Identification of gene targets that potentiate the action of rifampicin on Mycobacterium bovis BCG.

Tuberculosis (TB) caused by bacteria of the Mycobacterium tuberculosis complex remains one of the most important infectious diseases of mankind. Rifampicin is a first line drug used in multi-drug treatment of TB, however, the necessary duration of treatment with these drugs is long and development of resistance is an increasing impediment to treatment programmes. As a result, there is a requirement for research and development of new TB drugs, which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as rifampicin. This study describes a TnSeq analysis to identify mutants with enhanced sensitivity to sub-minimum inhibitory concentrations (MIC) of rifampicin. The rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups: firstly, genes that were involved in DNA/RNA metabolism, secondly, genes involved in sensing and regulating mycobacterial cellular systems, and thirdly, genes involved in the synthesis and maintenance of the cell wall. Selection at two concentrations of rifampicin (1/250 and 1/62 MIC) demonstrated a dose response for mutants with statistically significant sensitivity to rifampicin. The dataset reveals mechanisms of how mycobacteria are innately tolerant to and initiate an adaptive response to rifampicin; providing putative targets for the development of adjunctive therapies that potentiate the action of rifampicin.

Koumiss promotes Mycobacterium bovis infection by disturbing intestinal flora and inhibiting endoplasmic reticulum stress.

Mycobacterium bovis is the causative agent of bovine tuberculosis and also responsible for serious threat to public health. Koumiss is a fermented mare's milk product, used as traditional drink. Here, we explored the effect of koumiss on gut microbiota and the host immune response against M bovis infection. Therefore, mice were treated with koumiss and fresh mare milk for 14 days before M bovis infection and continue for 5 weeks after infection. The results showed a clear change in the intestinal flora of mice treated with koumiss, and the lungs of mice treated with koumiss showed severe edema, inflammatory infiltration, and pulmonary nodules in M bovis-infected mice. Notably, we found that the content of short-chain fatty acids was significantly lower in the koumiss-treated group compared with the control group. However, the expression of endoplasmic reticulum stress and apoptosis-related proteins in the lungs of koumiss-treated mice were significantly decreased. Collectively, these findings suggest that koumiss treatment disturb the intestinal flora of, which is associated with disease severity and the possible mechanism that induces lungs pathology. Our current findings can be exploited further to establish the "gut-lung" axis which might be a novel strategy for the control of tuberculosis.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Design and synthesis of novel 5-alkynyl pyrimidine nucleosides derivatives: Influence of C-6-substituent on antituberculosis activity.

We herein report new 5-substituted uridine derivatives as potent inhibitors of mycobacteria - causative agents of tuberculosis. A series of new 5-alkynyl-substituted uridine derivatives were synthesised via palladium-catalysed Sonogashira cross-coupling reaction of 5-iodo-6-methylpyrimidine base with terminal acetylenes with good yields in DMF at room temperature. It was found that methyl group in C-6 position of pyrimidine ring had no impact on yields of target compounds. All obtained compounds were evaluated for their antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis at concentrations of 1-100 µg/ml using MABA test. Synthesized nucleosides showed high antimycobacterial activity against M. bovis and M. Tuberculosis. The MIC50 values of 11 and 13 were similar or close to that of the reference drug rifampicin.

Identification of inhibitors of UDP-galactopyranose mutase via combinatorial in situ screening.

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.

Synthesis of new 2-(thiazol-4-yl)thiazolidin-4-one derivatives as potential anti-mycobacterial agents.

To search for potent antimycobacterial lead compounds, a new series of 3-substituted phenyl-2-(2-(substituted phenyl)thiazol-4-yl) thiazolidin-4-one (5a-t) derivatives have been synthesized by the condensation of 2-substituted phenyl thiazole-4-carbaldehyde with aromatic amine followed by cyclocondensation with thioglycolic acid. The structure of the newly synthesized 2-(thiazol-4-yl)thiazolidin-4-one derivatives were characterized by the spectroscopic analysis. The synthesized compounds were screened for antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MTB) (ATCC 25177) and Mycobacterium bovis BCG (BCG, ATCC 35743). Most of the 2-(thiazol-4-yl)thiazolidin-4-one derivatives showed good to excellent antimycobacterial activity against both the Mtb strains. Nine derivatives 5c, 5g, 5j, 5m, 5n, 5o, 5p, 5s, and 5t showed excellent activity against M. bovis BCG with MIC 4.43 to 24.04 µM were further evaluated for the cytotoxicity activity against HeLa A549, and HCT-116 cell lines and showed no significant cytotoxic activity at the maximum concentration evaluated. The potential antimycobacterial activities enforced that the thiazolyl-thiazolidin-4-one derivatives could lead to compounds that could treat tuberculosis.

Loperamide exerts a direct bactericidal effect against M. tuberculosis, M. bovis, M. terrae and M. smegmatis.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis. TB is highly prevalent, characterized by the constant occurrence of drug-resistant cases, and confounded by the incidence of respiratory disease caused by non-tuberculous mycobacteria (NTB). Expanding the spectrum of drugs for the treatment of TB is indispensable. Loperamide, an antidiarrhoeal drug, enhances immune-driven antimycobacterial activity, and we aimed to evaluate its bactericidal activity against M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium terrae and Mycobacterium smegmatis. Loperamide exhibited an inhibitory effect against all mycobacterial species tested, with MICs of 100 and 150 µg ml-1 . Thus, loperamide is a mycobactericidal drug with potential as adjunctive therapy for TB and NTB infections.

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG.

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.

Silicea terra and Zincum metallicum Modulate the Activity of Macrophages Challenged with BCG In Vitro.

BACKGROUND: The homeopathic medicines Silicea terra (Sil) and Zincum metallicum (Zinc) modulate macrophage activity and were assessed in an experimental study in-vitro for their effects on macrophage-BCG (Bacillus Calmette-Guérin) interaction. METHODS: RAW 264.7 macrophages were infected with BCG, treated with different potencies of Sil and Zinc (6cH, 30cH and 200cH) or vehicle, and assessed 24 and 48 h later for bacilli internalization, hydrogen peroxide (H2O2) and cytokine production, and lysosomal activity. RESULTS: Treatment with vehicle was associated with non-specific inhibition of H2O2 production to the levels exhibited by uninfected macrophages. Sil 200cH induced significant reduction of H2O2 production (p < 0.001) compared with the vehicle and all other treatments, as well as higher lysosomal activity (p ≤ 0.001) and increased IL-10 production (p ≤ 0.05). Such effects were considered specific for this remedy and potency. The number of internalized bacilli was inversely proportional to Zinc potencies, with statistically significant interaction between dilution and treatment (p = 0.003). Such linear-like behavior was not observed for Sil dilutions: peak internalization occurred with the 30cH dilution, accompanied by cellular degeneration, and IL-6 and IL-10 increased (p ≤ 0.05) only in the cells treated with Sil 6cH. CONCLUSION: Sil and Zinc presented different patterns of potency-dependent effect on macrophage activity. Bacterial digestion and a balanced IL-6/IL-10 production were related to Sil 6cH, though reduced oxidative stress with increased lysosomal activity was related to Sil 200cH. Degenerative effects were exclusively related to Sil 30cH, and potency-dependent phagocytosis was related only to Zinc.

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms.

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae - Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum - and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis. The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

The synthesis and antituberculosis activity of 5-alkynyl uracil derivatives.

A series of new 5-alkynyl-substituted uracil and uridine derivatives were synthesised via palladium-catalysed Sonogashira cross-coupling reaction of 5-bromo-pyrimidine base with terminal acetylenes with good yields in DMF at room temperature. All obtained compounds were tested for antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis (H37Ra) at concentrations of 1-100 µg/ml using MABA test. Obtained results revealed that most of tested uracil derivatives exhibited high antimycobacterial activity (MIC50 = 1.1-19.2 µg/ml) in comparison with therapeutic agents such as rifampicin, isoniazid and d-cycloserine, excluding compounds having alkyl substituent at triple alkyne bond.

Synthesis of Novel Sulfamethaoxazole 4-Thiazolidinone Hybrids and Their Biological Evaluation.

A search for potent antitubercular agents prompted us to design and synthesize sulfamethaoxazole incorporated 4-thiazolidinone hybrids (7a-l) by using a cyclocondensation reaction between 4-amino-N-(5-methylisoxazol-3-yl)benzenesulfonamide (4), aryl aldehyde (5a-l), and mercapto acetic acid (6) resulting in good to excellent yields. All the newly synthesized 4-thiazolidinone derivatives were screened for their in vitro antitubercular activity against M. Bovis BCG and M. tuberculosis H37Ra (MTB) strains. The compounds 7d, 7g, 7i, 7k, and 7l revealed promising antimycobacterial activity against M. Bovis and MTB strains with IC90 values in the range of 0.058-0.22 and 0.43-5.31 µg/mL, respectively. The most active compounds were also evaluated for their cytotoxicity against MCF-7, HCT 116, and A549 cell lines and were found to be non-cytotoxic. Moreover, the synthesized compounds were also analyzed for ADME (absorption, distribution, metabolism, and excretion) properties and showed potential as good oral drug candidates.

A Rapid Unraveling of the Activity and Antibiotic Susceptibility of Mycobacteria.

The development of antibiotic-resistant bacteria is a worldwide health-related emergency that calls for new tools to study the bacterial metabolism and to obtain fast diagnoses. Indeed, the conventional analysis time scale is too long and affects our ability to fight infections. Slowly growing bacteria represent a bigger challenge, since their analysis may require up to months. Among these bacteria, Mycobacterium tuberculosis, the causative agent of tuberculosis, has caused more than 10 million new cases and 1.7 million deaths in 2016 only. We employed a particularly powerful nanomechanical oscillator, the nanomotion sensor, to characterize rapidly and in real time tuberculous and nontuberculous bacterial species, Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium abscessus, respectively, exposed to different antibiotics. Here, we show how high-speed and high-sensitivity detectors, the nanomotion sensors, can provide a rapid and reliable analysis of different mycobacterial species, obtaining qualitative and quantitative information on their responses to different drugs. This is the first application of the technique to tackle the urgent medical issue of mycobacterial infections, evaluating the dynamic response of bacteria to different antimicrobial families and the role of the replication rate in the resulting nanomotion pattern. In addition to a fast analysis, which could massively benefit patients and the overall health care system, we investigated the real-time responses of the bacteria to extract unique information on the bacterial mechanisms triggered in response to antibacterial pressure, with consequences both at the clinical level and at the microbiological level.

Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.

NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G.

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.

The prominent alteration in transcriptome and metabolome of Mycobacterium bovis BCG str. Tokyo 172 induced by vitamin B1.

BACKGROUND: Vitamin B1 (VB1) is a crucial dietary nutrient and essential cofactor for several key enzymes in the regulation of cellular and metabolic processes, and more importantly in the activation of immune system. To date, the precise role of VB1 in Mycobacterium tuberculosis remains to be fully understood. RESULTS: In this study, the transcriptional and metabolic profiles of VB1-treated Mycobacterium. bovis BCG were analyzed by RNA-sequencing and LC-MS (Liquid chromatography coupled to mass spectrometry). The selection of BCG strain was based on its common physiological features shared with M. tuberculosis. The results of cell growth assays demonstrated that VB1 inhibited the BCG growth rate in vitro. Transcriptomic analysis revealed that the expression levels of genes related to fatty acid metabolism, cholesterol metabolism, glycolipid catabolism, DNA replication, protein translation, cell division and cell wall formation were significantly downregulated in M. bovis BCG treated with VB1. In addition, the metabolomics LC-MS data indicated that most of the amino acids and adenosine diphosphate (ADP) were decreased in M. bovis BCG strain after VB1 treatment. CONCLUSIONS: This study provides the molecular and metabolic bases to understand the impacts of VB1 on M.bovis BCG.

Case report: systemic tuberculosis caused by Mycobacterium bovis in a cat.

BACKGROUND: The diagnosis of previous cases of feline tuberculosis in Turkey has been made based solely on pathological changes without isolation of the causative agent. This case report details the first case of feline tuberculosis in Turkey for which the causative agent (Mycobacterium bovis) was confirmed with microbiological isolation, morphological evaluation, molecular (PCR) characterization and antibiotic sensitivity. CASE PRESENTATION: Systemic tuberculosis was diagnosed via postmortem examination of a 5-year-old stray male cat. Mycobacterium bovis was isolated from the lungs, bronchial and gastrointestinal lymph nodes, kidney and liver. The isolate was defined as M. bovis using the Genotype MTBC assay (Hain Lifescience, Germany), which allows differentiation of species within the Mycobacterium tuberculosis complex with an easy-to-perform reverse hybridization assay. Pathological changes were characterized by multifocal to coalescing granulomatous inflammation in the lungs, liver, lymph nodes and kidneys. Further pathological changes included severe, diffuse, hepatocytic atrophy, periportal fibrosis with lymphohistiocytic infiltration, multifocal lymphohistiocytic interstitial nephritis, mild focal pulmonary anthracosis and mild renal and hepatic amyloidosis. Infection by immunosuppressive viral pathogens including feline herpes virus-1, feline immunodeficiency virus and feline parvovirus virus were ruled out by polymerase chain reaction assay (PCR). The isolated mycobacteria were susceptible to isoniazid, ethambutol, rifampicin or streptomycin. CONCLUSION: Disseminated M. bovis is a rare infection in cats. Involvement of submandibular lymph nodes suggested that primary transmission might have been the oral route in the present case.

Cell Wall Hydrolytic Enzymes Enhance Antimicrobial Drug Activity Against Mycobacterium.

Cell wall hydrolases are enzymes that cleave bacterial cell walls by hydrolyzing specific bonds within peptidoglycan and other portions of the envelope. Two major sources of hydrolases in nature are from hosts and microbes. This study specifically investigated whether cell wall hydrolytic enzymes could be employed as exogenous reagents to augment the efficacy of antimicrobial agents against mycobacteria. Mycobacterium smegmatis cultures were treated with ten conventional antibiotics and six anti-tuberculosis drugs-alone or in combination with cell wall hydrolases. Culture turbidity, colony-forming units (CFUs), vital staining, and oxygen consumption were all monitored. The majority of antimicrobial agents tested alone only had minimal inhibitory effects on bacterial growth. However, the combination of cell wall hydrolases and most of the antimicrobial agents tested, revealed a synergistic effect that resulted in significant enhancement of bactericidal activity. Vital staining showed increased cellular damage when M. smegmatis and Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) were treated with both drug and lysozyme. Respiration analysis revealed stress responses when cells were treated with lysozyme and drugs individually, and an acute increase in oxygen consumption when treated with both drug and lysozyme. Similar trends were also observed for the other three enzymes (hydrolase-30, RipA-His6 and RpfE-His6) evaluated. These findings demonstrated that cell wall hydrolytic enzymes, as a group of biological agents, have the capability to improve the potency of many current antimicrobial drugs and render ineffective antibiotics effective in killing mycobacteria. This combinatorial approach may represent an important strategy to eliminate drug-resistant bacteria.

New insight into structure-activity of furan-based salicylate synthase (MbtI) inhibitors as potential antitubercular agents.

Starting from the analysis of the hypothetical binding mode of our previous furan-based hit (I), we successfully achieved our objective to replace the nitro moiety, leading to the disclosure of a new lead exhibiting a strong activity against MbtI. Our best candidate 1 h displayed a Ki of 8.8 µM and its antimycobacterial activity (MIC99 = 250 µM) is conceivably related to mycobactin biosynthesis inhibition. These results support the hypothesis that 5-phenylfuran-2-carboxylic derivatives are a promising class of MbtI inhibitors.