L-Form Switching in Escherichia coli as a Common beta-Lactam Resistance Mechanism

Cell wall deficient bacterial L-forms are induced by exposure to cell wall-targeting antibiotics and immune effectors such as lysozyme. L-forms of different bacteria (including Escherichia coli) have been reported in human infections, but whether this is a normal adaptive strategy or simply an artifact of antibiotic treatment in certain bacterial species remains unclear. Here we show that members of a representative, diverse set of pathogenic E. coli readily proliferate as L-forms in supratherapeutic concentrations of the broad-spectrum antibiotic meropenem. We report that they are completely resistant to antibiotics targeting any penicillin-binding proteins in this state, including PBP1A/1B, PBP2, PBP3, PBP4, and PBP5/6. Importantly, we observed that reversion to the cell-walled state occurs efficiently, less than 20 h after antibiotic cessation, with few or no changes in DNA sequence. We defined for the first time a logarithmic L-form growth phase with a doubling time of 80 to 190 min, followed by a stationary phase in late cultures. We further demonstrated that L-forms are metabolically active and remain normally susceptible to antibiotics that affect DNA torsion and ribosomal function. Our findings provide insights into the biology of L-forms and help us understand the risk of beta-lactam failure in persistent infections in which L-forms may be common. IMPORTANCE Bacterial L-forms require specialized culture techniques and are neither widely reported nor well understood in human infections. To date, most of the studies have been conducted on Gram-positive and stable L-form bacteria, which usually require mutagenesis or long-term passages for their generation. Here, using an adapted osmoprotective growth media, we provide evidence that pathogenic E. coli can efficiently switch to L-forms and back to a cell-walled state, proliferating aerobically in supratherapeutic concentrations of antibiotics targeting cell walls with few or no changes in their DNA sequences. Our work demonstrates that L-form switching is an effective adaptive strategy in stressful environments and can be expected to limit the efficacy of beta-lactam for many important infections.