Aminoglycoside potentiation via activation of carbohydrate transporters

Abstract

The present invention relates to the potentiation of aminoglycosides, particularly by the use of nucleosides, through the activation of carbohydrate transporters. The present invention encompasses the combination of a nucleoside and an aminoglycoside antibiotic for treating bacterial infections, wherein the nucleoside potentiates bacterial killing by the antibiotic, and preferably the nucleoside limits the development of antibiotic-resistant bacteria or resensitizes antibiotic-resistant bacteria to killing by the antibiotic.

Description

[Technical Field]

[0001] The present invention relates to the potentiation of aminoglycosides through the activation of carbohydrate transporters, particularly by the use of nucleosides. The present invention encompasses the combination of a nucleoside and an aminoglycoside antibiotic for the treatment of bacterial infections, where the nucleoside potentiates bacterial killing by the antibiotic, particularly where the nucleoside limits the development of aminoglycoside-resistant bacteria and / or resensitizes aminoglycoside-resistant bacteria to killing by the antibiotic. [Background technology]

[0002] Antibiotics save many lives and play a key role in modern medicine. However, the use of new antibiotics is accompanied by the spread of resistance in bacterial populations. 1 As outlined by the World Health Organization (WHO), studies have shown the prevalence of resistant Gram-negative bacteria. 2 , which is mainly due to the double membrane barrier of Gram-negative bacteria 3 A recent report estimated that 1.27 million deaths were directly attributable to bacterial AMR in 2019, with 95% of those deaths attributed to five pathogens: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. This suggests that the burden of AMR exceeds the death toll caused by malaria and HIV combined, highlighting the urgency of developing new treatments. All but one of these pathogens (S. aureus) are Gram-negative bacteria.

[0003] Aminoglycosides (AGs) constitute a family of broad-spectrum antibiotics that are able to penetrate the double membrane barrier of Gram-negative bacteria. They are used clinically to treat infections such as pneumonia, sepsis, and urinary tract infections (UTIs) caused by a variety of Gram-negative pathogens. 5 However, AGs treatment is ototoxic.6 and nephrotoxicity 7,8 Related to.

[0004] Entry of AGs into Gram-negative cells has been proposed to occur in three steps. 9,10 The first step is considered a "passive" uptake step, facilitated by the cationic nature of AGs. 9 :AGs interact electrostatically with negatively charged components of the outer membrane (lipopolysaccharides, phospholipids) in an energy-independent manner 11,12 This leads to membrane disruption and subsequent "self-promoted" uptake into the periplasm. 13 Nonspecific uptake by the outer membrane porins OmpF and OmpC has also been observed in E. coli. 14 During the second step, an energy-dependent step allows AG to enter the cytoplasm from the periplasm. AG uptake in bacterial cells is thus described as dependent on the proton motive force (PMF), which can be reduced by inhibitors of electron transport and oxidative phosphorylation. 15,16 Finally, AGs target ribosomes, slowing translation. 17 and mistranslation 18,19 It is generally accepted that mistranslated proteins, especially membrane proteins, alter membrane integrity and allow the uptake of many AGs at the final stage, causing further damage to the cell.

[0005] Aminoglycosides are molecules that have shown good efficacy in humans in the treatment and prevention of infectious diseases, but they also cause serious side effects. 20 Nephrotoxicity and ototoxicity have been observed at varying rates across studies, with an average incidence ranging from 3 to 15% of cases. 21,22 These values ​​are based on the patient's general health, the presence or absence of mutations (e.g., mutations in the mitochondrial gene MTRNR1 associated with aminoglycoside-induced hearing loss), and 23 ), and the type of infection, especially the duration of treatment, making it difficult to determine. In fact, the occurrence of these side effects is accelerated by long-term treatment. 22Aminoglycosides, by their chemical nature, bind to negatively charged sites on the brush border cells of the proximal tubules of the kidney. 24 They accumulate in cells before being translocated to the vacuole, where they cause excessive accumulation of phospholipids, alter lysosomal membranes, and impair mitochondrial metabolism. 25 In a study combining data from 10,000 patients, the average incidence of nephrotoxicity for gentamicin and tobramycin was estimated to be 14% and 12%, respectively, and 9.4% for amikacin. 21 The incidence of nephrotoxicity has been shown to be area under the curve dependent, therefore single doses at 12-hour intervals are recommended. 26,27 .

[0006] The use of aminoglycosides can cause significant loss of high-frequency hearing, dizziness, and even irreversible hearing loss 28 Side effects increase with increasing duration of treatment. For example, hearing loss has been observed in 4-15% of patients treated with 1 g / day streptomycin for 7 days or more. Symptoms usually appear after a 7-10 day incubation period. 29 This ototoxicity is due to the high affinity of aminoglycosides for the cochlear hair cell membrane, first damaging the outer hair cells of the first to third rows, and then spreading to the inner hair cells. 30 Absorption by the organ of Corti in the cochlea is rapid (saturation is reached in 3 hours), and the effect on hair cells is concentration-dependent. 31 .

[0007] Residual concentrations (Cmin) are predictive of toxicity. Dosage determination for long-term treatment should be performed 48 hours after treatment. For amikacin, the toxicity threshold is 2.5–5 μg / ml, and for tobramycin and gentamicin, it is 0.5–1 μg / ml. The recommended maximum aminoglycoside concentration (Cmax / MIC) is 8–10 times the minimum inhibitory concentration (MIC).

[0008] International application WO 2020 / 227530 and the corresponding scientific paper (Yang et al., Cell, 2019, 177, 1649-1661) disclose a machine learning approach using 206 metabolites from the Biolog phenotype microarray (PMs 1-4, Bochner, FEMS Microbiol Rev, 2009, 33-191-205) included in the iJ0136 genome-scale model of E. coli metabolism to identify metabolic pathways involved in the lethality of three antibiotics: ampicillin (AMP, a β-lactam), ciprofloxacin (CIP, a fluoroquinolone), and gentamicin (GENT, an aminoglycoside). Model-guided machine learning predictions identified the purine biosynthetic pathway as a novel pathway associated with antibiotic lethality, with shared directionality between AMP and CIP and reverse directionality for GENT (Example 5, pp. 79-80; Figure 3). This hypothesis was tested in wild-type E. coli by genetic deletion of enzymes involved in purine or pyrimidine biosynthesis, biochemical inhibition with purine biosynthetic enzyme inhibitors, and biochemical supplementation of purine biosynthetic substrates (ribosylpyrophosphate (prpp) and glutamine (gln)) (Example 6, p81; Figure 4A). Stimulation of the purine biosynthetic pathway increased the lethality of AMP and CIP and decreased that of GENT (Figure 4E). Conversely, inhibition of the purine biosynthetic pathway decreased the lethality of AMP and CIP and increased that of GENT (Figures 4B, 4C, and 4D). Based on these predictions and observations, we hypothesized that (i) purine supplementation rescues antibiotic-induced purine depletion and reduces antibiotic lethality, and (ii) pyrimidine supplementation, such as with uracil, inhibits pyrimidine biosynthesis and promotes purine biosynthesis via the accumulation of prpp, resulting in increased antibiotic lethality (Figure 5A). This hypothesis does not fit the model tested for gentamicin (GENT), which clearly shows that GENT has the opposite effect to AMP or CIP and that the lethality of GENT is reduced by stimulation of the purine biosynthetic pathway ( Fig. 4E ).Therefore, it was not surprising that, with respect to gentamicin, no significant increase in antibiotic lethality was observed 4 h after the addition of all tested pyrimidines (cytosine, thymine, uracil, and uridine) (Figures 5B and 5C), indicating that pyrimidines do not improve bacterial killing by gentamicin or other aminoglycoside antibiotics under the conditions tested. This was confirmed by measuring the half-maximal inhibitory concentration (IC50) of the antibiotics 4 h after treatment using Biolog plates. For all pyrimidines tested (PM1: negative control (1,31E-07); uridine (1,55E-07); PM3: negative control (9,98E-09); cytidine (1,27E-08); cytosine (1,14E-08); thymine (1,20E-08); thymidine (1,02E-08); uracil (1,17E-08); uridine (1,12E-08); a 2-fold change was considered significant), no significant changes in the IC50 of gentamicin were observed compared to the control group. The inventors reproduced these experiments and found no significant changes in antibiotic lethality for all pyrimidine compounds tested under the test conditions (Figure 21 of the present application).

[0009] Application EP 3 027 213 discloses the use of a combination of an antibiotic with both a uridine compound and pyruvate to suppress the side effects of the antibiotic, and discloses that (i) the effect of the antibiotic depends only on the antibiotic compound and not on the co-administered uridine and / or pyruvate compounds, and (ii) the presence of both the uridine and pyruvate compounds is necessary to neutralize the side effects of the antibiotic compound.

[0010] Therefore, there is a need in the art for compositions and methods for enhancing the efficacy of aminoglycosides against Gram-negative bacteria by increasing their uptake across bacterial membranes. The present invention fulfills this need by improving uptake in bacteria, thereby potentially improving the efficacy of AGs therapy. Summary of the Invention [Means for solving the problem]

[0011] The present invention encompasses compositions, uses of these compositions to kill bacteria, and methods of killing bacteria. The present invention encompasses combinations of nucleosides and aminoglycoside antibiotics, where the nucleoside enhances bacterial killing by the antibiotic, and preferably the nucleoside limits the emergence of bacteria resistant to the aminoglycoside antibiotic and / or resensitizes bacteria resistant to the aminoglycoside antibiotic to killing by the antibiotic.

[0012] In some embodiments, the nucleoside is selected from uridine, inosine, guanosine, cytidine, thymidine, or xanthosine. Preferably, the nucleoside is uridine.

[0013] In some embodiments, the nucleoside is administered to the urinary tract, digestive tract, lungs, eye, ear, nose, brain, heart, blood, or skin.

[0014] In some embodiments, the nucleoside is 1-20 g / m 2 is administered at a dose of

[0015] In some embodiments, the method is for preventing or limiting the development of antibiotic-resistant bacteria by killing the bacteria more rapidly by co-administering a nucleoside with an aminoglycoside antibiotic at concentrations already in clinical use.

[0016] In some embodiments, the method is for killing antibiotic-resistant bacteria, and co-administration of a nucleoside and an aminoglycoside antibiotic together allows for killing of antibiotic-resistant bacteria, whereas the same concentration of the aminoglycoside antibiotic alone is ineffective.

[0017] In some embodiments, the method is for reducing the toxicity of a reference treatment by co-administering a nucleoside with an aminoglycoside antibiotic, with the aminoglycoside administered at a lower concentration than the reference treatment, hi some embodiments, the level of bacterial kill by the antibiotic is equivalent to the reference treatment.

[0018] In some embodiments, the antibiotic is selected from tobramycin, gentamicin, and amikacin.

[0019] In some embodiments, the bacteria is an enterobacterium.

[0020] In some embodiments, the bacterium is selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii.

[0021] In some embodiments, the bacteria causing the infection include bacteria that are resistant to aminoglycoside antibiotics. [Brief explanation of the drawings]

[0022] [Figure 1] A-C: CmtA and Crp are involved in tobramycin resistance. E-test of E. coli WT, ΔcmtA, and Δcrp. Deletion of CmtA increases the MIC by 4-fold, while deletion of Crp increases the MIC by 15-fold despite impaired growth. B. Growth curves of E. coli WT, ΔcmtA, and Δcrp in the presence or absence of 0.4 μg / ml tobramycin. C. E-test of E. coli harboring an empty vector (p0+) compared to a vector overexpressing cmtA (pcmtA+). Inducer was added to the medium. [Figure 2]AC: A. Overexpression of 11 carbohydrate transporters specifically sensitizes to AGs. See also Table 3. Response of E. coli carrying an empty vector (p0) compared to vectors overexpressing carbohydrate transporters to tobramycin (0.1 μg / ml), carbenicillin (3 μg / ml), ciprofloxacin (0.005 μg / ml), or gentamicin (0.08 μg / ml). Cultures of each strain were grown overnight. Sensitivity was assessed by serial dilution (ND: undiluted) and by spotting 5 μl of each dilution onto plates with or without 0.1 μg / ml tobramycin. Inducers were added to the medium. A. Overexpression of 16 transporters sensitizes to tobramycin. B. Overexpression of five transporters also sensitizes to carbenicillin or ciprofloxacin. C. Overexpression of 11 transporters also sensitizes to gentamicin (AG). [Figure 3] CmtA is involved in the differential uptake of neocy5. Uptake of neocy5 assessed by flow cytometry for E. coli ΔcmtA and Δcrp compared with the WT strain, and for E. coli harboring a plasmid overexpressing cmtA compared with a strain carrying an empty vector (p0), is shown as the fold change in mean fluorescence per cell (compared to WT for mutants or to empty vector for overexpression). [Figure 4] GFP expression under the control of the cmtA promoter. Quantification of GFP fluorescence using flow cytometry was shown as the fold change in mean fluorescence per cell (mutants compared to WT or carbon source compared to substrate-free conditions) depending on the strain (WT, ΔcmtA, Δcrp, or Δcra) or substrate added to the medium (whether supplemented with glucose or mannitol 0.5%). [Figure 5]AD: Uridine increases cmtA expression and reduces the MIC of tobramycin by promoting its uptake. A. GFP expression from the cmtA promoter depends on the conditions (whether supplemented with glucose, uridine, ribose, uracil, or 0.5% mannitol). Growth in response to substrate is shown as OD600nm on the left. B. Quantification of GFP fluorescence using flow cytometry in response to substrates added to the medium (whether supplemented with glucose, uridine, ribose, uracil, or mannitol 0.5%) is shown as the mean percent change in fluorescence intensity per cell compared to the substrate-free condition. The MIC of tobramycin in E. coli WT, measured by the CE test, is shown in μg / ml depending on the substrates added to the medium (whether supplemented with glucose, ribose, or uridine 0.5%). D. neocy5 uptake assessed by flow cytometry for E. coli WT growth supplemented with glucose (MIC 1.5 μg / ml) or 0.5% uridine (MIC 0.1 μg / ml) is shown as the fold change in mean fluorescence per cell compared to glucose. [Figure 6] AB: Substrate-dependent GFP expression from the fruA or btuB promoter. A. Quantitation of GFP fluorescence from the fruA promoter using flow cytometry as a function of substrates added to the medium (whether supplemented with 0.5% glucose, uridine, ribose, fructose, or mannose) is shown as the fold change in mean fluorescence per cell. B. Quantitation of GFP fluorescence from the btuB promoter using flow cytometry as a function of substrates added to the medium (whether supplemented with 0.5% glucose, uridine, ribose, fructose, or mannose) is shown as the fold change in mean fluorescence per cell. [Figure 7]AB: Uridine-mediated AG sensitivity is not related to uridine uptake, catabolism, or stress response. A. Tobramycin sensitivity of E. coli harboring an empty plasmid (p0) compared with vectors overexpressing the two uridine transporters nupG or nupC. Cultures of each strain were grown overnight. Sensitivity was assessed by serial dilution and spotting 5 μl of each dilution onto plates containing or without 0.1 μg / ml tobramycin and 0.5% uridine. Inducer was added to the medium. B. Quantification of GFP fluorescence in logarithmic-phase P1rrnB-expressing cells using flow cytometry in response to substrates added to the medium (whether supplemented with glucose, uridine, ribose, or maltose 0.5%) was shown as fold change relative to mean fluorescence per cell. Stationary-phase (Stat) cultures and cells treated with submicrogram tobramycin (Tob, 0.06 μg / ml) were used as positive controls for stringent response activity. [Figure 8] AB: Uridine causes rapid killing and prevents the emergence of resistant mutants. A. Time-kill curves of E. coli WT in mid-exponential phase in liquid cultures supplemented with or without glucose, maltose, or 0.5% uridine. Lethal treatment with tobramycin (10 μg / ml) was administered, and survival after 1, 2, 3, 4, 6, and 20 hours was assessed by plating and counting CFU / ml. B. Photograph of a plate showing small and normal colonies 20 hours after tobramycin and maltose treatment. [Figure 9]AB: The uridine effect is PMF-dependent but not mediated by changes in PMF. A. Mitotracker Red uptake assessed by flow cytometry for E. coli ΔcmtA and Δcrp strains compared with the WT strain; E. coli harboring a cmtA overexpressing plasmid compared with a strain carrying an empty vector (p0); and E. coli WT grown in medium supplemented with glucose or 0.5% uridine. TCS (tetrachlorosalicylanilide) treatment was used as a negative control. B. Survival to 4 μg / ml tobramycin treatment in synthetic urine medium supplemented with or without 0.5% uridine, in the presence or absence of 15 μM of the protonophore carbonyl cyanide m-chlorophenylhydrazine (CCCP). Survival was assessed by plating and counting CFU 16 hours after treatment. [Figure 10] AB: Overexpression of the carbohydrate transporter MtlFGK in P. aeruginosa sensitizes it to tobramycin by increasing uptake. A. E-test of P. aeruginosa harboring an empty vector (p0+) compared to a vector overexpressing MtlFGK (pmtlFGK+). Inducer was added to the medium. B. Neocy5 uptake assessed by flow cytometry for P. aeruginosa harboring a plasmid overexpressing MtlFGK (MtlFGK+) compared to a strain with an empty vector (p0+), shown as the fold change in mean fluorescence per cell. [Figure 11]AD: Uridine enhances AGs in synthetic urine medium by promoting uptake. A. Survival of E. coli after 20 hours of low-dose tobramycin (0.5 μg / ml) treatment in response to the addition of different concentrations (expressed as %) of uridine. The significance above each point indicates the significance of the reduction in survival induced by the addition of the indicated percentage of uridine compared to the no-uridine condition (0%), except for the comparison between 0.0315% and 1%, as indicated. B. Liquid MIC tests performed on E. coli in synthetic urine medium using concentrations of AG ranging from 0 to 100 μg / ml with or without 0.5% uridine added to the medium by the drop-through method as described in the Methods section. C. Neocy5 uptake assessed by flow cytometry in E. coli grown in synthetic urine medium with or without supplementation of 0.5% uridine, shown as the fold change in mean fluorescence per cell compared to unsupplemented. D. Time-kill curves of E. coli in synthetic urine medium after addition of 4 μg / ml tobramycin, 4 μg / ml gentamicin, or 8 μg / ml amikacin to medium supplemented with or without 0.031% or 0.0009% uridine. Survival was assessed by plating and counting CFU / ml at 0, 1, 2, 4, 6, and 24 hours after treatment. Limit of detection: no colonies on pure culture plates. [Figure 12] Determination of the lowest effective uridine concentration. Survival of E. coli after 20 hours of treatment with 4 μg / ml tobramycin as a function of added uridine concentration (expressed as %). (MIC=10 μg / ml in synthetic urine). The significance above each point indicates the significance of the decrease in survival induced by the addition of that percentage of uridine compared to the no uridine condition (0%). One-way ANOVA was used to calculate statistical significance. *** means p<0.001, * means p<0.05. Number of replicates: n=3. [Figure 13]Effect of nucleosides on the bactericidal activity of aminoglycosides in synthetic urine. Survival of E. coli K12 strains 20 hours after treatment with tobramycin 0.5 μg / ml (MIC = 10), gentamicin 1 μg / ml (MIC = 10), amikacin 6 μg / ml (MIC = 50), or P. aeruginosa after treatment with tobramycin 10 μg / ml (MIC = 15), with or without 0.031% supplementation of uridine, cytidine, adenosine, thymidine, or inosine. Statistical significance was calculated using two-way ANOVA with Bonferroni correction for multiple hypothesis testing. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05, and ns means not significant. Number of replicates for each experiment: n=3. Number of replicates for each experiment: n=3. #: AG-resistant strain. MIC is against synthetic urine. [Figure 14] Uridine enhances tobramycin against E. coli K12 in human plasma-like medium. Survival of E. coli K12 after 20 hours of treatment with 1 μg / ml tobramycin with or without 0.031% uridine. The dotted line indicates the limit of detection (no colonies on pure culture plates). [Figure 15] Uridine enhances AGs in human blood, including AG-sensitive strains. Survival of E. coli CFT073 after 1 hour of treatment with 0.1 μg / ml gentamicin with or without 0.05% uridine supplementation in human blood. One-way ANOVA was used to calculate statistical significance. *** means p<0.001. Number of replicates: n=3. [Figure 16] Uridine enhances AGs in human blood, including AG (amikacin)-resistant strains. Survival of E. coli 932 in human blood 1 hour after treatment with 100 μg / ml amikacin, with or without 0.05% uridine supplementation. One-way ANOVA was used to calculate statistical significance. *** indicates p<0.001. Number of replicates: n=3. The right panel shows the same results for AMI and AMI+U as the left panel, but on a linear scale. [Figure 17]Uridine enhances the effect of AGs against clinical E. coli strains in synthetic urine medium. Survival of sensitive and resistant E. coli after 20 hours of treatment with tobramycin, supplemented with or without 0.031% uridine. Tobramycin concentrations were: 10 μg / ml for sensitive strains; 50, 200, or 400 μg / ml for resistant strains, and are marked with a "#". Two-way ANOVA was used to calculate statistical significance. **** means p<0.0001, ** means p<0.01, ns: not significant. Number of replicates for each experiment: n=3. Number of replicates for each experiment: n=3. #: AG-resistant strain. [Figure 18] Uridine enhances urinary AGs in a mouse bladder infection model. A. Uridine enhances UTI 89 strain in synthetic urine. B. In vivo. CFU / whole bladder homogenate in C57BI / 6 female mice infected intravesically with UTI 89 strain for 24 hours and then treated with PBS, gentamicin, or uridine and gentamicin for an additional 24 hours. One-way ANOVA was used to calculate statistical significance. ** denotes p<0.01. ns: not significant. Results are from two pooled experiments with n=5-7 per group. [Figure 19] Uridine induces rapid killing in synthetic medium (tryptone) and prevents the emergence of resistant mutants. Survival of E. coli WT growing in mid-logarithmic phase in liquid cultures supplemented with or without glucose, maltose, or 0.5% uridine. MIC=1 μg / ml in tryptone. Survival was assessed 1, 2, 3, 4, 6, and 20 hours after lethal treatment with tobramycin (10 μg / ml) by plating and counting CFU / ml. Geometric means and geometric standard deviations of three biological replicates are shown. Welch's t-test was used to calculate statistical significance. ** denotes p<0.01. Number of replicates: n=3-7 [Figure 20]Neo-Cy5 uptake assessed by flow cytometry in E. coli grown in synthetic urine medium supplemented with or without 0.5% uridine is shown as the fold change in mean fluorescence per cell without supplementation. Welch's parametric t-test was used to calculate statistical significance. * means p<0.05. ns: not significant. Number of replicates: n=3. [Figure 21] Reproduction of the pyrimidine-supplemented antibiotic lethality test disclosed in WO 2020 / 227530 and the corresponding scientific paper (Yang et al., Cell, 2019, 177, 1649-1661). The method by Yang et al., 2019, was reproduced as follows: E. coli K12 cells were grown overnight in triplicate in MOPS minimal medium (MM) (Teknova) supplemented with 0.2% glucose. The culture was diluted 500-fold with 10 ml of MOPS MM + 0.2% glucose in a 125 ml baffled flask. The culture was grown at 37°C with 300 rpm agitation until the OD600nm reached approximately 0.3, then rediluted to an OD600nm of 0.1 with MOPS MM + 0.2% glucose. For antibiotic treatment, 1 ml of culture was dispensed into 14 ml tubes and treated with the appropriate antibiotic (gentamicin 48 ng / mL) and the indicated biochemical supplement (1 mM). Samples were taken before treatment and after 1, 2, 3, and 4 hours of treatment, serially diluted in PBS, and plated for colony counts. Results are presented as CFU / ml before treatment and after 1, 2, 3, and 4 hours of treatment. In killing assays performed in the presence of glucose, no significant changes in the antibiotic lethality of gentamicin were observed for all pyrimidine compounds tested. DETAILED DESCRIPTION OF THE INVENTION

[0023] We investigated the uptake of aminoglycosides (AGs) via carbohydrate transporters, focusing specifically on E. coli. The sugar transporters responsible for AG uptake were identified, and it was shown that such uptake is not due to changes in the proton motive force. This provides evidence for a previously undescribed mechanism of AG uptake in Gram-negative bacteria. AGs are substrates for the excess carbohydrate transporters in Gram-negative pathogens, likely due to their osidic structure, allowing this molecule to hijack the bacterial sugar transport system. Uridine was identified as a substrate that can increase the number of these transporters under laboratory conditions in rich medium and synthetic human urine. Increasing AG transporters in vivo using select carbohydrate boosters could be a means to exploit this novel mechanism, enhancing AG therapy by reducing the effective dose and associated side effects.

[0024] In addition to uridine, other nucleosides, particularly cytidine, thymidine, and inosine, also enhance the efficacy of aminoglycosides (AGs). Aminoglycoside potentiation by nucleosides, such as uridine, is effective in various biological media, including synthetic urine, synthetic plasma medium, human blood, and in vivo, as demonstrated in mouse models of urinary tract infection. AG potentiation by nucleosides, such as uridine, is effective against a variety of pathogens, including the major pathogens Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Furthermore, potentiation of AGs by nucleosides, such as uridine, is effective against clinical strains resistant to AGs. Additionally, the combination of AGs with nucleosides, such as uridine, induces rapid killing and prevents the selection of resistant mutants.

[0025] The use of metabolites to resensitize patients to aminoglycosides is a strategy already employed in other studies. 32,33 The role of the addition of compounds, such as mannitol, in stimulating PMF and thereby allowing aminoglycosides to enter surviving bacterial cells. 33In this study, sensitive cells were studied, and the addition of uridine did not alter the PMF, suggesting that increased aminoglycoside entry depends on the presence of a proton gradient but uses an alternative pathway. Furthermore, the addition of mannitol has little or no effect on the MIC under the conditions used. Also, the combination of aminoglycosides with nucleosides, such as uridine, induces rapid killing and prevents the emergence of resistant mutants. On the other hand, maltose-treated bacteria show regrowth due to the selection of AG-resistant mutants.

[0026] This research allows the use of many molecules to potentiate aminoglycosides, with two distinct axes of improvement: (1) maintaining the same therapeutic effect with a lower dose of aminoglycoside, thereby lowering the toxicity threshold and reducing the amount of antibiotic ingested and therefore rejected; (2) enabling rapid eradication of cells (shorter treatment, limiting the emergence of resistance); and (3) resensitization of aminoglycoside-resistant bacteria. Although this is an "old" class of antibiotic, it is undergoing reevaluation. Indeed, the WHO's predictions regarding the emergence of MDR bacteria indicate that the next few years will be critical, making each functional molecule crucial. The screening system employed to identify uridine used the cmtA gene, which showed increased induction in the presence of uridine. While the use of biolog plates is a first step in the search for activated substrates, larger-scale compound screening could identify other substrates that could be used in therapy, and even combine them. Carbohydrate administration in humans has been shown to be effective in combination with other potentiators, such as the primary alcohol n-butanol. 34 ) suggesting lower toxicity compared to

[0027] Studies on MICs depending on the carbon source in MH medium (traditionally used for MIC determination in diagnostics) were performed, followed by 1% bactotryptone and 0.5% NaCl to avoid the presence of complex glucose (starch) in MH. When searching for active compounds, the MIC values ​​induced by the addition of the compounds were compared with those induced by the addition of glucose (corresponding to transporter inhibition). In fact, the addition of uridine did not induce any difference in MIC in MH medium (the MIC of tobramycin is equal to 0.1 μg / ml with or without the addition of 0.5% uridine) or in 0.2% glucose-supplemented MOPS medium. Due to the inhibition of AG transporters in glucose-containing culture media, as demonstrated for the first time in this invention, the potentiation of gentamicin with uridine could not be observed in the cited prior art (WO 2020 / 227530 and Yang et al., Cell, 2019, 177, 1649-1661). The unexpected effect of uridine on the AG transporter could not be revealed by referring to the cited prior art that discloses the potentiation of antibiotics by stimulating purine biosynthesis using pyrimidine compounds (pyrimidine bases or nucleosides). Therefore, it is possible that the composition of the culture medium induces different responses that lead to regulatory pathways that can counteract the effects of the tested molecules. 35 It is possible that the cells elicit a response, or simply fail to elicit a response, which reflects the relevance of the culture medium used during screening of the molecules. The use of synthetic urine medium allowed for the observation of differences in conditions with or without uridine without the need for comparison with glucose. Results were also validated in synthetic human plasma-like medium, human blood, and in vivo (a mouse urinary tract infection (UTI) model). Growth in human urine shows a large variability, and recent studies have suggested the use of synthetic medium to standardize results between laboratories. 36 The medium used here may provide a basis for future studies.

[0028] According to the present invention, nucleosides are used to enhance bacterial killing by aminoglycoside antibiotics. Thus, according to the present invention, a combination of an aminoglycoside antibiotic and a nucleoside is used to treat bacterial infections. Thus, the combination of the present invention, which is a combination of (only) two compounds, can be defined as a combination consisting of an aminoglycoside antibiotic and a nucleoside.

[0029] How to kill bacteria The present invention includes a method for killing bacteria. In one embodiment, the method includes administering a nucleoside with an aminoglycoside antibiotic. Preferably, the nucleoside enhances the killing of bacteria by the antibiotic. More preferably, the nucleoside limits the development of antibiotic-resistant bacteria. More preferably, the nucleoside resensitizes antibiotic-resistant bacteria to killing by the antibiotic.

[0030] The efficacy of nucleosides in antibiotic killing of bacteria can be assessed by various testing methods known in the art and disclosed in the Examples herein, such as bactericidal tests or MIC determinations, among others. The efficacy of nucleosides in antibiotic killing of bacteria can be: (i) increased killing of antibiotic-susceptible bacteria, (ii) reduced development (or selection) of antibiotic-resistant bacteria, and / or (iii) killing of antibiotic-resistant bacteria, as disclosed in the Examples herein.

[0031] According to the present invention, antibiotic-resistant bacteria include bacteria that are resistant to aminoglycoside antibiotics and bacteria that are genetically resistant to aminoglycoside antibiotics.

[0032] In one embodiment, the method is for the prevention of the development of antibiotic-resistant bacteria by more rapid bacterial killing involving co-administration of a nucleoside with an aminoglycoside antibiotic, hi a preferred embodiment, the method is for the prevention of bacteria that are genetically resistant to antibiotics.

[0033] In some embodiments, the method is for killing antibiotic-resistant bacteria. Co-administration of a nucleoside with an aminoglycoside antibiotic can kill antibiotic-resistant bacteria, whereas the same concentration of the aminoglycoside antibiotic alone is ineffective. In a preferred embodiment, the method is for killing bacteria that are genetically resistant to antibiotics.

[0034] In one embodiment, the co-administration of a nucleoside with an aminoglycoside antibiotic, with the aminoglycoside administered at a lower concentration than the reference treatment, is to reduce the toxicity of the reference treatment.

[0035] Preferably, administration of the nucleoside allows for a 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold reduction in the amount of aminoglycoside antibiotic to achieve the same degree of kill as without the nucleoside.

[0036] Preferably, the nucleoside and aminoglycoside antibiotic are administered simultaneously, hi some embodiments, the nucleoside is administered 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, or 24 hours before or after administration of the aminoglycoside antibiotic.

[0037] Nucleosides Preferably, the nucleoside is uridine. In various embodiments, the nucleoside is selected from uridine, inosine, guanosine, cytidine, adenosine, thymidine, or xanthosine. Preferably, the nucleoside is not adenosine. Preferably, the nucleoside is selected from uridine, inosine, guanosine, cytidine, thymidine, or xanthosine.

[0038] In some embodiments, the nucleoside is 0.5 to 20 g / m 2 In various embodiments, the dose of uridine is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 g / m 2In various embodiments, the dose of uridine is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 g / m 2 The following is the result.

[0039] antibiotics Preferably, the aminoglycoside antibiotic is selected from paromomycin, amikacin, gentamicin, streptomycin, neomycin, tobramycin, plazomycin, and kanamycin. Most preferably, the antibiotic is selected from tobramycin, gentamicin, and amikacin.

[0040] In various embodiments, the aminoglycoside antibiotic is administered at a dose of 0.3-30 mg / kg body weight. In some embodiments, the aminoglycoside antibiotic is administered at a dose of 1.0-7.0 mg / kg, 1.0-5.0 mg / kg, or 1-2.5 mg / kg body weight. Preferably, the aminoglycoside antibiotic is administered at a dose of less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, or 3 mg / kg body weight when used in combination with a nucleoside, preferably to achieve the same degree of kill as higher doses.

[0041] In some embodiments, the aminoglycoside antibiotic is administered every 8, 12, 24, or 36 hours. In some embodiments, the aminoglycoside antibiotic is administered as a single monodose. In various embodiments, the aminoglycoside antibiotic is administered at a dose of 1-2.5 mg / kg / dose every 8 hours or every 12 hours. In various embodiments, the aminoglycoside antibiotic is administered at a dose of 7 mg / kg every 24 hours or every 36 hours. In some embodiments, the dose of the aminoglycoside antibiotic, when used in combination with a nucleoside, is reduced to less than 50%, 40%, 30%, or 20% of these doses, preferably to achieve the same degree of kill as a higher dose.

[0042] Preferred combinations include tobramycin and uridine; gentamicin and uridine; amikacin and uridine; tobramycin and thymidine; gentamicin and thymidine; amikacin and thymidine; tobramycin and cytidine; gentamicin and cytidine; amikacin and cytidine; tobramycin and inosine; gentamicin and inosine; amikacin and inosine.

[0043] In some embodiments, when the nucleoside is uridine, the antibiotic is different from gentamicin.

[0044] More preferred combinations include tobramycin and uridine; amikacin and uridine; tobramycin and thymidine; gentamicin and thymidine; amikacin and thymidine; tobramycin and cytidine; gentamicin and cytidine; amikacin and cytidine; tobramycin and inosine; gentamicin and inosine; amikacin and inosine.

[0045] bacteria Preferably, the bacteria killed are gram-negative bacilli (aerobic or anaerobic), Staphylococci, or Mycobacterium tuberculosis.

[0046] In various embodiments, the bacterium belongs to the Enterobacteriaceae family and includes Escherichia coli, Klebsiella pneumoniae and K. oxytoca, Enterobacter cloacae and E. aerogenes, Providencia spp., Proteus spp., Morganella spp., and Serratia spp. In various embodiments, the bacterium is Yersinia pestis or Francisella tularensis. In various embodiments, the bacterium is Staphylococcus aureus, P. aeruginosa, or Acinetobacter baumannii, including methicillin-resistant and vancomycin-intermediate-resistant and resistant isolates. In various embodiments, the bacterium is Mycobacterium spp., including Mycobacterium tuberculosis, M. fortuitum, M. chelonae, and M. avium. In various embodiments, the bacterium is Streptococcus spp., including S. pneumoniae, S. pyogenes, S. gallolyticus, S. saprophyticus, and S. agalactiae. In various embodiments, the bacterium is Vibrio cholerae.

[0047] Preferably, the bacterium is selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.

[0048] In some embodiments, the bacteria disclosed herein comprise or consist of bacteria that are resistant to aminoglycoside antibiotics, which may be resistant or genetically resistant to the antibiotic, and preferably, the resistant bacteria comprise genetically resistant bacteria.

[0049] In some preferred embodiments, a combination selected from tobramycin and uridine, amikacin and uridine, tobramycin and thymidine, gentamicin and uridine, gentamicin and thymidine, amikacin and thymidine, tobramycin and cytidine, gentamicin and cytidine, or amikacin and cytidine is used to kill E. coli, for example, in blood infections or urinary tract infections; preferably, the combination is selected from tobramycin and uridine, amikacin and uridine, tobramycin and thymidine, gentamicin and thymidine, amikacin and thymidine, tobramycin and cytidine, gentamicin and cytidine, or amikacin and cytidine. In some preferred embodiments, a combination selected from tobramycin and uridine, tobramycin and adenosine, tobramycin and thymidine, gentamicin and uridine, or tobramycin and inosine is used to kill P. aeruginosa, for example, in pulmonary infections; preferably, the combination is selected from tobramycin and uridine, tobramycin and adenosine, tobramycin and thymidine, or tobramycin and inosine.

[0050] Administration In various embodiments, the nucleoside and / or antibiotic is administered systemically or to the urinary tract, gastrointestinal tract, lungs, eyes, ears, nose, brain, heart, blood, or skin. Preferably, the nucleoside and / or antibiotic is administered systemically or to the lungs, eyes, ears, nose, or skin.

[0051] In various embodiments, administration is via a parenteral route, such as subcutaneous (sc), intradermal (id), intramuscular (im), intraperitoneal (ip) or intravenous (iv) injection, etc. In various embodiments, administration is via a route such as inhalation, oral, topical, ocular, rectal or vaginal.

[0052] In various embodiments, the nucleoside and / or antibiotic are administered in one or more doses.

[0053] In various embodiments, the level of glucose in the bacterial environment is minimized. Preferably, the patient does not have hyperglycemia or renal glycosuria.

[0054] The amount to be administered (single dose) depends on the subject of treatment, including the patient's health condition, the state of the individual's immune system, the route of administration, and the size of the host, etc. The range of an appropriate single dose can be determined depending on the circumstances and can be modified by those skilled in the art.

[0055] The present invention encompasses different therapeutic applications. First, its application to the treatment of urinary tract infections. In a synthetic urine medium, the concentration of uridine that induced the most significant enhancing effect was shown to be 0.031% or 1.27 mM. Ingestion of a purine-rich beverage increased urinary uridine excretion from 0.21 μM to 0.23 μM, a concentration shown to be effective (0.031%). 37 Therefore, the mere ingestion of a purine-enriched product was not sufficient to achieve sufficient concentrations of uridine in the bladder to see the enhancing effect of aminoglycosides.

[0056] In animal models, intravenous administration of 0.5 g / kg in rats produces a peak of 13 mM uridine in plasma but induces a fall in blood pressure within 1 minute of injection. 38 In rabbits, 7.7% of intravenously injected uridine is recovered in the urine, compared with only 1% when given orally. The plasma peak is approximately 25 μg / ml or 100 mM at 0.1 g / kg. 39 These values ​​show large variability in animal models and must be taken into consideration in preclinical studies.

[0057] In humans, urinary excretion appears to be more favorable, especially in humans. Uridine can be administered to counteract the toxicity of fluorouracil, a chemotherapy drug, and studies on the PK / PD and toxicity of the molecule have been carried out. Initial studies have shown that uridine is administered at doses of 1-12 g / m 2 It was administered as an intravenous infusion in doses ranging from 1.5 m to 1.5 m (the average human body surface area is 1.5 m).2 In this way, after 1 hour of introduction, the dose is 8-12 g / m 2 In this study, plasma uridine concentrations increased from 1-8 μM to a peak of 2 mM. It was also shown that 24% of uridine was excreted in the urine within 24 hours of treatment. Regarding toxicity, one of two patients receiving the highest dose of uridine experienced 15 minutes of shivering 1 hour after the end of the infusion, but without fever or neurological impairment. A second study involving continuous or intermittent exposure to uridine also provided interesting data: continuous exposure caused fever, but this did not occur when the dose was administered at 3-hour intervals. Uridine was rapidly excreted from plasma from mM to μM concentrations during treatment. Urinary excretion was estimated to be 15-40%.

[0058] Based on a low urinary excretion of 15% uridine, 12 g of uridine (8 g / m 2 ) infused over a one-hour period, it can be estimated that 1.8 g will be excreted in the urine. Assuming a large bladder volume of 2 liters, 0.9 g / L or 0.09% uridine can potentially be detected in the urine. It can be estimated that a dose of 0.031% will produce a strong enhancing effect. These estimates are based on two highly informative studies involving approximately 20 patients.

[0059] Oral administration of uridine has already been used clinically. This dosage form has a single dose limit (8-12 g / m 2 ) has been attributed to the occurrence of diarrhea. After oral ingestion, the plasma uridine dose can reach values ​​of 60-80 μM, which is 10 times lower than after intravenous administration and is associated with very low urinary excretion (1%). 40 Uridine triacetate granules are already commercially available under the name Vistogard (https: / / www.vistogard.com / Professional / Data / Pre-Clinical). Ingestion of the uridine-based drug PN401 produces a peak of 200 μM uridine in plasma at a dose of 9.9 g. 41However, there is no information on urinary excretion. Therefore, the best way to achieve increased concentrations may be intravenous injection, similar to antibiotics. If the infusion is well tolerated, a "switch" of treatments can be performed, starting with a uridine infusion followed by an aminoglycoside injection. Preliminary results suggest that the lowest dose of uridine tested (0.0009% or 0.036 mM) is effective, although the inoculum is less than 0.031% when more significant. This could be due to consumption of uridine added to the medium in limited amounts. Therefore, daily intake of uridine may be sufficient to observe an enhancing effect.

[0060] Uridine can also be used in the treatment of pyelonephritis (defined by accumulation in the kidney), endocarditis, and in the context of the topical treatment of eye, ear, and skin infections, where uridine concentration is not limited because it can be administered locally. Recently, inhaled tobramycin therapy has been developed for the treatment of cystic fibrosis patients with S. aureus or P. aeruginosa infections. 42 Mannitol 43 or uridine triphosphate 44 Similarly, uridine can be administered by inhalation to enhance tobramycin against P. aeruginosa in the setting of pulmonary infection. In fact, underdosing of aminoglycosides in nebulized therapy has been the cause of such treatment failure. Finally, uridine can also be used in cases of blood infection, as it has been proposed to administer a single dose of aminoglycoside before additional treatment with other antibiotics in patients hospitalized with sepsis.

[0061] Compositions and Uses Thereof The present invention encompasses compositions for killing bacteria, comprising a nucleoside and an aminoglycoside antibiotic, wherein the nucleoside potentiates the killing of bacteria by an antibiotic as disclosed herein. Preferably, the nucleoside inhibits the development of antibiotic-resistant bacteria. Preferably, the nucleoside resensitizes antibiotic-resistant bacteria to killing by the antibiotic.

[0062] In various embodiments, the nucleoside is selected from uridine, inosine, guanosine, cytidine, adenosine, thymidine, or xanthosine. In some embodiments, the nucleoside is selected from uridine, inosine, guanosine, cytidine, thymidine, or xanthosine. Preferably, the nucleoside is uridine.

[0063] Preferably, the aminoglycoside antibiotic is selected from paromomycin, amikacin, gentamicin, streptomycin, neomycin, tobramycin, plazomycin, apramycin, and kanamycin. Most preferably, the antibiotic is selected from tobramycin, gentamicin, and amikacin.

[0064] In various embodiments, the nucleoside is not adenosine and / or the aminoglycoside antibiotic is not gentamicin.

[0065] The present invention encompasses the use of nucleosides to enhance bacterial killing by aminoglycoside antibiotics.

[0066] In various embodiments, the nucleoside is selected from uridine, inosine, guanosine, cytidine, adenosine, thymidine, or xanthosine. In some embodiments, the nucleoside is selected from uridine, inosine, guanosine, cytidine, thymidine, or xanthosine. Preferably, the nucleoside is uridine.

[0067] Preferably, the aminoglycoside antibiotic is selected from paromomycin, amikacin, gentamicin, streptomycin, neomycin, tobramycin, plazomycin, apramycin, and kanamycin. Most preferably, the antibiotic is selected from tobramycin, gentamicin, and amikacin.

[0068] In various embodiments, the nucleoside is not adenosine and / or the aminoglycoside antibiotic is not gentamicin.

[0069] In various embodiments, administration is performed without co-administration of glucose, and thus, in some embodiments, glucose concentrations are minimal. [Example]

[0070] 1. Materials and Methods 1.1 Strains, plasmids and primers The strains, plasmids, and primers used in this study are listed in Table 1 .

[0071] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0072] 1.2 Growth conditions MH medium was used to determine the MICs of deletion or overexpression strains. MOPS Rich (Teknova EZ rich defined medium) was used for screening assays to avoid the inherent fluorescence of other media and for the neocy5 assay. For substrate-specific response studies, substrates were added to a medium containing 1% bactotryptone and 0.5% NaCl. To mimic urinary tract infection, a synthetic urine medium was used. 45 The five species were prepared according to the method described in the previous section. To mimic human plasma, we used HPLM medium (Human Plasma-Like Medium, Thermofischer Scientific), which is formulated to resemble the natural cellular environment found in the body and mimics the metabolic profile of human plasma. The five species were cultured at 37°C with shaking (100-150 rpm).

[0073] 1.3 Gene deletion All E. coli strains used in this study were derivatives of E. coli MG1655 and were constructed by transduction using the Keio knockout strain. The kanamycin resistance cassette aph was inserted using the FLP / FRT system. 46 was removed using

[0074] 1.4 Transporter overexpression Overexpression was achieved using 1 mM sodium benzoate as an inducer under the Pm promoter-dependent conditions in the pSEVA-238 vector. 47 This was carried out by cloning the target gene into 48 The primers and restriction enzyme cleavage sites used for cloning are shown in Table 1.

[0075] 1.5 MIC evaluation Etests: Stationary-phase cultures of MH, except for the Δcrp strain, were diluted 20-fold with PBS and 300 μl was plated onto the appropriate medium: MH medium for gene deletion strains; MH medium supplemented with kanamycin and sodium benzoate for plasmid maintenance and induction in overexpression strains; 1% amino acids, 0.5% NaCl, and 0.5% substrate (e.g., glucose, ribose, uridine, etc.) for assessing the influence of carbon source. Plates were allowed to dry for 10 minutes. Etests (Biomerieux) were placed on the plates and incubated overnight at 37°C.

[0076] Liquid culture: MIC is an initial inoculum of 5.10 5 CFU / ml were determined by microtiter broth dilution. For MH medium, the MIC was interpreted as the lowest antibiotic concentration that inhibited visible growth. For synthetic urine medium, because interpretation of macroscopic growth was difficult, 5 μl of pure culture of each antibiotic dilution was plated, and the MIC was interpreted as the lowest concentration that inhibited growth.

[0077] 1.6 RNA-seq E. coli cultures were diluted 1000-fold and grown in triplicate in MH medium supplemented with or without 0.1 μg / ml tobramycin, reaching an OD of 0.4, corresponding to 25% of the MIC in liquid culture. 600nm was cultured until

[0078] Cultures of E. coli, P. aeruginosa, K. pneumoniae, and A. baummannii were diluted 1000-fold and measured in triplicate at OD in MH medium supplemented or not with 0.5% uridine. 600nm The culture was continued until the β-amyloid ratio reached 0.4.

[0079] All RNA was purified using the RNAeasy mini kit (Qiagen) according to the manufacturer's instructions. Briefly, 4 ml of RNA-protect reagent (Qiagen) was added to 2 ml of bacterial culture for 5 min. After centrifugation, the precipitate was stored at -80°C until extraction. Protocol 2 of the RNAprotect Bacteria Reagent Handbook was followed, with an additional proteinase K digestion step as described in Protocol 4. RNA quality was controlled using a Bioanalyzer.

[0080] Count data were analyzed using R and the Bioconductor package DESeq2. Data were normalized in DESeq2. Raw p-values ​​were adjusted for multiple comparisons according to the Benjamini-Hochberg method, and genes with adjusted p-values ​​<0.005 were considered differentially expressed.

[0081] 1.7 Neocy5 uptake assay Quantification of fluorescent neomycin (Neo-cy5) uptake 49 Neo-cy5 is an aminoglycoside (neomycin) linked to the fluorophore Cy5, which maintains the activity and mode of uptake in Gram-negative bacteria. 50The overnight culture was diluted 100-fold in rich MOPS (Teknova EZ rich defined medium). The OD of the bacterial culture was 600 When the β-actin concentration reached 0.25, the cells were treated with 0.4 μM Cy5-labeled neomycin under aluminum foil at 37°C for 15 minutes. For assays using different substrates, the cultures were washed once with PBS before treatment. 20 μl of each treated culture was used for flow cytometry and diluted with 200 μl of PBS before reading the fluorescence. Flow cytometry experiments were performed using the following methods: 51 Each experiment was performed as described in. 50,000 events were counted on a Miltenyi MACSquant instrument.

[0082] 1.8 PMF Evaluation PMF quantification was performed using Mitotracker Red CMXRos stain (Invitrogen). 52 A parallel assay was performed using the same bacterial cultures as the neocy5 uptake assay. 50 μl of each culture was mixed with 60 μl of PBS. The protonophore tetrachlorosalicylanilide (TCS) (Thermofischer) was used as a negative control and treated at 500 μM for 10 minutes at room temperature. 25 nM Mitotracker Red was then added to each sample and the sample was left under aluminum foil for 15 minutes at room temperature. 20 μl of the treated culture was used for flow cytometry and diluted with 200 μl of PBS before fluorescence reading. Flow cytometry was performed as described above.

[0083] 1.9 Carbon Source Screening Assay GFPmut3 as the promoter of interest 53The plasmid was fused to and cloned into pSC101. For the initial screening, overnight cultures of strains carrying the screening system were diluted 200-fold into MOPS Rich (Teknova EZ Rich Defined Medium) supplemented with carbenicillin for plasmid maintenance. For substrate screening, phenotype Microarray (Biolog) plates PM1, PM2B, and PM3B were used. Each well was filled with 100 μl of inoculated medium and mixed by pipetting. The medium was transferred to a 96-well dark-bottom plate (Thermo Scientific). GFP fluorescence was measured at 37°C for 8 hours using a Tecan Infinite 200 PRO (Life Science). Fluorescence induction by the substrate was measured as a function of growth (t8h-t0h OD). 600nm The fluorescence intensity was calculated as the ratio of fluorescence (t8h-t0h) to fluorescence intensity (t0h).

[0084] For flow cytometry quantification, overnight cultures of strains carrying the screening system were diluted 200-fold into rich MOPS (Teknova EZ rich defined medium) supplemented with carbenicillin for plasmid maintenance and subjected to substrate testing at 0.5% after overnight incubation. Fluorescence was read on 5 μl of the culture diluted to 200 μl with PBS.

[0085] 1.10 Austerity Response The P1rrnB-gfp fusion was constructed using the gfp ASV and cloned into the plasmid pSC101. Cultures were grown overnight (positive control) or in MH medium supplemented with carbenicillin and 0.5% of the above substrates or 0.1 μg / ml tobramycin for plasmid maintenance to reach an OD. 600nm The cultures were diluted 100-fold to a fluorescence intensity of 0.4. Fluorescence was read by flow cytometry on 20 μl of the culture diluted to 200 μl with PBS.

[0086] 1.11 Killing test Bactotryptone medium: Overnight cultures were diluted 1:1000 in 25 ml of medium containing 1% bactotryptone and 0.5% NaCl, supplemented with or without 0.5% glucose, maltose, or uridine. Cultures were grown to an OD of 0.3–0.4. 600nm Cultures were grown to 0°C, and 5 ml aliquots were treated with a lethal concentration of tobramycin (10 μg / ml). Cultures were plated at 0 h (t0), 1, 2, 4, 6, and 20 h after treatment, and survival was calculated by counting the number of CFU / ml after treatment divided by the initial number of CFU / ml (t0).

[0087] Synthetic urine medium: An overnight culture grown in MH medium was diluted 10-fold with PBS. Approximately 5.10 6 or 5.10 7 CFU / ml were diluted in 200 μl of urine-like medium with or without uridine, cytidine, thymidine, or inosine, treated with AGs, and incubated in 96-well plates with shaking at 100–120 rpm at 37°C. Cultures were plated at 0 h (t0), 1, 2, 4, 6, and 24 h or 20 h after treatment, and survival was calculated by counting the post-treatment CFU / ml divided by the initial number of CFU / ml (t0).

[0088] 1.12 Dose-Response Studies Two-fold dilutions of uridine in synthetic urine medium were prepared in columns of a 96-well plate, ranging from 2% to 0.0018%. The last column was filled with synthetic urine medium alone, without uridine. Next, 100 μl of a solution containing a two-fold concentration of the antibiotic to be tested was added, resulting in a two-fold dilution of uridine and antibiotic, bringing the total volume per well to 200 μl. The last column was filled with synthetic urine medium alone, without antibiotic. Each well was filled with approximately 5.10 μl of uridine from a stationary-phase MH culture. 6 CFU were inoculated and incubated for 20 hours with agitation (100 rpm) at 37° C. CFU were counted by plating onto MH medium before and after treatment.

[0089] 1.13 Whole-genome sequencing gDNA was extracted from 500 μl of an overnight culture in MH using a Blood and Tissue Extraction Kit (Qiagen) according to the manufacturer's instructions. The presence of variants (single nucleotide polymorphisms) was analyzed using SnpEff 5.0. 54 was analyzed.

[0090] 1.14 Urinary tract infection model Urinary tract infection was induced in 6-7 week-old female C57BL / 6J mice from Charles River, France, as previously reported. Briefly, the human UPEC cystitis isolate UTI89 (UPEC-RFP), engineered to express the fluorescent protein RFP and antibiotic resistance to kanamycin, was statically cultured in Luria-Bertani (LB) broth in the presence of kanamycin (50 μg / ml) at 37°C for 18 hours. The culture was diluted to 2 × 10 in PBS. 8 Adjust to CFU / mL and add 50 μL (10 7 The virus (CFU / mouse) was administered directly via catheter into the bladder of mice anesthetized with an intraperitoneal injection of 100 mg / kg ketamine and 5 mg / kg xylazine. At 24 h postinfection, mice were treated with 100 μL of PBS, 0.2 mg / kg gentamicin, or 0.2 mg / kg gentamicin plus 0.5 g / kg uridine via retroorbital intravenous injection. At 48 h postinfection (24 h postinjection), mice were sacrificed by isoflurane inhalation followed by cervical dislocation. To count CFU, bladders were aseptically removed and homogenized in 1 mL of PBS. Serial dilutions were plated on LB agar plates containing kanamycin. All animals used in this study had free access to standard laboratory chow and water at all times. Infection experiments were carried out at the Institut Cochin under approval number APAFIS #34290 of the SC3-CEEA34-Université de Paris in application of the European Directive 2010 / 63 EU.

[0091] 1.15 Human bloodborne infections This study was conducted by QIMA Life Science (1 bis rue des plantes 86160 GENCAY - France). Blood was collected from a 39-year-old male donor. E. coli CFT073 (ATCC® 700928™) or strain 932 was cultured overnight in MH and then diluted with PBS to an OD 600nm of 1. Blood (500 μl) was inoculated with 2.10 4 CFU / ml (double the amount), treated with uridine (0.05%), gentamicin (0.1 μg / ml) for the sensitive strain CFT073, or amikacin (100 μg / ml) for the resistant strain 932, or a combination of uridine and antibiotics (500 μl mixture; prepared in blood, double the amount). Blood with or without compounds and / or E. coli bacteria was incubated at 37 °C for 1 hour under stirring (150 rpm). Bacterial counts were performed on the blood treated under each condition before (t0) and after incubation. For each condition, the treated or untreated blood was plated on two MH agar plates (100 μl and the sediment of the remaining blood (900 μl)).

[0092] 1.16 Statistical analysis An F-test was performed to determine whether the variances between conditions were equal or different. For conditions with equal variances, Student's t-test was used. For conditions with significantly different variances, Welch's correction was applied. One-way ANOVA or two-way ANOVA was used for multiple comparisons. Statistical differences between groups were determined using GraphPad Prism. **** means p < 0.0001, *** means p < 0.001, ** means p < 0.01, and * means p < 0.05. The number of repetitions for each experiment was 3 < n < 7. The mean and standard deviation of the growth curves and survival rates, and the mean and geometric mean of the logarithmic values were calculated using GraphPad Prism.

[0093] 2. Results 2.1 cmtA deletion reduces sensitivity to AGs Deletion mutants of E. coli carbohydrate transporter genes were constructed, and the effect of a single deletion on tobramycin susceptibility was tested. In the case of multicomponent systems, membrane-resident proteins were deleted. The response to tobramycin was evaluated for 30 tested mutants (Table 3) using serial dilution and Etests (Table 3). One of these deletions, CmtA, exhibited a phenotype of reduced susceptibility to tobramycin: the deletion strain exhibited a 4-fold increase in the minimum inhibitory concentration (MIC) compared to the wild-type (WT) strain (Fig. 1A), and also exhibited reduced susceptibility in the presence of 4x the MIC of tobramycin (Fig. 1B). CmtA is related to MtlA. 55 It has 52% similarity to and is annotated as a mannitol E11C PTS cryptic enzyme. CmtA is capable of complementing mannitol transport in the ΔmtIA strain only when expressed under a heterologous promoter. 56 Increases in the MIC of ΔcmtA with other AGs (kanamycin, gentamicin) were also observed, but not with antibiotics from other families, such as trimethoprim, ciprofloxacin, amoxicillin, or chloramphenicol (Table 1). The susceptibility of ΔcmtA was unchanged to spectinomycin (Table 1). Here, spectinomycin is an aminocyclotitol antibiotic with a similar chemical structure to AGs but lacking an amino sugar or glycosidic bond. The reduced susceptibility of ΔcmtA was therefore specific to AGs. Other transporter deletion mutants either had little effect on serial dilution tests or showed a slight increase in the MIC of tobramycin (Table 3), consistent with specificity for AGs.

[0094] Crp, a key regulator of carbon metabolic repression and a transcriptional activator of nonselective sugar transporters 57 Deletion of α-glucan has previously been associated with streptomycin resistance. 58 Despite a significant growth defect, the Δcrp strain exhibited a 10-fold higher MIC than the WT strain (Fig. 1A) and was insensitive to 0.4 μg / ml tobramycin treatment (Fig. 1B). The effect of each transport system was tested by overexpressing them in trans.

[0095] 2.2 Overexpression of various carbohydrate transporters increases susceptibility to AGs First, we cloned the cmtAB PTS transporter gene into a plasmid under the control of an inducible promoter and evaluated its response to tobramycin. Deletion of this transporter increased the concentration of AGs required for bacterial killing (increased the MIC), whereas its overexpression increased susceptibility to tobramycin and gentamicin but not to spectinomycin (Fig. 1C and Table 2). This response was specific to AGs, as there was no effect on ciprofloxacin, chloramphenicol, trimethoprim, or amoxicillin, which were used as negative controls (Table 2).

[0096] Next, the 23 identified carbohydrate transporters were cloned into inducible plasmids. Survival by serial dilution was first determined in media containing tobramycin, gentamicin, chloramphenicol, and carbenicillin. MICs were then assessed for strains with reduced resistance to AGs (Table 3). Overexpression of 16 carbohydrate transporters increased susceptibility to tobramycin compared with the empty vector (Figure 2A and Table 3). Growth of frwBC+ strains was impaired more strongly in media containing serial dilutions of tobramycin than in the empty plasmid control (Figure 2A), but they did not exhibit MICs to tobramycin lower than p0. Overexpression of the lamB porin in the outer membrane showed no response to AGs, nor did chiP (titin porin), ptsG (glucose PTS), xylEFG (xylose Major Facilitator Superfamily (MFS) protein), galP (galactose MFS protein), frvAB (fructose-like PTS), and mtlA (mannitol PTS) (Fig. 2A ). These increased sensitivity profiles were AG-specific for 11 transporters: cmtAB, chbCB (chitobiose PTS), srlEAB (glucitol PTS), ascF (cellobiose / arbutin PTS), malEFG (maltose ABC transporter), fruBKA (fructose PTS), frwBC (fructose-like PTS), mngA (fructose-like PTS), ypdGH (fructose-like PTS), bglF (β-glucoside PTS), and malX (maltose PTS) (Figure 2C). Strains overexpressing manXYZ (mannose PTS), treB (trehalose PTS), bglH (beta-glucoside porin), gatABC (galactochol PTS), and glpTQ (glycerol-3-phosphate permease already known to promote fluoroquinolone 59 uptake) showed a susceptibility phenotype to AGs, as well as to ciprofloxacin or carbenicillin (Figure 2B).

[0097] [Table 2]

[0098] 2.3 Carbohydrate transporters facilitate the uptake of AGs. To test whether the effect of carbohydrate transporter expression was related to differential uptake, we monitored the cellular uptake of Neo-cy5, a fluorescent AG synthesized for uptake studies in bacteria, as previously performed. 49 , with AG characteristics in terms of uptake, mechanism of action, and activity against Gram-negative bacteria 50 We found that the cmtA deletion reduced the amount of intracellular fluorescence after treatment, indicating reduced uptake of AG (Figure 3). The Δcrp mutant showed a significant decrease in fluorescence, consistent with the increased MIC value (Figures 1 and 3A). As a corollary, we next tested whether overexpression of CmtAB caused increased entry of Neo-cy5. No difference was observed between the WT strain carrying p0 and the WT strain carrying the pcmtAB overexpression vector during the 15-minute treatment (not shown). This may be due to the membrane already being saturated with CmtA during this observation period. Therefore, we tested the ΔcmtA strain carrying an empty plasmid or a strain overexpressing CmtA, and confirmed that uptake of Neo-cy5 was increased in the presence of CmtA (Figure 3).

[0099] Taken together, these observations suggest that redundant transporters are involved in the uptake of AGs and that increasing the expression of these transporters may enhance therapeutic AGs.

[0100] 2.4 CmtA as a screening tool for activators of AG transporters The transcriptome of growing E. coli was analyzed in the presence or absence of tobramycin. Transcriptome analysis under sub-MIC (25% of the MIC) tobramycin treatment in MH medium did not show induction of carbohydrate transporters. Some transporters, such as mglA (galactose permease), PTS malX, manXY, nagE, or rbsC (ribose permease), were even repressed by the addition of tobramycin (Table 4).

[0101] To define the conditions favoring AG uptake by the sugar transporter, a screening system was constructed based on a plasmid-delivered fusion between the promoter of the cmtAB operon and gfp. In a crp-deficient mutant, fluorescence was abolished, confirming that Crp is a positive regulator in this transport system (Fig. 4). A cmtA-deficient strain showed a two-fold increase in fluorescence, suggesting a potential positive feedback control (Fig. 4). Among the regulators associated with sugar utilization, the Cra protein has also been reported to be a major player: it regulates genes that undergo catabolite activation, such as fruBKA and mtlADR. 60 Deletion of cra also increased fluorescence by two-fold. As a corollary, the addition of glucose causes catabolic repression of non-glucose transporters, increasing the AG MIC. 61 62 63 (Table 3) significantly reduces the fluorescence. 56 , failed to increase the expression of cmtA (Figure 4). These observations demonstrate the use of cmtA as a tool to screen for conditions that induce transporter expression.

[0102] [Table 3-1] [Table 3-2]

[0103] 2.5 Uridine can activate the expression of CtmA To search for carbohydrate sources that could activate expression from the cmtA promoter, two Biolog 96-well plates (PM1 and PM2B) containing 180 carbon sources from the Biolog Phenotype Microarray system were used. These plates were inoculated with medium containing WT E. coli harboring the PcmtA-gfp plasmid. OD 600nm Growth and GFP production were monitored for 8 hours (Table 5). Growth (t8h-t0h OD 600nm Using the ratio of fluorescence (t8h-t0h) to fluorescence (t8h-t0h), uridine was identified as the most potent activator of PcmtA-GFP (ratio 9959) (Table 5), followed by bromosuccinate and inosine. Glucose was identified as one of the compounds with the lowest activation of cmtA (ratio 698). Nucleosides were tested with PM3B. Again, uridine (ratio 7601) was followed by inosine (ratio 6235), guanosine (ratio 5669), cytidine (ratio 5177), adenosine (ratio 5154), thymidine (ratio 4152), xanthine (ratio 3907), and finally glucose (ratio 839) as a control. The above data obtained from the Biolog plate were confirmed in triplicate, this time using standard media and solutions, by plate reader (Figure 5A) and flow cytometry (Figure 5B). Again, a six-fold increase in fluorescence production was measured when the medium was supplemented with uridine compared to conditions containing no added carbohydrate.

[0104] We next tested whether uridine could activate the promoters of other carbohydrate transporters involved in the uptake of AGs. Because fruA is a PTS system present in both Enterobacteriaceae and Pseudomonas, we chose fruA and annotated it as a fructose-specific transporter. The pfruA-GFP construct also showed increased fluorescence when uridine was added to the medium, suggesting that uridine can activate the transcription of one or more sugar transporters. As a negative control to rule out pleiotropic effects of uridine on gene expression in our experimental conditions, we fused the promoter of the vitamin B12 transporter btuB to GFP: the pbtuB-GFP construct did not show increased GFP expression when the medium was supplemented with uridine (Figure 6).

[0105] 2.6 Uridine as a carbon source decreases aminoglycoside MICs by increasing incorporation Because uridine is a substrate that strongly activates the cmtA promoter, we tested the effect of uridine on the MICs of several antibiotics in E. coli. If uridine can increase the expression of AGs transporters, a lower dose of AGs is required to kill the bacteria, thus reducing the MICs.

[0106] All MICs measured for the different substrates in this study are shown in Table 3. Regarding AGs, uridine reduced the MICs of tobramycin and gentamicin by 10-fold compared to glucose. As expected from the AG specificity of these transporters, this phenotype was not observed with amoxicillin or chloramphenicol (Table 3).

[0107] Uridine is composed of a sugar moiety, ribose, and a nucleotide moiety, uracil. Interestingly, the 10-fold decrease in MIC caused by the addition of uridine to the medium was not observed when ribose was added (MIC 1 μg / ml for ribose vs. 1.5 μg / ml for glucose; Figure 5C and Table 3).

[0108] To examine whether the enhancing effect of uridine is dependent on Crp, we measured the MICs in the presence of glucose or uridine in the Δcrp mutant. The MIC of the Δcrp strain increased to 6 μg / ml in the presence of glucose and remained unchanged in the presence of uridine. Thus, in the absence of Crp, the enhancing effect of uridine on AGs was abolished (6 μg / ml in Δcrp versus 0.1 μg / ml in the WT strain). These results support the hypothesis that uridine-stimulated carbohydrate transport is Crp-dependent. Similar to uridine, cytidine, adenosine, and guanosine also sensitized AGs (MIC 0.1 μg / ml) compared with glucose supplementation, whereas inosine did not (MIC 1 μg / ml) (Table 3).

[0109] To test whether the effect of uridine on AG sensitivity was related to increased AG uptake, we monitored Neo-Cy5 uptake in cells treated with uridine or glucose. Addition of uridine to the medium increased fluorescence from 1.92 with glucose to 2.82 with uridine during a 15-minute sub-MIC treatment (Figure 5D).

[0110] 2.7 Uridine-mediated AG sensitivity is not related to uridine transporter, uridine catabolism, or stress response-mediated uptake Uridine has been shown to induce the expression of at least two carbohydrate transporters (cmtA and fruA), which results in increased uptake and sensitivity of AG. To test whether the uptake and utilization of uridine itself is responsible for these effects, we investigated whether nucleoside transporters are also involved in AG uptake. nupG and nupC, which can transport uridine and other nucleosides, were also involved in AG uptake. 64We overexpressed nucleoside transporters. These overexpressions did not affect tobramycin sensitivity (Table 3), indicating that AG cannot enter the cells via the nucleoside transporters. Furthermore, adding uridine to the medium when the nucleoside transporters were overexpressed increased the amount of uridine in the cells, but did not induce an increase in tobramycin sensitivity (Figure 7A). This suggests that uridine uptake itself is not the cause of the increased sensitivity to tobramycin.

[0111] To test whether uridine catabolism is required for the AG-potentiating phenotype, we deleted udk (the decomposition of uridine to uracil and ribose-1-phosphate) and udp (the decomposition of uridine to uridine monophosphate by reduction of guanosine triphosphate). These deletions had no effect on the MIC of tobramycin (Table 3). Together with the results of overexpression of nucleoside transporters, these data suggest that uridine utilization is not required for the AG-potentiating effect of uridine. Furthermore, the fact that other nucleosides can also reduce the MIC of AGs suggests that this phenotype is not related to the uridine utilization pathway.

[0112] Finally, we investigated whether the effects of uridine could be related to the stringent response. Previous studies have shown that the stringent response is related to the uptake of streptomycin. 65 and AGs susceptibility 66,67 It has also been shown that the synthesis of ppGpp by SpoT is regulated by the carbon source. 68 To test whether uridine supplementation could increase or decrease the stringent response, we used a reporter plasmid containing the rrnB 16S ribosomal RNA promoter 1 fused to GFP, where a decrease in fluorescence corresponds to the induction of a stringent response. As a control for inducing a stringent response, we used tobramycin sub-MIC 69,70 and stationary-phase cultures were used. Treatment with various carbon sources, including uridine, did not induce a stringent response under the experimental conditions (Figure 7B).

[0113] Taken together, these results support the effect of uridine at the level of carbohydrate transporters.

[0114] 2.8 Uridine causes rapid killing and prevents the development of resistant mutants The effect of the carbon source was assessed in time-kill curve experiments in which bacteria were grown to mid-logarithmic growth in medium containing only tryptone and substrate and then treated with a bactericidal dose of tobramycin. The addition of glucose inhibited bactericidal activity, even though tobramycin was added at 10-fold its MIC (Figure 8). The addition of maltose (MIC 0.4 μg / ml) or uridine (MIC 0.1 μg / ml) increased bactericidal activity at different levels (Figure 8A), with maltose being less effective. Regrowth was observed after 20 h of treatment with maltose. Since the number of surviving cells after 6 h of treatment was lower than after 20 h, the growing cells were likely suppressor mutants that arose after 6 h of treatment. Uridine supplementation was most effective, as it did not promote regrowth and all surviving cells had the same MIC as the wild-type strain.

[0115] The suppressor mutants that survived in maltose were divided into two groups: small colonies and normal colonies (Fig. 8B), which were present in equal proportions. Genomic DNA sequencing of these resistant mutants (MIC approximately 1 μg / ml) confirmed the appearance of mutations in the fusA (elongation factor G) and rplL (50S ribosomal protein L7 / L12) genes, both of which are involved in the translation process targeted by AG. Rapid killing by uridine may prevent the selection of these mutations within the population.

[0116] 2.9 Uridine does not cause changes in PMF, but its enhancing effect requires membrane potential. Changes in AG resistance have traditionally been associated with changes in membrane potential. 71 To examine whether AG uptake is related to changes in PMF, we treated WT, ΔcmtA, Δcrp, and p0 / pcmtA+ strains with Mitotracker Red, a probe whose accumulation depends on the membrane potential. 49,52No changes in PMF were observed in either the deletion strain or the cmtAB-overexpressing strain (Figure 9), indicating that carbohydrate transporters promote AG uptake without affecting PMF. Because the effects of metabolites on AG uptake have traditionally been associated with changes in PMF, it was essential to test whether uridine supplementation affected PMF. Addition of 0.5% uridine or glucose to the medium did not change PMF (Figure 9A). Therefore, the positive effect of uridine on AG uptake is not due to an increase in membrane potential.

[0117] However, the fact that uridine does not increase PMF does not mean that its effect on AG uptake is independent of PMF. To address this question, we investigated the effect of uridine addition on the MIC of tobramycin in the presence or absence of carbonyl cyanide m-chlorophenylhydrazine (CCCP). CCCP is a protonophore that disrupts the proton gradient and thus the PMF. Addition of uridine in the presence of 4 μg / ml tobramycin inhibited bacterial growth, while the absence of uridine prevented bacteria from growing at 10 -7 The cells could grow to UFC / ml (Figure 9B). Addition of 15 μg / ml CCCP significantly reduced the effectiveness of uridine as a potentiator of AGs. The potentiating effect of uridine could be observed in the presence of a proton gradient.

[0118] 2.10 The uptake of Ags by carbohydrate transporters and the effect of uridine are conserved among Gram-negative pathogens. Because carbohydrate transporters for the uptake of AGs are present in E. coli, we investigated whether this mechanism is conserved in pseudomonads, such as Pseudomonas aeruginosa and Acinetobacter baumannii, a Gram-negative pathogen involved in resistant infections. We overexpressed five P. aeruginosa transporters involved in the uptake of glucose, mannose, maltose, and fructose. Overexpression of any of these five transporters increased susceptibility to tobramycin and gentamicin (Figure 10A and Table 3), but not to ciprofloxacin or chloramphenicol (Table 3). Overexpression of gtsB, fruA, and mtlFGK reduced the MIC of tobramycin by 10-fold (MIC 0.4 μg / ml vs. 4 μg / ml for empty vector), and overexpression of oprB and homologous genes reduced the MIC by 2.5-fold (MIC to amoxicillin was not tested due to strain resistance). Neocy5 assays for strains overexpressing mtlFGK confirmed increased AG uptake compared to empty vector (approximately 1.3-fold after 15 minutes of sub-MIC treatment with Neocy5) (Figure 10B). In A. baumannii, overexpression of the PTS fruA reduced the MIC of tobramycin to <0.064 compared to 0.1 for empty vector.

[0119] Furthermore, the addition of uridine reduced the MIC of tobramycin for V. cholerae (10-fold), the ESKAPE pathogens K. pneumoniae (4-fold), P. aeruginosa (4-fold), and A. baumannii (8-fold) in MH medium compared with glucose (Table 3). The involvement of carbohydrate transporters in AG uptake is thus shared among Gram-negative bacteria and is not limited to a single genus. The enhancing effect of carbohydrate substrates appears to be shared among these pathogens.

[0120] 2.11 Uridine enhances the availability of AG in synthetic urine AG kills in a concentration-dependent manner 72This indicates that the active administration of AG improves the probability of successful treatment. 73 Therefore, the use of uridine to enhance AG uptake is a potential solution to increase the effective dose in bacteria without increasing toxicity to patients.

[0121] Gram-negative bacteria are frequently involved in urinary tract infections. To test this hypothesis, we developed a synthetic human urine medium that mimics the bacterial growth conditions in UTIs. 45 The effect of various uridine concentrations on AG sensitivity in 5.10% uridine-containing cultures was examined. 6 CFU / ml of bacteria were treated with low doses of tobramycin, and survival was assessed 24 hours after treatment in the presence or absence of uridine (two-fold dilutions of uridine from 1% to 0%) (Figure 11A). Using 0.5 μg / ml of tobramycin, we observed that 0.031% uridine induced the greatest effect, confirming the potentiation of tobramycin by the addition of uridine in synthetic urine medium. Furthermore, the potentiation effect increased between 0.0009% and 0.031% uridine and decreased between 0.031% and 1% uridine (Figure 11A). This may suggest competition for the transporter between uridine and tobramycin when uridine is present in excess relative to AG, thus preventing AG entry via the transporter. Importantly, a uridine concentration of 0.031% is achievable in vivo in humans. 74 , making this molecule a promising adjunct to improve AG treatment.

[0122] Liquid MICs were performed for other AGs: streptomycin, neomycin, and apramycin, and uridine addition reduced the MICs of the three AGs (FIG. 11B).

[0123] Neo-cy5 assay confirmed that uptake at the single-cell level increased approximately 2.5-fold in cells cultured in uridine-supplemented medium (Fig. 11C).

[0124] Finally, we observed time-kill curves using uridine concentrations of 0.0009% (0.036 mM) or 0.031% (1.27 mM) in the presence of three of the most commonly used clinical AGs: tobramycin or gentamicin (4 μg / ml) or amikacin (8 μg / ml) (Figure 11D). For all three AGs, uridine addition rapidly eradicated bacteria, significantly reducing survival after 24 hours compared with only a 1-2 log reduction in survival in the absence of uridine. The concentrations of AGs used here were lower than those seen in bladder treatments, which explains the weaker killing without uridine and highlights the effect of uridine addition (e.g., in humans, following a 1 mg / kg gentamicin dose, urinary concentrations ranged between 113 and 423 μg / ml 1 hour after treatment and between 12 and 271 μg / ml 2 hours after treatment). 75 ).

[0125] The lowest effective uridine concentration was determined to be 0.031% (Figure 12).

[0126] 2.12 In addition to uridine, other nucleosides also enhance the efficacy of AG in E. coli and P. aeruginosa in synthetic urine. Next, we compared the efficacy of other nucleosides (cytidine, thymidine, adenosine, and inosine) as potentiators of the aminoglycosides tobramycin, gentamicin, and amikacin against E. coli. In synthetic urine, cytidine and thymidine also showed significant potentiating effects (Figure 13).

[0127] P. aeruginosa is also an opportunistic pathogen that can cause serious UTI infections. The efficacy of nucleosides in combination with tobramycin was also tested on P. aeruginosa. Uridine, adenosine, thymidine, and inosine efficiently enhanced the killing of P. aeruginosa by tobramycin in synthetic urine.

[0128] 2.13 Aminoglycoside potentiation by uridine is effective in synthetic plasma media and human blood. To evaluate whether uridine could be used to treat blood infections such as sepsis, we performed similar killing experiments in a plasma-like medium (Human Plasma-Like Medium, HPLM). E. coli K12 or E. coli strain CFT073 (a pyelonephritic strain isolated from a patient with sepsis) were treated with tobramycin, and survival was assessed after 20 hours in response to the addition of 0.031% uridine. Although the medium also contained other sugars, we observed that uridine reduced survival at 1 μg / ml tobramycin (Figure 14).

[0129] These results were also confirmed using human blood. In these experiments, human blood was infused with either the aminoglycoside-susceptible E. coli strain CFT073 or the amikacin-resistant E. coli 932 at 1:10 4 CFU inoculation was performed. For the susceptible strain CFT073, treatment with gentamicin (0.1 μg / ml) for 1 hour resulted in the death of 30% of the initial bacterial population, while the combination of uridine and gentamicin killed 50% of the bacterial population (Figure 15). For the resistant strain 932, 932 was treated with 100 μg / ml amikacin (Figure 16), and the addition of uridine statistically significantly reduced the survival of the strain 1 hour after treatment.

[0130] These results support the hypothesis that uridine may enhance the effects of AGs in therapy.

[0131] 2.14 Uridine potentiates tobramycin in clinical E. coli strains Clinical E. coli strains were then used to evaluate the enhancing effect of uridine supplementation (predetermined 0.031%) in combination with tobramycin in the same synthetic urine medium. Synthetic urine medium was also used to evaluate the AG-enhancing effect of uridine on clinical strains of E. coli isolated from long-term hospitalized patients and on uropathogenic UPEC strains isolated from the NILS collection (low-passage natural isolates)

[99] , for which complete genome sequences were available.

[0132] [Table 6]

[0133] We first tested strains that lacked any known tobramycin resistance genes and were therefore susceptible to aminoglycosides: strains 886 and NILS 9, 10, 23, 24, 29, 31, 47, 49, and 78 (but with different levels of resistance); strains Ec019 and Ec068, which possess the MdfA efflux pump, responsible for a two- to three-fold increase in the MICs of AG

[76] ; and strain 1236, which possesses resistance genes to various other antibiotics (β-lactams, sulfonamides, spectinomycin, and streptomycin). In all strains, the addition of uridine (at 0.031%) significantly reduced survival after 20 hours of lethal tobramycin treatment (10–50 μg / ml) (Figure 17). AG-resistant clinical strains with aminoglycoside resistance (modifying enzymes) were also evaluated (Figure 17). Addition of uridine showed promising results at tobramycin concentrations of 200 and 400 μg / ml, which are normally ineffective against these strains. In strains 1193 and 1195, uridine supplementation reduced survival at 200 μg / ml. In strains 1215 and 932, and NILS 55 and 64, the effect of uridine was observed with 400 μg / ml tobramycin treatment. The urinary attainable aminoglycoside concentration was shown to be up to 423 μg / ml 1 hour after treatment with 1 mg / kg gentamicin. 75 , and the doses used in current treatments are on the order of 3–8 mg / kg.

[0134] This data indicates that uridine can be used in combination with AGs to treat selected infections with resistant E. coli. The effectiveness of uridine treatment was observed in strains characterized for resistance to tobramycin at concentrations of 200 μg / ml or greater.

[0135] These results support the idea that uridine can enhance AGs in the treatment of resistant strains.

[0136] 2.15 The aminoglycoside-enhancing effect of uridine is effective in vivo Using a mouse UTI model, we examined whether the effects of uridine could be detected in vivo. First, strain UTI 89

[0101] was treated with gentamicin in synthetic urine to examine whether the uridine-induced enhancement effect was effective in this strain, which was indeed the case (Figure 18). Six-week-old female C57BI / 6 mice were treated with gentamicin in a 10-mL dose. 7 Mice were infected transurethrally with CFU of E. coli UTI89 strain and treated 24 hours later with gentamicin (0.2 mg / kg) with or without 0.5 g / kg uridine. Bacterial survival was assessed 24 hours after treatment, i.e., 48 hours after infection. Gentamicin treatment had no significant effect compared to the PBS control, but its combination with uridine significantly reduced the number of bacteria in the bladder (Figure 18).

[0137] 2.16 Uridine causes rapid killing and prevents the selection of resistant mutants To assess the effect of different carbon sources on tobramycin efficacy, we quantified cell death kinetics by measuring viability at multiple time points after lethal tobramycin treatment. Supplementation with maltose or uridine enhanced the bactericidal effect of tobramycin, with maltose exhibiting the weakest efficacy. Notably, regrowth was observed in maltose-treated bacteria 20 hours after treatment, which was due to the selection of fusA- or rpIL-resistant mutants, which are known to be involved in the AG resistance mechanism [102, 103]. Conversely, tobramycin exhibited the highest efficacy against uridine-treated bacteria (Figure 19). The rapid bactericidal effect induced by uridine supplementation may have prevented the selection of these mutants within the bacterial population.

[0138] 2.17 Uridine supplementation increases aminoglycoside uptake and thereby reduces their MICs Because uridine increases the expression of carbohydrate / AG transporters, its addition to growth medium is expected to increase bacterial AG uptake, resulting in a lower dose of AG required to kill the bacteria, i.e., a lower MIC. The MIC values ​​of various antibiotics in E. coli were measured in the presence of different carbohydrate sources (Table 7). In the case of AGs, uridine supplementation reduced the MICs of tobramycin and gentamicin by 10-fold compared to glucose (Figure 5C). No increased susceptibility to amoxicillin or chloramphenicol was observed (Table 7).

[0139] [Table 7] All antibiotics except neomycin (microtiter broth dilution method) were measured by E-test in MH medium. (Tob: tobramycin; Kan: kanamycin; Gen: gentamicin; Neo: neomycin; Spec: spectinomycin; Cip: ciprofloxacin; Trim: trimethoprim; Amox: amoxicillin; Chlo: chloramphenicol). AG: aminoglycoside. R: plasmid resistance. "-" indicates a condition not tested.

[0140] Unlike uridine, no significant reduction in MIC was observed upon supplementation with ribose, the sugar moiety of uridine (Figure 20 and Table 7).

[0141] Uridine supplementation reduced the MIC of tobramycin compared with glucose for V. cholerae (10-fold), the ESKAPE pathogens K. pneumoniae (4-fold), P. aeruginosa (4-fold), and A. baumannii (8-fold) (Table 7). Thus, the involvement of carbohydrate transporters in AG uptake is a shared property among Gram-negative bacteria that extends beyond a single genus, and the enhancing effect of uridine was observed in all pathogens examined.

[0142] Similar to uridine, supplementation with cytidine, adenosine, and thymidine also increased susceptibility to AG compared with glucose supplementation (MICs of 0.1–0.4 μg / ml), whereas inosine did not (MIC: 1 μg / ml) (Table 7). Guanosine was less soluble.

[0143] The fact that uridine enhances sensitivity to AGs by increasing AG uptake was confirmed using Neo-Cy5 fluorescence per cell, which changed from 1.92 for glucose to 2.82 for uridine after 15 minutes of sub-MIC tobramycin treatment (Figure 20). These data support the idea that uridine supplementation leads to greater AG uptake via carbohydrate transporters in proliferating cells. Therefore, uridine may be an effective aminoglycoside enhancer for sensitivity not only to resistant or refractory strains but also to actively dividing cells. Importantly, uridine can resensitize resistant strains.

[0144] 2.18 Discussion AG uptake in Gram-negative cells has been evaluated for several decades, highlighting the key role of PMF 9、15、77 Therefore, the uptake mechanism of AGs has traditionally been proposed to be primarily dependent on membrane potential. In this study, we identified and elucidated an active mechanism for the uptake of AGs in Gram-negative bacteria via carbohydrate transporters.

[0145] Transporter overexpression in E. coli allowed the identification of at least 11 transport systems involved in the AG-specific response: mainly PTS systems and the ABC transporter MalEFG, which respond to different substrates. This diversity of transporters involved and the weak phenotypes resulting from single deletions suggest that this mechanism has so far been implicated in P. aeruginosa. 78 , A. baumannii 79 , or V. cholerae 80This may explain why these mutants have not been identified using high-throughput techniques such as Tn-seq in the past. Conversely, respiratory chain mutants, for example, are more easily identifiable due to the effect of single deletions on AGs uptake. 81 .

[0146] Although this is not the first report of antibiotic uptake by PTS, 82 No active mechanisms have been described for AGs. The influence of individual transporters on susceptibility to AGs is minor compared to genetic resistance, but simultaneous expression of many transporters (e.g., via the addition of substrates such as uridine) can play a major role. Collectively, these data support the hypothesis of an equilibrium between redundant systems capable of transporting diverse carbohydrates and AGs. Improved therapeutics based on carbohydrate transport pathways appear to be an effective means of circumventing resistance selection, both because single deletions have little or no effect on bacterial adaptation and because large-scale mutations in transporters are detrimental to bacterial growth.

[0147] Aminoglycoside molecules, as the name suggests, consist of a core structure consisting of two or more amino sugars linked via glycosidic bonds to an aminocyclitol. 10 The presence of sugar patterns could explain the fact that these molecules can be recognized as substrates for bacterial carbohydrate transporters. This is supported by the fact that spectinomycin, which has a similar structure but lacks the sugar moiety, does not induce the same response. Similar observations were made for the uptake of imipenem in P. aeruginosa, which can be transported by the amino acid porin OprD2: the authors also noted that imipenem shares structural similarities with amino acids that could explain transporter recognition. 83 .

[0148] In assessing the role of various carbohydrate transporters on AG uptake in E. coli, studies of single-transporter deletion strains identified ΔcmtA, which was responsible for a fourfold increase in the MIC of tobramycin.

[0149] Carbon sources and metabolites were determined from P. aeruginosa 84,85 , E. coli surviving cells, K. pneumoniae, Salmonella typhimurium 32,33 , Salmonella spp., E. coli and S. aureus 86 , and A. baumannii 87 It has been reported that ATP affects AG sensitivity through modulation of PMF. However, the effect of carbohydrate transporters is due to AG uptake, not PMF enhancement.

[0150] The fact that PMF remains unchanged during carbohydrate treatment does not mean that this mechanism is unrelated to PMF, but simply indicates that the mechanism is not dependent on an increased membrane potential. Indeed, the effect of uridine was shown to be conditioned by the functionality of the proton motive force. Regarding the PTS, NADH is required for the phosphorylation of EIA in E. coli. 89,90 This suggests a link between the redox state of the cell (and the electron transport chain) and the functional activity of the PTS. ABC transporters are also driven by ATP. 91 , which may be partially generated by PMF. The requirement for PMF in AG uptake may be related, at least in part, to the functionality of these transporters. These findings constitute an important element in understanding the uptake of bacterial AGs.

[0151] The role of Crp and cAMP in streptomycin uptake has already been observed, and a mechanism for AGs penetration via the polyamine transport system has been proposed. 58 Previous studies have predicted that Crp is a regulator of cmtA and multiple sugar transporters. 92 This study confirmed the involvement of Crp as a regulator of cmtA and a player in AG sensitivity in E. coli. However, an indirect effect of Crp cannot be excluded. This also does not exclude Crp / cAMP-independent regulation of AG uptake, particularly the cAMP-independent action of Crp.

[0152] The mechanism of AG uptake by carbohydrate transporters is shared with Pseudomonadales and other Gram-negative bacteria, as overexpression of carbohydrate transporters also enhances AG killing in P. aeruginosa and A. baumannii. Catabolic repression and carbon source utilization and selectivity in Pseudomonadales have been reported in enterobacteria such as E. coli. 93 This may represent a different regulation of AG entry.

[0153] AG is classified by the WHO as an antimicrobial agent of vital importance for human medicine. 94 , making research into improving such treatments relevant. 95 Using a screening tool based on cmtA expression, uridine was identified among 188 substrates as the most potent activator of cmtA transcription and a facilitator of E. coli killing by AGs. Because AGs are toxic to the host, combination therapy with uridine and AGs may offer a way to improve treatment by reducing the effective AG dose and thereby mitigating the side effects associated with AG therapy. For example, the use of mannitol against K. pneumoniae has been shown to render gentamicin effective below the toxicity threshold and protect the kidney from AG toxicity. 32 .

[0154] High-dose uridine (up to 12 g / m 2 , the average human body surface area is 1.7m 2 ) treatment with 10 g / m 2 Lower doses have no side effects, while higher doses cause shivering for 15 minutes without fever, making uridine suitable for use in human therapy. 74 Furthermore, plasma uridine levels peak at 2 mM (0.5 g / L) after injection (0.05%), with 15–40% of the administered dose being excreted in the urine. 96 These doses correspond to concentrations of uridine that have been shown to be effective in enhancing AGs in synthetic urine medium.

[0155] In mimicking bacterial growth conditions in the bladder during UTI, the killing effects of tobramycin, gentamicin, and amikacin were enhanced, and were shown to be due to increased AG uptake. The development of antibiotic resistance was reported in 5% of treatments. 97 , which has been proposed to be due to inadequate antibiotic dosage. 98 Since no spontaneous mutations were observed in the presence of uridine compared to maltose (4-fold higher MIC), the use of uridine accelerates killing kinetics under laboratory conditions and reduces the risk of spontaneous resistance development. Killing bacterial populations more rapidly may therefore prevent or limit the development and spread of AG resistance and recurrent infections. Uridine combination therapy with AGs may be useful in cases of UTIs, e.g., cystitis, but also in cases of pulmonary infections, e.g., in combination with inhaled tobramycin. 42 Similarly, the dose can be easily adjusted and administered, so it may also be useful for pyelonephritis, otitis, or eye infections. Furthermore, the enhancing effect of uridine was observed in plasma-mimicking media (containing sugar compositions) even in the presence of glucose in the blood, which may be useful in the treatment of blood infections (sepsis).

[0156] 2.19 Conclusion This study identified carbohydrate transporters as players in a novel mechanism that may regulate aminoglycoside (AG) uptake in various Gram-negative bacteria.

[0157] AG resistance is usually associated with genomic AG modifiers or genetic mutations that inactivate AG molecules. These mutations may affect the target of AGs (ribosomes) or, more frequently, impaired PMF-mediated uptake [102, 104, 105]. The diverse array of transporters capable of transporting AGs may explain why mutations in carbohydrate transporters have not previously been associated with AG resistance. By systematically overexpressing sugar transporters, we identified at least 11 transport systems specifically involved in AG uptake in E. coli. These systems include phosphotransferase systems (PTS) for various sugars (chitobiose, glucitol, cellobiose / arbutin, fructose and fructose-like sugars, β-glucosides, and maltose) and the ABC transporter MalEFG for maltose.

[0158] Concurrent activation of multiple transporters can have a significant impact on sensitization to AGs. In this study, we demonstrated that such upregulation could be achieved by supplementing with uridine, the most potent activator of cmtA transcription among 188 substrates tested. Uridine enhanced the killing effects of the clinical AGs tobramycin, gentamicin, and amikacin in synthetic urine, a standardized medium that mimics bacterial growth conditions in UTIs. This enhancement was attributed to increased uptake of the AGs by the bacteria.

[0159] The uptake of AGs via carbohydrate transporters has been shown to be a shared property among Pseudomonadales

[0106] , and overexpression of carbohydrate transporters enhanced the killing of P. aeruginosa and A. baumannii by AGs. Therefore, considering the potential synergistic effects of AGs and sugars, improving therapeutic approaches utilizing carbohydrate / AG transport represents a promising strategy for combating antibiotic resistance.

[0160] In clinical practice, the development of AG resistance has been detected in 5.5% of treated cases

[97] , and insufficient antibiotic administration has been identified as a major cause of resistance

[98] . Uridine supplementation may reduce the selection of AG resistance by accelerating the kinetics of bacterial killing, as observed with synthetic urine, potentially limiting the occurrence of reinfection. Combination therapy of AGs and uridine may be particularly useful in UTIs, but may also be applied to otitis and ocular infections, as the dosage can be easily adjusted depending on the drug delivery format. In pulmonary infections, combining uridine with, for example, inhaled tobramycin may improve outcomes

[42] . Treatment with high doses of uridine (up to 10 g / m 2 ) does not cause side effects in humans

[0107] and is positioned as a promising adjuvant to AG.

[0161] The World Health Organization has classified AGs as critically important antimicrobial agents for human medicine,

[0108] highlighting the importance of pursuing improvements in AG treatment.

[95] The concentrations of AGs used in these experiments were lower than those typically achieved in the bladder during treatment. In humans, a 1 mg / kg dose of gentamicin results in urinary concentrations of 113–423 μg / ml 1 hour after treatment and 12–271 μg / ml 2 hours after treatment.

[75] Despite this, the addition of uridine still demonstrated significant efficacy, suggesting that its use for UTIs is theoretically feasible.

[0162] The use of uridine as an adjunct to AGs may be a powerful approach to improve treatment outcomes by reducing the required dose of AGs and alleviating associated side effects, or by limiting the development of AG resistance and resensitizing AG-resistant strains. Treatment with high doses of uridine (up to 10 g / m 2 ) does not cause side effects in humans.

[0107] AGs exhibit concentration-dependent killing

[72] and are therefore more effective in treating bacterial infections when administered in high doses

[73] . Uridine offers a solution by enhancing AG uptake, which may increase the effective dose in bacteria without increasing toxicity to patients. [Table 4-1] Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 5-1 Table 5-2

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Claims

1. A combination of a nucleoside and an aminoglycoside antibiotic for use in treating a bacterial infection, wherein the nucleoside is administered with the aminoglycoside antibiotic and the nucleoside enhances bacterial killing by the antibiotic.

2. 2. The combination for use according to claim 1, wherein said nucleoside inhibits the development of bacteria resistant to said aminoglycoside antibiotics.

3. 2. The combination for use according to claim 1, wherein said nucleoside resensitizes bacteria resistant to said aminoglycoside antibiotic to killing by said antibiotic.

4. The combination for use according to any one of claims 1 to 3, wherein the nucleoside is selected from uridine, inosine, guanosine, cytidine, thymidine, or xanthosine.

5. 5. The combination for use according to claim 4, wherein the nucleoside is uridine.

6. The combination for use according to any one of claims 1 to 5, wherein the nucleoside is administered to the urinary tract, gastrointestinal tract, lungs, eyes, heart, brain, blood, ears, nose, or skin.

7. The nucleoside is 1 to 20 g / m 2 The combination for use according to any one of claims 1 to 6, wherein the combination is administered in a dose of

8. 8. The combination for use according to any one of claims 1 to 7 for preventing the development of antibiotic-resistant bacteria by killing said bacteria more quickly by co-administration of said nucleoside and said aminoglycoside antibiotic together.

9. 9. The combination for use according to any one of claims 1 to 8 for reducing the toxicity of a reference treatment by co-administration of the nucleoside with the aminoglycoside antibiotic, with the aminoglycoside being administered at a lower concentration than in the reference treatment.

10. 10. The combination for use according to claim 9, wherein the level of killing of said bacteria by said antibiotic is equivalent to a reference treatment.

11. The combination for use according to any one of claims 1 to 10, wherein the antibiotic is selected from tobramycin, gentamicin, and amikacin.

12. The combination for use according to any one of claims 1 to 11, wherein said bacteria causing the infection are enterobacteria.

13. The combination for use according to any one of claims 1 to 12, wherein the bacteria causing the infection are selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii.

14. The combination for use according to any one of claims 1 and 3 to 13, wherein the bacteria causing the infection comprise bacteria that are resistant to the aminoglycoside antibiotic.

15. A composition for killing bacteria comprising a nucleoside and an aminoglycoside antibiotic, wherein the nucleoside enhances the killing of bacteria by the antibiotic.

16. 16. The composition of claim 15, wherein the nucleoside is selected from uridine, inosine, guanosine, cytidine, thymidine, or xanthosine.

17. 17. The composition of claim 16, wherein the nucleoside is uridine.

18. The composition of any one of claims 15 to 17, wherein the antibiotic is selected from tobramycin, gentamicin, and amikacin.

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