Isolation of L-form Bacteria from Urine using Filtration Method.

Transition of bacteria to the L-form state is thought to play a possible role in immune evasion and bacterial persistence during treatment with cell wall-targeting antibiotics. However, isolation and handling of L-form bacteria is challenging, mainly due to their high sensitivity to changes in osmolarity. Here, we describe detailed protocols for the preparation of L-form medium, isolation of L-forms from urine using a filtration method, detection of L-forms in urine samples by phase contrast microscopy and induction of L-forms in vitro. The exact requirements for survival and growth of L-forms may vary from strain to strain. Therefore, the methods presented here are intended to act as basic guidelines for establishing L-form protocols within individual laboratories, rather than as precise instructions. The filtration method can lead to a reduction in the number of L-forms in a sample and should not be used for quantification. However, it is the only method used so far for effective separation of cell wall-deficient variants from their walled counterparts and for identification of bacterial strains, which are capable of L-form switching in patients with urinary tract infections. The filtration method has the potential to be adapted for the isolation of L-forms from patients with other categories of bacterial infections and from environmental samples.

Author Correction: Possible role of L-form switching in recurrent urinary tract infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Possible role of L-form switching in recurrent urinary tract infection.

Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E. coli strains from patient samples readily transition from the walled state to L-form during challenge with a cell wall targeting antibiotic. Following antibiotic withdrawal, they then efficiently transition back to the walled state. E. coli switches between walled and L-form states in a zebrafish larva infection model. The results suggest that L-form switching is a physiologically relevant phenomenon that may contribute to the recurrence of infection in older patients with rUTI, and potentially other infections.