Antimicrobial resistance (particularly through extended-spectrum β-lactamase and
aminoglycoside-modifying
enzyme production) in
neonatal sepsis is a global problem, particularly in low- and middle-income countries, with significant mortality rates. High rates of resistance are reported for the current WHO-recommended first-line
antibiotic regimen for
neonatal sepsis, i.e.,
ampicillin and
gentamicin. We assessed the utility of
fosfomycin and
amikacin as a potential alternative regimen to be used in settings of increasingly prevalent antimicrobial resistance. The combination was studied in a 16-arm dose-ranged hollow-fiber
infection model (HFIM) experiment. The combination of
amikacin and
fosfomycin enhanced bactericidal activity and prevented the emergence of resistance, compared to monotherapy with either
antibiotic. Modeling of the experimental quantitative outputs and data from checkerboard assays indicated synergy. We further assessed the combination regimen at clinically relevant doses in the HFIM with nine Enterobacterales strains with high
fosfomycin and
amikacin MICs and demonstrated successful kill to sterilization for 6/9 strains. From these data, we propose a novel combination breakpoint threshold for microbiological success for this antimicrobial combination against Enterobacterales strains, i.e., MICF ×
MICA < 256 (where MICF and
MICA are the
fosfomycin and
amikacin MICs, respectively). Monte Carlo simulations predict that a standard
fosfomycin-
amikacin neonatal regimen would achieve >99% probability of pharmacodynamic success for strains with MICs below this threshold. We conclude that the combination of
fosfomycin with
amikacin is a viable regimen for the empirical treatment of
neonatal sepsis and is suitable for further clinical assessment in a randomized controlled trial.