Inoculum Effect in Multidrug-Resistant Gram-Negative Isolates: Impact on Ceftazidime and Cefepime Susceptibility.

Abstract

Multidrug- or extensively drug-resistant (MDR or XDR) Gram-negative infections remain a global challenge, further complicated by the inoculum effect (IE), which can significantly alter the in vitro activity of β-lactam antibiotics and may contribute to treatment failure in infections with a high bacterial burden. In this study, we evaluated the IE of ceftazidime (CAZ) and cefepime (FEP) against multidrug-resistant Gram-negative clinical isolates. Fifty isolates, including Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Proteus mirabilis (P. mirabilis), were included in the current study. Minimum inhibitory concentrations (MICs) of CAZ and FEP were determined at standard (10⁵ CFU/mL) and high (10⁷ CFU/mL) inoculum levels. The IE was defined as an increase of ≥8-fold in MIC. Five β-lactamase genes, bla_CTXM-9, bla_OXA-2, bla_OXA-10, bla_tem-1, and bla_SHV, were screened by polymerase chain reaction. Tested isolates were mainly MDR (74.0%) or XDR (26.0%). A significant increase in MIC values at high inoculum was observed for both CAZ and FEP in E. coli and K. pneumoniae (p<0.001), with a marked shift in susceptibility. In K. pneumoniae, CAZ exhibited significantly greater stability against IE than FEP (p = 0.041). In contrast, no significant IE was detected in P. aeruginosa or P. mirabilis. The presence of β-lactamase resistance genes is a significant predictor of FEP IE (OR=14.7, p=0.026). Significant positive correlations between MICs at standard and high inoculum, alongside inverse correlations between standard MIC and fold change in E. coli and K. pneumoniae, were observed. The IE significantly compromises the activity of CAZ and FEP in E. coli and K. pneumoniae, with important clinical implications in high bacterial burden infections. However, CAZ may be superior compared to FEP in IE conditions, particularly for K. pneumoniae. The IE is primarily organism-dependent and may be influenced by β-lactamase gene patterns such as bla_oxa genes with bla_CTXM-9, bla_tem-1, or bla_SHV, particularly for FEP. These findings highlight the limitations of standard susceptibility testing and emphasize the need for careful antibiotic selection in severe, life-threatening Gram-negative infections.