Exploring the Role of Unstable L-Forms in the Survival of Uropathogenic Escherichia coli Under Fosfomycin Exposure

Urinary tract infections (UTIs) are common bacterial infection primarilly caused by Uropathogenic E. coli (UPEC). A major concern with UTIs their elevated rate of recurrence. The principal mechanisms for recurrence are believed to be reinfection from an endogenous reservoir or a persistent infection. Uropathogenic bacteria's capacity to invade the bladder tissue as intracellular bacterial communities are believed to be a major contributor in persistent infection. Recently, L-forms were found in urine samples from patients with recurrent UTIs suggesting that cell wall deficient bacteria may play a role in UTI recurrence. L-forms are proliferating bacteria with a deficient cell wall. Normally rod shapped bacteria can be induced into the L-form state by cell wall targetting bacteria. While the L-form state is fragile and requires osmotic support, L-forms are also refractory to killing by the inducing cell wall targetting antibiotic. Once the cell wall synthesis inhibitor is removed, L-forms can revert to their original rod-shape morphology and grow rapidly. Therefore, there is a clear meachnism outlining how L-forms could represent a form of persistent infection. Here, we investigated how L-forms could cause recurrence in the UTI context. We used synthetic human urine and UPEC as an axenic model for UTIs. We exposed UPEC to fosfomycin, a cell wall synthesis inhibitor that is recommended for treating UTIs. We found that the vast majority of cells which survived fosfomycin exposure were converted to L-forms before they regrew as rod-shapped bacteria. We identified that colanic acid synthesis is highly upregulated by UPEC during fosfomycin exposure. We found that the ability to produce colanic acid was not highly protective for fosfomycin treated UPEC. However, UPEC mutants lacking the colanic acid exporter Wza showed significantly decreased survival to fosfomycin. We demonstrate that the Wza is present in approximately 90% of UPEC genomes and represents a general target for reducing L-from survival in fosfomycin treat UTIs. Timelapse microscopy was essential to the findings detailed above. L-forms lack easily identifiable marker and are usually identified by visual analysis. Therefore, we developped an image analysis pipeline to segment UPEC L-forms. Furthermore, to identify if growth rate correlated with UPEC's ability to survive L-form induction we created TrackEcoli which created lineages for cells pre-antibiotic exposure. In this thesis, we provide insight on the expected frequency of L-form survivors in fosfomycin treated UTIs. Furthermore, we determined that inhibiting colanic acid exporter Wza is a generalized target which can help reduce UPEC L-form survival.