结核分枝杆菌对吡嗪酰胺耐药的相关基因及耐药机制研究进展

吡嗪酰胺是结核病最重要的一线治疗药物之一,但结核分枝杆菌对其耐药性在逐年增长,目前对于结核分枝杆菌吡嗪酰胺的耐药基因的分子机制尚无定论。作者对结核分枝杆菌吡嗪酰胺的相关耐药基因pncA、rpsA、panD、fadD2及FAS-I的研究进展进行综述。     

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。     

125株耐多药结核分枝杆菌的吡嗪酰胺耐药及pncA基因突变分析

目的 了解125株耐多药结核分枝杆菌对吡嗪酰胺(PZA)的耐药水平和pncA基因的突变特征,为PZA耐药的快速诊断提供依据。方法 通过简单随机抽样方法选取2010—2019年福建省9个耐药监测点的125株耐多药菌株,采用BACTEC MGIT 960液体培养进行PZA药物敏感性试验(简称“药敏试验”),通过PCR扩增pncA目的基因,分析耐多药菌株对PZA的耐药性及pncA基因突变情况。结果 50株耐多药菌株对PZA耐药,PZA耐药率为40.0%(50/125)。男性患者PZA耐药率为39.0%(37/95),女性患者PZA耐药率为43.3%(13/30),差异无统计学意义(χ²=0.183,P=0.669);15~25岁、26~45岁、46~60岁、61~80岁患者PZA耐药率分别为58.3%(7/12)、 32.7%(16/49)、39.5%(17/43)、 47.6%(10/21),PZA耐药率在年龄组之间的差异无统计学意义(χ²=3.294,P=0.348);初治患者PZA耐药率为41.5%(21/65),复治患者PZA耐药率为38.3%(23/60),差异无统计学意义(χ²=0.134,P=0.715)。30株PZA耐药菌株检出pncA基因突变,突变类型22种,突变检出率为60.0%(30/50);PZA耐药株的pncA基因突变检出率[60.0%(30/50)]高于PZA敏感株的pncA基因突变检出率[5.3%(4/75)],差异有统计学意义(χ²=45.276,P=0.000)。结论 福建省耐多药结核分枝杆菌的PZA耐药率较高,且 pncA基因突变检出率较高,临床上应关注PZA的耐药问题。     

福建省耐多药结核分枝杆菌对吡嗪酰胺的耐药水平与基因型特征分析

目的 了解福建省耐多药结核分枝杆菌(MDR-MTB)对吡嗪酰胺(PZA)的耐药水平及基因型分布情况,分析PZA耐药水平及基因型之间的关系。方法 收集2007—2008年和2010—2011年福建省耐药监测点共119株MDR-MTB临床分离株,采用WHO推荐的方法进行耐药性检测,应用间隔区寡核苷酸分型技术(Spoligotyping)进行基因分型,对分型结果上传至SpolDB4.0数据库,并进行聚类分析。结果 119株MDR-MTB菌株对PZA的耐药率为36.97%(44/119)。耐PZA菌株对链霉素(Sm)的耐药率为70.45%(31/44),高于PZA敏感菌株对Sm的耐药率[50.67%(38/75)],差异有统计学意义(χ ²=4.45,P=0.035);耐PZA菌株对乙胺丁醇(EMB)的耐药率为81.82%(36/44),高于PZA敏感菌株对EMB的耐药率[52.00%(39/75)],差异有统计学意义(χ ²=1.58,P=0.001)。北京基因型菌株占67.23%(80/119);非北京基因型菌株占32.77%(39/119),共有16个基因型(含8个新发现的基因型),其中常见的是H家族(含H和H3)和T家族(含T1、T2、T3和T5)。北京基因型菌株对PZA的耐药率(38.75%,31/80)与非北京基因型菌株对PZA的耐药率(33.33%,13/39)间差异无统计学意义(χ ²=0.33,P=0.566)。 结论 福建省耐多药结核病患者临床分离株对PZA耐药的问题应引起重视,对PZA耐药与对Sm和EMB耐药有关;MDR-MTB菌株主要为北京基因型,北京基因型与非北京基因型菌株对PZA的耐药率未见差异。     

吡嗪酰胺耐药性结核分枝杆菌的噬菌体检测技术研究

目的 建立噬菌体生物扩增法（PhaB）快速测定吡嗪酰胺耐药性，并探讨其在结核分枝杆菌吡嗪酰胺耐药性测定中的应用价值。方法 应用建立的PhaB测定108株结核分枝杆菌临床分离株的吡嗪酰胺耐药性，并与绝对浓度法的药敏结果进行比较，对不符合的菌株进行最低抑菌浓度（MIC）测定和序列测定分析。结果 PhaB检测吡嗪酰胺耐药性的最佳测定条件为pH值5．5、药物浓度200μg／ml、37℃作用48h。用绝对浓度法检测108株结核分枝杆菌临床分离株，其中吡嗪酰胺敏感33株、耐药75株；用PhaB检测该108株结核分枝杆菌临床分离株，若以噬菌斑减少95％为判断标准。则吡嗪酰胺敏感32株，耐药76株。2种方法检测均为敏感的为28株，均为耐药的为71株，符合率为91．7％；2种方法检测结果不符的为9株，其中5株的PhaB结果与MIC结果相符，4株的结果不符，测序结果表明9株中有7株的PhaB结果与测序结果相符。如以绝对浓度法药敏结果为判断标准，PhaB检测吡嗪酰胺耐药性的敏感性为94．7％，特异性为84．8％，阳性预测值为93．4％，阴性预测值为87．5％，准确性为91．7％。结论 PhaB测定吡嗪酰胺耐药性简便、快速，3d即可获得药敏结果，可作为吡嗪酰胺耐药性的快速筛选方法。     

Fluoroquinolone and pyrazinamide resistance in multidrug-resistant tuberculosis

In a study performed in Cambodia, a higher number of tuberculosis (TB) strains with mutations in the pncA gene associated with pyrazinamide resistance (PZA-R) was found in fluoroquinolone-resistant (FQ-R) multidrug-resistant (MDR) strains (93%), compared with 47% in MDR and 3% in non-MDR strains. This emphasises the need for easy and rapid tests for identification of PZA-R for efficient treatment of MDR-TB.     

颗粒显色指示技术快速检测结核分枝杆菌对吡嗪酰胺耐药性的价值

目的 探讨颗粒显色指示技术快速测定结核分枝杆菌（Mycobacterium tuberculosis,Mtb）对吡嗪酰胺耐药性的临床应用价值。方法 应用颗粒显色指示法测定102株Mtb对吡嗪酰胺耐药性,并与美国BD公司BACTEC MGIT-960分枝杆菌检测系统结果进行比较。 结果 颗粒显色指示法测定102株Mtb临床分离株,结果对吡嗪酰胺敏感70株、耐药32株,BACTEC MGIT-960法测定结果敏感69株、耐药33株;两法测定均敏感67株、均耐药30株。如以BACTEC MGIT-960法药敏结果为判断标准,则颗粒显色指示法测定吡嗪酰胺耐药性的敏感度为90.9%(30/33),特异度为97.1%（67/69),阳性预测值为93.8%(30/32),阴性预测值为95.7%(67/70),准确性为95.1%(97/102)。结论 颗粒显色指示技术快速测定Mtb吡嗪酰胺耐药性简便快速,操作不需特殊仪器设备,具有很高的临床应用价值。     

三种方法检测结核分枝杆菌吡嗪酰胺耐药性的比较

目的 探讨绝对浓度法、液体培养基最低抑菌浓度（MIC）法和噬菌体生物扩增（PhaB）法检测结核分枝杆菌（MTB）吡嗪酰胺（PZA）耐药性的临床应用价值。方法 应用3种方法同时检测90株MTB临床分离株PZA耐药性，并比较检测结果的敏感度、特异度、阳性和阴性预测值及准确度。结果 90株MTB临床分离株用绝对浓度法、液体培养基MIC法和PhaB法3种方法分别测得21株、33株、22株敏感株和69株、57株、68株耐药株。若以绝对浓度法测定结果为判断标准，则PhaB法检测PZA耐药性的敏感度、特异度、阳性和阴性预测值及准确度分别为94．2％、85，7％、95．6％、81．8％、92．2％；若以液体培养基MIC法测定结果为判断标准，则PhaB法检测的敏感度、特异度、阳性和阴性预测值及准确度分别为98．2％、63．6％、82．4％、95．5％、85．6％；若以绝对浓度法和液体培养基MIC法测定结果为判断标准，则PhaB法检测的敏感度、特异度、阳性和阴性预测值及准确度分别为96．4％、90．0％、96．4％、90．0％、94．7％。结论 绝对浓度法尽管是目前国内常用的方法，但影响因素较多；液体培养基MIC法测定虽能提早报告药物敏感结果，但检测条件尚需优化；PhaB法检测快速、简便、安全，有一定的应用价值。     

Characterization of pyrazinamide and ofloxacin resistance among drug resistant Mycobacterium tuberculosis isolates from Singapore.

OBJECTIVES: To evaluate rapid molecular approaches for the detection of pyrazinamide (PZA) and ofloxacin resistance, by screening 100 known drug-resistant Mycobacterium tuberculosis isolates. METHODS: Mycobacterium tuberculosis isolates were tested for phenotypic resistance to pyrazinamide and ofloxacin using the BACTEC 460 radiometric method and the E-test, respectively. Mutation screening was done by amplifying the pncA, gyrA, and gyrB genes by the polymerase chain reaction (PCR) and direct automated sequencing. RESULTS: Twelve isolates were PZA-resistant and 8 of 12 (66.7%) isolates had missense mutations or deletions at the pncA gene, suggesting that mutation or deletion at the pncA gene is the major molecular mechanism of PZA resistance among the Singaporean isolates. Using the E-test, 48 isolates were resistant to ofloxacin, with minimum inhibitory concentrations of 4 microg/mL or higher. No mutations were observed at the quinolone resistance-determining region (QRDR) of gyrA in all isolates. At the QRDR of gyrB, mutations were present in 1 of 48 ofloxacin-resistant isolates and 0 of 19 ofloxacin-susceptible isolates. CONCLUSIONS: In Singapore, genotypic analysis of resistance to PZA and ofloxacin is inadequate and should be complemented by conventional methods.     

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.     

Mycobacterium tuberculosis gene Rv2136c is dispensable for acid resistance and virulence in mice

The gene Rv2136c is annotated to encode the Mycobacterium tuberculosis (Mtb) homolog of Escherichia coli’s undecaprenyl pyrophosphate phosphatase. In previous work, a genetic screen of 10,100 Mtb transposon mutants identified Rv2136c as being involved in acid resistance in Mtb. The Rv2136c:Tn strain was also sensitive to sodium dodecyl sulfate, lipophilic antibiotics, elevated temperature and reactive oxygen and nitrogen intermediates and was attenuated for growth and persistence in mice. However, none of these phenotypes could be genetically complemented, leading us to generate an Rv2136c knockout strain to test its role in Mtb pathogenicity. Genetic deletion revealed that Rv2136c is not responsible for any of the phenotypes observed in the transposon mutant strain. An independent genomic mutation is likely to have accounted for the extreme attenuation of this strain. Identification of the mutated gene will further our understanding of acid resistance mechanisms in Mtb and may offer a target for anti-tuberculosis chemotherapy.     

Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis.

SETTING: Pyrazinamide (PZA) is an effective sterilising drug in tuberculosis, but its mode of action is controversial. OBJECTIVE: To test the bactericidal activity of 1.56-100 microg/ml PZA in Hu/Coates models of dormant and rifampicin (RMP) tolerant Mycobacterium tuberculosis. METHODS: In model 1, bactericidal activity was tested in pH 5.5 medium against 4-day, 30-day or 100-day static, hypoxic cultures. In models 2 and 3, 100 microg/ml RMP was added to a 100-day culture and PZA was added either during incubation with RMP in model 3, or after resuspension in RMP-free medium in model 2. RESULTS: Model 1: cfu counts on the 100-day and 30-day cultures fell by a maximum of about 1.6 log cfu/ml with increasing culture age, PZA concentration and incubation period, while counts on the 4-day culture showed little change. Model 2: cfu counts at the end of 7 days of recovery showed little bactericidal activity. Model 3: viable bacilli were almost completely eliminated. Bactericidal activity in these models increased with decreasing metabolic bactericidal activity, as measured by the uptake of [3H] uridine into bacterial RNA. CONCLUSION: PZA differs from other anti-tuberculosis drugs in showing greater bactericidal activity the slower the bacillary metabolic activity, hence its great value as a sterilising drug, likely to remain as an effective companion drug with newer sterilising drugs.