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Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (www.specs.net). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 μg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 μM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Isoxazoline derivatives of the natural products eugenol, 1’- S-acetoxychavicol acetate and sclareol are prepared through 1,3-dipolar cycloaddition reactions in an aqueous buffered system. The compounds are evaluated for their antitubercular and antibacterial activities. Compounds 2, 2a and 3f display strong antitubercular activity with minimum inhibitory concentration values of 26.68, 17.89 and 14.58 µM, respectively. Furthermore, derivative 3f exhibits antibacterial activity against Bacillus cereus (minimum inhibitory concentration value of 29.16 µM). Isoxazoline derivatives of 1’- S-acetoxychavicol acetate demonstrate improvements in cytotoxicity, and derivative 3f of sclareol demonstrates improved antitubercular and antibacterial activities. Isoxazolines derived from natural products exhibit Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) inhibitory activity, and molecular modelling predicts that they form hydrogen bonding and hydrophobic interactions with NADH and with the key residues of the InhA binding site.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski’s rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 μg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 μM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

A new series of mangostin analogs of natural α-mangostin from mangosteen was prepared and their antimycobacterial activity was evaluated in vitro against Mycobacterium tuberculosis H(37)Ra. The results showed that the monoalkyl tetrahydro α-mangostin analogs displayed increased antimycobacterial activity as compared with the lead natural xanthone, α-mangostin. Among the tested compounds, 6-methoxytetrahydro α-mangostin (16) exhibited the most potent antimycobacterial activity with minimum inhibitory concentration (MIC) of 0.78 µg/mL. The activity of the monoalkylated and monoacylated tetrahydro α-mangostins decreases as the length of carbon chain increases. The methyl ether analog was also active against the multidrug-resistant (MDR) strains with pronounced MICs of 0.78-1.56 µg/mL.

In the process of evaluating the effect of several plant extracts against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA), an extract of Thai herb Alpinia galanga rhizome and its major component, 1′-acetoxychavicol acetate (ACA), exhibited marked anti-tuberculosis activity. The minimal inhibition concentrations (MICs) of the S-enantiomer of ACA (S-ACA) against M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294 strains were 0.2 µg/mL and 0.7 µg/mL, respectively. More than 95% of 100 drug-sensitive and 50 drug-resistant mycobacterial clinical isolates were inhibited by extracted S-ACA at 1.0 µg/mL. All of the remaining isolates were inhibited at 2.0 µg/mL. In contrast to the S-enantiomer, synthetic racemic 1′-R,S-ACA (rac-ACA) showed MICs of 0.5 µg/mL and 2.7 µg/mL for M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294, respectively, suggesting that the anti-tuberculosis effect might be primarily due to the S-form. These observations were in line with the MICs of rac-ACA against 98% of 93 drug-resistant clinical isolates, which showed the effective inhibitory dose at 2.0 µg/mL. After exposure to 2.7 µg/mL of rac-ACA for at least 3 h, the tubercle bacilli were completely killed. These demonstrated that ACA had potent anti-TB activity.

Introduction: Mycobacterium haemophilum is one of the non‐tuberculous mycobacteria (NTM) that can cause cutaneous infection. As acid‐fast staining cannot distinguish NTM from Mycobacterium tuberculosis, and as skin culture for M. haemophilum is not performed routinely, the diagnosis of M. haemophilum infection in Thailand is rarely made. Case presentation: Between 2006 and 2009, five patients with M. haemophilum infection were diagnosed in Ramathibodi Hospital, a tertiary care centre in Bangkok, Thailand. The patients were aged 3, 29, 47, 75 and 76 years, and four were immunocompromised. Three patients received immunosuppressive medication. Most patients presented with subacute cutaneous infection. A suboptimal response to conventional antibiotics raised suspicions of M. haemophilum cutaneous infections, which can occur in immunocompromised patients. Diagnoses of these cases were made by skin culture for mycobacteria at an incubating temperature of around 30 °C with iron supplementation, DNA sequencing, or PCR/restriction enzyme analysis. Rifampicin, ofloxacin and clarithromycin were active against all isolates, whereas ethambutol and streptomycin were inactive. Conclusion: Skin culture should be performed under special conditions or molecular technique should be used to identify M. haemophilum in susceptible patients.

A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of Mycobacterium tuberculosis complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evaluated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as well as Mycobacteria Growth Indicator Tube (MGIT) liquid cultures from 80 AFB-positive sputum samples. Mycobacterium species could be identified from a total of 53 LJ cultures and 77 MGIT cultures. The diagnostic specificities of the MTB and NTM probes were 100% for both cultures. The diagnostic sensitivities of the MTB probe for the LJ and MGIT cultures were 98 and 99%, respectively, whereas the sensitivities of the NTM probe were 57 and 100%, respectively. The relatively low sensitivity of the NTM probe was due to a high proportion of M. fortuitum , which is not identified by the probe.

TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum , which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC ( n = 46) or NTM ( n = 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.

A new series of mangostin analogs of natural α-mangostin from mangosteen was prepared and their antimycobacterial activity was evaluated in vitro against Mycobacterium tuberculosis H(37)Ra. The results showed that the monoalkyl tetrahydro α-mangostin analogs displayed increased antimycobacterial activity as compared with the lead natural xanthone, α-mangostin. Among the tested compounds, 6-methoxytetrahydro α-mangostin (16) exhibited the most potent antimycobacterial activity with minimum inhibitory concentration (MIC) of 0.78 µg/mL. The activity of the monoalkylated and monoacylated tetrahydro α-mangostins decreases as the length of carbon chain increases. The methyl ether analog was also active against the multidrug-resistant (MDR) strains with pronounced MICs of 0.78-1.56 µg/mL.

The in vitro activities of cloxyquin (5-chloroquinolin-8-ol) against 9 standard strains and 150 clinical isolates of Mycobacterium tuberculosis were studied. The MICs ranged from 0.062 to 0.25 μg/ml. The MIC 50 and MIC 90 were 0.125 and 0.25 μg/ml, respectively. These indicate that cloxyquin exhibited good antituberculosis activity, even for multidrug-resistant isolates.

Existing macrolides have never shown definitive clinical efficacy in tuberculosis. Recent reports suggest that ribosome methylation is involved in macrolide resistance in Mycobacterium tuberculosis , a mechanism that newer macrolides have been designed to overcome in gram-positive bacteria. Therefore, selected macrolides and ketolides (descladinose) with substitutions at positions 9, 11,12, and 6 were assessed for activity against M. tuberculosis , and those with MICs of ≤4 μM were evaluated for cytotoxicity to Vero cells and J774A.1 macrophages. Several compounds with 9-oxime substitutions or aryl substitutions at position 6 or on 11,12 carbamates or carbazates demonstrated submicromolar MICs. For the three macrolide-ketolide pairs, macrolides demonstrated superior activity. Four compounds with low MICs and low cytotoxicity also effected significant reductions in CFU in infected macrophages. Active compounds were assessed for tolerance and the ability to reduce CFU in the lungs of BALB/c mice in an aerosol infection model. A substituted 11,12 carbazate macrolide demonstrated significant dose-dependent inhibition of M. tuberculosis growth in mice, with a 10- to 20-fold reduction of CFU in lung tissue. Structure-activity relationships, some of which are unique to M. tuberculosis , suggest several synthetic directions for further improvement of antituberculosis activity. This class appears promising for yielding a clinically useful agent for tuberculosis.

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium . In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.