Possible multidrug-resistant (MDR) mechanisms of four resistant strains of Escherichia coli to a model β-lactam, ampicillin, were investigated using contact angle measurements of wettability, crystal violet assays of permeability, biofilm formation, fluorescence imaging, and nanoscale analyses of dimensions, adherence, and roughness. Upon exposure to ampicillin, one of the resistant strains, E. coli A5, changed its phenotype from elliptical to spherical, maintained its roughness and biofilm formation abilities, decreased its length and surface area, maintained its cell wall integrity, increased its hydrophobicity, and decreased its nanoscale adhesion to a model surface of silicon nitride. Such modifications are suggested to allow these cells to conserve energy during metabolic dormancy. In comparison, resistant strains E. coli D4, A9, and H5 elongated their cells, increased their roughness, increased their nanoscale adhesion forces, became more hydrophilic, and increased their biofilm formation upon exposure to ampicillin. These results suggest that these strains resisted ampicillin through biofilm formation that possibly introduces diffusion limitations to antibiotics. Investigations of how MDR bacterial cells modify their surfaces in response to antibiotics can guide research efforts aimed at designing more effective antibiotics and new treatment strategies for MDR bacterial infections.

An experimental X-ray powder diffraction pattern was produced and analyzed for imipenem monohydrate, an antimicrobial pharmaceutical agent. Although there are no experimental powder patterns in the ICDD PDF-4/Organics Database, there is one powder pattern calculated with single-crystal X-ray diffraction data from the Cambridge Structural Database. Here, we report the refined experimental powder diffraction data for imipenem monohydrate. These data for imipenem monohydrate are consistent with an orthorhombic crystal system having reduced unit-cell parameters of a = 8.2534(3) Å, b = 11.1293(4) Å, and c = 15.4609(6) Å. The resulting unit-cell volume, 1420.15(15) Å3, indicates four formula units per unit cell. Observed peaks are consistent with the P212121 space group.

Despite decades of intense study, the complementarity of β-lactams for β-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, β-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free β-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of β-lactams for the β-lactamase AmpC through reversible denaturation of adducts of the enzyme with β-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 °C and a van't Hoff enthalpy of unfolding (ΔHVH) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (ΔGuH2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (ΔTm = +5.8 °C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (ΔTm = +4.9 °C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (ΔTm = −3.2 °C) and 0.7 kcal/mol (ΔTm = −1.2 °C), respectively. These β-lactams, which share nonhydrogen substituents in the 6(7)α position of the β-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (ΔTm = −1.3 °C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the β-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for β-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other β-lactam binding proteins as well.