katG基因二个不同区域基因变异与结核分枝杆菌异烟肼耐药的相关性研究

目的 分析结核分枝杆菌katG基因2个不同区域的基因变异,并确定与INH耐药的相关性.方法 从痰液分离并鉴定结核分枝杆菌耐INH菌株53株,用PCR扩增katG基因的2个区域:区域1为第1位密码子至150位密码子,区域2为第227位密码子至470位密码子,并分别测序.结果 3株对INH耐药但2个区域都不发生突变.14株区域1存在突变,其中5株只在区域1存在突变,5株在区域1出现缺失突变,并呈现高度耐药.点突变是区域2的主要特点,特别是S315位密码子,60.4%(32/53)S315发生突变,最常见的是S315N(AGC→AAC)(18株);katG S315在高度INH耐药和低度INH耐药的结核分枝杆菌中突变率分别是84.4%(27/32)、15.6%(5/32),两组间差异有统计学意义(x2=30.25,P＜0.01).27株S315突变呈高度耐药,占S315突变菌株总数的84.4%,其余18株至少有一个非S315点突变的耐药株中高度耐药只有5株,占27.7%,两组间差异有统计学意义(x2=16.02,P＜0.01).对INH耐药的结核分枝杆菌区域2的突变发生率为84.9%.5株只在区域1存在突变,通过检测基因突变诊断INH耐药的检出率上升至94.3%.结论 S315突变发生率最高,突变类型和位置与耐药程度密切相关,分析区域1能使检出率提高9.4%.

Abstract：

Objective To analyze and compare the mutations in two different regions of the katG gene and study the relevance of Mycobacterium tuberculosis isoniazid-resistance and mutations in two different regions of the katG gene. Methods Fifty-three INH-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from Zhejiang province were analyzed. PCR was used to amplify two regions of the katG gene (GenBank accession no. U06258) region 1 (from codon 1 to codon 150) and region 2 ( from codon 227 to codon 470) which were then sequenced in order to identify mutations. Results Three strains resistant to INH did not contain mutations in either region. Fourteen strains carried mutations in region 1. Among them 5 strains barbered deletions, and showed high-level resistance to isoniazid. Five strains had mutations only in region 1. Region 2 carried multiple point mutations, especially at codon 315, and there were S315 N ( AGC→AAC ) substitution in 18 of those cases. The frequency of mutations in the katG S315 of high-level INH-resistance isolates ( 84. 4%, 27/32) was significantly higher than those of low-level INH-resistance isolates( 15.6%, 5/32 ), there was statistically significant difference (x2 = 30. 25, P ＜ 0. 01 ).katG S315 mutations in high-level INH-resistance frequency (84. 4%, 27/32) was significantly higher than the other mutations of katG gene of high-level INH-resistance frequency (27. 7%, 5/18 ), there was significant difference (x2 = 16.02, P ＜ 0. 01 ). The analysis of region 2 allowed INH resistance to be diagnosed in 84. 9% of the strains. Five strains had mutations only in region 1 ,which allowed the proportion of INH-resistant strains identified to be increased to 94. 3%. Conclusions The number of mutations at codon 315 was high. Mutation type and location closely related with drug resistance and the analysis of region 1 resulted in a 9. 4% increase in the rate at which mutations were identified.     

耐异烟肼结核杆菌Kat G463condon点突变的分子信标快速检测

目的应用分子信标探针检测结核杆菌耐异烟肼kat G463condon点突变,并与测序结果比较以验证该检测方法。方法运用软件对Beacondesigner设计,katG基因包含463condon的分子信标,建立其扩增体系及分子信标芯片检测方法;对扩增产物测序并作比较。结果通过CDC相机观测到结核标准株及耐异烟肼PCR产物与分子信标杂交后荧光信号区别明显;16株耐异烟肼组与10株H37RV标准株对照组荧光信号强度比较,耐异烟肼组463condon突变检出率为37％,分子信标检测方法与测序法符合率达93％。结论分子信标技术是一种具有高灵敏核酸点突变检测技术;分子信标芯片检测方法与测序法符合率较好。     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia

Elucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katG S315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosis isolates collected in Australia were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katG S315T substitution and fabG1-inhA -15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katG mutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katG codon 315, fabG1-inhA regulatory region, and inhA structural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katG S315T nor fabG1-inhA -15 C-to-T mutation clustered with any one genotypic group, but that the -15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315 and the fabG1-inhA regulatory region.     

Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea

The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p > 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, >1 microg/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, >0.2 to 1 microg/ml). The previously undescribed GGT-->GAT (Gly-->Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea.     

耐异烟肼结核杆菌KatG463condon点突变的分子信标快速检测

目的应用分子信标探针检测结核杆菌耐异烟肼kat G463condon点突变,并与测序结果比较以验证该检测方法。方法运用软件对Beacondesigner设计,katG基因包含463condon的分子信标,建立其扩增体系及分子信标芯片检测方法;对扩增产物测序并作比较。结果通过CDC相机观测到结核标准株及耐异烟肼PCR产物与分子信标杂交后荧光信号区别明显;16株耐异烟肼组与10株H37RV标准株对照组荧光信号强度比较,耐异烟肼组463condon突变检出率为37％,分子信标检测方法与测序法符合率达93％。结论分子信标技术是一种具有高灵敏核酸点突变检测技术;分子信标芯片检测方法与测序法符合率较好。     

基因芯片诊断耐多药结核病的临床多中心研究

目的 评估基因芯片法检测耐多药结核临床分离株的临床意义。方法采取分层抽样的方法分别从北京胸科医院、同济大学附属上海市肺科医院和广州市胸科医院保存的临床菌株库的耐药组和敏感组中,随机抽取利福平耐药株800株,异烟肼耐药株797株,耐多药株791株,利福平/异烟肼双敏感380株。用基因芯片法检测包括rpoB基因的511(T→C)、513(A→C,C→A)、516( G→T,A→T,A→G)、526( C→T,C→G,A→T,A→G)、531( C→T,C→G)、533( T→C)位点、katG的315(G→C,G→A)位点和inhA的-15(C→T)位点的耐药突变。以绝对浓度法药敏结果为金标准,计算基因芯片法的符合率、敏感度和特异度。同时对基因芯片法的核酸扩增产物进行测序,以验证基因芯片对核酸序列检测的准确性。结果以绝对浓度法药敏结果作为标准,基因芯片法检测利福平、异烟肼耐药和耐多药的符合率分别是93.7％(1 108/1 183)、83.8％ (994/1 186)、82.4％ (975/1 183)。检测利福平耐药的敏感度为92.0％ (733/797),特异度为97.2％( 375/386);检测异烟肼的敏感度为77.4％ (617/797),特异度为96.9％ (377/389);检测耐多药的敏感度为74.6％( 588/788),特异度为98.0％(387/395)。在利福平基因芯片检测为突变的菌株中,突变频率最高的位点是531( TCG),突变率为64.5％(480/744);在katG/ inhA突变菌株中,基因芯片检测为katG 315( AGC)单突变的为77.4％(487/629);且与测序结果基本一致,仅有5例菌株中不完全相符。其中1株异烟肼耐药菌基因芯片法检测为katG 315(G→C)突变,而测序结果为野生型,其余4株为基因芯片法未包含的突变类型。结论基因芯片法可快速可靠地检测结核临床分离株利福平和异烟肼的耐药性,有望在临床诊断中广泛应用。     

Experimental in vitro efficacy study on the interaction of epiroprim plus isoniazid against Mycobacterium tuberculosis

Thirty Mycobacterium tuberculosis strains (8: INH(R)/INH(R), 12: INH(R)/RIF(S), 10: INH(S)/RIF(S)) were examined against MICs of epiroprim (EPM) and isoniazid (INH) separately or in association. EPM alone proved to be insufficiently active against the various mycobacterial isolates (MIC > or =256 microg/ml). The observed average sensitivity to the association of EPM plus INH was, in contrast, considerably increased, as reflected by reduced MICs and lower percentages of resistant strains. MICs ranged between 16 and 32 microg/ml EPM and 2 and 4 microg/ml INH for INH(R) strains. All INH(S) isolates were inhibited by a concentration of 0.125 microg/ml EPM and 0.06 microg/ml INH. The fractional inhibitory concentration indices indicated an additive activity on INH(R)/RIF(R) strains and a synergistic activity on INH(R)/RIF(S) and INH(S)/RIF(S) strains. The synergistic activity of this drug association needs to be confirmed in an animal model.     

Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 microg/ml to 7, 7, 8.5, and 14 microg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 microg/ml or 28 microg/ml, respectively, to 3 microg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.     

Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol

In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 microg/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 microg/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 microg/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 microg/ml EMB (61 of 67=91.0%) than in isolates either susceptible to EMB or resistant to 2.5 microg/ml EMB (39 of 60=65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 microg/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 microg/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52=94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.     

Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.     

Contribution of kasA Analysis to Detection of Isoniazid-Resistant Mycobacterium tuberculosis in Singapore

Genotypic analysis of resistance to isoniazid (INH) in Mycobacterium tuberculosis is complex due to the various genes potentially involved. Mutations in ketoacyl acyl carrier protein synthase (encoded by kasA) were present in 16 of 160 (10%) INH-resistant isolates (R121K [n = 1], G269S [n = 3], G312S [n = 11], G387D [n = 1]). However, G312S was also present in 6 of 32 (19%) susceptible strains. kasA analysis contributed marginally to the performance of INH genotypic testing in Singapore. The significance of kasA polymorphisms in INH resistance should be carefully established.     

Isoniazid-induced transient high-level resistance in Mycobacterium tuberculosis

An American Type Culture Collection reference strain and eight clinical strains of Mycobacterium tuberculosis, all of which were susceptible to isoniazid (INH) (mean MIC, 0.06 mg/liter) and negative for the Ser315Thr katG mutation, were left in their BACTEC 12B vials (for use with the BACTEC 460-TB method) containing 0.1 mg of INH per liter for periods of up to 28 days after the completion of the antibiotic susceptibility test. Each eventually grew to levels compatible with those of INH-resistant strains. Successive passages in INH-containing BACTEC 12B vials and onto solid media showed that the resistance noted above was maintained. Successive passages of these M. tuberculosis strains in which INH resistance had been induced into BACTEC 12B vials or solid media containing stepwise increases in INH concentrations eventually yielded organisms resistant to 20 mg of INH per liter. Transfer of cells in which INH resistance had been induced to drug-free medium followed by repeated passages in that medium eventually yielded organisms whose susceptibility to INH was identical to that of the original parent strains. The cycle of induced INH resistance could be repeated with these now INH-susceptible cells. The use of M. tuberculosis identification probes and IS6110-based restriction fragment length polymorphism analyses of cultures throughout the induction of INH resistance and the reversal of resistance in drug-free medium eliminated the possibility that the culture was contaminated or that the initial specimen had a mixed type of infection. Induced high-level resistance to INH (20 mg/liter) could be reduced 100-fold with a subinhibitory concentration of reserpine but not with verapamil. These results collectively suggest that high-level resistance to INH can be induced in INH-susceptible M. tuberculosis strains by the induction of a reserpine-sensitive efflux mechanism.     

Detection of ciprofloxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing

A total of 27 strains of Campylobacter jejuni (24 clinical strains and three laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis with silver stain. Direct DNA sequencing of the polymerase chain reaction (PCR)-amplified DNA fragments confirmed the results obtained by non-RI SSCP analysis and revealed that in clinical strains high-level quinolone resistance [minimal inhibitory concentration (MIC) to ciprofloxacin ≥ 16 μg/ml] was closely associated with one type of single-point mutation at codon 86 (Thr-lle). Two strains with MICs of 8 and 1 μg/ml showed point mutations at codons 86 and 70, respectively. Furthermore, transitions at codon 119 of the gyrA QRDR were identified in 17 strains. Six types of bands were separated in a single electrophoretic step with silver stain within 2 hours after PCR amplification of the gyrA QRDR as follows: type I associated to mutation at codon 70 (Ala-Thr), type II to mutation at codon 90 (Asp-Asn), type III to variant with transition at 119, type IV to wild-type, type V to mutation at codon 86 (Thr-lle), and type VI to mutation at codon 86 (Thr-lle) and transition at codon 119. Using four DNA extracts from Cambylobacter coli organisms as templates for amplification of the gyrA QRDR, no PCR products were obtained. Non-RI SSCP was proved to be a simple, rapid, and useful screening method for detecting gyrA mutations associated with ciprofloxacin resistance in C. jejuni. © 1996 Wiley-Liss, Inc.     

2010-2012年浙江省衢州市涂阳肺结核患者结核菌株耐药状况分析

目的 分析结核分枝杆菌的耐药情况与耐药谱,为制订结核病控制对策提供依据。 方法 对2010-2012年期间在浙江省衢州市各个结核病定点医院就诊新发和复治涂阳患者进行结核杆菌培养,鉴定为结核分枝杆菌的菌株采用比例法进行6种抗结核药物[异烟肼(H)、利福平(R)、乙胺丁醇(E)、链霉素(S)、左氧氟沙星(OFX)、卡那霉素(KM)]耐药性测试。 结果 入选1169株结核分枝杆菌中,总体耐药率为22.80%,总体耐多药率为5.13%,广泛耐多药率为0.43%;单耐药谱位于前3位的是异烟肼、链霉素、氧氟沙星,多耐药谱位于前3位的是INH+SM、INH+SM+OFX、RFP +OFX;耐多药谱位于前3位的是INH+RFP+S、INH+RFP+SM+OFX和INH+RFP。 结论 耐药结核病已对衢州市结核病控制造成较大威胁,相关部门必须采取针对性的措施控制耐药结核病的流行。     

Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities.

Automated DNA sequencing was used to analyze the oxyR-ahpC region in 229 Mycobacterium tuberculosis complex isolates recently recovered from diseased humans and animals. The entire 1,221-bp region was studied in 118 isolates, and 111 other isolates were sequenced for oxyR, ahpC, or the 105-bp oxyR-ahpC intergenic region. The sample included isoniazid (INH)-susceptible and -resistant organisms in which the katG gene and inhA locus had previously been sequenced in their entirety to identify polymorphisms. A total of 16 polymorphic sites was identified, including 5 located in oxyR, 2 in ahpC, and 9 in the 105-bp intergenic region. All polymorphic sites located in the intergenic region, and the two missense substitutions identified in ahpC, occurred in INH-resistant organisms. In contrast, there was no preferential association of polymorphisms in oxyR, a pseudogene, with INH-resistant organisms. Surprisingly, most INH-resistant strains with KatG codon 315 substitutions that substantially reduce catalase-peroxidase activity and confer high MICs of INH lacked alterations in the ahpC gene or oxyR-ahpC intervening region. Taken together, the data are consistent with the hypothesis that some polymorphisms located in the ahpC-oxyR intergenic region are selected for after reduction in catalase or peroxidase activity attributable to katG alterations arising with INH therapy. These mutations are uncommon in recently recovered clinically significant organisms, and hence, there is no strict association with INH-resistant patient isolates. The ahpC compensatory mutations are apparently uncommon because strains with a KatG null phenotype are relatively rare among epidemiologically independent INH-resistant organisms.     

MPT64在MTB利福平和异烟肼耐药性检测方法中的应用

目的 建立一种以MPT64为指标的结核分枝杆菌（MTB）耐药性检测的快速方法,探讨MPT64在MTB利福平（RFP）和异烟肼（INH）耐药性检测中的应用。方法 应用胶体金免疫层析法（GICA）分别检测MTB敏感株和耐多药株在含（观察组）与不含（对照组）RFP、INH的Middlebrook 7H9液体培养基上培养3、7、10 d的培养液中的MPT64,通过凝胶成像仪白光源拍照进行条带灰度分析（Image Jab Software 3.0）,比较敏感株和耐多药株间的灰度比值的差异,依据受试者工作特征（ROC）曲线下面积（AUC）分析确定RFP、INH耐药的灰度比值界值;以比例法检测结果为金标准,评价新建方法的准确度、灵敏度、特异度、阳性预测值和阴性预测值。结果 对照组中,随着培养时间的延长,12株MTB敏感株和11株耐多药株的灰度比值增大;观察组中,敏感株在不同的培养时间的灰度比值没有显著变化,而耐多药株则明显增大。3、7、10 d敏感菌与耐多药菌在药物培养基中MPT64检测灰度比值差异均有统计学意义（P均< 0.05）。依据观察组灰度比值分别绘制以MPT64指示的RFP和INH耐药性检测的ROC曲线,两者的3 d AUC分别为0.84和0.77,灰度比值界值均为0.05;7、10 d AUC均为1.00,7 d的灰度比值界值分别为0.23与0.20;10 d的灰度比值界值分别为0.49和0.48。GICA检测MPT64用于34株MTB的RFP、INH耐药性鉴定的准确度分别为97%和94%,灵敏度分别为93%和86%,特异度均为100%,阳性预测值均为100%,阴性预测值分别为95%和91%。结论 GICA检测MPT64于MTB培养7 d时能准确检测抗结核药物RFP、INH耐药性,MPT64可作为MTB药敏试验的有效检测指标。     

C1 inhibitor suppresses the endotoxic activity of a wide range of lipopolysaccharides and interacts with live gram-negative bacteria

Human C1 inhibitor (C1INH) prevents endotoxin shock via a direct interaction with Gram-negative bacterial lipopolysaccharide (LPS) and improves survival in animal models of sepsis. In this report, we further characterize the interaction of C1INH with LPS and whole live bacteria. We investigate C1INH interactions with LPS from five different strains of Gram-negative enteric bacteria known to participate in the pathogenesis of human sepsis. Treatment with C1INH improved survival in mice with endotoxin shock induced by LPS from Salmonella enterica serovar typhimurium as previously shown, as well as LPS from Escherichia coli O55:B5 and Pseudomonas aeruginosa, and a trend to improved survival was observed when Klebsiella pneumoniae and Serratia marcescens LPS were used. Enzyme-linked immunosorbent assay and native polyacrylamide gel electrophoresis shift experiments demonstrated a direct interaction of C1INH with LPS from all the strains studied. The binding of both native and reactive center-cleaved, inactive C1INH results in inhibition of LPS-induced proinflammatory cytokine production. Furthermore, we demonstrate the ability of C1INH to bind at the surface of only a restricted number of whole live Gram-negative bacteria as well as mutant bacteria expressing a truncated LPS lacking the O-antigen. These data reveal the interaction of C1INH with a wide range of enteric bacterial LPS and strongly suggest that the interaction between C1INH and the surface of Gram-negative microorganisms is determined by the length of the polysaccharide chain of the endotoxin molecule.     

Mutations in katG gene sequences in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis are rare.

In this study, a battery of oligonucleotides was directed toward the katG gene and PCR-single-stranded conformation polymorphism (SSCP) analysis was used to search for katG gene deviations in clinical isolates of Mycobacterium tuberculosis from different geographical regions. Since a complete deletion of the katG gene was not found, it is suggested that deletion is not a major mechanism of isoniazid (isonicotinic acid hydrazide; INH) resistance in these isolates. However, 7 of 39 isolates (4 of 25 from South Africa and 3 of 14 from other geographical regions) showed mobility shifts by SSCP analysis, suggesting aberrations in the katG gene. Direct sequence analysis confirmed that the mobility shifts were due to Thr-275-->Ala (Thr275Ala), Arg409Ala, Arg463Leu, and Asp695Ala mutations and a 12-bp deletion in the 5' region of the katG gene. Mutations at codons 275, 463, and 695 created altered restriction sites for HhaI, MspI, and HaeIII, respectively, and sequence results, supported by restriction fragment length polymorphism analysis, suggested that the PCR-SSCP procedure is a good indicator of mutations in PCR-amplified fragments. Identical mutations at codons 463 and 275 were found in isolates from different geographical regions. This may suggest a common evolutionary event, but one of the control isolates (susceptible to INH [3%; n = 30]) also had a mutation at codon 463. The results suggest that variations in the katG coding gene sequences of INH-resistant isolates of M. tuberculosis are infrequent and that defects in other regions of the M. tuberculosis genome are of equal or greater importance in contributing to the acquisition of resistance to INH.     

Relationship between genetic alteration of the rpsL gene and streptomycin susceptibility of Mycobacterium tuberculosis in Japan

We have investigated the effect of genetic alterations in the rpsL gene on the MICs of streptomycin for Mycobacterium tuberculosis strains. Direct DNA sequencing showed a point mutation in 23/121 strains; in 18 strains the mutation was associated with an amino acid change. The MICs of streptomycin in 22 out of 23 point-mutated strains were > or = 256 mg/L. Restriction fragment length polymorphism (RFLP) analysis showed mutations at codon 43 in all 18 strains with point mutations in the same codon. Our results suggest that both RFLP and base sequencing analysis of the rpsL gene are useful for the rapid prediction of highly streptomycin-resistant strains of M. tuberculosis.