Molecular Basis of Enrofloxacin Affinity to a Membrane Channel of E. Coli - When Binding Does Not Imply Translocation

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Optical investigation of individual red blood cells for determining cell count and cellular hemoglobin concentration in a microfluidic channel

We demonstrate a blood analysis routine by observing red blood cells through light and digital holographic microscopy in a microfluidic channel. With this setup a determination of red blood cell (RBC) concentration, the mean corpuscular volume (MCV), and corpuscular hemoglobin concentration mean (CHCM) is feasible. Cell count variations in between measurements differed by 2.47% with a deviation of −0.26×106 μL to the reference value obtained from the Siemens ADVIA 2120i. Measured MCV values varied by 2.25% and CHCM values by 3.78% compared to the reference ADVIA measurement. Our results suggest that the combination of optical analysis with microfluidics handling provides a promising new approach to red blood cell counts

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

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

Molecular Basis of Enrofloxacin Translocation through OmpF, an Outer Membrane Channel of Escherichia coli - When Binding Does Not Imply Translocation

The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation