Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate ‘Streptomyces maritimus’: evidence for the derailment of an aromatic polyketide synthase

Background: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-β-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain ‘Streptomyces maritimus’ deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides.Results: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from ‘S. maritimus’ has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis.Conclusions: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Relative stereochemical assignment of C-33 and C-35 in the antibiotic gladiolin

Gladiolin is a macrolide antibiotic isolated from Burkholderia gladioli BCC0238 with promising activity against Mycobacterium tuberculosis, including several multidrug resistant strains. The configuration of all but one of the stereogenic centers of gladiolin has previously been elucidated using a combination of NOESY NMR experiments and predictive sequence analysis of the polyketide synthase responsible for its assembly. However, it was not possible to assign the configuration of the C-35 methyl group using such methods. Here we report the synthesis of C-33/C-35-syn and C-33/C-35-anti mimics of the C-30 to C-38 fragment of gladiolin from (R) and (S)-citronellol, respectively. Comparison of HSQC NMR data for the mimics and the natural product showed that the C-35 methyl is anti to the C-33 hydroxyl group, indicating that gladiolin has the 35S configuration.     

The use of NIR as a multi-parametric in situ monitoring technique in filamentous fermentation systems

The use of Fourier transform near infrared (FT-NIR) spectroscopy for simultaneous determination of multiple properties in an active pharmaceutical ingredient (API) fermentation process is described, together with procedures for developing accurate NIR calibrations with a performance independent of scale and the specific bioreactor used. Measurements were made in situ, by insertion of transflection probes into pilot and industrial bioreactors providing direct contact with the fermentation culture media. The ultimate goal was to establish methods for real time process monitoring aimed at enhanced process supervision, fault detection diagnosis and control of bioreactors. The in situ acquired spectra were related to lab results of samples taken from the reactors during the course of the manufacturing process. Suitable spectral wavenumber regions were selected and calibration models based on partial least squares (PLS) were developed. The root mean square errors of prediction for API content, viscosity, nitrogen source and carbon source concentration were all within acceptable ranges as compared to the off-line lab measurements, respectively, 0.03% (w/w), 150 cp, 0.01% (w/w), and 0.4% (w/w).     

Food safety evaluation: Detection and confirmation of chloramphenicol in milk by high performance liquid chromatography-tandem mass spectrometry

A simple and rapid procedure for extraction of chloramphenicol (CAP) in milk and analysis by high-performance liquid chromatography coupled with quadrupole mass spectrometry in tandem was developed. The method consisted of one step of liquid-liquid extraction using ethyl acetate and acidified water (10 mmol L⁻¹ formic acid) and HPLC-MS/MS detection. CAP-D5 was used as internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear, with typical r² values higher than 0.98. Absolute recovery of CAP from milk proved to be more than 95%, however CAP-D5 absolute recovery was 75%. The method was accurate and reproducible, being successfully applied to the monitoring of CAP in milk samples obtained from the Brazilian market. Decision limit (CCα) was 0.05 ng mL⁻¹ and detection capability (CCβ) was 0.09 ng mL⁻¹.     

基于微流控芯片的尿路感染细菌鉴定及抗生素敏感性测试

发展了一种微流控芯片纸基细菌分析技术,用于多重细菌鉴定与抗生素敏感性测试.制备了阵列培养池芯片,以滤纸作为衬底固定显色培养基和抗生素.利用PVDF疏水薄膜止流阀,将尿液样品引入芯片并分隔于不同培养池.借助于培养池阵列的空间分辨力,实现多重细菌分析.根据特异性显色结果实现细菌鉴定,通过实时显色强度分析实现细菌定量,依据抑制显色反应的最低抗生素浓度确定抗生素敏感性.以3种泌尿系统感染常见病原菌(大肠杆菌、金黄色葡萄球菌和粪肠球菌)为模拟测试对象进行分析,结果表明,芯片方法可以在18 h内实现对3种细菌的同时鉴定及6种抗生素敏感性测试.对照实验显示,芯片法与传统方法细菌鉴定和抗生素敏感性测试结果一致性分别为94.1％和93.9％.本研究建立的微流控芯片细菌分析方法简便快速,非常适合于医疗资源匮乏条件下的细菌分析.     

超高效液相色谱同时测定奶粉和牛奶中β-内酰胺、喹诺酮和大环内酯类抗生素的残留

建立了牛奶和奶粉中β-内酰胺类头孢族,喹诺酮类和大环内酯类20种抗生素残留的超高效液相色谱-二极管阵列检测器测定的方法.采用0.5 mol/LHClO4沉淀牛奶或奶粉中的蛋白质成分后,样品用乙腈+水(2+8,V/V)提取,HLB固相萃取小柱净化,以含有2g/L甲酸的乙腈和0.006 mol/,L乙酸铵为流动相,BEH ...     

Synthetic studies of incednine: synthesis of C1-C13 pentaenoic acid segment

Stereoselective synthesis of the C1-C13 pentaenoic acid segment (4) of the novel antibiotic incednine (1) is described.     

Evaluation of a Microbiological Multi-Residue System on the detection of antibacterial substances in ewe milk

To protect both, public health and the dairy industry, from the presence of antibiotic residues in milk, control programmes have been established, which include the needed screening tests. This work focuses on the application of a Microbiological Multi-Residue System in ewe milk, a method based on the use of six different plates, each seeded with one of the following bacteria: Geobacillus stearothermophilus var. calidolactis (beta-lactams), Bacillus subtilis at pH 8.0 (aminoglycosides), Kocuria rhizophila (macrolides), Escherichia coli (quinolones), B. cereus (tetracyclines) and B. subtilis at pH 7.0 (sulphonamides), respectively. Twenty-three antimicrobial substances were analysed and a logistic regression was established for each substance assayed to relate the antibiotic concentration and the zone of microbial growth inhibition. Great linearity in the response was observed (regression coefficients of over 0.97). This fact suggests the possibility of establishing a decision level of antibiotic concentrations near to the Maximum Residue Limits (MRL). Zones of inhibition were suggested as proposed action levels for the different antimicrobial groups (diameters of inhibition of 18 mm for the aminoglycoside, beta-lactam and sulphonamide plates; 19 mm for the tetracycline plate, 21 mm for the macrolide plate, and 24 mm for the quinolone plate). Specificity and cross-reactivity were also assayed.     

Analysis of antimicrobial agents in animal feed

We review current trends in the analysis of antimicrobial agents in animal feeds. After a brief introduction to feed-industry figures and the unavoidable problem of cross-contamination, we provide an overview of the European Union legislative framework for feedingstuffs. We devote the core of the article to analytical methodology developed over the past 10 years for monitoring residues of antibiotics and coccidiostats in feedingstuffs in order to ensure that feeds comply with current legislation and are of high quality and safety for both livestock and consumers. We consider the potential and the limitations of analytical methods and devote special attention to their validation and performance characteristics.     

表面增强拉曼光谱技术在农产品药物残留检测中的应用

近年来,农产品安全领域中的药物残留问题引起了人们的广泛关注,为了保障国家食品安全和国民健康、促进经济贸易发展,对农产品中的农药、抗生素等有害残留物进行检测是非常必要的。表面增强拉曼散射技术（SERS）作为一种新兴的检测手段,具有操作简单、耗时短、灵敏度高等优点。对SERS技术概况、增强理论和增强基底进行了简要介绍,以药物残留检测领域常用的金属溶胶类基底、固体活性基底和柔性活性基底为切入点,重点介绍了表面增强拉曼光谱技术在农产品（肉类、水产品、果蔬和其他部分农产品等）药物残留检测领域中的研究现状。传统溶胶基底具有成本低、易合成和SERS性能优异等特点,为了提高增强效果,众多研究者从尺寸和形态入手进行基底优化,开发出花状纳米结构、星状纳米结构、棒状纳米结构和链状纳米结构等形态的溶胶颗粒;基于复合材料互补协同效应发展了一系列的核壳纳米结构,有助于提升胶体基底的拉曼性能和稳定性。固体基底结构稳定,具有较好的一致性、重复性。柔性基底主要有基于柔性器件的、柔性聚合物的、以及柔性碳材料的三类SERS基底,其具有机械性能优良、不易损坏、成本低等优点,有利于实现微创或无损检测。通过对比分析,发现基底类型、待检农产品的基质、农药类型、检测环境均会对检测结果产生影响,当前研究的灵敏度较高,表明了SERS技术结合纳米基底在检测复杂基质中农药残留的应用潜力。同时指出应用SERS技术进行农产品药物残留检测的挑战：（1）农产品体内的药物残留量低、分布不均匀,导致拉曼信号相对较弱且易受荧光干扰和背景噪声干扰;（2）农产品基质复杂,对SERS光谱数据的影响不容小觑;（3）当前SERS检测方法尚未标准化,不同检测方案的结果差异较大。随着表面增强拉曼散射的支撑理论和检测技术的不断进步,SERS将在食品安全领域具有更广阔的应用前景。