BioSAXS Measurements Reveal That Two Antimicrobial Peptides Induce Similar Molecular Changes in Gram-Negative and Gram-Positive Bacteria.

Andreas Von Gundlach,Axel Rosenhahn,Sharon Ann Huws,Christoph Rumancev,Martin P Ashby,Ralf Mikut,Vasil M Garamus,Paula Matilde Lopez-Perez,Alan Roy Cookson,Jurnorain Gani,Kai Hilpert

doi:10.3389/fphar.2019.01127

Abstract

Two highly active short broad-spectrum AMPs (14D and 69D) with unknown mode of action have been investigated in regards to their effect against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria methicillin-resistant Staphylococcus aureus (MRSA). Minimal inhibitory concentration (MIC) measurements using a cell density of 108 cfu/ml resulted in values between 16 and 32 µg/ml. Time-kill experiments using 108 cfu/ml revealed complete killing, except for 69D in combination with MRSA, where bacterial load was reduced a million times. Small-angle X-ray scattering of biological samples (BioSAXS) at 108 cfu/ml was applied to investigate the ultrastructural changes in E. coli and MRSA in response to these two broad-spectrum AMPs. In addition, electron microscopy (EM) was performed to visualize the treated and non-treated bacteria. As expected, the scattering curves generated using BioSAXS show the ultrastructure of the Gram-positive and Gram-negative bacteria to be very different (BioSAXS is not susceptible to the outer shape). After treatment with either peptide, the scattering curves of E. coli and MRSA cells are much more alike. Whereas in EM, it is notoriously difficult to observe changes for spherical Gram-positives; the BioSAXS results are superior and reveal strongly similar effects for both peptides induced in Gram-positive as well as Gram-negative bacteria. Given the high-throughput possibility and robust statistics, BioSAXS can support and speed up mode of action research in AMPs and other antimicrobial compounds, making a contribution toward the development of urgently needed drugs against resistant bacteria.

Highlights

The World Health Organization (WHO) has classified antimicrobial resistance as one of the biggest threats to global health and food security
The aims of this study are (1) demonstrating that BioSAXS can be used for Gram-positive bacteria and (2) a comparison of modes of action of two antimicrobial peptides against Grampositive and Gram-negative bacteria
Bacterial strains used for antimicrobial activity testing in this project were methicillin-resistant Staphylococcus aureus (S. aureus) HO 5096 0412; a methicillin-sensitive S. aureus (ATCC 29213); Escherichia coli (E. coli, UB1005, F, LAM, gyrA37, relA1, spoT1, metB1, LAMR); E. coli (ATCC 25922); E. coli (68610Y); a clinical isolate from St

Summary

Introduction

The World Health Organization (WHO) has classified antimicrobial resistance as one of the biggest threats to global health and food security. The extent of the threat requires action from researchers, but from governments and society. Antibiotics are misused in ton scale in agriculture for growth promotion or prevention of disease, but actions are taken to reduce this—for example, the European Union has banned the use of antibiotics for growth promotion in 2006. About 700,000 to 1,000,000 people die worldwide each year because of antibiotic-resistant infections. In the O’Neil Report, it is estimated that, by 2050, the numbers increase to 10,000,000 more people than are currently killed by cancer It was estimated that the additional healthcare cost worldwide for antibiotic-resistant infections will reach US$100 trillion. FDA approval of new antimicrobials has dropped to three new molecular entities (NME) in this decade

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