The spectrum of mutations in genes associated with resistance to rifampicin,isoniazid, and fluoroquinolones in the clinical strains of <Emphasis Type="Italic">M. tuberculosis</Emphasis> reflects the transmissibility of mutant clones

To study the transmissibility of drug resistant mutant clones, M. tuberculosis samples were isolated from the patients of the clinical department and the polyclinic of the Central TB Research Institute (n = 1455) for 2011–2014. A number of clones were phenotypically resistant to rifampicin (n = 829), isoniazid (n = 968), and fluoroquinolones (n = 220). We have detected 21 resistance-associated variants in eight codons of rpoB, six variants in three codons of katG, three variants in two positions of inhA, four variants in four positions of ahpC, and nine variants in five codons of gyrA, which were represented in the analyzed samples with varied frequencies. Most common mutations were rpoB 531 Ser→Leu (77.93%), katG 315 (Ser→Thr) (94.11%), and gyrA 94 (Asp→Gly) (45.45%). We found that the mutations at position 15 of inhA (C→T) (frequency of 25.72%) are commonly associated with katG 315 (Ser→Thr). This association of two DNA variants may arise due to the double selection by coexposure of M. tuberculosis to isoniazid and ethionamide. The high transmissibility of mutated strains was observed, which may be explained by the minimal influence of the resistance determinants on strain viability. The high transmissibility of resistant variants may also explain the large populational prevalence of drug-resistant TB strains.     

Triacylglycerol: nourishing molecule in endurance of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The ability of Mycobacterium tuberculosis (M. tuberculosis) to accumulate lipid-rich molecules as an energy source obtained from host cell debris remains interesting. Additionally, the potential of M. tuberculosis to survive under different stress conditions leading to its dormant state in pathogenesis remains elusive. The exact mechanism by which these lipid bodies generated in M. tuberculosis infection and utilized by bacilli inside infected macrophage for its survival is still not understood. In this, during bacillary infection, many metabolic pathways are involved that influence the survival of M. tuberculosis for their own support. However, the exact energy source derived from infecting host cells remain elusive. Therefore, this study highlights several alternative energy sources in the form of triacylglycerol (TAG) and fatty acids, i.e. oleic acids accumulation, which are essential in dormancy-like state under M. tuberculosis infection. The prominent stage in tuberculosis (TB) infection is re-establishment of M. tuberculosis under stress conditions and deployment of a confined strategy to utilize these biomolecules for its persistence survival. So, growing in our understanding of these pathways will help us in accelerating therapies, which could reduce TB prevalence world widely.     

Antimicrobial Efficacy of Synthetic Pyranochromenones and (Coumarinyloxy)acetamides

Four (1, 2, 4 and 6) synthetic quaternary ammonium derivatives of pyranochromenones and (coumarinyloxy)acetamides were synthesized and investigated for their antimicrobial efficacy on MRSA (Methicillin-resistant Staphylococcus aureus), and multi-drug resistant Pseudomonas aeruginosa, Salmonella enteritidis and Mycobacterium tuberculosis H37Rv strain. One of the four compounds screened i.e. N,N,N-triethyl-10-((4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahydropyrano[3,2-g]chromen-10-yl)oxy)decan-1-aminium bromide (1), demonstrated significant activity against S. aureus, P. aeruginosa and M. tuberculosis with MIC value of 16, 35, and 15.62 µg/ml respectively. The cytotoxicity evaluation of compound 1 on A549 cell lines showed it to be a safe antimicrobial molecule, TEM study suggested that the compound led to the rupture of the bacterial cell walls.     

Transposition mechanism,molecular characterization and evolution of IS<Emphasis Type="Italic">6110</Emphasis>, the specific evolutionary marker of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.     

Activity of phosphino palladium(II) and platinum(II) complexes against HIV-1 and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Treatment of human immunodeficiency virus (HIV) is currently complicated by increased prevalence of co-infection with Mycobacterium tuberculosis. The development of drug candidates that offer the simultaneous management of HIV and tuberculosis (TB) would be of great benefit in the holistic treatment of HIV/AIDS, especially in sub-Saharan Africa which has the highest global prevalence of HIV-TB coinfection. Bis(diphenylphosphino)-2-pyridylpalladium(II) chloride (1), bis(diphenylphosphino)-2-pyridylplatinum(II) chloride (2), bis(diphenylphosphino)-2-ethylpyridylpalladium(II) chloride (3) and bis(diphenylphosphino)-2-ethylpyridylplatinum(II) (4) were investigated for the inhibition of HIV-1 through interactions with the viral protease. The complexes were subsequently assessed for biological potency against Mycobacterium tuberculosis H37Rv by determining the minimal inhibitory concentration (MIC) using broth microdilution. Complex (3) showed the most significant and competitive inhibition of HIV-1 protease (p = 0.014 at 100 µM). Further studies on its in vitro effects on whole virus showed reduced viral infectivity by over 80 % at 63 µM (p < 0.05). In addition, the complex inhibited the growth of Mycobacterium tuberculosis at an MIC of 5 µM and was non-toxic to host cells at all active concentrations (assessed by tetrazolium dye and real time cell electronic sensing). In vitro evidence is provided here for the possibility of utilizing a single metal-based compound for the treatment of HIV/AIDS and TB.     

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.     

Incidence of metallo-beta-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolates from hospital setting in Pakistan

The aim of this study was monitoring and surveillance in different wards of the PIMS hospital, Islamabad, to understand emerging challenges of antibiotic resistance in particular association with most virulent serotypes of Klebsiella pneumoniae. The study was conducted during March 2015 to September 2015. The study showed that rate of isolation of K. pneumoniae was 37% (103 positives out of a total of 277 clinical samples) and 7.7% (8) were phenotypically and genotypically confirmed to be metallo-β-lactamase resistant (carbapenem resistant) and all of them were multidrug resistant (MDR). These carbapenem-resistant isolates were isolated from blood, endotracheal tubes, and pus. Molecular screening for the presence of integrons indicated that distribution of class I integrons (87.5% of carbapenem-resistant K. pneumoniae isolates) was higher than class II integrons (1.25%) among given isolates. The study indicated that exposure of metallo-beta-lactamase-producing strains through hospitalizations increases the chances of spread of MDR pathogens. There is an urgent need for effective surveillance and monitoring strategies to control the spread of extremely resistant K. pneumoniae implicated in nosocomial infections leading to the increased health burden and enforcement of policy guideline on appropriate antibiotics usage.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Structural and inhibition analysis of novel sulfur-rich 2-mercaptobenzothiazole and 1,2,3-triazole ligands against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> DprE1 enzyme

Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (MtbDprE1) acts in concert with decaprenylphosphoryl-β-d-ribose 2-epimerase (MtbDprE2) and catalyzes the epimerization of DPR into DPA. DPA is the sole precursor for synthesis of arabinogalactan and lipoarabinomannan in the mycobacterial cell wall. MtbDprE1 is a unique antimalarial drug target and many covalent and non-covalent inhibitors against MtbDprE1 have been studied for their antituberculosis activities. In the current study, we have purified MtbDprE1 enzyme and synthesized six sulfur-rich 2-mercaptobenzothiazole and 1, 2, 3-triazole conjugated ligands and performed binding analysis with MtbDprE1. All ligands have shown competitive binding, as observed for other covalently and noncovalently bound MtbDprE1 inhibitors. Molecular docking analysis of six ligands with MtbDprE1 shows that they occupy the substrate binding pocket of MtbDprE1 and are stabilized by hydrogen bonds and van der Waals interactions. Our study shows that sulfur-rich 2-mercaptobenzothiazole ligands act as specific inhibitors against MtbDprE1 and could be used as antituberculosis agents.     

Basidiospores of <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> succeed to infect barberry,while Urediniospores are blocked by non-host resistance

Stripe rust (Yellow rust) caused by Puccinia striiformis f. sp. tritici (Pst) is a major disease of wheat worldwide. The use of resistant cultivars to control Pst has been very effective, low-cost, and ecologically sound. However, virulence patterns of Pst can quickly change, which may render resistant cultivars susceptible. The discovery of infection of Berberis spp. by basidiospores of Pst in 2010 raised important concerns about the evolution of new virulent races of the pathogen. Little is known about the infection process of Berberis spp. by basidiospores of Pst and the interaction between Berberis spp. and asexual urediniospores. In this study, the interaction between Pst urediniospores and Berberis spp. was investigated at histological and cytological levels. Our results indicate that Berberis spp. expresses a continuum of layered defenses comprised of structural and chemical changes in the cell wall as well as post-haustorial hypersensitive responses to urediniospore infection. Our study also re-examines in detail the infection process of Pst basidiospores on Berberis spp. and provides useful information for further research on the molecular mechanisms governing the interaction between Berberis spp. and Pst.     

Madurastatin B3, a rare aziridine derivative from actinomycete <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. LS150010 with potent anti-tuberculosis activity

Since the discovery of the first antibiotic, natural products have played an important role in chemistry, biology and medicine. To explore the potential of bioactive compounds from microbes isolated from the southeast of Tibet, China, a crude extract library was constructed and screened against Staphylococcus aureus. The strain Nocardiopsis sp. LS150010 was scaled up and subjected to further chemical studies, resulting in the identification of N-salicyloyl-2-aminopropan-1,3-diol (2) and its rare aziridine derivative, madurastatin B3 (1). Their structures were determined by detailed analysis of 1D, 2D NMR and HRMS data. Compounds 1 and 2 displayed significant inhibitory activity against S. aureus and methicillin resistant S. aureus, with MIC values of 6.25 µg/mL. Compound 1 also showed potent inhibitory activity against Bacillus subtilis and Escherichia coli, as well as activity in a Mycobacterium tuberculosis Bacillus Calmette-Guérin infected THP-1 cell model.     

The role of thiol redox systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV₂₅₄) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.     

New Targets for Growth Inhibition of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: Why Do Natural Terpenoids Exhibit Antitubercular Activity?

The article draws the attention of chemists to the literature data reporting the discovery of new targets for growth inhibition of Mycobacterium tuberculosis, namely, diterpene cyclase (Rv3377c) and tuberculosinol phosphatase (Rv3378c), which produce diterpenoids of tuberculosinols in the cell membrane of M. tuberculosis, and these diterpenoids ensure the pathogenicity and the virulence of M. tuberculosis. For the first time, by the example of diterpenoid of isosteviol, its binuclear derivatives, triterpenoid betulinic, oleanolic, and ursolic acids, it has been shown by the molecular docking method that the antitubercular activity of natural terpenoids is caused by their ability to bind to the active site of tuberculosinol phosphatase (Rv3378c) of M. tuberculosis. It is suggested that natural and semisynthetic terpenoids represent a promising platform for design of a new generation of antitubercular agents that affect this enzyme.     

Transferable Carbenicillin Resistance in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

IN 1969, after carbenicillin had been in use for three years in this unit, highly resistant strains of Pseudomonas aeruginosa were isolated for the first time¹. Because these resistant strains included, from their first appearance, representatives of two unrelated types, it seemed likely that the resistance was transferable; this hypothesis was supported by experiments showing the transfer of carbenicillin resistance between Ps. aeruginosa and Escherichia coli K12 in vitro and in vivo^2–4;. The resistant Ps. aeruginosa produced a penicillinase (β lactamase) similar to that normally produced by some strains of Enterobacteria and different from that normally produced by Ps. aeruginosa^2,3, so it seemed likely that the Ps. aeruginosa had initially acquired resistance by the transfer of an R factor from a carbenicillin-resistant member of the Enterobacteriaceae colonizing the same burn. This hypothesis is now supported by a study on strains of Enterobacteria and Ps. aeruginosa isolated in a number of hospitals. We have also found evidence suggesting that Ps. aeruginosa which has acquired this R factor may not show resistance until it has been exposed repeatedly to carbenicillin.     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

The protective effect of a novel antioxidant gene from <Emphasis Type="Italic">Mycobacterium avium</Emphasis> against nitrosative and oxidative stress in <Emphasis Type="Italic">E. coli</Emphasis>

The production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) is an important host defense mechanism in response to infection by Mycobacterium tuberculosis. A variety of genes have been implicated in resistance to ROI and RNI, including noxR1. However, studies in Mycobacterium avium, an important pathogen among nontuberculous mycobacteria, are limited. We aim to investigate the role of a novel gene cloned from M. avium with high similarity to noxR1, noA, in resistance against RNI and ROI in M. tuberculosis. After subcloning noA into vector for expression in E. coli, we performed survival rate analysis in the bacteria transformed with noA (pET-noA) and without noA (pET-his) after exposure to nitrosative stresses by S-nitrosoglutathione (GSNO) and sodium nitrite, and oxidative stresses by H₂O₂. Compared with pET-his, the survival rate of pET-noA was 1 log₁₀-fold higher after exposure to GSNO and sodium nitrite. We observed 1 log₁₀-fold, 2 log₁₀-fold and 3 log₁₀-fold higher survival rate in pET-noA than pET-his after exposure to H₂O₂ for 3, 6 and 9 h, respectively. With the combined treatment of H₂O₂ and GSNO, we found more than 2 log₁₀-fold increase in survival rate in pET-noA comparing with pET-his, suggesting a possible synergistic effect. In summary, noA gene cloned from M. avium has been shown to protect E. coli from both RNI and ROI.