Author: Evita Chrisnayanti
Chrisnayanti, Evita, 2018 Bioactive Compounds against Multidrug Resistant Bacteria Produced by an Endophytic Actinomadura sp., Flinders University, College of Nursing and Health Sciences
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The emergence of antibiotic resistance compels the continuing efforts to discover new effective antibiotics. However, the antibiotic pipeline is starving because natural products as the sources for high quality compounds in drug discovery and one of the keys for success in pharmaceutical discovery programs have been overlooked. This study aims to find bioactive compounds produced by endophytic actinobacteria that are active against resistant bacteria overexpressing drug efflux pumps.
The isolation of endophytic actinobacteria residing in wood from deep within the trunks of Australian eucalyptus trees was conducted to search for rare actinobacteria that were proposed to potentially produced novel antibiotics. Applying a rational approach to improve the isolation of actinobacteria ,i.e., powdering the wood to expose the microorganisms, extending the incubation of isolation plates for 16 weeks, using isolation media with low nutrients, and plating more samples on a larger number of isolation plates, a total of 59 endophytic actinobacteria were isolated from wood of Australian eucalyptus trees: Sydney blue gum (Eucalyptus saligna), Lemon scented gum (Corimbia citriodora) and a tentatively classified eucalyptus as stringy bark (Eucalyptus obliqua). Most of them (51) were obtained from wood powder of stringy bark eucalyptus. It is interesting that rare genera of actinobacteria were identified among the isolates including Asanoa, Actinoplanes, Actinomadura and Actinoallomurus.
The methanol extracts of endophytic actinobacteria isolated from wood in the present study and endophytic actinobacteria that have been isolated before from the root, leaves and stem of native Australian plants were screened for their activity using a whole cell target based assay against well defined Staphylococcus aureus overexpressing Nor A efflux pump. One extract of an actinobacterium isolated from the inner part of the wood of Sydney Blue Gum designated as DG1 produced clear zones of inhibition. The HPLC/DAD analysis showed the extract contains at least 3 peaks at the retention time of 4.45, 6.84, and 7.02 that have unique UV spectra compared to a proprietary database of compounds produced by actinobacteria. Strain DG1 was identified as Actinomadura sp.
Submerged fermentation up to 4 L was done using F40 medium for the production of active compounds by strain DG1 compared to other 5 media. Bioassay guided fractionation was then conducted to separate the active compounds. The mycelial extract from scaled up submerged fermentation was purified using High Performance Centrifugal Partition Chromatography (HPCPC). However, it was difficult to achieve further purification due to its instability and resultant low quantity of active compound. More stable active compounds were detected in the agar culture derived extract. Based on TLC patterns, the active compounds produced in solid-state fermentation were different from those produced in submerged fermentation but retained their higher activity against the multidrug resistant Staphylococcus aureus compared to the sensitive strain. It is interesting that the LC/MS of the ethyl acetate extract after liquid partition of solid fermentation derived extract showed the presence of stable active compounds with unique UV spectra compared to a data base of compounds produced by actinobacteria. Larger scale up of the solid state growth conditions are required to generate sufficient pure compounds for structure elucidation.
Despite the lack of structure elucidation of the pure active compounds, this study suggested that uncommon actinobacteria are potential producers of novel antimicrobial compounds to reverse antibiotic resistance. The whole cell-target based screening employing a well-defined resistant strain overexpressing efflux pumps led to the detection of potentially new antibiotics active against multidrug resistant bacteria. The isolation of actinobacteria from neglected habitats has enormous promise for the discovery of new antibiotics and subsequently allow the antibiotic pipeline to flow once more.
Keywords: Bioactive compounds, multidrug resistant bacteria, endophytic actinobacteria
Subject: Medical Biotechnology thesis
Thesis type: Doctor of Philosophy
Completed: 2018
School: College of Nursing and Health Sciences
Supervisor: Professor Chris Franco