Hydroxamate-Containing Bisphosphonates as Fosmidomycin Analogues: Design, Synthesis, and Proherbicide Activity.

Fosmidomycin (FOS) is a natural product inhibiting the DXR enzyme in the MEP pathway and has stimulated interest for finding more suitable FOS analogues. Herein, two series of FOS analogue hydroxamate-containing bisphosphonates as proherbicides were designed, with bisphosphonate replacing the phosphonic unit in FOS while retaining the hydroxamate (BPF series) or replacing it with retro-hydroxamate (BPRF series). The BPF series were synthesized through a three-step reaction sequence including Michael addition of vinylidenebisphosphonate, N-acylation, and deprotection, and the BPRF series were synthesized with a retro-Claisen condensation incorporated into the reaction sequence. Evaluation on model plants demonstrated several compounds having considerable herbicidal activities, and in particular, compound 8m exhibited multifold activity enhancement as compared to the control FOS. The proherbicide properties were comparatively validated. Furthermore, DXR enzyme assay, dimethylallyl pyrophosphate rescue, and molecular docking verified 8m to be a promising proherbicide candidate targeting the DXR enzyme. In addition, 8m also displayed good antimalarial activities.

Bacterial Persistence in Urinary Tract Infection Among Postmenopausal Population.

IMPORTANCE: Urinary tract infections (UTIs) are common in older-aged women. Our study examined bacterial persistence with commonly prescribed antibiotics. Bacterial growth was demonstrated despite antibiotic treatment. OBJECTIVES: The aims of this study were to quantify the bacterial persister phenotype in urine collected from postmenopausal women with acute and recurrent UTI and to determine the capabilities of first-line antibiotics to effectively treat persister cells. STUDY DESIGN: This was an institutional review board-approved cross-sectional analysis within a large academic referral center. Uropathogens were cultured from postmenopausal women with acute or recurrent UTI and screened for persister cells using persistence assays. Demographic and clinical variables were collected and analyzed. The entire experimental process was repeated in triplicate. Data were analyzed for significance (P < 0.05) between the persister culture and antibiotic treatments using a 1-way analysis of variance with multiple comparisons in Prism 9.3.0. RESULTS: Forty participants were included: 62.5% White, 22.5% Black, 3% Asian, and 2% Hispanic with a mean age of 72.3 ± 11.62 years. The persister phenotype was demonstrated in all of Escherichia coli isolates. Treatment with fosfomycin demonstrated reduced colony-forming units per milliliter compared with control (P < 0.01). Among recurrent isolates, there was a statistically significant decrease in colony-forming units per milliliter after antibiotic treatment with all 4 antibiotics (P < 0.05). CONCLUSIONS: This study demonstrated in vitro bacterial persistence in uropathogens from urogynecology patients despite treatment with commonly prescribed antibiotics. Fosfomycin generated the least amount of persister cells. Results suggest that persistence may be one bacterial defense mechanism involved in UTIs. Further research is needed to understand the clinical implications.

In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153.

BACKGROUND: The emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues. OBJECTIVE: The objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153. METHODOLOGY: Enterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation. RESULTS: The Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 µg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 µg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA. CONCLUSION: The findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

Pharmacokinetics/pharmacodynamics cut-off determination for fosfomycin using Monte Carlo simulation in healthy horses.

Fosfomycin (FOM) is an approved veterinary medicinal product for large animals in Japan, but Clinical breakpoint (CBP) for antimicrobial susceptibility test (AST) is not defined for animals. This study aimed at conducting a pharmacokinetics/pharmacodynamics (PK/PD) analysis to determine the PK/PD cutoff for the CBP in horses. Drug concentrations following single intravenous administration (IV) of 20 mg/kg body weight (BW) FOM in nine horses were measured using liquid chromatography/mass spectrometry. The data were modelled using a nonlinear mixed-effects model, followed by Monte Carlo simulations. A 90% probability of target attainment for a PK/PD target of the ratio of Area Under the free plasma concentration-time curve divided by the minimal inhibitory concentration (MIC) >24 hr was set as PK/PD cut-off. The PK/PD cutoff for FOM 20 mg/kg BW q12 hr IV was estimated with the MIC value of ≤16.0 mg/L, and this regimen was considered effective against E. coli (MIC90; 16.0 mg/L) in healthy horses based on the MIC90 values of the wild population. Owing to the relevance of FOM to human health, veterinarians should use q 12 hr FOM 20 mg /kg against E. coli infections with an MIC <16 µg/mL, as suggested by our PK/PD cutoff after AST.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Pharmacokinetic/pharmacodynamic analysis of ceftazidime/avibactam and fosfomycin combinations in an in vitro hollow fiber infection model against multidrug-resistant Escherichia coli.

IMPORTANCE: Mechanistic understanding of pharmacodynamic interactions is key for the development of rational antibiotic combination therapies to increase efficacy and suppress the development of resistances. Potent tools to provide those insights into pharmacodynamic drug interactions are semi-mechanistic modeling and simulation techniques. This study uses those techniques to provide a detailed understanding with regard to the direction and strength of the synergy of ceftazidime-avibactam and ceftazidime-fosfomycin in a clinical Escherichia coli isolate expressing extended spectrum beta-lactamase (CTX-M-15 and TEM-4) and carbapenemase (OXA-244) genes. Enhanced killing effects in combination were identified as a driver of the synergy and were translated from static time-kill experiments into the dynamic hollow fiber infection model. These findings in combination with a suppression of the emergence of resistance in combination emphasize a potential clinical benefit with regard to increased efficacy or to allow for dose reductions with maintained effect sizes to avoid toxicity.

Role of the phosphotransferase system in the transport of fosfomycin in Escherichia coli.

The inducible inner membrane transporters, UhpT and GlpT are considered to be unique fosfomycin transporters. Glucose-6-phosphate, the substrate for UhpT, enhances fosfomycin activity. Previous work indicates that the fructose phosphotransferase system (PTS) might be involved in fosfomycin transport in the bacterial species, Stenotrophomonas maltophilia. Fosfomycin transport in Escherichia coli has been extensively studied and characterised. The current paper addresses the potential fosfomycin transport activity of the fructose PTS in E. coli. Notably, the deletion of both fructose-specific and general PTS proteins in E. coli increases fosfomycin resistance, which indicates that fructose PTS is involved in fosfomycin transport in E. coli. Further, although inactivation of UhpT, the canonical fosfomycin transporter, in E. coli increases fosfomycin resistance by 2-fold, inactivation of genes encoding the PTS increases it by up to 256-fold. Moreover, intracellular accumulation declines in the absence of both transporters, being mutations in the PTS associated with a larger decline. The results presented in this paper re-open the study of fosfomycin transport and reveal the role of the PTS in the transport of this bactericidal antibiotic in E. coli.

Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

Design and synthesis of fosmidomycin analogs containing aza-linkers and their biological activity evaluation.

BACKGROUND: The enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway are attractive targets of a new mode of action for developing anti-infective drugs and herbicides, and inhibitors against 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), the second key enzyme in the pathway, have been intensively investigated; however, few works are reported regarding IspC inhibitors designed for new herbicide discovery. RESULTS: A series of fosmidomycin (FOS) analogs were designed with nitrogen-containing linkers replacing the trimethylene linker between the two active substructures of FOS, phosphonic acid and hydroxamic acid. Synthesis followed a facile three-step route of sequential aza-Michael addition of α-amino acids to dibenzyl vinylphosphonate, amidation of the amino acid carboxyl with O-benzyl hydroxylamine, and simultaneous removal of the benzyl protective groups. Biological activity evaluation of IspC and model plants revealed that some compounds had moderate enzyme and model plant growth inhibition effects. In particular, compound 10g, which has a N-(4-fluorophenylethyl) nitrogen-containing linker, exhibited the best plant inhibition activities, superior to the control FOS against the model plants Arabidopsis thaliana, Brassica napus L., Amaranthus retroflexus and Echinochloa crus-galli. A dimethylallyl pyrophosphate rescue assay on A. thaliana confirmed that both 10g and FOS exert their herbicidal activity by blocking the MEP pathway. This result consistent with molecular docking, which confirmed 10g and FOS binding to the IspC active site in a similar way. CONCLUSION: Compound 10g has excellent herbicidal activity and represents the first herbicide lead structure of a new mode of action that targets IspC enzyme in the MEP pathway. © 2023 Society of Chemical Industry.

Fosfomycin Uptake in Escherichia coli Is Mediated by the Outer-Membrane Porins OmpF, OmpC, and LamB.

The antibiotic fosfomycin (FOS) is widely recognized for the treatment of lower urinary tract infections with Escherichia coli and has lately gained importance as a therapeutic option to combat multidrug-resistant bacteria. However, resistance to FOS frequently develops through mutations reducing its uptake. Although the inner-membrane transport of FOS has been extensively studied in E. coli, its outer-membrane (OM) transport remains insufficiently understood. While evaluating minimal inhibitory concentrations in OM porin-deficient mutants, we observed that the E. coli ΔompFΔompC strain is four times more resistant to FOS than the wild type and the respective single mutants. Continuous monitoring of FOS-induced lysis of porin-deficient strains additionally highlighted the importance of LamB. The relevance of OmpF, OmpC, and LamB to FOS uptake was confirmed by electrophysiological and transcriptional analysis. Our study gives for the first time in-depth insight into the transport of FOS through the OM in E. coli.

fosA11, a novel chromosomal-encoded fosfomycin resistance gene identified in Providencia rettgeri.

This study investigated resistance genes corresponding to the fosfomycin resistance phenotype in clinical isolate Providencia rettgeri W986, as well as characterizing the enzymatic activity of FosA11 and the genetic environment. Antimicrobial susceptibility testing was performed using the agar microdilution method based on the Clinical and Laboratory Standards Institute guidelines. The whole genomic sequence of Providencia rettgeri W986 was obtained using Illumina sequencing and the PacBio platform. The fosA-11 gene was amplified by PCR and cloned into the pUCP20 vector. The recombinant strain pCold1-fosA11-BL21 was expressed to extract the target protein, and absorbance photometry was applied for enzymatic parameter determination. Minimal inhibitory concentration (MIC) tests showed that W986 conferred fosfomycin resistance and was inhibited by phosphonoformate, thereby indicating the presence of a FosA protein. A novel resistance gene designated as fosA11 was identified by whole-genome sequencing and bioinformatics analysis, and it shared 54.41%-64.23% amino acid identity with known FosA proteins. Cloning fosA11 into Escherichia coli obtained a significant increase (32-fold) in the MIC with fosfomycin. Determination of the enzyme kinetics showed that FosA11 had a high catalytic effect on fosfomycin, with Km = 18 ± 4 and Kcat = 56.1 ± 3.2. We also found that fosA11 was located on the chromosome, but the difference in the GC content between the chromosome and fosA11 was dubious, and thus further investigation is required. In this study, we identified and characterized a novel fosfomycin inactivation enzyme called FosA11. The origin and prevalence of the fosA11 gene in other bacteria require further investigation.IMPORTANCEFosfomycin is an effective antimicrobial agent against Enterobacterales strains. However, the resistance rate of fosfomycin is increasing year by year. Therefore, it is necessary to study the deep molecular mechanism of bacterial resistance to fosfomycin. We identified a novel chromosomal fosfomycin glutathione S-transferase, FosA11 from Providencia rettgeri, which shares a very low identity (54.41%-64.23%) with the previously known FosA and exhibits highly efficient catalytic ability against fosfomycin. Analysis of the genetic context and origin of fosA11 displays that the gene and its surrounding environments are widely conserved in Providencia and no mobile elements are discovered, implying that FosA11 may be broadly important in the natural resistance to fosfomycin of Providencia species.

Susceptibility to Fosfomycin and Nitrofurantoin of ESBL-Positive Escherichia coli and Klebsiella pneumoniae Isolated From Urine of Pediatric Patients.

BACKGROUND: Pediatric urinary tract infection (UTI) caused by extended-spectrum ß-lactamase (ESBL)-positive gram-negative bacilli (GNB) has limited options for oral antibiotic treatment. The purpose of this study was to investigate the susceptibility of ESBL-positive Escherichia coli and Klebsiella pneumoniae isolates from pediatric urine samples to two oral antibiotics (fosfomycin and nitrofurantoin). METHODS: From November 2020 to April 2022, ESBL-positive E. coli and K. pneumoniae isolates from urine samples were collected at Samsung Medical Center, Seoul, Korea. Patients over 18 years of age or with malignancy were excluded. For repeated isolates from the same patient, only the first isolate was tested. Minimum inhibitory concentrations (MICs) were measured using agar (fosfomycin) or broth (nitrofurantoin) dilution methods. MIC50 and MIC90 were measured for fosfomycin and nitrofurantoin in both E. coli and K. pneumoniae. RESULTS: There were 117 isolates from 117 patients, with a median age of 7 months (range, 0.0-18.5 years). Among 117 isolates, 92.3% (108/117) were E. coli and 7.7% (9/117) were K. pneumoniae. Isolates from the pediatric intensive care unit (PICU) and general ward (GW) was 11.1% (13/117) and 88.9% (104/117), respectively. Among 108 E. coli isolates, MIC50 and MIC90 for fosfomycin were 0.5 µg/mL and 2 µg/mL, respectively. Fosfomycin susceptibility rate was 97.2% (105/108) with a breakpoint of 128 µg/mL. Fosfomycin susceptibility rate was significantly lower in PICU isolates than in GW isolates (81.8% vs. 99.0%, P = 0.027). For nitrofurantoin, both the MIC50 and MIC90 were 16 µg/mL. Nitrofurantoin susceptibility rate was 96.3% (104/108) with a breakpoint of 64 µg/mL based on Clinical and Laboratory Standards Institute guidelines. Among the nine K. pneumoniae isolates, the MIC50 and MIC90 for fosfomycin was 2 µg/mL and 32 µg/mL, respectively. MIC50 and MIC90 for nitrofurantoin were 64 µg/mL and 128 µg/mL, respectively. CONCLUSION: For uncomplicated UTI caused by ESBL-positive GNB in Korean children, treatment with fosfomycin and nitrofurantoin for E. coli infections can be considered as an effective oral therapy option.

The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa.

The identification of targets whose inactivation increases the activity of antibiotics helps to fight antibiotic resistance. Previous work showed that a transposon-insertion mutant in the gene PA14_27940 increases Pseudomonas aeruginosa susceptibility to aminoglycosides. Since polar effects may affect the phenotype, in the present work, we generated an in-frame PA14_27940 deletion mutant. A PA14_27940 deletion increased the susceptibility to aminoglycosides, tetracycline, tigecycline, erythromycin and fosfomycin. Excepting fosfomycin, the other antibiotics are inducers of the MexXY efflux pump. MexXY induction is required for P. aeruginosa resistance to these antibiotics, which is post-transcriptionally regulated by the anti-repressor ArmZ. Although mexXY is inducible by tobramycin in ΔPA14_27940, the induction level is lower than in the parental PA14 strain. Additionally, armZ is induced by tobramycin in PA14 and not in ΔPA14_27940, supporting that ΔPA14_27940 presents an ArmZ-mediated defect in mexXY induction. For its part, hypersusceptibility to fosfomycin may be due to a reduced expression of nagZ and agmK, which encode enzymes of the peptidoglycan recycling pathway. ΔPA14_27940 also presents defects in motility, an element with relevance in P. aeruginosa's virulence. Overall, our results support that PA14_27940 is a good target for the search of adjuvants that will increase the activity of antibiotics and reduce the virulence of P. aeruginosa.

Synergistic Antimicrobial Activity of Eugenol in Combination with Fosfomycin to Combat Escherichia coli and Potential Effect on Plasmid-Mediated Fosfomycin Resistance Genes.

The presence of multidrug-resistant pathogenic microorganisms makes it challenging to cure bacterial illnesses. Syzygium aromaticum has been used for medicinal purposes since ancient times. The objective of this study was to investigate the potential synergistic effect of the combination of Eugenol and Fosfomycin against clinically Uropathogenic Escherichia coli (UPEC) and their possible co-treatment as well as their contribution to plasmid-mediated Fosfomycin resistance (fosA3 and fosA4) genes using molecular assays. Eugenol was extracted from clove (Syzygium aromaticum) plants using steam distillation by Clevenger and analyzed by high-performance liquid chromatography (HPLC). UPEC accounted for 63.6 % of all isolates. Specifically, 99.3 % of the UPEC isolates exhibited resistance to multiple types of antibiotics [multidrug-resistant (MDR)]. The MIC for Eugenol was 1.25-5 µg/mL, and Fosfomycin was 512-1024 µg/mL, while the MBC for Eugenol was 5-10 µg/mL and Fosfomycin was 2048 µg/mL. The synergistic effects were considerable, with 1/4 MIC of Eugenol resulting in 1/8 MIC Fosfomycin. Eugenol inhibited most of the UPEC isolates at 4-8 hours, Fosfomycin at 8-12 hours, and co-treatment at 4-8 hours. The fosA3 and fosA4 genes were detected in 5.7 % and 2.9 % of the isolates, respectively. The results showed variable gene expression changes in response to the different treatments.

Design, Synthesis and Bioactivity Evaluation of Heterocycle-Containing Mono- and Bisphosphonic Acid Compounds.

Fosmidomycin (FOS) is a naturally occurring compound active against the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and using it as a template for lead structure design is an effective strategy to develop new active compounds. In this work, by replacing the hydroxamate unit of FOS with pyrazole, isoxazole and the related heterocycles that also have metal ion binding affinity, while retaining the monophosphonic acid in FOS or replacing it with a bisphosphonic acid group, heterocycle-containing mono- and bisphosphonic acid compounds as FOS analogs were designed. The key steps involved in the facile synthesis of these FOS analogs included the Michael addition of diethyl vinylphosphonate or tetraethyl vinylidenebisphosphonate to ß-dicarbonyl compounds and the subsequent cyclic condensation with hydrazine or hydroxylamine. Two additional isoxazolinone-bearing FOS analogs were synthesized via the Michaelis-Becker reaction with diethyl phosphite as a key step. The bioactivity evaluation on model plants demonstrated that several compounds have better herbicidal activities compared to FOS, with the most active compound showing a 3.7-fold inhibitory activity on Arabidopsis thaliana, while on the roots and stalks of Brassica napus L. and Echinochloa crus-galli in a pre-emergence inhibitory activity test, the activities of this compound were found to be 3.2- and 14.3-fold and 5.4- and 9.4-fold, respectively, and in a post-emergency activity test on Amaranthus retroflexus and Echinochloa crus-galli, 2.2- and 2.0-fold inhibition activities were displayed. Despite the significant herbicidal activity, this compound exhibited a DXR inhibitory activity lower than that of FOS but comparable to that of other non-hydroxamate DXR inhibitors, and the dimethylallyl pyrophosphate rescue assay gave no statistical significance, suggesting that a different target might be involved in the inhibiting process. This work demonstrates that using bioisosteric replacement can be considered as a valuable strategy to discover new FOS analogs that may have high herbicidal activities.

Efficacy of antibiotic combinations in an experimental sepsis model with Pseudomonas aeruginosa.

This study aimed to compare the efficacy of fosfomycin, colistin, tobramycin and their dual combinations in an experimental sepsis model. After sepsis was established with a Pseudomonas aeruginosa isolate (P1), antibiotic-administered rats were divided into six groups: Fosfomycin, tobramycin, colistin and their dual combinations were administered by the intravenous or intraperitoneal route to the groups. The brain, heart, lung, liver, spleen and kidney tissues of rats were cultured to investigate bacterial translocation caused by P1. Given the antibiotics and their combinations, bacterial colony counts in liver tissues were decreased in colistin alone and colistin plus tobramycin groups compared with control group, but there were no significant differences. In addition, a non-statistical decrease was found in the spleen tissues of rats in the colistin plus tobramycin group. There was a > 2 log10 CFU/ml decrease in the number of bacterial colonies in the kidney tissues of the rats in the fosfomycin group alone, but the decrease was not statistically significant. However, there was an increase in the number of bacterial colonies in the spleen and kidney samples in the group treated with colistin as monotherapy compared to the control group. The number of bacterial colonies in the spleen samples in fosfomycin plus tobramycin groups increased compared to the control group. Bacterial colony numbers in all tissue samples in the fosfomycin plus colistin group were found to be close to those in the control group. Colistin plus tobramycin combinations are effective against P. aeruginosa in experimental sepsis, and clinical success may be achieved. New in vivo studies demonstrating the ability of P. aeruginosa to biofilm formation in tissues other than the lung are warranted in future.

Discovery of 1,2-diaryl-3-oxopyrazolidin-4-carboxamides as a new class of MurA enzyme inhibitors and characterization of their antibacterial activity.

The cytoplasmic steps of peptidoglycan synthesis represent an important targeted pathway for development of new antibiotics. Herein, we report the synthesis of novel 3-oxopyrazolidin-4-carboxamide derivatives with variable amide side chains as potential antibacterial agents targeting MurA enzyme, the first committed enzyme in these cytosolic steps. Compounds 15 (isoindoline-1,3-dione-5-yl), 16 (4-(1H-pyrazol-4-yl)phenyl), 20 (5-cyanothiazol-2-yl), 21 and 31 (5-nitrothiazol-2-yl derivatives) exhibited the most potent MurA inhibition, with IC50 values of 9.8-12.2 µM. Compounds 15, 16 and 21 showed equipotent inhibition of the C115D MurA mutant developed by fosfomycin-resistant Escherichia coli. NMR binding studies revealed that some of the MurA residues targeted by 15 also interacted with fosfomycin, but not all, indicating an overlapping but not identical binding site. The antibacterial activity of the compounds against E. coli ΔtolC suggests that inhibition of MurA accounts for the observed effect on bacterial growth, considering that a few potent MurA inhibitors could not penetrate the bacterial outer membrane and were therefore inactive as proven by the bacterial cell uptake assay. The most promising compounds were also evaluated against a panel of Gram-positive bacteria. Remarkably, compounds 21 and 31 (MurA IC50 = 9.8 and 10.2 µM respectively) exhibited a potent activity against Clostridioides difficile strains with MIC values ranging from 0.125 to 1 µg/mL, and were also shown to be bactericidal with MBC values between 0.25 and 1 µg/mL. Furthermore, both compounds were shown to have a limited activity against human normal intestinal flora and showed high safety towards human colon cells (Caco-2) in vitro. The thiolactone derivative (compound 5) exhibited an interesting broad spectrum antibacterial activity despite its weak MurA inhibition. Altogether, the presented series provides a promising class of antibiotics that merits further investigation.

Activity of polymyxin B combinations against genetically well-characterised Klebsiella pneumoniae producing NDM-1 and OXA-48-like carbapenemases.

BACKGROUND: Combination therapy can enhance the activity of available antibiotics against multidrug-resistant Gram-negative bacteria. This study assessed the effects of polymyxin B combinations against carbapenemase-producing Klebsiella pneumoniae (K. pneumoniae). METHODS: Twenty clinical K. pneumoniae strains producing NDM-1 (n = 8), OXA-48-like (n = 10), or both NDM-1 and OXA-48-like (n = 2) carbapenemases were used. Whole-genome sequencing was applied to detect resistance genes (e.g. encoding antibiotic-degrading enzymes) and sequence alterations influencing permeability or efflux. The activity of polymyxin B in combination with aztreonam, fosfomycin, meropenem, minocycline, or rifampicin was investigated in 24-hour time-lapse microscopy experiments. Endpoint samples were spotted on plates with and without polymyxin B at 4 x MIC to assess resistance development. Finally, associations between synergy and bacterial genetic traits were explored. RESULTS: Synergistic and bactericidal effects were observed with polymyxin B in combination with all other antibiotics: aztreonam (11 of 20 strains), fosfomycin (16 of 20), meropenem (10 of 20), minocycline (18 of 20), and rifampicin (15 of 20). Synergy was found with polymyxin B in combination with fosfomycin, minocycline, or rifampicin against all nine polymyxin-resistant strains. Wildtype mgrB was associated with polymyxin B and aztreonam synergy (P = 0.0499). An absence of arr-2 and arr-3 was associated with synergy of polymyxin B and rifampicin (P = 0.0260). Emergence of populations with reduced polymyxin B susceptibility was most frequently observed with aztreonam and meropenem. CONCLUSION: Combinations of polymyxin B and minocycline or rifampicin were most active against the tested NDM-1 and OXA-48-like-producing K. pneumoniae. Biologically plausible genotype-phenotype associations were found. Such information might accelerate the search for promising combinations and guide individualised treatment.