The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.
Summary. To study genotype and phenotype correlation of haemophilia A in Thai patients, molecular defects of the factor VIII ( FVIII ) gene were examined and their correlation with clinical phenotypes were evaluated. The molecular pathologies of FVIII in Thai patients were found to be heterogeneous. The most common mutation was FVIII intron 22 inversion accounting for about 30% of the severe cases while gene deletion was rare. Sixteen point mutations were identified, comprising two nonsense mutations (R‐5X and R1966X), five missense mutations (T233I, D542Y, G1850V, W2229S and G2325C), five nucleotide deletions (1145delT, 1187–8delACAC, 1191–4delA, 1458delGA and 1534delA), three nucleotide insertions (1439–41insA, 1934insTA and 2245insACTA) and one splicing defect (IVS15+1G>T). Nine mutations (T233I, D542Y, 1145delT, 1458delGA, 1534delA, 1934insTA, W2229S, 2245insACTA and G2325C) were novel, firstly identified in Thai patients. The genotypes were found to correlate with clinical phenotypes in a majority of cases. However, in five patients the molecular defects did not correlate with clinical severity and FVIII:C level. Cellular and molecular mechanisms were proposed to be responsible in amelioration of clinical severity caused by deleterious mutations. Carrier detection by direct mutation analysis was also demonstrated.
The aim of the study is to investigate the antibacterial activity of 10 volatile oils extracted from medicinal plants, including galangal (Alpinia galanga Linn.), ginger (Zingiber officinale), plai (Zingiber cassumunar Roxb.), lime (Citrus aurantifolia), kaffir lime (Citrus hystrix DC.), sweet basil (Ocimum basilicum Linn.), tree basil (Ocimum gratissimum), lemongrass (Cymbopogon citratus DC.), clove (Syzygium aromaticum), and cinnamon (Cinnamomum verum) against four standard strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and 30 clinical isolates of multidrug-resistant A. baumannii (MDR-A. baumannii).Agar diffusion, minimum inhibitory concentration, and minimum bactericidal concentration (MBC) were employed for the determination of bactericidal activity of water distilled medicinal plants. Tea tree oil (Melaleuca alternifolia) was used as positive control in this study.The results indicated the volatile oil extracted from cinnamon exhibited potent antibacterial activity against the most common human pathogens, S. aureus, E. coli, P. aeruginosa, and A. baumannii. Most of volatile oil extracts were less effective against non-fermentative bacteria, P. aeruginosa. In addition, volatile oil extracted from cinnamon, clove, and tree basil possessed potent bactericidal activity against MDR-A. baumannii with MBC90 of 0.5, 1, and 2 mg/mL, respectively.The volatile oil extracts would be useful as alternative natural product for the treatment of the most common human pathogens and MDR-A. baumannii infections.
Abstract Tuberculosis (TB) is a global health problem caused by Mycobacterium tuberculosis (MTB) infection. The main problem of TB treatment is the emergence of drug resistance, which can occur by inappropriate of antibiotic used. Isoniazid (INH) is the first-line anti-TB drug that inhibits mycolic acid synthesis, an important part of the mycobacterial cell wall. Mannose-capped lipoarabinomannan (ManLAM) is an essential cell wall part that plays a role as an immunomodulator and acts as a virulence factor. In this study, MTB clinical isolates with different drug resistant profiles were used to determine the expression of ManLAM related genes including pimB, mptA, mptC, dprE1, dprE2 and embC by qRT-PCR. Stress-related genes including hspX, tgs1 , and sigE were determined by multiplex real-time PCR with probe assay. Sanger sequencing of ManLAM related genes and genes associated with drug resistance ( inhA, katG , and rpoB ) were analyzed. In response to INH, the expression pattern of ManLAM related genes was different among four strains. Interestingly, MDR-TB markedly up-regulated ManLAM related genes greater than others. Stress-related genes hspX and tgs1 were significantly upregulated in MDR response to INH, whereas sigE was significantly upregulated in MDR response to RIF and INH-R. DprE1 is crucial for MTB and it is a valuable target for anti-TB drugs. RIF-R and MDR isolates show C→T mutation at nucleotide position 459 of the dprE1 gene leading to the same amino acid at codon 153. Codon usage analysis for DprE1 showed that RIF-R and MDR preferred ACT codon over drug sensitive strains. This work provides the expression pattern of ManLAM related genes and stress responder genes, which are key factors in the interaction between MTB and host. Moreover, ManLAM is a possible factor that plays an important role in the adaptive mechanism and the drug resistance mechanism of mycobacteria. Author summary The adaptive mechanism of mycobacteria in response to stressors is an important strategy to promote their virulence and pathogenesis. This study determined the effect of antibiotic stress on Mycobacterium tuberculosis (MTB) focusing on mannose-capped lipoarabinomannan (ManLAM), which is one of the virulence factors that modulate host immune response. Multiplex real-time PCR with probe assay targeting stress responder genes and qRT-PCR targeting ManLAM related genes were performed. Isoniazid acts as a stressor to induce stress response in mycobacteria, as shown in the up-regulation of stress-related genes including hspX, tgs1 , and sigE . The expression pattern of ManLAM related genes in drug resistant and drug sensitive-MTB in response to INH was different, causing a unique pattern. ManLAM related genes respond to isoniazid mostly in drug resistant strains and are present at high expression levels in INH-R and MDR. The results suggest that ManLAM is one factor involved in the adaptive mechanism of MTB response to antibiotic stress and probably associated with the emergence of MTB drug resistance. This work provides new insights into the adaptive mechanism of mycobacterial response to isoniazid that will improve understanding of how mycobacteria develop drug resistance.
Background: Mycobacterium tuberculosis is the most common causative agent of tuberculosis. It releases secretory proteins, especially the Ag85 complex, from actively growing mycobacteria, which can be detected in mycobacterial liquid culture. Ag85B is the most abundant in the Ag85 complex and is an interesting target for the detection of tuberculosis. In addition, measuring Ag85 level is beneficial for comparing growing and non-growing mycobacteria in liquid culture. Objectives: To detect Ag85B protein from active growing M. tuberculosis in liquid culture for the diagnosis of tuberculosis and compare the level of Ag85B between growing and non-growing mycobacteria. Materials and methods: A sandwich ELISA assay using anti-Ag85B monoclonal antibody was performed to detect Ag85B protein. Drug-susceptible mycobacteria, M. tuberculosis H37Ra and M. tuberculosis H37Rv, as well as 12 other M. tuberculosis complex strains isolated from clinical specimens were cultured in liquid media. In addition, mycobacterial culture was separated into two conditions: untreated and treated with streptomycin. Then the liquid media were collected, filtered for sterility, and used for the detection of Ag85B. The levels of Ag85B between treated and untreated conditions were analyzed. Results: In untreated mycobacterial culture, Ag85B protein was detected and continuously increased each day. However, Ag85B in treated mycobacterial culture increased slightly in the early days and then stabilized. These results demonstrate that the growth of mycobacteria was inhibited after culturing with streptomycin. Thus, the increase of Ag85B was not observed because this protein cannot be secreted by dead mycobacteria. On the other hand, living mycobacteria can secrete the Ag85B protein continuously. Moreover, Ag85B accumulated and increased with each passing day. Conclusion: M. tuberculosis can produce and release Ag85B protein. In this study, detection of Ag85B in liquid culture by a sandwich ELISA assay was used to identify M. tuberculosis. When M. tuberculosis was suppressed or killed by anti-TB medications, it stopped growing and the increase in Ag85B proteins disappeared. As a result, the levels of Ag85B could be used to detect living mycobacteria. Furthermore, this knowledge can be further applied to monitor mycobacterial growth affected by testing mycobacteria with anti-TB drugs.
Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTC), is the leading cause of infectious disease-related mortality worldwide. The standard method for TB diagnosis usually requires long periods of mycobacteria cultivation, leading to delayed diagnosis, inefficient treatment and widespread occurrence of the disease. Therefore, a rapid method for the detection and differentiation of MTC from other mycobacteria is essential for disease diagnosis. Here, we describe the potential of using the type I signal peptidase (lepB) gene as a novel target for TB diagnosis, based on confronting two-pair primers PCR (PCR-CTPP) that can detect MTC and simultaneously differentiate M. bovis. The limit of detection of the developed technique was equivalent to 12–120 bacilli. PCR-CTPP was highly specific to only MTC and M. bovis, and no cross-reaction was detected in 27 DNA of the non-tuberculous mycobacterial and bacterial strains tested. Thirty-nine blinded clinical isolates and 72 sputum samples were used to validate the PCR-CTPP in comparison with the standard mycobacterial culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP were equal to 95, 100, 100 and 95 %, respectively, when tested with clinical isolates. Furthermore, upon testing with the sputum samples, the sensitivity, specificity, PPV and NPV were observed to be 84, 76, 90 and 67 %, respectively. Hence, this highly sensitive novel technique, which is rapid, easy to conduct and cost-effective, is a potential method for TB diagnosis and epidemiological studies, especially in resource-limited countries with a high TB burden.
