结核分枝杆菌Rv3737抑制巨噬细胞自噬促进耻垢分枝杆菌在巨噬细胞内的存活

目的 了解结核分枝杆菌跨膜蛋白Rv3737对耻垢分枝杆菌感染巨噬细胞自噬的作用。方法 通过PCR扩增结核分枝杆菌Rv3737目的基因，T4连接酶连接Rv3737及穿梭质粒pMV261，构建pMV261-Rv3737，双酶切及测序验证pMV261-Rv3737，通过电转将pMV261-Rv3737及pMV261-Vector电转至耻垢分枝杆菌感受态细胞中以构建过表达Rv3737的耻垢分枝杆菌菌株（MS::pMV261-Rv3737）和对照菌株（MS::pMV261）,PCR及Western blot验证过表达Rv3737的耻垢分枝杆菌菌株。利用分光光度计检测OD600评估过表达Rv3737对耻垢分枝杆菌体外生长的影响。将MS::pMV261-Rv3737及MS::pMV261感染巨噬细胞，通过菌落形成单位（CFU）计数检测耻垢分枝杆菌在巨噬细胞内的存活，用Western blot及激光共聚焦检测自噬相关蛋白LC3II的变化。结果 Rv3737不影响耻垢分枝杆菌的体外生长，但促进耻垢分枝杆菌在巨噬细胞内的存活，过表达Rv3737明显抑制LC3II的表达，激光共聚焦结果显示过表达Rv3737...     

噬菌体生物扩增法检测涂阴肺结核患者痰标本的临床应用价值

目的 探讨噬菌体生物扩增法对涂阴肺结核临床诊断的应用价值.方法 对110例涂阴肺结核和20例非结核性其他肺部疾病患者的痰标本进行噬菌体生物扩增法检测,并同时进行分枝杆菌罗氏培养.结果 110例涂阴痰标本中46例噬菌体生物扩增法阳性(41.82%);27例罗氏培养阳性(24.55%).结论 噬蒲体生物扩增法检测涂阴痰标本中结核分枝杆菌只需2天时间,操作简便、灵敏度高、特异性强,可作为诊断涂阴肺结核的一种检测方法.     

病原生物学检验与临床噬菌体生物扩增法快速检测结核分枝杆

孕妇B型链球菌感染率及药物敏感性分析，粪肠球菌与屎肠球菌药敏表型和耐药基因的比较，噬菌体生物扩增法、DNA序列分析及单链构象多态性分析检测结核分枝杆菌耐药性，噬菌体生物扩增法快速检测结核分枝杆菌标准化研究，噬菌体生物扩增法快速检测结核分枝杆菌标准化研究，噬菌体生物扩增法快速测定结核分枝杆菌利福平耐药性……     

噬菌体生物扩增法在白带标本结核分枝杆菌检测中的应用价值

目的 探讨噬菌体生物扩增法在检测白带标本结核分枝杆菌中的应用价值.方法 应用噬菌体生物扩增法检测30例女性生殖器结核疑似患者白带标本,以改良罗氏培养法为对照,评价噬菌体生物扩增法检测白带标本中结核分枝杆菌的应用价值.结果 30例白带标本中,噬菌体生物扩增法检测阳性率为20.00%(6/30),改良罗氏培养法阳性率为13.33%(4/30),2种方法检测结果阳性符合率为75.00%(3/4),阴性符合率为88.46%(23/26);总符合率为86.67%(26/30).2种方法检测结果比较差异无统计学意义(χ2=0.25,P>0.05).结论噬菌体生物扩增法检测白带标本中的结核分枝杆菌具有较高的特异度和准确度,适于临床推广应用.     

体液结核分枝杆菌三种检测方法临床应用价值

目的比较体液结核分枝杆菌三种方法检测结果,探究其临床应用价值。方法应用EL ISA法检测结核分枝杆菌抗体、结核分枝杆菌噬菌体生物扩增法、直接涂片抗酸染色法分别对临床可疑结核感染体液标本进行检测。结果结核分枝杆菌噬菌体生物扩增法、结核抗体EL ISA法检测与直接涂片抗酸染色法有统计学差别(P<0.05),结核分枝杆菌噬菌体生物扩增法与结核抗体EL ISA法检测二者无显著性差异(P>0.05),噬菌体生物扩增法与结核抗体EL ISA法阳性符合率为80.3%,阴性符合率为97.2%。结论结核抗体EL ISA法检测和结核分枝杆菌噬菌体扩增法比直接涂片法阳性率高,具有一定的临床应用价值。     

噬菌体生物扩增法检测涂阴肺结核患者痰标本的临床应用价值

目的探讨噬菌体生物扩增法对涂阴肺结核临床诊断的应用价值。方法对110例涂阴肺结核和20例非结核性其他肺部疾病患者的痰标本进行噬菌体生物扩增法检测,并同时进行分枝杆菌罗氏培养。结果110例涂阴痰标本中46例噬菌体生物扩增法阳性（41．82％）;27例罗氏培养阳性（24．55％）。结论噬菌体生物扩增法检测涂阴痰标本中结核分枝杆菌只需2天时间,操作简便、灵敏度高、特异性强,可作为诊断涂阴肺结核的一种检测方法。     

噬菌体生物扩增法检测结核性胸膜炎患者胸水临床应用研究

目的 评价噬菌体生物扩增法检测胸水中结核分枝杆菌对诊断结核性胸膜炎的临床应用价值.方法 采用噬菌体生物扩增法技术对73例结核性胸膜炎患者,20例非结核性胸膜炎患者胸水中的结核分枝杆菌进行检测.同份标本同时进行涂片抗酸染色、罗氏培养.结果 73例结核性胸膜炎胸水.噬菌体生物扩增法阳性率为49.3%(36/73)、涂片抗酸染色阳性率为2.7%(2/731、罗氏培养阳性率为4.1%(3/73).20例非结核性胸膜炎,噬菌体生物扩增法、涂片抗酸染色、罗氏培养都未检出阳性.结论 噬菌体生物扩增法检测胸水中结核分枝杆菌只需2天时间,操作简便、灵敏度高、特异性强,不需要特殊仪器设备,可作为诊断结核性胸膜炎的一种好方法 .     

噬菌体生物扩增法检测结核性胸膜炎患者胸水临床应用研究

目的评价噬菌体生物扩增法检测胸水中结核分枝杆菌对诊断结核性胸膜炎的临床应用价值。方法采用噬菌体生物扩增法技术对73例结核性胸膜炎患者．20例非结核性胸膜炎患者胸水中的结核分枝杆菌进行检测．同份标本同时进行涂片抗酸染色、罗氏培养。结果73例结核性胸膜炎胸水,噬菌体生物扩增法阳性率为49．3％（36／73）、涂片抗酸染色阳性率为2.7％（2/73）、罗氏培养阳性率为4．1％（3／73）。20例非结核性胸膜炎。噬菌体生物扩增法、涂片抗酸染色、罗氏培养都未检出阳性。结论噬菌体生物扩增法检测胸水中结核分枝杆菌只需2天时间,操作简便、灵敏度高、特异性强,不需要特殊仪器设备,可作为诊断结核性胸膜炎的一种好方法．     

噬菌体法直接检测痰标本中结核分枝杆菌利福平耐药性的研究

目的 应用噬菌体生物扩增(PhaB)法直接检测痰标本中结核分枝杆菌(MTB)利福平(RFP)耐药性,探讨其在临床MTB耐药性测定中的应用价值.方法 应用PhaB法检测临床40例痰标本中MTB对利福平(RFP)的耐药性,并与绝对浓度法检测结果 进行比较.结果 加例痰标本PhaB法与绝对浓度法RFP耐药性检测均阳性25例,均阴性7例,两法不相符8例.以绝对浓度法检测结果 为判断标准,则PhaB法RFP耐药性检测的敏感性、特异性、准确性分别是78.13%、87.50%、80.00%.结论 PhaB法检测痰标本中结核分枝杆菌(MTB)对RFP的耐药性具有较高的敏感性和特异性,只需要3天时间,简便快速,不需要特殊仪器设备,费用低廉,可作为MTB耐药性检测的快速筛选方法 .     

噬菌体法直接检测痰中结核分枝杆菌敏感性的研究

目的探讨噬菌体生物扩增法结合痰涂片在快速测定结核药敏中的可行性和实用性。方法收集痰厚涂片抗酸染色3＋阳性标本与一定浓度的利福平作用后，加入结核分枝杆菌噬菌体，观察噬菌体斑数量，来测定对利福平的敏感性，同时以改良罗氏法作结核药敏对照。结果从收到标本到药敏报告需时仅3d，93份标本中85份噬菌体生物扩增药敏试验成功，污染2份，6份失败，有效率91．4％（85／93），与改良罗氏法药敏试验比较，敏感性96．9％（31／32），特异性92．5％（49／53），报告时间缩短了近20倍。结论应用噬菌体法直接测定痰中结核分枝杆菌利福平敏感试验，快速、敏感、特异性高，适宜基层正常开展。     

Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD(+)-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD(+)-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD(+)-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD(+)-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.     

Effects of antituberculosis and antileprosy drugs on mycobacteriophage D29 growth.

The minimal inhibitory concentrations of antituberculosis and antileprosy drugs were determined for Mycobacterium aurum. The concentrations that reduced the final yield of bacteriophage D29R1 by 50% and the time during the replication cycle at which the drugs completely inhibited phage production were estimated. THe 50% inhibitory concentration/minimal inhibitory concentration ratios were close to 1.0 for clofazimine, colistin, rifampin, and streptomycin; these ratios were high for dapsone (diaminodiphenylsulfone) and isoniazid. Ethambutol (minimal inhibitory concentration, 1.0 micrograms/ml) was without effect on viral growth.     

Phenoxazine compounds produced by the reactions with bovine hemoglobin show antimicrobial activity against non-tuberculosis mycobacteria

We studied the anti-microbial effects of phenoxazines produced by the reaction of o-aminophenol or its derivatives with bovine hemoglobin, on seven species of mycobacteria such as Mycobacterium tuberculosis, Mycobacterium marinum, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium fortuitum, Mycobacterium kansasii and Mycobacterium smegmatis and some bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Listeria monocytogeneses. These phenoxazines, including 2-amino-4, 4alpha-dihydro-4alpha, 7-dimethyl-3H-phenoxzine-3-one (Phx-1), 3-amino-1, 4alpha-dihydro-4alpha, 8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3), prevented the proliferation of four non-tuberculosis mycobacteria including M. scrofulaceum, M. kansasii, M. marinum, and M. intracellulare dose-dependently, though the inhibitory effects of these phenoxazines differed according to the species of mycobacteria. However these phenoxazines failed to prevent the proliferation of M. tuberculosis, M. fortuitum, and M. smegmatis, and the concerned bacteria other than mycobacteria. The present results may contribute to development of novel antibiotics against non-tuberculolsis mycobacteria.     

Genomic approach to identifying the putative target of and mechanisms of resistance to mefloquine in mycobacteria

The emergence of mycobacterial resistance to multiple antimicrobials emphasizes the need for new compounds. The antimycobacterial activity of mefloquine has been recently described. Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium tuberculosis are susceptible to mefloquine in vitro, and activity was evidenced in vivo against M. avium. Attempts to obtain resistant mutants by both in vitro and in vivo selection have failed. To identify mycobacterial genes regulated in response to mefloquine, we employed DNA microarray and green fluorescent protein (GFP) promoter library techniques. Following mefloquine treatment, RNA was harvested from M. tuberculosis H37Rv, labeled with 32P, and hybridized against a DNA array. Exposure to 4x MIC resulted in a significant stress response, while exposure to a subinhibitory concentration of mefloquine triggered the expression of genes coding for enzymes involved in fatty acid synthesis, the metabolic pathway, and transport across the membrane and other proteins of unknown function. Evaluation of gene expression using an M. avium GFP promoter library exposed to subinhibitory concentrations of mefloquine revealed more than threefold upregulation of 24 genes. To complement the microarray results, we constructed an M. avium genomic library under the control of a strong sigma-70 (G13) promoter in M. smegmatis. Resistant clones were selected in 32 microg/ml of mefloquine (wild-type M. avium, M. tuberculosis, and M. smegmatis are inhibited by 8 microg/ml), and the M. avium genes associated with M. smegmatis resistant to mefloquine were sequenced. Two groups of genes were identified: one affecting membrane transport and one gene that apparently is involved in regulation of cellular replication.     

AsnB is involved in natural resistance of Mycobacterium smegmatis to multiple drugs

Mycobacteria are naturally resistant to most common antibiotics and chemotherapeutic agents. The underlying molecular mechanisms are not fully understood. In this paper, we describe a hypersensitive mutant of Mycobacterium smegmatis, MS 2-39, which was isolated by screening for transposon insertion mutants of M. smegmatis mc2155 that exhibit increased sensitivity to rifampin, erythromycin, or novobiocin. The mutant MS 2-39 exhibited increased sensitivity to all three of the above mentioned antibiotics as well as fusidic acid, but its sensitivity to other antibiotics, including isoniazid, ethambutol, streptomycin, chloramphenicol, norfloxacin, tetracycline, and beta-lactams, remained unchanged. Uptake experiment with hydrophobic agents and cell wall lipid analysis suggest that the mutant cell wall is normal. The transposon insertion was localized within the asnB gene, which is predicted to encode a glutamine-dependent asparagine synthetase. Transformation of the mutant with wild-type asnB of mc2155 or asnB of Mycobacterium tuberculosis complemented the drug sensitivity phenotype. These results suggest that AsnB plays a role in the natural resistance of mycobacteria.     

鸟分枝杆菌脂类诱导的人肺泡巨噬细胞抗结核免疫反应性的改变及机制的初步研究

目的研究鸟分枝杆菌脂类(MALs)对人肺泡巨噬细胞抗结核免疫反应性的影响。方法平板计数法评价MALs对人肺泡巨噬细胞杀结核菌效应的影响;ELISA检测肿瘤坏死因子-α(TNF-α)水平;Greisse法检测一氧化氮(NO)水平。结果MALs减弱了人肺泡巨噬细胞对牛结核分枝杆菌(BCG)的杀灭。MALs刺激后,结核分枝杆菌纯蛋白衍生物(PPD)和干扰素γ(IFN-γ)诱导的人肺泡巨噬细胞TNF-α和NO的分泌均有所下降。结论 MALs减弱了人肺泡巨噬细胞对结核分枝杆菌的免疫反应性。     

海南省肺部感染患者非结核分枝杆菌的菌种鉴定分析

目的 通过对海南省肺部感染患者非结核分枝杆菌（NTM）进行菌种鉴定,探讨海南省NTM肺部感染菌种的类型及人群分布情况。方法 收集2016年7月至2021年6月期间就诊于海南医学院第二附属医院疑似肺结核患者的呼吸道标本进行分枝杆菌培养,对培养阳性标本进行对硝基苯甲酸（PNB）/噻吩-2-羧酸肼（TCH）培养菌型初步鉴定,采用DNA微阵列芯片技术进行分枝杆菌菌种鉴定,无法确定菌种的菌株进一步采用基因测序法鉴定。结果 共收集8 507例疑似肺结核患者的呼吸道标本,剔除重复病例后,有318例经PNB/TCH培养初步鉴定为NTM,315例经DNA微阵列芯片技术和热休克蛋白65(hsp65)基因测序鉴定为NTM。其中308例患者为单一感染模式,6例MTB+NTM和1例2种不同NTM的混合感染模式。快速生长分枝杆菌128株占40.5%,以龟/脓肿分枝杆菌为主（占32.9%）;缓慢生长分枝杆菌188株占59.5%,以胞内分枝杆菌为主（占39.6%）。女性患者多于男性且好发于中老年人,男女性别比为0.79∶1。男女性患者年龄分布差异有统计学意义（Z=2.200,P<0.05）,>40岁的患者...     

Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis

The characteristics of the accumulation of 2 mg/L [(14)C]rifampicin by wild-type strains of Mycobacterium aurum (A(+)), Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis (H37Rv) were determined. After 10 min exposure M. aurum had accumulated 220 ng rifampicin/mg cells, M. smegmatis had accumulated 120 ng rifampicin/mg cells and M. tuberculosis had accumulated 154 ng rifampicin/mg cells. A steady-state concentration (SSC) of rifampicin was accumulated rapidly by M. aurum and M. tuberculosis within minutes of drug exposure, unlike M. smegmatis, which accumulated rifampicin more slowly. With an increase in the concentration of rifampicin from 0.12 mg/L to 2 mg/L there was an increase in the concentration of rifampicin accumulated by M. tuberculosis, with no detectable loss of viability over the 20 min of the accumulation experiment. With an increase in temperature there was also an increase in the concentration of rifampicin accumulated by M. tuberculosis; between 15 and 30 degrees C the increase was linear. For all three species sub-inhibitory concentrations of ethambutol increased the concentration of rifampicin accumulated. However, both growth and accumulation of rifampicin were lower in the presence of 0.05% Tween 80. Accumulation of rifampicin by M. smegmatis was unaffected by the presence of the proton motive force inhibitor, 2,4-dinitrophenol (1 mM), whether added before or after the addition of rifampicin to the mycobacterial culture. For all three species, the Gram-positive bacterial efflux inhibitor reserpine (20 mg/L) slightly increased the SSC of rifampicin, but the increase was not statistically significant. Addition of glucose to energize a putative efflux pump had little effect on the accumulation of rifampicin in the presence or absence of reserpine for M. tuberculosis; however, for M. aurum and M. smegmatis the reserpine effect was abolished by the addition of glucose. These data suggest that rifampicin may be removed from wild-type mycobacteria by efflux, but that the pump(s) is expressed at low level.     

Rapid drug susceptibility testing of mycobacteria in tissue culture medium

Mycobacteria grew rapidly in FST medium (tissue culture medium F12 supplemented with 5% serum and 0.05% Tween 80). Growth of Mycobacterium tuberculosis and other niacin-negative mycobacteria in flat-bottomed, 96-well tissue culture plates was estimable by the naked eye within 3 to 5 days when mycobacteria were inoculated at 0.1 to 0.01 Klett units (5 X 10(4) to 0.5 X 10(4) CFU) per well. Spontaneous resistant variants of M. tuberculosis to isoniazid arose and grew in the medium within 2 weeks of culture. A total of 56 clinically isolated mycobacteria whose drug susceptibilities had been tested by a conventional method were tested in FST medium for minimal inhibitory concentrations of streptomycin, ethambutol, rifampin, and isoniazid. The minimal inhibitory concentrations of these drugs in FST medium strictly coincided with the drug susceptibility patterns obtained by a conventional method, except for 3 of 224 estimations.     

预防性MPT64和ESAT6疫苗保护力的评价

目的利用BACTEC-960仪分析动物试验模型主要脏器的结核分枝杆菌生长情况,分析结核分枝杆菌MPT64和ESAT6 DNA疫苗预防结核病的效力。方法将BALB/C小鼠随机分成五组,分别用A组生理盐水、B组载体质粒、C组卡介苗、D组重组质粒JW4303-MPT64DNA、E组JW4303-ESAT6DNA免疫9周后,用H37RV结核分枝杆菌强毒株攻击小鼠。分别在攻击一个月后和二个月后观察结果。结果 C、D、E组比B组生长天数总体向后推迟,生长时间明显集中,生长曲峰强度比B组明显减弱,C组肝脏中的感染率比D、E组有所下降。D组肺脏中感染率下降明显。D、E组鼠脾淋巴细胞转化率较C组高,B组明显降低。结论与B组比,小鼠免疫力的短期预防是有所增强,其中卡介苗前期效果明显;MPT64、ESAT6是持续效果好。