Mycobacterial Strains Research Articles

Improving theantimicrobial activity of oldantibacterial drug mafenide: Schiff bases and their bioactivity targeting resistant pathogens.

Background: Increasing rates of acquired resistance have justified the critical need for novel antimicrobial drugs. One viable concept is the modification of known drugs. Methods & results: 21 mafenide-based compounds were prepared via condensation reactions and screened for antimicrobial efficacy, which demonstrated promising activity against both Gram-positive and Gram-negative pathogens, pathogenic fungi and mycobacterial strains (minimum inhibitory concentrations from 3.91μM). Importantly, they retained activity against a panel of superbugs (methicillin- and vancomycin-resistant staphylococci, enterococci, multidrug-resistant Mycobacterium tuberculosis) without any cross-resistance. Unlike mafenide, most of its imines were bactericidal. Toxicity to HepG2 cells was also investigated. Conclusion: Schiff bases were significantly more active than the parent drug, with iodinated salicylidene and 5-nitrofuran/thiophene-methylidene scaffolds being preferred in identifying the most promising drug candidates.

Comparison of Autof Ms1000 and EXS3000 MALDI-TOF MS Platforms for Routine Identification of Microorganisms.

Matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF) has recently been widely used in clinical microbiology laboratories, with the advantages of being reliable, rapid, and cost-effective. Here, we reported the performance of two MALDI-TOF MS instruments, EXS3000 (Zybio, China) and Autof ms1000 (Autobio, China), which are commonly used in clinical microbiology field. A total of 209 common clinical common isolates, including 70 gram-negative bacteria strains, 58 gram-positive bacteria strains, 33 yeast strains, 15 anaerobic bacteria strains, and 33 mold strains, and 19 mycobacterial strains were tested. All strains were identified by EXS3000 (Zybio, China) and Autof ms1000 (Autobio, China). Sequence analysis of 16S rRNA or ITS regions was used to verify all strains. Current study found that species-level discrimination was found to be 191 (91.39%) and 190 (90.91%) by EXS3000 and Autof ms1000, respectively. Genus-level discrimination was 205 (98.09%) by the EXS3000 and 205 (98.09%) by the Autof ms1000, respectively. The correct results at species level of the EXS3000 were 91.43% (64/70) for gram-negative bacteria, 93.1% (54/58) for gram-positive cocci, 93.94% (31/33) for yeast, 100% (15/15) for anaerobes and 81.82% (27/33) for filamentous fungi. The correct results at species level of the Autof ms1000 were 92.86% (65/70) for gram-negative bacteria, 91.38% (53/58) for gram-positive cocci, 93.94% (31/33) for yeast, 100% (15/15) for anaerobes and 78.79% (26/33) for filamentous fungi. Although the results show that the EXS3000 and Autof ms1000 systems are equally good choices in terms of analytical efficiency for routine procedures, the test result of EXS3000 is slightly better than Autof ms1000. It's worth mentioning that the target plate of the EXS 3000 instrument is reusable, but the target plate of the Autof ms1000 is disposable, making the EXS3000 more effective in reducing costs.

Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway

4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δkstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Hybridization Approach Toward Novel Antituberculars: Design, Synthesis, and Biological Evaluation of Compounds Combining Pyrazinamide and 4-Aminosalicylic Acid.

Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our long-term research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4-aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11') had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colony-forming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11' opens in human plasma to its parent compound 11 (t1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t1/2 of compound 11 overcomes the main drawback of PAS (short t1/2 necessitating frequent administration of high doses of PAS).

Fighting Tuberculosis: In Search of a BCG Replacement.

Tuberculosis is one of the most threatening infectious diseases and represents an important and significant reason for mortality in high-burden regions. The only licensed vaccine, BCG, is hardly capable of establishing long-term tuberculosis protection and is highly variable in its effectiveness. Even after 100 years of BCG use and research, we still cannot unequivocally answer the question of which immune correlates of protection are crucial to prevent Mycobacterium tuberculosis (Mtb) infection or the progression of the disease. The development of a new vaccine against tuberculosis arises a nontrivial scientific challenge caused by several specific features of the intracellular lifestyle of Mtb and the ability of the pathogen to manipulate host immunity. The purpose of this review is to discuss promising strategies and the possibilities of creating a new vaccine that could replace BCG and provide greater protection. The considered approaches include supplementing mycobacterial strains with immunodominant antigens and genetic engineering aimed at altering the interaction between the bacterium and the host cell, such as the exit from the phagosome. Improved new vaccine strains based on BCG and Mtb undergoing clinical evaluation are also overviewed.

Synthesis, Biological Evaluation and Computational Studies of New Hydrazide Derivatives Containing 1,3,4-Oxadiazole as Antitubercular Agents

To extend our screening for novel antimycobacterial molecules, we have designed, synthesized, and biologically evaluated a library of 14 new hydrazide derivatives containing 1,3,4-oxadiazole core. A variety of mycobacterial strains, including some drug-resistant strains, were tested for antimycobacterial activity. Among the compounds tested, five showed high antimycobacterial activity (MIC values of 8 μg/mL) against M. tuberculosis H37Ra attenuated strain, and two derivatives were effective (MIC of 4 µg/mL) against pyrazinamide-resistant strains. Furthermore, the novel compounds were tested against the fungal C. albicans strain, showing no antimycotic activity, and thus demonstrating a good selectivity profile. Notably, they also exhibited low cytotoxicity against human SH-SY5Y cells. The molecular modeling carried out suggested a plausible mechanism of action towards the active site of the InhA enzyme, which confirmed our hypothesis. In conclusion, the active compounds were predicted in silico for ADME properties, and all proved to be potentially orally absorbed in humans.

Design, Synthesis and Antimicrobial Properties of New Tetracyclic Quinobenzothiazine Derivatives.

A new method for modifying the structure of tetracyclic quinobenzothiazinium derivatives has been developed, allowing introduction of various substituents at different positions of the benzene ring. The method consists of reacting appropriate aniline derivatives with 5,12-(dimethyl)thioquinantrenediinium bis-chloride. A series of new quinobenzothiazine derivatives was obtained with propyl, allyl, propargyl and benzyl substituents in 9, 10 and 11 positions, respectively. The structure of the obtained compounds was analyzed by 1H and 13C NMR (HSQC, HMBC) and X-ray analysis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, and representatives of multidrug-resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). In addition, all the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. 9-Benzyloxy-5-methyl-12H-quino [3,4-b][1,4]benzothiazinium chloride (6j), 9-propoxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6a) and 9-allyloxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6d) demonstrated high activity against the entire tested microbial spectrum. The activities of the compounds were comparable with oxacillin, tetracycline and ciprofloxacinagainst staphylococcal strains and with rifampicin against both mycobacterial strains. Compound 6j had a significant effect on the inhibition of bacterial respiration as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity, but also bactericidal activity. Preliminary in vitro cytotoxicity screening of the compounds performed using normal human dermal fibroblasts (NHDF) proved that the tested compounds showed an insignificant cytotoxic effect on human cells (IC50 > 37 µM), making these compounds interesting for further investigation. Moreover, the intermolecular similarity of novel compounds was analyzed in the multidimensional space (mDS) of the structure/property-related in silico descriptors by means of principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The distance-oriented structure/property distribution was related with the experimental lipophilic data.

Advantages of Real-time PCR and RD4-PCR as Molecular Methods for Rapid Detection of Bovine Tuberculosis in Bulgaria

Representatives of Mycobacterium tuberculosis complex (MTBC) are potential causative agents of tuberculosis in animals and humans, and Mycobacterium bovis is considered a major one among domestic and wild ruminants. In the last twenty years, the role of another representative of MTBC – Mycobacterium caprae, has been proven in the countries of Central and Southern Europe. Study sample included 121 diagnostic materials from lymph nodes and lungs from cattle, positively and doubtfully PPD tuberculin-reacted, originating from 6 farms belonging to the 4 regions in Northern and Southern Bulgaria. The bacteriological examination showed typical growth for mycobacteria for 110 (90.9%) samples, which was confirmed by qPCR. By RD4-PCR we proved that 102 (92.7%) of the mycobacterial strains were M. caprae and the remaining 8 strains (7.3%) were M. bovis. This defines M. caprae as a dominant species in the etiology of tuberculosis in cattle in Bulgaria.
 In conclusion, we confirmed that the application of real-time PCR is an accurate and convenient method for rapid detection of M. bovis from cattle in the Bulgarian settings. RD4-PCR provides a sufficiently high differentiation and can be used for first-line typing of M. bovis isolates in Bulgaria. This proves the need for the simultaneous application of both methods in the diagnosis of bovine tuberculosis in Bulgaria.

Novel oxazolidinones harbor potent in vitro activity against the clinical isolates of multidrug-resistant Mycobacterium tuberculosis in China.

To investigate the in vitro activities of five oxazolidinones in parallel against the reference strains of different mycobacterial species and clinical isolates of Mycobacterium tuberculosis (Mtb), and shed light on the differences in the efficacy of these homolog drugs. The minimum inhibitory concentrations (MICs) of linezolid, tedizolid, sutezolid, delpazolid, and contezolid against 16 mycobacterial reference strains and 69 M. tuberculosis clinical isolates, including 17 drug-susceptible isolates and 52 multidrug-resistant (MDR) isolates, were determined by microplate alamarBlue assay (MABA). The intracellular killing activities of contezolid and linezolid against Mtb H37Rv were compared. In addition, mutations in the linezolid resistance-related genes (rplC, rplD, and 23S rRNA) of the Mtb clinical isolates were also analyzed. Tedizolid exhibited the strongest inhibitory activities against the reference strains of both rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM), among the tested oxazolidinones. In contrast, sutezolid only manifested potent activity against reference strains of SGM. Linezolid, delpazolid, and contezolid were less active against the non-tuberculous mycobacterial references. For the Mtb clinical isolates, the antimicrobial action was ranked as: sutezolid > tedizolid > contezolid and linezolid > delpazolid, whereas no difference between drug-sensitive and multiple drug-resistant isolates was observed. Notably, contezolid demonstrated obviously superior intracellular antimicrobial activity than linezolid. Few strains harbored mutations in rrl gene or rplD genes, although these strains had drug susceptible profiles to linezolid. Different oxazolidinones can have discrepant antimicrobial activity against different mycobacterial species, or have different manifestations out of cell or in cell. Understanding these differences would be helpful in choosing the appropriate drug in clinical practice.

A Systematic Review on Antituberculosis Drug Discovery and Antimycobacterial Potential of Biologically Synthesized Silver Nanoparticles: Overview and Future Perspectives

Abstract Rapid emergence and quick evolution of drug-resistant and aggressive mycobacterial strains have resulted in the present antimycobacterial drug crisis and the persistence of tuberculosis as a major public health problem. Green/biological nanotechnologies constitute an interesting area of research for discovering antimycobacterial agents. This review focused on the biological (green) synthesis of silver nanoparticles (AgNPs) as an alternative source of antimycobacterial agents. Data for this study were searched and screened from three electronic databases (Google Scholar, PubMed and ScienceDirect) following the Preferred Reporting Items for Systematic Reviews and Meta-analyses flowchart. Data from in total 17 eligible studies were reported in this systematic review. Twelve of the 17 studies used plants to fabricate AgNPs, whereas the remaining five studies used microorganisms (bacteria and/or fungi). Silver as part of silver nitrate (AgNO3) was the metal precursor reported for the synthesis of AgNPs in these studies. Silver nanoparticles were mostly spherical, with sizes ranging from 12 to 140 nm. Results based on minimum inhibitory concentrations varied between studies and were divided into three groups: (i) those more effective than the antibiotic (controls), (ii) those more effective than plant extracts, and (iii) those less effective than the antibiotic controls. In addition, little or no cytotoxicity effects were reported. Silver nanoparticles were also shown to be highly specific or selective toward mycobacterial strains. This systematic review highlights the antimycobacterial potential of biologically synthesized AgNPs, underscoring the possibility of discovering/developing new antimycobacterial agents using biological synthesis approaches with less toxicity and high selectivity.

Performance of lipid fingerprint by routine matrix-assisted laser desorption/ionization time of flight for the diagnosis of Mycobacterium tuberculosis complex species

ObjectivesRapid detection of bacterial pathogens at species and sub-species levels is crucial for appropriate treatment, infection control, and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial lipid-based approach to identify MTBC cultures and sub-species. MethodsA blinded study was conducted using 90 mycobacterial clinical isolate strains comprising MTBC strains sub-cultured in Middlebrook 7H11 medium supplemented with 10% oleic-acid, dextrose, catalase growth supplement and incubated for up to 6 weeks at 37°C and using the following seven reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, and M. bovis BCG) grown under the same conditions, to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the matrix-assisted laser desorption/ionization target followed by the addition of the matrix. Acquisition of the data was performed using the positive ion mode. ResultsBased on the identification of clear and defined lipid signatures from the seven reference strains, the method that we developed was fast (<10 minutes) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the matrix-assisted laser desorption/ionization time of flight were 94.4 (95% CI, 86.4–98.5) and 94.4 (95% CI, 72.7–99.9), respectively. ConclusionsMycobacterial lipid profiling provides a means of rapid, safe, and accurate discrimination of species within the MTBC.

Comparative genome analysis of mycobacteria focusing on tRNA and non-coding RNA

BackgroundThe Mycobacterium genus encompasses at least 192 named species, many of which cause severe diseases such as tuberculosis. Non-tuberculosis mycobacteria (NTM) can also infect humans and animals. Some are of emerging concern because they show high resistance to commonly used antibiotics while others are used and evaluated in bioremediation or included in anticancer vaccines.ResultsWe provide the genome sequences for 114 mycobacterial type strains and together with 130 available mycobacterial genomes we generated a phylogenetic tree based on 387 core genes and supported by average nucleotide identity (ANI) data. The 244 genome sequences cover most of the species constituting the Mycobacterium genus. The genome sizes ranged from 3.2 to 8.1 Mb with an average of 5.7 Mb, and we identified 14 new plasmids. Moreover, mycobacterial genomes consisted of phage-like sequences ranging between 0 and 4.64% dependent on mycobacteria while the number of IS elements varied between 1 and 290. Our data also revealed that, depending on the mycobacteria, the number of tRNA and non-coding (nc) RNA genes differ and that their positions on the chromosome varied. We identified a conserved core set of 12 ncRNAs, 43 tRNAs and 18 aminoacyl-tRNA synthetases among mycobacteria.ConclusionsPhages, IS elements, tRNA and ncRNAs appear to have contributed to the evolution of the Mycobacterium genus where several tRNA and ncRNA genes have been horizontally transferred. On the basis of our phylogenetic analysis, we identified several isolates of unnamed species as new mycobacterial species or strains of known mycobacteria. The predicted number of coding sequences correlates with genome size while the number of tRNA, rRNA and ncRNA genes does not. Together these findings expand our insight into the evolution of the Mycobacterium genus and as such they establish a platform to understand mycobacterial pathogenicity, their evolution, antibiotic resistance/tolerance as well as the function and evolution of ncRNA among mycobacteria.

Targeted dose delivery of Mycobacterium tuberculosis in mice using silicon antifoaming agent via aerosol exposure system.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that forms aggregates (clumps) on solid agar plates and in liquid media. Detergents such as Tween 80/Tyloxapol are considered the gold standard to disrupt clump formation in Mtb cultures. The presence of detergent, however, may generate foam and hinder Mtb aerosolization thus requiring addition of an antifoam agent for optimal Mtb aerosol-based procedures. Aerosol inhalation can be technically challenging, in particular to achieve a reproducible inhaled target dose. In this study, the impact of an antifoam, the silicon antifoaming agent (SAF), on Mtb aerosolization and whole-body mouse aerosol infection was investigated. A comparative study using SAF in a liquid suspension containing Mycobacterium bovis BCG (M. bovis BCG) or Mtb H37Rv did not cause any adverse effect on bacterial viability. Incorporation of SAF during mycobacteria inhalation procedures revealed that aerosolized mycobacterial strains were maintained under controlled environmental conditions such as humidity, temperature, pressure, and airflow inside the aerosol chamber. In addition, environmental factors and spray factors were not affected by the presence of SAF in mycobacterial cultures during aerosolization. Spray factor was significantly less during aerosol procedures with a low-input dose of mycobacteria in comparison to high-dose, as predicted. The mycobacterial load recovered in the biosampler (AGI) was ~2–3 logs lower than nebulizer or input bacterial load. A consistent Mtb bacillary load determined in mouse lungs indicates that SAF does not affect mycobacteria aerosolization during the aerosol generation process. These data confirmed that 1) SAF prevents formation of excessive foam during aerosolization, 2) SAF had no negative impact on mycobacterial viability within aerosol droplets, 3) Mtb droplets within aerosol-generated particles are well within the range required for reaching and depositing deep into lung tissue, and 4) SAF had no negative impact on achieving a target dose in mice exposed to Mtb aerosol.

Total Synthesis and Biological Evaluation of Modified Ilamycin Derivatives.

Ilamycins/rufomycins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclic peptides target the AAA+ protein ClpC1 that, together with the peptidases ClpP1/ClpP2, forms an essential ATP-driven protease. Derivatives of the ilamycins with a simplified tryptophane unit are synthesized in a straightforward manner. The ilamycin derivative 26 with a cyclic hemiaminal structure is active in the nM-range against several mycobacterial strains and shows no significant cytotoxicity. In contrast, derivative 27, with a glutamic acid at this position, is significantly less active, with MICs in the mid µM-range. Detailed investigations of the mode of action of 26 indicate that 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. The consequences of 26 on ClpC1 proteolytic activities were substrate-specific, suggesting dual effects of 26 on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation, and the negative to competition with substrates for binding to the ClpC1 NTD.

Comparison of two mycobacterial strains in performance of the whole blood mycobacterial growth inhibition assay in Indian children

A major challenge in tuberculosis is identifying correlates of a protective immune response. The Mycobacterial Growth Inhibition Assay (MGIA) is a functional assay providing an integrated measure of the host immune response to mycobacteria. However, its feasibility is limited by reliance on biosafety level 3 facilities, and its performance has not been widely evaluated in TB-endemic settings. Here, we compared two mycobacterial strains (M. tuberculosis H37Rv versus attenuated M. bovis BCG) in the performance of whole-blood MGIA in 30 TB-exposed children (median age 2 years) in Chennai, India. The time-to-positivity in both assays was similar (5.7 days vs 6 days) and the mycobacterial growth of M. tuberculosis H37Rv and M. bovis BCG were correlated (r = 0.64, p<0.0001). In Bland-Altman analysis, the bias was −0.54 days (95% limit of agreement −2.08, 0.99). Collectively, our results indicate that M. tuberculosis H37Rv can be substituted with the less virulent M. bovis BCG strain to improve feasibility of the MGIA assay, particularly in low-income settings.

Effect of biogenic polyamines on sliding motility of mycobacteria in the presence of antibiotics.

Nowadays, sliding is the least investigated mode of bacterial motility. Sliding is a process of passive movement on the surface of semi-liquid mediums which was originally described for mycobacteria and other bacterial species deprived of the organelles specialized for movement. Some mycobacteria are able to colonize surfaces, including tissues of macro-organisms, using glycopeptidolipids localized in the cell envelope for this aim. This is a serious problem for effective therapy of mycobacteriosis caused by nontuberculosis mycobacteria. Furthermore, animal tissues contain biogenic polyamines, which can increase tolerance of microorganisms to stresses, including antibiotics, and modulate cell motility. Therefore, studying mutual effects of biogenic polyamines and antibiotics on the expansion of mycobacteria is important for medicine. Mycobacterial strains, including the parent Mycolicibacterium smegmatis mc2 155 and strains containing single (ΔrelMsm) or double (ΔrelMsmΔrelZ) deletions, were used as the objects of this study. The content of glycopeptidolipids was determined using thin layer chromatography. Sliding motility was assessed by measuring the area of the sliding colony. The effectiveness of antibiotics was measured by comparison of the areas of sliding colonies in the presence of comparable concentrations of antibiotics. The polyamines spermidine and spermine had different effects on the sliding of mycobacteria through an increase or decrease in the colony areas. At the same time, polyamines had neither bactericidal nor bacteriostatic effects. The polyamines contained in the medium decreased the bactericidal effects of the antibiotics streptomycin or isoniazid, but enhanced the effects of DMNP, a synthetic analogue of the natural antibiotic erogorgiaene. Rifampicin was the most effective of all antibiotics investigated here. Moreover, we found that glycopeptidolipids are, apparently, not the only regulators of mycobacterial sliding.

Transition between Mycobacterium tuberculosis and nontuberculous mycobacteria in recurrent "tuberculosis" patients.

Recurrence of tuberculosis (TB) is still a key issue in the control of tuberculosis. The presence of nontuberculous mycobacteria (NTM) complicates the diagnosis of recurrent TB due to similarity in clinical presentation. Herein, we have used molecular genotyping methods to identify mycobacteria species, and analyzed the characteristics of patients with transition between MTB and NTM. Eighty-nine patients with recurrent tuberculosis over the past 12years were included in our analysis. We found that 9 patients had NTM infections during the study period. Six patients were infected with different mycobacterial strains, half of which were transformed from NTM to MTB, and the other half from MTB to NTM. In addition, the other 3 patients were infected with the same NTM species. Further WGS analysis showed that only one patient had a relapse and the remaining two were classified as reinfection. In conclusion, our results demonstrate that a proportion of previously diagnosed recurrent TB cases are attributed to the transition between MTB and NTM, highlighting the significance of species identification prior to initiation of treatment. The recurrence of mycobacterial diseases is majorly noted within 1year after treatment completion.

Increased Interferon-Induced Protein With Tetracopeptides (IFITs) Reduces Mycobacterial Growth.

ObjectivesThe host immune response towards Mycobacterium tuberculosis (M. tb) is known to vary with the virulence of mycobacterial species. While the majority of M. tb-exposed individuals develop latent TB infection (LTBI), a small proportion develops active TB disease. The milieu of understudied immune factors is believed to play an important role against host immune response towards mycobacteria. Here, we investigate the role of antiviral factors of the interferon-induced proteins with tetracopeptides (IFITs) family, which, in our previous research, have shown to be upregulated in response to pathogenic M. tb, but as yet have no established role in host response to bacterial infections. MethodsWe performed vector-driven overexpression and siRNA-mediated downregulation of IFITs in THP-1 cells infected with different mycobacterial species. Also, we investigated the mRNA levels of IFITs in the LTBI and active-TB cases.ResultsOverexpression of IFITs reduces CFUs by ~32% (30%–43%) [Median (IQR)] across three different mycobacterial strains, while knock-down increases CFUs by ~57% (41%–78%). Compared to IFN-γ, treatment of infected THP-1 cells with IFN-β significantly increases the expression of IFITs, while the overexpression of IFITs had higher mRNA expression of IFN-β than IFN-γ. Cytokines like IDO-1, IL-6, IL-23, and IFN- γ are observed to play key roles in mycobacterial survival upon IFITs intervention. mRNA expression levels of IFITs were higher in LTBI cases as compared to active TB.ConclusionHigher expression levels of IFITs reduce in vitro survival of different drug-susceptible and drug-resistant mycobacteria and correlates with latent TB infection in infected individuals, hence emerging as an immuno-therapeutic target against M. tb.

Resistance Profile of Mycobacterial Strains Causing Cluster Pulmonary Infection in Bayelsa State, Nigeria

Aim: The aim of this study was to profile the anti-microbial resistant of Mycobacterial strains causing cluster pulmonary infections in Bayelsa State, Nigeria.&#x0D; Study Design: Cross sectional facility based.&#x0D; Place and Duration: This study was carried out in all the in tuberculosis centers in all the eight local government area in Bayelsa State, Nigeria between January 2019 to February 2021.&#x0D; Materials and Methods: A total of 102 tuberculosis patients who had been previously diagnosed of Tuberculosis (both new and old cases) participated in the study. Two sputum samples were collected from each subject in a wide mouth screw cape container and falcon tube container respectively. The first was used for GenXpert technique while the second was used for culture on Lowenstein Jensen solid Media. The visible and confirmed Mycobacterium growth was subjected to Line Probe Assay (MTBDRplus assay) and genetic sequencing. The DNA of the isolates where extracted using Genolyse method. The extracted DNA was used to perform a gene mutation profiling using MTBRplus Assay. The 16Sr RNA sequencing was done on the amplified genes using the BigDye terminator kit on a 3510 AB1 sequencer.&#x0D; Results: Out of the 102 sputum samples analyzed a total of 91(89.2%) were GeneXpert positive. Drug resistant profiling by GeneXpert shows 8(7.8) strain mutation at the rpoB gene only while the resistant profiling of the isolated on Lowenstein Jensen solid media had 14(13.7) strain with mutation on genes responsible for first and second line drug resistant. Two non-tuberculosis Mycobacterium species which are Mycobacteriode abscessus subsp. abscessus st and Mycobacterium Kansasii strain FDAARGOS_1534 where isolated among Tuberculosis patients, both are multidrug-resistant strains. The Mycobacterium tuberculosis strains in circulation causing cluster TB infection in Yenagoa especially and other parts of Bayelsa State, Nigeria are MG003 and R2092 strains, but the most predominant strain that is traced to cluster TB infection is MG003. The extraction of MTB gene from a pure culture of a Lowenstein Jensen selected media reduced the possibility of contamination and enhanced the reliability of gene sequencing and Bioinformatics analysis which includes comparing strains of MTB with the once in the National center for Biotechnology information (BLAST) data base.&#x0D; Conclusion: The resistance profiling of MTB reveals that most strains were resistant to rifampicin. This means that there were more mutation on the rifampicin activation gene (rpoB) than any other gene. Most cases of multi drug resistance is associated with rifampicin resistance while cases of extensive drug resistance is not common.

Alkaloids of Dicranostigma franchetianum (Papaveraceae) and Berberine Derivatives as a New Class of Antimycobacterial Agents.

Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis. The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has created a need for new antiTB agents with new chemical scaffolds to combat the disease. Thus, the key question is: how to search for new antiTB and where to look for them? One of the possibilities is to search among natural products (NPs). In order to search for new antiTB drugs, the detailed phytochemical study of the whole Dicranostigma franchetianum plant was performed isolating wide spectrum of isoquinoline alkaloids (IAs). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. Alkaloids were screened against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. avium, M. kansasii, and M. smegmatis). Alkaloids 3 and 5 showed moderate antimycobacterial activity against all tested strains (MICs 15.625–31.25 µg/mL). Furthermore, ten semisynthetic berberine (16a–16k) derivatives were developed and tested for antimycobacterial activity. In general, the derivatization of berberine was connected with a significant increase in antimycobacterial activity against all tested strains (MICs 0.39–7.81 μg/mL). Two derivatives (16e, 16k) were identified as compounds with micromolar MICs against M. tuberculosis H37Ra (MIC 2.96 and 2.78 µM). All compounds were also evaluated for their in vitro hepatotoxicity on a hepatocellular carcinoma cell line (HepG2), exerting lower cytotoxicity profile than their MIC values, thereby potentially reaching an effective concentration without revealing toxic side effects.