Virtual Screening and In Vitro Experimental Verification of LuxS Inhibitors for Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 is an important foodborne pathogen that forms biofilms. In this study, three quorum-sensing (QS) inhibitors (M414-3326, 3254-3286, and L413-0180) were obtained through virtual screening, and their in vitro antibiofilm activities were validated. Briefly, the three-dimensional structure model of LuxS was constructed and characterized using the SWISS-MODEL. High-affinity inhibitors were screened from the ChemDiv database (1,535,478 compounds) using LuxS as a ligand. Five compounds (L449-1159, L368-0079, M414-3326, 3254-3286, and L413-0180) with a good inhibitory effect (50% inhibitory concentration <10 µM) on type II QS signal molecule autoinducer-2 (AI-2) were obtained using a AI-2 bioluminescence assay. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties predicated that the five compounds had high intestinal absorption levels (high) and plasma protein binding (absorbent strong) and did not inhibit the metabolism of CYP2D6 metabolic enzymes. In addition, molecular dynamics simulation showed that compounds L449-1159 and L368-0079 could not stably bind with LuxS. Thus, these compounds were excluded. Furthermore, surface plasmon resonance results showed that the three compounds could specifically bind to LuxS. IN addition, the three compounds could effectively inhibit the biofilm formation without affecting the growth and metabolism of the bacteria. Finally, the reverse transcription-quantitative PCR results showed that the three compounds downregulated the expression of the LuxS gene. Overall, these results revealed that the three compounds obtained through virtual screening could inhibit biofilm formation of E. coli O157:H7 and are potential LuxS inhibitors that can be used to treat E. coli O157:H7 infections. IMPORTANCE E. coli O157:H7 is a foodborne pathogen of public health importance. Quorum sensing (QS) is a form of bacterial communication that can regulate various group behaviors, including biofilm formation. Here, we identified three QS AI-2 inhibitors (M414-3326, 3254-3286, and L413-0180) that can stably and specifically bind to LuxS protein. The three QS AI-2 inhibitors inhibited biofilm formation without affecting the growth and metabolic activity of E. coli O157:H7. The three QS AI-2 inhibitors are promising agents for treating E. coli O157:H7 infections. Further studies to identify the mechanism of the three QS AI-2 inhibitors are needed to develop new drugs to overcome antibiotic resistance.

Novel quorum sensing inhibitor Echinatin as an antibacterial synergist against Escherichia coli.

A new antibacterial strategy based on inhibiting bacterial quorum sensing (QS) has emerged as a promising method of attenuating bacterial pathogenicity and preventing bacterial resistance to antibiotics. In this study, we screened Echinatin (Ech) with high-efficiency anti-QS from 13 flavonoids through the AI-2 bioluminescence assay. Additionally, crystal violet (CV) staining combined with confocal laser scanning microscopy (CLSM) was used to evaluate the effect of anti-biofilm against Escherichia coli (E. coli). Further, the antibacterial synergistic effect of Ech and marketed antibiotics were measured by broth dilution and Alamar Blue Assay. It was found that Ech interfered with the phenotype of QS, including biofilm formation, exopolysaccharide (EPS) production, and motility, without affecting bacterial growth and metabolic activity. Moreover, qRT-PCR exhibited that Ech significantly reduced the expression of QS-regulated genes (luxS, pfs, lsrB, lsrK, lsrR, flhC, flhD, fliC, csgD, and stx2). More important, Ech with currently marketed colistin antibiotics (including colistin B and colistin E) showed significantly synergistically increased antibacterial activity in overcoming antibiotic resistance of E. coli. In summary, these results suggested the potent anti-QS and novel antibacterial synergist candidate of Ech for treating E. coli infections.

14-Day Repeated Intraperitoneal Toxicity Test of Ivermectin Microemulsion Injection in Wistar Rats.

To evaluate the safety of ivermectin microemulsion injection, 100 Wistar rats were injected intraperitoneally at 0.38 g/kg, 0.19 g/kg, and 0.1 g/kg for 14 days. The 14-day repeated toxicity test of ivermectin microemulsion injection was systematically evaluated by clinical observation, organ coefficient, hematological examination, clinical chemistry examination, and histopathological examination. The results showed that no rats died during the test. At the initial stage of treatment, the rats in the high dose group had mild clinical reaction, which disappeared after 4 days. Clinical chemistry showed that the high dose of ivermectin microemulsion could cause significant changes in ALT and LDH parameters in male rats; high and medium doses could increase the liver coefficients of male and female rats. The toxic target organ may be the liver as indicated by histopathological findings. No significant toxic injury was found in the heart, liver, spleen, lung, kidney, brain, ovary, and testes of all groups of rats. No drug-related toxic effects were found at low doses, and thus the NOVEL of ivermectin microemulsion injection was 0.19 g/kg.

Efficacy of enteric-coated tilmicosin granules in pigs artificially infected with Actinobacillus pleuropneumoniae serotype 2.

BACKGROUND: Porcine infectious pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one of the most serious infectious diseases in pigs and has brought huge economic losses to the world pig industry. The aim of this trial was to evaluate the effect of enteric-coated tilmicosin granule in the treatment and control of artificial infection of App. METHODS: Sixty Duroc and Yorkshire crossbred pigs (50 of which were artificially infected) were divided into six groups: BCG (Blank control group), ICG (Infection-only control group), HDG (High-dose enteric-coated tilmicosin granules), MDG (Medium-dose enteric-coated tilmicosin granules), LDG (Low-dose enteric-coated tilmicosin granules) and TPG (Tilmicosin premix drug control group). The cure rate, mortality, clinical respiratory score, body temperature score, weight gain, lung score and so on were recorded. RESULTS: The cure rate of HDG and MDG was as high as 90%, the mortality was 10%, and the clinical signs recovered quickly. CONCLUSION: The results showed that enteric-coated tilmicosin granules had obvious therapeutic effect on artificial infection, which could reduce the damage caused by the disease and reduce the mortality.

New life for an old drug: In vitro and in vivo effects of the anthelmintic drug niclosamide against Toxoplasma gondii RH strain.

Toxoplasma gondii is the causative agent of toxoplasmosis and causes serious public health problems. However, the current treatment drugs have many limitations, such as serious side effects. Niclosamide is a salicylanilide drug commonly used to treat worm infections. Herein, the effectiveness of niclosamide for the treatment of T. gondii infection was demonstrated. This study was to evaluate the in vitro and in vivo activities of niclosamide against T. gondii and to explore its mechanism of action. The in vitro cytotoxicity of niclosamide on human foreskin fibroblast cells was evaluated by MTT test. Niclosamide displayed low host toxicity and its 50% inhibitory concentration was 8.3 µg/mL. The in vitro anti-proliferation and anti-invasion effects of niclosamide on T. gondii were determined by quantitative PCR and Giemsa staining. Niclosamide also inhibited T. gondii tachyzoite proliferation, with a 50% effective concentration of 45.3 ng/mL, and reduced the invasion of cells by tachyzoites (17.8% for the parasite control versus 1.9% for the niclosamide group treated with 100 ng/mL). A model was established by infecting BALB/c mice with the virulent RH strain of T. gondii and used to determine the in vivo effects of niclosamide on acute infection. The mice infected with tachyzoites and treated with 160, 200 or 240 mg/kg·bw niclosamide for 7 days exhibited 20%, 40% and 50% survival, respectively. In addition, niclosamide reduced the parasite burden in the blood and tissues of acutely infected mice, and niclosamide induced decreases in the mitochondrial membrane potential (ΔΨm) and adenosine triphosphate (ATP) levels in extracellular tachyzoites, as assessed by laser confocal microscopy and a multilabel reader. These findings indicated that the mechanism of action of niclosamide might be associated with T. gondii mitochondria oxidative phosphorylation. In conclusion, our results support the efficacy of niclosamide as a potential compound for the treatment of T. gondii infection.

High prevalence of fasciolosis and evaluation of the efficacy of anthelmintics against Fasciola hepatica in buffaloes in Guangxi, China.

Fasciola hepatica is a common parasite of grazing livestock in Guangxi Zhuang Autonomous Region in China, but its prevalence has not been studied. While triclabendazole is commonly used to treat F. hepatica infection in China, oxyclozanide has never been used. This study investigated the prevalence of F. hepatica infections in buffaloes in the Guangxi and evaluated the efficacy of oxyclozanide and triclabendazole as treatments. In the prevalence study, a total of 767 individual faecal samples were obtained from 58 farms in Guangxi to detect the prevalence of F. hepatica, and the total rate of infection was 87.35%. A subset of 277 infected buffaloes from these farms were randomly divided into 3 groups. Group 1 (n = 101) was treated with oxyclozanide at 10 mg/kg.bw; group 2 (n = 94) was treated with triclabendazole (12 mg/kg.bw); and group 3 (n = 82) was untreated. Faecal samples were taken on days 0, 7, 14, 21 and 28. Whole blood and serum were collected on days 0 and 14. Anthelmintic efficacy was assessed using faecal egg count reduction (FECR), buffaloes positive by coprology reduction (BPCR) as well as post-treatment improvement in biochemical and haematological indicators. After 28 days treatment, group 1 and 2 showed FECR% values above 98%, and BPCR% values of 97.03% and 77.66%, respectively. In addition, the biochemical indicators and haematological parameters were improved at 14 days post-treatment compared with those before treatment. These results indicate a high prevalence of F. hepatica in Guangxi, demonstrate that oxyclozanide and triclabendazole are effective against F. hepatica infection in buffaloes, and indicate that oxyclozanide could be used in China as an alternative drug.

EFFICACY AND SAFETY OF BAN HUANG ORAL LIQUID FOR TREATING BOVINE RESPIRATORY DISEASES.

BACKGROUND: Ban Huang oral liquid was developed as a veterinary compound preparation by the Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences (CAAS). The purpose of this study was to determine whether the oral liquid preparation of traditional Chinese medicine, Ban Huang, is safe and effective for treating respiratory diseases in cattle. MATERIALS AND METHODS: Acute oral toxicity experiments were conducted in Wistar rats and Kunming mice via oral administration. The minimum inhibitory concentration of the drug against Mycoplasma bovis in vitro with the double dilution method was 500 mg/mL, indicating good sensitivity. The results of laboratory pathogen testing, analysis of clinical symptoms, and analysis of pathological anatomy were combined to diagnose bovine respiratory diseases in 147 Simmental cattle caused by mixed infections of M. bovis, bovine respiratory syncytial virus, bovine parainfluenza virus type 3, and Mannheimia haemolytica. These cattle were randomly divided into three groups: drug treatment group 1 (treated via Tilmicosin injection), drug treatment group 2 (treated with Shuang Huang Lian oral liquid combined with Tilmicosin injection), and drug treatment group 3 (treated with Ban Huang oral liquid combined with Tilmicosin injection). Treatment effects were observed within 7 days. RESULTS: The results showed no toxicity and a maximum tolerated dose greater than 20 g/kg BW. For the 87 cattle in drug-treatment group, the cure rate was 90.80%, whereas the response rate was 94.25%. The cure rate of drug treatment group was increased by 14.13% in comparison with that of drug control group 1 and by 7.47% in comparison with that of drug control group 2 (both P < 0.05). CONCLUSION: This study demonstrates that Ban Huang oral liquid is a safe and effective treatment for bovine respiratory diseases, especially for mixed infection caused by M. bovis, bacteria, and viruses.

Acaricidal activities of the essential oil from Rhododendron nivale Hook. f. and its main compund, δ-cadinene against Psoroptes cuniculi.

In this paper, the acaricidal activities of Rhododendron nivale Hook. f. and its main compound, δ-cadinene were investigated, and the chemical composition of the essential oil was analyzed. The results showed that among aqueous, 70% ethanols, acetic ether, chloroform, petroleum ether and essential oil extracts from the shoots and leaves, the essential oil showed the best in vitro acaricidal activity against adult P. cuniculi, which occurred in a concentration- and time-dependent manner. The median lethal time (LT50) values of four concentrations (33.33-4.17mg/ml) of the essential oil ranged from 1.476 to 25.900h, respectively. After the treatment of P. cuniculi with the essential oil and ivermectin, infected rabbits were free of scabs or secretions in the ear canal by day 20. Then, the percent yield of essential oil from the leaves and shoots was 2.45% (w/w), which includes 50 compounds. The primary component identified was terpenes, and among of compounds identified from the essential oil of R. nivale the highest relative content was δ-cadinene, which also presented the marked acaricidal activity against Psoroptes cuniculi in vitro. These findings provide evidence for the use of acaricides as a traditional medicine and indicate that the essential oil and δ-cadinene could be used to control mites in livestock.

Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column (2.1 × 30 mm, 3.5 µm) using acetonitrile-water (90:10, v/v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r2 = 0.9998). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC-MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability.

Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneous quantify artesunate and its metabolite in sheep plasma. The plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column (250×4.6mm, 5µm) using methanol: water (60:40, v/v) (the water included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 1ng/mL to 400ng/mL (r(2)=0.9992 for artesunate, r(2)=0.9993 for its metabolite). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations for both artesunate and its metabolite. The recoveries ranged from 92% to 98% at the three concentrations for both. In summary, the LC-MS/MS metho described herein was fully successfully applied to pharmacokinetic studies of artesunate nanoemulsion after intramuscular delivery to sheep.

Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with ß-pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6×75mm, 3.5µm) using methanol: 5mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2-500ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC-MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration.

Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis.

The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme has been characterized in other species, but not in the genus Babesia, which causes major losses in the livestock industries worldwide. Therefore, we isolated, cloned and expressed the wild-type B. bovis dxs cDNA in Escherichia coli and evaluated its enzymatic activity in vitro. DNA sequence analysis revealed an open reading frame of 2061 bp capable of encoding a polypeptide of 686 amino acid residues with a calculated isoelectric point of pH 6.93 and a molecular mass of 75 kDa. The expressed soluble recombinant fusion DXS protein was approximately 78 kDa, which is similar to the native enzyme identified from the parasite merozoite using anti-rDXS serum. The recombinant fusion DXS enzyme exhibited Km values of 380 ± 46 µM and 790 ± 52 µM for D,L-glyceraldehyde 3-phosphate and pyruvate, respectively. In this work, we present the first cloning, expression and characterization of DXS enzyme from B. bovis.