Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans, secretes toxin called mycolactone that triggers inflammation and ...immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo1,5-apyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log
CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU.
Klebsiella pneumoniae is an opportunistic pathogen that is responsible for community acquired infections and nosocomial infections. Antibiotic-resistant K. pneumoniae and/or hypervirulent K. ...pneumoniae are emerging as a serious threat to public health. For the sake of alleviating and conquering current dilemma, discovery of effective new drugs against K. pneumoniae is a tough challenge. However, traditional anti-K. pneumoniae drug discovery methods cost considerable amount of time, animals, labor and so on. So an efficient technique for in vitro and in vivo drug screening with the least time duration, animals and labor cost is highly needed for the discovery of new effective compounds. Hence, in this study we constructed a selectable marker-free autoluminescent K. pneumoniae (SfAlKp) harboring luxCDABE by combining Tn7 transposon and Xer-dif system. SfAlKp can be used for discovery of new drugs via detecting luminescence intensity as a surrogate marker. The energy-consuming autoluminescent reaction catalyzed by the LuxAB enzymes which use the substrates produced by LuxCDE using the metabolites of the bacteria. Tn7 can insert exogenous genes into the bacterial genome and the DNA fragment in between dif sequences can be recognized and removed by endogenous XerCD recombinases of K. pneumoniae. The drug susceptibility and growth rate of SfAlKp are identical to its parent strain, meanwhile the luminescence intensity and stability are also significant characteristics of SfAlKp. Compared to conventional techniques, the autoluminescence-based measurement is more applicable to high throughput screening for compounds both in vitro as well as in vivo in animal model.
•A stable selectable marker-free autoluminescent K. pneumoniae (SfAlKp) was created.•The fragment in between dif sequences in K. pneumoniae can be removed efficiently.•SfAlKp is suitable for high-throughput drug screening in vitro within 2 h.•Outlines of mice and sites of SfAlKp can be recorded simultaneously with dual light.•Rapid testing drug activities in the same live animals using SfAlKp was realized.
Drug-resistant tuberculosis （TB） poses a significant challenge to the successful treatment and control of TB worldwide. Resistance to anti-TB drugs has existed since the beginning of the chemotherapy ...era. New insights into the resistant mechanisms of anti-TB drugs have been provided. Better understanding of drug resistance mechanisms helps in the development of new tools for the rapid diagnosis of drug- resistant TB. There is also a pressing need in the development of new drugs with novel targets to improve the current treatment of TB and to prevent the emergence of drug resistance in Mycobacterium tuber- culosis. This review summarizes the anti-TB drug resistance mechanisms, furnishes some possible novel drug targets in the development of new agents for TB therapy and discusses the usefulness using known targets to develop new anti-TB drugs. Whole genome sequencing is currently an advanced technology to uncover drug resistance mechanisms in M. tuberculosis. However, further research is required to unravel the significance of some newly discovered gene mutations in their contribution to drug resistance.
Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by
has ...been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers
resistance to PZA. Expression of the mutant allele but not the wild-type allele in
recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.
ABSTRACT
Over the last few decades, weather and climate extremes have become a major focus of researchers, the media and general public due to their damaging effects on human society and ...infrastructure. Trends in indices of climate extremes are studied for the South Asian region using high‐quality records of daily temperature and precipitation observations. Data records from 210 (265) temperature (precipitation) observation stations are analysed over the period 1971–2000 (1961–2000). Spatial maps of station trends, time series of regional averages and frequency distribution analysis form the basis of this study. Due to the highly diverse geography of the South Asian region, the results are also described for some specific regions, such as the island of Sri Lanka; the tropical region (excluding Sri Lanka); the Greater Himalayas above 35°N, the Eastern Himalayas (Nepal) and the Thar Desert. Generally, changes in the frequency of temperature extremes over South Asia are what one would expect in a warming world; warm extremes have become more common and cold extremes less common. The warming influence is greater in the Eastern Himalayas compared with that in the Greater Himalayas. The Thar Desert also shows enhanced warming, but increases are mostly less than in the Eastern Himalayas. Changes in the indices of extreme precipitation are more mixed than those of temperature, with spatially coherent changes evident only at relatively small scales. Nevertheless, most extreme precipitation indices show increases in the South Asia average, consistent with globally averaged results. The indices trends are further studied in the context of Atmospheric Brown Clouds (ABCs) over the region. Countries falling within the ABC hotspot namely Indo‐Gangetic Plain (IGP) have shown a different behaviour on the trends of extreme indices compared with the parts outside this hotspot. IGP has increased temperature and decreased rainfall and tally closely with the actual trends.
Tuberculosis (TB), an infectious disease, has been a leading cause of morbidity and mortality for decades. The causative agent of TB is the
Mycobacterium tuberculosis
(Mtb) which can infects various ...parts of the body, mainly the lungs in pulmonary TB cases.
Mycobacterium bovis
Bacillus Calmette–Guerin (BCG) is the only approved vaccine for TB, but its efficiency to combat pulmonary TB is limited. Multidrug-resistant (MDR) TB and extensive drug-resistant (XDR) TB requires the evolution of more potent vaccines. Therefore, this research aims to generate a universal TB subunit vaccine using advanced immunoinformatics techniques. In generating a novel multiepitope subunit vaccine, we selected the conserved and experimentally confirmed antigens Rv0058, Rv0101, and Rv3343. After a rigorous evaluation, the top candidates from predicted Helper T-lymphocytes (HTL), Cytotoxic T-lymphocytes (CTL), and B-cell epitopes were considered potential vaccine candidates. Immunogenicity was enhanced by the addition of an adjuvant to the ultimate construct of the vaccine. B-cell epitopes predictions guaranteed the eventual induction of a humoral response. Thereafter, dynamics simulations and molecular docking validated the vaccine-receptor complex’s stability and high affinity for the immune receptor TLR-3. Also, immune simulations revealed the significantly elevated levels of immunoglobulins such as IgM, cytokines such as interleukin-2, helper T (Th) cells, and cytotoxic T-cell populations. These results agreed with the actual inflammatory response and showed rapid antigen clearance after manifold exposure. Finally, the
E. coli
K12 strain was confirmed
via in-silico
cloning for quality expression. Nevertheless,
in vivo
experiments should be performed to validate the safety of the proposed vaccine and its inherent ability to prevent TB infection.
TB47, a new drug candidate targeting QcrB in the electron transport chain, has shown a unique synergistic activity with clofazimine and forms a highly sterilizing combination. Here, we investigated ...the sterilizing effects of several all-oral regimens containing TB47 plus clofazimine and linezolid as a block and the roles of fluoroquinolones and pyrazinamide in them. All these regimens cured tuberculosis within 4 to 6 months in a well-established mouse model, and adding pyrazinamide showed a significant difference in bactericidal effects.
Objective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations ...pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF. Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay. Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to >64 microg/mL) of which 57 were MDR and 81 pre- XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426-452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284- 1289 CCAGCT,1295-1303 AATTCATGG), and insertion at (1300-1302 TTC) were detected within RRDR of three RIFr strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIFr strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively. Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIFr clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings. Keywords: rifampicin-resistance, Mycobacterium tuberculosis, rpoB, GeneXpert MTB/RIF, resistance-determining region
Pseudomonas aeruginosa is an increasingly prevalent pathogen that has become a serious health concern due to an increasing incidence of multidrug-resistant (MDR) hospital-acquired infections. The ...emergence of MDR-P. aeruginosa coupled with shrinking antibiotic pipelines has increased the demand for new antimicrobials and therapeutics. An effective tool for drug screening both in vitro and in vivo can facilitate the discovery of drugs and regimens for treating P. aeruginosa infection. Here, for the first time, we combined the mini-Tn7 system and Xer/dif recombinase system to construct a stable and selectable marker-free autoluminescent P. aeruginosa (SfAlPa) by one step. Afterwards, in vitro and in vivo activities of several antibiotics including amikacin, biapenem, levofloxacin and polymyxin B were assessed using SfAlPa. This study demonstrated that the use of SfAlPa could significantly facilitate rapid real-time evaluating the activities of compounds. Compared to prevailing methods, this method reduces the time, effort, animals and costs consumed in the discovery of new drugs against P. aeruginosa. Additionally, the methodology described in this study could be easily modified for construction of selectable marker-free reporter strain in other Gram-negative bacteria.
•A selectable marker-free autoluminescent P. aeruginosa (SfAlPa) was constructed.•SfAlPa was suitable for high-throughput anti-P. aeruginosa drug screening.•SfAlPa could be used for non-invasive evaluation of drug activity in live animals.•The genetic system could be used to construct other Gram-negative reporter bacteria.
is intrinsically resistant to most antimicrobial agents. The emerging infections caused by
and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable ...marker-free autoluminescent
strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating
The UAlMab strain was constructed using the
/Xer recombinase system.
and
activities of several drugs, including clofazimine and TB47, a recently reported cytochrome
inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical
isolates. The UAlMab strain enabled us to evaluate drug efficacy both
and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both
and
This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against
, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.
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