In silico genome-scale metabolic modeling and in vitro static time-kill studies of exogenous metabolites alone and with polymyxin B against Klebsiella pneumoniae

Multidrug-resistant (MDR) Klebsiella pneumoniae is a top-prioritized Gramnegative pathogen with a high incidence in hospital-acquired infections. Polymyxins have resurged as a last-line therapy to combat Gram-negative superbugs, including MDR K. pneumoniae. However, the emergence of polymyxin resistance has increasingly been reported over the past decades when used as monotherapy, and thus combination therapy with non-antibiotics (e.g., metabolites) becomes a promising approach owing to the lower risk of resistance development. Genome- scale metabolic models (GSMMs) were constructed to delineate the altered metabolism of New Delhi metallo-beta-lactamase- or extended spectrum beta-lactamase-producing K pneumoniae strains upon addition of exogenous metabolites in media. The metabolites that caused significant metabolic perturbations were then selected to examine their adjuvant effects using in vitro static time-kill studies. Metabolic network simulation shows that feeding of 3-phosphoglycerate and ribose 5-phosphate would lead to enhanced central carbon metabolism, ATP demand, and energy consumption, which is converged with metabolic disruptions by polymyxin treatment. Further static time-kill studies demonstrated enhanced antimicrobial killing of 10 mM 3-phosphoglycerate (1.26 and 1.82 log(10) CFU/ml) and 10 mM ribose 5-phosphate (0.53 and 0.91 log(10 )CFU/ml) combination with 2 mg/L polymyxin B against K. pneumoniae strains. Overall, exogenous metabolite feeding could possibly improve polymyxin B activity via metabolic modulation and hence offers an attractive approach to enhance polymyxin B efficacy. With the application of GSMM in bridging the metabolic analysis and time-kill assay, biological insights into metabolite feeding can be inferred from comparative analyses of both results. Taken together, a systematic framework has been developed to facilitate the clinical translation of antibiotic-resistant infection management.

Automated summary

This study investigates the metabolic changes of polymyxin-resistant Klebsiella pneumoniae strains upon the addition of exogenous metabolites using genome-scale metabolic models. The results show that feeding specific metabolites can improve the activity of polymyxin B against K. pneumoniae. In vitro experiments demonstrate enhanced antimicrobial killing when combining 3-phosphoglycerate and ribose 5-phosphate with polymyxin B against K. pneumoniae strains.