Evaluation of cefiderocol trailing and other unclear susceptibility testing endpoints for Acinetobacter spp

Broth microdilution (BMD) susceptibility testing with Acinetobacter spp. can be challenging with skipped wells and trailing observed for some agents. This also applies to cefiderocol, where isolates can display different growth patterns, such as trailing, regrowth, or hazy growth. To better understand the growth patterns observed with cefiderocol BMD testing, the content of wells was evaluated microscopically. Analysis of the trailing, hazy, and regrowth wells above the MIC endpoints showed disturbed growth with long filamentous and spherical cells characteristic of cell envelope instability observed with other β-lactams. When a cell metabolic activity reagent was used to evaluate the viability of the cells in wells above the MIC, inconclusive results were obtained, but when plated, substantial growth was noted for most wells, demonstrating that those cells were able to escape cefiderocol's bactericidal activity. Replicate testing (n = 5) showed that aberrant growth patterns were reproducible, with some MIC variability observed with isolates showing regrowth or trailing, and did not correlate with growth patterns that can be noted in cefiderocol disk diffusion testing (inner colonies, ghost zones, or fuzzy edges). When the prevalence of the different BMD growth patterns was evaluated with 455 isolates from Europe and the United States tested as part of a surveillance program for cefiderocol, about 60% of the isolates exhibited unclear endpoints, and these were noted in most countries, Acinetobacter species, and different genetic backgrounds. Unclear endpoints are not uncommon when testing Acinetobacter spp. against cefiderocol, and tolerability of cells in the presence of cefiderocol seems to be the cause of this.

Cefiderocol susceptibility testing for Acinetobacter spp. is challenging due to the aberrant growth patterns that occur in the broth microdilution method, including trailing, regrowth, and haziness. We initiated this study to better understand these phenomena and assess if these growth patterns were reproducible. We concluded that cefiderocol is not bactericidal against certain isolates and that, despite the cell membrane biosynthesis process being impaired, the cells do not undergo lysis as expected with β-lactam agents. We also evaluated the prevalence of these aberrant growth patterns and evaluated the genetic traits of the isolates producing them, showing that these patterns are common and not related to geographic origin or specific genetic traits.