Environmental Stress Shapes Persistence-like Phenotypes and Genomic Changes in Escherichia coli and Morganella morganii: An Exploratory Study

Tosin Senbadejo^1,2Samuel Ntiamoah Osei^1,2Elizabeth Bugase^1,2Christina M. Bourne³Abiola Isawumi^1,2*

• ¹West African Centre for Cell Biology of Infectious Pathogens, College of Basic and Applied Sciences, University of Ghana, Accra, Ghana
• ²AMR Research Group, West African Centre for Cell Biology of Infectious Pathogens, College of Basic and Applied Science, LG 54 Volta Road, University of Ghana, Legon Accra, Legon, Ghana
• ³Department of Chemistry and Biochemistry, University of Oklahoma, Oklahoma, U.S, Norman, United States

Enterobacterales, including E. coli and M. morganii, employ adaptive mechanisms to withstand environmental and host-related stressors, including extreme temperatures, osmotic pressure changes, acidic conditions, and antibiotic pressures. Survival in these conditions can consequently enhance their antibiotic tolerance and persistence. Bacterial persistence contributes to chronic infections and antibiotic treatment failure. This exploratory study investigates the impact of stress conditions (pH, temperature, osmotic, and antibiotic stress) on persistence and genomic adaptations in E. coli and M. morganii. Clinical and environmental M. morganii and E. coli strains from Ghanaian tertiary hospitals were exposed to extreme temperatures (cold ≈ 4°C, and heat ≈ 45°C), extreme pH (pH 3, pH 9, pH 10), and hyperosmolarity (1.71 M NaCl). Growth kinetics were monitored by OD600 and CFU determinations, and persister-like cell formation was assessed using time-kill assays at 2× MIC antibiotic conditions. Genomic changes associated with stress recovery including both adaptive mutations and enrichment of pre-existing variants were captured using comparative whole genome sequencing (WGS) of stress-recovered isolates to parental strains. Strains exhibited variation in growth kinetics under different stress conditions compared to controls. Temperature, pH, and osmotic stress each affected bacterial growth to varying degrees. Heat stress in particular promoted increased persister-like cell formation in E. coli (with strain-specific differences) and, to a lesser extent, in M. morganii upon exposure to meropenem as observed in the isolates examined in this study. WGS analysis revealed that all four studied strains harbored virulence and resistance genes, with missense mutations detected in stress-recovered variants. Most of the mutated genes encode proteins that may play key roles in metabolic processes, transport functions and transcriptional regulations. This exploratory suggest that environmental stress drives both phenotypic and genotypic changes, and that these enhance subsequent survival upon challenge with antibiotics. These adaptive responses may contribute to antibiotic tolerance and chronic infection, emphasizing the need for therapeutic strategies targeting stress response pathways.