cyp2E1 and genes related to lipogenesis (SREBP-1c, FAS, SCD-1, and ACC) were significantly (P < 0.05) downregulated following treatment with the FHDE. Histopathological investigation demonstrated a slight degree of inflammatory cell infiltration and occasional fatty changes in the FHDE-treated groups. CONCLUSION: The GABA-enriched fermented H. dulcis extract prevented ethanol-induced hepatic damage by enhancing the antioxidant defense system, fatty acid oxidation, and reducing lipogenesis.

Tunable regulatory activities of 1,10-phenanthroline derivatives towards acid sphingomyelinase and Zn(ii)-amyloid-ß.

We report a new series of small molecules able to achieve the tunability of modulatory activities against acid sphingomyelinase (ASM) and Zn(ii)-bound amyloid-ß [Zn(ii)-Aß], two pathological targets found in the brain affected by Alzheimer's disease. Rational tuning of the hydrophobicity and Zn(ii) binding affinity of the 1,10-phenanthroline (phen) framework successfully yielded compounds as chemical modulators for ASM (4 and 5), Zn(ii)-Aß (phen, 1, and 2), or both (3).

In silico analysis of putative drug and vaccine targets of the metabolic pathways of Actinobacillus pleuropneumoniae using a subtractive/comparative genomics approach.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that resides in the respiratory tract of pigs and causes porcine respiratory disease complex, which leads to significant losses in the pig industry worldwide. The incidence of drug resistance in this bacterium is increasing; thus, identifying new protein/gene targets for drug and vaccine development is critical. In this study, we used an in silico approach, utilizing several databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database of Essential Genes (DEG), DrugBank, and Swiss-Prot to identify non-homologous essential genes and prioritize these proteins for their druggability. The results showed 20 metabolic pathways that were unique and contained 273 non-homologous proteins, of which 122 were essential. Of the 122 essential proteins, there were 95 cytoplasmic proteins and 11 transmembrane proteins, which are potentially suitable for drug and vaccine targets, respectively. Among these, 25 had at least one hit in DrugBank, and three had similarity to metabolic proteins from Mycoplasma hyopneumoniae, another pathogen causing porcine respiratory disease complex; thus, they could serve as common therapeutic targets. In conclusion, we identified glyoxylate and dicarboxylate pathways as potential targets for antimicrobial therapy and tetra-acyldisaccharide 4'-kinase and 3-deoxy-D-manno-octulosonic-acid transferase as vaccine candidates against A. pleuropneumoniae.

Enhancement of Lipid Metabolism and Hepatic Stability in Fat-Induced Obese Mice by Fermented Cucurbita moschata Extract.

The aim of this study was to evaluate the potentials of fermented Cucurbita moschata extract (FCME) in the treatment of obesity and nonalcoholic fatty liver disease (NAFLD). Five-week-old male C57BL/6 mice were assigned to 6 groups and treated for 8 weeks by feeding the normal diet (ND) and high fat diet (HFD) with and without FCME. Changes in body weight gain and consumption of feed and water were recorded. Major organs, adipose tissues, and blood samples were collected after the experimental period. The serum lipid profile, histological features of liver and adipose tissues, and mRNA expression of different adipogenic/lipogenic genes from liver tissue were evaluated. The supplementation of FCME in HFD significantly prevented HFD-induced increment of bodyweight. The adipose tissue mass, liver enzymes, and plasma lipids were also reduced significantly (p < 0.05) by the consumption of FCME. The mRNA expressions of adipogenic/lipogenic genes (PPARγ, C/EBPα, C/EBP ß , C/EBPγ, and SREBP-1C) in FCME-treated obese mice were considerably (p < 0.05) suppressed. FCME showed its antiobesity potential by suppressing the body weight gain and by modulating the plasma lipids and liver enzymes through the regulation of adipogenic/lipogenic transcriptional factors. Fermented Cucurbita moschata could be an opportunistic agent in controlling obesity and fatty liver changes.

The phenotypic and molecular resistance induced by a single-exposure to sub-mutant prevention concentration of marbofloxacin in Salmonella Typhimurium isolates from swine.

In the present study, the molecular mechanisms of antibiotic resistance in Salmonella Typhimurium clinical isolates from pigs were investigated using a single-step mutation model of exposure to sub-mutant prevention concentrations (MPCs) of marbofloxacin. The minimum inhibitory concentrations (MICs) of seven antibacterial drugs were evaluated against 30 S. Typhimurium clinical isolates from different pigs. MPCs of marbofloxacin were also determined. The mechanism of marbofloxacin-resistance was investigated by sequencing analysis of target gene mutations and quantifying the overexpression of efflux pumps and their regulators by quantitative RT-PCR. Marbofloxacin showed the highest potency against all isolates (23.3%), including multi-drug resistant isolates. The MPC50 (0.5µg/mL) and MPC90 (2µg/mL) of marbofloxacin were determined, as were MPC/MIC ratios of 2.5 to 8. A gyrA mutation (Ser83Phe or Asp87His) was detected in isolates with an MIC>0.06µg/mL and all single-step mutants. Moreover, expression of acrAB-tolC and marA/soxS/ramA increased following a single-step mutation, but only ramA expression showed a positive correlation with the resistance phenotype of clinical isolates and single-step mutants (p<0.05). Furthermore, the acrR mutation was detected in two clinical isolates and 50% of single-step mutants, regardless of whether the gyrA mutation was present. This is the first report of acrR mutations in S. Typhimurium isolates from pigs in Korea. Our findings suggest that a single-exposure to sub-MPCs of marbofloxacin was sufficient to reduce the susceptibility of Salmonella isolates. Therefore, optimized dosing based on application with the MPC concept is required to reduce the chances of marbofloxacin resistance.

Biofilm formation and determination of minimum biofilm eradication concentration of antibiotics in Mycoplasma hyopneumoniae.

The study was aimed to investigate biofilm forming ability of Mycoplasma hyopneumoniae and to determine the minimum biofilm eradication concentrations of antibiotics. Biofilm forming ability of six strains of M. hyopneumoniae was examined using crystal violet staining on coverslips. The results demonstrated an apparent line of biofilm growth in 3 of the strains isolated from swine with confirmed cases of enzootic pneumonia. BacLight bacterial viability assay revealed that the majority of the cells were viable after 336 hr of incubation. Moreover, M. hyopneumoniae persists in the biofilm after being exposed to 10 fold higher concentration of antibiotics than the minimum inhibitory concentrations in planktonic cells. To the best of our knowledge, this is the first report of biofilm formation in M. hyopneumoniae. However, comprehensive studies on the mechanisms of biofilm formation are needed to combat swine enzootic pneumonia caused by resistant M. hyopneumoniae.

Probiotic properties and adsorption of Enterococcus faecalis PSCT3-7 to vermiculite.

The probiotic properties of Enterococcus (E.) faecalis PSCT3-7, a new strain isolated from the intestines of pigs fed dietary fiber containing 50% sawdust, were investigated. E. faecalis PSCT3-7 tolerated a pH range of 3 to 8 and 0.3% bile salts, and it inhibited the growth of Salmonella Typhimurium in a concentration-dependent manner. In addition, E. faecalis showed resistance to several antibacterial agents. Vermiculite, a nutrient and microbial carrier, increased the bile tolerance of the strain. Scanning electron microscope images revealed good adsorption of E. faecalis PSCT3-7 onto vermiculite. E. faecalis PSCT3-7 represents a potential probiotic candidate to administer with vermiculite to swine.

Impact of phenolic compounds in the acyl homoserine lactone-mediated quorum sensing regulatory pathways.

Quorum sensing (QS) is a cell density-dependent regulation of virulent bacterial gene expression by autoinducers that potentially pertains in the epidemic of bacterial virulence. This study was initially designed to evaluate the effect of 5 phenolic compounds in the modulation of QS and virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa, and to determine the mechanisms of their effects. Biosensor strains were used to assess antibacterial and anti-QS effect of these compounds. Only methyl gallate (MG) among these compounds demonstrated profound anti-QS effect in the preliminary study, and thus only MG was utilized further to evaluate the effects on the synthesis and activity of acyl homoserine lactone (AHL) in C. violaceum and on the modulation of biofilm, motility, proteolytic, elastase, pyocyanin, and rhamnolipid activity in P. aeruginosa. Finally, the effect of MG on the expression of QS-regulated genes of P. aeruginosa was verified. MG suppressed both the synthesis and activity of AHL in C. violaceum. It also restricted the biofilm formation and other QS-associated virulence factor of P. aeruginosa. MG concentration-dependently suppressed the expression of lasI/R, rhlI/R, and pqsA of P. aeruginosa and was non-toxic in in vitro study. This is the first report of the anti-QS mechanism of MG.