Variations in the Morphology, Mechanics and Adhesion of Persister and Resister E. coli Cells in Response to Ampicillin: AFM Study

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

The Role of Growth Temperature in the Adhesion and Mechanics of Pathogenic L. monocytogenes: An AFM Study

The adhesion strengths of pathogenic L. monocytogenes EGDe to a model surface of silicon nitride were quantified using atomic force microscopy (AFM) in water for cells grown under five different temperatures (10, 20, 30, 37, and 40 °C). The temperature range investigated was chosen to bracket the thermal conditions in which L. monocytogenes survive in the environment. Our results indicated that adhesion force and energy quantified were at their maximum when the bacteria were grown at 30 °C. The higher adhesion observed at 30 °C compared to the adhesion quantified for bacterial cells grown at 37, 40, 20, and 10 °C was associated with longer and denser bacterial surface biopolymer brushes as predicted from fitting a model of steric repulsion to the approach distance–force data as well from the results of protein colorimetric assays. Theoretically predicted adhesion energies based on soft-particle DLVO theory agreed well with the adhesion energies computed from AFM force–distance retraction data (<i>r</i>^<i>2</i> = 0.94); showing a minimum energy barrier to adhesion at 30 °C

Combined Poisson and Soft-Particle DLVO Analysis of the Specific and Nonspecific Adhesion Forces Measured between <i>L. monocytogenes</i> Grown at Various Temperatures and Silicon Nitride

Adhesion forces between pathogenic <i>L. monocytogenes</i> EGDe and silicon nitride (Si₃N₄) were measured using atomic force microscopy (AFM) under water and at room temperature for cells grown at five different temperatures (10, 20, 30, 37, and 40 °C). Adhesion forces were then decoupled into specific (hydrogen bonding) and nonspecific (electrostatic and Lifshitz-van der Waals) force components using Poisson statistical analysis. The strongest specific and nonspecific attraction forces were observed for cells grown at 30 °C, compared to those observed for cells grown at higher or lower temperatures, respectively. By combining the results of Poisson analysis with the results obtained through soft-particle Derjaguin–Landau–Verwey–Overbeek (DLVO) analysis, the contributions of the Lifshitz-van der Waals and electrostatic forces to the overall nonspecific interaction forces were determined. Our results showed that the Lifshitz-van der Waals attraction forces dominated the total nonspecific adhesion forces for all investigated thermal conditions. However, irrespective of the temperature of growth investigated, hydrogen bonding forces were always stronger than the nonspecific forces. Finally, by combining Poisson analysis with soft-particle analysis of DLVO forces, the closest separation distances where the irreversible bacterial adhesion takes place can be determined relatively easily. For all investigated thermal conditions, the closest separation distances were <1 nm

Enhancing adipose stem cell chondrogenesis: A study on the roles of dexamethasone, transforming growth factor β3 and ascorbate supplements and their combination

Varied exogenous chondrogenic factors (CFs) are implicated in promoting differentiation of stem cells along a chondrocyte lineage in the field of regenerative tissue engineering for articular cartilage repair. The effects of dexamethasone, transforming growth factor &beta;3 (TGF-&beta;3), ascorbate, and their combinations, on mRNA expression in micromass-cultured human adipose derived stem cells (hADSCs) were investigated as a function of time. Indices include chondrogenic, hypertrophic, angiogenic, fibrogenic and osteogenic markers along with mechanical properties, assessed by atomic force microscopy. Early in the culture, i.e., at day three, no significant differences in mRNA expression of SOX9, aggrecan, lubricin, Col XI, Col X, vascular endothelial growth factor, Col I, and alkaline phosphatase were observed among samples treated with different CFs. However, significant differences in mRNA expression levels of pre-mentioned markers among samples treated with each CF exist when samples were supplied with the CFs for more than three days. A new indexing scheme summing expression of chondrogenic and subtracting non-chondrogenic angiogenic, fibrogenic and osteogenic marker levels shows dexamethasone is the overall leading CF among the factors and their combinations. Based on this scheme, we have projected not only the possible signaling pathways which might be affected by addition of CFs but also hypothetical indexes that may occur upon temporal variation of growth factor regimens

Forces Governing the Transport of Pathogenic and Nonpathogenic Escherichia coli in Nitrogen and Magnesium Doped Biochar Amended Sand Columns

Background: Access to safe drinking water remains a global issue with fecal indicator bacteria being major pollutants. Biochars offer low-cost adsorbents for bacterial pathogens. A fundamental understanding of how biochars interact with bacterial pathogens is essential to designing effective biofilters. Methods: Water-saturated sand columns amended with Magnesium and Nitrogen-doped biochars produced by pyrolysis at 400, 500, 600, and 700 &deg;C were used to Quantify the transport of pathogenic Escherichia coli O157:H7 and nonpathogenic E. coli k12 strains in porous media. Measured data were modeled using DLVO theory of colloidal stability. were explored. Results: (1) Biochar is hydrophobic while sand and bacteria are hydrophilic; (2) all Gibbs free energy values quantified between E. coli O157:H7 and biochar were negative except for biochar produced at 700 &deg;C; (3) all types of forces investigated (van der Waals, electrostatic, and acid-base interactions) played a role in governing the interactions between bacteria and biochar. Conclusions: (1) Adding doped biochar to sand at a 2% weight ratio enhanced the retention of bacterial cells in the sand/biochar columns; (2) bacterial transport is strain-dependent and mediated by various types of forces resulting from interactions between the various functional groups displayed on bacteria and biochar/sand. Our findings emphasize the importance of monitoring biochar&rsquo;s functionality to eliminate bacterial pollutants from contaminated water