Impact of Nitric Oxide Donors on Capsule, Biofilm, and Resistance Profiles of Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) is a bacterial species currently considered a critical public health threat due to its ability to cause fatal, multidrug-resistant infections in the bloodstream and key organs. The polysaccharide-based capsule layer that shields K. pneumoniae from clearance via innate immunity is a prominent virulence factor. K. pneumoniae also forms biofilms on biotic and abiotic surfaces. These biofilms significantly reduce penetration by and antibacterial activity from traditional antibiotics. Nitric oxide (NO), an endogenous molecule involved in the innate immune system, is equally as effective at eradicating bacteria but without engendering resistance. Herein, we investigated the effects of NO-releasing small molecules capable of diverse release kinetics on the capsule and biofilm formation characteristics of multiple K. pneumoniae strains. The use of NO donors with moderate and extended NO-release properties (i.e., half-life >1.8 h) inhibited bacterial growth. Additionally, treatment with NO decreased the capsule mucoviscosity in K. pneumoniae strains that normally exhibit hypermucoviscous capsule. The NO donors were also effective against K. pneumoniae biofilms at the same minimum biocidal concentrations that eliminated planktonic bacteria, while meropenem showed little antibacterial action in the same experiments. These results represent the first account of exogenous NO affecting biomarkers involved in K. pneumoniae infections and may therefore inform future development of NO-based therapeutics for treating such infections.

Nitric Oxide-Induced Morphological Changes to Bacteria.

Antimicrobial resistance poses a serious threat to global health, necessitating research for alternative approaches to treating infections. Nitric oxide (NO) is an endogenously produced molecule involved in multiple physiological processes, including the response to pathogens. Herein, we employed microscopy- and fluorescence-based techniques to investigate the effects of NO delivered from exogenous NO donors on the bacterial cell envelopes of pathogens, including resistant strains. Our goal was to assess the role of NO donor architecture (small molecules, oligosaccharides, dendrimers) on bacterial wall degradation to representative Gram-negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium) upon treatment. Depending on the NO donor, bactericidal NO doses spanned 1.5-5.5 mM (total NO released). Transmission electron microscopy of bacteria following NO exposure indicated extensive membrane damage to Gram-negative bacteria with warping of the cellular shape and disruption of the cell wall. Among the small-molecule NO donors, those providing a more extended release (t1/2 = 120 min) resulted in greater damage to Gram-negative bacteria. In contrast, rapid NO release (t1/2 = 24 min) altered neither the morphology nor the roughness of these bacteria. For Gram-positive bacteria, NO treatments did not result in any drastic change to cellular shape or membrane integrity, despite permeation of the cell wall as measured by depolarization assays. The use of positively charged quaternary ammonium (QA)-modified NO-releasing dendrimer proved to be the only NO donor system capable of penetrating the thick peptidoglycan layer of Gram-positive bacteria.

Biofilm Dispersal, Reduced Viscoelasticity, and Antibiotic Sensitization via Nitric Oxide-Releasing Biopolymers.

Compared to planktonic bacteria, biofilms are notoriously difficult to eradicate due to their inherent protection against the immune response and antimicrobial agents. Inducing biofilm dispersal to improve susceptibility to antibiotics is an attractive therapeutic avenue for eradicating biofilms. Nitric oxide (NO), an endogenous antibacterial agent, has previously been shown to induce biofilm dispersal, but with limited understanding of the effects of NO-release properties. Herein, the antibiofilm effects of five promising NO-releasing biopolymer candidates were studied by assessing dispersal, changes in biofilm viscoelasticity, and increased sensitization to tobramycin after treatment with NO. A threshold level of NO was needed to achieve biofilm dispersal, with longer-releasing systems requiring lower concentrations. The most positively charged NO-release systems (from the presence of primary amines) led to the greatest reduction in viscoelasticity of Pseudomonas aeruginosa biofilms. Co-treatment of tobramycin with the NO-releasing biopolymer greatly decreased the dose of tobramycin required to eradicate tobramycin-susceptible and -resistant biofilms in both cellular and tissue models.

Antimicrobial effects of nitric oxide in murine models of Klebsiella pneumonia.

RATIONALE: Inhalation of nitric oxide (NO) exerts selective pulmonary vasodilation. Nitric oxide also has an antimicrobial effect on a broad spectrum of pathogenic viruses, bacteria and fungi. OBJECTIVES: The aim of this study was to investigate the effect of inhaled NO on bacterial burden and disease outcome in a murine model of Klebsiella pneumonia. METHODS: Mice were infected with Klebsiella pneumoniae and inhaled either air alone, air mixed with constant levels of NO (at 80, 160, or 200 parts per million (ppm)) or air intermittently mixed with high dose NO (300 ppm). Forty-eight hours after airway inoculation, the number of viable bacteria in lung, spleen and blood was determined. The extent of infiltration of the lungs by inflammatory cells and the level of myeloperoxidase activity in the lungs were measured. Atomic force microscopy was used to investigate a possible mechanism by which nitric oxide exerts a bactericidal effect. MEASUREMENTS AND MAIN RESULTS: Compared to control animals infected with K. pneumoniae and breathed air alone, intermittent breathing of NO (300 ppm) reduced viable bacterial counts in lung and spleen tissue. Inhaled NO reduced infection-induced lung inflammation and improved overall survival of mice. NO destroyed the cell wall of K. pneumoniae and killed multiple-drug resistant K. pneumoniae in-vitro. CONCLUSIONS: Intermittent administration of high dose NO may be an effective approach to the treatment of pneumonia caused by K. pneumoniae.