Altered Antibiotic Transport in OmpC Mutants Isolated from a Series of Clinical Strains of Multi-Drug Resistant E. coli

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.

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…On the other hand, imipenem binding in P. aeruginosa OprD necessitates the presence of intact loops 2 and 3 (185), while external loops 5, 7, and 8 served to constrict the OprD channel entrance and prevent the nonspecific passage of antibiotics (96). Mutations in the constriction zone reduced the passage of cefotaxime and other ␤-lactams through the OmpC channel of several resistant E. coli isolates (138). A third type of mutation includes those affecting the regulatory proteins that control the expression of porin-encoding genes.…”

“…For example, E. coli isolates from a patient undergoing treatment with several antimicrobials showed changes in the OmpC protein that resulted in four different variants, all of which conferred increased resistance to cefotaxime (138). Likewise, Oteo et al (192) described the recovery of three consecutive isolates of E. coli from a patient treated with ertapenem.…”

One ring to rule them all: Current trends in combating bacterial resistance to the β‐lactams

“…For instance, b-lactam resistance in E. coli can involve the loss of OmpF 82 or mutations in OmpC around the point of pore constriction. 83 A further example is seen in P. aeruginosa carbapenem resistant isolates where the facilitating porin, OprD, is lost due to various disruptions including deletion, nonsense mutations, gene insertion, or decreased transcription due to mutation in regulatory elements. [84][85][86] Although resistance is often accompanied by a fitness cost, a recent study has challenged this paradigm by demonstrating the association of OprD loss and concurrent carbapenem resistance with enhanced in vivo fitness and virulence.…”

Compensation of the Metabolic Costs of Antibiotic Resistance by Physiological Adaptation in Escherichia coli

“…When resistant cells were exposed to amoxicillin, upregulation was observed for cusR (2.3-fold), a known regulator of genes related to copper efflux (38); cusF, coding for the copper efflux system (3.7-fold); cusC (23.5-fold); the multicopper oxidase gene, cueO (2.5-fold); and the Cu ϩ efflux ATPase gene, copA (2.3-fold). Altered expression of porins or restriction of their functions due to point mutations has been linked to antibiotic resistance (39). However, amoxicillin-resistant cells showed only 2.1-fold-increased expression of ompW in response to the antibiotic.…”