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

Abstract

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 β-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 β-lactam for many important infections.

Keywords: Escherichia coli; L-forms; antibiotic resistance; refractory infections; β-lactams.

FIG 1

Meropenem promotes L-form growth from the walled state under aerobic conditions. (A) Model illustrating how pathogenic E. coli can switch in and out of the L-form state in response to the antibiotic challenge. (B) E. coli L-form strain WH62 switch in the presence of meropenem and reversion to the cell walled state after meropenem withdrawal. (C) Time-lapse DIC microscopy of WH62 L-forms; individual micrograph frames are extracted from Movie S1, posted at https://figshare.com/s/09b4bbc18c62c1d6aadd. (D) Susceptibility of E. coli L-forms to β-lactams, aminoglycosides, and fluoroquinolones by standard Etest. MEM, meropenem; FOX, cefoxitin; ATM, aztreonam; CRO, ceftriaxone; AMX, amoxicillin; GEN, gentamicin; CIP, ciprofloxacin.

FIG 2

WH62 E. coli L-form growth kinetics and proliferation rates in LFA medium supplemented with the antibiotic meropenem. (A) Growth curves of J53 (ST10) and WH62 (ST127) L-forms. (B) Bacterial cell area fluctuations between division measured in 22 cells. (C) Length of L-form cell cycle measured in 22 different L-form cells. (D) Mode of cell division of L-forms, division by budding; see also Movie S1, posted at https://figshare.com/s/09b4bbc18c62c1d6aadd. Time 0 (origin) indicates the first division event in the L-form state.

FIG 3

Effects of the L-form switch on susceptibility to myoviruses. (A) Comparative analysis of phage genomes. Schematics show the genomic organization of phages vB_EcoM_OMNI-2 (Eco2), 6 (Eco6), and 12 (Eco12; GenBank OL362041) compared to available reference genomes (T4-like phage NC_000866.4 and V5-like phage DQ832317.1) (left). (B) Phage susceptibility of E. coli L-forms using standard spot assay and modified LFA; WH62 and JIE4799 meropenem-induced L-forms displaying resistance (no lysis) to T4-like phages (Eco2 and Eco12) (bottom right) and sensitivity to V5-like phages (Eco6) (top right), respectively. Control involved cell-walled counterpart on standard LBA without meropenem (left top and bottom) lysed by all three phages. (C) Growth curves of walled bacteria (WH62 on LBA) and L-forms (L-WH62 on LFA supplemented with meropenem) in the presence of Eco12 phage at MOI 1.

Fig 4

The double-layer method was used to test the L-form switching in pathogenic strains of E. coli.