Antibacterial Action of Nitric Oxide-Releasing Chitosan Oligosaccharides against Pseudomonas aeruginosa under Aerobic and Anaerobic Conditions

Abstract

Chitosan oligosaccharides were modified with N-diazeniumdiolates to yield biocompatible nitric oxide (NO) donor scaffolds. The minimum bactericidal concentrations and MICs of the NO donors against Pseudomonas aeruginosa were compared under aerobic and anaerobic conditions. Differential antibacterial activities were primarily the result of NO scavenging by oxygen under aerobic environments and not changes in bacterial physiology. Bacterial killing was also tested against nonmucoid and mucoid biofilms and compared to that of tobramycin. Smaller NO payloads were required to eradicate P. aeruginosa biofilms under anaerobic versus aerobic conditions. Under oxygen-free environments, the NO treatment was 10-fold more effective at killing biofilms than tobramycin. These results demonstrate the potential utility of NO-releasing chitosan oligosaccharides under both aerobic and anaerobic environments. Pseudomonas aeruginosa is an opportunistic human pathogen that frequently colonizes people with compromised immune systems, such as those with cystic fibrosis (CF) or severe burn wounds (1). The success of P. aeruginosa as a pathogen is related to its multitude of virulence factors, which increase adherence to the host cells, induce inflammation, and disrupt the host immune response (1). Furthermore, P. aeruginosa is intrinsically resistant to many antibiotics due to low membrane permeability and increased expression of ␤-lactamases and efflux pumps (2-4). In addition to this native resistance, P. aeruginosa readily adapts to antibiotic challenge by acquiring resistance genes (3, 5) and forming protective, cooperative communities known as biofilms (6, 7).While all antibiotic resistance mechanisms are not fully understood, at least three main factors reduce antibiotic efficacy against bacterial biofilms compared to planktonic cells. First, P. aeruginosa in biofilms secretes a protective layer of exopolysaccharides that prevent the diffusion of antibiotics (7). In the context of cystic fibrosis, biofilm-bound P. aeruginosa exists predominantly as the mucoid phenotype, characterized by a secreted alginate matrix that provides a physical barrier against the host immune response and antibiotics (8). This exopolysaccharide matrix also prevents the diffusion of oxygen into biofilms, causing P. aeruginosa to switch from aerobic to anaerobic respiration (9). The reduced metabolic activity of P. aeruginosa undergoing anaerobic respiration protects the bacterium against traditional antibiotics that are most effective against rapidly dividing cells, including aminoglycosides and ␤-lactams (10, 11). Finally, biofilms produce persister cells (i.e., dormant bacteria that are highly resistant to chemical disinfectants and exhibit multidrug tolerance) much more frequently than planktonic bacterial cultures (3, 7).The failure of conventional antibiotics to treat P. aeruginosa biofilms and infections necessitates the development of new antibacterial agents. Nitric oxide (NO), an endogenously produced free radical that can disperse (12, 13) and ...

Study of crystal formation and nitric oxide (NO) release mechanism from S-nitroso-N-acetylpenicillamine (SNAP)-doped CarboSil polymer composites for potential antimicrobial applications

“…Infections caused by P. aeruginosa also often occur in patients already battling with more severe illness, e.g., extensive trauma, burns, or malignancy [59]. It is very important to examine the antimicrobial effect of NO against this particular strain of bacteria because P. aeruginosa possesses NO reductase enzyme that may enable the bacteria cells to metabolize and deactivate NO, by converting NO to nitrous oxide (N 2 O) and ultimately to nitrogen [60–62]. On the other hand, the primary bacterium associated with persistent and complicated catheter-associated urinary tract infections (CAUTIs) is P. mirabilis [3,63,64], which is also known for its ability to elevate urine pH, induce calcium/magnesium phosphate precipitation and produce mature crystalline biofilms [3,63,65].…”

Recent advances in thromboresistant and antimicrobial polymers for biomedical applications: just say yes to nitric oxide (NO)

“…66,103 It has been clearly demonstrated that NO releasing/generating polymers can have strong bactericidal effects, 19,146,147,162,201,227,283,284 even for bacteria that are able to metabolize and deactivate NO, such as P. aeruginosa , that possesses NO reductase enzyme that converts NO to nitrous oxide (N 2 O) and ultimately nitrogen (N 2 ). 86,201,219 Of note, NO possesses broad-spectrum antibacterial activity against both gram-positive and gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). 219,281 Moreover, the dose of NO required to kill bacteria (e.g., 200 ppm of gaseous NO) 281,282,285 does not show any cytotoxic effects in human dermal fibroblasts when exposed for 48 h. 216,220 In addition to its bactericidal activity at high dosage, low levels of NO (picomolar to nanomolar range in solution phase) 286 also serve as a key mediator that minimize planktonic bacteria adhesion and colonization, as well as disperse mature biofilm and release the bacteria trapped in the EPS film back to their planktonic state.…”

Nitric oxide diffusion through cystic fibrosis-relevant media and lung tissue

“…Our lab has previously developed a number of biopolymer scaffolds capable of storing and releasing NO over extended periods to provide targeted therapeutic doses. 8,37,38,47 For example, NO donor-modied b-cyclodextrin exhibited the ability to eradicate bacteria while eliciting minimal cytotoxicity towards mammalian cells, 38 making it an appealing method for therapeutic NO delivery. In this work, the NO-releasing propylamine-modied variant (CD-PA/NO) was selected due to its ability to deliver high NO payloads (0.61 AE 0.05 mmol mg À1 ) with a moderate half-life (1.73 AE 0.24 h).…”

“…1,3,5 Biolms hinder drug diffusion, alter bacterial metabolism, and increase tolerance towards traditional antibiotics. 1,8,9 As such, severe chronic infections persist in the lungs of CF patients, requiring the constant administration of antibiotics (e.g., colistin and tobramycin) to mitigate infections and prolong the lives of those afflicted with CF. 3,10,11 The continuous administration of antibiotics required to combat these chronic infections has led to the rapid rise of multi-drug resistant (MDR) bacteria in the CF community, necessitating the development of novel therapeutics.…”