Are envelope stress responses essential for persistence to β-lactams in Escherichia coli?

Bacterial persistence to antibiotics defines the ability of small sub-populations of sensitive cells within an isogenic population to survive high doses of bactericidal antibiotics. Here, we investigated the importance of the five main envelope stress responses (ESRs) of Escherichia coli in persistence to five bactericidal β-lactam antibiotics by combining classical time-kill curve experiments and single-cell analysis using time-lapse microscopy. We showed that the survival frequency of mutants for the Bae, Cpx, Psp, and Rcs systems treated with different β-lactams is comparable to that of the wild-type strain, indicating that these ESRs do not play a direct role in persistence to β-lactams. Since the σ(E)-encoding gene is essential, we could not directly test its role. Using fluorescent reporters to monitor the activation of ESRs, we observed that σ(E) is induced by high doses of meropenem. However, the dynamics of σ(E) activation during meropenem treatment did not reveal any difference in persister cells compared to the bulk of the population, indicating that σ(E) activation is not a hallmark of persistence. The Bae, Cpx, Psp, and Rcs responses were neither induced by ampicillin nor by meropenem. However, pre-induction of the Rcs system by polymyxin B increased survival to meropenem in an Rcs-dependent manner, suggesting that this ESR might confer some yet uncharacterized advantages during meropenem treatment or at the post-antibiotic recovery step. Altogether, our data suggest that ESRs are not key actors in E. coli persistence to β-lactams in the conditions we tested.