The development of an antibacterial assay and its use in the investigation of the combined effect of tetracycline and synthetic antimicrobial peptides on strains of Escherichia coli

Du Toit, Etienne Andre (1999-11)

Thesis (M.Sc.) -- University of Stellenbosch, 1999.

Thesis

ENGLISH SUMMARY: Magainin 2 is a 23-residue antimicrobial peptide secreted on the skin of Xenopus laevis. In our study on antimicrobial peptides, this cationic, α-helical peptide and two N-terminal deletion analogues were synthesised. The first of the deletion peptides differed from the full length peptide by the omission of two amino acid residues from the N-terminus of rnagainin 2, while the second deletion peptide had four residues omitted from the N-terminus (termed magainin 2 N21 , and magainin 2 N19 respectively). Peptides were purified by gel permeation chromatography and high performance liquid chromatography (HPLC). The synthetic peptides were of high chemical purity as verified with' electro spray ionisation mass spectrometry (ESI-MS) and analytical HPLC. To further evaluate the antimicrobial activity of the peptides in this study, a highly sensitive micro-gel well diffusion assay was developed. This assay was compared to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus lute us as the indicator organism. The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method. Data analysis to calculate minimum inhibitory concentration (MIC), 50% microbial growth inhibition (IC50), and maximum bactericidal concentration (MBC) was refined by generating dose-response curves with Prism® 2.01 (Graphpad Software Inc.). The MICs, determined by the three methods, were significantly different (P<O.OOI), highlighting the limitations involved in comparing data obtained from different methods. Furthermore, the synergistic antimicrobial activity of magainin 2 and tetracycline on tetracycline resistant Escherichia coli was investigated. The two N-terminal deletion analogues were also used in the synergism studies. The resistance towards tetracycline is caused by expulsion from resistant cells through efflux pumps. Magainin 2 was selected as antimicrobial peptide because of its ability to form channels in the target membrane, which may help in the circumvention of this form of resistance. All tests were done using the microgel well diffusion assay, with the test organism being tetracycline resistant E. coli. Magainin 2, at MIC and IC50 concentrations, improved the activity of tetracycline by ±12% and ±29% respectively. Magainin 2 N21 improved the activity of tetracycline by ±19% at its IC50, and showed no effect at its MIC. Magainin 2 N19 exhibited no antimicrobial activity on its own, but still succeeded in improving the activity of tetracycline by approximately ±9% when used at 138 nmol/mL concentration. In all three cases, this improvement was only observed at tetracycline concentrations below or near its MIC. At higher tetracycline concentrations the improvement of activity, attributed to magainin 2, was not detected. The loss in synergism could be the consequence of competition between tetracycline and the peptide for the magnesium binding sites in the lipopolysaccharide layer of the gram-negative organism. Intensive research is necessary to alleviate the problem of resistance, and our research indicates that the combination of conventional antibiotics and antimicrobial peptides in antibiotic preparations may be a viable option.

AFRIKAANSE OPSOMMING: Magainin 2 is 'n 23-residue peptied wat op die vel van Xenopus laevis uitgeskei word. In ons studies, wat handel oor antimikrobiese peptiede, is hierdie kationiese, α-heliese peptied, sowel as twee N-terminaal delesie analoe gesintetiseer. Twee residue is van die N-terminaal weggelaat om die 21-residu delesie peptied te produseer, terwyl vier residue weggelaat is om die 19-residu peptied te produseer (onderskeidelik magainin 2 N21 en magainin 2 NI9 genoem). Peptiede is gesuiwer met behulp van gelpermeasie chromatografie en hoe doeltreffendheid vloeistof chromatografie (HPLC). Die hoe chemiese suiwerheid van die sintetiese produkte is deur elektrosproei-ionisasie massaspektrometrie (ESI-MS) en analitiese HPLC bevestig. Om die antimikrobiese aktiwiteit van die gesintetiseerde peptiede te evalueer was dit nodig om 'n hoogs sensitiewe mikro-gel dispersie essaY te ontwikkel. Hierdie essai is met 'n radiale difussie- en 'n mikrotiter-mediumverdunning-essai vergelyk. Die antimikrobiese peptied, gramicidin S, as toetspeptied en Micrococcus luteus as die indikator organisme, is gebruik in die ontwikkeling van die essai. Die mikro-gel dispersie-essal se sensitiwiteit was vergelykbaar met die mikrotiter-mediumverdunning-essai, terwyl dit ongeveer twee keer so sensitief was as die radiale difussie-essai. Die berekening van die minimum inhibitoriese konsentrasie (MIC), 50% inhibisie konsentrasie (ICs50, en maksimum bakterisidale konsentrasie (MBC) is verfyn deur die opstel van dosisrespons-kurwes met behulp van Prism®2.01 (Graphpad Software Inc.). Die MICs, wat in al drie metodes bereken is, het betekenisvol van mekaar verskil, wat aandui dat resultate van verskillende metodes nie met mekaar vergelyk kan word nie. Verder, is die sinergistiese antimikrobiese aktiwiteit van magainin 2 en tetrasiklien op tetrasiklien-weerstandbiedende Escherichia coli ondersoek. Die twee N-terminale delesie analoe is ook in die sinergisme studies gebruik. Die weerstandbiedendheid teenoor tetrasiklien word veroorsaak deur die uitpomp van die antibiotika uit die sel deur middel van efflukspompe. Magainin 2 is gebruik as antimikrobiese peptied omdat dit die vermoe het om kanale in die teikenmembraan te vorm, en moontlik hierdie vorm van weerstand kan verminder. Alle toetse is gedoen met behulp van die mikro-gel dispersie-essai, waarin tetrasiklien-weerstandbiedende Escherichia coli as toetsorganisme gebruik is. Magainin 2, gebruik by MIC en IC50 konsentrasies, het die aktiwiteit van tetrasiklien met ±12% en ±29% onderskeidelik verbeter. Magainin 2 N21 by IC50, het die aktiwiteit van tetrasiklien met ±19% verbeter, maar het geen effek by sy MIC getoon. Magainin 2 NI9 het geen antimikrobiese aktiwiteit getoon nie, maar het wel die aktiwiteit van tetrasiklien met ±9% verbeter as dit by 138 nmol/mL gebruik word. In al drie gevalle is sinergisme net gevind by konsentrasies laer of naby die MIC van tetrasiklien. By hoer tetrasiklienkonsentrasies het die verbetering in aktiwiteit, as gevolg van die teenwoordigheid van magainin 2, verlore gegaan. Die verlies in sinergisme is moontlik as gevolg van kompetisie tussen tetrasikIien en magainin 2 vir magnesiumbindingplekke op die lipopolisakkariedlaag van Gram-negatiewe organismes. Baie navorsing moet egter nog gedoen word om die probleem van weerstand teen antibiotika te oorkom. Hierdie resuitate bewys dat die kombinasie van konvensionele antibiotika en antimikrobiese peptiede wel weerstand kan verminder.

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