重庆地区耐多药结核分枝杆菌吡嗪酰胺耐药基因突变的特征分析

分析重庆地区分离的耐多药结核分枝杆菌(MDR-MTB)菌株pncA和rpsA基因突变特征及其与吡嗪酰胺(PZA)耐药的相关性。     

结核分枝杆菌吡嗪酰胺耐药相关基因研究进展

吡嗪酰胺是治疗结核病的重要抗结核药物之一,特别是用于耐多药结核病的治疗。近年来结核分枝杆菌对吡嗪酰胺的耐药逐渐增多,其耐药机制尚未明确。本文针对结核分枝杆菌吡嗪酰胺耐药及其与基因变异之间的关系研究进展进行综述,以供研究者参考。     

结核分枝杆菌吡嗪酰胺耐药相关蛋白的原核表达及纯化

目的构建结核分枝杆菌pncA基因的原核表达质粒,获得结核分枝杆菌吡嗪酰胺酶的表达蛋白。方法制备结核分枝杆菌基因组DNA,采用聚合酶链反应扩增目的基因片段;通过pET28a构建表达载体pET28a-pncA,序列测定证实正确后转化大肠埃希菌DH10b,经IPTG诱导表达His-吡嗪酰胺酶融合蛋白,采用SDS-PAGE和Western blot分析重组蛋白。结果扩增出结核分枝杆菌pncA基因并构建了具有正确基因序列的质粒载体pET28a-pncA,转化大肠埃希菌BL21后经诱导产生了分子质量单位约20ku的表达产物,并得到纯化的带His标签的目的蛋白。结论构建了结核分枝杆菌pncA基因原核表达质粒,并诱导表达了His-吡嗪酰胺酶融合蛋白,为进一步研究吡嗪酰胺耐药性奠定了基础。     

应用部分变性高效液相色谱法检测结核分枝杆菌的耐药基因突变

目前耐药问题给结核病的控制造成很大威胁。KatG、rpoB、embB、gyrA、pncA和rpsL基因在异烟肼、利福平、乙胺丁醇、氟喹诺酮类、吡嗪酰胺和链霉素的耐药中起关键作用。我们采用温度介导的异源双链高效液相色谱分析法（DHPLC）检测了结核分枝杆菌耐药基因的突变。     

中国异烟肼耐药结核分枝杆菌基因突变特征分析

目的: 明确中国异烟肼(INH)耐药结核分枝杆菌基因突变的分子特征。方法: 在PubMed、中国知网、万方、维普数据库中检索有关中国结核分枝杆菌耐INH基因突变的研究文献,对纳入文献的突变基因的突变位点、突变类型,以及氨基酸改变数量等信息进行整合分析。结果: 共纳入69篇中英文文献,共6393株INH耐药结核分枝杆菌。95.71%(6119/6393)检测到基因突变或缺失,其中katG、inhA、aphC突变数量最多,分别占77.57%(4959/6393)、15.20%(972/6393)、3.69%(236/6393)。单基因单位点突变占87.80%(5613/6393),联合突变占12.20%(780/6393)。katG315突变占INH耐药菌株总数的56.22%(3594/6393),katG463突变占10.03%(641/6393),inhA15突变占10.10%(646/6393)。突变形式最常见的是C→T,占10.03%(641/6393)。结论:中国INH耐药结核分枝杆菌突变基因最多的是katG、inhA、ahpC,突变密码子最多的是katG315、katG463、inhA15。     

结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的影响

目的研究结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的机制和作用靶点。方法将饥饿3、5和10d的结核分枝杆菌及未经过营养饥饿的结核分枝杆菌分别加入100μg／ml吡嗪酰胺和脂肪酸、苯甲酸和水杨酸进行处理，观察营养饥饿处理过程对于吡嗪酰胺抗结核分枝杆菌作用的影响，以及酸性物质对吡嗪酰胺抗菌活性的影响。同时用流式细胞仪检测结核分枝杆菌膜电位和平均荧光强度，以探讨吡嗪酰胺的作用机制。结果未经过营养饥饿的结核分枝杆菌和饥饿3、5和10d的结核分枝杆菌经吡嗪酰胺处理后，菌落形成单位（CFU）分别减少23．08％、37．75％、82．32％和81．03％；营养饥饿5d后，结核分枝杆菌的膜电位明显降低，经吡嗪酰胺处理后的膜电位则大幅降低，加入能量物质葡萄糖后，因吡嗪酰胺作用而降低的膜电位可得到恢复。脂肪酸、苯甲酸和水杨酸对于正常生长的和营养饥饿后的结核分枝杆菌均有促进吡嗪酰胺抗菌效果的作用，对营养饥饿后的结核分枝杆菌作用更明显。结论饥饿状态和弱酸能增强吡嗪酰胺对结核分枝杆菌的抗菌作用，吡嗪酰胺可能是通过于扰结核分枝杆菌的膜电位及其能量代谢而产生作用。     

重庆地区耐多药结核分枝杆菌对氟喹诺酮类药物耐药的相关基因特征分析

目的 分析重庆地区耐多药结核分枝杆菌对氟喹诺酮类(FQs)药物耐药的相关基因特征,以及与结核分枝杆菌基因型的相关性。 方法 收集2015年1月至2017年6月重庆市39个区(县)967例耐多药结核病(MDR-TB)可疑患者的所有耐多药(MDR)结核分枝杆菌临床分离株229株,通过微孔板Alamar blue显色法检测4种FQs药物[氧氟沙星(Ofx)、左氧氟沙星(Lfx)、莫西沙星(Mfx)、加替沙星(Gfx)]的耐药性,用PCR测序方法对FQs药物耐药相关基因gyrA、gyrB进行分析,并采用实时荧光定量熔解曲线方法进行北京基因型鉴定。 结果 在229株MDR菌株中,94株(41.0%,94/229)对任一FQs药物耐药。其中,Ofx耐药率最高(41.0%,94/229);Lfx、Mfx耐药率居中,分别为31.4%(72/229)、30.6%(70/229);Gfx耐药率最低(20.1%,46/229)。94株对FQs耐药菌株中,81株(86.2%,81/94)发生gyrA基因突变,第94位密码子突变最常见(60.6%,57/94);10株(10.6%,10/94)发生gyrB基因突变。7株gyrA基因双位点突变均显示为高水平耐药;9株gyrA与gyrB联合突变中,2株为高水平耐药。重庆地区对FQs耐药菌株中北京基因型80株(85.1%,80/94),其中,现代北京基因型占60.0%(48/80)。 结论 重庆地区MDR结核分枝杆菌 对FQs耐药的菌株以现代北京基因型为主,其耐药相关基因突变主要发生于gyrA基因。     

结核分枝杆菌耐药机制

耐多药结核病和广泛耐药结核病在人类免疫缺陷病毒感染时代的不断增长是有效结核病控制的主要威胁。来自染色体自发突变的结核分枝杆菌耐药概率很低。疾病治疗过程中由于不稳定的药品供应、不合理的治疗方案以及差的患者依从性等人为选择因素导致临床耐药结核病大量发生。已经阐明主要的一线和二线药物包括利福平、异烟肼、吡嗪酰胺、乙胺丁醇、氨基糖甙类和氟喹诺酮类的耐药分子机制。结核分枝杆菌菌株耐药性与毒性／传染性之间的关系需要进一步研究。理解结核分枝杆菌耐药机制将有助于快速分子诊断工具的发展并且可能进一步指导结核病治疗的新药的研制。     

广东省地区结核分枝杆菌寡核苷酸基因分型及耐药相关性

目的 研究结核分枝杆菌(MTB)不同基因型在广东省各地区的流行情况及其耐药相关性。方法 随机数字法抽取广州市胸科医院2014-2015年临床分离的来自于广东省不同地区的504株结核分枝杆菌菌株,采用比例法进行药物敏感性实验,选用间隔区寡核苷酸分型方法(Spoligotyping)对结核分枝杆菌进行基因分型。基因型耐药率差异比较采用χ²检验。结果 (1)在504株结核分枝杆菌临床分离株中北京家族和非北京家族分别是386株和118株。非北京家族中,T家族高达48株;其余是H3家族、EAI家族、MANU家族、AMBIGO家族、LAM家族以及新发基因型。北京家族构成比在性别、年龄上无统计学差异(P>0.05),但在汕头、汕尾、揭阳、东莞四个地级市的构成比低于广州市,差异有统计学意义(P<0.05)。(2)504株结核分枝杆菌中,453株在SPOIDB 4.0数据库中分为49种寡核苷酸类型归类于30个簇,其主要流行簇依次为:SIT1、SIT53、SIT52、SIT19、SIT523。SIT1基因型的异烟肼(INH)、利福平(RFP)、链霉素(SM)、乙胺丁醇(EMB)、 吡嗪酰胺(PZA)、 阿米卡星(AMK)、左氧氟沙星(LVFX)、耐多药(MDR)、泛耐药(XDR) 的耐药率均高于SIT53、 SIT52、SIT19,而SIT523在 RFP、SM、EMB、MDR的耐药率方面高于SIT1 (3)主要流行基因型北京家族的INH、PZA、AMK的耐药率高于非北京家族,差异有统计学意义(P<0.05),其余药物的耐药性无显著性差异(P>0.05)。结论 广东省结核分枝杆菌呈基因多态性,北京基因型菌株为本省结核分枝杆菌主要流行株,而且与异烟肼、吡嗪酰胺、阿米卡星耐药相关。非北京基因型在潮汕和东莞等华侨之乡比例有所增加。     

MTBDRplus技术快速检测结核分枝杆菌耐多药的临床应用评价

目的 评价线性探针杂交技术(简称MTBDRplus技术)对福州地区耐药结核杆菌的检测效果,并了解该地区耐药结核分枝杆菌的耐药基因特征方法 选取246株临床结核分枝杆菌分离株,以传统罗氏药敏试验为金标准,评价MTBDRplus技术在临床上应用的检测效果,分析耐药基因突变分布频率结果 与传统罗氏药敏试验相比较,MTBDRplus检测利福平(RIF)和异烟肼(INH)耐药性灵敏度分别为91.7%(11/12)和83.3%(15/18),MTBDRplus检测RIF和INH耐药性特异度分别为99.6%(233/234)和95.2%(217/228)。12例rpoB基因存在突变的结核分枝杆菌中,58.3%rpoB基因S531L突变;26例katG和inhA基因存在突变的结核分枝杆菌中,57.7%(15/26)katG基因315突变;存在C15T位点突变的菌株仅9.1%(1/11)为INH耐药菌株结论 MTBDRplus技术是一个敏感、特异的快速诊断耐多药结核病的有效方法,该技术在福州地区具有较好的应用前景。福州市耐药结核分枝杆菌以rpoB S531L和katG S315T突变型为主。建议谨慎判断结核分枝杆菌inhA基因C15T位点突变引起的INH耐药。     

Genetic diversity and evidence for acquired antimicrobial resistance in Mycobacterium tuberculosis at a large hospital in South India.

OBJECTIVE: To assess genetic diversity and drug resistance of Mycobacterium tuberculosis isolates collected at Christian Medical College Hospital (CMCH), Vellore, India, between July 1995 and May 1996. MATERIALS AND METHODS: Isolates were subjected to IS6110-based restriction fragment length polymorphism (RFLP) analysis and tested for resistance to isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide, and DNA from selected strains was sequenced in regions associated with drug resistance. RESULTS: One hundred and one M. tuberculosis isolates were collected from 87 patients with pulmonary tuberculosis. Charts of 69 patients were reviewed for history of tuberculosis illness and treatment. DNA from 29 strains was sequenced in katG, rpoB, and gyrA, and sometimes pncA regions. Analysis by RFLP revealed a high degree of genetic diversity, with no identifiable clusters of infection. Of the strains tested, 51% were resistant to at least one antibiotic, and 43% were resistant to more than one drug. There was a high rate of resistance observed in patients whose charts indicated a history of improperly administered tuberculosis treatment, whereas little drug resistance was observed in patients never previously treated for tuberculosis. Sequencing of genes associated with drug resistance revealed several previously unreported mutations in resistant strains. CONCLUSIONS: This analysis suggests that the cases of tuberculosis in the sample are largely reactivation of long-standing infections and that the drug resistance among patients in CMCH is largely acquired or secondary rather than attributable to the spread of drug-resistant strains.     

Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance

Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M.?tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M.?tuberculosis strains fell within a specific range, and M.?tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for M.?smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M.?tuberculosis strains. No significant variability was observed in the pyrazinamide flux rate. The pyrazinoic acid efflux rate explained 61% of the variability in Bactec pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast, pyrazinamidase activity accounted for only 27% of the Bactec pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of pyrazinamidase activity) are also implicated in pyrazinamide resistance, and that pyrazinoic acid efflux rate acts as a better proxy for pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.     

Drugs with activity against Mycobacterium tuberculosis

Treatment for Mycobacterium tuberculosis has to be lengthy, since populations of this bacillus differ in metabolic activity, and it has to consist of various associated drugs, since spontaneous chromosome mutations can give rise to drug resistance. The multiresistant phenotype emerges with sequential acquisition of mutations in several loci of separate genes. Knowledge of the mechanisms of resistance permits the development of molecular techniques for the early detection of resistant strains, thereby making proper control possible. Tuberculosis treatment includes isoniazid, rifampicin and pyrazinamide during the first two months and isoniazid and rifampicin to complete six months of treatment. In specific situations, a fourth drug is added, ethambutol for adults and streptomycin for children in whom visual acuity cannot be monitored. This review describes the characteristics, activity, resistance mechanisms and side effects associated with the various antituberculosis drugs.     

结核分枝杆菌异烟肼和丙硫异烟胺耐药及其交叉耐药相关机制研究进展

结核病是一种严重危害人类健康的呼吸道传染病。据世界卫生组织估算,2018年全球耐多药结核病患者数为48.4万。异烟肼是重要的一线抗结核药物,但其目前耐药情况比较严重。丙硫异烟胺,作为异烟肼耐药患者的替代治疗药物,与异烟肼存在部分的交叉耐药性。本文综述了异烟肼和丙硫异烟胺交叉耐药的相关机制,为异烟肼耐药及耐多药结核病患者的治疗提供科学依据。     

Evidence of 'amplifier effect' in pulmonary multidrug-resistant tuberculosis: report of three cases.

INTRODUCTION: A cluster of three related cases of tuberculosis (TB) with primary multidrug resistance was investigated at the Centre Hospitalier Universitaire of Kigali (CHUK) in Rwanda. The patients were HIV-1/2 seronegative. Patients 1 and 2 were hospitalized in the same room of CHUK for one month. Patient 3 was a younger sibling of patient 2. METHODS: Drug susceptibility of two consecutive Mycobacterium tuberculosis isolates from each patient was tested by the BACTEC 460 radiometric method. DNA fingerprinting was performed using spoligotyping and mycobacterial interspersed repetitive units of variable numbers of tandem repeats (MIRU-VNTR) analysis. All patients initially received the World Health Organization category I regimen. RESULTS: The isolates collected during the first TB episode were resistant to isoniazid, rifampin and ethambutol. After subsequent retreatment regimens with rifampin, isoniazid, streptomycin, pyrazinamide (8 months) and rifampin, isoniazid, streptomycin, pyrazinamide, ciprofloxacin (21 months), patients 1 and 2 developed additional resistance to streptomycin and quinolones. Patient 3 received only the category I regimen and consecutive isolates retained the initial drug susceptibility pattern. All isolates were genetically indistinguishable by spoligotyping and MIRU-VNTR, indicating the same origin. CONCLUSIONS: These observations highlight the risk of nosocomial transmission of multidrug-resistant (MDR) TB and the possible selection of secondary resistance to second-line drugs if a single new drug is added at the time of retreatment of MDR TB patients.     

结核分枝杆菌贝达喹啉和氯法齐明耐药及其交叉耐药机制研究进展

目前,全球结核病耐药现象严重,给结核病的防控工作带来了极大的困难与挑战,并亟需新型抗结核药物来阻止耐药结核病的蔓延.贝达喹啉和氯法齐明分别是世界卫生组织推荐的A组和B组核心抗结核药物,主要用于耐多药结核病的治疗且疗效较好.但近年来贝达喹啉和氯法齐明耐药的现象也时有报道,且二者存在交叉耐药的情况.本文综述了与结核分枝杆菌...     

Pharmacokinetics of pyrazinamide in children suffering from pulmonary tuberculosis.

SETTING: The Paediatric and Clinical Pharmacology unit of Maulana Azad Medical College and Associated Lok Nayak Hospital, New Delhi, India. OBJECTIVE: The pharmacokinetics of the anti-tuberculosis drug pyrazinamide was evaluated in 10 children aged 6 to 12 years suffering from pulmonary tuberculosis. METHODS: Serial blood samples were collected at 0, 1, 2, 4, 6, 12 and 24 hours after administration of pyrazinamide in a dose of 35 mg/kg. Serum pyrazinamide levels were analysed by spectrophotometry. RESULTS: The serum concentrations of pyrazinamide were above the minimum inhibitory concentration of 20 microg/ml of pyrazinamide for Mycobacterium tuberculosis up to 6 hours after drug administration in all the patients, and up to 12 hours in six patients. The mean peak serum concentration of pyrazinamide was 41.2+/-11.8 microg/ml, and this was attained in (Tmax) 2.9+/-1.7 hours. The elimination half life was 10.9+/-4.5 hours, the volume of distribution 16.1+/-10.9 litres and clearance 20.2+/-16.3 ml/minute. The corresponding mean residence time was 19.9+/-14.6 hours. CONCLUSION: The serum pyrazinamide concentrations achieved with a dose of 35 mg/kg were above the minimum inhibitory concentration of pyrazinamide for M. tuberculosis for over 6 hours after drug administration. It appears that the absorption and the clearance of pyrazinamide is slower, the elimination half life longer and the volume of distribution higher in children compared with the reported values in the adult population.     

Should isoniazid be used in retreatment of tuberculosis despite acquired isoniazid resistance?

The use of high-dose isoniazid in retreatment regimens for tuberculosis, despite acquired isoniazid resistance, could possibly improve therapeutic results if all or part of the organisms were resistant to only low concentrations of that drug. Furthermore, organisms resistant to low concentrations of isoniazid have been shown, on occasion, to be resistant to 2 of the retreatment drugs, ethionamide and pyrazinamide, whereas higher degrees of isoniazid resistance are associated with susceptibility to these drugs. Use of high-dose isoniazid might improve results in retreatment with ethionamide and pyrazinamide by eliminating any organisms with low degrees of isoniazid resistance that have associated ethionamide and pyrazinamide resistance. Two clinical trials concerning this topic have been reported. A controlled retreatment trial with various combinations of ethionamide, cycloserine, and pyrazinamide with and without conventional "low" doses of isoniazid (300 mg per day) showed no benefit when isoniazid was added. However, a noncontrolled trial using ethionamide and pyrazinamide with and without high doses of isoniazid, 1 to 1.5 g per day, showed marked benefit with the added isoniazid. In view of these conflicting data, the use of high-dose isoniazid in retreatment regimens needs further study, which could probably be carried out in the developing countries.     

Clinical and operational value of the extensively drug-resistant tuberculosis definition.

Currently, no information is available on the effect of resistance/susceptibility to first-line drugs different from isoniazid and rifampicin in determining the outcome of extensively drug-resistant tuberculosis (XDR-TB) patients, and whether being XDR-TB is a more accurate indicator of poor clinical outcome than being resistant to all first-line anti-tuberculosis (TB) drugs. To investigate this issue, a large series of multidrug-resistant TB (MDR-TB) and XDR-TB cases diagnosed in Estonia, Germany, Italy and the Russian Federation during the period 1999-2006 were analysed. Drug-susceptibility testing for first- and second-line anti-TB drugs, quality assurance and treatment delivery was performed according to World Health Organization recommendations in all study sites. Out of 4,583 culture-positive TB cases analysed, 361 (7.9%) were MDR and 64 (1.4%) were XDR. XDR-TB cases had a relative risk (RR) of 1.58 to have an unfavourable outcome compared with MDR-TB cases resistant to all first-line drugs (isoniazid, rifampicin ethambutol, streptomycin and, when tested, pyrazinamide), and an RR of 2.61 compared with "other" MDR-TB cases (those susceptible to at least one first-line anti-TB drug among ethambutol, pyrazinamide and streptomycin, regardless of resistance to the second-line drugs not defining XDR-TB). The emergence of extensively drug-resistant tuberculosis confirms that problems in tuberculosis management are still present in Europe. While waiting for new tools which will facilitate management of extensively drug-resistant tuberculosis, accessibility to quality diagnostic and treatment services should be urgently ensured and adequate public health policies should be rapidly implemented to prevent further development of drug resistance.