胞内分枝杆菌临床株分枝菌酸指纹谱变异性研究

近年来,非结核分枝杆菌(NTM)病较常见,分枝杆菌菌种鉴定不仅在流行病学上,而且在临床诊断和治疗上均有重要意义[1-3]。在前期工作的基础上[4-5],用本实验室所建立的HPLC分析分枝菌酸(Mycolic acids,MA)的方法及所构建的50种分枝杆菌标准菌株的MA HPLC指纹谱库,鉴定了部分分枝杆...     

Spoligotyping结合多位点PCR用于牛分枝杆菌卡介苗的快速鉴定

目的鉴别分枝杆菌临床分离菌株中的牛分枝杆菌卡介苗,为临床确诊提供依据。方法采用PNB／TCH鉴别培养基和分子生物学技术（间隔寡核苷酸分型Spoligotyping和多位点PCR）对从一例疑似牛分枝杆菌卡介苗感染的结核病患者分离的菌株（FJ07111）进行鉴定。结果临床分离分枝杆菌菌株FJ07111具有与牛分枝杆菌93006一致的生长特性,Spoli—gotyping和多位点PCR结果与注射用BCG完全一致。结论Spoligotyping和多位点PCR方法可简便、快速、准确鉴定牛分枝杆菌卡介苗。     

结核分枝杆菌复合群的进展和新成员

结核分枝杆菌复合群（M.tuberculosis-complex，MTC）是分枝杆菌5个复合群之一，也是3大致病性复合分枝杆菌群（鸟胞内和偶然分枝杆菌）之一，简称结核分枝杆菌群或结核杆菌群。上世纪70年代有4个成员（Wayne，1982）即人型结核分枝杆菌、牛型结核分枝杆菌、非洲分枝杆菌和田鼠分枝杆菌，80年代把卡介苗（BCG，减毒牛分枝杆菌）也独立列入，于是MTC有5个老成员。     

DNA指纹技术检测耐多药结核分枝杆菌的应用研究

目的应用结核分枝杆菌DNA指纹技术,了解耐多药结核分枝杆菌的基因型状况并探讨耐多药结核病的分子流行病学。方法以结核分枝杆菌插入序列IS6110序列为模板,设计一对特异外向引物,建立一种结核分枝杆菌DNA指纹技术方法,对临床标本中分离的220株耐多药结核分枝杆菌进行DNA指纹基因分析。同时对实验数据进行聚类分析,进行耐多药结核病分子流行病学研究。结果根据结核分枝杆菌DNA指纹图谱分析,220株耐多药结核分枝杆菌均产生特征DNA指纹图谱,DNA指纹图谱变异很大。每一菌株DNA指纹的拷贝数介于3至18之间。其中大部分菌株,即205株（93.2%）包含613个拷贝,平均为11个带。这205株DNA指纹图谱中,162株为特异的,提示它们在流行病学上是独立的。有58株（26.4%）指纹图谱组成了15个簇,每簇28株,它们的IDNA指纹图谱相同,可能代表了近期的传播。尤其是在此DNA指纹图谱中,其中分子量为200 bp扩增带频率最大,在220株耐多药结核分枝杆菌均出现。结论结核杆菌DNA指纹技术检测可应用耐多药结核分枝杆菌DNA指纹图谱分型,并可用于耐多药结核病分子流行病学调查。     

聚合酶链反应-反向线点杂交技术快速鉴定34种分枝杆菌

依靠传统方法鉴定分枝杆菌的生长特性、色素、生长速度、菌落形态及用生化试验鉴定分枝杆菌的方法费时、费力且没有标准化试剂，缺乏敏感性和特异性，导致很多结果不易判断。高效液相色谱测定分枝菌酸及气相色谱测定脂肪酸均存在菌种鉴定的局限性，受菌株的生长环境、营养状况的影响，并且需要特殊的仪器。     

结核分枝杆菌和牛分枝杆菌TaqMan荧光PCR检测的建立

目的对结核分枝杆菌和牛分枝杆菌分别建立荧光PCR快速检测方法。结核分枝杆菌荧光PCR对H37Rv标准菌株、结核分枝杆菌临床分离株的检测结果呈典型阳性反应，牛分枝杆菌荧光PCR对牛分枝杆菌标准菌株、BCG菌株的检测结果呈典型阳性反应，对其它分枝杆菌菌株以及常见微生物样品为阴性反应。两种荧光PCR对对应阳性重组质粒模板的检测灵敏度可达单个基因拷贝，对结核分枝杆菌或BCG标准菌株的检测灵敏度可达单个菌细胞。对临床样品的检测结果显示，荧光PCR对模拟感染奶样、血样的检测灵敏度可达单个菌细胞。所建立的方法，全过程包括血样、奶样、痰液等临床样品的前处理、核酸提取和荧光PCR检测，可在5～6h内完成。采用上述荧光PCR方法从临床采集的疑似病人痰液中检出多份结核分枝杆菌阳性样品。     

北京结核菌株分子流行病学的研究

目的：北京结核分枝杆菌分子流行病学的方法。方法（1）使用标准化的RFLP法检测并分析从128位初治患者分离的结核分支杆菌IS6110指纹多态性;（2）对选例患者进行社会人口学和流行病学调查问卷;（3）分析结核分枝杆菌IS6110指纹多态特征与流行病学的关系。结果（1）根据IS6110指纹特征的同源性,检测的128株结核分枝杆菌可基本分为3组,A、B组的菌株间因具有较高的同源性而被称为：北京基因型”菌株,并且此类菌株在检测菌株中所占的比例较高（103／128,80．5％）;C组菌株间同源性较低,多态性变化多而被称为“非北京基因型”,仅占19．5％。（2）从较低年龄（＜40岁）,患者分离菌株的多态性变化,与从较高年龄（＞40岁）患者分离菌株多态性变化比较存在差异,提示“北京基因型”菌株不是由于BCG接种而造成的选择性优势。（3）检测菌株的多态性变化与患者结核病接触史、患者长期居住地、菌株药物敏感性情况无明显联系。结论：结核分枝杆菌的指纹分析对结核病现代流行病学的研究是一个有用的工具。     

滇桂艾纳香的高效液相色谱指纹图谱鉴别方法的研究

目的：建立滇桂艾纳香的高效液相色谱（ HPLC）指纹图谱鉴别方法。方法采用高效液相色谱法。色谱条件包括Phenomenex 溍Synergi 4 u Polar-RP 80A色谱柱（250 mm×4.6 mm,4μm）,甲醇-水（用冰乙酸调pH至2.8）流动相系统,梯度洗脱：0～17 min,10％～40％甲醇;18～37 min,40％～60％甲醇,检测波长256 nm,柱温25℃,体积流量1 ml／min。测定12批滇桂艾纳香的指纹图谱,并作相似度比较分析。结果12批药材依法检测得到256 cm的HPLC指纹图谱,确定了11个特征峰构成的滇桂艾纳香指纹图谱,其中2号峰为原儿茶酸,4号峰为绿原酸。12批的相似度＞0.99,说明12批药材性能良好。结论该方法提供滇桂艾纳香的鉴别方法,有助于推广滇桂艾纳香药材的应用以及为中成药的中间体质量控制打下良好的基础。     

耐异烟肼结核分枝杆菌临床分离株耐药基因突变及其分子流行病学调查研究

目的阐明结核分枝杆菌耐异烟肼临床分离株katG基因突变特点以及Spoligotyping分型和结核病流行关系。方法对123株结核分枝杆菌临床分离株，其中有药敏结果的98株，（耐异烟肼菌株45株；异烟肼敏感株53株）的katG基因进行DNA片断扩增然后进行SSCP分析；同时对其中的121株菌株进行Spoligotyping分型。结果45株耐INH结核杆菌株中，27株（60％）第315位点突变，低耐药菌株（1mg／L）第315位点突变率显著高于高耐药菌株（10mg／L；53株敏感株中无KatG基因315位密码子突变。对121株临床株的Spoligotyping DNA指纹分析，1型，（北京型）103株占84％（103／121），其它基因型18株，分散在13种基因型中。结论本项研究进一步证实了结核分枝杆菌耐异烟肼与katG基因突变之间的关系；北京型感染是北京地区结核病流行的重要原因。     

全身播散性BCG感染1例

全身播散性卡介苗感染,其诊断依赖于标本培养有结核杆菌生长,菌型鉴定为卡介苗株^[1]。本例经抽取淋巴结炎内脓液,经间歇区寡核苷酸（SPO-LIGOTYPING）和PCR对临床分离株与结核分枝杆菌标准株（M.TB H37Rv）、牛结核分枝杆菌（M.bovis）标准株和BCG株作比对,证实临床分离株为BCG株。采用联合二线抗结核药治疗获临床治愈出院。     

Identification of Mycobacteria and measurement of whole cell fatty acids using a gas chromatography computer system

The technique of combination gas chromatography with computer used in measuring fatty acids of 28 species standard Mycobacteria was introduced in this study. The data of components of fatty acids in this genus and GC graphs with satisfaction were gained. Several peaks with good reproducibility were distinguished automatically, based on which, M. tuberculosis, M. bovis and other comment atypical Mycobacteria may be separated respectively. It was showed that this method have the characteristics of accurate, sensitive and rapid by means of the identification of clinical strains, also have certain practical value in clinical laboratory.     

Identification of Mycobacterium tuberculosis by the method of gas-liquid chromatography

Gas-liquid chromatography was used to study the composition of fatty acids and hydrocarbons of different types of Mycobacteria, including M. tuberculosis, opportunistic Mycobacteria and acid-fast saprophytes. In terms of composition of fatty acids and hydrocarbons, clinical and laboratory strains of M. tuberculosis are very similar. The cellular higher fatty acids of M. tuberculosis differ much from those of opportunistic Mycobacteria and acid-fast saprophytes. The findings can be used for the identification and differentiation of different types of Mycobacteria.     

A gas-liquid and thin-layer chromatographic study of Mycobacterium fortuitum

Forty-two strains of Mycobacterium fortuitum were examined for fatty acid composition by gas-liquid chromatography and for mycolic acid pattern by two dimensional thin-layer chromatography of whole cell acid methanolysates. The strains studied contained saturated and mono-unsaturated fatty acids from 12 to 24 carbon atoms and tuberculostearic acid, and they showed a thin-layer chromatographic pattern of mycolic acids similar to the pattern previously reported for this species and characterised by the presence of alpha and alpha'-mycolates and several more polar components. The heterogeneity within the species M. fortuitum, of its antigenic, biochemical and chemical properties, previously noted by several authors was slightly reflected (but not correlated) in the fatty acid composition found in the strains studied; the mycolic acid pattern of all of them was, however, very stable.     

A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a beta-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the alpha-branch of mycolic acids, followed by the joining of these two branches.     

Mycolic acids and ancient DNA confirm an osteological diagnosis of tuberculosis

SETTING: The underlying trends in the past epidemiology of tuberculosis (TB) are obscure, requiring recourse to the archaeological record. It would therefore be of value to develop methods for reliable TB diagnosis in ancient populations. OBJECTIVE: To test the capability of two biomarkers, Mycobacterium tuberculosis complex mycolic acids and a DNA target (IS6110), for confirming an osteological diagnosis of TB in medieval individuals, based on the presence of Pott's disease and/or rib lesions. DESIGN: Osteological examination of three archaeological individuals (Medieval: approximately 1000 years old) revealed a Pott's disease case, one with no changes consistent with TB and one with rib lesions. Rib samples from these individuals were examined for the presence of Mycobacterium tuberculosis complex mycolic acids and mycobacterial DNA. RESULTS: Mycobacterium tuberculosis complex mycolic acids and the DNA target were detected in the Pott's disease case, whilst mycolic acids (insufficient for confirmation) alone were detected in the rib lesion case. CONCLUSIONS: Biomarkers provide a sensitive tool to detect ancient TB. Mycobacterium tuberculosis DNA is not distributed homogeneously, making multiple sampling essential. Mycolic acids seem more reliable for ancient TB diagnosis than IS6110. The demonstrated stability of mycolic acids show that they may be of value in tracing the palaeoepidemiology of tuberculosis back into antiquity.     

Effect of low temperature on growth, viability, and synthesis of mycolic acids of Mycobacterium tuberculosis strain H37Ra.

Cultures of Mycobacterium tuberculosis strain H37Ra were grownto early logarithmic phase at 37 degrees C and were incubated at 16 degrees, 20 degrees, and 25 degrees C. The decrease in this ability was more rapid at 20 degrees C than at 16 degrees C. Low-temperature incubation caused decreases in the ratios of mycolic acids and monounsaturated C16-19 fatty acids relative to the total of fatty acids synthesized. It also caused an increase in the ratio of saturated C24-26 fatty acids relative to the total of fatty acids synthesized. These ratios were based on the incorporation of radiolabel from 14C-acetate into fatty acids. These results showed that when M. tuberculosis H37Ra was incubated at low temperatures, it did not adapt by increasing the ratio of unsaturated to saturated fatty acids synthesized. The ability of the cells to synthesize mycolic acids was sharply decreased. These changes may lead to the loss of viability of M. tuberculosos H37Ra. Mycolic acid synthesis is similarly affected by exposure of cells to isoniazid, an antimycobacterial drug.     

应用气相色谱技术分析全细胞脂肪酸快速鉴定分枝杆菌

目的探讨应用气相色谱技术分析全细胞脂肪酸进行分枝杆菌菌种鉴定方法的准确性和实用性。方法应用美国MIDI公司开发的基于细胞脂肪酸成分鉴定细菌菌种的软件系统MIS4．0，对14株分枝杆菌参考菌株和727株分枝杆菌临床分离株进行菌种鉴定，同时采用传统方法对相同菌株进行菌种鉴定，比较两种方法的检测结果。结果(1)参考菌株鉴定：应用传统方法对所有菌株均获得正确鉴定结果，应用脂肪酸分析方法则除母牛分枝杆菌外，其他13株均获得正确鉴定结果。(2)临床分离株鉴定：传统方法鉴定为结核分枝杆菌、牛分枝杆菌的625株中，应用脂肪酸分析方法有45株鉴定为非结核分枝杆菌，对结核分枝杆菌鉴定的正确率为93％；对传统方法鉴定为非结核分枝杆菌的102株菌株，脂肪酸分析法亦全部鉴定为非结核分枝杆菌，但其中7株的菌种鉴定结果不同，其鉴定正确率为93％。(3)脂肪酸分析法对结核分枝杆菌鉴定错误者主要是将其鉴定为胃分枝杆菌、次要分枝杆菌和耻垢分枝杆菌，对非结核分枝杆菌则主要是将瘰疬分枝杆菌鉴定为戈登分枝杆菌。结论应用气相色谱技术分析全细胞脂肪酸进行分枝杆菌菌种鉴定，和传统鉴定方法结果具有良好的一致性，且可通过一次性实验操作将临床中常见的分枝杆菌鉴定到种，是一种准确度高、实用性强的分枝杆菌菌种鉴定方法。     

Human plasma apolipoprotein C-II: total solid-phase synthesis and chemical and biological characterization.

The complete amino acid sequence of human plasma apolipoprotein C-II (apoC-II) has been synthesized chemically by the solid-phase method using phenylacetamidomethyl-resin. All amino acids were coupled to the peptide-resin in the presence of 1-hydroxybenzotriazole; tert-butyloxycarbonyl-protected amino acids with the appropriate side-chain-protecting groups that are stable to the reaction conditions used in the solid-phase methodology were used. After cleavage and deprotection, the crude apoC-II was purified by ion-exchange chromatography and then by reverse-phase high-performance liquid chromatography. The purified apolipoprotein was found to elute as a single peak under various chromatographic conditions, and the overall yield of the final purified protein was 20.7%. Synthetic apoC-II was characterized by several complementary analytical techniques including amino acid composition, Edman phenylisothiocyanate degradation, polyacrylamide gel electrophoresis, and high-performance liquid chromatography. The final product was found to be homogeneous and to activate normal human post-heparin lipase to the same extent as native apoC-II. The synthetic protein is also equally immunoreactive as native apoC-II.     

液体培养法联合菌种鉴定技术诊断HIV与分枝杆菌双重感染

目的 利用分枝杆菌液体培养法联合菌种鉴定技术诊断HIV与分枝杆菌双重感染,以了解目前华北地区HIV与分枝杆菌双重感染的情况。 方法 对2009-2011年收治的53例疑似HIV与分枝杆菌双重感染患者的可疑感染部位标本进行分枝杆菌液体培养及菌种鉴定。收集所有经确诊为HIV与分枝杆菌双重感染患者的临床资料、影像学及相关实验室检查结果进行分析与总结。 结果 53例疑似HIV与分枝杆菌双重感染的患者中,病原学证实存在分枝杆菌感染的患者为19例,11例(57.9%)为单纯肺脏累及,2例(10.5%)为肺外累及,4例(21.1%)为分枝杆菌血症, 2例(10.5%)为肺脏累及和分枝杆菌血症并存。16例患者为结核分枝杆菌感染,3例患者为鸟胞内分枝杆菌复合体感染。15例（78.9%,15/19）患者CD4⁺ T淋巴细胞计数<100个/μl。 结论 联合应用液体培养法与菌种鉴定技术对于诊断HIV与分枝杆菌双重感染是有效的,值得在临床推广应用。     

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.