Optimisation of the therapeutic potential of fosfomycin against Enterobacteriaceae: characterisation of genetic and physiological factors related to resistance and antimicrobial activity

Abstract

Since the discovery of the first antimicrobials, bacteria with resistance mechanisms against them have been detected. The appearance of bacterial resistance is a natural phenomenon, which has increased as a result of the use of antimicrobials. Therefore, the availability of antimicrobials does not ensure therapeutic success. Moreover, in recent decades, a progressive increase in antimicrobial resistance has occurred, and it has become a global public health problem, since there is an increase in deaths caused by or related to bacteria that present resistance mechanisms. As a result of this problem and the scarcity of new effective molecules for the treatment of multidrug-resistant bacteria, various organizations (such as WHO and FAO) are developing plans with different strategies to address the problem. These strategies include optimizing of the use of existing antimicrobials and the rescue of old antibiotics that are still active, such as fosfomycin. Fosfomycin is an old antimicrobial that can be a good therapeutic option, since it many bacteria of clinical interest remain sensitive to this antibiotic. Fosfomycin is a derivative of phosphonic acid, a hydrophilic, low molecular weight molecule. It has three carbon atoms, is soluble in water, and is similar to phosphoenolpyruvate. It is a broad-spectrum bactericidal antimicrobial that acts in the growth phase of bacteria, inhibiting the first step of cell wall peptidoglycan synthesis by binding to the enzyme MurA. Fosfomycin must penetrate the cytoplasm to reach its target, MurA, producing bactericidal effect. For this purpose, two membrane transporters GlpT and UhpT are described, whose physiological function in bacteria is the uptake of phosphorylated carbon sources and expel inorganic phosphate (Pi). The regulation and activity of these transporters is fundamental to the mechanism of action of fosfomycin and, therefore, to fosfomycin resistance. The transcription of both transporters is induced by their own substrate, in addition to the AMPc-CRP metabolism regulator complex, and they are also activated by the FNR regulator, a bacterial regulator under anaerobic conditions. The GlpT transporter has the function of introducing glycerol-3-phosphate (G3P), this molecule binds to the GlpR repressor, causing the loss of affinity for promoters of the glp regulon genes, such as glpT. On the other hand, UhpT transports hexose-phosphate, mainly glucose-6-phosphate (G6P). This molecule is detected by a two-component system, UhpB and C, and when this occurs, it phosphorylates UhpA, which binds to the uhpT promoter, inducing its transcription. Thus, in the susceptibility assays G6P must be added to induce the presence of this transporter, as the susceptibility results obtained in this way are more consistent with susceptibility breakpoints and therapeutic success. Fosfomycin resistance mechanisms can be plasmid and chromosomal mediated, as modifications of MurA, the presence of peptidoglycan recycling pathways, alteration of fosfomycin permeability or the presence of fosfomycin-modifying enzymes. Chromosomal mediated fosfomycin resistance usually occurs in a stepwise mode, often generating complex phenotypes difficult to interpret. In this sense, to better understand the mechanisms of resistance to fosfomycin in Klebsiella pneumoniae and to optimize the use of this antimicrobial, the following study was carried out. The objectives were to characterize the role of the genes uhpT, glpT, and fosA in resistance to fosfomycin in K. pneumoniae and to evaluate the use of phosphonoformate sodium (PPF) due to its ability to inhibit the FosA enzyme, in combination with fosfomycin. For this purpose, seven clinical isolates of K. pneumoniae and the reference strain (ATCC 700721) were used, and their genomes were sequenced. Mutants for transporters and fosA were constructed from two isolates of K. pneumoniae ATCC 700721. The susceptibility test to fosfomycin was performed using the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed with SynergyFinder. Spontaneous frequencies of occurrence of fosfomycin and PPF mutants, in vitro activity by growth curves with gradient concentrations of fosfomycin with and without PPF, and time-kill assays with and without PPF were also evaluated. The fosfomycin MICs of the clinical isolates ranged from 16 to 1,024mg/L. The addition of 0.623 mM PPF reduced the MIC by 2 to 8-times. Deletion of fosA gene led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623mM). The lowest frequencies of fosfomycin resistant mutants were found in ΔfosA mutants with frequency ranging from 1.69x10-1 to 1.60x10-5 for 64 mg/L fosfomycin. Finally, the growth monitoring and time-kill assays, fosfomycin showed bactericidal activity only against fosA mutants and not with the addition of PPF. The study concludes that inactivation of the fosA gene results in decreased resistance to fosfomycin in K. pneumoniae. The pharmacological approach using PPF did not achieve sufficient activity and the effect decreased with the presence of other fosfomycin resistant mutations. The second chapter of the Thesis follows the line of optimizing the use of fosfomycin with the addition of an adjuvant, and to better understand how the regulation of fosfomycin transporters may affect their activity. The main objective was to evaluate the role of glycerol at therapeutically relevant concentrations in combination with fosfomycin in Escherichia coli, since this molecule is clinically used as a treatment for example for elevated intracranial pressure and can induce glpT expression. For this purpose, a collection of isogenic mutants of fosfomycinrelated genes was evaluated in E. coli strains. The induction of fosfomycin transporters was evaluated and susceptibility tests, interaction assays, and time-to-death assays were performed. Our results showed that glycerol allows the activation of the GlpT transporter, but this induction is delayed in time and is not homogeneous in all E. coli strains throughout the bacterial population, leading to contradictory results in terms of fosfomycin activity. The susceptibility assays showed increased fosfomycin activity with glycerol in the disc diffusion assay, but not in the agar dilution or broth microdilution assays. Similarly, in time-kill assays, the effect of glycerol was absent because of the appearance of fosfomycin-resistant subpopulations. In conclusion, glycerol may not be a good candidate for use as an adjuvant to fosfomycin. Finally, to better understand physiological factors that affect fosfomycin transporters activity, the aim of third chapter was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). For this purpose, the wild-type strain BW25113, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT and ΔphoB) and six clinical isolates of E. coli with different fosfomycin susceptibilities were used. Susceptibility was assessed by agar dilution using Mueller-Hinton agar (Pi=1mM) and supplemented with Pi (13 and 42mM, minimum and maximum urinary concentrations of Pi) and/or glucose-6-phosphate (25mg/L). The promoter activity of the fosfomycin transporter was assessed by monitoring fluorescence accumulation using pUA66-PglpT::gfpmut2 or pUA66-PuhpT::gfpmut2 plasmids in standard Mueller-Hinton broth (MHB) supplemented with Pi (13 or 42mM) ± glucose-6-phosphate. Fosfomycin activity was quantified spectrophotometrically at 24 hours as before with glucose-6-phosphate, and fosfomycin ranged from 1 to 1024mg/L. The EC50 of fosfomycin was estimated and compared. Time-kill assays were performed with fosfomycin concentrations of 307 (plasma Cmax), 1053 and 4415mg/L (urinary Cmax range), using MHB with 28mM Pi (mean urinary concentration) +25mg/L glucose-6-phosphate. The results showed that all strains decreased fosfomycin susceptibility linked to increasing Pi concentrations: 1-4-log2 dilution differences from 1 to 13mM, and 1-8-log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporter promoters. Also, increasing Pi concentrations resulted in a higher bacterial viability EC50 of fosfomycin, except against the ΔglpT-uhpT mutant strain. Therefore, the present study concludes that Pi variations in physiological fluids may reduce the activity of fosfomycin against E. coli. Also, the elevated urinary Pi concentrations may explain the failure of oral fosfomycin in non-wild but fosfomycin-susceptible E. coli strains.