Background The respiratory microbiome plays a crucial role in respiratory health and influences the onset and progression of tuberculosis (TB). However, changes in the respiratory microbiota of patients with rifampicin-resistant TB (RR-TB) during the intensive-phase treatment have not been assessed. This study aimed to investigate the impact of a six-month intensive-phase treatment of second-line anti-TB drugs on the respiratory microbiota of RR-TB patients. Methods Sputum samples were collected from 14 RR-TB patients and 14 healthy controls. Microbiota composition was analyzed using 16S rRNA gene sequencing, and functional predictions were performed to assess metabolic pathway changes. Results RR-TB patients exhibited significantly lower alpha diversity compared to healthy controls, but no significant changes were observed after six months of treatment. Beta diversity analysis revealed distinct clustering patterns between RR-TB patients and healthy controls, with no significant differences between pre-and post-treatment groups. Functional analysis showed reduced microbial functions related to pyruvate fermentation and amino acid metabolism in RR-TB patients. Conclusions These findings highlight the specific effects of second-line anti-TB drugs on the respiratory microbiota and suggest potential roles of respiratory ecological imbalance in RR-TB pathogenesis. Future studies could explore microbiome-based diagnostic and therapeutic strategies for RR-TB.

Respiratory microbiota is closely related to tuberculosis (TB) initiation and progression. However, the dynamic changes of respiratory microbiota during treatment and its association with TB progression remains unclear. A total of 16 healthy individuals and 16 TB patients (10 drug-sensitive TB (DS-TB) and 6 drug-resistant TB (DR-TB)) were recruited. Sputum samples were collected at baseline for all anticipants and after anti-TB treatment at Month-6 for TB patients. High throughput 16 S RNA sequencing was used to characterize the respiratory microbiota composition. Compared to the healthy individuals, TB patients exhibited lower respiratory microbiota diversity (p < 0.05). This disruption was alleviated after anti-TB treatment, especially for DS-TB patients. Parvimonas spp. numbers significantly increased after six months of anti-TB treatment in both DS-TB and DR-TB patients (p < 0.05). Rothia spp. increase during treatment was associated with longer sputum-culture conversion time and worse pulmonary lesion absorption (p < 0.05). Besides, Moraxella spp. prevalence was associated with longer sputum-culture conversion time, while Gemella spp. increase was associated with worsening resolving of pulmonary lesions (p < 0.05). Dynamic changes of respiratory microbiota during anti-TB treatment is closely related to TB progression. The involvement of critical microorganisms, such as Parvimonas spp., Rothia spp., Moraxella, and Gemella spp., appears to be associated with pulmonary inflammatory conditions, particularly among DR-TB. These microorganisms could potentially serve as biomarkers or even as targets for therapeutic intervention to enhance the prognosis of tuberculosis patients.

Abstract Background: Pulmonary tuberculosis is a chronic infectious disease of the respiratory system. It is still one of the causes of death from a single infectious disease, but it has been stuck in the study of a single pathogen. Recent studies have shown that many diseases are related to the microbiota. We clarified the occurrence of tuberculosis and the correlation between drug resistance and respiratory flora. High-throughput 16S rRNA gene sequencing was used to characterize the respiratory microbiota composition of 30 tuberculosis (TB) patients and compared with 30 health (H) controls. According to their Gene Xpert results, 30 pulmonary tuberculosis patients were divided into 12 persons in the sensitive group (DS0) and 18 persons in the drug-resistant group (DR0). The flora of the two were compared with the H group. Results: The data generated by sequencing showed that Firmicutes, Proteus, Bacteroides, Actinomyces and Fusobacterium were the five main bacterial phyla detected, and they constituted more than 96% of the microbial community. The relative abundance of Fusobacterium, Haemophilus, Porphyromonas, Neisseria, TM7, Spirochetes, SR1, and tunica in the TB group was lower than that of the H group, and Granulicatella was higher than the H group. The PcoA diagrams of the two groups had obvious clustering differences. The Alpha diversity of the TB group is lower than that of the H group, and the Beta diversity is higher than that of the H group (P<0.05). The relative abundance of Streptococcus in the DS0 group was much higher than that in the DR0 group (P<0.005).Conclusion: Pulmonary tuberculosis can cause disorders of the respiratory tract flora, in which the relative abundance of Streptococcus was significantly different between rifampicin-sensitive and drug-resistant patients.

Background Pulmonary tuberculosis is a chronic infectious disease of the respiratory system. It is still one of the leading causes of death from a single infectious disease, but it has been stuck in the study of a single pathogen. Recent studies have shown that many diseases are associated with disruption of the native microbiota. In this study we investigated the occurrence of tuberculosis and the correlation between drug resistance and respiratory flora. High-throughput 16 S rRNA gene sequencing was used to characterize the respiratory microbiota composition of 30 tuberculosis (TB) affected patients and compared with 30 healthy (H) controls. According to their Gene Xpert results, 30 pulmonary tuberculosis patients were divided into 12 persons in the drug-sensitive group (DS0) and 18 persons in the drug-resistant group (DR0). The microbial flora of the two were compared with the H group. Results The data generated by sequencing showed that Firmicutes , Proteus , Bacteroides , Actinomyces and Fusobacterium were the five main bacterial phyla detected, and they constituted more than 96% of the microbial community. The relative abundances of Fusobacterium , Haemophilus , Porphyromonas , Neisseria , TM7 , Spirochetes , SR1 , and Tenericutes in the TB group was lower than that of the H group, and Granulicatella was higher than the H group. The PcoA diagrams of the two groups had obvious clustering differences. The Alpha diversity of the TB group was lower than that of the H group, and the Beta diversity was higher than that of the H group ( P < 0.05). The relative abundance of Streptococcus in the DS0 group was significantly higher than that in the DR0 group ( P < 0.05). Conclusion Pulmonary tuberculosis can cause disorders of the respiratory tract microbial flora, in which the relative abundance of Streptococcus was significantly different between rifampicin-sensitive and rifampicin-resistant patients.

目的 探讨初治肺结核患者与健康人群呼吸道细菌与真菌微生态的异同.方法 以70例初治肺结核患者为观察组、38例健康人群为对照组,检测呼吸道分泌物的细菌和真菌,应用MALDI-TOF方法鉴定菌种,比较分析两组间检出细菌(除外结核分枝杆菌)、真菌分类与密度的差异.结果(1)在"门"水平上,观察组在拟杆菌门、厚壁菌门、变形菌门、放线菌门、梭杆菌门和真菌子囊菌门的检出率分别为64.29％(45/70)、100％(70/70)、100％(70/70)、70.00％(49/70)、55.71％(39/70)和54.29％(38/70),菌落密度(CFU/mL)分别为(5.48±5.62)、(6.81±6.56)、(6.41±5.74)、(5.50±5.65)、(4.79±4.68)和(6.01±5.85);对照组各门的检出率分别为76.32％(29/38)、100％(38/38)、100％(38/38)、92.11％(35/38)、60.53％(23/38)和15.79％(6/38),菌落密度(CFU/mL)分别为(5.78±5.12)、(6.29±6.45)、(6.56±6.63)、(5.09±5.16)、(5.33±5.50)和(3.40±3.53);统计分析显示放线菌门、子囊菌门检出率以及厚壁菌门、放线菌门、子囊菌门、梭杆菌门的菌落密度在两组间差异均有统计学意义(P<0.05).(2)在"种"水平上,两组共检测到67个菌种,其中26个菌种为观察组单独检出,9个菌种为对照组单独检出,32个菌种为两组共同检出;对于在任何一组人群中检出率高于50％的菌种,其在观察组与对照组的检出率的对比分别为口腔链球菌/缓症链球菌95.71％vs.100％、奈瑟菌91.43％vs.100％、小韦荣球菌/不典型韦荣球菌61.43％vs.71.05％、粘滑罗斯菌60.00％vs.86.84％、副流杆嗜血杆菌58.57％vs.94.74％、产黑色素普雷沃菌45.71 vs.68.42％、生痰二氧化碳嗜纤维菌38.57％vs.60.53％和口腔纤毛菌44.29％vs.57.89％.结论 初治肺结核患者呼吸道细菌与真菌微生态与健康人群比较,优势菌种变化不大,但菌种及菌落密度发生紊乱,主要表现在菌种类型增多,特别是条件致病菌的增加,此将可能增大合并感染的机会,值得临床密切关注.

目的 探讨脓肿分枝杆菌复合群(MABC)对克拉霉素的耐药机制,为MABC肺病诊治提供依据.方法 对广州市胸科医院冻存的191株龟-脓肿分枝杆菌复合群进行hsp65、rpoB、ITS多靶位基因菌种鉴定,确定为MABC的菌株进行克拉霉素最低抑菌浓度(MIC)药敏,延长培养时间至14 d,记录3、7、10、14 d的MIC值,观察MABC对克拉霉素诱导耐药情况,并对耐药相关基因rrl和erm(41)进行测序,研究克拉霉素耐药表型和基因型的关系.结果 191株龟-脓肿分枝杆菌复合群经多靶位基因法鉴定出2株龟分枝杆菌,189株MABC,其中111株脓肿分枝杆菌,78株马赛分枝杆菌.111株脓肿分枝杆菌中,克拉霉素获得性耐药1株,获得性耐药率0.9％;克拉霉素诱导耐药78株,诱导耐药率70.3％;总耐药株79株,总耐药率71.2％;78株马赛分枝杆菌无诱导耐药,克拉霉素获得性耐药5株,获得性耐药率6.4％;总耐药株5株,总耐药率为6.4％;脓肿分枝杆菌和马赛分枝杆菌总耐药率和获得性耐药率差异均有统计学意义(P＜0.05).6株克拉霉素获得性耐药菌株中,rrl突变的菌株5株,突变率为83.3％;所有的脓肿分枝杆菌erm(41)序列完整,所有的马赛分枝杆菌erm(41)序列有短缺;111株脓肿分枝杆菌,T28序列型菌株84株、C28序列型菌株27株.78株马赛分枝杆菌皆为T28序列型.结论 MABC为我国较为常见的非结核分枝杆菌,耐药率高,可用药物有限,治疗困难,主要菌种为脓肿分枝杆菌,根本的原因在于克拉霉素诱导耐药,准确的菌种鉴定、可靠的药敏试验对指导MABC肺病的临床治疗有重要意义.

目的 研究吡嗪酰胺酶活性与耐多药肺结核(multidrug resistant pulmonary tuberculosis,MDR PTB)近期疗效的相关性,为MDR-PTB患者化学药物治疗的选择提供指导.方法 采用前瞻性队列研究的方法,将广州市胸科医院2018年7月至2019年12月收治的85例MDR-PTB患者作为研究对象,均接受世界卫生组织推荐的6Am-Mfx-PZA-Pto-Cs/18Mfx-PZA-Pto-Cs(Am∶阿米卡星∶Mfx∶莫西沙星;PZA∶吡嗪酰胺;Pto∶丙硫异烟胺;Cs∶环丝氨酸)治疗方案.85例患者中,剔除9例丢失、中断治疗、失访患者,共76例患者纳入最终研究.以WaNe方法检测吡嗪酰胺酶的活性,以BACTEC MGIT 960方法检测吡嗪酰胺药物敏感性;比较吡嗪酰胺酶阳性患者与阴性患者治疗2、4、6个月末痰菌阴转、病灶吸收和空洞闭合情况.结果 76例患者中,吡嗪酰胺酶阳性32例(42.1％),吡嗪酰胺酶阴性44例(57.9％);吡嗪酰胺表型耐药36例(47.4％),吡嗪酰胺表型敏感40例(52.6％).吡嗪酰胺药物敏感性检测与吡嗪酰胺酶活性检测的一致性较高(Kappa=0.687,P=0.000).吡嗪酰胺酶阳性患者治疗4个月末病灶吸收率(59.4％,19/32)及空洞闭合有效率(65.5％,19/29)均高于吡嗪酰胺酶阴性患者[分别为36.4％(16/44)和36.8％(14/38)],差异均有统计学意义(x2=3.949,P=0.047;x2 =5.411,P=0.020).吡嗪酰胺酶阳性患者治疗6个月后的空洞闭合有效率(86.2％,25/29)高于吡嗪酰胺酶阴性患者(63.2％,24/38),差异有统计学意义(x2 =4.447,P=0.035).结论 MDR-PTB患者的疗效与吡嗪酰胺酶活性有关,吡嗪酰胺酶阳性患者应用含吡嗪酰胺的MDR-PTB标准方案治疗后,病灶吸收及空洞闭合的效果优于吡嗪酰胺酶阴性患者.

目的 探讨外周血中性粒细胞和淋巴细胞比值(NLR)及胸腔积液NLR在积液性质及是否耐药结核鉴别中的临床价值.方法 回顾性分析135例胸腔积液患者,其中恶性胸腔积液、类肺炎胸腔积液、结核性胸腔积液各45例,结核性胸腔积液病例根据结核菌培养及药敏结果,进一步分为耐药组20例和非耐药组25例.采集胸腔积液及外周血进行血常规检测中性粒细胞(Neu)、淋巴细胞计数(Lym)和外周血血小板计数(PLT),获得外周血NLR、血小板与淋巴细胞比值(PLR),以及胸腔积液NLR并进行各组间的比较分析.结果 外周血Neu、NLR、Lym及PLR在恶性、肺炎性与结核性胸腔积液组间差异有统计学意义(P<0.05).其中结核性胸腔积液组外周血NLR为2.88±1.28,明显低于恶性胸腔积液组的4.83±1.16及类肺炎胸腔积液组的5.12±1.36(P<0.05).胸腔积液Neu、WBC、NLR在恶性、类肺炎性与结核性胸腔积液组间呈递减趋势,Lym呈递增趋势(P<0.05),其中恶性胸腔积液组NLR为3.71±1.26,明显高于类肺炎胸腔积液组的2.38±0.71及结核性胸腔积液组的1.31±0.24,而后者同时也明显低于类肺炎胸腔积液组(P<0.05).受试者工作特征(ROC)曲线下面积(AUC)结果显示在判断良(结核与肺炎)恶性中,AUC胸腔积液NLR=0.897,AUC外周血NLR=0.768,AUC外周血PLR=0.702,AUC胸腔积液NLR最高.耐药组血常规PLT及PLR水平分别为(241.13±53.17)×109·L-1和133.22±24.19,明显低于非耐药组的(291.56±53.08)×109·L-1和168.21±34.93,差异有统计学意义(P<0.01).耐药组胸腔积液Neu及PLR水平分别为(3.04±0.72)×109·L-1和1.35±0.31,明显高于非耐药组的(2.52±0.63)×109·L-1和1.17±0.21,差异有统计学意义(P<0.05).结论 外周血NLR及胸腔积液NLR均可较为准确地预测积液性质,也与耐药关系密切,且在判断积液良恶性中的表现更佳,值得推广.

Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STR (R)) isolates were Beijing lineage strains. Mutations were observed in STR (R) MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STR (R) isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STR (R) isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.

OBJECTIVES:Amikacin is the only second-line injectable antituberculosis (anti-TB) drug still recommended for multidrug-resistant tuberculosis (MDR-TB) treatment when a short MDR-TB regimen is designed. Mutations in rrs and eis are reported to be associated with resistance to amikacin. In this study, we investigated the incidence of rrs, eis, tap and whiB7 mutations in amikacin-resistant Mycobacterium tuberculosis clinical isolates to find the proportion of different mutations related to amikacin resistance. METHODS:A total of 395 clinical isolates of M. tuberculosis were used for phenotypic drug susceptibility testing (DST) to 10 drugs with the Löwenstein-Jensen (L-J) method. We sequenced rrs, eis, tap and whiB7 genes in 178 M. tuberculosis clinical isolates (89 amikacin-resistant isolates and 89 of 306 amikacin-susceptible isolates). RESULTS:Our data showed that 22.53% (89/395) M. tuberculosis clinical isolates were resistant to amikacin. Of the 89 amikacin-resistant isolates, 89.89% (80/89) were MDR-TB, of which 12.36% (11/89) were pre-extensively drug-resistant TB (pre-XDR-TB) and 77.53% (69/89) were XDR-TB. The rrs mutations were found in 82% (73/89) in amikacin-resistant M. tuberculosis clinical isolates. The A1401G alteration in the rrs gene was the most dominant mutation (80.90%; 72/89). Five mutations were detected as new in rrs, tap and whiB7. Notably, 13.48% (12/89) amikacin-resistant isolates had no known mutation in these genes. CONCLUSIONS:Our data reveal that the rrs mutation is a predominant molecular marker of amikacin resistance in southern China. Analysis of the rrs gene mutations will significantly reduce the time and cost to diagnose amikacin resistance in TB patients. Other unknown amikacin resistance mechanism(s) exist.

Mycobacterium abscessus is a fast growing Mycobacterium species mainly causing skin and respiratory infections in human. M. abscessus is resistant to numerous drugs, which is a major challenge for the treatment. In this study, we have sequenced the genomes of two clinical M. abscessus strains having rough and smooth morphology, using the single molecule real-time and Illumina HiSeq sequencing technology. In addition, we reported the first comparative methylome profiles of a rough and a smooth M. abscessus clinical strains. The number of N4-methylcytosine (4mC) and N6-methyladenine (6mA) modified bases obtained from smooth phenotype were two-fold and 1.6 fold respectively higher than that of rough phenotype. We have also identified 4 distinct novel motifs in two clinical strains and genes encoding antibiotic-modifying/targeting enzymes and genes associated with intracellular survivability having different methylation patterns. To our knowledge, this is the first report about genome-wide methylation profiles of M. abscessus strains and identification of a natural linear plasmid (15 kb) in this critical pathogen harboring methylated bases. The pan-genome analysis of 25 M. abscessus strains including two clinical strains revealed an open pan genome comprises of 7596 gene clusters. Likewise, structural variation analysis revealed that the genome of rough phenotype strain contains more insertions and deletions than the smooth phenotype and that of the reference strain. A total of 391 single nucleotide variations responsible for the non-synonymous mutations were detected in clinical strains compared to the reference genome. The comparative genomic analysis elucidates the genome plasticity in this emerging pathogen. Furthermore, the detection of genome-wide methylation profiles of M. abscessus clinical strains may provide insight into the significant role of DNA methylation in pathogenicity and drug resistance in this opportunistic pathogen.

目的 探讨吡嗪酰胺(PZA)耐药与否对耐多药结核病(MDR-TB)患者采用含PZA的标准MDR-TB方案近远期临床疗效的影响关系.方法 采用回顾性队列研究方法,选取2015年1-12月在广州市胸科医院就诊并顺利完成疗程,且采用BACTEC MGIT 960法检测有结核分枝杆菌生长,并同时采用比例法进行药物敏感性试验(简称“药敏试验”)证实对异烟肼、利福平和PZA耐药的MDR TB患者(ZR-MDR组)60例;采用1∶1配对原则,选择同期治疗药敏试验结果显示对PZA敏感、年龄相差5岁以内的MDR-TB患者(Zs-MDR组)60例.所有患者均给予化疗方案6Am-Lfx(Mfx)-PZA-Pto-Cs (PAS)/18Lfx(Mfx)-PZA-Pto-Cs (PAS)进行治疗.记录两组患者痰菌阴转率、病灶吸收率、空洞缩小率、治愈率及失败率等,并进行统计学分析.结果 ZS-MDR组患者在2、6、12、24个月末时痰菌阴转率分别为70.00％ (42/60)、90.00％ (54/60)、86.67％ (52/60)、86.67％ (52/60),高于ZR-MDR组的46.67％(28/60)、66.67％(40/60)、70.00％(42/60)、70.00％ (42/60),差异均有统计学意义(x2值分别为6.720、9.624、4.910、4.910,P值均＜0.05);疗程结束时ZS-MDR组患者病灶吸收率为81.67％(49/60),高于ZR-MDR组的65.00％(39/60),差异有统计学意义(x2=4.264,P=0.019);ZS-MDR组患者空洞缩小率为76.92％(40/52),与ZR-MDR组的62.00％(31/50)间比较,差异无统计学意义(x2=2.681,P=0.098);Zs MDR组的治愈率为86.67％(52/60),高于ZR-MDR组的70.00％(42/60),差异有统计学意义(x2=4.910,P=0.016).ZS-MDR组的失败率为13.33％(8/60),低于ZR MDR组的30.00％ (18/60),差异有统计学意义(x2=4.910,P=0.016).结论 PZA表型耐药的MDR-TB患者使用全程含PZA的标准耐多药方案治疗,其疗效较PZA敏感者差,建议对耐PZA的MDR-TB患者尽量选择不含PZA的耐多药化疗方案治疗.

Mycobacterium abscessus is intrinsically resistant to most antimicrobial agents. The emerging infections caused by M. abscessus and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable marker-free autoluminescent M. abscessus strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating M. abscessus. The UAlMab strain was constructed using the dif/Xer recombinase system. In vitro and in vivo activities of several drugs, including clofazimine and TB47, a recently reported cytochrome bc(1) inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical M. abscessus isolates. The UAlMab strain enabled us to evaluate drug efficacy both in vitro and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both in vitro and in vivo. This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against M. abscessus, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.

Objectives: TB-EASM (Howsome, Shanghai, China), an automated system combining smear preparation, staining and microscopy in a single platform, was evaluated for tuberculosis (TB) diagnosis in a high disease-burden setting. Methods: Sputum samples from individuals with pulmonary TB were processed in parallel using conventional manual smear microscopy (MS), TB-EASM, liquid culture and GeneXpert. Method sensitivity and specificity were compared with Mycobacterium tuberculosis detection by mycobacteria growth indicator tube (MGIT) and/or GeneXpert MTB/RIF. Results: Of 524 samples, 496 met evaluation criteria for study inclusion. The proportion of M. tuberculosis detected by TB-EASM was 28.2% (150/496), significantly higher than for MS (111/496, 21.2%, p 0.01) and comparable to the rate for MGIT (163/496, 32.9%, p > 0.05). For 190 M. tuberculosis-positive cases identified using MGIT and/or GeneXpert MTB/RIF, the reference standard detection methods, TB-EASM detected 140 positive cases, for an overall sensitivity rate of 73.7% (140/190, 95% CI 67.4-79.9), which was significantly higher than for MS (105/190, 55.3%, 95% CI 48.2-62.3, p < 0.01). Specificities were 96.7% (296/306, 95% CI 94.7e98.7) for TB-EASM and 98.0% (300/306, 95% CI 96.5-99.6) for MS. Conclusion: TB-EASM outperformed conventional MS for M. tuberculosis detection in sputum specimens. (C) 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans , secretes toxin called mycolactone that triggers inflammation and immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo[1,5-a]pyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log 10 CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU.

Toxin-antitoxin (TA) modules widely exist in bacteria, and their activities are associated with the persister phenotype of the pathogen Mycobacterium tuberculosis (M. tb). M. tb causes tuberculosis, a contagious and severe airborne disease. There are 10 MazEF TA systems in M. tb that play important roles in stress adaptation. How the antitoxins antagonize toxins in M. tb or how the 10 TA systems crosstalk to each other are of interest, but the detailed molecular mechanisms are largely unclear. MazEF-mt9 is a unique member among the MazEF family due to its tRNase activity, which is usually carried out by the VapC toxins. Here, we present the cocrystal structure of the MazEF-mt9 complex at 2.7 Å. By characterizing the association mode between the TA pairs through various techniques, we found that MazF-mt9 bound not only its cognate antitoxin but also the noncognate antitoxin MazE-mt1, a phenomenon that could be also observed in vivo. Based on our structural and biochemical work, we propose that the cognate and heterologous interactions among different TA systems work together in vivo to relieve the toxicity of MazF-mt9 toward M. tb cells.

目的探究结核分枝杆菌RNA恒温扩增实时荧光检测技术（SAT-TB）检测支气管肺泡灌洗液（BALF）对涂阴肺结核的诊断价值。方法对本院涂阴疑似肺结核患者进行筛查,共有80例患者进入最后分析,痰涂片连续3次阴性者进入研究,行培养法（Bactec MGIT 960）及SAT-TB检测,同时进行诊断。分别以培养法（BD960）及以临床诊断作为参考标准,计算SAT-TB法诊断涂阴肺结核的敏感度、特异度、阳性预测值及阴性预测值。结果以培养法（BD960）作为参考标准,SAT-TB诊断涂阴肺结核的敏感度为53.3%（8/15）,特异度为88.2%（15/17）,阳性预测值80.0%（8/10）,阴性预测值68.1%（15/22）。以临床诊断作为参考标准,SAT-TB诊断涂阴肺结核的敏感度31.7%（20/63）、特异度88.2%（15/17）、阳性预测值91.0%（20/22）、阴性预测值25.9%（15/58）。SAT-TB的检测时间约2 h,快于培养法（BD960）的平均阳性报告时间14.1 d。结论 SAT-TB可通过检测BALF为涂阴肺结核患者提供一项敏感度相对于快速培养法（BD960法）较有优势的实验室辅助诊断方法。

Objective To study the clinical value of simultaneous amplification and testing for detection of Mycobacteria tuberculosis(SAT-TB)in sputum samples and bronchoalveolar lavage fluid(BALF)samples. Methods Totally 169 sputum samples and 151 BALF samples from suspected pulmonary tuberculosis patients were detected by both SAT and Bactec MGIT960.The sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)of the samples using SAT-TB were calculated. Results Taken the results of BD960 as the reference,the sensitivity,specificity,PPV and NPV using SAT-TB of sputum samples were 84.00% (42/50),93.06%(67/72),89.36%(42/47)and 89.33%(67/75)respectively;and those of BALF samples 89.19% (33/37),95.12%(39/41),94.29%(33/35)and 42.39%(39/92)respectively.Taken clinical diagnostic results as reference standard,the sensitivity,specificity,PPV and NPV using SAT-TB of the sputum samples were 57.73% (56/97),93.06%(67/72),91.80%(56/61),and 62.04%(67/108)respectively;and those of BALF samples 51.82%(57/110),94.29%(39/41),96.61%(57/59)and、42.39%(39/92)respectively.The sensitivity,specificity, PPV and NPV using BD960 of the sputum samples were 51.55%(50/97),95.83%(69/72),94.34%(50/53),and 59.48%(69/116)respectively;and those of BALF samples 33.64%(37/110),90.24%(37/41),90.24%(37/41) and 33.64%(37/110)respectively.Conclusion SAT-TB is a rapid and sensitive method for the detection of Myco-bacteria tuberculosis in sputum and BALF samples.It can improve the detection rate of mycobaterium tuberculosis.

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.

BACKGROUND:Pulmonary nontuberculous mycobacteria (NTM) disease is of increasing public health concern in China. Information is limited regarding risk factors associated with this disease in China. The objective of this study was to describe the epidemiology of pulmonary disease due to NTM in Southern China.METHODS:We retrospectively reviewed the medical records of pulmonary NTM patients registered in the Guangzhou Chest Hospital with positive mycobacterial cultures during 2013-2016. We described sex, age, residence, treatment history, laboratory examination results and comorbidities of pulmonary NTM patients.RESULTS:Among the 607 NTM cases, the most prevalent species were Mycobacterium avium complex (44.5%), Mycobacterium abscessus complex (40.5%), Mycobacterium kansasii (10.0%) and Mycobacterium fortuitum (2.8%). The male:female ratio was significantly lower among patients infected with rapidly growing mycobacteria (RGM) than among those with slowly growing mycobacteria (SGM). The risk of developing SGM disease significantly increased with advancing age. In addition, pulmonary RGM diseases were more common in migrant population than resident population. Notably, patients with pulmonary RGM diseases were significantly more likely to have bronchiectasis underlying noted than those with SGM diseases. No significant difference was observed in in vitro drug susceptibility among NTM species.CONCLUSION:Our data illustrate that the M. avium complex is the most predominant causative agent of pulmonary NTM disease in Southern China. Female, migrant population, the presence of bronchiectasis are independent risk factors for pulmonary diseases due to RGM. In addition, the prevalence of SGM increases significantly with advancing age.