ABSTRACTβ-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis.

Understanding the factors that influence the airborne survival of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARS-CoV-2 in aerosol droplets (∼5 to 10 µm equilibrated radius) over timescales spanning 5 s to 20 min using an instrument to probe survival in a small population of droplets (typically 5 to 10) containing ∼1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to ∼10% of the starting value was observable for SARS-CoV-2 over 20 min, with a large proportion of the loss occurring within the first 5 min after aerosolization. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallize at relative humidities (RHs) below 50%, leading to a near-instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH, the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 min, beyond which it decays to only 10% remaining infectious after 10 min. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilization of CO 2 from bicarbonate buffer within the droplet. Four different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.

Understanding the factors that influence the airborne survival of viruses such as SARSCoV2 in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARSCoV2 in aerosol droplets (5 to 10 micrometres equilibrated radius) over timescales spanning from 5 seconds to 20 minutes using a novel instrument to probe survival in a small population of droplets (typically 5-10) containing ~1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to 10 % of the starting value was observable for SARS-CoV-2 over 20 minutes, with a large proportion of the loss occurring within the first 5 minutes after aerosolisation. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallise at RHs below 50% leading to a near instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 minutes, beyond which it decays to only 10% remaining infectious after 10 minutes. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilisation of CO2 from bicarbonate buffer within the droplet. Three different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.

The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate theC-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of C-NMR for studying lysine carbamylation in solution.

Emerging outbreaks of airborne pathogenic infections worldwide, such as the current SARS-CoV-2 pandemic, have raised the need to understand parameters affecting the airborne survival of microbes in order to develop measures for effective infection control. We report a novel experimental strategy, TAMBAS (Tandem Approach for Microphysical and Biological Assessment of Airborne Microorganisms Survival), to explore the synergistic interactions between the physicochemical and biological processes that impact airborne microbe survival in aerosol droplets. This innovative approach provides a unique and detailed understanding of the processes taking place from aerosol droplet generation through to equilibration and viability decay in the local environment, elucidating decay mechanisms not previously described. The impact of evaporation kinetics, solute hygroscopicity and concentration, particle morphology and equilibrium particle size on airborne survival are reported, using Escherichia coli (MRE162) as a benchmark system. For this system, we report that the particle crystallisation does not directly impact microbe longevity, bacteria act as crystallization nuclei during droplet drying and equilibration, and the kinetics of size and compositional change appear to have a larger effect on microbe longevity than equilibrium solute concentration. IMPORTANCE A transformative approach to identify the physicochemical processes that impact the biological decay rates of bacteria in aerosol droplets is described. It is shown that the evaporation process and changes in the phase and morphology of the aerosol particle during evaporation impact microorganism viability. The equilibrium droplet size was found to affect airborne bacterial viability. Furthermore, the presence of Escherichia coli (MRE162) in a droplet does not affect aerosol growth/evaporation, but influences the dynamic behaviour of the aerosol through processing the culture media prior to aerosolization affecting the hygroscopicity of the culture medium; this highlights the importance of the inorganic and organic chemical composition within the aerosolised droplets that impact hygroscopicity. Bacteria act also as a crystallisation nucleus. The novel approach and data has implications for increased mechanistic understanding of aerosol survival and infectivity in bioaerosol studies spanning medical, veterinary, farming, and agricultural fields, including the role of micro-organisms in atmospheric processing and cloud formation.