1株产NDM-9和MCR-1的鸡源大肠杆菌的分子及生物学特征

为探究质粒介导耐药性的传播特征,本试验以1株分离自山东某肉鸡养殖场的产新德里金属β-内酰胺酶NDM-9和磷酸乙醇胺转移酶MCR-1的大肠杆菌(25R)为研究对象,通过全基因组序列分析、质粒接合转移试验、抗菌药物敏感性试验、大蜡螟毒性感染试验、质粒稳定性试验和生长动力学方法研究其质粒分子和生物学特征。结果显示,携带bla_NDM-9和mcr-1的质粒可各自亦可共同转移至其他菌株,并且携带上述耐药基因的质粒对宿主菌造成的适应性代价和毒性较低,更有利于耐药基因的传播。本试验为更深入了解质粒介导的细菌耐药性传播机制提供理论依据。     

“超级细菌”NDM-1的研究现状

金属-β-内酰胺酶是β-内酰胺酶中的B亚群,NDM-1是一种近来发现的新的金属-β-内酰胺酶,携带NDM-1基因的细菌几乎对所有抗生素耐药,所以被称为"超级细菌"。论文从"超级细菌"NDM-1的起源、分子特点、传播途径以及预防措施等方面进行了介绍。     

金不换水提取物与抗菌药联合对含NDM-1不动杆菌的体外作用研究

本试验旨在探讨金不换水提取物与抗菌药联合对含新德里金属β-内酰胺酶-1 (New Delhi metallo-β-lactamase-1,NDM-1)基因不动杆菌传代的体外抑菌活性,为金不换的综合利用提供参考依据。试验采用二倍微量稀释法体外分别检测金不换水提取物与抗菌药对受试菌的最小抑菌浓度(MIC),并以亚抑菌浓度(即1/2 MIC)的金不换水提取物与抗菌药联合作用对含NDM-1不动杆菌进行传代。结果显示,金不换水提取物的MIC为0.25 g/mL,以0.125 g/mL的金不换水提取物与头孢曲松钠、阿莫西林、头孢噻肟、痢菌净、磺胺间甲氧嘧啶、头孢他啶、头孢噻呋钠、美罗培南8种抗菌药联合作用传代后MIC明显降低。综上所述,金不换水提取物部分联合抗菌药诱导耐药细菌体外抗菌活性明显增强,对含NDM-1的不动杆菌具有一定的抑菌作用。     

NDM-1肺炎克雷伯菌裂解性噬菌体的分离鉴定及其对小鼠菌血症的治疗研究

噬茵体疗法在对抗多重耐药病原茵引起的感染方面具有很大的应用潜力。本研究从污水中分离到一株新的噬茵体,命名为NKP-1,并对噬茵体NKP-1的主要生物学特性进行研究。通过电镜观察确定NKP-1为短尾噬茵体,裂解试验表明该噬茵体为烈性噬茵体,具有很高的增值效率。体外试验表明它能够快速感染超强耐药的肺炎克雷伯菌株BAA-2146(NDM-1)并将其裂解。通过腹腔注射单一剂量的NKP-1(2×10~7 pfu/mL)就可以有效地保护BAA-2146(4×10~9 cfu/mL)感染引起的小鼠茵血症模型。在肺炎克雷伯菌BAA-2146与噬菌体NKP-1体外共同培养的过程中,我们分离到对该噬茵体具有抗性的突变菌株,通过动物试验确定该突变株表现为极低的毒力,且不影响NKP-1的治疗效果。本研究为利用噬菌体治疗多重耐药菌感染提供了理论基础与试验依据。     

中药对细菌R质粒消除作用的研究概况

质粒是细菌内染色体外具有独立复制能力的对细菌生存非必需的小型共价闭合环状双链DNA分子,作为遗传因子,在细菌细胞内普遍存在。细菌的某些表型特征,包括抗性、代谢能力、致病性、共生现象、接合转移等往往由质粒控制(吴乃虎等,1998)。细菌质粒包括F因子(fertility factor)、R     

质粒介导的细菌抗药基因水平转移及应对策略

据统计,全球畜禽饲用抗生素在2010年超过了6300万千克[1].过去的半个多世纪里在人类选择性使用抗生素的压力下,导致细菌耐药基因的快速转移和耐药菌株的急剧增加.为了减轻和控制细菌耐药性的产生和传播,一种有效的方法就是从基因水平上解析细菌产生耐药性以及耐药性传播的分子机制.     

超级细菌bla_(NDM-1)基因TaqMan-MGB荧光定量PCR检测方法的建立

为建立快速检测超级细菌编码新德里金属β-内酰胺酶1(NDM-1)的bla_(NDM-1)基因的方法,本研究针根据bla_(NDM-1)及其变异体的基因序列设计一对特异性引物和一条MGB探针,以构建的含有bla_(NDM-1)基因片段的重组质粒作为阳性标准品,经条件优化,建立了检测bla_(NDM-1)基因的TaqMan-MGB荧光定量PCR方法。结果显示该方法可以特异性扩增bla_(NDM-1)基因重组质粒标准品,而对鸡白痢沙门氏菌、大肠杆菌、多杀性巴氏杆菌和鼠伤寒沙门氏菌的标准菌株扩增均为阴性;检出下限为2.59拷贝/μL,比常规PCR敏感性高100倍;组内及组间重复性试验的变异系数均小于1.5%。利用所建立的方法对34株鸡源致病性沙门氏菌分离株进行检测,结果均未检测到目的片段,表明所有分离株均未携带bla_(NDM-1)基因。本研究建立的TaqMan-MGB荧光定量PCR方法特异性强、敏感性高、重复性好,可用于监测携带bla_(NDM-1)基因及其变异体的超级细菌。     

铜可阻止超级细菌传播

【西班牙《数码报》9月9日报道】英国南安普敦大学的一项最新研究表明,铜可阻止一种含有“NDM-1”的新细菌的传播。这种细菌由于对几乎所有抗生素都具有耐药性,并且已经传播到欧洲,因而被命名为“超级细菌”。     

亚抑菌浓度万古霉素对质粒接合转移的促进作用

为了分析亚抑菌浓度万古霉素对质粒接合转移的影响,本试验建立了以大肠杆菌(E.coli)J53作为受体菌,携带RP4-7质粒的E.coli DH5α和携带mcr-1阳性IncI2质粒的临床菌株E.coli LD67-1为供体菌的接合转移模型,测定亚抑菌浓度的万古霉素对接合转移的影响,并通过细胞膜通透性检测、扫描电子显微镜观察、活性氧(ROS)检测和实时荧光定量PCR(RT-qPCR)方法探究其内在机制。结果显示,与空白对照组相比,亚抑菌浓度万古霉素可显著提高RP4-7质粒和mcr-1阳性IncI2质粒的接合转移频率(P<0.05);N-苯基-1-萘胺(NPN)和碘化丙啶(PI)探针检测显示,亚抑菌浓度万古霉素可使受体菌细胞内、外膜通透性增加,供体菌细胞外膜通透性增加;扫描电子显微镜观察显示,经1/4最小抑菌浓度(MIC)万古霉素处理后接合细菌细胞膜发生损伤;ROS检测结果显示,与空白对照组相比,虽然经1/4 MIC万古霉素处理的受体菌的ROS表达水平显著上调(P<0.05),但1/8 MIC万古霉素处理后,供体菌和受体菌的ROS水平均无显著变化(P>0.05);RT-q...     

耐药蛋白NDM-1的双抗体夹心ELISA检测方法研究

旨在建立一种检测细菌中新德里金属β-内酰胺酶(NDM)耐药蛋白双抗体夹心ELISA方法.采用化学合成的方法获得优化后的NDM-1蛋白基因序列,构建表达载体pET-28a-NDM-1(G29-R270),并在大肠杆菌中进行IPTG诱导表达,用SDS-PAGE和Western blot验证重组蛋白.以纯化的NDM-1蛋白作...     

Study on the Effects of Stephania delavayi Diels Water Extracts Combined with Antibiotics on NDM-1-producing Acinetobacter in Vitro

The study was aimed to investigate the antibacterial activity of NDM-1-producing Acinetobacter in vitro induced by water extracts of Stephania delavayi Diels combined with antibacterial agents, and provide reference for the utilization of Stephania delavayi Diels.The minimum inhibitory concentration (MIC) of water extracts of Stephania delavayi Diels and antibacterial agents were tested through twice micro-dilution method.NDM-1-producing Acinetobacter were induced and subcultured by the 1/2 MIC of water extracts of Stephania delavayi Diels combined with antibacterial agents.The results showed that the MIC of water extracts of Stephania delavayi Diels was 0.250 g/mL.The MIC of NDM-1-producing Acinetobacter was decreased obviously after inducing by 0.125 g/mL of water extracts of Stephania delavayi Diels combined with 8 antibacterial agents, such as ceftriaxone sodium, amoxicillin, cefotaxime sodium, mequindox, sulfamonomethoxine, ceftazidime, ceftiofur sodium and meropenem.Antibacterial activities of antibacterial agents against NDM-1-producing Acinetobacter were enhanced obviously by combining with water extracts of Stephania delavayi Diels, and this combination could inhibit NDM-1-producing Acinetobacter to a certain degree.     

水平显微注射法制备转基因小鼠的研究

由于传统显微注射法具有操作繁琐、技术要求高和外源基因导入率低等局限性,本试验对常规显微注射法进行改进,旨在建立一套比较完善的水平显微注射方法。采用水平微注射系统,将外源DNA片段导入胚胎的雄原核中,获得子代鼠,分娩后3周提取鼠尾基因组DNA,PCR法筛选转基因阳性鼠。本试验采用Puc/BLG IN S和Chym os in基因,共使用710枚鼠原核胚进行研究,胚胎经注射后移植了25只受体,产下仔鼠77只;妊娠率分别为33.3%(5/15)和50%(5/10),胚胎成活率分别为9.47%(41/433)和l3.00%(36/277),阳性率分别为4.88%(2/41)和8.33%(3/36)。将阳性雌鼠配种妊娠后,对外源基因整合情况进行检测,结果表明初步获得了转基因阳性小鼠。     

规模化养牛场地上式青贮池设计问题探讨

青贮池建设是规模化养牛场必备并且投资较大的工程。在分析青贮工艺对青贮池建筑要求的基础上,提出了地上式青贮池建筑设计的要点,并提出牛场建设环境影响评价中应评价青贮饲料流出液对环境影响的建议。     

Horizontal moment around the hoof's centre of pressure during walking in a straight line

Reasons for performing study: Joint congruity and ligaments restrain the distal limb joints from excessive motion in the transverse and frontal planes, but the magnitudes and direction of the horizontal twisting moments around the hoof's centre of pressure (CoP) that induce these motions are unknown. Objectives: To quantify the horizontal moment around the vertical axis through the hoof's CoP at walk, and to determine whether these are symmetric. Methods: Nine sound Thoroughbred horses (mean age 5.3 years; mean mass 502 kg) were led at walk in a straight line across a Kistler force platform. Five trials were collected for each fore and hindlimb. The resultant moment around the hoof's CoP was calculated from the horizontal moment arms between the calculated CoP and the 4 horizontal forces in the transverse (X) and cranio‐caudal (Y) directions. Results: The calculated moments were consistent within limbs and horses, but variable between horses. Hindlimbs demonstrated a biphasic moment pattern and the largest moments were typically in the first half of stance. Mean ± s.d. peak moments were internal under both hindlimbs (L: Int 14.1 ± 4.6 Nm; R: Int 13.3 ± 5.5 Nm). In the forelimbs, 7/9 horses demonstrated an asymmetric moment pattern, with the left forelimb exerting an internal moment (L: Int 6.9 ± 2.9 Nm) and the right forelimb an external moment (R: Ext 8.4 ± 4.4 Nm), while the remaining 2 horses exerted internal moments in both forelimbs (L: Int 11.7 ± 1.4 Nm; R: Int 6.6 ± 1.9 Nm). Conclusion: In 7/9 horses, the forelimbs exerted asymmetric horizontal moments around the hoof CoP. The hindlimbs appear to behave with mechanical symmetry during stance, exerting an internal moment during retraction. Potential relevance: Extrasagittal joint motions in the forelimb are unlikely to be symmetric and future studies should account for possible bilateral variations.     

堆型艾美耳球虫重组DNA质粒pcDNA3-3-1E的免疫原性

用E.acervulina重组质粒pcDNA3-3-1E免疫雏鸡,观察其对雏鸡细胞免疫和体液免疫功能的影响。将7日龄雏鸡随机分为4组,即空白对照组、pcDNA3.1空质粒组、重组质粒免疫组和卵囊免疫组,每组30只。pcDNA3-3-1E经肌肉注射途径免疫雏鸡,剂量为50μL/只（含质粒1g/L）。7日龄免疫1次,14日龄加强免疫1次。检测雏鸡血液T淋巴细胞增殖率,测定血清抗体水平。结果显示,与空质粒组和健康对照组相比,重组质粒组淋巴细胞的增殖能力明显增强（P〈0.05）,外周血中IgG的含量均显著升高（P〈0.05）,表明pcDNA3-3-1E裸质粒DNA能有效的提高雏鸡的细胞免疫和体液免疫水平。     

Horizontal moment around the hoof centre of pressure during walking on right and left circles

Reasons for performing study: Recent research indicates that the digital joints experience some degree of extrasagittal motion during stance and that the moments under the hoof are asymmetric in horses walking in a straight line. On a circle, these have not been defined. Objectives: To quantify the amplitude and symmetry of horizontal twisting moments around the vertical axis through the hoof's centre of pressure on left and right circles at walk. Methods: Six Thoroughbred horses were led at walk across a Kistler force platform on a left and a right circle of 5 m radius. The resultant moment around the hoof was calculated from the 4 horizontal forces and their moment arms. Results: Five of the 6 horses exerted an internal moment around their left forehoof, and 4 exerted an internal moment around their right forehoof on the left circle. On the right circle, 5 of the 6 exerted an internal moment around the left forehoof and a weak external moment around the right forehoof. The moments under the hind hooves were bilaterally similar for right and left circles. Conclusion: Intrahorse variability in the applied moments is low, but there is some interhorse variability, especially in the forelimb moments, that indicates future studies of movements of the distal limb joints should be bilateral to account for mechanical asymmetry. Potential relevance: The finding that horizontal moments vary between forelimbs in some horses will apply to how exercise on a circle is approached, especially in rehabilitation programmes for horses with orthopaedic injury of the distal limb.     

Horizontal transmission and phylogenetic analysis of bovine leukemia virus in two districts of Miyazaki,Japan

Horizontal transmission is recognized as a major infection route for bovine leukemia virus (BLV), and cattle with high viral loads are considered to be a major infectious source in a herd. However, a correlation between viral loads and the risk of infection has been insufficient to use as a foundation for BLV control strategies. In this report, we examined the epidemiology of BLV infection and the infectious source in a local area. In 2013–2014, BLV infection was investigated in 1,823 cattle from 117 farms in two adjacent districts, Miyazaki, Japan. Seropositive samples for BLV were detected with 88 cattle and in 14 farms. Phylogenetic analysis revealed that 94% of the isolates clustered into genotype I and the remaining isolate into genotype III. Among genotype I, genetically distinct strains were spread at each farm, and cattle infected with less than 3 copies/100 cells did not transmit BLV to other cattle for more than thirty months. This is the first report of concrete data of viral load in relation to viral horizontal transmission under the field condition. The data facilitate farmers and veterinarians understanding the status of BLV infected cattle. This research contributes to BLV infection control and the development of effective BLV eradication programs.