Characterization of a novel antibiotic isolated from Xenorhabdus khoisanae and encapsulation of vancomycin in nanoparticles

Booysen, Elzaan (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

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.

AFRIKAANSE OPSOMMING: Post-chirurgiese infeksie van ‘n prostetiese gewrig is die mees algemene oorsaak vir onsuksesvolle totale gewrigs vervangings. Hierdie infeksies word meestal deur metisillien-weerstandige Staphylococcus aureus (MWSA) veroorsaak. Die vermoeë van hierdie bakterieë om vinnige weerstandigheid teen antibiotika op te bou, maak dit bykans onmoonlik om weerstandige infeksies te behandel. In die laaste 50 jaar het die aantal antibiotika wat kliniese toetse suksesvol voltooi het, vinnig gedaal. Die soektog na unieke en nuwe antibiotika en alternatiewe middels is dus van uiterste belang. Die entomopatogeniese genus, Xenorhabdus, wat in ‘n eng gemeenskaplike verwantskap met nematodes leef, is ‘n moontlike bron van unieke antibiotika. Xenorhabdus spp. produseer n verskeidenheid sekondêre metaboliete, insluitend antimikrobiese verbindings. Die meederheid van hierdie verbindings is teen ‘n verskeidenheid veelvuldige antibiotika-weerstandige patogene aktief. Daar, is dus ‘n moontlikheid dat antibiotika van Xenorhabdus spp. as behandeling teen prostetiese gewrigs-infeksies kan dien. As gevolg van onoplaasbaarheid, toksisiteit en onstabiliteit teen farmakologies-aktiewe vlakke, misluk veelvuldige antibiotia tydens fase II and III kliniese toetse. Hierdie negatiewe punte kan oorkom word deur die terapeutiese middels in nanopartikels te omsluit. Die polimeer “poly(DL- lactide-co-glycolide) (PLGA)” geniet baie aandag as ‘n kolloïdale toedieningsisteem en is wêreldbekend as ‘n biologies-versoenbare polimeer. In hierdie studie is Xenorhabdus khoisanae vir die producksie van unieke antibiotika getoets. Drie antibiotika is van ‘n X. khoisanae kultuur geïsoleer, twee is soortgelyk aan xenocoumacin- 2 en een is ‘n unieke verbinding met ‘n massa-tot-lading verhouding van 671, aangewys as rhabdin. Rhabdin is aktief teen twee klinies-verwante S. aureus rasse (insluitend MWSA). Die osteogeniese en sitotoksiese effek van rhabdin is teen twee populasies van rot femur-afgeleide mesenkiem-stamselle (MSS) getoets. Rhabdin is sitotoksies vir beenmurg-afgeleide mesenkiem-stamselle (bmMSS) teen konsentrasies hoër as 3.5 μg/ml, maar het geen anti-osteogeniese effek gehad nie. In teenstelling hiermee, is rhabdin heeltemal toksies vir proksimaal femur-afgeleide mesenkiem-stamselle (MSS). Vankomisien word tradisioneel gebruik om MWSA infeksies te behandel en is vir toksisiteit en osetogeniese effek geëvalueer. Geen toksiese effek is waargeneem in bmMSS en pfMSS kulture nie, maar vankomisien het wel ‘n antiosteogeniese effek teen pfMSS getoon. Vankomisien is in PLGA nanoparticles (VNP) deur middel van elektrosproei omsluit. Die hidrodinamiese deursnit van VNP is 247 nm. Die antimikrobiese aktiwiteit van vrye vankomisien en PLGA-omsluite vankomisien is vergelyk. Vankomisien omsluite partikels het meer antimikrobiese aktiwiteit getoon.. Vankomisien vrystelling is vir 10 dae gemonitor en het eerste-orde vrystelling getoon. Na 10 dae was 50% van die omsluite vankomisien vrygestel. So ver ons kennis strek is hierdie die eerste studie wat die werking van ‘n antibiotikum, geproduseer deur X. khoisanae, raporteer. Hierdie is ook die eerste studie op die anti- osteogeniese effek van vankomisien en die omsluiting van vankomisien in ‘n PLGA polimeer deur middle van die elektrosproei tegniek.

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