中国耐多药结核分枝杆菌二线抗结核药物敏感性分析

目的分析中国耐多药结核分枝杆菌(MDR-TB)菌株对二线抗结核药物的敏感性,为耐多药结核病防治政策的制定提供科学依据。方法从全国2007—2008年耐药基线调查收集的菌株中选取2008年4—7月期间耐药基线调查点收集的126株MDR菌株,对其进行2种一线抗结核药物(链霉素和乙胺丁醇)和7种二线抗结核药物(氧氟沙星、卡那霉素、卷曲霉素、乙硫异烟胺、丙硫异烟胺、环丝氨酸和对氨基水杨酸)的敏感性进行检测,分析对不同药物耐药率以及交叉耐药的情况。结果一线药物链霉素和乙胺丁醇,总体耐药率分别为73.0%(92/126)和58.7%(74/126);对于二线药物,氧氟沙星和乙硫异烟胺耐药率最高,分别为25.4%(32/126)和23.0%(29/126);其次为卡那霉素和环丝氨酸,其耐药率分别为17.5%(22/126)和13.5%(17/126);最后为卷曲霉素、丙硫异烟胺和对氨基水杨酸,其耐药率均为3.2%(4/126)。初治患者对卡那霉素(χ2=20.025,P<0.01)和环丝氨酸(χ2=6.558,P=0.017)的耐药率(卡那霉素:20/60,33.3%;环丝氨酸:13/60,21.7%)显著高于复治患者(卡那霉素:2/66,3.0%;环丝氨酸:4/66,6.1%)。此外,卡那霉素和卷曲霉素,乙硫异烟胺和丙硫异烟胺间均存在单向交叉耐药,在对卷曲霉素耐药的4株中,有3株同时对卡那霉素耐药;4株对丙硫异烟胺耐药菌株均同时对乙硫异烟胺耐药。结论我国耐多药结核分枝杆菌菌株对二线抗结核药物具有较高的耐药率,特别是氧氟沙星和乙硫异烟胺,这对我国结核病控制工作,特别是耐多药结核病控制工作的实施带来严峻的挑战。

地中海拟无枝酸杆菌甲基丙二酰CoA变位酶和消旋酶的纯化及性质

采用原生质体裂解方法确定甲基丙二酰CoA变位酶(MCM)和消旋酶(MCR)均是胞浆内酶。各经过四步纯化得到电泳纯酶。纯化MCM酶的比活力为12.84u/mg,纯化倍数为528,酶活回收为60％,纯化的MCM酶服从典型的米氏底物饱和曲线,对琥珀酰CoA和辅酶B_(12)的K_m值分别为9.723#mol/L和0.1277#mol/L。经SephadexG-150测定MCM分子量约为134.000±2000,SDS-聚丙烯酰胺凝胶电泳显示两条分子量分别为67000和65000的蛋白带,说明该酶由两个大小不等亚基组成。吸收光谱测定每摩尔纯化全酶含两摩尔辅酶B_(12)。纯化MCR酶比活力为2.305u/mg,纯化倍数96,酶活回收46.7％。MCR酶由两个分子量均为17500的亚基组成。MCR酶活性能被二价金属离子Cu^(2+)、Co^(2+)、Mg^(2+)、Mn^(2+)和Fe^(2+)所促进。两酶的酶学性质和其他生物来源的MCM、MCR酶明显相似。

地中海拟无枝酸杆菌甲基丙二酰CoA转羧基酶的纯化及酶学特性

经硫酸铵沉淀，ＤＥＡＥ－纤维素吸附，磷酸纤维素吸附和Ｓｅｐｈａｒｏｓｅ４Ｂ分子筛层析四步从地中海拟无枝酸杆菌纯化得到电泳纯ＭＣＴ酶，酶比活力为３．２１Ｕ／ｍｇ，纯化倍数１７８，酶活回收１４．９％。酶反应的最适ｐＨ和温度分别为７．０和３５℃。纯化ＭＣＴ酶对底物丙酰ＣｏＡ和草酰乙酸的米氏常数分别为０．０２７ｍｍｏｌ／Ｌ０．０３５０９ｍｍｏｌ／Ｌ．经ＳｅｐｈａｄｅｘＧ－１５０测定酶分子量为２０００００，ＳＤＳ－取丙烯酰胺电泳凝胶显示一条分子量６８０００的亚基蛋白带，说明该酶由三个等大小亚基组成．薄层等电聚焦测定酶等电点为ｐＩ６．０．二价金属离子Ｃｏ￣（２＋）和Ｆｅ￣（３＋）促进酶活力．采用原生质体裂解的方法发现ＭＣＴ酶是可能分布于胞浆和细胞膜上．

烟草黑胫病菌拮抗菌的筛选及鉴定

从番茄种植地中分离获得1株对烟草黑胫病菌等多种植物病原菌和金黄色葡萄球菌有明显抑制作用的菌株ZJUT-K15,测定发现该菌株的发酵液及其无菌滤液对烟草黑胫病菌菌丝的延伸具有良好而持续的抑制作用,稀释100倍后其抑制率分别为92.29%和69.79%;24 h内可抑制烟草黑胫病菌孢子的萌发,48 h后抑制效果有所下降.通过形态特征观察、生理生化特征测定及16S rDNA序列分析,初步鉴定为枝芽孢杆菌属(Virgibacillus sp.),16S rDNA序列提交GenBank,序列号为EU639686.

山羊源无枝菌酸棒状杆菌(Corynebacterium amycolatum)的分离鉴定与耐药性分析

为了对1起羊腹泻病例进行诊断,从腹泻病羊粪便中分离到10株革兰阳性棒状杆菌YN21083~YN210812,对分离株进行形态学和16S rDNA基因鉴定并研究其药物敏感性和耐药基因特征。NCBI数据库BLAST比对鉴定结果显示,10株分离株与无枝菌酸棒状杆菌(Corynebacterium amycolatum)德国分离株FDAARGOS_991的同源性为99.3%~100.0%。16S rDNA基因进化分析显示10株分离株与C.amycolatum参考株形成同一个进化分支,与C.amycolatum参考株之间的同源性为97.2%~100.0%,与C.amycolatum模式菌株ATCC49368之间的同源性为98.3%~99.2%,将10株分离株鉴定为C.amycolatum。药敏试验结果显示,10株分离菌株对青霉素、氨苄西林、阿莫西林克拉维酸、头孢噻吩、头孢呋辛、氟苯尼考、阿米卡星和四环素敏感,对卡那霉素和磺胺甲恶唑耐药。耐药基因PCR检测结果显示,共检测到8种耐药基因aadA1、aadA2、tetA、tetM、qnrS、Sul1、Sul2和Sul3,其检出率分别为30.0%,30.0%,100.0%,60.0%,60.0%,100.0%,10.0%和20.0%。本研究首次报道羊C.amycolatum感染,对羊C.amycolatum感染的预防和控制具有重要指导意义。

蜢虾酱中生物胺降解菌的特性及应用

从蜢虾酱中筛选出一株脱羧酶阴性菌株——盐反硝化枝芽孢杆菌,分析了其对生物胺的降解特性。研究发现,此菌降解生物胺的酶为细胞膜上的胺氧化酶。安全性评估(抗生素敏感性、生物膜活性和溶血活性)结果显示,这株生物胺降解菌可作为发酵剂。盐反硝化枝芽孢杆菌在蜢虾酱中组胺、酪胺、腐胺和尸胺的降解率分别为11.1%、11.3%、15.5%和4.1%。为考察此菌对其他发酵产品中生物胺的降解情况,选择生物胺含量较高的奶酪和纳豆作为研究对象。在奶酪中,组胺、酪胺和腐胺的降解率分别为37.03%、15.67%、23.70%;在纳豆中,组胺、酪胺、腐胺和尸胺的降解率分别为26.47%、14.23%、10.50%和10.40%。结果表明,盐反硝化枝芽孢杆菌可以作为发酵剂,以控制蜢虾酱及其他发酵产品中生物胺的积累。

Differentiation of Crohn’s disease from intestinal tuberculosis in India in 2010

Differentiating intestinal tuberculosis from Crohn’s disease (CD) is an important clinical challenge of considerable therapeutic significance. The problem is of greatest magnitude in countries where tuberculosis continues to be highly prevalent, and where the incidence of CD is increasing. The final clinical diagnosis is based on a combination of the clinical history with endoscopic studies, culture and polymerase chain reaction for Mycobacterium tuberculosis, biopsy pathology, radiological investigations and response to therapy. In a subset of patients, surgery is required and intraoperative findings with pathological study of the resected bowel provide a definitive diagnosis. Awareness of the parameters useful in distinguishing these two disorders in each of the different diagnostic modalities is crucial to accurate decision making. Newer techniques, such as capsule endoscopy, small bowel enteroscopy and immunological assays for Mycobacterium tuberculosis, have a role to play in the differentiation of intestinal tuberculosis and CD. This review presents currently available evidence regarding the usefulness and limitations of all these different modalities available for the evaluation of these two disorders.

Thalidomide induces mucosal healing in Crohn's disease: Case report

Crohn's disease is a chronic inflammatory disorder of the gastrointestinal tract that is defi ned by relapsing and remitting episodes. Tumor necrosis factor alpha (TNF-α) appears to play a central role in the pathophysiology of the disease. Standard therapies for inflammatory bowel disease fail to induce remission in about 30% of patients. Biological therapies have been associated with an increased incidence of infections, especially infection by Mycobacterium tuberculosis (Mtb). Thalidomide is an oral immunomodulatory agent with anti-TNF-α properties. Recent studies have suggested that thalidomide is effective in refractory luminal and fistulizing Crohn's disease. Thalidomide costimulates T lymphocytes, with greater effect on CD8+ than on CD4+ T cells, which contributes to the protective immune response to Mtb infection. We present a case of Crohn's disease with gastric, ileal, colon and rectum involvement as well as steroid dependency, which progressed with loss of response to infliximabafter three years of therapy. The thorax computed tomography scan demonstrated a pulmonary nodule suspected to be Mtb infection. The patient was started on thalidomide therapy and exhibited an excellent response.

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified 10 patients(4 males and 6 females;mean age = 65.1 ± 13.6 years) with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen(ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction(PCR) for tubercle bacilli DNA,as well as Tuberculin skin test(TST) and QuantiFERON-TB gold test(QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded(FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients(60%) had a positive TST,and 4 patients(40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings(linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients(70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples.The histopathological data revealed that tuberculous granulomas were present in 4 cases(40%).IHC staining in archived FFPE samples with anti-M.tuberculosis monoclonal antibody revealed positive findings in 4 patients(40%);the same patients in which granulomas were detected by hematoxylin and eosin staining.M.tuberculosis antigens were found to be mostly intracellular,granular in pattern,and primarily located in the CD68 + macrophages of the granulomas.CONCLUSION:IHC staining with a monoclonal antibody to M.tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.

NOD2 and ATG16L1 polymorphisms affect monocyte responses in Crohn's disease

AIM： TO assess whether polymorphisms in NOD2 and ATG16L1 affect cytokine responses and mycobacterium avium subspecies paratuberculosis （MAP） survival in monocytes from Crohn＇s disease （CD） patients METHODS： Monocytes were isolated from peripheral blood of CD patients of known genotype for common single nucleotide polymorphisms of NOD2 and ATG16L1, Monocytes were challenged with MAP and bacterial per- sistence assessed at subsequent time-points. Cytokine responses were assayed using a Milliplex multi-analyte profiling assay for 13 cytokines. RESULTS： Monooltes heterozygous for a NOD2 polymorphism （R702W, P268S, or 1007fs） were more permissive for growth of MAP （P = 0.045） than those without. There was no effect of NOD2 genotype on subsequent cytokine expression. The T300A polymorphism of ATG16L1 did not affect growth of MAP in our model （P = 0.175）, but did increase expression of cytokines interleukin （IL）-10 （P = 0.047） and IL-6 （P = 0.019）. CONCLUSION： CD-associated polymorphisms affected the eliminaUon of MAP from ex v/vo monooltes （NOD2）, or expression of certain oltokines （ATG16LI）, implying independent but contributory roles in the pathogenesis of CD.

Role for mycobacterial infection in pathogenesis of primary biliary cirrhosis?

Primary biliary cirrhosis （PBC） is a progressive cho- lestatic liver disease characterized by the immune- mediated destruction of biliary epithelial cells in small intrahepatic bile ducts. The disease is characterized by circulating antimitochondrial antibodies （AMAs） as well as disease-specific antinuclear antibodies, cholestatic liver function tests, and characteristic histological fea- tures, including granulomas. A variety of organisms are involved in granuloma formation, of which mycobacte- ria are the most commonly associated. This has led to the hypothesis that mnycobacteria may be involved in the pathogenesis of PBC, along with other infectious agents. Additionally, AMAs are found in a subgroup of patients with rnycobacterial infections, such as lep- rosy and pulmonary tuberculosis. Antibodies against species-specific mycobacterial proteins have been re- ported in patients with PBC, but it is not clear whether these antibodies are specific for the disease. In addi- tion, data in support of the involvement of the role of molecular mimicry between rnycobacterial and human mitochondrial antigens as triggers of cross-reactive im- mune responses leading to the loss of immunological tolerance, and the induction of pathological features have been published. Thus, antibodies against myco- bacterial heat shock protein appear to cross-recognize AMA-specific autoantigens, but it is not clear whether these autoantibodies are mycobacterium-species-spe- cific, and whether they are pathogenic or incidental. The view that mycobacteria are infectious triggers of PBC is intriguing, but the data provided so far are not conclusive.

Animal models to study Mycobacterium tuberculosis and HIV co-infection

Mycobacterium tuberculosis(M.tb) and human immunodeficiency virus(HIV) co-infection has become a public health issue worldwide. Up to now, there have been many unresolved issues either in the clinical diagnosis and treatment of M.tb/HIV coinfection or in the basic understanding of the mechanisms for the impairments to the immune system by interactions of these two pathogens. One important reason for these unsolved issues is the lack of appropriate animal models for the study of M.tb/HIV coinfection. This paper reviews the recent development of research on the animal models of M.tb/HIV co-infection, with a focus on the non-human primate models.

Pathotype Structure of Magnaporthe grisea in the Fields of Chongyang and Yuan'an in Hubei Province,China

[Objective] This study aimed to explore the pathotype structure of Magnaporthe grisea in Chongyang and Yuan'an in Hubei Province,China.[Method] From the rice-growing fields of Chongyang and Yuan'an in Hubei Province where rice blast occurs frequently,60 isolates which were pathotyped against two sets of host differentials:Chinese host differentials and CO39 NILs,were obtained.Then,20 pathotypes with the six indica host differentials(CO39 NILs) were observed,while 13 pathotypes in four race groups were observed out of the same single spore isolates with Chinese host differentials which consists of three indica cultivars and four japonica cultivars.The diversity of the pathotypes of M.grisea populations tested by CO39 NILs was 2.54 and the pathotype 137.1 occurred at predominantly high frequency(21.67%).The diversity of physiological races of M.grisea populations tested by Chinese host differentials was 1.22 and the race group ZA occurred at predominantly high frequency(73.33%).The diversity of physiological races of M.grisea in Chongyang and Yuan'an were also calculated.Overall,the diversity of pathotypes of M.grisea in Yuan'an was higher than that in Chongyang with the two sets of the host differentials.[Conclusion] This study provided current information on the pathotype spectrum of M.grisea populations in the rice fields of Hubei Province to allow the formulation of viable strategies for blast resistance breeding programs in Hubei Province.

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconiosis workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.

Choroidal Tuberculoma in an Immunocompetent Young Patient

TUBERCULOSIS （TB） remains one of the leading causes of preventable morbidity and mortality from infectious disease worldwide.1 It is a chronic progressive granulomatous infection caused by Mycobacterium tuberculosis （M. tuberculosis）.

An epidemiological study of resistant tuberculosis in Chongqing,China

Background The epidemiological characteristics of drug-resistant tuberculosis （DR-TB） is fundamental to improving the prevention and control of DR-TB. Mutations in katG315 is thought to be the most predictive molecule markers for Isoniazid （INH） resistance in Mycobacterium tuberculosis （MTB）. However, mutations to these genes have not been thoroughly studied in China, and epidemiological evidence of their expression levels are especially lacking in the southwest of China, which has a high TB burden within the population. Methods MTB isolates were obtained from patients with active pulmonary tuberculosis at the TB dispensary and Chest hospital in Chongqing city between June 2003 and June 2006. Proportion methods were used to test the sensitivity to INH, RFP, SM and EMB of cultured MTB. A total of 100 MTB isolates were also randomly selected for analysis of the molecular mutation spectrum of katG by DNA sequencing. Results Totally 1 089 MTB isolates that completed positive sputum cultures and evaluated for their sensitivity to the four first-line drugs among 2 777 patients with TB. The prevalence of DR-TB and multi-drug resistant tuberculosis （MDR-TB） were 27.7% （302/1 089） and 7.3% （79/1 089）, respectively. The resistance to anti-TB drugs was found to be highest for SM （16.3%） and INH （14.0%）. There was also a significant increase in the prevalence of resistance to RFP and EMB （P〈0.01）, and an increase in MDR-TB between June 2003 and June 2004 and between July 2005 and June 2006. The total mutation rate of katG315 was 75＂5% （37/49） in INH-resistant MTB, and mutation sites included $315T, $315N and $315I with mutation rates of 81.1% （30/37）, 13.5% （5/37） and 5.4% （2/37）, respectively No katG315 mutants were found in any of the 48 INH-sensitive MTB. Our preliminary diagnostic results suggest that mutations in katG315 may potentially serve as molecular markers that can be used to diagnose the resistance to anti-TB drug of INH. Conclusion In the Chongqing, DR-TB and MDR-TB are increasing, and are becoming key problems for tuberculosis control. The use of katG315 mutations as potential molecule markers for drug resistance to INH may help improve patient treatment and decrease the spread of the disease

Nontuberculous Mycobacteria Infections in Katavi Rukwa Ecosystems

A study on nontuberculous mycobacteria （NTM） was carried out in wildlife-livestock interface of Katavi Rukwa ecosystem （KRE）. 328 livestock tissues and 178 wild animals were cultured, wild animals were sampled opportunistically during professional hunting and game cropping operations in the KRE protected areas. The objective of the study was to generate data on epidemiology of NTM in the wildlife-livestock interface of the KRE. Methods used to identify the NTM were： culture and isolation, polymerase chain reaction, protein heat shock 65 kilodalton （hsp65） and sequencing. Mycobacteria were detected on 25.9% and 11.9% of livestock and wildlife tissue cultures, respectively. The most NTM isolated were M. kansasii （30%）, M. gastri （30%）, M. fortuitum （1%）, M. intracellulare （4%）, M. indicuspranii （4%）, M. nonchromogenicum （6%） and M. lentiflavum （6%）. Other NTM in smaller percentages were M. hibernae, M. engbaekii, M. septicum, M. arupense and 34.. godii. Due to rise of NTM infection in both human and animals, it is recommended that awareness and laboratory facilities be improved to curb the underreporting especially in TB-endemic countries. For species specific identification, a network of national and regional laboratories is promoted.

Characterization of the Mycobacterium Tuberculosis Protein Rv2302

The Mycobacterium tuberculosis protein Rv2302 has been characterized using nuclear magnetic resonance （NMR） and circular dichroism （CD） spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparalle β-sheet core with one C-terminal α-helix （A61 to A75） nestled against its side. Hydrophobic interactions between residues in the α-helix and β-strands 3 and 4 hold the α-helix near the β-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {^1H}-^15N heteronuclear nuclear Overhauser effects indicate that the protein＇s core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G 13 to H 19 in the ^1H-^15N heteronuclear single quantum correlation spectrum suggests that this region, a loop between β-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.

Molecular Cloning of PhoR Sensor Domain from Mycobacterium tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular

The emergence of multidrug-resistant strains （MDR-TB） and extensively drug-resistant strains （XDR-TB） has fuelled the quest for novel drugs and drug targets for its successful treatment. One of the potential candidates as novel TB drug target is the PhoR sensor domain, an extracellular domain of PhoR histidine kinase. PhoR sensor domain is part of the two-component system PhoR-PhoP that senses environmental stimuli and relays the signal to control the expression of 78 virulent associated genes in Mycobacterium tuberculosis. 3D structure of the PhoR sensor domain will facilitate the structure based drug discovery of novel anti- tubercular. In this study, we successfully predicted and isolated the gene encoding PhoR sensor domain from Mycobacterium tuberculosis H37Rv, cloned it in pGEM-T vector and subcloned it in pRSET emGFP expression vector. PhoR sensor domain was successfully cloned and would be used for further expression, purification and crystallization studies.