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High throughput method to determine the surface activity of antimicrobial polymeric materials.

ABSTRACT: Surface colonization by microorganisms, combined with the rise in antibiotic resistance, is the main cause of production failures in various industries. Self-sterilising materials are deemed the best prevention of surface colonization. However, current screening methods for these sterilising materials are laborious and time-consuming. The disk diffusion antimicrobial assay and the Japanese industrial standard method for antimicrobial activity on solid surfaces, JIS Z 2801, were compared to our modified solid surface antimicrobial assay in terms of detecting the activity of antibiotic-containing cellulose disks against four bacterial pathogens. Our novel assay circumvents the long incubation times by utilising the metabolic active dye, resazurin, to shorten the time in which antibacterial results are obtained to less than 4 h. This assay allows for increased screening to identify novel sterilising materials for combatting surface colonisation.•Disk diffusion assay could only detect the activity of small compounds that leached from the material over 20-24 h.•JIS Z 2801 was also able to detect the surface activity of non-polar compounds, thought to be inactive based on the disk diffusion results.•The resazurin solid surface antimicrobial assay could obtain the same results as the JIS Z 2801, within a shorter time and in a high-throughput 96-well plate setup.

SUBMITTER: van Rensburg W

PROVIDER: S-EPMC8720914 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTo facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.ResultsTwo or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.ConclusionsThe method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.

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High throughput method to determine the surface activity of antimicrobial polymeric materials.

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