Research from the University of Cincinnati College of Medicine shows that use of a topical drug called AB569, a combination of acidified nitrite and EDTA (or ethylenediaminetetraacetic acid) promotes killing of antibiotic-resistant bacteria while enhancing the healing of wounds in a variety of burn injuries.
The study was published in the journal Infection and Immunity.
AB569 was patented in the United States in 2018 by Daniel Hassett, Ph.D., professor in the Department of Molecular Genetics, Biochemistry and Microbiology at the UC College of Medicine. Hassett’s research has found that AB569 kills virtually all pathogenic bacteria tested with no observed acquired resistance.
These bacteria, including multidrug resistant Pseudomonas aeruginosa (MDR-PA), are some of the most serious pathogens exhibiting multidrug resistance and enhanced virulence properties, according to the Centers for Disease Control and Prevention.
“Multidrug resistant bacteria, often called ‘superbugs,’ are an ever-burgeoning global health problem,” says Hassett. “As a result, there is a critical need to develop novel and effective antimicrobials for the prevention, treatment and eradication and healing of such wounds that are complicated by the most formidable pathogen of burn patients, known as MDR-PA. Injury severity is predominantly due to potentially lethal sepsis caused by MDR-PA.”
Hassett, who is the co-corresponding author on this study with Latha Satish, Ph.D., director of clinical lab operations at Cincinnati Children’s Hospital Medical Center, says the research shows AB569 acts synergistically to kill all human burn wound strains of the pathogen in vitro.
“AB569 not only kills the bug, it also enhances the wound-healing process,” says Hassett. “At the same time, AB569 topical application significantly increases the anti-inflammatory mediators … that allow improved epidermal restoration and the healing process to occur more rapidly and efficiently.”
In addition to tackling COPD and cystic fibrosis, AB569 may also be effective in addressing infections related to severe burns, urinary tract disorders, endocarditis and diabetes, said Hassett.
“This study provides solid foundational evidence that AB569 can be used topically to treat highly problematic dermal [injuries] including wound, burn, blast and likely diabetic infections in civilian and military populations and help relieve the economical burden that MDR organisms have on the global health care system,” says Hassett.
Acinetobacter baumannii (Ab) infections have historically been a major clinical challenge for both military and civilian health professionals, especially during the Iraq/Afghanistan conflicts. In the context of combat medical care and acquired wound, burn, blast and ventilator-associated pneumonia (VAP) infections, Ab represents a formidable multi-drug resistant (MDR-AB) pathogen and, as such, is a top 10 CDC priority organisms [1, 2].
Joint Program Committee 2, Military Infectious Disease Research Program (JPC-2/MIDRP) is a congressionally directed committee with a charter to research wound infection prevention as well as antimicrobial countermeasures. The focus research area for JPC-2/MIDRP includes poly-trauma and blast wound injuries [3, 4]. Ab, a common blast wound pathogen, is recognized as a nosocomial isolate that readily acquires resistance genes that especially plagues immunocompromised patients.
In a retrospective study of wounded combat evacuees in 2003, Petersen et al. [5] found that of 56 patients that acquired infections, 84% were infections resulting from blast wound type injuries. Furthermore, 36% of the infections were caused by Ab, making it the predominant wound pathogen. Such organisms were also resistant to 80% of tested drugs [5], colloquially designating Ab as “Iraqibacter, [6]”.
The 2003 and 2004 Acinetobacter baumannii-calcoaceticus complex (ABC) outbreak among U.S. military personnel treated in Military Treatment Facilities was investigated and discovered that 7 of 7 military field hospitals sampled recovered ABC strains [7].
This discovery further highlights the importance of Ab as a significant nosocomial pathogen as well as a top-priority, “serious threat” according to the CDC (https://www.cdc.gov/drugresistance/biggest_threats.html). In addition, mortality rates for nosocomial infections with multi-drug resistant Acinetobacter spp. were 26% higher than the mortality rate of uninfected patients [8].
Further compounding this problem is the fact that Ab possesses genomic resistance islands (Ab antibiotic resistance, AbaR) enabling the bacteria to rapidly acquire drug antibiotic resistance [9]. Furthermore, this bacterium is also naturally competent, thereby being highly proficient at the uptake of stable plasmids [10].
These characteristics make Ab and Acinetobacter spp. ideal model organisms and a huge challenge for the development of novel antibiotics or non-cytotoxic biocides, especially considering that the development of novel therapeutics has dramatically slowed in recent years.
AB569: A novel, two component formulation for the treatment of Ab infections
i. The EDTA component.
AB569 is a non-toxic, bactericidal combination of EDTA and acidified nitrite (herein A-NO2–) buffered to pH of 5.5 to 6.5. The EDTA component of AB569 is widely known as a chelator of di/tri-valent cations with a binding preference prioritizing iron (Fe2/3+), calcium (Ca2+), and magnesium (Mg2+) [11]. EDTA also increases the permeability of the bacterial cell wall by binding Ca2+ and Mg2+ ions that bridge the vital lipopolysaccharide (LPS) component within the outer membrane of Gram-negative bacteria [12].
The ability of EDTA to also chelate metals, especially Fe, dramatically influences the ability of Ab to form complex, antibiotic-resistant, highly organized communities known as biofilms [13]. Biofilm formation by Ab is considered a significant virulence property, thereby enhancing its ability to cause disease [14].
Fe also influences the robustness of Ab biofilm formation [15] and we speculate that its sequestration may play a translational role in future clinical studies designed to treat highly problematic Ab and Acinetobacter spp. infections. Taken together, the role of EDTA in altering the permeability of bacterial cell walls, its ability to sequester Fe, and its effect on biofilm formation are just a few of the actions that contribute to the bactericidal status of EDTA that has been observed in multiple bacterial pathogens [16–18].
ii. The acidified nitrite (A-NO2–) component.
Bacteria can also be exposed to potentially toxic doses of reactive nitrogen species (RNS) during the course of human infection [19]. The endogenous metabolic production of or exposure to exogenous RNS has been shown to enable bacteria to acquire resistance to some antibiotics [20]. Treatment of bacteria with A-NO2– has been shown to increase RNS formation in the form of nitric oxide (NO) production [21]. Given its ability to diffuse through bacterial cell membranes [22], NO can potentially combine with the one-electron reduction product of molecular oxygen, superoxide (O2–), to form peroxynitrite (OONO–), an exceedingly powerful oxidant that can react with virtually all known biomolecules at diffusion-limited rates (~1010 M-1s-1, [23]). Still, the predominant means by which OONO– causes cell death is thought to be predominantly due to DNA damage [24]. In addition, excess A-NO2– can lead to lipid peroxidation and increased cell permeability or tyrosine nitration that can alter cell function, structure or homeostasis [25]. NO also binds to cysteine residues on proteins, forming S-NO proteins (via S–nitrosylation/nitrosation), potentially inhibiting proper protein processing and overall cellular function [26].
Despite the fact that the genus Acinetobacter are strict aerobes, they still have the ability to reduce nitrate to nitrite using reductase enzymes [27, 28], further “recycling” A-NO2–. Ab also converts nitrate to ammonia and uses nitrogen as an energy source in the assimilatory nitrate reduction pathway. Ab is also capable of converting nitrite (NO2–) to ammonium hydroxide (NH3OH) via an NADH/NADPH nitrite reductase [29]. As there is likely a myriad of targets/processes adversely affected during the process of A-NO2– mediated bacterial killing, the direct mechanism of bactericidal action, as with any biocide (e.g., HOCl, H2O2, F–, etc.), is unknown. Finally, in a previous study, Yoon et al. [21] showed that at slightly acidic pH levels, A-NO2– can give rise to HNO2 that is unstable and spontaneously generates NO, N2O3, NO2. and ultimately NO3–.
Though many bacteria are sensitive to A-NO2–, a previous study by McDaniel et. al. [30] showed increased sensitivity of the ESKAPE pathogen Pseudomonas aeruginosa (Pa) to AB569 compared to using either EDTA or A-NO2– alone in a synergistic fashion. Pa differs from Ab in the metabolism of nitrate, due to its ability to grow under both aerobic and anaerobic conditions, the latter of which is by the process of denitrification. Moreover, current studies have also revealed sensitivity to AB569 in bacteria such as other ESKAPE pathogens including methicillin-resistant Staphylococcus aureus (MRSA) strain USA300, Enterococcus faecium, Klebsiella pneumoniae and Enterobacter sp., as well as Escherichia coli and many other Gram-positive and Gram-negative organisms [31].
The promise of AB569 as a therapeutic bacterial biocide is particularly relevant considering that bacteria are unable to develop resistance to it over time. In another combat wound/blast pathogen, Pa, AB569 significantly compromised >30 vital pathways including those involved in the biosynthesis of DNA, RNA, protein and ATP, leading to rapid killing of such bacteria [32].
Thus, given the interests of military and civilian practitioners in antibiotic resistance mechanisms and wound infection prevention and treatment, the goals of this study were to test AB569 against clinical isolates of Ab and Acinetobacter spp. with seven separate experimental challenges that are described in detail below. We found that all Iraq and Afghanistan battlefield clinical isolates as well as reference strains were sensitive to AB569, some of which were in a synergistic fashion.
In addition, bactericidal concentrations of AB569 were non-toxic to primary adult human skin (dermal) fibroblasts. Regarding human use, both the NaNO2 and/or EDTA component(s) of AB569 have separately proven to be safe in studies related to the treatment of urinary tract infection [33], burn wounds [34], cystic fibrosis lung infection [35], chelation therapy [36], soaps [37] and cosmetics [38]. Thus, our results suggest that AB569 has great potential as a therapeutic agent form the treatment of battlefield wound/burn/blast infections as well as problematic ventilator-associated pneumonia (VAP) infections by Ab.
reference link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247513
More information: Amanda Barry et al, AB569, a Novel, Topical Bactericidal Gel Formulation, Kills Pseudomonas aeruginosa and Promotes Wound Healing in a Murine Model of Burn Wound Infection, Infection and Immunity (2021). DOI: 10.1128/IAI.00336-21
