Impact of the inoculum effect on cefepime activity against AmpC-hyperproducing Enterobacter spp.: insights into resistance mechanisms

Abstract Background Antimicrobial resistance among Enterobacter spp. is a significant concern in healthcare settings due to their ability to hyperproduce AmpC β-lactamase, thereby limiting therapeutic options. Cefepime is recommended for treating these infections; however, its efficacy might be compromised under high bacterial inoculum conditions. Objectives To evaluate the inoculum effect of cefepime on AmpC-hyperproducing Enterobacter spp. clinical isolates. Methods MICs were determined in 62 Enterobacter spp. clinical isolates using broth microdilution at standard (5 × 105 cfu/mL) and high (5 × 106 cfu/mL) inocula. The inoculum effect was defined as an 8-fold or greater MIC increase. Kaplan–Meier survival analysis and time–kill assays were conducted, and β-lactamase activity was measured to investigate the mechanism. Results At standard inoculum, cefepime showed a MIC50 of 1 mg/L and a MIC90 of 8 mg/L. High inoculum conditions resulted in a significant increase in MIC50 (8 mg/L) and MIC90 (256 mg/L), with an inoculum effect observed in 61.3% of isolates. This effect led to a substantial rise in cefepime-resistant isolates, from 6.5% to 54.8% (P < 0.001). Kaplan–Meier analysis confirmed a 16-fold increase in median inhibitory concentration under high inoculum conditions. Time–kill assays and β-lactamase activity measurements revealed that isolates with the inoculum effect displayed significantly higher β-lactamase activity, contributing to cefepime hydrolysis and resistance. Conclusions These findings highlight the critical impact of bacterial inoculum size on cefepime efficacy in AmpC-hyperproducing Enterobacter spp. infections. Tailored antimicrobial strategies are needed, especially for infections with high bacterial loads, to optimize clinical outcomes and minimize resistance development.