Susceptibility of Mycobacterium tuberculosis to pyrazinamide and its relationship to pyrazinamidase activity.

Pyrazinamidase activity has been associated with pyrazinamide-susceptible Mycobacterium tuberculosis strains. The detection of pyrazinamidase activity by the Wayne method was found to be of limited value when compared with the results of standard pyrazinamide susceptibility tests, especially when a high level of pyrazinamide resistance was found. When resistance to pyrazinamide reached a level of 150 to 200 micrograms/ml, there was too much variability in Wayne test results to accurately define pyrazinamide susceptibility.     

Use of pyrazinamidase activity on Mycobacterium tuberculosis as a rapid method for determination of pyrazinamide susceptibility.

Pyrazinamidase activity in clinical isolates of Mycobacterium tuberculosis has been previously found to correlate with susceptibility to the antituberculosis drug pyrazinamide. The Wayne method for determining pyrazinamidase activity, a technique also utilized as an aid in identification of mycobacteria, and thin-layer chromatography method were found to be useful screening methods for susceptibility testing, since resistant strains are pyrazinamidase negative. These simple methods overcome the difficulty in growing M. tuberculosis at pH 5.5, as is required in the conventional method of susceptibility testing.     

Influence of antituberculosis drug resistance and Mycobacterium tuberculosis lineage on outcome in HIV-associated tuberculous meningitis

HIV-associated tuberculous meningitis (TBM) has high mortality. Aside from the devastating impact of multidrug resistance (MDR) on survival, little is understood about the influence of other bacterial factors on outcome. This study examined the influence of Mycobacterium tuberculosis drug resistance, bacterial lineage, and host vaccination status on outcome in patients with HIV-associated TBM. Mycobacterium tuberculosis isolates from the cerebrospinal fluid of 186 patients enrolled in two studies of HIV-associated TBM in Ho Chi Minh City, Vietnam, were tested for resistance to first-line antituberculosis drugs. Lineage genotyping was available for 122 patients. The influence of antituberculosis drug resistance and M. tuberculosis lineage on 9-month mortality was analyzed using Kaplan-Meier survival analysis and Cox multiple regression models. Isoniazid (INH) resistance without rifampin resistance was associated with increased mortality (adjusted hazard ratio [HR], 1.78, 95% confidence interval [CI], 1.18 to 2.66; P = 0.005), and multidrug resistance was uniformly fatal (n = 8/8; adjusted HR, 5.21, 95% CI, 2.38 to 11.42; P < 0.0001). The hazard ratio for INH-resistant cases was greatest during the continuation phase of treatment (after 3 months; HR, 5.05 [95% CI, 2.23 to 11.44]; P = 0.0001). Among drug-susceptible cases, patients infected with the "modern" Beijing lineage strains had lower mortality than patients infected with the "ancient" Indo-Oceanic lineage (HR, 0.29 [95% CI, 0.14 to 0.61]; P = 0.001). Isoniazid resistance, multidrug resistance, and M. tuberculosis lineage are important determinants of mortality in patients with HIV-associated TBM. Interventions which target these factors may help reduce the unacceptably high mortality in patients with TBM.     

Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates

Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.     

In vitro susceptibility of Mycobacterium kansasii to clarithromycin.

The MICs of the macrolide clarithromycin for 31 clinical isolates of Mycobacterium kansasii were determined by three different methods. The methods employed were the proportion resistance method on 7H10 agar, the radiometric (BACTEC) method, and the T100 method of datum analysis. All methods gave similar results. The MICs were in a narrow range from 0.16 to 0.50 microgram/ml, with the MICs for 90% of isolates tested of 0.50 microgram/ml for the agar dilution and radiometric methods and 0.37 microgram/ml for the T100 method. The MBCs were determined for nine representative isolates by the radiometric broth method. The MBCs were equal to the MICs for four isolates, and the MBCs were twofold higher than the MICs for five isolates. Killing of 99.9% of the bacterial population was achieved at a clarithromycin concentration of 2.0 micrograms/ml for all nine isolates tested.     

Multiple drug resistance of Mycobacterium tuberculosis

Mycobacterium tuberculosis acquires drug resistance by chromosomal mutation resulting in alterations of target molecules of drugs.     

Evaluation of BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA): compared with the results of pyrazinamidase assay and Kyokuto PZA test

The fully automated BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA) was evaluated using 101 Mycobacterium tuberculosis clinical isolates. The results obtained with the system were compared with those of the pyrazinamidase (PZase) assay and the Kyokuto PZA test based on a broth culture, which is commercially available in Japan. The overall concordance rate was 90.1% (91/101) among the three methods in the initial test. The concordance rates between the BACTEC MGIT 960 PZA medium vs the PZase assay, the BACTEC MGIT 960 PZA medium vs the Kyokuto PZA test, and the PZase assay vs the Kyokuto PZA test were 93.1, 91.1, and 96.0%, respectively. On the repeat test of the 10 strains with discrepant results among the three methods, the concordance rates reached over 97% between each of the two systems. The results of the repeat test were confirmed by MIC testing and sequencing analysis of the pncA gene encoding PZase of M. tuberculosis. The mean turnaround times from incubation for PZA susceptibility testing were almost similar for the two methods based on liquid media, the BACTEC MGIT 960 PZA medium and the Kyokuto PZA test (7.7 and 7.4 days, respectively). These results indicate that both methods based on liquid media, the fully automated BACTEC MGIT 960 PZA medium and the Kyokuto PZA test for susceptibility testing to PZA, are useful for rapid diagnosis of PZA resistant tuberculosis.     

Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms.

A gene (pncA) with mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis complex members was characterized in 67 pyrazinamide-resistant and 51 pyrazinamide-susceptible isolates recovered from diverse geographic localities and anatomic sites and typed by IS6110 profiling. All pyrazinamide-susceptible organisms had identical pncA alleles. In striking contrast, 72% of the 67 resistant organisms had pncA mutations that altered the primary amino acid sequence of pyrazinamidase. A total of 17 previously undescribed mutations were found, including upstream mutations, missense changes, nucleotide insertions and deletions, and termination mutations. The mutations were arrayed along virtually the entire length of the gene. These data are further evidence that most drug resistance in M. tuberculosis is due to simple mutations occurring in chromosomally encoded genes rather than to acquisition of resistance genes by horizontal transfer events.     

Simple fibroblast-based assay to test the pyrazinamide susceptibility of Mycobacterium tuberculosis

A simple fibroblast-based assay (SFA) was found to be efficient in evaluating the susceptibilities of clinical isolates of Mycobacterium tuberculosis to pyrazinamide (PZA). Forty-five clinical isolates were examined. The MICs of PZA for susceptible strains in an SFA were between 3.13 and 12.5 microg/ml, and the MICs of PZA for resistant strains were more than 100 microg/ml.     

Correlation between pyrazinamide activity and pncA mutations in Mycobacterium tuberculosis isolates in Taiwan

A total of 76 clinical Mycobacterium tuberculosis isolates from Taiwan were tested for pyrazinamidase activity, pyrazinamide susceptibility, and pncA mutations. Frequency of resistance to PZA rose with increases in resistance to first-line drugs. Of 17 pyrazinamide-resistant strains, 7 (3 of which had not been previously described) possessed mutations in the pncA gene.     

In vitro activity of linezolid, clarithromycin and moxifloxacin against clinical isolates of Mycobacterium kansasii

OBJECTIVES: To compare the activity of linezolid with a range of drugs used in the treatment of Mycobacterium kansasii infections. RESULTS: The percentages of resistant isolates against isoniazid, rifampicin and ethambutol were 2.9%, 1.9% and 2.9%, respectively. All isolates were susceptible to clarithromycin and moxifloxacin both with MIC(90) values of 0.125 mg/L. Linezolid was active against all isolates with MIC(50) and MIC(90) values of 0.5 and 1 mg/L, respectively, both below the susceptibility breakpoint established for mycobacteria. CONCLUSION: Linezolid, clarithromycin or moxifloxacin, could be used as alternative drugs for treatment of infections due to rifampicin-resistant isolates as well as short-course or intermittent therapy of M. kansasii lung disease.     

In-vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium kansasii to amoxycillin and ticarcillin in combination with clavulanic acid

The in-vitro susceptibility of Mycobacterium tuberculosis, M. bovis, and M. kansasii to amoxycillin alone and in combination with 2 mg/l of clavulanic acid was evaluated by broth dilution. The MIC90 of amoxycillin plus clavulanic acid was 4 mg/l compared with greater than 32 mg/l for amoxycillin alone when tested against M. tuberculosis (n = 27). M. bovis (n = 8) was the most susceptible species with an MIC90 of amoxycillin 8 mg/l, compared with 0.5 mg/l for the combination. M. kansasii (n = 6), with an MIC90 of 16 mg/l for amoxycillin plus clavulanic acid was more resistant than either M. tuberculosis or M. bovis. Ticarcillin plus clavulanic acid with an MIC90 of 32 mg/l was less active against M. tuberculosis (n = 28) than amoxycillin plus clavulanic acid. The addition of clavulanic acid to amoxycillin greatly improves its in-vitro activity against M. tuberculosis and M. bovis.     

Structure of the Mycobacterium tuberculosis D-alanine:D-alanine ligase, a target of the antituberculosis drug D-cycloserine

D-alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 ?. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC(50)) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.     

Salicylate reduces susceptibility of Mycobacterium tuberculosis to multiple antituberculosis drugs

Salicylate induces multiple antibiotic resistance in various bacterial species. Here we investigated the effect of salicylate on the susceptibility of Mycobacterium tuberculosis to a range of antituberculosis (anti-TB) drugs. In the presence of salicylate, the killing effects of isoniazid (INH), rifampin (RMP), ethambutol (EMB), streptomycin (STR), and p-aminosalicylate (PAS) were reduced, as shown with a tetrazolium redox dye viability assay and a bacterial survival assay. Salicylate-induced resistance was more pronounced for PAS, STR, and EMB but was not apparent for INH and RMP when salicylate and the anti-TB agents were incorporated into 7H11 plates. The significance of these findings for TB treatment needs to be further evaluated in vivo.     

显微镜观察药物敏感性试验检测结核分枝杆菌对吡嗪酰胺的敏感性

目的:评价显微镜观察药物敏感性(microscopic-observation drug-susceptibility,MODS)试验检测结核分枝杆菌对吡嗪酰胺(pyrazinamide,PZA)敏感性的价值。方法:采用pncA基因测序法、MGIT 960 PZA法、Wayne法及MODS法检测53株结核分枝杆菌对PZA的敏感性。结果:①pncA基因测序结果显示,53株结核分枝杆菌中30株发生了不同位点的突变,另23株为野生型;MGIT 960 PZA法检测结果显示,其中22株对PZA敏感,31株对PZA耐药;经Wayne法检测,28株为PZase阳性,25株为PZase阴性。②MODS法检测结果显示,53株结核分枝杆菌中,12株在规定时间内未生长,41株在5~14 d获得结果,平均时间为7.5 d。③MODS法检测结果与pncA测序法、MGIT960 PZA法及Wayne法比较,符合率分别为95.3%、97.6%及87.8%。结论:MODS法可快速、准确、经济地检测结核分枝杆菌对PZA的敏感性,但小部分菌株会受pH影响而无法生长。对于无法生长的菌株可联合Wayne法、pncA基因测序法、MGIT 960 PZA法弥补检测的不足,以实现用最低的检测成本完成准确快捷的检测。     

微量液体培养硅胶显色法快速检测结核分枝杆菌吡嗪酰胺耐药性

目的建立用24孔微量液体培养硅胶显色板对结核分枝杆菌(MTB)吡嗪酰胺(PZA)药物敏感性判断的方法,并评价该方法临床应用价值。方法以MGIT960药敏结果作为标准对照,应用硅胶显色板对30株已知PZA药敏结果 MTB临床分离株进行PZA药敏检测,观察不同pH值,不同接种浓度对其结果的影响,并对最佳检测条件进行探讨。最后应用硅胶显色板和MGIT960同时对98株未知PZA药敏结果的MTB临床分离株进行检测,判断灵敏度、特异度、准确度。结果 24孔微量液体培养显色板,液体培养基最佳pH值为5.8~5.9,最佳接种菌量为2.5×10-1 mg/mL,7~14d即可报告结果。PZA临界浓度100μg/mg时灵敏度达95.50%,特异度为96.30%,准确度为98.21%;PZA临界浓度200μg/mg时灵敏度均可达90.90%,特异度为92.59%,准确度为91.84%。结论使用24孔微量液体培养硅胶显色板能对MTB的PZA药物敏感性进行快速鉴定,结果准确度高,操作简便,成本低廉。     

Dose-ranging activity of the newly registered antituberculosis drug bedaquiline (TMC207)

Evaluation of: Diacon AH, Dawson R, Von Groote-Bidlingmaier F et al. Randomized dose-ranging study of the 14-day early bactericidal activity of bedaquiline (TMC207) in patients with sputum microscopy smear-positive pulmonary tuberculosis. Antimicrob. Agents Chemother. 57(5), 2199–2203 (2013).

During the past decade considerable efforts have been made to develop and register new anti-TB drugs, making them available for patients in need. Bedaquiline (BDQ), approved by the US FDA in December 2012, is the first new anti-TB drug available for treatment of this disease since rifampicin became available in 1967. BDQ has the peculiarity of being a drug with a very long half-life and potent antimicrobial activity that becomes noticeable only after the first 4 days of treatment. Consequently, Diacon et al. have conducted a 14-day dose-ranging study aimed at assessing the early bactericidal activity of BDQ in TB patients who received loading doses of the drug during the first 2 days of treatment. The loading doses partially overcame the delayed antimicrobial activity only in patients treated daily with as much as 400 mg.