(504) Pseudomonas aeruginosa populations in synthetic cystic fibrosis media evolve cooperative behavior in response to cefiderocol pressure

Background: P. aeruginosa infection in CF patients remains the most relevant factor promoting morbidity and mortality in people with CF. Its pathogenicity comes from genetic/metabolic plasticity, its ability to adapt rapidly, and also thrive in high stress environments, including under continuous antibiotic treatment or low iron availability in host. The trojan-horse antibiotic strategy hijacks bacterial outer membrane iron transporters to facilitate bacterial antibiotic uptake. Using this approach, cefiderocol (CFDC), a novel siderophore cephalosporin, emerges as a potential candidate for treating chronic P. aeruginosa CF infections. However, the mechanisms of CFDC resistance are not completely understood. Previously, our lab showed that planktonic and biofilm P. aeruginosa SCFM2-adapted populations evolved CFDC resistance by acquiring several non-synonymous mutations. Mutations in the cpxS gene (T163P, S227G, V235A), piuA, piuC and the deletion of 21,938 bp (including mexEF-oprN efflux pump) arose in multiple CFDC-resistant populations. To further investigate the molecular mechanisms of cefiderocol resistance, the present study aimed to decipher the phenotypic consequences of evolving CFDC resistance.

Methods: We compared the susceptibility profiles and growth rates of ancestral and CFDC-evolved populations in SCFM2. In addition, we evaluated population dynamics during competitions between susceptible and resistant populations using mApple and eYFP fluorescent tags. Planktonic competitions were assayed by fluorimeter, and aggregates were visualized by confocal laser scanning microscopy.

Results: We observed that populations showed cross-resistance against other cephalosporins and colistin, potentially mediated by

mexAB induction and a shift to metabolic nitrite respiration, respectively. In addition, CFDC resistance evolved through fitness trade-offs in which the acquisition of CFDC-mutations also caused growth defects in the absence of CFDC. Under CFDC pressure, most of the CFDC populations outcompeted the ancestral susceptible population. Surprisingly, we identified populations that evolved cooperative behavior, protecting ancestral populations from CFDC’s bactericidal effect. Protective effects were also observed in some aggregate competitions in which resistant populations grow surrounding ancestral populations, presumably shielding them from CFDC action. RNAseq analysis revealed that protective and non-protective competitions displayed distinct gene expression profiles, where genes of CPX two-component system and MuxABC-OpmB were up-regulated while phenazine biosynthesis genes were downregulated.

Conclusions: In summary, the current study demonstrated that P. aeruginosa evolves CFDC resistance paying a fitness cost. However, planktonic and aggregate CFDC-resistant sub-populations can act as cooperators protecting susceptible populations from the effects of the antibiotic. Understanding what drives the cooperative behavior might help researchers and clinicians improve the usage of cefiderocol and delay the spread of CFDC resistance.

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