Research on storage stability differences between ceftriaxone sodium products.

The conditions and mechanisms leading to stability differences between ceftriaxone sodium products were examined to ensure drug quality and efficacy. We used a combination of powder X-ray diffraction and thermogravimetric analysis to examine the differences between preparations for injection from different pharmaceutical processes to elucidate the changed processes by exposing samples to different humidity and high-temperature conditions. Water loss or absorption due to varying environmental humidity levels did not adversely affect the crystal structure, but could lead to the reversible redistribution of hepta-hydrate in the unit cell of generic products, causing its stability change. The irreversible distribution of hydrate may occur when generic drugs stored at 25 °C, whereas the brand-name products remained stable at 40 °C. Therefore, generic ceftriaxone sodium and its powder preparations would be acceptable by better controlled sealing and storing under cool conditions during storage period to meet the efficacy and stability.

Imaging-Guided Delivery of a Hydrophilic Drug to Eukaryotic Cells Based on Its Hydrophobic Ion Pairing with Poly(hexamethylene guanidine) in a Maleated Chitosan Carrier.

Imaging-guided delivery is developed for hydrophobic drugs, and to a much lesser extent, hydrophilic ones. In this work we have designed a novel strategy for real-time monitoring of hydrophilic drug delivery. Traditionally, the drug and the dye are covalently attached to a nanocarrier or are electrostatically adsorbed. Recently, we found an efficient way to bind the drug by ion-paring with an appropriate counter-ion to form the aggregate that embeds a hydrophobic dye with a considerable fluorescence enhancement. We synthesized a series of carbocyanine dyes of hydrophobicity sufficient for solubilization in hydrophobic ion pairs, which restores their emission in the near-infrared (NIR) region upon the formation of the ternary aggregates. To avoid using toxic surfactants, we applied an amphiphilic polymer-oligomer poly(hexamethylene guanidine) (PHMG) as a counter-ion. Сeftriaxone was used as a model hydrophilic drug ensuring the highest fluorescent signal. The so-formed drug-counter-ion-dye aggregates were encapsulated into a cross-linked maleated chitosan carrier. Confocal laser scanning microscopy (CLSM) studies have demonstrated internalization of the encapsulated model drug by breast adenocarcinoma cells at 40 min after treatment. These results suggest the potential application of hydrophobic ion pairs containing an NIR dye in imaging-guided delivery of hydrophilic compounds.

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the ß3-ß4 loop to favor a low-affinity drug-binding state.

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended ß3-ß4 loop conformation and a high-affinity state with an inward ß3-ß4 loop conformation. Histidine-514, which is located at the boundary of the ß4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward ß3-ß4 loop conformation or stabilizing the extended ß3-ß4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.

Synthesis of pH responsive, photocrosslinked gelatin-based hydrogel system for control release of ceftriaxone.

Stimuli-responsive or smart nanocarriers are emerging class of materials used for drug delivery and tissue engineering applications. The objective of this study was to develop environment friendly, economic and smart gelatin based hydrogel for controlled delivery of ceftriaxone. Gelatin was modified with furfuryl amine and converted to hydrogel via photo oxidation reaction, initiated with visible light in the presence of crosslinking agent (eosin Y and riboflavin) to enhance the mechanical properties and increase structural integrity of the hydrogel. The conjugated gelatin-furfuryl amine (GFA) was characterized by 1H NMR and FTIR techniques. An antimicrobial drug (ceftriaxone) was encapsulated in hydrogels systems (HG-E and HG-R) and its encapsulation efficiency was found as 88 % and 74 %, respectively. The in-vitro swelling property and release profile of the drug reveals that the behavior of hydrogel was pH dependent for both the hydrogels systems. Antibacterial study performed on HG-R hydrogel system via disc diffusion method revealed a greater zone of inhibition for Staphylococcus aureus. Findings of the presented work revealed the formation of an excellent, smart hydrogel system using Eosin-Y as cross-linker having greater encapsulation efficiency that can potentially be used for drug delivery application of ceftriaxone to enhance its therapeutic effects.

Structure and elemental composition of Ceftriaxone induced pediatric nephrolithiasis.

Ceftriaxone is a widely used antibiotic because to its broad-spectrum gram-negative coverage, safety, and biological half life (5-9 h) permit dose once-daily administration. It is specifically used in pediatric patients in developing countries. Ceftriaxone forms insoluble sludge/stone when combined with calcium in the urinary system. In this study, Ceftriaxone induced sludge/stones from pediatric patients were collected to identify its microstructure and composition to gather insights into the mechanism of Ceftriaxone induced sludge/stone formation. The results illustrated that Ceftriaxone induced stones formed rapidly following antibiotic administration. Ceftriaxone calcium salt crystals could easily be broken with minimal intervention. However, Ceftriaxone combined with calcium phosphate formed an insoluble stone aggregate.

Comparing cefotaxime and ceftriaxone in combating meningitis through nose-to-brain delivery using bio/chemoinformatics tools.

Bio/chemoinformatics tools can be deployed to compare antimicrobial agents aiming to select an efficient nose-to-brain formulation targeting the meningitis disease by utilizing the differences in the main structural, topological and electronic descriptors of the drugs. Cefotaxime and ceftriaxone were compared at the formulation level (by comparing the loading in gelatin and tripalmitin matrices as bases for the formation of nanoparticulate systems), at the biopharmaceutical level (through the interaction with mucin and the P-gp efflux pumps) and at the therapeutic level (through studying the interaction with S. pneumoniae bacterial receptors). GROMACS v4.6.5 software package was used to carry-out all-atom molecular dynamics simulations. Higher affinity of ceftriaxone was observed compared to cefotaxime on the investigated biopharmaceutical and therapeutic macromolecules. Both drugs showed successful docking on mucin, P-gp efflux pump and S. pneumoniae PBP1a and 2b; but ceftriaxone showed higher affinity to the P-gp efflux pump proteins and higher docking on mucin. Ceftriaxone showed less out-of-matrix diffusion and higher entrapment on the gelatin and the tripalmitin matrices. Accordingly, Ceftriaxone gelatin nanospheres or tripalmitin solid lipid nanoparticles may pose a more feasible and efficient nose-to-brain formulation targeting the meningitis disease compared to the cefotaxime counterparts.

Neo-antigens for the serological diagnosis of IgE-mediated drug allergic reactions to antibiotics cephalosporin, carbapenem and monobactam.

New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for ß-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty ß-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-ß-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-ß-lactam IgE antibodies with high sensitivity (< 0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of ß-lactam antibiotics renders improved molecular recognition events when the 4-member ß-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.

Bactericidal Effect of 5-Mercapto-2-nitrobenzoic Acid-Coated Silver Nanoclusters against Multidrug-Resistant Neisseria gonorrhoeae.

Neisseria gonorrhoeae is among the most multidrug-resistant bacteria in circulation today, and new treatments are urgently needed. In this work, we demonstrate the ability of 5-mercapto-2-nitrobenzoic acid-coated silver nanoclusters (MNBA-AgNCs) to kill strains of Neisseria gonorrhoeae. Using an in vitro bactericidal assay, MNBA-AgNCs had been found to show significantly higher anti-gonococcal bioactivity than the antibiotics ceftriaxone and azithromycin and silver nitrate. These nanoclusters were effective against both planktonic bacteria and a gonococcal infection of human cell cultures in vitro. Treatment of human cells in vitro with MNBA-AgNCs did not induce significant release of lactate dehydrogenase, suggesting minimal cytotoxicity to eukaryotic cells. Our results suggest that MNBA-AgNCs hold great potential for topical treatment of localized gonorrhoeae.

Enhanced adsorption of Levofloxacin and Ceftriaxone antibiotics from water by assembled composite of nanotitanium oxide/chitosan/nano-bentonite.

A novel NBent-NTiO2-Chit nanocomposite has been synthesized from the crosslinking combination of nanotitanium oxide-chitosan (NTiO2-Chit) and nanotitanium oxide-bentonite (NTiO2-Bent) via formaldehyde. The characterization of NBent-NTiO2-Chit was confirmed by different instrumentations. The particle size (34-97 nm) and surface area (16.385 m2 g-1) were confirmed from the scanning electron microscopy and BET measurement, respectively. The FT-IR of NBent-NTiO2-Chit confirmed the presence of OH, N-H, Si-O-Al and Si-O-Si functional groups. Four thermal degradation steps were characterized from the TGA of NBent-NTiO2-Chit with a total loss = 23.514% in the temperature range 30-600 °C. The assembled nanocomposite enhanced the removal of two important classes of antibiotics including Levofloxacin (LEVO) Ceftriaxone (CFT) in the forms of Fluoroquinolone and Cephalosporin, respectively. The various factors that affected the percentage of extraction were applied and optimized such as pH, dosage, initial concentration of LEVO and CFT, contact time and interfering ions. Maximum percentage values of 90.2% (pH 4) and 93.5% (pH 5) for LEVO and CFT, respectively, were achieved at 10 min. The enhancement in removal percentage of LEVO to 92.4% was mainly established by increasing the dose of NBent-NTiO2-Chit to 60.0 mg. The extraction mechanisms of LEVO and CFT were positively explained by three models of adsorption isotherm including Langmuir, Freundlich and Temkin. The obtained correlation coefficients (R2) by Langmuir model were 0.952 and 0.987 for LEVO and CFT, respectively to offer excellent fit to the adsorption processes. The thermodynamics parameters of NBent-NTiO2-Chit were evaluated and referred that the reaction is spontaneous and endothermic. The kinetic studies of NBent-NTiO2-Chit with LEVO and CFT antibiotics were better fitted by the pseudo-second-order based on the acquired R2 values as 0.999 and 0.997 for LEVO and CFT, respectively. The results proved that the designed NBent-NTiO2-Chit was successively implemented for extraction of LEVO and CFT from industrial wastewater providing percentage values 83.2 and 79.0% using 10.0 and 150.0 ±â€¯1.0 mg NBent-NTiO2-Chit, respectively.

New Perspective Enteric-Coated Tablet Dosage Form for Oral Administration of Ceftriaxone: In Vitro and In Vivo Assessments.

Ceftriaxone (CTX) is a widely used injectable third-generation cephalosporin that exhibits broad-spectrum antibacterial activity. Unfortunately, the oral route of this drug suffers different encumbrances, such as instability in the upper part of the GIT and enzymatic degradation, as well as poor permeability. There is no reported tablet dosage form for this drug. In this respect, the authors investigated the possibility of developing an enteric-coated oral tablet of CTX that would be helpful for better patient compliance. The tablet consists of directly compressed core of CTX, citric acid (CA), sodium chloride (NaCl), and two biopolymers-chitosan (CH), a permeation enhancer, and silicified microcrystalline cellulose (SMCC), a wicking agent. Both biopolymers are naturally occurring polysaccharides that are biodegradable in the colon and able to incorporate acid labile drugs. CA is a pH modulator to protect CTX from protease enzymes, while NaCl is a translocation enhancer that helps drug penetration. The enteric coat of the core was shellac (SH) with plasticizer glycerol tristearate (GTS) and CA that was applied by direct compression (dry coating). The solventless heat curable coat resulted in an enteric-coated tablet that complies with the USP pharmacopeia. The optimized formula was further subjected to in vitro release and stability studies, as well as ingredient compatibility. In vivo oral bioavailability of the enteric-coated tablets in rabbits gave promising results (absolute bioavailability of about 80%). Synergistically, all ingredients together augmented oral bioavailability of CTX. This developed formula could be a perspective delivery system for those drugs intended to be absorbed from the colon such as peptides and peptide-like drugs.

Appliance of fungal chitosan/ceftriaxone nano-composite to strengthen and sustain their antimicrobial potentiality against drug resistant bacteria.

Nano-biopolymers could be employed for the delivery of active compounds to increase their stability, bioavailability, efficacy and sustainability. The bioactive chitosan polymer (Cts) was extracted from grown fungus, Cunninghamella elegans, and used for loading ceftriaxone (CFT) and forming the nano-conjugates using tripolyphosphate (TPP) - ionic crosslinking method. The characterization of synthesized CFT/chitosan nanoparticles (NCT) revealed that they chemically crosslinked and had particles' size mean of 56 nm. The CFT loading capacity onto NCT was 54.37%, while its entrapment efficiency was apparently high (79.43%); the maximum released of CFT was 78% from NCT composite after 90 h from dialysis. The CFT/NCT antibacterial activity was confirmed against 3 strains of Staphylococcus aureus (methicillin resistants), using disc diffusion and scanning images of electron microscope, which elucidate that CFT/NCT nano-composite had a vigorous action toward bacterial cells; most cells were ruptured and exploded after 6 h of exposure and entirely lysed after 9 h. The formulation of CFT/NCT nano-composite is exceedingly recommended for enhancing drug biocidal activity, especially against resistant bacterial strains.

Synergistic evaluation of AgO2 nanoparticles with ceftriaxone against CTXM and blaSHV genes positive ESBL producing clinical strains of Uro-pathogenic E. coli.

The silver oxide nanoparticles (AgO2-NPs) were synthesised using silver foil as a new precursor in wet chemical method. X-ray diffraction analysis shows crystallographic structures of AgO2-NPs with crystallite size of 35.54 nm well-matched with standard cubic structure. Scanning electron microscopy analysis clearly shows the random distribution of spherical-shaped nanoparticles. Energy dispersive X-ray analysis confirmed the purity of the samples as it shows no impurity element. Fourier transforms infra-red analysis confirmed the formation of AgO2-NPs with the presence of Ag-O-Ag stretching bond. All the techniques also confirmed the loading of ceftriaxone drug on the surface of AgO2-NPs. This study also described the effect of AgO2-NPs having synergistic activity with ß lactam antibiotic i.e. ceftriaxone against ESBL generating Escherichia coli (E. coli). Among isolated strains of E. coli, 60.0% were found to be ESBL producer. The synergistic activities of AgO2-NPs with ceftriaxone suggest that these combinations are effective against MDR-ESBL E. coli strains as evident by increase in zone sizes. The present study observed rise in MDR-ESBL E. coli with polymorphism of blaCTXM and blaSHV causing UTI infections in Pakistani population. The antibiotic and AgO2-NPs synergistic effect can be used as an efficient approach to combat uro-pathogenic infections.

Photo-induced oxidation of ceftriaxone by persulfate in the presence of iron oxides.

The present study focuses on degradation and mineralization of a third generation cephalosporin antibiotic ceftriaxone (CTA) in UVA- and UVC-induced persulfate (PS) system combined with heterogeneous (α-FeO(OH) and Fe3O4) activators. The CTA oxidation efficiency was investigated in buffered solution (pH 7.4) to stimulate the inhibitory properties of environmental and processed water matrices. Irrespective of the studied UV-induced persulfate system, the mineralization was less effective than CTA degradation. In turn, UVC-induced systems proved to be more effective than UVA-induced processes for decomposition of the target compound and removal of TOC. Accordingly, 2-h oxidation in UVA-induced systems resulted in partial decomposition and negligible mineralization of CTA. While the application of UVC-activated persulfate processes resulted in complete CTA degradation during the first 15 min of oxidation with the most efficient kobs of 0.53 min-1 and 38.3% TOC removal obtained in the UVC/PS system at [PS]0 = 500 µM. Groundwater (GW) trials results clearly indicated the inhibitory effect of the GW composition on the effectiveness of CTA degradation in the studied UV-induced PS-based systems, while the potential treatment efficacy in GW proved predictable based on the results obtained in the buffered UW trials. Adjusting the pH to 3 considerably improved the removal of TOC and the use of PS in both of the water matrices studied. The results of radicals scavenging experiments indicated that both SO4- and HO contributed to the CTA decomposition efficacy in the UV-induced persulfate systems, but the former was the predominant radical in all studied processes. The findings of the study strongly suggest that the UV-induced PS systems are promising treatment technologies for the abatement of cephalosporin antibiotics pollution in natural aqueous matrices.

Formulation of Ceftriaxone Conjugated Gold Nanoparticles and Their Medical Applications against Extended-Spectrum ß-Lactamase Producing Bacteria and Breast Cancer.

Gold nanoparticles (AuNP) and their conjugates have been gaining a great deal of recognition in the medical field. Meanwhile, extended-spectrum ß-lactamases (ESBL)-producing bacteria are also demonstrating a challenging problem for health care. The aim of this study was the biosynthesis of AuNP using Rosa damascenes petal extract and conjugation of ceftriaxone antibiotic (Cef-AuNP) in inhibiting ESBL-producing bacteria and study of in vitro anticancer activity. Characterization of the synthesized AuNP and Cef-AuNP was studied. ESB-Lproducing strains, Acinetobacter baumannii ACI1 and Pseudomonas aeruginosa PSE4 were used for testing the efficacy of Cef-AuNP. The cells of MCF-7 breast cancer were treated with previous AuNP and Cef-AuNP at different time intervals. Cytotoxicity effects of apoptosis and its molecular mechanism were evaluated. Ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy established the formation of AuNP and Cef-AuNP. Transmission electron microscope demonstrated that the formed nanoparticles were of different shapes with sizes of 15~35 nm and conjugation was established by a slight increase in size. Minimum inhibitory concentration (MIC) values of Cef-AuNP against tested strains were obtained as 3.6 and 4 µg/ml, respectively. Cef-AuNP demonstrated a decrease in the MIC of ceftriaxone down to more than 27 folds on the studied strains. The biosynthesized AuNP displayed apoptotic and time-dependent cytotoxic effects in the cells of MCF-7 at a concentration of 0.1 µg/ml medium. The Cef-AuNP have low significant effects on MCF-7 cells. These results enhance the conjugating utility in old unresponsive ceftriaxone with AuNP to restore its efficiency against otherwise resistant bacterial pathogens. Additionally, AuNP may be used as an alternative chemotherapeutic treatment of MCF-7 cancer cells.

Coordination and redox interactions of ß-lactam antibiotics with Cu2+ in physiological settings and the impact on antibacterial activity.

An increase in the copper pool in body fluids has been related to a number of pathological conditions, including infections. Copper ions may affect antibiotics via the formation of coordination bonds and/or redox reactions. Herein, we analyzed the interactions of Cu2+ with eight ß-lactam antibiotics using UV-Vis spectrophotometry, EPR spectroscopy, and electrochemical methods. Penicillin G did not show any detectable interactions with Cu2+. Ampicillin, amoxicillin and cephalexin formed stable colored complexes with octahedral coordination environment of Cu2+ with tetragonal distortion, and primary amine group as the site of coordinate bond formation. These ß-lactams increased the solubility of Cu2+ in the phosphate buffer. Ceftazidime and Cu2+ formed a complex with a similar geometry and gave rise to an organic radical. Ceftriaxone-Cu2+ complex appears to exhibit different geometry. All complexes showed 1:1 stoichiometry. Cefaclor reduced Cu2+ to Cu1+ that further reacted with molecular oxygen to produce hydrogen peroxide. Finally, meropenem underwent degradation in the presence of copper. The analysis of activity against Escherichia coli and Staphylococcus aureus showed that the effects of meropenem, amoxicillin, ampicillin, and ceftriaxone were significantly hindered in the presence of copper ions. The interactions with copper ions should be taken into account regarding the problem of antibiotic resistance and in the selection of the most efficient antimicrobial therapy for patients with altered copper homeostasis.

Multifunctional Scaffolds with Improved Antimicrobial Properties and Osteogenicity Based on Piezoelectric Electrospun Fibers Decorated with Bioactive Composite Microcapsules.

The incorporation of bioactive compounds onto polymer fibrous scaffolds with further control of drug release kinetics is essential to improve the functionality of scaffolds for personalized drug therapy and regenerative medicine. In this study, polymer and hybrid microcapsules were prepared and used as drug carriers, which are further deposited onto polymer microfiber scaffolds [polycaprolactone (PCL), poly(3-hydroxybutyrate) (PHB), and PHB doping with the conductive polyaniline (PANi) of 2 wt % (PHB-PANi)]. The number of immobilized microcapsules decreased with increase in their ζ-potential due to electrostatic repulsion with the negatively charged fiber surface, depending on the polymer used for the scaffold's fabrication. Additionally, the immobilization of the capsules in dynamic mechanical conditions at a frequency of 10 Hz resulted in an increase in the number of the capsules on the fibers with increase in the scaffold piezoelectric response in the order PCL < PHB < PHB-PANi, depending on the chemical composition of the capsules. The immobilization of microcapsules loaded with different bioactive molecules onto the scaffold surface enabled multimodal triggering by physical (ultrasound, laser radiation) and biological (enzymatic treatment) stimuli, providing controllable release of the cargo from scaffolds. Importantly, the microcapsules immobilized onto the surface of the scaffolds did not influence the cell growth, viability, and cell proliferation on the scaffolds. Moreover, the attachment of human mesenchymal stem cells (hMSCs) on the scaffolds revealed that the PHB and PHB-PANi scaffolds promoted adhesion of hMSCs compared to that of the PCL scaffolds. Two bioactive compounds, antibiotic ceftriaxone sodium (CS) and osteogenic factor dexamethasone (DEXA), were chosen to load the microcapsules and demonstrate the antimicrobial properties and osteogenesis of the scaffolds. The modified scaffolds had prolonged release of CS or DEXA, which provided an improved antimicrobial effect, as well as enhanced osteogenic differentiation and mineralization of the scaffolds modified with capsules compared to that of individual scaffolds soaked in CS solution or incubated in an osteogenic medium. Thus, the immobilization of microcapsules provides a simple, convenient way to incorporate bioactive compounds onto polymer scaffolds, which makes these multimodal materials suitable for personalized drug therapy and bone tissue engineering.

UV-Denaturation Assay to Assess Protein Photostability and Ligand-Binding Interactions Using the High Photon Flux of Diamond B23 Beamline for SRCD.

Light irradiation with high photon flux in the vacuum and far-UV region is known to denature the conformation of biopolymers. Measures are in place at Diamond Light Source B23 beamline for Synchrotron Radiation Circular Dichroism (SRCD) to control and make this effect negligible. However, UV denaturation of proteins can also be exploited as a novel method for assessing biopolymer photostability as well as ligand-binding interactions. Usually, host⁻ligand binding interactions can be assessed monitoring CD changes of the host biopolymer upon ligand addition. The novel method of identifying ligand binding monitoring the change of relative rate of UV denaturation using SRCD is especially important when there are very little or insignificant secondary structure changes of the host protein upon ligand binding. The temperature study, another method used to determine molecular interactions, can often be inconclusive when the thermal effect associated with the displacement of the bound solvent molecules by the ligand is also small, making the determination of the binding interaction inconclusive. Herein we present a review on the UV-denaturation assay as a novel method to determine the relative photostability of protein formulations as well as the screening of ligand-binding interactions using the high photon flux Diamond B23 beamline for SRCD.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

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.