H2dpa derivatives containing pentadentate ligands: An acyclic adjuvant potentiates meropenem activity in vitro and in vivo against metallo-ß-lactamase-producing Enterobacterales.

The emergence and dissemination of metallo-ß-lactamases (MBLs) producing Enterobacterales is a great concern for public health due to the limited therapeutic options. No MBL inhibitors are currently available in clinical practice. Herein, we synthesized a series of H2dpa derivatives containing pentadentate-chelating ligands and evaluated their inhibitory activity against MBLs. Related compounds inhibited clinically relevant MBLs (Imipenemase, New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase) with IC50 values of 1-4.9 µM. In vitro, the most promising compounds, 5b and 5c, which had a chiral methyl at the acid adjacent to 5a, demonstrated potent synergistic activity against engineered strains, with fractional inhibitory concentration index values as low as 0.07-0.18. The addition of 5b and 5c restored meropenem efficacy against 42 MBL-producing Enterobacterales and Pseudomonas aeruginosa to satisfactory clinical levels. In addition, safety tests revealed that 5b/5c showed no toxicity in red blood cells, cell lines or mouse model. Further studies demonstrated that compounds 5b and 5c were non-competitive MBL inhibitors. In vivo compounds 5b and 5c potentiated meropenem efficacy and increased the survival rate from 0 to at least 83% in mice with sepsis caused by an NDM-1-positive clinical strain. The activity of the compounds exhibited consistency at the molecular, cellular, and in vivo levels. These data indicated that H2dpa derivatives 5b and 5c containing pentadentate-chelating ligands may be worthy of further study.

Activity of carbonyl cyanide-3-chlorophenylhydrazone on biofilm formation and antimicrobial resistance in Pseudomonas aeruginosa using quantum dots-meropenem conjugates as nanotools.

Hospital infections associated with multidrug-resistant (MDR) Pseudomonas aeruginosa are a worldwide public health problem. Efflux systems and biofilm formation are mechanisms related to resistance to carbapenemics. In this study, quantum dots (QDs) were used to evaluate the effect of carbonyl cyanide-3-chlorophenylhydrazone (CCCP), an efflux pump system inhibitor, on biofilm formation and antimicrobial resistance profile of P. aeruginosa strains. For this, QDs were covalently conjugated to meropenem (MPM) and incubated with a P. aeruginosa resistant isolate (P118) or a control sensitive strain (ATCC Pa27853). P118 was also analyzed with conjugates after previous CCCP efflux inhibitor incubation. Fluorescence microscopy images showed that both sensitive and resistant bacteria were efficiently labeled. Nevertheless, P118 isolates presented fluorescent cell agglomerates, suggesting biofilm formation. The addition of the CCCP changed the labeling profile of the resistant isolate, and the absence of agglomerates was observed, indicating no biofilm formation. Genetic assays revealed the presence of MexA and MexE genes encoding channel proteins from efflux pump systems in both resistant and sensitive strains. Disk-diffusion and broth microdilution tests determined drug susceptibility profiles in the presence and absence of CCCP for P118 isolates. We verified that the CCCP efflux system inhibitor may contribute to P. aeruginosa resistant phenotype reduction for some antimicrobials. This study verified the efficiency of QD-MPM conjugates to trigger and study biofilm formation, or its inhibition, before and after CCCP addition. QDs conjugated to antimicrobials can be used as nanotools to investigate multidrug-resistant bacterial strains on biofilm formation.

Investigation of meropenem stability after reconstitution: the influence of buffering and challenges to meet the NHS Yellow Cover Document compliance for continuous infusions in an outpatient setting.

Objectives: To determine the influence of different buffers, pH and meropenem concentrations on the degradation rates of meropenem in aqueous solution during storage at 32°C, with the aim of developing a formulation suitable for 24-hour infusion in an ambulatory elastomeric device, compliant with the latest National Health Service Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD) requirements. Methods: Meropenem was diluted to 6.25 mg/mL and 25 mg/mL in aqueous solutions adjusted to various pH with phosphate or citrate buffer and assessed for stability. Meropenem concentrations were determined using a validated stability-indicating high-performance liquid chromatography method at time 0 and following storage for up to 24 hours at 32°C as per the YCD requirements. Results: Degradation was observed to be slowest in citrate buffer around pH 7 and at a meropenem concentration of 6.25 mg/mL; however, losses exceeded 10% after storage for 24 hours at 32°C in all of the diluents tested in the study. Conclusions: Meropenem at concentrations between 6.25 mg/mL and 25 mg/mL as tested is not sufficiently stable to administer as a 24-hour infusion in ambulatory device reservoirs. If the YCD 95% minimum content limit is applied, the infusion period must be reduced to less than 6 hours for body-worn devices, especially at the higher concentration studied (25 mg/mL). This limits the possibility of using elastomeric devices to deliver continuous infusions of meropenem as part of a wider outpatient parenteral antimicrobial therapy service.

Novel approach to quorum quenching: rational design of antibacterials in combination with hexahistidine-tagged organophosphorus hydrolase.

N-acyl homoserine lactones (AHLs) are quorum sensing (QS) signal molecules used by most Gram-negative pathogenic bacteria. In this article the lactonase activity of the preparations based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) towards AHLs was studied. Initially, three of the most interesting ß-lactam antibiotics were selected from seven that were trialed during molecular docking to His6-OPH. Combinations of antibiotics (meropenem, imipenem, ceftriaxone) and His6-OPH taken in the native form or in the form of non-covalent enzyme-polyelectrolyte complexes (EPCs) with poly(glutamic acid) or poly(aspartic acid) were obtained and investigated. The lactonase activity of the preparations was investigated under different physical-chemical conditions in the hydrolysis of AHLs [N-butyryl-D,L-homoserine lactone, N-(3-oxooctanoyl)-D,L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone]. An increased efficiency of catalytic action and stability of the lactonase activity of His6-OPH was shown for its complexes with antibiotics and was confirmed in trials with bacterial strains. The broadening of the catalytic action of the enzyme against AHLs was revealed in the presence of the meropenem. Results of molecular docking of AHLs to the surface of the His6-OPH dimer in the presence of antibiotics allowed proposing the mechanism of such interference based on a steric repulsion of the carbon chain of hydrolyzed AHLs by the antibiotics bounded to the enzyme surface.