Nitrate potentiates kanamycin activity in bacteria through metabolic reprogramming and CpxR-associated respiratory activation

Abstract

ABSTRACT Antibiotic resistance in foodborne bacteria profoundly compromises the effectiveness of antimicrobial therapies and represents a persistent global threat to public health and livestock production. There is therefore an urgent need to develop adjuvant strategies that can improve the activity of existing antibiotics against drug-resistant pathogens. In this study, comparative proteomic profiling of kanamycin-susceptible and laboratory-passaged kanamycin-resistant Salmonella enterica serovar Enteritidis (S. Enteritidis) revealed coordinated differences in the tricarboxylic acid (TCA) cycle, nitrogen metabolism, and the respiratory electron transport chain. The resistant strain displayed reduced NADH production, proton motive force (PMF), and intracellular kanamycin accumulation. Because these proteomic analyses were performed under antibiotic-free conditions and whole-genome sequencing was not conducted, these differences are interpreted as physiological features associated with the resistant phenotype rather than as evidence of the specific genetic mechanism responsible for the increased kanamycin MIC. Mechanistically, nitrate acted as a potent kanamycin adjuvant by increasing cellular bioenergetic activity through the nitrate-glutamine-glutamate-associated pathway. Nitrate increased NADH production, enhanced TCA cycle activity, and elevated PMF, thereby increasing intracellular kanamycin accumulation. Nitrate also altered CpxR-associated regulation of respiratory genes, providing an additional route by which respiratory activity could be enhanced. Genetic disruption of nitrate transport or selected metabolic and regulatory components attenuated nitrate-mediated potentiation, supporting contributions from both metabolic flux and CpxR-associated respiratory regulation. Importantly, nitrate enhanced kanamycin-mediated killing not only in the laboratory-derived Kana-R strain but also in kanamycin-susceptible Kana-S, indicating a general kanamycin-potentiating effect rather than specific reversal of an acquired resistance mechanism. The nitrate-kanamycin combination also improved treatment outcomes against clinically resistant S. Enteritidis isolates, including increased survival in murine and chicken infection models. Collectively, these findings identify nitrate as a metabolic adjuvant that can potentiate kanamycin activity by increasing bacterial bioenergetics and drug accumulation. They further illustrate how bacterial physiological state can modulate aminoglycoside efficacy without establishing that nitrate reverses the underlying genetic mechanism of kanamycin resistance. IMPORTANCE Antibiotic efficacy is influenced not only by inherited resistance determinants but also by the physiological and metabolic state of bacterial cells. Our study shows that nitrate can potentiate kanamycin activity in Salmonella enterica serovar Enteritidis by enhancing central metabolism, respiratory activity, proton motive force, and intracellular drug accumulation. This potentiation was observed in both a kanamycin-resistant laboratory-passaged strain and a kanamycin-susceptible strain, supporting a general adjuvant effect rather than specific reversal of a defined resistance mechanism. The nitrate-kanamycin combination also improved outcomes in animal infection models using resistant strains. These findings identify inorganic nitrogen metabolism and CpxR-associated respiratory regulation as potential leverage points for developing aminoglycoside adjuvant strategies. Antibiotic efficacy is influenced not only by inherited resistance determinants but also by the physiological and metabolic state of bacterial cells. Our study shows that nitrate can potentiate kanamycin activity in Salmonella enterica serovar Enteritidis by enhancing central metabolism, respiratory activity, proton motive force, and intracellular drug accumulation. This potentiation was observed in both a kanamycin-resistant laboratory-passaged strain and a kanamycin-susceptible strain, supporting a general adjuvant effect rather than specific reversal of a defined resistance mechanism. The nitrate-kanamycin combination also improved outcomes in animal infection models using resistant strains. These findings identify inorganic nitrogen metabolism and CpxR-associated respiratory regulation as potential leverage points for developing aminoglycoside adjuvant strategies.