UHPLC-Q/TOF MS结合HPLC-DAD定性和定量分析硫酸黏菌素可溶性粉中的未知添加物

定性和定量分析一批兽药硫酸黏菌素可溶性粉中的未知添加物。照《中国兽药典》2010年版一部对该批检品用微生物检定法进行含量测定时,发现该样品的抑菌圈为虚圈,用薄层色谱鉴别该样品,未显示与标准品溶液一致的主斑点,怀疑该样品中有处方外非法添加物。采用超高效液相色谱-四级杆-飞行时间质谱(UHPLC-Q/TOF MS)对该样品进行筛查,发现疑似添加物,并使用液相色谱-二极管阵列检测(HPLC-DAD)法进行了双重确证和含量测定。该样品中非法添加物确证为磺胺氯达嗪和甲氧苄啶,添加量分别为58.5 mg/g和13.4 mg/g。本研究通过建立筛查方法为监管部门提供技术支撑,通过分析非法添加物的可能原因为打击兽药处方外非法添加提供了思路。     

阿莫西林和硫酸粘菌素对猪鸡大肠杆菌和沙门氏菌的体外联合抗菌作用

探讨了阿莫西林与硫酸粘菌素对猪鸡的大肠杆菌和沙门氏菌的联合抗菌效果。采用微量稀释法分别测定阿莫西林与硫酸粘菌素单药对大肠杆菌和沙门氏菌的最低抑菌浓度（MIC）,采用棋盘稀释法测定阿莫西林与硫酸粘菌素联用对大肠杆菌和沙门氏菌的MIC并计算联合指数（FIC）。试验结果显示,对14株试验菌株体外联合抗菌呈协同与相加作用的占78.6%,呈无关作用的占21.4%,无拮抗作用,表明阿莫西林和硫酸粘菌素可联合应用于治疗猪鸡大肠杆菌和沙门氏菌感染。     

鸡蛋中粘杆菌素残留含量消除药代动力学研究简

鸡蛋中粘杆菌素残留含量消除规律研究结果表明。鸡蛋中药物浓度-时间的药动学主要参数为：鸡蛋中药物的达峰浓度（Cmax）为（242．5641±158．7074）μg／kg,达峰时间（Tmax为（10．20668±0．93777）d,零阶矩曲线下面积（AUC）（1668．298±686．8698）μg／（ks·d）,吸收半衰期（K01-HL）（2．459439±2．255054）d,消除半衰期（K10-HL）（2．387610±1．889865）d,清除率（CL）（0．130340±0．029891）L／（kg·d）。     

硫酸粘杆菌素的急性毒性及对小鼠骨髓细胞的微核效应

为评价国产硫酸粘杆菌素的安全性 ,进行了改良寇氏法测定LD50 小鼠骨髓细胞的微核试验。结果显示 ,国产硫酸粘杆菌素的LD50 为 71 1 45mg/kg ,属于低毒类化合物 ;小鼠骨髓微核试验结果为阴性 ,表明其不具致突变作用     

Evaluation of the ability of colistin,amoxicillin (components of Potencil®), and fluoroquinolones to attenuate bacterial endotoxin‐ and Shiga exotoxin‐mediated cytotoxicity—In vitro studies

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS‐ and Stx‐mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS‐mediated cytotoxicity in an experiment mimicking “natural infection,” (c) the effect of antibiotics to decrease Stx2e‐mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS‐ and Stx2e‐induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS‐ and Stx2e‐mediated cytotoxicity.     

CpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ调控作用的研究

为了探索 cpxR 对鼠伤寒沙门菌的黏菌素耐药相关基因 pmrB 和 phoQ 的调控作用,用overlapping PCR扩增得到 cpxR 、 pmrB 和 cpxR 、 phoQ 共表达基因 cpxR - pmrB 、 cpxR - phoQ ,用2倍微量肉汤稀释法对构建的 pmrB 、 phoQ 单基因过表达菌株JSΔ acrB Δ cpxR :: kan /p pmrB (JSΔΔ/p B )、JSΔ acrB Δ cpxR :: kan /p phoQ (JSΔΔ/p Q )和 cpxR - pmrB 、 cpxR - phoQ 共表达菌株JSΔ acrB Δ cpxR :: kan /p cpxR - pmrB (JSΔΔ/p RB )、JSΔ acrB Δ cpxR :: kan /p cpxR - phoQ (JSΔΔ/p RQ )进行黏菌素最小抑菌浓度(MICs)的测定,同时以各菌株在LB中的OD 600 nm 值绘制生长曲线,以各菌株在不同浓度的黏菌素中的存活率绘制杀菌曲线。结果:成功构建的鼠伤寒沙门菌 pmrB 、 phoQ 的单基因过表达菌株JSΔΔ/p B 、JSΔΔ/p Q 和 cpxR - pmrB 、 cpxR - phoQ 共表达菌株JSΔΔ/p RB 、JSΔΔ/p RQ 的MIC结果显示,JSΔ acrB Δ cpxR :: kan /p cpxR (JSΔΔ/p R )的MIC值较JS下降93.75%,与本实验室前期研究结果一致。JSΔΔ/p B 、JSΔΔ/p Q 的MIC值较JS均上升3倍,而JSΔΔ/p RB 、JSΔΔ/p RQ 的MIC值较JS分别降低50%和75%。生长曲线结果显示JSΔΔ/p R 的生长活性最低,JSΔΔ/p B 、JSΔΔ/p Q 、JSΔΔ/p RB 和JSΔΔ/p RQ 的生长活性均低于JSΔ acrB Δ cpxR :: kan /pHisA(JSΔΔ/pHisA)。黏菌素的杀菌曲线结果显示JSΔΔ/p B 和JSΔΔ/p Q 在不同浓度的黏菌素中的存活率显著高于黏菌素较敏感的JSΔΔ/p RB 和JSΔΔ/p RQ 。上述结果表明:CpxR能够通过PmrB和PhoQ调控鼠伤寒沙门菌对黏菌素的敏感性。     

Improving clinical outcomes via responsible antimicrobial use in horses

The growing problem of antimicrobial resistance affects veterinarians on a daily basis. Antimicrobial stewardship and responsible prescribing are essential for a future with effective antimicrobials, as it is unlikely that new antimicrobials will become available for use in horses in the near future. Extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella spp. are pathogens of significant concern but there are also other opportunistic pathogens such as Pseudomonas spp., α-haemolytic Streptococcus spp., Enterococcus spp. and Acinetobacter spp. which, due to their high intrinsic resistance, have limited treatment options in adult horses. It is essential that highest priority critically important antimicrobials such as ceftiofur, enrofloxacin, rifampicin and polymyxin B are used prudently in horses and ideally based on culture and antimicrobial susceptibility testing (AST). For example, the use of polymyxin B at a low anti-endotoxic dose rather than at a higher antimicrobial dose in horses for the treatment of systemic inflammatory response syndrome is a potential driver for resistance to colistin (polymyxin E), an antimicrobial used as a last resort in the treatment of multidrug resistant (MDR) Enterobacteriaceae infections in humans. Serum procalcitonin levels are used in humans to distinguish noninfectious inflammatory conditions from inflammation caused by bacteria and other infectious agents and are also used to guide cessation of antimicrobial treatment. Although no such studies have been performed in horses, this or other markers may prove to be helpful in guiding antimicrobial treatment decisions in the future. Optimising sampling techniques and good communication with the microbiology laboratory are essential for generating the accurate culture and AST results that underpin appropriate antimicrobial use. Additionally, there is clearly a need for national and international harmonisation of laboratory methods in order to improve the reliability and consistency of results reported by different laboratories.     

硫酸黄连素与硫酸粘菌素的体外联合抑菌研究

为了考察硫酸黄连素与硫酸粘菌素的联合抑菌作用,对硫酸黄连素和硫酸粘菌素单一和联合应用分别对仔猪大肠杆菌(C83901),猪多杀性巴氏杆菌(C44-1),鸡白痢沙门氏菌(C79-6),禽多杀性巴氏杆菌(C48-1)进行药物敏感性试验;结果:硫酸黄连素对以上4株菌的M IC值分别为:32,128,64,128μg/mL;硫酸粘菌素对以上4株菌的M IC值分别为:64,128,128,256μg/mL;联合应用对上述4株菌的FIC指数分别为:0.375,0.375,0.25,0.5。结果显示上述4株菌对硫酸黄连素与硫酸粘菌素联用的FIC指数均小于0.5,表现为协同作用。二者最佳比例为1∶2~1∶4,这为复方制剂的开发提供了一定的理论依据。     

粘杆菌素研究及其应用

本文阐述了粘杆菌素的理化性质、药理作用、体内代谢、毒性及对免疫器官的影响等方面及其应用前景。     

硫酸粘杆菌素对獭兔肠道菌群及免疫功能的影响

为探讨硫酸粘杆菌素对獭兔盲肠菌群和免疫器官组织TLR2、TLR4mRNA相对表达量及外周血细胞因子含量的影响,试验分为2组,Ⅰ组(对照组)饲喂基础全价饲料;Ⅱ组饲喂含20mg/kg硫酸粘杆菌素的基础全价饲料。试验期为28d。试验应用PCR-DGGE及Real-Time PCR技术比较分析獭兔盲肠菌群结构的差异,采用ELISA检测外周血中细胞因子的含量,Real-Time PCR检测TLR2、TLR4mRNA相对表达量。结果表明:Ⅱ组盲肠菌群多样性与Ⅰ组相似(P0.05);Ⅱ组盲肠中梭菌类群XIVa、肠球菌属、链球菌属数量明显高于Ⅰ组(P0.05),梭菌类群Ⅳ明显低于Ⅰ组(P0.05);Ⅱ组盲肠TLR2/4和回肠TLR4的相对表达量高于Ⅰ组,盲肠TLR4差异显著(P0.05),其余差异不显著(P0.05)。与Ⅰ组相比,Ⅱ组獭兔脾脏指数和脾脏TLR2/4表达量增加,肝脏指数和肝脏TLR4表达量减少,均无显著差异(P0.05),但肝脏TLR2显著增加(P0.05);Ⅱ组外周血细胞因子IL-4的浓度显著高于Ⅰ组(P0.05)。饲料中添加20mg/kg的硫酸粘杆菌素可在一定程度上促进断奶幼兔脾脏发育并提高机体免疫力。