Life And Death In A Bacterial Biofilm Under Antibiotic Attack Characterized By Fluorescence And Atomic Force Microscopy

With the increase in bacteria developing resistance to traditional antibiotics, there is a pressing need for new antibiotic compounds. Researchers are currently exploring the use of antimicrobial peptides as a new class of antibiotic drugs. Antimicrobial peptides are typically small and occur naturally as components of many immune systems. These peptides kill bacteria in one of two mechanisms. In the first mechanism, multiple peptide subunits join together to form pores in the bacterial membranes. In the second, the peptide crosses the membrane and binds to some molecule in the cell, preventing its action. Magainin II, first isolated from the skin of the African clawed toe frog Xenopus leavis, is a classic example of a pore-forming peptide and has been investigated in lipid micelles, bacterial spheroplasts, and various bacteria. However, much of the research on magainin II and other antimicrobial peptides has focused on free-swimming, planktonic bacteria, yet many bacteria live not as planktonic cells, but in an organized community called a biofilm. Due to their complex architecture and excreted exopolymeric substances (EPS), cells in the biofilm are notoriously hard to kill. Thus it is essential to evaluate the effectiveness and mechanism of action of any new classes of antibiotics on both planktonic and biofilm cells. Here we use fluorescence microscopy and atomic force microscopy (AFM) to evaluate the changes that occur in both planktonic and biofilm Escherichia coli cells when exposed to this antimicrobial peptide in native conditions. Notably, using AFM we find distinct changes in both cell stiffness and the morphology of the outer membrane after treatment of both planktonic and biofilm cells, but we also observe that the EPS protects some cells in the biofilm.