分枝杆菌同源可控表达系统的建立及其应用

目的 构建分枝杆菌可控表达载体,利用其过表达结核分枝杆菌(Mycobacterium tu-berculosis,Mtb)的免疫保护性抗原,亲和层析纯化后分析免疫原性.方法 应用PCR扩增耻垢分枝杆菌(Mycobacterium smegmatis,Ms)乙酰胺酶编码基因启动子(pACE),构建分枝杆菌可控表达载体pMF系列;克隆Mtb嵌合抗原编码基因并用乙酰胺进行诱导表达,以Ni~(2+)亲和层析纯化重组蛋白;并用该重组嵌合抗原Ag856A2对卡介苗(BCG)免疫的小鼠脾细胞进行体外刺激,以IFN-γ酶联免疫斑点技术(ELISPOT)分析其免疫原性.结果 成功构建了分枝杆菌可控表达载体pMF系列,利用0.02%乙酰胺进行诱导可实现目标抗原在Ms中的高水平表达;同时利用引入的6×His标签可方便实现重组抗原的一步纯化,且该同源表达重组抗原可诱导更高水平IFN-γ的分泌.结论 以Ms乙酰胺酶编码基因启动子pACE为基础构建的分枝杆菌可控表达系统可实现Mtb抗原在Ms中的高水平表达与纯化,与大肠杆萧异源表达相比,该同源表达重组抗原具有更好的免疫原性.     

Mycobacterium abscessus: an emerging rapid-growing potential pathogen

Mycobacterium abscessus is the most pathogenic and chemotherapy-resistant rapid-growing mycobacterium. It is commonly associated with contaminated traumatic skin wounds and with post-surgical soft tissue infections. It is also one of the mycobacteria that are most often isolated from cystic fibrosis patients. It is essential to differentiate this species from the formerly indistinct "M. chelonae-complex", as chemotherapy is especially difficult in M. abscessussenso strictu. Clarithromycin or azithromycin are the only regular oral antimycobacterial agents with an effect on M. abscessus, and should preferably be supplemented with other drugs since long-term monotherapy may cause resistance. Amikacin is a major parenteral drug against M. abscessus that should also be given in combination with another drug. The recently introduced drug tigecycline may prove to be an important addition to chemotherapy, but has yet to be fully clinically evaluated as an antimycobacterial agent. Surgery can be curative, or at least helpful, in the healing of M. abscessus infection, and if conducted, it should include the removal of all foreign or necrotic material. There is increasing awareness of M. abscessus as an emerging pathogen.     

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn²⁺fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.     

Pulmonary nontuberculous mycobacterial infections: Antibiotic treatment and associated costs

Recent studies suggest an increasing prevalence of pulmonary nontuberculous mycobacteria (NTM) disease. In the absence of prevalence and cost data, the public health burden of pulmonary NTM disease is difficult to assess. The goal of this study was to assess costs associated with NTM disease treatment and to identify risk factors associated with increased costs. Records from subjects with pulmonary NTM disease enrolled in a natural history protocol were abstracted for presenting symptoms, comorbidities, microbiology, and treatment histories. Antibiotic frequency, duration, adverse reaction, and costs were noted, the total antibiotic burden and cost were calculated, and risk factors associated with high costs were analyzed. From Jan 2004 to Dec 2005, 33 subjects were enrolled; 27 met disease criteria and had sufficient data to assess antibiotic use. Mycobacterium avium complex was present in 89% and Mycobacterium abscessus was present in 21% of subjects. Subjects received a median of 5 (1–10) antibiotics. Adverse effects were common seen in up to 50% with common antibiotics and up to 100% with uncommonly used antibiotics. Median burden of treatment was 2638 (84–7689) drug-days and the median total cost per patient was $19,876 ($398–70,917). Subjects with high treatment costs had an adjusted 9.5 fold (95% CI 1.5–97.2) likelihood of having M. abscessus and a 4.2 fold (95% CI 0.6–59.3) increased likelihood of having more extensive disease. Pulmonary NTM represent an underappreciated disease burden in the US population, with an associated treatment cost comparable to that for other chronic diseases of infectious origin such as HIV/AIDS.     

Development of leprosy in a patient with ankylosing spondylitis during the infliximab treatment: reactivation of a latent infection?

The use of tumor necrosis factor α as a treatment for chronic inflammatory conditions has been shown to be associated with an increased risk of developing infections, especially Mycobacterium tuberculosis, atypical mycobacteria, and other microorganisms. We report the case of a 58-year-old man with ankylosing spondylitis, receiving infliximab treatment, who presented with multiple plaques on the face, chest, and extremities, a thickened, tender ulnar nerve, and severe neuritis of the feet. The results of a biopsy of these lesions revealed histopathological features of lepromatous Hansen disease. The use of anti-tumor necrosis factor biologic agent on this patient may have resulted in either a new infection or reactivation of a latent infection of Mycobacterium leprae.     

Minocycline: old and new therapeutic uses

Since the introduction of minocycline HCl in the late 1960's, it has been used for disease states that have ranged from typical community-acquired infectious diseases to others that are non-infectious, such as resistant rheumatoid arthritis. Owing to its high penetration characteristics throughout the body, minocycline can be used in the treatment of a wide variety of extracellular and intracellular pathogens. This review examines the known and potential therapeutic uses of minocycline in a clinical setting.     

结核分枝杆菌肝素结合血凝黏附素研究进展

结核分枝杆菌肝素结合血凝黏附素是一种表面蛋白,有三个功能区,它与人补体成分C3结合可介导单核细胞黏附和吞噬结核杆菌,也与黏附宿主细胞、肺外传播等相关,还可作为有前景的候选疫苗。     

BACTEC MGIT960分枝杆菌分析系统结果分析

目的评价美国BD公司生产的BACTECMGIT960分枝杆菌分析系统（960）快速分离分枝杆菌效果。方法采用BACTECMGIT960分枝杆菌分析系统对临床标本进行分析，同时用《结核病诊断细菌学检验规程》规定的中性改良罗氏培养基法（L—J法）进行平行对照，比较其对不同来源的样本中分枝杆菌的分离率、阳性报告时间。结果对1449份标本用960和传统的中性改良罗氏培养基进行平行接种，960培养的阳性率为32．9％（482／1449），传统的L—J法培养的阳性率为25．5％（370／1449），前者明显高于后者（X^2=61．65，P〈0．005）。960的污染率为2．3％，L—J法的污染率为6．5％，前者明显小于后者（X^2=29．42，P〈0．005）。阳性报告时间方面，14d内支纤镜提取物（BF液）晚于其它标本；而对链霉素（STR）、异烟肼（INH）、利福平（RIF）和乙胺丁醇（EMB）全耐药菌株早于其它耐药菌株。结论应用BACTEC MGIT960系统分离培养分枝杆菌可以明显提高分离分枝杆菌的阳性率和减少污染。     

Evaluation of the MB/BACT automated mycobacteria culture system versus culture on Lowenstein medium

Objective: To evaluate the growth time and recovery rate of mycobacteria, and the percentage of contamination in the MB/BACT system versus traditional culture, on Lowenstein medium.
Methods: One thousand one hundred and fifty-nine samples for mycobacterial analysis were cultured in Lowenstein medium and the MB/BACT system: 0.5-mL aliquots of the sample were inoculated into tubes containing either medium and incubated for 49 days. The mycobacterial isolates were identified by means of the Accu-Probe (Gen-Probe) or a system based on chromogenesis, time and temperature of growth and principal biochemical differentiation analyses. Fischer's test was performed.
Results: Ninety-three mycobacterial strains were isolated: 80 Mycobacterium tuberculosis , seven strains of the M. avlum-intracellulare complex, four M. gordonae and two M. smegmatis. For M. tuberculosis , 76 of 80 isolates grew in MB/BACT, while 60 of 80 grew on Lowenstein medium. A total of 88 of the mycobacterial strains grew in the MB/BACT system, while 66 grew on the Lowenstein solid medium. Growth in the MB/BACT averaged 16.51 days, as opposed to 22.71 days on Lowenstein medium.
Conclusions: The MB/BACT system is a suitable complement to culturing on Lowenstein medium, while the workload does not significantly increase in comparison with culturing on solid media only and still allows major recovery of mycobacteria with a significant time-saving.