Abstract
Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.
Highlights
Multi-drug resistance is increasingly prevalent in clinically important pathogens and threatens to hinder treatment of diseases hitherto thought ‘‘cured’’
To establish whether there was a role for the OmpC function in altering the minimum inhibitory concentrations (MIC) for different antibiotics clinical isolates were transformed with a plasmid expressing OmpC K12
We infer that OmpC mediated transport of antibiotics into the cell is an important component of clinical resistance. (As a control we tested gentamicin resistance, which does not require transport through the porin to the periplasm and the MIC was unchanged by the presence OmpC.) Our purpose is not to compare OmpC K12 with OmpC20
Summary
Multi-drug resistance is increasingly prevalent in clinically important pathogens and threatens to hinder treatment of diseases hitherto thought ‘‘cured’’. Bacterial resistance occurs by a combination of a reduction in drug passage through the outer membrane into the cell, modification of drug targets (protein or the metabolic pathway), antibiotic degradation or increased transport of antibiotic out of the cell. Porins, which are beta-barrel proteins that traverse the bacterial outer membrane, facilitate the uptake of small polar nutrients and are the main entry pathway for many antibiotics [1,2]. Several clinical studies have linked antibiotic resistance to altered expression of porins, or their restricted function due to point mutation(s) (reviewed in [3]). The selection pressure for such mutants must balance increased resistance against the loss of capacity for nutrient access to the periplasm
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