Joint effects of predatory protists and predatory bacteria on driving the evolution of bacterial antibiotic resistance

Biological factors, especially predator-prey interactions, are crucial drivers of ecological processes. However, their roles in driving the evolution of antibiotic resistance, a global health concern, remain poorly understood in complex natural soil ecosystems. Predatory protists and predatory bacteria are primary bacterial predators, however, mechanisms underlying the influence of their predation on the richness and abundance of bacterial antibiotic resistance within a multitrophic soil ecosystem are still poorly known. Here, we assessed effects of soil predatory protists on predatory bacteria and antibiotic resistance genes (ARGs) across a gradient of protist concentrations under the control of bacteria's competitors – fungi. Our findings reveal that high-protist predation pressure significantly amplified the relative abundance of predatory bacteria Streptomycetales and Myxococcales between days 15 and 45 of the microcosm incubation. Aligning with a rising abundance of predatory bacteria and antibiotic-producing bacteria, the relative abundance and diversity of soil ARGs significantly increased under high protist concentrations, regardless of fungal effects. Many ARGs encoding key resistance mechanisms (antibiotic deactivation and efflux pumps) were enriched in response to predatory protists within complex inter-group interactions. Moreover, these enriched ARGs were strongly associated with both predatory bacteria and predatory protists. More profound effects of predatory protists on the predatory bacteria and soil ARGs were identified in the presence of fungi. Our study provides novel evidence about crucial effects of predatory protists on shaping the predatory bacterial community and driving bacterial antibiotic resistance under complex multitrophic interactions in a natural soil. These findings pave the way for future research aimed at mitigating this global health issue and uncovering other ecological processes. • High-protist predation increased relative abundance of predatory bacteria. • High-protist treatment enhanced the ARGs profile regardless of fungal effect. • Enriched ARGs were strongly associated with predatory bacteria and protists. • Protists' effects on predatory bacteria and ARGs were greater in presence of fungi.