The new attenuated strain of Mycobacterium tuberculosis BN. Characteristics and vaccine properties

The objective of the study: to obtain a live attenuated strain and investigate its properties by multiple cultures of the virulent strain of Mycobacterium tuberculosis H37Rv.Subjects and Methods. The original virulent strain H37Rv was subcultured 70 times in 7H9 liquid medium. Genetic properties of the new strain, degree of avirulence, and vaccine properties were studied.Results. Mycobacteria of the new strain MtbBN lost their virulence to inbred mice. Eight mutations were identified by whole genome sequencing: single nucleotide insertions and deletions (in/del) distinguishing the MtbBN and H37Rv strains. The MtbBN strain demonstrated vaccine potential at the BCG level. Additionally, in some genetic models, the attenuated strain was highly effective in protecting inbred mice when infected with Mtb H37Rv as opposed to BCG.

Effect of Thymoquinone on Th1 and Th2 Balance in Rats Infected with Mycobacterium tuberculosis

Thymoquinone is an active compound in Nigella sativa which has potential immunomodulatory effect. Mycobacterium tuberculosis could alter the Th1 and Th2 balance by stimulating phagocyte IL-1β production, and subsequent Th2 differentiation. We aim to evaluate the effect of thymoquinone (TQ) in restore the Th1 and Th2 balance in Mycobacterium tuberculosis infection. Four groups of rats were infected with virulent Mycobacterium tuberculosis strain H37Rv. Thymoquinone at different doses was given to three groups, and one group left without treatment. Additional one group was either infected or treated with TQ. We measure IL-1β, IL-4 and IFN-γ levels using ELISA 14 days after TQ treatment. We found there were increased IL-1β, IL-4 and IFN-γ level after Mycobacterium tuberculosis infection, but we observe no significant effect of TQ treatment to Th1 and Th2 balance. We conclude that TQ could not restore Th1 and Th2 balance in rats infected with Mycobacterium tuberculosis.

Black Cumin (Nigella sativa) Against Mycobacterium tuberculosis Strain H37RV And MDR-TB

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis. 10 million people suffer from TB Every year. Although TB is a preventable and treatable disease, 1.5 million people die every year due to TB. Alternative treatments continue to be pursued, and treatment with the latest TB drugs that are continuously being encouraged. Black cumin (Nigella sativa) seed contains essential oils with active compounds such as thymohydroquinone, Oleoresins, flavonoids, alkaloids, saponins, tannins, and terpenoids that act as antibacterial drugs. This study aims to determine the sensitivity of N. sativa seed extract in inhibiting the growth of M. tuberculosis strain H37RV and MDR-TB (Multidrug Resistance-TB). This research using Microscopic-Observation and Drug-Susceptibility Assay (MODS) method. Extraction of N. sativa was carried out by the maceration method using 70% methanol as a solvent. The results showed that the M. tuberculosis strain H37RV and MDR-TB were sensitive to N. sativa extract at concentrations of 5 and 10% but resistant to N. sativa extract at concentrations of 1 and 3%.Abstrak: Tuberkulosis (TB) adalah penyakit menular yang disebabkan oleh Bakteri Mycobacterium tuberculosis. Penyakit ini menimbulkan dampak kematian yang cukup mengkhawatirkan. Penyakit tersebut dapat dicegah dan diobati. Salah satu sumber pengobatannya menggunakan biji jintan hitam (Nigella sativa) yang mengandung minyak atsiri dengan senyawa aktif seperti timohidrokuinon, oleoresin, flavonoid, alkaloid, saponin, tanin, dan terpenoid yang berfungsi sebagai obat antibakteri. Penelitian ini bertujuan untuk mengetahui sensitivitas ekstrak biji N. sativa dalam menghambat pertumbuhan M. tuberculosis strain H37RV and MDR-TB (Multidrug-Resistance-TB). Penelitian ini menggunakan metode Microscopic-Observation and Drug-Susceptibility Assay (MODS). Ekstraksi N. sativa dilakukan dengan metode maserasi menggunakan pelarut metanol 70%. Hasil yang diperoleh menunjukkan bahwa bakteri M. tuberculosis strain H37RV dan TB-MDR, kedua strain tsb sensitif terhadap ekstrak N. sativa konsentrasi 5 dan 10%, tetapi resisten terhadap ekstrak N. sativa konsentrasi 1 dan 3%.

Synthesis of 2-Hydroxynaphthyl Pyrazolines Containing Isoniazid Moiety: A Potential Antitubercular Agents

: The new series of pyrazolines derivatives containing isoniazid moiety were synthesized from 2-hydroxynaphthyl functionalized chalcones and isoniazid using sodium hydroxide as a base in 2-ethoxy ethanol.We are evaluated for their antitubercular activity against Mycobacterium tuberculosis strain (H37Rv) by Microplate Alamar Blue Assay (MABA) some of the tested compounds 3a, 3b, and 3c, were found to have higher antitubercular activity than the selected standard drugs, where as the compounds 3d, 3e, 3i and 3j found to have higher antitubercular activity than Streptomycin and same as that of drug Pyrazinamide and Ciprofloxacin. While remaining compound showed moderate activity. Where as it is found that the disubstituted halogen compound and electron withdrawing group on the phenyl ring are important substitution for increase in antitubercular activity.

Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

Sterilization by Adaptive Immunity of a Conditionally Persistent Mutant of Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can enter into a persistent state that confers resistance to antibacterial agents. Many observations suggest that persistent M. tuberculosis cells also evade the antimycobacterial immune mechanisms, thereby reducing the effectiveness of the current tuberculosis vaccine. Understanding the factors that contribute to persistence may enable the rational design of vaccines that stimulate effective immune killing mechanisms against persister cells. Independent mutations targeting the methionine and arginine biosynthetic pathways are bactericidal for M. tuberculosis in mice. However, in this study, we discovered that the addition of leucine and pantothenate auxotrophy altered the bactericidality of methionine auxotrophy. Whereas the leucine/pantothenate/methionine auxotrophic M. tuberculosis strain H37Rv ΔleuCD ΔpanCD ΔmetA was eliminated in immunocompetent mice, this strain persisted in multiple organs of immunodeficient Rag1−/− mice for at least a year. In contrast, the leucine/pantothenate/arginine auxotroph H37Rv ΔleuCD ΔpanCD ΔargB was eliminated in both immunocompetent and immunodeficient Rag1−/− mice. Our results showed that leucine and pantothenate starvation metabolically blocked the sterilization mechanisms of methionine starvation but not those of arginine starvation. These triple-auxotrophic strains should be invaluable tools for unravelling the bacterial and host factors that enable persistence and for vaccine development studies to assess the efficacy of vaccines that boost immune recognition of M. tuberculosis in the persistent state. The sterilization of the ΔleuCD ΔpanCD ΔmetA auxotroph in immunocompetent mice, but not in mice lacking an adaptive immune response, could provide a new system for studying the antimycobacterial killing mechanisms of adaptive immunity. IMPORTANCE The bacterial pathogen Mycobacterium tuberculosis can enter into a persistent state in which M. tuberculosis can evade host immunity, thereby reducing the effectiveness of current tuberculosis vaccines. Understanding the factors that contribute to persistence would enable the rational design of vaccines effective against persisters. We previously generated two attenuated, triple-auxotrophic M. tuberculosis strains that are safe to use in a biosafety level 2 laboratory. Herein, we discovered that the triple-auxotrophic strain H37Rv ΔleuCD ΔpanCD ΔmetA persisted in immunodeficient Rag1−/− mice, which lack adaptive immunity, but not in immunocompetent mice. The conditional persistence of this auxotrophic mutant, which is susceptible to the sterilizing effect of the adaptive immune response over time, provides an important tool to dissect the mycobactericidal effector mechanisms mediated by adaptive immunity. Furthermore, because of its remarkable safety attributes, this auxotrophic mutant can potentially be used to develop a practical human challenge model to facilitate vaccine development.

Efficacy and immunogenicity of different BCG doses in BALB/c and CB6F1 mice when challenged with H37Rv or Beijing HN878

In this study, 2 strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guérin (BCG) Danish doses from 3×105 to 30 CFU/mouse, followed by either immunogenicity evaluation or aerosol infection with Mycobacterium tuberculosis (a laboratory strain H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3×105 – 3000 CFU (BALB/c) and 3×105-300 CFU (CB6F1). Whereas, both strains of mice when infected with H37Rv, significant protection was seen in BCG doses at 3×105 - 300 CFU. Immunological evaluation revealed interesting results; i) both strains of mice demonstrated a significant increase in the frequencies of BCG-specific IFNγ+ IL2+ TNFα+ CD4 T cells in the BCG doses at 3×105 – 3000 CFU (BALB/c) and 3×105 - 300 CFU (CB6F1); ii) secretion of IL2 and IFNγ were correlated with the bacterial burden in the lungs of HN878 infected CB6F1 mice. The study demonstrated a BCG dose at 3000 CFU (an equivalent single human dose in the mice by body weight index) is protective in both strains of mice and the use of a virulent clinical isolate in testing new tuberculosis vaccine/advancing research is recommended.

Effects of Cigarette Smoke Condensate on Growth and Biofilm Formation by Mycobacterium tuberculosis

Background and Objectives. Cigarette smoke (CS) is a major risk factor contributing to the burden of tuberculosis. Little is known, however, about the effects of CS exposure on growth and persistence of Mycobacterium tuberculosis (Mtb) organisms. This issue has been addressed in the current study, which is focused on the effects of cigarette smoke condensate (CSC) on the growth and viability of Mtb planktonic and biofilm-forming cultures. Materials and Methods. The planktonic and biofilm-forming cultures were prepared in Middlebrook 7H9 and Sauton broth media, respectively, using Mtb strain, H37Rv. The effects of CSC at concentrations of 0.05-3.12 mg/L on growth, biofilm formation and structure were evaluated using microplate Alamar Blue assay, spectrophotometric procedure and scanning electron microscopy (SEM), respectively. Involvement of reactive oxygen species in CSC-mediated biofilm formation was investigated by including catalase in biofilm-forming cultures. Results. CSC did not affect the growth of planktonic bacteria, but rather led to a statistically significant increase in biofilm formation at concentrations of 0.4-3.12 mg/L, as well as in the viability of biofilm-forming bacteria at CSC concentrations of 0.2-1.56 mg/L. SEM confirmed an agglomerated biofilm matrix and irregular bacterial morphology in CSC-treated biofilms. Inclusion of catalase caused significant attenuation of CSC-mediated augmentation of biofilm formation by Mtb, implying involvement of oxidative stress. These findings demonstrate that exposure of Mtb to CSC resulted in increased biofilm formation that appeared to be mediated, at least in part, by oxidative stress, while no effect on planktonic cultures was observed. Conclusion. Smoking-related augmentation of biofilm formation by Mtb may contribute to persistence of the pathogen, predisposing to disease reactivation and counteracting the efficacy of antimicrobial chemotherapy.

1-(1-Arylethylpiperidin-4-yl)thymine Analogs as Antimycobacterial TMPK Inhibitors

A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 μM).

DESENVOLVIMENTO DE PRIMERS IN SILICO DE Mycobacterium tuberculosis DA REGIÃO 16S PARA O DIAGNÓSTICO DA TUBERCULOSE

Mycobacterium tuberculosis é uma bactéria agente causadora responsável por grande parte dos casos de tuberculose (TB), mostrando-se como um grande problema de saúde pública. A TB é uma doença infectocontagiosa que atinge principalmente os pulmões, além de outros órgãos como rins, intestinos e meninges. O objetivo deste estudo foi desenhar e analisar primers de M. tuberculosis strain H37Rv da região 16S do rDNA para fins de diagnóstico molecular da tuberculose pela Reação em Cadeia da Polimerase (PCR). Foi utilizado a plataforma NCBI para obter a sequência completa do gene específico de M. tuberculosis, em seguida, cinco primers foram desenhados pela ferramenta BLAST do NCBI e analisados pela IDT integrated DNA Technologies, utilizando a ferramenta OligoAnalyzer. A partir da análise e desenho in silico dos cinco primers, foi observado que todos eles estavam dentro dos parâmetros estabelecidos. Sendo assim, os primers em teoria são eficientes para amplificar pares de bases e ser útil para o diagnóstico da tuberculose.