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Non-genetic mechanisms of intrinsic antimicrobial resistance

Final Report Summary - NONANTIRES (Non-genetic mechanisms of intrinsic antimicrobial resistance)

The World Health Organization has singled out antibiotic resistance as one of the “greatest threats facing the world”. This threat is further aggravated by the emergence of Gram-negative bacterial pathogens with high-level multidrug resistance (e.g. Acinetobacter sp., Burkholderia cepacia complex, Stenotrophomonas maltophilia, and the Enterobacteriaceae). Current research on antibiotic action and resistance focuses on targeting essential functions within the bacterial cell. However, whether bacteria produce molecules that interfere with antibiotics outside the bacterial cell has been overlooked.
We recently discovered a novel and general mode of bacterial antibiotic resistance operating in the extracellular space. "Extracellular" resistance depends on molecules produced and released by bacteria in response to sublethal antibiotic concentrations. This mechanism protects less resistant bacterial cells of the same or different species from killing by antibiotics. The molecules involved are the polyamine putrescine and secreted lipocalin proteins. Further, we also discovered that certain vitamins, essential food micronutrients, overcome in vitro and in vivo antibiotic resistance by bacterial lipocalins, providing an unexpected potential solution to combat antimicrobial resistance.
Initial observations were made with the intrinsically multidrug resistant and epidemic cystic fibrosis (CF) pathogen, B. cenocepacia K56-2, which secretes putrescine, a polyamine, and the bacterial lipocalin protein BcnA (formerly BCAL3311, YceI) upon bactericidal antibiotic challenge. BcnA is remarkably conserved, being encoded by ~4000 bacterial genomes and annotated as a "conserved hypothetical protein" referred to as YceI, which denotes a large family of proteins of unknown function. Some of the YceI members are proposed to bind amphiphilic molecules and sequester toxic fatty acids or amides, while others may bind isoprenoid lipids and chlorophenoxy herbicides.
Putrescine and BcnA protect the producing bacterial cells and more sensitive bystander cells of other species from antibiotic killing. Putrescine protects extracellularly by preventing antibiotic binding to the bacterial surface, and intracellularly by ameliorating oxidative damage. Clinically, putrescine can be detected in sputum samples from infected CF patients undergoing exacerbations that require antibiotic treatment, and preliminary data show that the most common CF pathogen, P. aeruginosa, also overproduces putrescine upon antibiotic challenge. In contrast, published and extensive preliminary results revealed that Bcn-like proteins (BcnLPs) scavenge antibiotics in the extracellular space.