Failed collaborations between host immunity and antibiotic treatment enable the evolution of generalized drug tolerance in Streptococcus pneumoniae

To understand the contribution of antibiotic- and immune state-driven selective pressures on pneumococcal adaptation, we evolved Streptococcus pneumoniae in mice with differing immune states repeatedly exposed to various antibiotics. Strikingly, evolved pneumococcal populations failed to develop significant resistance to most antibiotics, with the exception of rifampicin, where resistance mutations arose regardless of host immune status. Pneumococcal strains carrying antibiotic resistance mutations-except for those conferring rifampicin resistance-were highly attenuated in vivo, explaining their scarcity and failure to fix in evolved populations. Instead, many populations acquired parallel mutations in rny (RNase Y) that conferred multi-antibiotic tolerance and enhanced fitness against drugs with disparate cellular targets. Single-cell RNA sequencing revealed that the rny mutant rapidly remodels its transcriptome architecture during antibiotic stress and recovery, partitioning into transcriptionally distinct subpopulations that either enter a quiescent, low-RNA state or maintain ribosome-associated transcriptional activity. This dual survival strategy is mediated by selective and reversible degradation of key transcripts-including those essential for ribosome function-highlighting RNase Y-dependent control of RNA turnover as a central determinant of stress resilience.