Reclaiming The Efficacy of β-Lactam–β-Lactamase Inhibitor Combinations: Avibactam Restores The Susceptibility of CMY-2-Producing Escherichia Coli to Ceftazidime

CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying blaCMY, novel β-lactam–β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry blaCMY-2 or blaCMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC90, ≤0.5 mg/liter); in comparison, ceftazidime had a MIC90 of \u3e128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 β-lactamase, as demonstrated by a second-order onset of acylation rate constant (k2/K) of (4.9 ± 0.5) × 104 M−1 s−1 and the off-rate constant (koff) of (3.7 ± 0.4) ×10−4 s−1. Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2–avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69

Reclaiming The Efficacy of β-Lactam–β-Lactamase Inhibitor Combinations: Avibactam Restores The Susceptibility of CMY-2-Producing Escherichia Coli to Ceftazidime

CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying blaCMY, novel β-lactam–β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry blaCMY-2 or blaCMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC90, ≤0.5 mg/liter); in comparison, ceftazidime had a MIC90 of \u3e128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 β-lactamase, as demonstrated by a second-order onset of acylation rate constant (k2/K) of (4.9 ± 0.5) × 104 M−1 s−1 and the off-rate constant (koff) of (3.7 ± 0.4) ×10−4 s−1. Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2–avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69

A Kinetic Analysis of The Inhibition of FOX-4 β-Lactamase, A Plasmid-Mediated AmpC Cephalosporinase, By Monocyclic β-lactams and Carbapenems

Abstract: Objectives: Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC). Methods: The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described. Results: The Ki values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. Conclusions: Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Evaluation of a Commercial Microarray System for Detection of SHV-, TEM-, CTX-M-, and KPC-Type β-Lactamase Genes in Gram-Negative Isolates ▿ †

We evaluated the ability of a commercial microarray system (Check KPC/ESBL; Check-Points Health BV) to detect clinically important class A β-lactamase genes. A total of 106 Gram-negative strains were tested. The following sensitivity and specificity results were recorded, respectively: for blaSHV, 98.8% and 100%; for blaTEM, 100% and 96.4%; and for blaCTX-M and blaKPC, 100% and 100%

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-β-lactam β-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major β-lactam resistance determinant expressed in <i>B. multivorans</i>, the class A carbapenemase, PenA was readily inhibited by avibactam with a high <i>k</i>₂/<i>K</i> of (2 ± 1) × 10⁶ μM^–1 s^–1 and a slow <i>k</i>_off of (2 ± 1) × 10^–3 s^–1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA–avibactam revealed several interactions that stabilized the acyl–enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime–avibactam was shown to significantly (<i>p</i> < 0.001) improve survival of larvae infected with <i>B. multivorans</i>. To further support the translational impact, the ceftazidime–avibactam combination was evaluated using susceptibility testing against other strains of <i>Burkholderia</i> spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime–avibactam may serve as a preferred therapy for people that have CF and develop <i>Burkholderia</i> spp. infections and should be considered for clinical trials

Stress and human health in diabetes: A report from the 19^th Chicago Biomedical Consortium symposium

Stress and diabetes coexist in a vicious cycle. Different types of stress lead to diabetes, while diabetes itself is a major life stressor. This was the focus of the Chicago Biomedical Consortium’s 19th annual symposium, “Stress and Human Health: Diabetes,” in November 2022. There, researchers primarily from the Chicago area met to explore how different sources of stress – from the cells to the community – impact diabetes outcomes. Presenters discussed the consequences of stress arising from mutant proteins, obesity, sleep disturbances, environmental pollutants, COVID-19, and racial and socioeconomic disparities. This symposium showcased the latest diabetes research and highlighted promising new treatment approaches for mitigating stress in diabetes