Browsing by Author "Booysen, Elzaan"

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    Characterization of a novel antibiotic isolated from Xenorhabdus khoisanae and encapsulation of vancomycin in nanoparticles
    (Stellenbosch : Stellenbosch University, 2018-03) Booysen, Elzaan; Dicks, Leon Milner Theodore; Sadie-Van Gijsen, Hanel; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Periprosthetic joint infection (PJI) is the major cause of total joint arthroplasty failures and is often caused by methicillin-resistant Staphylococcus aureus (MRSA). The ability of these bacteria to rapidly acquire resistance against antibiotics has made it nearly impossible to treat these persistent infections. The number of novel antibiotics that have successfully completed clinical trials has declined rapidly in the last 50 years. The search for novel antibiotics and alternative delivery routes is thus of utmost importance. Entomopathogenic bacteria, living in close association with nematodes, are a potential source of novel antibiotics. One such genus, Xenorhabdus, produces a variety of secondary metabolites, including antimicrobial compounds. The majority of these compounds are active against numerous so-called multidrug resistant pathogens. Antibiotics produced by Xenorhabdus spp. may thus be an alternative treatment for PJI. Numerous drugs fail phase II and III clinical trials due to insolubility, toxicity and instability at pharmaceutically active levels. This can be overcome by encapsulating the therapeutic drugs in nanoparticles. The polymer poly(DL-lactide-co-glycolide) (PLGA) has significant attention as a colloidal drug delivery device and is well-known for its biocompatibility. In this study, Xenorhabdus khoisanae was screened for the production of novel antibiotics. Three antibiotics were isolated from a X. khoisanae culture, two were similar to xenocoumacin-2 and one a novel antibiotic with a mass-to-charge ratio of 671, designated rhabdin. Rhabdin is active against two clinical strains of S. aureus (including MRSA). The osteogenic and cytotoxic effects of rhabdin were evaluated on two populations of rat femora-derived mesenchymal stem cells (MSC). Rhabdin was cytotoxic to the bone marrow-derived mesenchymal stem cells (bmMSC) at concentrations exceeding 3.5 μg/ml, but had no anti-osteogenic effects. In contrast, rhabdin was completely cytotoxic to proximal femur-derived mesenchymal stem cells (pfMSC). Vancomycin, traditionally used to treat MRSA, was also evaluated and no cytotoxicity was observed in bmMSC or pfMSC, but vancomycin had an anti-osteogenic effect on pfMSC. Vancomycin was encapsulated in PLGA nanoparticles (VNP) by electrospraying. The mean hydrodynamic diameter of VNP was 247 nm. The antimicrobial activity of free vancomycin and encapsulated vancomycin was compared and VNP showed enhanced antimicrobial activity. Vancomycin release was monitored for 10 days and followed first-order release. After10 days, only 50% of the encapsulated vancomycin was released from the nanoparticles. To our knowledge, this is the first study to report on antibiotics produced by X. khoisanae, the anti-osteogenic effects of vancomycin and the encapsulation of vancomycin in PLGA nanoparticles by electrospraying.
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    Profiling of the secondary metabolites and the characterization two novel antilisterial peptides, xenopep and rhabdin, produced by xenorhabdus khoisanae
    (Stellenbosch : Stellenbosch University, 2022-04) Booysen, Elzaan; Dicks, Leon Milner Theodore; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: In the early 1900’s the discovery of sulfonamides and penicillin paved the way for antibiotics and led to a boom in the discovery of other antibiotics. Unfortunately, this boom was short lived and soon the discovery and approval of novel antibiotics by the food and drug association and other similar associations dwindled. With the ever-increasing prevalence of antibiotic resistant pathogens this soon became a problem that was not foreseen. Most antibiotics currently on the market have been isolated from a select few genera. With nearly all the antibiotics from such few sources, bacteria were able to acquire resistance at an enhanced pace. This study focused on a relatively unexplored niche for novel antibiotics, from the genus Xenorhabdus. Species of this genus is mutually associated with Steinernema nematodes and have a unique life cycle. Xenorhabdus spp. are known to produce various secondary metabolites (SMs) that have antimicrobial, insecticidal, antiviral, immunosuppressant and proteolytic properties. Species from this genus use different synthesis machineries to produce these compounds, although the majority are produced via the non-ribosomal peptide synthesis. The ability of non-ribosomal peptides to incorporate non-proteogenic amino acids, D-amino acids, fatty chains, or polyketide chains result in unique resistance to proteinases and environmental stressors. Xenorhabdus khoisanae J194 is mutually associated with Steinernema jeffreyense J194, a nematode that was isolated from soil in the Eastern Cape. Culture conditions, especially oxygen, greatly affected SM production of X. khoisanae J194. PAX peptides, xenocoumacins and xenoamicins were identified in the cell-free crude extract of X. khoisanae J194 cultures. Two novel antilisterial peptides, xenopep and rhabdin, were also detected in the cell-free crude extract of. Xenopep has a narrow spectrum of activity and inhibited the growth of only, Listeria monocytogenes and Staphylococcus epidermidis, while rhabdin is active against both Gram-positive and Gram-negative bacteria. Xenopep and rhabdin share numerous characteristics and both contain a tetra-peptide in their structure including a tetra-peptide in their structure. Both peptides share the same amphipathic characteristic and behave similar suspension. Membrane potential and ATP release assays have shown that xenopep formed pores/lesions in the cell membrane of L. monocytogenes within minutes, followed by a rapid decrease in cell numbers over 3 hours. Scanning electron microscopy (SEM) images of L. monocytogenes treated with xenopep became elongated and formed filaments. This suggests that xenopep may inhibit penicillin binding protein three. This is the first study reporting on SMs produced by X. khoisanae when cultured under different conditions and is the first detailed description of antilisterial peptides produced by the species.
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    Profiling the production of antimicrobial secondary metabolites by xenorhabdus khoisanae J194 under different culturing conditions
    (Frontiers Media, 2020-03-30) Booysen, Elzaan; Rautenbach, Marina; Stander, Marietjie A.; Dicks, Leon M. T.
    Species from the genus Xenorhabdus, endosymbiotic bacteria of Steinernema nematodes, produce several antibacterial and antifungal compounds, some of which are anti-parasitic. In this study, we report on the effect growth conditions have on the production of antimicrobial compounds produced by Xenorhabdus khoisanae J194. The strain was cultured in aerated and non-aerated broth, respectively, and on solid media. Production of antimicrobial compounds was detected after 24 h of growth in liquid media, with highest levels recorded after 96 h. Highest antimicrobial activity was obtained from cells cultured on solid media. By using ultraperformance liquid chromatography linked to mass spectrometry and HPLC, a plethora of known Xenorhabdus compounds were identified. These compounds are the PAX lipopeptides (PAX 1′, PAX 3′, PAX 5, and PAX 7E), xenocoumacins and xenoamicins. Differences observed in the MS-MS fractionation patterns collected in this study, when compared to previous studies indicated that this strain produces novel xenoamicins. Three novel antimicrobial compounds, khoicin, xenopep and rhabdin, were identified and structurally characterized based on MS-MS fractionation patterns, amino acid analysis and whole genome analysis. The various compounds produced under the three different conditions indicates that the secondary metabolism of X. khoisanae J194 may be regulated by oxygen, water activity or both. Based on these findings X. khoisanae J194 produce a variety of antimicrobial compounds that may have application in disease control.