Biology subjects

Bermudez, T.

Publications and source records attributed to Bermudez, T..

3 recordsLinked to original sources

bioRxiv2023

Drug Resistance without a cost? Common and uncommon routes to fosfomycin resistance in Uropathogenic Escherichia coli

Fosfomycin kills bacteria by blocking the binding of phosphoenolpyruvate (PEP) to the bacterial enzyme MurA and halting peptidoglycan synthesis. While its use has increased with the emergence of antibiotic resistance, the mechanisms leading to fosfomycin resistance remain relatively unexplored. In uropathogenic Escherichia coli (UPEC) that accounts for >75% of urinary tract infections (UTIs), fosfomycin enters the cell primarily through UhpT, which transports glucose-6-phosphate (G6P) glycolysis intermediate into the cell. Mutations in uhpT lead to fosfomycin resistance and have been identified during antimicrobial susceptibility testing (AST) in non-susceptible inner colonies that form within the zone of inhibition. However, EUCAST and CLSI guidelines differ in how to read fosfomycin AST when such resistant colonies arise. Work from our lab and others demonstrated that glycolysis is dispensable during acute UTI. Moreover, G6P is scarce in urine, prompting us to test the hypothesis that uhp mutations may not impart a fitness cost to the pathogen. We report that loss of uhp indeed does not impair UPEC pathogenesis and that clinical isolates exist that lack the uhp locus altogether. Analysis of non-susceptible inner colonies revealed a suite of novel genes involved in fosfomycin resistance. One of them is PykF that converts PEP to pyruvate during glycolysis. Single deletions of pykF or its anaerobic homolog pykA do not attenuate UPEC. Based on our data, we raise the alarm that multiple routes lead to fosfomycin resistance and do not affect pathogenesis and propose that the current EUCAST and CLSI guidelines unify into how they evaluate fosfomycin AST. IMPORTANCEWhile fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here we report that the most common route to fosfomycin resistance, as well as novel routes described in this work do not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

microbiology↗

bioRxiv2023

Mapping Niche-specific Two-Component System Requirements in Uropathogenic Escherichia coli

Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify - for the first time - a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct. IMPORTANCEWhile two-component system (TCS) signaling has been investigated at depth in model strains of E. coli, there have been no studies to elucidate - at a systems level - which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.

microbiology↗

bioRxiv2023

The QseB response regulator imparts tolerance to positively charged antibiotics by controlling metabolism and minor changes to LPS

The modification of lipopolysaccharide (LPS) in Escherichia coli and Salmonella spp. is primarily controlled by the two-component system PmrAB. LPS modification allows bacteria to avoid killing by positively charged antibiotics like polymyxin B. We previously demonstrated that in uropathogenic E. coli (UPEC), the sensor histidine kinase PmrB also activates a non-cognate transcription factor, QseB, and this activation somehow augments polymyxin B tolerance in UPEC. Here, we demonstrate - for the first time - that in the absence of the canonical LPS transcriptional regulator, PmrA, QseB can direct some modifications on the LPS. In agreement with this observation, transcriptional profiling analyses demonstrate regulatory overlaps between PmrA and QseB in terms of regulating LPS modification genes. However, both PmrA and QseB must be present for UPEC to mount robust tolerance to polymyxin B. Transcriptional and metabolomic analyses also reveal that QseB transcriptionally regulates the metabolism of glutamate and 2-oxoglutarate, which are consumed and produced during the modification of lipid A. We show that deletion of qseB alters glutamate levels in the bacterial cells. The qseB deletion mutant, which is susceptible to positively charged antibiotics, is rescued by exogenous addition of 2-oxoglutarate. These findings uncover a previously unknown mechanism of metabolic control of antibiotic tolerance that may be contributing to antibiotic treatment failure in the clinic. IMPORTANCEAlthough antibiotic prescriptions are guided by well-established susceptibility testing methods, antibiotic treatments oftentimes fail. The presented work is significant, because it uncovers a mechanism by which bacteria transiently avoid killing by antibiotics. This mechanism involves two closely related transcription factors, PmrA and QseB, which are conserved across Enterobacteriaceae. We demonstrate that PmrA and QseB share regulatory targets in lipid A modification pathway and prove that QseB can orchestrate modifications of lipid A in E. coli in the absence of PmrA. Finally, we show that QseB controls glutamate metabolism during the antibiotic response. These results suggest that rewiring of QseB-mediated metabolic genes can lead to stable antibiotic resistance in subpopulations within the host, thereby contributing to antibiotic treatment failure.

microbiology↗