Molecular and pharmacodynamic insights into β-lactam therapy for high-inoculum <i>Enterobacter cloacae</i> complex infections

High-inoculum <i>Enterobacter cloacae</i> complex (ECC) infections challenge β-lactam therapy due to β-lactamase-mediated resistance and the inoculum effect, where high bacterial densities amplify resistance and worsen outcomes. We evaluated the pharmacodynamic efficacy of cefepime, meropenem, ertapenem, meropenem-vaborbactam, and ceftazidime-avibactam against three high-inoculum (10⁹ colony-forming unit [CFU]/g) ECC isolates (0008, 0032, and 0060) harboring diverse β-lactamases using a 96 h simulated endocardial vegetation (SEV) model with humanized exposures. Bactericidal activity, resistance emergence, and β-lactamase expression were assessed. Cefepime failed in all isolates, with SEV growth from +0.4 to +1.6 log₁₀ CFU/g and minimum inhibitory concentrations (MICs) increasing from 2-8 µg/mL to >64 µg/mL in 0008, 0032, and 0060, coinciding with AmpC β-lactamase (ACT) induction up to 16.1-fold (0032). In 0060, cefepime failed, whereas meropenem displayed infusion-dependent efficacy: the 3 h infusion achieved bactericidal killing (-4.6 log₁₀ CFU/g), while the 30 min infusion produced partial killing (-2.9 log₁₀ CFU/g). Meropenem failed in 0032 (+1.1 log₁₀ CFU/g), with ACT-16 induction reaching 14.6-fold. Ertapenem produced initial bactericidal activity in 0060 (-3.2 log₁₀ CFU/g) but regrowth to -1.9 log₁₀ CFU/g by 96 h, accompanied by ACT-89 expression rising 26-fold. Meropenem-vaborbactam was bactericidal in 0008 (-4.6 log₁₀ CFU/g) and 0032 (-3.6 log₁₀ CFU/g) with stable MICs and minimal β-lactamase induction. Ceftazidime-avibactam achieved bactericidal activity in 0008 (-4.9 log₁₀ CFU/g) but only bacteriostatic activity in 0032 (-1.2 log₁₀ CFU/g), with modest KPC-3 and ACT-16 upregulation (~1.7- to 2.3-fold). β-Lactamase diversity and inoculum effects limit β-lactam efficacy in high-inoculum ECC infections. Meropenem-vaborbactam showed consistent activity, and prolonged meropenem infusion benefited select isolates. Variable ceftazidime-avibactam efficacy reinforces aligning β-lactam selection with underlying β-lactamase mechanisms.