Tivadar Mach

• The outer membrane of Gram--negative bacteria, acting as a selectively permeable barrier, is a major factor in broad--spectrum antibiotic resistance. This selective permeability may be investigated extit[in vitro], by focussing on the individual constituents of the outer membrane. It has been shown that a weak affinity of hydrophilic antimicrobials to the interior of general diffusion porins in the bacterial wall enhances their translocation through the pore, resulting in greater accumulation of the antibiotic in the bacterium. In this work, a method is developed to ascertain with molecular detail the membrane translocation route of antibiotics, using a combination of high--resolution conductance measurements on a single porin in an artificial bilayer, fluorescence resonance energy transfer measurements and molecular dynamics simulations. A porin--based route of entry is shown for moxifloxacin, and a pathway through the lipid for nalidixic acid (a hydrophilic and hydrophobic quinolone). The translocation kinetics and affinity of other quinolone antibiotics have been measured by pore conductance fluctuations. An understanding of the detailed molecular interactions between the antibiotic and its entry channel may be used to develop new antibiotics with improved uptake kinetics. To facilitate future measurements a novel microfluidics--based planar lipid bilayer measurement system has been developed capable of high--resolution, low noise recordings and double sided perfusion. The system has the potential to screen a large number of specific outer membrane channels against a library of antibiotics.