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Background Nitrofurantoin has been re-introduced as a first-choice antibiotic to treat uncomplicated acute urinary tract infections in England and Wales. Highly effective against common uropathogens such as Escherichia coli, its use is accompanied by a low incidence (<10%) of antimicrobial resistance. Resistance to nitrofurantoin is predominantly via the acquisition of loss-of-function, step-wise mutations in the nitroreductase genes nfsA and nfsB. Objective To explore the in situ evolution of NitR in E. coli isolates from 17 patients participating in AnTIC, a 12-month open label randomized controlled trial assessing the efficacy of antibiotic prophylaxis in reducing urinary tract infections (UTIs) incidence in clean intermittent self-catheterizing patients. Methods The investigation of NitR evolution in E. coli used general microbiology techniques and genetics to model known NitR mutations in NitS  E. coli strains. Results Growth rate analysis identified a 2%–10% slower doubling time for nitrofurantoin resistant strains: NitS: 20.8 ± 0.7 min compared to NitR: 23 ± 0.8 min. Statistically, these data indicated no fitness advantage of evolved strains compared to the sensitive predecessor (P-value = 0.13). Genetic manipulation of E. coli to mimic NitR evolution, supported no fitness advantage (P-value = 0.22). In contrast, data argued that a first-step mutant gained a selective advantage, at sub-MIC (4–8 mg/L) nitrofurantoin concentrations. Conclusion Correlation of these findings to nitrofurantoin pharmacokinetic data suggests that the low incidence of E. coli NitR, within the community, is driven by urine-based nitrofurantoin concentrations that selectively inhibit the growth of E. coli strains carrying the key first-step loss-of-function mutation.

BACKGROUNDNitrofurantoin has been re-introduced as a first-choice antibiotic to treat uncomplicated acute urinary tract infections in England and Wales. Its mode of action involves initial reduction by nitroreductases, to generate electrophilic intermediates that inhibit protein and nucleic acid synthesis. Highly effective against common uropathogens such as Escherichia coli, its use is accompanied by a low incidence (<10%) of antimicrobial resistance. Resistance to Nitrofurantoin is predominantly via the acquisition of loss-of-function, step-wise mutations in the nitroreductase genes nfsA and nfsB.OBJECTIVETo explore the in situ evolution of NitR, longitudinal uropathogenic E. coli isolates recovered from two rUTI patients.RESULTSGrowth rate analysis identified a 2-10% slower doubling time for Nitrofurantoin resistant strains, but statistically, these data suggested there was no fitness advantage of evolved strains over their sensitive predecessor (ANOVA P-value = 0.13). Genetic manipulation of E. coli to mimic nitrofurantoin resistance evolution, again confirmed no fitness advantages (ANOVA P-value = 0.22). Rather, further analysis argued that a first-step mutant gained a selective advantage, at sub-MIC (4-8 mg/L) nitrofurantoin concentrations.CONCLUSIONCorrelation of these findings to Nitrofurantoin pharmacokinetic data suggests that the low incidence of E. coli NitR, within the community, is driven by urine-based nitrofurantoin concentrations that selectively inhibit the growth of E. coli strains carrying the key first-step loss-of-function mutation.

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Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 40 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NFκB luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NFκB signalling. Although two isolates were associated with swarm sizes of >7 cm and NFκB activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NFκB signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4±1.8 to 46.1±2.5 fold, P = NS) NFκB signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

Background The mainstay treatments for urinary tract infections (UTIs) are nitrofurantoin and trimethoprim. The common route to acquire trimethoprim resistance (TriR) is via horizontal transfer of the trimethoprim-insensitive dihydrofolate reductase: dfrA. In contrast, nitrofurantoin resistance (NitR) requires the inactivation of two chromosomally encoded nitroreductases: nfsA and nfsB. The difference in antimicrobial resistance (AMR) evolution is reflected in community surveillance data: NitR incidence = ∼10% and for TriR &gt; 30%. Longitudinal clinical UTI trials provide a unique opportunity to monitor in situ evolution of AMR and correlate its appearance to trial outcomes and the impact on uropathogens. Objectives To explore the in situ AMR evolution and its impact in Escherichia coli isolates from patients participating in the clinical trials: AnTIC and ALTAR. AnTIC was an open label randomized trial assessing the efficacy of antibiotic prophylaxis use in clean intermittent self-catheterizing patients. ALTAR compared the efficacy of the non-antibiotic alternative methenamine hippurate to antibiotic prophylaxis to treat recurrent UTIs. Methods The investigation of the evolution of nitrofurantoin and trimethoprim resistance in E. coli used general microbiology techniques, bioinformatic genome analysis and genetics to model known AMR mutations in susceptible E. coli strains. Results Trimethoprim resistance amongst E. coli trial isolates was driven by 14 out of ∼45 known dfrA allelic variants; the most common being dfrA1 and dfrA17. Growth analysis identified an allelic bias when strains were exposed to sub-MIC concentrations of trimethoprim. Growth rate analysis identified a 2%–10% slower doubling time for nitrofurantoin-resistant strains: NitS: 20.8 ± 0.7 min compared to NitR: 23 ± 0.8 min. Statistically, these data suggested no fitness advantage of evolved strains compared to the sensitive predecessor (P value = 0.13). Genetic manipulation of E. coli to mimic NitR evolution, however, supported a selective advantage. Conclusions Longitudinal sampling during antibiotic based clinical trials has provided a new perspective to the response of E. coli to UTI-related antibiotics. Correlation of nitrofurantoin response to pharmacokinetic data suggests that the low incidence of E. coli NitR is driven by selection not fitness. However, fitness correlates to dfrA allele carriage and could potentially be exploited to benefit the use of trimethoprim if other antibiotics are impeded by AMR.

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