Crohn's disease is accompanied by a persistent imbalance of the gut microbiota. Among the bacteria associated with this pathology, adherent-invasive Escherichia coli (AIEC) strains have been implicated in the mechanisms of the disease: they colonize intestinal epithelial cells but, above all, survive and replicate within macrophages. The reference strain of this pathovar, AIEC LF82, forms microcolonies inside phagolysosomes, a compartment that is nonetheless toxic and prevents the proliferation of commensal E. coli. Until now, the growth state of LF82 in this environment, as well as the signals that trigger its intracellular replication, were poorly understood.
To address these questions, the authors combined single-cell analysis, genetic dissection, and mathematical modeling in order to track the growth status and cell-cycle regulation of internalized bacteria. Infections of human macrophages derived from the THP1 cell line were carried out at a multiplicity of infection of 30, resulting in an average of three bacteria per macrophage one hour after infection. Fluorescent biosensors, a fluorescence dilution assay, the TIMER tool, and gentamicin protection assays were used to quantify growth, viability, and antibiotic tolerance, while a three-differential-equation model reconstructed the infection kinetics.
The work shows that, inside macrophages, bacteria can either replicate or switch to a non-proliferating phenotype. This switch results from two successive mechanisms. Immediately after internalization, the stringent response blocks the replicative cycle of AIEC LF82 for a few hours; during this period, a substantial fraction of the bacteria acquire the ability to tolerate antibiotic treatment. When replication resumes, in a second phase, it requires DNA repair, suggesting that the bacteria accumulated lesions during the first hours of infection. This genotoxic damage is accompanied by induction of the SOS response, and non-proliferating bacteria frequently emerge from the growing population. Overall, the proportion of antibiotic-tolerant bacteria reaches up to 10% of the population. Tolerance assays, performed with ciprofloxacin, cefotaxime, and ofloxacin, confirm that these dormant cells resist antibiotics of various classes.
Thus, the constraints encountered in the intracellular environment induce phenotypic heterogeneity in AIEC LF82 and generate non-proliferating bacteria. The authors propose that this subpopulation constitutes a reservoir of antibiotic-tolerant bacteria that may underlie recurrent infections.