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Biological activity of macrofungi in South Africa against respiratory and lung disease
- Authors: Didloff, Jenske
- Date: 2018
- Subjects: Macrofungi , Microbiology , Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/30138 , vital:30835
- Description: Macrofungi represent an untapped source of natural bioactive compounds for various diseases, which have been targeted as potential therapeutic agents. The medicinal uses of macrofungi discovered to date include anticancer, antidiabetic, antioxidant, antimicrobial, and immunomodulatory properties. The knowledge regarding the medicinal uses of macrofungi in Africa is limited; however, it is believed that Africa may contain a large number of unidentified species of macrofungi. The objectives of this study were to: (i) screen the macrofungal extracts for antimicrobial activity against microorganisms responsible for respiratory diseases (e.g. Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium tuberculosis, HIV-1), (ii) determine the effect of macrofungal extracts on bacterial morphology, (iii) investigate the cytotoxicity of macrofungal extracts against human lung carcinoma cells, and to elucidate the mechanism/s of action of cytotoxicity/anticancer activity. In vitro bioassays for antimicrobial activity included: ρ-iodonitrotetrazolium chloride assays and microplate alamar blue assay (MABA), while the HIV-1 reverse transcriptase colorimetric ELISA and HIV-1 protease fluorometric assay kits were used for anti-HIV activity. Cytotoxicity of the macrofungal species against A549 lung cancer cell line was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the IC50 values determined for the most cytotoxic macrofungal spp. The mechanism of cytotoxicity was investigated by cell cycle analysis and fluorescent staining to observe morphological and biochemical changes (i.e. acridine orange, caspase activation). Ethanol extracts of Amanita foetidissima, Gymnopilus junonius, Pisolithus tinctorius, Fomitopsis lilacinogilva, Stereum hirsutum and Pycnoporus sanguineus showed higher antimicrobial activity against the Gram-positive bacteria than aqueous extracts, with S. pneumoniae being the most susceptible. The ethanol extracts of Agaricus campestris, Chlorophyllum molybdites, Gymnopilus penetrans, Pseudophaeolus baudonii and Laetiporus sulphureus exhibited anti-TB (tuberculosis) activity with minimum inhibitory concentrations (MICs) ranging between 500-1,000 μg/mL. C. molybdites ethanol extract inhibited HIV-1 protease activity (IC50: 49.7 μg/mL). The macrofungal extracts did not inhibit HIV-1 reverse transcriptase activity. Ethanol extracts of F. lilacinogilva, G. junonius, P. sanguineus and the aqueous extract iv of P. baudonii were cytotoxic against A549 lung cancer cells at IC50 values of 69.2±3.6, 57±5, 7.4±1.1 and 53.6±1.1 μg/mL, respectively. Cell cycle arrest was observed in the G2 phase for both P. sanguineus and P. baudonii, and G2/M and early M phases for G. junonius and F. lilacinogilva, respectively. Apoptosis induced by macrofungal extracts was confirmed by fluorescent staining. Morphological and biochemical changes included chromatin condensation, membrane blebbing, loss of cytoskeletal structure, caspase activation and phosphatidylserine translocation. This study demonstrates the biological activities of selected macrofungal extracts and their potential mechanisms of action. Isolation and identification of active compounds require further analysis.
- Full Text:
- Authors: Didloff, Jenske
- Date: 2018
- Subjects: Macrofungi , Microbiology , Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/30138 , vital:30835
- Description: Macrofungi represent an untapped source of natural bioactive compounds for various diseases, which have been targeted as potential therapeutic agents. The medicinal uses of macrofungi discovered to date include anticancer, antidiabetic, antioxidant, antimicrobial, and immunomodulatory properties. The knowledge regarding the medicinal uses of macrofungi in Africa is limited; however, it is believed that Africa may contain a large number of unidentified species of macrofungi. The objectives of this study were to: (i) screen the macrofungal extracts for antimicrobial activity against microorganisms responsible for respiratory diseases (e.g. Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium tuberculosis, HIV-1), (ii) determine the effect of macrofungal extracts on bacterial morphology, (iii) investigate the cytotoxicity of macrofungal extracts against human lung carcinoma cells, and to elucidate the mechanism/s of action of cytotoxicity/anticancer activity. In vitro bioassays for antimicrobial activity included: ρ-iodonitrotetrazolium chloride assays and microplate alamar blue assay (MABA), while the HIV-1 reverse transcriptase colorimetric ELISA and HIV-1 protease fluorometric assay kits were used for anti-HIV activity. Cytotoxicity of the macrofungal species against A549 lung cancer cell line was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the IC50 values determined for the most cytotoxic macrofungal spp. The mechanism of cytotoxicity was investigated by cell cycle analysis and fluorescent staining to observe morphological and biochemical changes (i.e. acridine orange, caspase activation). Ethanol extracts of Amanita foetidissima, Gymnopilus junonius, Pisolithus tinctorius, Fomitopsis lilacinogilva, Stereum hirsutum and Pycnoporus sanguineus showed higher antimicrobial activity against the Gram-positive bacteria than aqueous extracts, with S. pneumoniae being the most susceptible. The ethanol extracts of Agaricus campestris, Chlorophyllum molybdites, Gymnopilus penetrans, Pseudophaeolus baudonii and Laetiporus sulphureus exhibited anti-TB (tuberculosis) activity with minimum inhibitory concentrations (MICs) ranging between 500-1,000 μg/mL. C. molybdites ethanol extract inhibited HIV-1 protease activity (IC50: 49.7 μg/mL). The macrofungal extracts did not inhibit HIV-1 reverse transcriptase activity. Ethanol extracts of F. lilacinogilva, G. junonius, P. sanguineus and the aqueous extract iv of P. baudonii were cytotoxic against A549 lung cancer cells at IC50 values of 69.2±3.6, 57±5, 7.4±1.1 and 53.6±1.1 μg/mL, respectively. Cell cycle arrest was observed in the G2 phase for both P. sanguineus and P. baudonii, and G2/M and early M phases for G. junonius and F. lilacinogilva, respectively. Apoptosis induced by macrofungal extracts was confirmed by fluorescent staining. Morphological and biochemical changes included chromatin condensation, membrane blebbing, loss of cytoskeletal structure, caspase activation and phosphatidylserine translocation. This study demonstrates the biological activities of selected macrofungal extracts and their potential mechanisms of action. Isolation and identification of active compounds require further analysis.
- Full Text:
Incidence of bacterial infections and colonisation in patients admitted to a tuberculosis hospital
- Authors: Annear, Dale John
- Date: 2018
- Subjects: Medical microbiology , Microbiology , Bacteriology , Tuberculosis -- Hospitals -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/21482 , vital:29526
- Description: Patients with drug resistant tuberculosis (TB) are treated with multiple antibiotics including moxifloxacin, linezolid, and meropenem, which puts them at greater risk for colonisation by multi-drug resistant (MDR) bacteria. The objectives of this study were to: (i) assess the antimicrobial prescribing patterns practiced within the hospital by retrospective patient file review; (ii) determine the spectrum of bacterial colonisation in TB patients upon admission and during hospitalisation; (iii) identify bacterial isolates and evaluate antimicrobial susceptibility profiles; (iv) detect antimicrobial resistance genes in the bacterial isolates by PCR and DNA sequencing; and (v) investigate genetic relatedness of Klebsiella pneumoniae isolates using Multi Locus Sequence Typing. Nasal, groin and rectal swabs [for the detection of extended spectrum beta lactamases (EBSLs), carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA)] were analysed from a cohort of patients (n=37) admitted either from the community (n = 28) or from other healthcare facilities (n=9) to a TB hospital. Swab samples were collected at admission and at four week intervals thereafter during hospitalization. Identification and antimicrobial susceptibility testing of bacterial isolates (n=62) were determined at the National Health Laboratory Services (NHLS) by the VITEK-MS and Vitek 2 systems respectively. Additional antimicrobial susceptibility testing was conducted by Sensititre Gram Negative Xtra (GNFX2) MIC plates. PCR and DNA sequencing were used for detection of resistance genes. Patients (n=13/37; 35%) were colonized by MDR bacteria (ESBLs [n=11], MRSA [n=2]) on admission. Colonization rates were lower in patients admitted from the community (9/28; 32%) compared to those transferred from other healthcare facilities (4/9; 44%). All admitted patients who did not exhibit colonization at baseline and who were resident within the hospital for longer than 4 weeks (17/37; 46% of total patients) became colonised by an ESBL-producing Enterobacteriaceae species. No patients acquired MRSA during hospitalisation. Among ESBL Enterobacteriaceae, Escherichia coli (41/62; 66%) and K. pneumoniae [14/62; 23%]) predominated. Nineteen percent (7/37) of patients demised during their hospitalization. Both the Vitek system and Sensititre Gram Negative Xtra (GNFX2) MIC plates susceptibilities were similar for most antimicrobials, however there were discrepancies for tigecycline susceptibility profiles. A high number of isolates exhibited resistance to aminoglycosides and fluoroquinolones. Genes encoding for ESBLs (CTX-M-14, CTX-M-15, SHV-28, OXA-1, and OXY-2-9) were detected among ESBL Enterobacteriaceae. Two Enterobacteriaceae isolates with reduced carbapenem susceptibility did not contain carbapenemase-encoding genes. MLST revealed unique sequence types and genetic diversity among the K. pneumoniae isolates from hospitalised patients. However, the source and colonization routes of these isolates could not be determined, which requires further investigation. This study provides insight into the spectrum of bacterial pathogen colonisation in hospitalised TB patients and suggests a review of infection control programs and practices at the TB hospital.
- Full Text:
- Authors: Annear, Dale John
- Date: 2018
- Subjects: Medical microbiology , Microbiology , Bacteriology , Tuberculosis -- Hospitals -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/21482 , vital:29526
- Description: Patients with drug resistant tuberculosis (TB) are treated with multiple antibiotics including moxifloxacin, linezolid, and meropenem, which puts them at greater risk for colonisation by multi-drug resistant (MDR) bacteria. The objectives of this study were to: (i) assess the antimicrobial prescribing patterns practiced within the hospital by retrospective patient file review; (ii) determine the spectrum of bacterial colonisation in TB patients upon admission and during hospitalisation; (iii) identify bacterial isolates and evaluate antimicrobial susceptibility profiles; (iv) detect antimicrobial resistance genes in the bacterial isolates by PCR and DNA sequencing; and (v) investigate genetic relatedness of Klebsiella pneumoniae isolates using Multi Locus Sequence Typing. Nasal, groin and rectal swabs [for the detection of extended spectrum beta lactamases (EBSLs), carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA)] were analysed from a cohort of patients (n=37) admitted either from the community (n = 28) or from other healthcare facilities (n=9) to a TB hospital. Swab samples were collected at admission and at four week intervals thereafter during hospitalization. Identification and antimicrobial susceptibility testing of bacterial isolates (n=62) were determined at the National Health Laboratory Services (NHLS) by the VITEK-MS and Vitek 2 systems respectively. Additional antimicrobial susceptibility testing was conducted by Sensititre Gram Negative Xtra (GNFX2) MIC plates. PCR and DNA sequencing were used for detection of resistance genes. Patients (n=13/37; 35%) were colonized by MDR bacteria (ESBLs [n=11], MRSA [n=2]) on admission. Colonization rates were lower in patients admitted from the community (9/28; 32%) compared to those transferred from other healthcare facilities (4/9; 44%). All admitted patients who did not exhibit colonization at baseline and who were resident within the hospital for longer than 4 weeks (17/37; 46% of total patients) became colonised by an ESBL-producing Enterobacteriaceae species. No patients acquired MRSA during hospitalisation. Among ESBL Enterobacteriaceae, Escherichia coli (41/62; 66%) and K. pneumoniae [14/62; 23%]) predominated. Nineteen percent (7/37) of patients demised during their hospitalization. Both the Vitek system and Sensititre Gram Negative Xtra (GNFX2) MIC plates susceptibilities were similar for most antimicrobials, however there were discrepancies for tigecycline susceptibility profiles. A high number of isolates exhibited resistance to aminoglycosides and fluoroquinolones. Genes encoding for ESBLs (CTX-M-14, CTX-M-15, SHV-28, OXA-1, and OXY-2-9) were detected among ESBL Enterobacteriaceae. Two Enterobacteriaceae isolates with reduced carbapenem susceptibility did not contain carbapenemase-encoding genes. MLST revealed unique sequence types and genetic diversity among the K. pneumoniae isolates from hospitalised patients. However, the source and colonization routes of these isolates could not be determined, which requires further investigation. This study provides insight into the spectrum of bacterial pathogen colonisation in hospitalised TB patients and suggests a review of infection control programs and practices at the TB hospital.
- Full Text:
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