鲍曼不动杆菌和铜绿假单胞菌在低碱性氨基酸培养基中对抗菌药物的敏感性

为了探讨鲍曼不动杆菌和铜绿假单胞菌在低碱性氨基酸培养基中对抗菌药物的敏感性,本研究利用常规培养基和低碱性氨基酸培养基,采用琼脂稀释法检测140株鲍曼不动杆菌和60株铜绿假单胞菌对亚胺培南、帕尼培南和罗美培南的最低抑菌浓度,利用K-B纸片扩散法进行药敏实验并计算敏感率。结果显示,在低碱性培养基中,铜绿假单胞菌和鲍曼不动杆菌对三种药物的最低抑菌浓度(MIC)值显著降低,铜绿假单胞菌在低碱性氨基酸培养基中对帕尼培南的敏感率显著上升,而鲍曼不动杆菌对三种药物的敏感性均显著上升。研究表明,在低碱性氨基酸培养基中,鲍曼不动杆菌和铜绿假单胞菌对帕尼培南、亚胺培南和美罗培南的敏感性有所增强,在临床检验中需考虑由培养基导致的敏感性差异。     

耐药结核分枝杆菌感染动物模型所用菌株的筛选

本研究通过小鼠体内实验检测我国部分地区结核分枝杆菌耐药菌株的毒力,以筛选耐药结核分枝杆菌感染动物模型所用菌株。收集从我国部分省份98例结核病患者痰培养液中分离出的结核分枝杆菌,用比例法药敏试验进行结核分枝杆菌一线和二线药物的药敏试验,筛选出对二线药物敏感而对一线药物利福平或异烟肼耐药或敏感的菌株,然后进行小鼠体内毒力实验,对异烟肼耐药相关基因katG和利福平耐药相关基因rpoB测序并进行基因突变分析。从98株菌中筛选出药物敏感谱清晰的40株,进行小鼠体内毒力实验。结果显示,共35株半数死亡时间≤H37Rv的半数死亡时间,其中18株耐利福平合并耐异烟肼、5株单耐利福平,7株单耐异烟肼、5株对利福平和异烟肼均敏感。通过小鼠毒力研究,分别筛选出基因背景清晰,半数死亡时间≤7d的耐利福平合并耐异烟肼的菌株1株、半数死亡时间≤7d单耐利福平和异烟肼的菌株各1株,作为耐药结核分枝杆菌感染小鼠模型所用菌株及进一步进行豚鼠等其他动物模型感染用候选菌株。     

痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

体液病原菌分布及革兰氏阴性杆菌药敏结果分析

目的：分析2005-2006年我院内无菌体液病原菌流行状况及革兰氏阴性杆菌对常用抗生素的敏感性,以便更好地为临床治疗感染性疾病提供用药参考。方法：所有菌株用美国BD公司BBLCRYSTAL AUTOREADER仪器进行鉴定,药敏用K-B法,培养基和药敏纸片为BD公司的。结果：80株致病菌中,肠杆菌科占65%,非发酵菌占12.5%,革兰氏阳性葡萄球菌占17.5%,酵母样真菌占2.5%,厌氧菌占2.5%。大肠埃希菌感染占首位（50株）其次为铜绿假单胞菌（3株）。嗜麦芽窄食单胞菌（3株）。肺炎克雷伯菌（2株）。大肠埃希菌中ESBL阳性菌18%（9/50）ESBL阳性菌和阴性菌对不同抗菌药物的敏感性有显著差异,前者耐药明显高于后者。结论：临床应根据药敏结果合理使用抗生素,减少院内感染的发生。     

结核分枝杆菌在自制培养基上快速生长初步观察

观察结核分枝杆菌标准株在5种自制培养基上的生长时间及最佳实验条件。将改良罗氏基的成分及传统的实验条件进行改进自制5种培养基,观察结核分枝杆菌标准株生长情况。并以第5号培养基3种成分不同浓度,做正交叉实验。结核分枝杆菌标准株在5种自制培养基上,菌落开始生长天数及典型菌落出现天数均比改良罗氏培养基短,两者差异均具有显著性(P<0.01)。第5号培养基以25%牛肉浸液,20%当归煎液,20%党参煎液的浓度最佳,结核分枝杆菌接种后第3~5 d开始生长,第7~9 d出现典型的“菜花状”菌落。结核分枝杆菌标准株在自制第5号培养基上生长快,培养时间短,为该菌培养提供了一种新的快速培养基,值得进一步研究。     

肺炎克雷伯菌的分布特点耐药性分析

目的分析2010年至2012年房县人民医院患者肺炎克雷伯菌的耐药特点,为临床合理用药提供依据。方法对2010年至2012年该院各类感染患者标本中分离获得的肺炎克雷伯菌,采用纸片扩散法（K—B法）检测抗菌药物的敏感性,产超广谱β-内酰胺酶（ESBLs）检测采用ESBLs表型筛选与确证试验,并用WHONET5．3软件对药敏结果进行统计分析。结果3年分离的肺炎克雷伯菌共计248株,分离出产ESBLs菌株102株,其对亚胺培南、美洛培南无耐药,对头孢哌酮／舒巴坦、哌拉西林／三唑巴坦、阿米卡星和头孢替坦的耐药率较低,对其他抗菌药物的耐药率均较高。结论肺炎克雷伯菌对多种抗菌药物均具有较高的耐药性,临床上治疗该菌感染时应根据药敏与ESBLs检测结果选择抗菌药物。     

126株淋病奈瑟菌耐药性分析及其所致感染的中西医结合治疗

目的研究近几年来淋病奈瑟菌(简称淋球菌)对抗生素的敏感性,探讨中西医结合治疗的有效性。方法对我科从2000年1月～2007年6月收治保存的126株淋球菌分别用纸片扩散法和快速药敏测试法进行15种药物的药敏检测,并用头孢曲松合并中药治疗。结果用快速定量药敏检测法测定的最小抑菌浓度(MIC)与用K-B法基本相符。对环丙沙星的耐药率为40%,1.50%的菌株耐大观霉素(MIC≥64μg/ml);所有菌株均对头孢曲松敏感。中西医结合治疗所有病例均获得痊愈。结论头孢曲松药物敏感性无明显变化,6年间未发现耐药菌株;环丙沙星耐药性显著上升。中西医结合在淋病治疗中有效可行。     

铜绿假单胞菌的分布特点耐药性分析

目的分析3年来我院患者铜绿假单胞菌的耐药特点,为临床合理用药提供依据,并有助于医院感染预防与控制。方法对2010年至2012年我院各类感染患者标本中分离获得的铜绿假单胞菌,采用纸片扩散法（K-B法）检测抗菌药物的敏感性,并用WHONET5．3软件对药敏结果进行统计分析。结果3年分离的铜绿假单胞菌共计369株,其对多黏菌素B无耐药,对亚胺培南、美罗培南、头孢哌酮／舒巴坦、哌拉西彬他唑巴坦和阿米卡星的耐药率较低（6．81％～22．73％）,对其他抗菌药物的耐药率较高（32．47％～73．38％）。结论铜绿假单胞菌对多种抗菌药物均具有较高的耐药性,临床上治疗该菌感染时应根据药敏检测结果选择抗菌药物。     

北京朝阳医院深部真菌感染的临床分析和药敏试验

目的对院内临床深部真菌感染的标本进行分离鉴定和药敏试验,为临床感染性疾病提供病原学诊断和合理使用抗真菌药物的依据。方法采用念珠菌显色培养基进行鉴定。对真菌(丝状菌)用棉蓝染色直接镜检和培养相结合的方法进行鉴定。采用纸片扩散法进行药敏试验。结果450株真菌标本中,念珠菌396株,占88.0%;真菌(丝状菌)54株,占12.0%。分离菌株对药物的敏感性分别为氟康唑90.15%,两性霉素B97.98%,伊曲康唑91.67%,氟胞嘧啶89.1%。结论院内真菌感染以念珠菌最为多见,特别是白念珠菌和热带念珠菌。     

快速尿菌计数培养盒的研究

该快速尿菌计数培养盒进行了４种选择性培养基功能试验,共试验２３种９７株细菌,Ｇ－菌１７种７２株,Ｇ＋菌４种１９株,真菌６株。结果表明４种培养基功能特异,即Ⅰ＃为菌落计数,Ⅱ＃只限Ｇ－菌生长,Ⅲ＃只限Ｇ＋菌生长,Ⅳ＃只限真菌生长。该法与微量加样器法进行对比检测１８４份尿菌标本,符合率９６．７％,与日本培养盒平行对比试验１２份,结果一致     

Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations

The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a ΔlysAΔpanCD M. tuberculosis strain and a ΔmmaA4 BCG vaccine mutant. For every vaccine tested, a significant correlation was observed between intracellular ATP concentrations and the number of viable attenuated bacilli. However, the extractable intracellular ATP levels detected per cell among the different live vaccines varied suggesting that validated ATP luminescence assays with specific appropriate standards must be developed for each individual live attenuated vaccine preparation. Overall, these data indicate that the ATP luminescence assay is a rapid, sensitive, and reliable alternative method for quantifying the viability of varying live attenuated mycobacterial vaccine preparations.     

Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

Potential application of dot-immunobinding assay as a rapid diagnostic test in tuberculous meningitis

A simple dot-immunobinding assay (Dot-Iba) in nitrocellulose paper was developed for the detection of specific IgG antibody to Mycobacterium tuberculosis antigen 5 and mycobacterial antigen in cerebrospinal fluid of patients with tuberculous meningitis (TBM). The assay gave 77.1% sensitivity for the detection of IgG antibody to M. tuberculosis antigen 5 and 48.6% sensitivity for the detection of mycobacterial antigen in patients with TBM.     

Studies on alpha(1-->5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(alpha-D-arabinofuranosyl)-alpha-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.     

利用ATP扩增反应与生物发光法结合检测微量微生物

摘要:【目的】腺苷酸激酶（adenylate kinase, ADK）和多聚磷酸盐激酶（polyphosphate kinase, PPK）偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇（Polyurethane）的磁珠（magnetic beads）,通过化学反应将腺苷酸双磷酸酶（apyrase）固定于磁珠表面,制备固相腺苷酸双磷酸酶（Beads-apyrase）,用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。     

Calmodulin-like activity in mycobacteria.

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.     

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

An efficient method for quantitative determination of cellular ATP synthetic activity

The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. Under the used condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatment was used to make Escherichia coli cells permeable. ATP was discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.     

An ATP Bioluminescence Assay Applicable to Rapid Fluconazole Susceptibility Testing of Dermatophytes

An ATP bioluminescence assay as a rapid reference method for fluconazole (FLCZ) susceptibility testing of dermatophytes, as well as yeasts, was developed and evaluated by comparing it with viability, turbidity and fungal protein content-based conventional methods. FLCZ susceptibility results obtained with strains of Candida albicans and dermatophytes by the bioluminescence method in high-resolution medium were well correlated with those obtained by conventional methods currently used in clinical microbiology laboratories or reported previously, including a broth dilution method by the National Committee for Clinical Laboratory Standards (NCCLS). Thus, ATP bioluminescence assay can be used to monitor fungal growth in liquid culture media. The procedure has considerable potential for the rapid testing of FLCZ susceptibility of dermatophytes and other fungi.