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 <i>Acanthamoeba</i>, allowing them to persist intracellularly and, in some cases, enhance their virulence. We hypothesize that <i>Acanthamoeba</i> species may also play a role in promoting antimicrobial resistance (AMR) in amoeba-resistant bacteria. This study investigated whether <i>Acanthamoeba castellanii</i> enhanced AMR development in <i>Pseudomonas putida</i> under lethal ciprofloxacin concentrations. <i>P. putida</i> was co-incubated with <i>A. castellanii</i> 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 <i>P. putida</i> and the development of resistance were monitored, and antimicrobial susceptibility tests were conducted using multiple antibiotics. <i>P. putida</i> co-incubated with <i>A. castellanii</i> 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 <i>P. putida</i>, suggesting that the combination of extracellular drug pressure and intracellular survival is important in driving resistance. These findings indicate that intracellular survival within <i>Acanthamoeba</i> can significantly accelerate AMR development in <i>P. putida</i> under fluoroquinolone pressure. Further research into the molecular mechanisms involved is warranted to inform strategies for mitigating AMR emergence in clinical and environmental contexts.