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.

Atomic force microscopy (AFM) was used to quantify both the nanomechanical properties of pathogenic (ATCC 51776 & EGDe) and non-pathogenic (ATCC 15313 & HCC25) Listeria monocytogenes strains and the conformational properties of their surface biopolymers. The nanomechanical properties of the various L. monocytogenes strains were quantified in terms of Young's moduli of cells. To estimate Young's moduli, the classic Hertz model of contact mechanics and a modified version of it that takes into account substrate effects were used to fit the AFM nanoindentation-force measurements collected while pushing onto the bacterial surface biopolymer brush. When compared, the classic Hertz model always predicted higher Young's moduli values of bacterial cell elasticity compared to the modified Hertz model. On average, the modified Hertz model showed that virulent strains are approximately twice as rigid (88.1 ± 14.5 KPa) as the avirulent strains (47.3 ± 7.6 kPa). To quantify the conformational properties of L. monocytogenes' strains surface biopolymers, two models were used. First, the entropic-based, statistical mechanical, random walk formulation, the wormlike chain (WLC) model was used to estimate the elastic properties of the bacterial surface molecules. The WLC model results indicated that the virulent strains are characterized by a more flexible surface biopolymers as indicated by shorter persistence lengths (L_p = 0.21 ± 0.08 nm) compared to the avirulent strains (L_p = 0.24 ± 0.14 nm). Second, a steric model developed to describe the repulsive forces measured between the AFM tip and a high density polymer surface indicated that the virulent strains are characterized by crowded and longer biopolymer brushes compared to those of the avirulent strains. Finally, scaling relationships developed for grafted polyelectrolyte brushes indicated that the L. monocytogenes strains' biopolymer brushes are charged. Collectively, our data indicate that the conformational properties of the bacterial surface biopolymers and their surface densities play an important role in controlling the overall bacterial cell elasticity.

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.