Abstract
Antibiotic resistance is emerging in Gram-negative bacteria worldwide. The outer membrane of Gram-negative bacteria is a formidable selective barrier, and a major factor in broad-spectrum antibiotic resistance. Influx of antibiotics into the Periplasm of bacteria is facilitated by proteins that form channels in the outer membrane. We studied the influx of several cephalosporin antibiotics through the major Escherichia coli porins OmpF and OmpC.Conductance measurements through purified single porins reconstituted in artificial lipid bilayers allowed us to count the passage of single antibiotic molecules. Statistical analysis of transport events yields the kinetic parameters at the single molecular level. Fluorescence steady-state measurements were used to quantify the interaction between the antibiotics and the porin channels and verify the calculation of translocation kinetics. For the first time, we have been able to characterize facilitated translocation of several β-lactams through OmpC (most expressed porin in vivo) and quantified the distinguishable permeation properties of ceftriaxone, ceftazidime and cefpirome through both outer membrane porins OmpF and OmpC - concluding a stronger interaction with OmpF than OmpC for all three cephalosporins -especially ceftazidime-.Our approach may be of great benefit to the understanding of porin-drug interaction at the molecular scale and may contribute to the rational design of more efficient antibiotics.
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