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Subject: search.filters.subject.Drug resistance

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    Investigation of antifungal compounds against Aspergillus species from respiratory infections of immunocompromised patients
    (2023-05) Juglal, Sarla; Mchunu, Nokuthula Peace; Mohanlall, Viresh
    The rapid emergence of invasive fungal infections correlates with the increasing population of immunocompromised individuals, with many cases leading to death. The progressive increase in the incidence of Aspergillus isolates is even more severe due to clinical challenges in treating invasive respiratory infections in immunocompromised patients. Azoles are the drugs of choice for the prevention and treatment of Aspergillus infections however, azole resistance in Aspergillus-related infections is an increasing concern, especially against Aspergillus fumigatus. This situation is further complicated by the use of azoles in agriculture, as increasing resistance has been associated with increased use in agriculture. Rapid initiation in detection, diagnosis and appropriate antifungal therapy is needed to reduce mortality among individuals with invasive Aspergillus-related infections. Improvements in all aspects of azole resistance management, from identification and antifungal therapy to the discovery of novel non-toxic drugs, impact clinical success. Hence, this study assessed the effect of routine and salvage drug therapy on Aspergillus respiratory infections and investigated the potential of new drugs in combination therapy in light of increasing antifungal resistance in the management of invasive fungal infections. Aspergillus species isolated from immunocompromised patients with respiratory infections at the Inkosi Albert Luthuli Hospital in Kwa-Zulu Natal were investigated. Conventional morphology identification methods were assessed for reliability and compared to molecular identification. In the search for reliable and rapid alternatives in fungal identification to impact diagnosis, phenotypic microarray (Biolog) for fungal species identification was explored. Phenotypic microarray (Biolog) was also used to analyse the isolates' nutritional patterns and drug sensitivity profiles to investigate the potential for new compounds in drug therapy. In addition, gliotoxin expression, which has been linked to increased pathogenesis, was quantified and analysed using high-performance liquid chromatography. This was investigated in vitro to correlate gliotoxin production as a possible virulence factor in azole resistance. The effect of pathogen-associated molecular patterns (PAMPS) in bacterial coinfection was also investigated, especially since the species were isolated from respiratory infections where co-infection is common. Conventional morphological techniques were generally similar to 18S rRNA identification, assigning twenty-six Aspergillus fumigatus species, eight A. niger and two A. flavus. Biolog technology only identified isolates correctly up to genus level and had no significant similarity matches at species level due to the Biolog fungal database not having adequate reference clinical species however, an updated database makes this technology a good alternative when reliability and speed in identification are considered. Antifungal profiles showed that 6% of the 36 isolates were resistant to the routine azole voriconazole, with 61% having moderate susceptibility. All isolates resistant to the salvage therapy drug, posaconazole pose a serious concern. Significantly, A.niger was the only species resistant (25%) to voriconazole and has recently been reported as the species isolated from patients with COVID-19-associated pulmonary aspergillosis (CAPA). Mutations in the cpy51A gene representing common mechanisms for azole resistance in clinical Aspergillus isolates were highlighted in the azole susceptibility profile. Phenotypic microarray showed that 83% of the isolates were susceptible to the 24 new compounds. Berberine and blasticidin hydrochloride were selected and further investigated for combination drug therapy, considering their non-toxicity upon oral administration currently with thirty of the thirty-six isolates showing susceptibility to the new agents. Carbon profiles demonstrated the consistent assimilation of alternate monosaccharides and disaccharides by all three Aspergillus species, accomplished by secreted enzymes which very likely contribute to nutrient acquisition during infection, impact persistent growth and survival to influence infection maintenance and disease progression. Of the twenty-six A. fumigatus isolates, 58% produced gliotoxin during the 48-hour incubation at 37℃. Forty per cent of those gliotoxin-producing isolates showed increased gliotoxin production after exposure to PAMPS, with the highest concentration of 2.6 mg/L and a mean of 0.6 mg/L. Compared to similar investigations, the gliotoxin concentrations for invasive aspergillosis were higher than for colonization in respiratory patients. This study found that different identification strategies in azole resistance treatment management are required for both developed and resource-challenged situations to influence diagnosis. Drug resistance in azole monotherapy was demonstrated. Thus this study supports the search for new agents in drug regimen strategy and found potential in novel compounds, berberine and blasticidin hydrochloride, for combination therapy.
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    Bioactive compounds from South African plants against Mycobacterium tuberculosis
    (2016) Singh, Alveera; Odhav, Bharti; Coovadia, Yacoob
    Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna.