Septic Arthritis Caused by Mycobacterium Kansasii in a Bone Marrow Transplant Recipient.

We report an 18-year-old female individual with septic arthritis due to Mycobacterium kansasii. Three years and 6 months before arthritis, the patient underwent bone marrow transplantation and developed severe chronic graft-versus-host disease. The arthritis was refractory to medication, and she underwent joint lavage of the right foot, hip joint, and elbow joint. After surgery, her joint symptoms were relieved, and chronic graft-versus-host disease remitted more easily. It is important that we maintain a high index of suspicion for mycobacterial arthritis and diagnose it early when immunosuppressed patients experience chronic pain and joint swelling.

In Vitro Activity of Bedaquiline and Delamanid against Nontuberculous Mycobacteria, Including Macrolide-Resistant Clinical Isolates.

We evaluated the in vitro activities of the antimicrobial drugs bedaquiline and delamanid against the major pathogenic nontuberculous mycobacteria (NTM). Delamanid showed high MIC values for all NTM except Mycobacterium kansasii However, bedaquiline showed low MIC values for the major pathogenic NTM, including Mycobacterium avium complex, Mycobacterium abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. kansasii Bedaquiline also had low MIC values with macrolide-resistant NTM strains and warrants further investigation as a potential antibiotic for NTM treatment.

Bioactivity of Methoxylated and Methylated 1-Hydroxynaphthalene-2-Carboxanilides: Comparative Molecular Surface Analysis.

A series of twenty-six methoxylated and methylated N-aryl-1-hydroxynaphthalene- 2-carboxanilides was prepared and characterized as potential anti-invasive agents. The molecular structure of N-(2,5-dimethylphenyl)-1-hydroxynaphthalene-2-carboxamide as a model compound was determined by single-crystal X-ray diffraction. All the analysed compounds were tested against the reference strain Staphylococcus aureus and three clinical isolates of methicillin-resistant S. aureus as well as against Mycobacterium tuberculosis and M. kansasii. In addition, the inhibitory profile of photosynthetic electron transport in spinach (Spinacia oleracea L.) chloroplasts was specified. In vitro cytotoxicity of the most effective compounds was tested on the human monocytic leukaemia THP-1 cell line. The activities of N-(3,5-dimethylphenyl)-, N-(3-fluoro-5-methoxy-phenyl)- and N-(3,5-dimethoxyphenyl)-1-hydroxynaphthalene-2-carbox- amide were comparable with or even better than the commonly used standards ampicillin and isoniazid. All promising compounds did not show any cytotoxic effect at the concentration >30 µM. Moreover, an in silico evaluation of clogP features was performed for the entire set of the carboxamides using a range of software lipophilicity predictors, and cross-comparison with the experimentally determined lipophilicity (log k), in consensus lipophilicity estimation, was conducted as well. Principal component analysis was employed to illustrate noticeable variations with respect to the molecular lipophilicity (theoretical/experimental) and rule-of-five violations. Additionally, ligand-oriented studies for the assessment of the three-dimensional quantitative structure-activity relationship profile were carried out with the comparative molecular surface analysis to determine electron and/or steric factors that potentially contribute to the biological activities of the investigated compounds.

Drug Susceptibility Profiling and Genetic Determinants of Drug Resistance in Mycobacterium kansasii.

Very few studies have examined drug susceptibility of Mycobacterium kansasii, and they involve a limited number of strains. The purpose of this study was to determine drug susceptibility profiles of M. kansasii isolates representing a spectrum of species genotypes (subtypes) with two different methodologies, i.e., broth microdilution and Etest assays. To confirm drug resistance, drug target genes were sequenced. A collection of 85 M. kansasii isolates, including representatives of eight different subtypes (I to VI, I/II, and IIB) from eight countries, was used. Drug susceptibility against 13 and 8 antimycobacterial agents was tested by using broth microdilution and Etest, respectively. For drug-resistant or high-MIC isolates, eight structural genes (rrl, katG, inhA, embB, rrs, rpsL, gyrA, and gyrB) and one regulatory region (embCA) were PCR amplified and sequenced in the search for resistance-associated mutations. All isolates tested were susceptible to rifampin (RIF), amikacin (AMK), co-trimoxazole (SXT), rifabutin (RFB), moxifloxacin (MXF), and linezolid (LZD) according to the microdilution method. Resistance to ethambutol (EMB), ciprofloxacin (CIP), and clarithromycin (CLR) was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The calculated concordance between the Etest and dilution method was 22.6% for AMK, 4.8% for streptomycin (STR), 3.2% for CLR, and 1.6% for RIF. For EMB, INH, and SXT, not even a single MIC value determined by one method equaled that by the second method. The only mutations disclosed were A2266C transversion at the rrl gene (CLR-resistant strain) and A128G transition at the rpsL gene (strain with STR MIC of >64 mg/liter). In conclusion, eight drugs, including RIF, CLR, AMK, SXT, RFB, MXF, LZD, and ethionamide (ETO), showed high in vitro activity against M. kansasii isolates. Discrepancies of the results between the reference microdilution method and Etest preclude the use of the latter for drug susceptibility determination in M. kansasii Drug resistance in M. kansasii may have different genetic determinants than resistance to the same drugs in M. tuberculosis.

Clofazimine for the Treatment of Mycobacterium kansasii.

Mycobacterium kansasii pulmonary infection is a global problem. Standard combination therapy consists of isoniazid at 300 mg/day, rifampin at 600 mg/day, and ethambutol at 15 mg/kg of body weight/day for 18 months. Coincubation of M. kansasii with different clofazimine concentrations over 7 days in test tubes resulted in a maximal kill (maximum effect [Emax]) of 2.03 log10 CFU/ml below the day 0 bacterial burden. The concentration associated with Emax was 110 times the MIC. Next, the effects of human-like concentration-time profiles of clofazimine human-equivalent doses ranging from 0 to 200 mg daily for 21 days were examined in the hollow-fiber model of intracellular M. kansasii (HFS-Mkn). On day 14, when the clofazimine microbial effect was maximal, the Emax was 2.57 log10 CFU/ml, while the dose associated with Emax was 100 mg/day. However, no dose killed M. kansasii to levels below the day 0 bacterial burden. Thus, the antimicrobial effect of clofazimine monotherapy in the HFS-Mkn was modest. Human-equivalent concentration-time profiles of standard combination therapy and doses were used as comparators in the HFS-Mkn On day 14, standard therapy killed to a level 2.32 log10 CFU/ml below the day 0 bacterial burden. The effect of standard therapy was consistent with a biexponential decline, with kill rate constants of 1.85 per day (half-life = 0.37 days) and 0.06 per day (half-life = 12.76 days) (r2 > 0.99). This means that standard therapy would take 9.3 to 12 months to completely eliminate M. kansasii in the model, which is consistent with clinical observations. This observation for standard therapy means that the modest to poor effect of clofazimine on M. kansasii identified here is likely to be the same in the clinic.

In Vitro Activity of Clofazimine against Nontuberculous Mycobacteria Isolated in Beijing, China.

Due to the natural resistance of nontuberculous mycobacteria (NTM) to many antibiotics, the treatment of diseases caused by NTM is often long-term but unsuccessful. The main goal of this study was to evaluate the in vitro susceptibilities to clofazimine of 209 isolates consisting of different NTM species isolated in Beijing, China. Furthermore, 47 reference strains were also tested, including 30 rapidly growing mycobacterium (RGM) species and 17 slowly growing mycobacterium (SGM) species. The potential molecular mechanism contributing to clofazimine resistance of NTM was investigated as well. Clofazimine exhibited excellent activity against both reference strains and clinical isolates of different SGM species, and most of the strains had MICs far below 1 µg/ml. Although the majority of the clinical isolates of Mycobacterium abscessus and Mycobacterium fortuitum had MICs higher than 2 µg/ml, 17 out of the 30 reference strains of different RGM species had MICs below 1 µg/ml in vitro According to the MIC distributions, the tentative epidemiological cutoff (ECOFF) values for Mycobacterium kansasii, Mycobacterium avium, and Mycobacterium intracellulare were defined at 0.5 µg/ml, 1 µg/ml, and 2 µg/ml, respectively. Intriguingly, single-direction cross-resistance between bedaquiline- and clofazimine (Cfz)-resistant isolates was observed among the tested NTM species. This study demonstrates that clofazimine had strong activity against most SGM species in vitro, as well as some RGM species. The data provide important insights into the possible clinical application of Cfz to treat NTM infections.

Dibasic Derivatives of Phenylcarbamic Acid against Mycobacterial Strains: Old Drugs and New Tricks?

In order to provide a more detailed view on the structure⁻antimycobacterial activity relationship (SAR) of phenylcarbamic acid derivatives containing two centers of protonation, 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium oxalates (1a⁻d)/dichlorides (1e⁻h) as well as 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(di-propylammonio)propyl]azepanium oxalates (1i⁻l)/dichlorides (1m⁻p; alkoxy = butoxy to heptyloxy) were physicochemically characterized by estimation of their surface tension (γ; Traube's stalagmometric method), electronic features (log ε; UV/Vis spectrophotometry) and lipophilic properties (log kw; isocratic RP-HPLC) as well. The experimental log kw dataset was studied together with computational logarithms of partition coefficients (log P) generated by various methods based mainly on atomic or combined atomic and fragmental principles. Similarities and differences between the experimental and in silico lipophilicity descriptors were analyzed by unscaled principal component analysis (PCA). The in vitro activity of compounds 1a⁻p was inspected against Mycobacterium tuberculosis CNCTC My 331/88 (identical with H37Rv and ATCC 2794, respectively), M. tuberculosis H37Ra ATCC 25177, M. kansasii CNCTC My 235/80 (identical with ATCC 12478), the M. kansasii 6509/96 clinical isolate, M. kansasii DSM 44162, M. avium CNCTC My 330/80 (identical with ATCC 25291), M. smegmatis ATCC 700084 and M. marinum CAMP 5644, respectively. In vitro susceptibility of the mycobacteria to reference drugs isoniazid, ethambutol, ofloxacin or ciprofloxacin was tested as well. A very unique aspect of the research was that many compounds from the set 1a⁻p were highly efficient almost against all tested mycobacteria. The most promising derivatives showed MIC values varied from 1.9 µM to 8 µM, which were lower compared to those of used standards, especially if concerning ability to fight M. tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 or M. avium CNCTC My 330/80. Current in vitro biological assays and systematic SAR studies based on PCA approach as well as fitting procedures, which were supported by relevant statistical descriptors, proved that the compounds 1a⁻p represented a very promising molecular framework for development of 'non-traditional' but effective antimycobacterial agents.

Biochemical Validation of a Third Guanidine Riboswitch Class in Bacteria.

Recently, it was determined that representatives of the riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II riboswitch RNAs, respectively. Moreover, we had previously noted that a third candidate riboswitch class, called ykkC-III, was associated with a distribution of genes similar to those of the other two motifs. Therefore, it was predicted that ykkC-III motif RNAs would sense and respond to the same ligand. In this report, we present biochemical data supporting the hypothesis that ykkC-III RNAs represent a third class of guanidine-sensing RNAs called guanidine-III riboswitches. Members of the guanidine-III riboswitch class bind their ligand with an affinity similar to that observed for members of the other two classes. Notably, there are some sequence similarities between guanidine-II and guanidine-III riboswitches. However, the characteristics of ligand discrimination by guanidine-III RNAs are different from those of the other guanidine-binding motifs, suggesting that the binding pockets have distinct features among the three riboswitch classes.

Synthesis and biological evolution of hydrazones derived from 4-(trifluoromethyl)benzohydrazide.

Reflecting the known biological activity of isoniazid-based hydrazones, seventeen hydrazones of 4-(trifluoromethyl)benzohydrazide as their bioisosters were synthesized from various benzaldehydes and aliphatic ketones. The compounds were screened for their in vitro activity against Mycobacterium tuberculosis, nontuberculous mycobacteria (M. avium, M. kansasii), bacterial and fungal strains. The most antimicrobial potent derivatives were also investigated for their cytostatic and cytotoxic properties against three cell lines. Camphor-based molecule, 4-(trifluoromethyl)-N'-(1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide, exhibited the highest and selective inhibition of M. tuberculosis with the minimum inhibitory concentration (MIC) of 4 µM, while N'-(4-chlorobenzylidene)-4-(trifluoromethyl)benzohydrazide was found to be superior against M. kansasii (MIC = 16 µM). N'-(5-Chloro-2-hydroxybenzylidene)-4-(trifluoromethyl)benzohydrazide showed the lowest MIC values for gram-positive bacteria including methicillin-resistant Staphylococcus aureus as well as against two fungal strains of Candida glabrata and Trichophyton mentagrophytes within the range of ≤0.49-3.9 µM. The convenient substitution of benzylidene moiety at the position 4 or the presence of 5-chloro-2-hydroxybenzylidene scaffold concomitantly with a sufficient lipophilicity are essential for the noticeable antimicrobial activity. This 5-chlorosalicylidene derivative avoided any cytotoxicity on two mammalian cell cultures (HepG2, BMMΦ) up to the concentration of 100 µM, but it affected the growth of MonoMac6 cells.

N-Alkoxyphenylhydroxynaphthalenecarboxamides and Their Antimycobacterial Activity.

A series of nineteen N-(alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides and a series of their nineteen positional isomers N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides were prepared and characterized. Primary in vitro screening of all the synthesized compounds was performed against Mycobacterium tuberculosis H37Ra, M. kansasii and M. smegmatis. Screening of the cytotoxicity of the compounds was performed using human monocytic leukemia THP-1 cells. Some of the tested compounds showed antimycobacterial activity comparable with or higher than that of rifampicin. For example, 2-hydroxy-N-(4-propoxyphenyl)-naphthalene-1-carboxamide showed the highest activity (MIC = 12 µM) against M. tuberculosis with insignificant cytotoxicity. N-[3-(But-2-yloxy)phenyl]- and N-[4-(but-2-yloxy)phenyl]-2-hydroxy-naphthalene-1-carboxamide demonstrated high activity against all tested mycobacterial strains and insignificant cytotoxicity. N-(Alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides demonstrated rather high effect against M. smegmatis and M. kansasii and strong antiproliferative effect against the human THP-1 cell line. Lipophilicity was found as the main physicochemical parameter influencing the activity. A significant decrease of mycobacterial cell metabolism (viability of M. tuberculosis H37Ra) was observed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. Structure-activity relationships are discussed.

Rapid drug tolerance and dramatic sterilizing effect of moxifloxacin monotherapy in a novel hollow-fiber model of intracellular Mycobacterium kansasii disease.

Mycobacterium kansasii is the second most common mycobacterial cause of lung disease. Standard treatment consists of rifampin, isoniazid, and ethambutol for at least 12 months after negative sputum. Thus, shorter-duration therapies are needed. Moxifloxacin has good MICs for M. kansasii. However, good preclinical models to identify optimal doses currently are lacking. We developed a novel hollow fiber system model of intracellular M. kansasii infection. We indexed the efficacy of the standard combination regimen, which was a kill rate of -0.08 ± 0.05 log10 CFU/ml/day (r(2) = 0.99). We next performed moxifloxacin dose-effect and dose-scheduling studies at a half-life of 11.1 ± 6.47 h. Some systems also were treated with the efflux pump inhibitor reserpine. The highest moxifloxacin exposure, as well as lower exposures plus reserpine, sterilized the cultures by day 7. This suggests that efflux pump-mediated tolerance at low ratios of the area under the concentration-time curve from 0 to 24 h (AUC0 - 24) to MICs is an early bacterial defense mechanism but is overcome by higher exposures. The highest rate of moxifloxacin monotherapy sterilization was -0.82 ± 0.15 log10 CFU/ml/day (r(2) = 0.97). The moxifloxacin exposure associated with 80% of maximal kill (EC80) was an AUC0-24/MIC of 317 (the non-protein-bound moxifloxacin AUC0-24/MIC was 158.5). We performed Monte Carlo simulations of 10,000 patients in order to identify the moxifloxacin dose that would achieve or exceed the EC80. The simulations revealed an optimal moxifloxacin dose of 800 mg a day. The MIC susceptibility breakpoint at this dose was 0.25 mg/liter. Thus, moxifloxacin, at high enough doses, is suitable to study in patients for the potential to add rapid sterilization to the standard regimen.

Synthesis and biological activity of new salicylanilide N,N-disubstituted carbamates and thiocarbamates.

The development of novel antimicrobial drugs represents a cutting edge research topic. In this study, 20 salicylanilide N,N-disubstituted carbamates and thiocarbamates were designed, synthesised and characterised by IR, (1)H NMR and (13)C NMR. The compounds were evaluated in vitro as potential antimicrobial agents against Mycobacterium tuberculosis and nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii) as well as against eight bacterial and fungal strains. Additionally, we investigated the inhibitory effect of these compounds on mycobacterial isocitrate lyase and cellular toxicity. The minimum inhibitory concentrations (MICs) against mycobacteria were from 4 µM for thiocarbamates and from 16 µM for carbamates. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited with MICs from 0.49 µM by thiocarbamates, whilst Gram-negative bacteria and most of the fungi did not display any significant susceptibility. All (thio)carbamates mildly inhibited isocitrate lyase (up to 22%) at a concentration of 10 µM. The (thio)carbamoylation of the parent salicylanilides led to considerably decreased cytotoxicity and thus improved the selectivity indices (up to 175). These values indicate that some derivatives are attractive candidates for future research.

An unusual lung mass post stem cell transplantation.

We report a case of Mycobacterium kansasii presenting as an obstructing endobronchial mass in a patient post stem cell transplant. The patient had a complete clinical, microbiological, and radiological response to anti-tuberculous treatment. To our knowledge, this is the first case of M. kansasii presenting post transplant with an obstructing lung mass simulating relapse or post-transplant lymphoma.

Diethyl 2-(phenylcarbamoyl)phenyl phosphorothioates: synthesis, antimycobacterial activity and cholinesterase inhibition.

A new series of 27 diethyl 2-(phenylcarbamoyl)phenyl phosphorothioates (thiophosphates) was synthesized, characterized by NMR, IR and CHN analyses and evaluated against Mycobacterium tuberculosis H37Rv, Mycobacterium avium and two strains of Mycobacterium kansasii. The best activity against M. tuberculosis was found for O-{4-bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (minimum inhibitory concentration of 4 µM). The highest activity against nontuberculous mycobacteria was exhibited by O-(5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}-phenyl) O,O-diethyl phosphorothioate with MIC values from 16 µM. Prepared thiophosphates were also evaluated against acetylcholinesterase from electric eel and butyrylcholinesterase from equine serum. Their inhibitory activity was compared to that of the known cholinesterases inhibitors galanthamine and rivastigmine. All tested compounds showed a higher (for AChE inhibition) and comparable (for BChE inhibition) activity to that of rivastigmine, with IC50s within the 8.04 to 20.2 µM range.

N-substituted 2-isonicotinoylhydrazinecarboxamides--new antimycobacterial active molecules.

This report presents a new modification of the isoniazid (INH) structure linked with different anilines via a carbonyl group obtained by two synthetic procedures and with N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation. All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses and were evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4-octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1-2 µM. Among the halogenated derivatives, the best anti-tuberculosis activity was found for 2-isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (MIC=4 µM). In silico modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl substituents are advantageous for the interactions and affinity to InhA. Most of the hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited significant activity against INH-resistant nontuberculous mycobacteria.

Omadacycline drug susceptibility testing for non-tuberculous mycobacteria using oxyrase to overcome challenges with drug degradation.

BACKGROUND: Drug susceptibility testing (DST) protocol of omadacycline against non-tuberculous mycobacteria has not yet been established. We developed a method to accurately determine MIC omadacycline MIC against Mycobacterium abscessus (Mab), Mycobacterium avium-complex (MAC), and Mycobacterium kansasii (Mkn). METHODS: First, we identified the oxyrase concentration not affecting Mab, MAC, and Mkn growth followed by omadacycline MIC experiments with and without oxyrase using reference and clinical strains. RESULTS: Oxyrase 0.5 % (v/v) stabilized omadacycline in the culture medium. The median omadacycline MIC was 1 mg/L for Mab and 8 mg/L for Mkn. For MAC, the median omadacycline MIC was 2 mg/L for M. avium, 256 mg/L for M. intracellulare, and 4 mg/L for M. chimaera (p < 0.0001). Wilcoxon matched-pairs signed rank test revealed statistically lower MICs with oxyrase for all MAC subspecies (p < 0.0001), all Mab subspecies (p < 0.0001), and Mkn (p = 0.0002). The decrease in MICs with oxyrase was 17/18 of Mab, 14/19 of Mkn, 8/8 of M. avium, 4/5 M. chimera, but only 11/18 of M. intracellulare (p < 0.013). CONCLUSION: Use of 0.5 % oxyrase could be a potential solution to reliable and reproducible omadacycline MIC of Mab. However, oxyrase demonstrated a variable effect in reducing MICs against MAC and Mkn.

Clinical and therapeutic features of pulmonary nontuberculous mycobacterial disease, Brazil, 1993-2011.

To identify clinical and therapeutic features of pulmonary nontuberculous mycobacterial (PNTM) disease, we conducted a retrospective analysis of patients referred to the Brazilian reference center, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, who received a diagnosis of PNTM during 1993­2011 with at least 1 respiratory culture positive for NTM. Associated conditions included bronchiectasis (21.8%), chronic obstructive pulmonary disease (20.7%), cardiovascular disease (15.5%), AIDS (9.8%), diabetes (9.8%), and hepatitis C (4.6%).Two patients had Hansen disease; 1 had Marfan syndrome. Four mycobacterial species comprised 85.6% of NTM infections: Mycobacterium kansasii, 59 cases (33.9%); M. avium complex, 53 (30.4%); M. abscessus, 23 (13.2%); and M. fortuitum, 14 (8.0%). A total of 42 (24.1%) cases were associated with rapidly growing mycobacteria. In countries with a high prevalence of tuberculosis, PNTM is likely misdiagnosed as tuberculosis, thus showing the need for improved capacity to diagnose mycobacterial disease as well as greater awareness of PNTM disease prevalence.

Mutations in the essential FAS II ß-hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii.

It has recently been shown that the anti-mycobacterial pro-drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build-up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that 'de novo' biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC-resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti-mycobacterial compound.

Drug susceptibility distributions in slowly growing non-tuberculous mycobacteria using MGIT 960 TB eXiST.

In general, uniform clinical antibiotic susceptibility breakpoints (CBPs) for slowly growing nontuberculous mycobacteria (NTM) have not been established. The aim of this study was to determine wild-type drug susceptibility distributions for relevant antibiotics using Bactec MGIT 960 equipped with EpiCenter TB eXiST and to derive epidemiological cut-offs (ECOFFs) from semi quantitative drug susceptibility measurements. One hundred and twenty-six NTM clinical isolates (Mycobacterium avium n=58, Mycobacterium intracellulare n=18, Mycobacterium kansasii n=50) were investigated in this study. Drug susceptibility distributions and MIC90 values were determined for clarithromycin, ethambutol, rifampicin, rifabutin, ofloxacin, moxifloxacin, and amikacin using Bactec MGIT 960/EpiCenter TB eXiST. For most species/drug combinations ECOFFs were determined. For some species/drug combinations ECOFFs were not defined as either the isolates were susceptible to the lowest drug concentration tested or because isolates, in part, had MIC levels exceeding the highest drug concentration tested. This study describes drug susceptibility distributions and MIC90 values of M. avium, M. intracellulare, and M. kansasii that may aid the definition of CBPs when correlating in vitro drug susceptibility with clinical outcomes in future studies.