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.

The roles of the thicknesses and grafting densities of the surface biopolymers of four multi-drug resistant (MDR) Escherichia coli bacterial strains that varied in their biofilm formation in controlling cellular elasticities after exposure to ampicillin were investigated using atomic force microscopy. Exposure to ampicillin was carried out at minimum inhibitory concentrations for different duration times. Our results indicated that the four strains resisted ampicillin through variable mechanisms. Strain A5 did not change its cellular properties upon exposure to ampicillin and as such resisted ampicillin through dormancy. Strain H5 increased its biopolymer brush thickness, adhesion and biofilm formation and kept its roughness, surface area and cell elasticity unchanged upon exposure to ampicillin. As such, this strain likely limits the diffusion of ampicillin by forming strong biofilms. At three hours’ exposure to ampicillin, strains D4 and A9 increased their roughness, surface areas, biofilm formation, and brush thicknesses and decreased their elasticities. Therefore, at short exposure times to ampicillin, these strains resisted ampicillin through forming strong biofilms that impede ampicillin diffusion. At eight hours’ exposure to ampicillin, strains D4 and A9 collapsed their biopolymers, increased their apparent grafting densities and increased their cellular elasticities. Therefore, at long exposure times to ampicillin, cells utilized their higher rigidity to reduce the diffusion of ampicillin into the cells. The findings of this study clearly point to the potential of using the nanoscale characterization of MDR bacterial properties as a means to monitor cell modifications that enhance “phenotypic antibiotic resistance”.

Persister bacterial cells are great at surviving antibiotics. The phenotypic means by which they do that are underexplored. As such, atomic force microscope (AFM) was used to quantify the contributions of the surface properties of the outer membrane of multidrug resistance (MDR)-Escherichia coli Strains (A5 and A9) in the presence of ampicillin at minimum inhibitory concentration (MIC) (resistant cells) and at 20× MIC (persistent cells). The properties quantified were morphology, root mean square (RMS) roughness, adhesion, elasticity, and bacterial surface biopolymers’ thickness and grafting density. Compared to untreated cells, persister cells of E. coli A5 increased their RMS, adhesion, apparent grafting density, and elasticity by 1.2, 3.4, 2.0, and 3.3 folds, respectively, and decreased their surface area and brush thickness by 1.3 and 1.2 folds, respectively. Similarly, compared to untreated cells, persister cells of E. coli A9 increased their RMS, adhesion and elasticity by 1.6, 4.4, and 4.5 folds, respectively; decreased their surface area and brush thickness by 1.4 and 1.6 folds, respectively; and did not change their grafting densities. Our results indicate that resistant and persistent E. coli A5 cells battled ampicillin by decreasing their size and going through dormancy. The resistant E. coli A9 cells resisted ampicillin through elongation, increased surface area, and adhesion. In contrast, the persistent E. coli A9 cells resisted ampicillin through increased roughness, increased surface biopolymers’ grafting densities, increased cellular elasticities, and decreased surface areas. Mechanistic insights into how the resistant and persistent E. coli cells respond to ampicillin’s treatment are instrumental to guide design efforts exploring the development of new antibiotics or renovating the existing antibiotics that may kill persistent bacteria by combining more than one mechanism of action.