Preliminary insights into the potential role of Acanthamoeba-Pseudomonas interactions in the development of antibiotic resistance

Interactions between environmental protists and bacteria play a crucial role in shaping bacterial survival strategies and pathogenic potential. Certain bacteria have evolved mechanisms to resist predation by protists such as Acanthamoeba, allowing them to persist intracellularly and, in some cases, enhance their virulence. We hypothesize that Acanthamoeba species may also play a role in promoting antimicrobial resistance (AMR) in amoeba-resistant bacteria. This study investigated whether Acanthamoeba castellanii enhanced AMR development in Pseudomonas putida under lethal ciprofloxacin concentrations. P. putida was co-incubated with A. castellanii and maintained in ciprofloxacin concentrations starting at 2 µg ml^-1, four times the planktonic MIC, which was incrementally increased as resistance emerged. The survival of the co-incubated P. putida and the development of resistance were monitored, and antimicrobial susceptibility tests were conducted using multiple antibiotics. P. putida co-incubated with A. castellanii in the presence of ciprofloxacin became increasingly resistant in a dose-dependent manner, with the MIC increasing from 0.5 to 20 µg ml^-1 after 17 days. Contrastingly, the naïve strain did not survive sustained exposure at 2 µg ml^-1. Co-incubated bacteria maintained under ciprofloxacin pressure developed resistance to ciprofloxacin, chloramphenicol, azithromycin and enrofloxacin while retaining susceptibility to streptomycin and tetracycline. Co-incubation in the absence of ciprofloxacin did not promote resistance in P. putida, suggesting that the combination of extracellular drug pressure and intracellular survival is important in driving resistance. These findings indicate that intracellular survival within Acanthamoeba can significantly accelerate AMR development in P. putida under fluoroquinolone pressure. Further research into the molecular mechanisms involved is warranted to inform strategies for mitigating AMR emergence in clinical and environmental contexts.