Bioanalytical Methods for Investigating Bacterial Adhesion and the Antibacterial Action of Nitric Oxide

Infection is a continuing problem in both hospital and community settings, further compounded by swift adaptation and rising emergence of more virulent and antibacterial-resistant pathogens. The complex mechanisms underlying the process of infection must be understood in order to develop preventative technologies. Simultaneously, effective solutions must be devised to counter infections as they arise. My dissertation research has contributed to both aspects by working to understand the process of adhesion as well as the development of novel therapeutic strategies. To investigate bacterial adhesion at a fundamental level, a quantitative method was developed for measuring the shear force required to detach individual adhered bacteria using atomic force microscopy (AFM) that featured both improved accuracy and higher-throughput data acquisition. This technique was employed to characterize the adhesion strength kinetics of Pseudomonas aeruginosa and Staphylococcus aureus. Both the magnitudes of detachment force and rates of increase in adhesion strength were greater for P. aeruginosa than for S. aureus. Furthermore, adhered cells demonstrated a range of adhesion forces that broadened with time, indicating that change in adhesion strength does not proceed uniformly. Morphological analyses were conducted to study the antimicrobial properties of nitric oxide (NO) against two Gram-negative pathogens. The effects of NO as a function of concentration, exposure time, and delivery format were studied using two materials with differing NO-release properties. Analysis of cell topography revealed that higher doses of NO correlated with increasing membrane roughness. Treatment with amoxicillin, an antibiotic that compromises the integrity of the cell wall, led to morphologies resembling those resulting from NO treatment. Our observations indicated cell wall deterioration is a consequence of NO-exposure for both species studied. The combination of NO and silver sulfadiazine (AgSD) was evaluated for bactericidal efficacy using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD proved synergistic against most pathogens, particularly the Gram-positive species. A highly synergistic effect was produced against S. aureus and E. faecalis, including a vancomycin-resistant strain. The synergistic activity of AgSD and NO against a broad range of pathogens advocates future investigation of this therapeutic combination for use as a topical anti-infective

Morphological analysis of the antimicrobial action of nitric oxide on Gram-negative pathogens using atomic force microscopy

Atomic force microscopy (AFM) was used to study the morphological changes of two Gram-negative pathogens, Pseudomonas aeruginosa and Escherichia coli, after exposure to nitric oxide (NO). The time-dependent effects of NO released from a xerogel coating and the concentration-dependent effects rendered by a small-molecule that releases NO in a bolus were examined and compared. Bacteria exhibited irregular and degraded exteriors. With NO-releasing surfaces, an increase in surface debris and disorganized adhesion patterns were observed compared to controls. Analysis of cell surface topography revealed that increasing membrane roughness correlated with higher doses of NO. At a lower total dose, NO delivered via a bolus resulted in greater membrane roughness than NO released from a surface via a sustained flux. At sub-inhibitory levels, treatment with amoxicillin, an antibiotic known to compromise the integrity of the cell wall, led to morphologies resembling those resulting from NO treatment. Our observations indicate that cell envelope deterioration is a visible consequence of NO-exposure for both Gram-negative species studied

Synergy of Nitric Oxide and Silver Sulfadiazine against Gram-Negative, Gram-Positive, and Antibiotic-Resistant Pathogens

The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver (I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be <0.5 Gram-negative species were generally more susceptible to the individual antimicrobial agents than the Gram-positive bacteria. The in vitro synergistic activity of AgSD and NO observed against a range of pathogens strongly supports future investigation of this therapeutic combination, particularly for its potential use in the treatment of chronic and burn wounds

Synergy of Nitric Oxide and Silver Sulfadiazine against Gram-Negative, Gram-Positive, and Antibiotic-Resistant Pathogens

The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver (I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be <0.5 Gram-negative species were generally more susceptible to the individual antimicrobial agents than the Gram-positive bacteria. The in vitro synergistic activity of AgSD and NO observed against a range of pathogens strongly supports future investigation of this therapeutic combination, particularly for its potential use in the treatment of chronic and burn wounds