The Structural Role of N170 in Substrate-Assisted Deacylation in KPC-2 ß-Lactamase.

The amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC-2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. Specific substitutions in the omega loop (R164-D179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop - more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and an altered spectrum of enzymatic activity.

Stability of Vancomycin and Ceftazidime With Prolonged Storage at -20°C.

BACKGROUND AND OBJECTIVE: Vancomycin and ceftazidime are commonly used intravitreal antibiotics for suspected bacterial endophthalmitis. Many retina surgical practices prepare aliquoted individual doses in syringes that are then stored frozen for future use, but this practice has not been well studied. This investigation aims to examine the stability of frozen vancomycin and ceftazidime. MATERIALS AND METHODS: Samples of drugs were reconstituted monthly and placed in a -20°C freezer. At the end of 3 months and again at 6 months, a newly reconstituted drug constant was created and compared to a newly created reference sample. The frozen samples were compared to a freshly produced drug solution. Using high-performance liquid chromatography (HPLC), the peak heights were compared to evaluate stability. RESULTS: The vancomycin reference sample was 100 ± 1.67%. Values over time were 97.4 ± 0.75%, 98.8 ± 0.44%, 102.1 ± 0.4%, 100.5 ± 0.12%, 101.8 ± 0.12, 101.5 ± 0.11, and 100.6 ± 1.87 for 1, 2, 3(A), 3(B), 4, 5, and 6 months, respectively. The ceftazidime reference sample was 100 ± 1.8%. Values over time were 100.7 ± 1.78%, 100.0 ± 1%, 102.3 ± 1.55%, 117.5 ± 11.6%, 112.8 ± 1.64%, 123 ± 2.8%, and 117 ± 2.5% for 1, 2, 3(A), 3(B), 4, 5, and 6 months, respectively. CONCLUSION: Both vancomycin and ceftazidime were stable over 6 months under frozen conditions at -20°C. [Ophthalmic Surg Lasers Imaging Retina 2023;54:281-283.].

Simultaneous determination of ceftazidime and pyridine in human plasma by LC-UV.

A sensitive, accurate and precise liquid chromatography (LC) method for the simultaneous determination of ceftazidime and pyridine in human plasma has been developed and validated. Acetonitrile (ACN) was employed to precipitate the proteins in the plasma samples. Chromatographic separation was performed with a Kinetex® C18 (150 mm × 3 mm, 2.6 µm) column with gradient elution. Ammonium formate 20 mM and ACN were mixed in a ratio of 98:2 (v/v) for mobile phase A and 85:15 (v/v) for mobile phase B. Both were adjusted to pH 4.5 with formic acid. The flow rate was 0.4 mL/min. UV detection was performed at 254 nm. Calibration curves were linear in the range from 0.3 to 225 µg/mL for ceftazidime and from 0.2 to 10 µg/mL for pyridine with correlation coefficients ≥ 0.999. Within- and between-run precision and accuracy were satisfactory with coefficients of variation (CV) ≤ 8.0% and deviations ≤ 7.0%, respectively. The method fulfilled all validation criteria prescribed by the European Medicines Agency guidelines. Next, it has been used successfully to analyze plasma samples of patients who received ceftazidime under intermittent and continuous administration. With intermittent administration, the concentration of the antibiotics reached a peak and then dropped quickly, which may be below the minimal inhibitory concentration (MIC). With continuous administration, the concentration of the antibiotics remained stable over 24 h, certainly above the MIC. Although the same tendency in ceftazidime concentration changes over time was observed, a difference in concentration amongst the patients was noticeable. The concentration of pyridine in plasma was negligible.

Sustained Release of Antifungal and Antibacterial Agents from Novel Hybrid Degradable Nanofibers for the Treatment of Polymicrobial Osteomyelitis.

This study aimed to develop a drug delivery system with hybrid biodegradable antifungal and antibacterial agents incorporated into poly lactic-co-glycolic acid (PLGA) nanofibers, facilitating an extended release of fluconazole, vancomycin, and ceftazidime to treat polymicrobial osteomyelitis. The nanofibers were assessed using scanning electron microscopy, tensile testing, water contact angle analysis, differential scanning calorimetry, and Fourier-transform infrared spectroscopy. The in vitro release of the antimicrobial agents was assessed using an elution method and a high-performance liquid chromatography assay. The in vivo elution pattern of nanofibrous mats was assessed using a rat femoral model. The experimental results demonstrated that the antimicrobial agent-loaded nanofibers released high levels of fluconazole, vancomycin, and ceftazidime for 30 and 56 days in vitro and in vivo, respectively. Histological assays revealed no notable tissue inflammation. Therefore, hybrid biodegradable PLGA nanofibers with a sustainable release of antifungal and antibacterial agents may be employed for the treatment of polymicrobial osteomyelitis.

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions.

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M ß-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried ß-sheet. The two mutations located in this ß-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M ß-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

Liquid chromatographic method to follow-up ceftazidime and pyridine in portable elastomeric infusion pumps over 24 h.

Portable infusion pumps are an interesting solution to continue outpatient parenteral antimicrobial therapy (OPAT) at the patient's home. However, the use of ceftazidime for such applications is challenging in view of its relatively poor stability in solution. In this study, elastomeric infusion pumps with 6 or 7 g of ceftazidime were deflated over 24 h in an oven at 33°C while ceftazidime and its degradation product, pyridine, were regularly monitored. Hereto, a fast and sensitive liquid chromatographic (LC) method has been developed using a Kinetex® C18 (150 × 3 mm, 2.6 µm) column with gradient elution. Ammonium formate 20 mM and acetonitrile (ACN) were mixed in a ratio of 98:2 v/v for mobile phase A and 85:15 v/v for mobile phase B. Both were adjusted to pH 4.5 with formic acid. The flow rate was set at 0.4 mL/min. The solution with a starting dose of 6 g ceftazidime was found to be degraded 10% after an average of 19 h 11 min so that an administration of 6 g to the patient was not reached. For the solution with a starting dose of 7 g of ceftazidime, 10% degradation was observed after an average of 18 h 42 min. However, by starting from a higher dose, an average of 6.56 g of ceftazidime could be administered over 24 h. In addition, 1.0% of pyridine versus ceftazidime pentahydrate with sodium carbonate (=mixture for injection) was formed over 24 h.

Evaluation of ceftazidime/avibactam alone and in combination with amikacin, colistin and tigecycline against Klebsiella pneumoniae carbapenemase-producing K. pneumoniae by in vitro time-kill experiment.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) poses a major threat to human health worldwide. Combination therapies of antibiotics with different mechanisms have been recommended in literatures. This study assessed in vitro antibacterial activities and synergistic activities of ceftazidime/avibactam alone and in combinations against KPC-Kp. In total, 70 isolates from 2 hospitals in Beijing were examined in our study. By using the agar dilution method and broth dilution method, we determined the minimum inhibitory concentration (MIC) of candidate antibiotics. Ceftazidime/avibactam demonstrated promising susceptibility against KPC-Kp (97.14%). Synergistic activities testing was achieved by checkerboard method and found ceftazidime/avibactam-amikacin displayed synergism in 90% isolates. Ceftazidime/avibactam-colistin displayed partial synergistic in 43% isolates, and ceftazidime/avibactam-tigecycline displayed indifference in 67% isolates. In time-kill assays, antibiotics at 1-fold MIC were mixed with bacteria at 1 × 105 CFU/ml and Mueller-Hinton broth (MHB). Combinations of ceftazidime/avibactam with amikacin and tigecycline displayed better antibacterial effects than single drug. Ceftazidime/avibactam-colistin combination did not exhibit better effect than single drug. In KPC-Kp infections, susceptibility testing suggested that ceftazidime/avibactam may be considered as first-line choice. However, monotherapy is often inadequate in infection management. Thus, our study revealed that combination therapy including ceftazidime/avibactam colistin and ceftazidime/avibactam tigecycline may benefit than monotherapy in KPC-Kp treatment. Further pharmacokinetic/pharmacodynamic and mutant prevention concentration studies should be performed to optimize multidrug-regimens.

Cyclodextrins Allow the Combination of Incompatible Vancomycin and Ceftazidime into an Ophthalmic Formulation for the Treatment of Bacterial Keratitis.

Ceftazidime (CZ) and vancomycin (VA) are two antibiotics used to treat bacterial keratitis. Due to their physical incompatibility (formation of a precipitate), it is not currently possible to associate both molecules in a single container for ophthalmic administration. We firstly characterized the incompatibility then investigated if 2-hydroxypropyl-beta (HPßCD) and 2-hydroxypropyl-gamma cyclodextrins (HPγCD) could prevent this incompatibility. The impact of pH on the precipitation phenomena was investigated by analysing the supernatant solution of the mixture using high performance liquid chromatography. A characterization of the inclusion of CZ with HPγCD using 1H nuclear magnetic resonance (NMR), and VA with HPßCD using 1H-NMR and a solubility diagram was performed. A design of experiment was built to determine the optimal conditions to obtain a formulation that had the lowest turbidity and particle count. Our results showed that VA and CZ form an equimolar precipitate below pH 7.3. The best formulation obtained underwent an in-vitro evaluation of its antibacterial activity. The impact of HPCDs on incompatibility has been demonstrated through the inclusion of antibiotics and especially VA. The formulation has been shown to be able to inhibit the incompatibility for pH higher than 7.3 and to possess unaltered antibacterial activity.

Time-Kill Evaluation of Antibiotic Combinations Containing Ceftazidime-Avibactam against Extensively Drug-Resistant Pseudomonas aeruginosa and Their Potential Role against Ceftazidime-Avibactam-Resistant Isolates.

Ceftazidime-avibactam (CZA) has emerged as a promising solution to the lack of new antibiotics against Pseudomonas aeruginosa infections. Data from in vitro assays of CZA combinations, however, are scarce. The objective of our study was to perform a time-kill analysis of the effectiveness of CZA alone and in combination with other antibiotics against a collection of extensively drug-resistant (XDR) P. aeruginosa isolates. Twenty-one previously characterized representative XDR P. aeruginosa isolates were selected. Antibiotic susceptibility was tested by broth microdilution, and results were interpreted using CLSI criteria. The time-kill experiments were performed in duplicate for each isolate. Antibiotics were tested at clinically achievable free-drug concentrations. Different treatment options, including CZA alone and combined with amikacin, aztreonam, meropenem, and colistin, were evaluated to identify the most effective combinations. Seven isolates were resistant to CZA (MIC ≥ 16/4 mg/liter), including four metallo-ß-lactamase (MBL)-carrying isolates and two class A carbapenemases. Five of them were resistant or intermediate to aztreonam (MIC ≥ 16 mg/liter). Three isolates were resistant to amikacin (MIC ≥ 64 mg/liter) and one to colistin (MIC ≥ 4 mg/liter). CZA monotherapy had a bactericidal effect in 100% (14/14) of the CZA-susceptible isolates. Combination therapies achieved a greater overall reduction in bacterial load than monotherapy for the CZA-resistant isolates. CZA plus colistin was additive or synergistic in 100% (7/7) of the CZA-resistant isolates, while CZA plus amikacin and CZA plus aztreonam were additive or synergistic in 85%. CZA combined with colistin, amikacin, or aztreonam was more effective than monotherapy against XDR P. aeruginosa isolates. A CZA combination could be useful for treating XDR P. aeruginosa infections, including those caused by CZA-resistant isolates. IMPORTANCE The emergence of resistance to antibiotics is a serious public health problem worldwide and can be a cause of mortality. For this reason, antibiotic treatment is compromised, and we have few therapeutic options to treat infections. The main goal of our study is to search for new treatment options for infections caused by difficult-to-treat resistant germs. Pseudomonas aeruginosa is a Gram-negative bacterium distributed throughout the world with the ability to become resistant to most available antibiotics. Ceftazidime-avibactam (CZA) emerged as a promising solution to the lack of new antibiotics against infections caused by P. aeruginosa strains. This study intended to analyze the effect of CZA alone or in combination with other available antibiotics against P. aeruginosa strains. The combination of CZA with other antibiotics could be more effective than monotherapy against extensively drug-resistant P. aeruginosa strains.

Determination of ceftazidime in plasma by RP-HPLC and ultraviolet detection.

A simple high-performance liquid chromatography method for the determination of ceftazidime in plasma has been developed. Using an ultrafiltration technique samples were separated by reverse-phase high-performance liquid chromatography on a Symmetry C18 4.6 × 250 mm column (5.0 µm) and ultraviolet absorbance was measured at 260 nm. The mobile phase was a mixture of 10 mm potassium phosphate monobasic pH 2.5 with phosphoric acid and acetonitrile (90:10). The standard curve ranged from 0.1 to 100 µg/ml. Intra- and inter-assay variability for ceftazidime was <12%, and the average recovery was 89%. The lower limit of quantification was 0.1 µg/ml. This method has been used successfully to analyze frog plasma samples at this institution and it could be applied to other small volume samples in a clinical or research setting.

Effect of the Association and Evaluation of the Induction to Adaptation of the (+)-α-pinene with Commercial Antimicrobials against Strains of Escherichia coli.

BACKGROUND: The increasing and inappropriate use of antibiotics has increased the number of multidrug-resistant microorganisms to these drugs, causing the emergence of infections that are difficult to control and manage by health professionals. As an alternative to combat these pathogens, some monoterpenes have harmful effects on the bacterial cell membrane, showing themselves as an alternative in combating microorganisms. Therefore, the positive enantiomer α -pinene becomes an alternative to fight bacteria, since it was able to inhibit the growth of the species Escherichia coli ATCC 25922, demonstrating the possibility of its use as an isolated antimicrobial or associated with other drugs. AIMS: The aim of this study is to evaluate the sensitivity profile of E. coli ATCC 25922 strain against clinical antimicrobials associated with (+) -α-pinene and how it behaves after successive exposures to subinhibitory concentrations of the phytochemicals. METHODS: The minimum inhibitory concentration (MIC) was determined using the microdilution method. The study of the modulating effect of (+) -α-pinene on the activity of antibiotics for clinical use in strains of E. coli and the analysis of the strain's adaptation to the monoterpene were tested using the adapted disk-diffusion method. RESULTS: The results demonstrate that the association of monoterpene with the antimicrobials ceftazidime, amoxicillin, cefepime, cefoxitin and amikacin is positive since it leads to the potentiation of the antibiotic effect of these compounds. It was observed that the monoterpene was able to induce crossresistance only for antimicrobials: cefuroxime, ceftazidime, cefepime and chloramphenicol. CONCLUSION: It is necessary to obtain more concrete data for the safe use of these combinations, paying attention to the existence of some type of existing toxicity reaction related to the herbal medicine and to understand the resistance mechanisms acquired by the microorganism.

Intravenous Compatibility of Ceftazidime-Avibactam and Aztreonam Using Simulated and Actual Y-site Administration.

PURPOSE: The objective of this communication was to determine the intravenous compatibility of ceftazidime/avibactam and aztreonam using simulated and actual Y-site administration. METHODS: Ceftazidime-avibactam was reconstituted and diluted to concentrations of 8, 25, and 50 mg/mL in 0.9% sodium chloride. Aztreonam was reconstituted and diluted to concentrations of 10 and 20 mg/mL. Each combination of concentrations was tested for compatibility using visual, Tyndall beam, microscopy, turbidity, and pH assessments. Microscopy results were compared to those from sodium chloride 0.9% in water, pH was compared to that at time 0, and turbidity of combinations was compared to that of individual agents. Actual Y-site mixing was conducted over 2-h infusions with samples collected at 0, 1, and 2 h. Test results were evaluated at 0, 1, 2, 4, 8, and 12 h after mixing. All experiments were completed in triplicate. FINDINGS: Across simulated and actual Y-site experiments, no evidence of incompatibility between combinations of ceftazidime-avibactam + aztreonam was observed. Visual and microscopic tests revealed no particulate matter, color changes, or turbidity. Tyndall beam tests were negative with all combinations. No evidence of incompatibility was observed in turbidity testing. The pH values were consistent across each of the 6 combinations, from immediately after mixing until 12 h after mixing. When the addition of agents was reversed in simulated Y-site experiments, no differences in compatibility were observed. No differences in compatibility between actual and simulated Y-site administration were observed, and there was minimal variability across all replicate experiments. IMPLICATIONS: Ceftazidime-avibactam, at concentrations of 8, 25, and 50 mg/mL, appeared compatible with aztreonam at concentrations of 10 and 20 mg/mL.

Antagonism between substitutions in ß-lactamase explains a path not taken in the evolution of bacterial drug resistance.

CTX-M ß-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-µs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

Interactions between Avibactam and Ceftazidime-Hydrolyzing Class D ß-Lactamases.

Class D ß-lactamases exhibit very heterogeneous hydrolysis activity spectra against the various types of clinically useful ß-lactams. Similarly, and according to the available data, their sensitivities to inactivation by avibactam can vary by a factor of more than 100. In this paper, we performed a detailed kinetic study of the interactions between two ceftazidime-hydrolyzing OXA enzymes and showed that they were significantly more susceptible to avibactam than several other class D enzymes that do not hydrolyze ceftazidime. From a clinical point of view, this result is rather interesting if one considers that avibactam is often administered in combination with ceftazidime.

Pyridine levels in ceftazidime - peritoneal dialysis admixtures stored at body temperature.

BACKGROUND: For the treatment of peritoneal dialysis-associated peritonitis (PDAP), ceftazidime is routinely admixed with peritoneal dialysis (PD) solutions before its intraperitoneal administration. One of the major degradation products of ceftazidime is pyridine, a potentially toxic compound. Depending on the type of PD solution, ceftazidime is exposed to an environment with acidic or basic pH, and depending on the type of dosing and individual unit practices related to preparation and storage, ceftazidime can be at body temperature for 4-10 h, resulting in potentially varying rates of degradation to pyridine by-product. No study has investigated whether the amount of generated pyridine exceeds the maximum daily exposure limit of 2 mg when ceftazidime-PD admixtures are kept at body temperature. Therefore, the current study aimed to determine the levels of pyridine generated in PD-ceftazidime admixtures kept at 37°C for various time points. METHODS: Ceftazidime was admixed with 2 L Dianeal (1.5%, 2.5% and 4.25% dextrose) and 2 L Physioneal (1.36%, 2.27% and 3.86% glucose) PD solutions to obtain a concentration of 125 mg/L (continuous dosing model) or 500 mg/L (intermittent dosing model). A total of 36 PD admixtures (3 bags for each type of PD solution and 3 bags for each type of dosing) were prepared and stored at 37°C for 10 h. An aliquot was withdrawn at time 0 (baseline) and after 2, 6, 8 and 10 h of storage. The withdrawn samples were then analysed to determine the concentrations of ceftazidime and pyridine using high-performance liquid chromatography. RESULTS: With the intermittent dosing model (500 mg/L), ceftazidime was found to be stable for only 2 and 6 h when admixed with 3.86% and 2.27% glucose Physioneal PD solutions, respectively. While ceftazidime (500 mg/L) retained more than 90% of its initial concentration in the three types of Dianeal and 1.36% dextrose Physioneal solutions for 10 and 8 h, respectively, the generated amount of pyridine ranged between approximately 290% and 371% more than the daily recommended limit. With the continuous dosing model (125 mg/L), ceftazidime was found to be stable for 6 h in all three types of Physioneal PD solutions, but the total amount of generated pyridine with four daily exchanges (6 h each) was estimated to be 170-360% over the daily recommended limit. Ceftazidime (125 mg/L) was chemically stable when admixed with three types of Dianeal PD solutions and stored at 37°C for 10 h, and the levels of pyridine were estimated to be less than the maximum recommended daily limit. CONCLUSIONS: Until the outcomes of this in vitro study are confirmed by appropriate in vivo studies, continuous dosing of ceftzadime-Dianeal admixtures for the treatment of PDAP may be preferred over continuous dosing of ceftazidime-Physioneal admixtures, and intermittent dosing of ceftazidime-Physioneal and ceftazidime-Dianeal admixtures, as ceftazidime remains stable and the generated levels of pyridine are below the maximum recommended daily exposure.

A Three-Dimensional Printed Polycaprolactone Scaffold Combined with Co-Axially Electrospun Vancomycin/Ceftazidime/Bone Morphological Protein-2 Sheath-Core Nanofibers for the Repair of Segmental Bone Defects During the Masquelet Procedure.

INTRODUCTION: Masquelet proposed a new solution for the healing of segmental bone defects, thus minimizing the disadvantages associated with traditional bone grafting. However, a major factor leading to the failure of this technique pertains to be the residual infection. Accordingly, we developed an antibiotic- and osteo-inductive agent-loaded composite scaffold to solve this problem. METHODS: A mesh-like polycaprolactone scaffold was prepared using a lab-exploited solution-type three-dimensional printer, and hybrid sheath-core structured poly(lactic-co-glycolic-acid) nanofibers were fabricated using co-axial electrospinning technology. Vancomycin, ceftazidime, and bone morphological protein (BMP)-2 were employed. The in vitro and in vivo (rabbit fracture model) release patterns of applied agents from the composite scaffold were investigated. RESULTS: The results revealed that the drug-eluting composite scaffold enabled the sustainable release of the medications for at least 30 days in vitro. Animal tests demonstrated that a high concentration of medications was maintained. Abundant growth factors were induced within the bioactive membrane stimulated by the applied scaffold. Finally, satisfactory bone healing potential was observed on radiological examination and biomechanical evaluation. DISCUSSION: The developed composite scaffold may facilitate bone healing by inducing bioactive membrane formation and yielding high concentrations of antibiotics and BMP-2 during the Masquelet procedure.

Identification of Inhibitors of Integrin Cytoplasmic Domain Interactions With Syk.

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin ß-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the ß-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin ß-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02-4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1ß and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.

Magnetic zeolite imidazole framework material-8 as an effective and recyclable adsorbent for removal of ceftazidime from aqueous solution.

Zeolitic imidazolate framework-8 (ZIF-8) was synthesized by solvothermal method and the adsorption pore size was adjusted by changing the amount of template agent. The ZIF-8@SiO2@Fe3O4 derived from self-assembly of ZIF-8 and SiO2@Fe3O4 were then synthesized and used for ceftazidime (CAZ) removal. ZIF-8 was a regular dodecahedral particle with uniform particle size. The pore diameter was 6.47 nm and the specific surface area was 1182.5 m²·g-1 in ZIF-8@SiO2@Fe3O4. The adsorption of CAZ on ZIF-8@SiO2@Fe3O4 as a function of adsorption temperature, contact time, ionic strength solution pH, and humic acid concentration were investigated. The error of equilibrium adsorption capacity between model fitting and actual experiments is only 1.19%. Kinetics for CAZ removal on ZIF-8@SiO2@Fe3O4 was found to follow pseudo-second-order kinetics. Langmuir, Freundlich and Sips isotherm fitted the adsorption data well and gave similar correlation coefficients, suggesting a single layer adsorption of CAZ on ZIF-8@SiO2@Fe3O4. The ZIF-8@SiO2@Fe3O4 showed no apparent loss in CAZ adsorption after five cycles. These features indicate that the ZIF-8@SiO2@Fe3O4 may be a promising adsorbent for CAZ removal from aqueous solution.

High Pyridine Generation in Ceftazidime-Icodextrin Admixtures Used to Treat Peritoneal Dialysis-associated Peritonitis.

PURPOSE: To investigate the amount of pyridine generated from degradation of ceftazidime in icodextrin peritoneal dialysis (PD) solutions. METHODS: PD solutions that contained 1 and 1.5 g of ceftazidime were stored at 25 °C for 12 hours and then at 37 °C for 14 hours. An aliquot was withdrawn at predefined time points and analyzed for the concentrations of ceftazidime and pyridine. FINDINGS: The amount of pyridine generated was >225% and 400% of its maximum recommended daily exposure in the 1- and 1.5-g ceftazidime-PD admixtures, respectively. IMPLICATIONS: Until these results are confirmed with appropriate in vivo studies, intermittent intraperitoneal dosing of ceftazidime admixed with icodextrin should be used with caution and appropriate clinical monitoring or a suitable alternative antibiotic should be used.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.