Impact of sodium nitroprusside concentration added to batch cultures of Escherichia coli biofilms on the c-di-GMP levels, morphologies and adhesion of biofilm-dispersed cells.

Biofilm dispersion can be triggered by the application of dispersing agents such as nitric oxide (NO)-donors, resulting in the release of biofilm-dispersed cells into the environment. In this work, biofilm-dispersed cells were obtained by adding different concentrations of NO-donor sodium nitroprusside (0.5, 5, 50 µM, and 2.5 mM of SNP) to batch cultures of pre-formed Escherichia coli biofilms. Except for those dispersed by 5 µM of SNP, biofilm-dispersed cells were found to be wider and longer than the planktonic cells and to have higher c-di-GMP levels and greater adhesion forces to silicon nitride surfaces in water as measured by atomic force microscope. Consequently, the optimum concentration of SNP to disperse E. coli biofilms was found to be 5 µM of SNP, whose addition to batch cultures resulted in a significant biofilm dispersion and the dispersed cells having c-di-GMP levels, morphologies and adhesion strengths similar to their planktonic counterparts.

Responses of Acinetobacter baumannii Bound and Loose Extracellular Polymeric Substances to Hyperosmotic Agents Combined with or without Tobramycin: An Atomic Force Microscopy Study.

In this work, contributions of extracellular polymeric substances (EPS) to the nanoscale mechanisms through which the multidrug-resistant Acinetobacter baumannii responds to antimicrobial and hyperosmotic treatments were investigated by atomic force microscopy. Specifically, the adhesion strengths to a control surface of silicon nitride (Si3N4) and the lengths of bacterial surface biopolymers of bound and loose EPS extracted from A. baumannii biofilms were quantified after individual or synergistic treatments with hyperosmotic agents (NaCl and maltodextrin) and an antibiotic (tobramycin). In the absence of any treatment, the loose EPS were significantly longer in length and higher in adhesion to Si3N4 than the bound EPS. When used individually, the hyperosmotic agents and tobramycin collapsed the A. baumannii bound and loose EPS. The combined treatment of maltodextrin with tobramycin collapsed only the loose EPS and did not alter the adhesion of both bound and loose EPS to Si3N4. In addition, the combined treatment was not as effective in collapsing the EPS molecules as when tobramycin was applied alone. Finally, the effects of treatments were dose-dependent. Altogether, our findings suggest that a sequential treatment could be effective in treating A. baumannii biofilms, in which a hyperosmotic agent is used first to collapse the EPS and limit the diffusion of nutrients into the biofilm, followed by the use of an antibiotic to kill the bacterial cells that escape from the biofilm because of starvation.

Atomic Force Microscopy Investigation of the Contributions of Listeria monocytogenes Cell-Wall Biomacromolecules to Their Adherence and Mechanics.

In this work, the contributions of the pathogenic Listeria monocytogenes cell-wall biomacromolecules to the bacterial mechanics and adhesion to a model inert surface of silicon nitride in water were investigated by atomic force microscopy. Chemical ethylenediaminetetraacetic acid (EDTA) and biological enzymatic trypsin treatments of cells were performed to partially or totally remove the bacterial cell-wall proteins and carbohydrates. Removal of 48.2% proteins and 29.2% of carbohydrates from the cell-wall of the bacterium by the EDTA treatment resulted in a significant decrease in the length of the bacterial cell-wall biomacromolecules and an increase in the rigidity of the bacterial cells as predicted from fitting a model of steric repulsion to the force-distance approach data and classic Hertz model to the indentation-force data, respectively. In comparison, removal of almost all the cell-wall proteins (99.5% removal) and 8.6% of cell-wall carbohydrates by the trypsin treatment resulted in an increase in the elasticity of the bacterial cells, an increase in the extension of the cell-wall biomacromolecules, and a significant decrease in their apparent grafting densities. In addition, adhesion strength of native-untreated L. monocytogenes to silicon nitride in water decreased by 30% on average after the EDTA treatment and further decreased by 60% on average after the trypsin treatment, showing a positive correlation with the% removal of cell-wall proteins by the EDTA and trypsin treatments, respectively.