Penicillanic Acid Sulfones Inactivate the Extended-Spectrum ß-Lactamase CTX-M-15 through Formation of a Serine-Lysine Cross-Link: an Alternative Mechanism of ß-Lactamase Inhibition.

ß-Lactamases hydrolyze ß-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) ß-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum ß-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cß closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of ß-lactamase inhibition by these agents. IMPORTANCE ß-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing ß-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to ß-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining ß-lactams with ß-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to ß-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of ß-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.

Magnetohydrodynamic Simulations of Self-Consistent Rotating Neutron Stars with Mixed Poloidal and Toroidal Magnetic Fields.

We perform the first magnetohydrodynamic simulations in full general relativity of self-consistent rotating neutron stars (NSs) with ultrastrong mixed poloidal and toroidal magnetic fields. The initial uniformly rotating NS models are computed assuming perfect conductivity, stationarity, and axisymmetry. Although the specific geometry of the mixed field configuration can delay or accelerate the development of various instabilities known from analytic perturbative studies, all our models finally succumb to them. Differential rotation is developed spontaneously in the cores of our magnetars which, after sufficient time, is converted back to uniform rotation. The rapidly rotating magnetars show a significant amount of ejecta, which can be responsible for transient kilonova signatures. However, no highly collimated, helical magnetic fields or incipient jets, which are necessary for γ-ray bursts, arise at the poles of these magnetars by the time our simulations are terminated.

Multimessenger Binary Mergers Containing Neutron Stars: Gravitational Waves, Jets, and γ-Ray Bursts.

Neutron stars (NSs) are extraordinary not only because they are the densest form of matter in the visible Universe but also because they can generate magnetic fields ten orders of magnitude larger than those currently constructed on earth. The combination of extreme gravity with the enormous electromagnetic (EM) fields gives rise to spectacular phenomena like those observed on August 2017 with the merger of a binary neutron star system, an event that generated a gravitational wave (GW) signal, a short γ -ray burst (sGRB), and a kilonova. This event serves as the highlight so far of the era of multimessenger astronomy. In this review, we present the current state of our theoretical understanding of compact binary mergers containing NSs as gleaned from the latest general relativistic magnetohydrodynamic simulations. Such mergers can lead to events like the one on August 2017, GW170817, and its EM counterparts, GRB 170817 and AT 2017gfo. In addition to exploring the GW emission from binary black hole-neutron star and neutron star-neutron star mergers, we also focus on their counterpart EM signals. In particular, we are interested in identifying the conditions under which a relativistic jet can be launched following these mergers. Such a jet is an essential feature of most sGRB models and provides the main conduit of energy from the central object to the outer radiation regions. Jet properties, including their lifetimes and Poynting luminosities, the effects of the initial magnetic field geometries and spins of the coalescing NSs, as well as their governing equation of state, are discussed. Lastly, we present our current understanding of how the Blandford-Znajek mechanism arises from merger remnants as the trigger for launching jets, if, when and how a horizon is necessary for this mechanism, and the possibility that it can turn on in magnetized neutron ergostars, which contain ergoregions, but no horizons.

Accretion onto a small black hole at the center of a neutron star.

We revisit the system consisting of a neutron star that harbors a small, possibly primordial, black hole at its center, focusing on a nonspinning black hole embedded in a nonrotating neutron star. Extending earlier treatments, we provide an analytical treatment describing the rate of secular accretion of the neutron star matter onto the black hole, adopting the relativistic Bondi accretion formalism for stiff equations of state that we presented elsewhere. We use these accretion rates to sketch the evolution of the system analytically until the neutron star is completely consumed. We also perform numerical simulations in full general relativity for black holes with masses up to nine orders of magnitude smaller than the neutron star mass, including a simulation of the entire evolution through collapse for the largest black hole mass. We construct relativistic initial data for these simulations by generalizing the black hole puncture method to allow for the presence of matter, and evolve these data with a code that is optimally designed to resolve the vastly different length scales present in this problem. We compare our analytic and numerical results, and provide expressions for the lifetime of neutron stars harboring such endoparasitic black holes.

Relativistic Bondi accretion for stiff equations of state.

We revisit Bondi accretion - steady-state, adiabatic, spherical gas flow on to a Schwarzschild black hole at rest in an asymptotically homogeneous medium - for stiff polytropic equations of state (EOSs) with adiabatic indices Γ > 5/3. A general relativistic treatment is required to determine their accretion rates, for which we provide exact expressions. We discuss several qualitative differences between results for soft and stiff EOSs - including the appearance of a minimum steady-state accretion rate for EOSs with Γ ≥ 5/3 - and explore limiting cases in order to examine these differences. As an example, we highlight results for Γ = 2, which is often used in numerical simulations to model the EOS of neutron stars. We also discuss a special case with this index, the ultrarelativistic 'causal' EOS, P = ρ. The latter serves as a useful limit for the still undetermined neutron star EOS above nuclear density. The results are useful, for example, to estimate the accretion rate on to a mini-black hole residing at the centre of a neutron star.

Gravitational waves from disks around spinning black holes: Simulations in full general relativity.

We present fully general-relativistic numerical evolutions of self-gravitating tori around spinning black holes with dimensionless spin a/M = 0.7 parallel or antiparallel to the disk angular momentum. The initial disks are unstable to the hydrodynamic Papaloizou-Pringle instability which causes them to grow persistent orbiting matter clumps. The effect of black hole spin on the growth and saturation of the instability is assessed. We find that the instability behaves similarly to prior simulations with nonspinning black holes, with a shift in frequency due to spin-induced changes in disk orbital period. Copious gravitational waves are generated by these systems, and we analyze their detectability by current and future gravitational wave observatories for a large range of masses. We find that systems of 10 M ⊙-relevant for black hole-neutron star mergers-are detectable by Cosmic Explorer out to ~300 Mpc, while DECIGO (LISA) will be able to detect systems of 1000 M ⊙ (105 M ⊙)-relevant for disks forming in collapsing supermassive stars-out to cosmological redshift of z ~ 5 (z ~ 1). Computing the accretion rate of these systems we find that these systems may also be promising sources of coincident electromagnetic signals.

Neutron stars harboring a primordial black hole: Maximum survival time.

We explore in general relativity the survival time of neutron stars that host an endoparasitic, possibly primordial, black hole at their center. Corresponding to the minimum steady-state Bondi accretion rate for adiabatic flow that we found earlier for stiff nuclear equations of state (EOSs), we derive analytically the maximum survival time after which the entire star will be consumed by the black hole. We also show that this maximum survival time depends only weakly on the stiffness for polytropic EOSs with Γ ≥ 5/3, so that this survival time assumes a nearly universal value that depends on the initial black-hole mass alone. Establishing such a value is important for constraining the contribution of primordial black holes in the mass range 10-16 M ⊙ ≲ M ≲ 10-10 M ⊙ to the dark-matter content of the Universe.

Sigmoid Emax Modeling To Define the Fixed Concentration of Enmetazobactam for MIC Testing in Combination with Cefepime.

The use of carbapenem antibiotics to treat infections caused by Enterobacterales expressing increasingly aggressive extended-spectrum ß-lactamases (ESBLs) has contributed to the emergence of carbapenem resistance. Enmetazobactam is a novel ESBL inhibitor being developed in combination with cefepime as a carbapenem-sparing option for infections caused by ESBL-producing Enterobacterales. Cefepime-enmetazobactam checkerboard MIC profiles were obtained for a challenge panel of cefepime-resistant ESBL-producing clinical isolates of Klebsiella pneumoniae. Sigmoid maximum effect (Emax) modeling described cefepime MICs as a function of enmetazobactam concentration with no bias. A concentration of 8 µg/ml enmetazobactam proved sufficient to restore >95% of cefepime antibacterial activity in vitro against >95% of isolates tested. These results support a fixed concentration of 8 µg/ml of enmetazobactam for MIC testing.

HIV Vaccine Development: 35 Years of Experimenting in the Funding of Biomedical Research.

Funding vaccine development research is more complicated than simply putting out an announcement of funds available. The funders must decide whether product development can be accomplished by purely applied research, or whether more fundamental knowledge is needed before product development can be started. If additional basic knowledge is needed, identifying the specific area of the knowledge gap can be a challenge. Additionally, when there appears to be a clear path of applied research sometimes obstacles are encountered that require a return to more basic work. After deciding on the work to be done, funders must attract the scientists with the broad range of needed skills to cover all the stages of development. Collaborations must be promoted and alliances with other funders and industry must be developed. Funders use multiple tools and strategies to accomplish these tasks with varying success.

Great Impostors: Extremely Compact, Merging Binary Neutron Stars in the Mass Gap Posing as Binary Black Holes.

Can one distinguish a binary black hole undergoing a merger from a binary neutron star if the individual compact companions have masses that fall inside the so-called mass gap of 3-5 M_{⊙}? For neutron stars, achieving such masses typically requires extreme compactness and in this work we present initial data and evolutions of binary neutron stars initially in quasiequilibrium circular orbits having a compactness C=0.336. These are the most compact, nonvacuum, quasiequilibrium binary objects that have been constructed and evolved to date, including boson stars. The compactness achieved is only slightly smaller than the maximum possible imposed by causality, C_{max}=0.355, which requires the sound speed to be less than the speed of light. By comparing the emitted gravitational waveforms from the late inspiral to merger and postmerger phases between such a binary neutron star vs a binary black hole of the same total mass we identify concrete measurements that serve to distinguish them. With that level of compactness, the binary neutron stars exhibit no tidal disruption up until merger, whereupon a prompt collapse is initiated even before a common core forms. Within the accuracy of our simulations the black hole remnants from both binaries exhibit ringdown radiation that is not distinguishable from a perturbed Kerr spacetime. However, their inspiral leads to phase differences of the order of ∼5 rad over an ∼81 km separation (1.7 orbits) while typical neutron stars exhibit phase differences of ≥20 rad. Although a difference of ∼5 rad can be measured by current gravitational wave laser interferometers (e.g., aLIGO/Virgo), uncertainties in the individual masses and spins will likely prevent distinguishing such compact, massive neutron stars from black holes.

Magnetohydrodynamic simulations of binary neutron star mergers in general relativity: Effects of magnetic field orientation on jet launching.

Binary neutron star mergers can be sources of gravitational waves coincident with electromagnetic counterpart emission across the spectrum. To solidify their role as multimessenger sources, we present fully 3D, general relativistic, magnetohydrodynamic simulations of highly spinning binary neutrons stars initially on quasicircular orbits that merge and undergo delayed collapse to a black hole. The binaries consist of two identical stars modeled as Γ = 2 polytropes with spin χ NS = 0.36 aligned along the direction of the total orbital angular momentum L. Each star is initially threaded by a dynamical unimportant interior dipole magnetic field. The field is extended into the exterior where a nearly force-free magnetosphere resembles that of a pulsar. The magnetic dipole moment µ is either aligned or perpendicular to L and has the same initial magnitude for each orientation. For comparison, we also impose symmetry across the orbital plane in one case where µ in both stars is aligned along L. We find that the lifetime of the transient hypermassive neutron star remnant, the jet launching time, and the ejecta (which can give rise to a detectable kilonova) are very sensitive to the magnetic field orientation. By contrast, the physical properties of the black hole + disk remnant, such as the mass and spin of the black hole, the accretion rate, and the electromagnetic (Poynting) luminosity, are roughly independent of the initial magnetic field orientation. In addition, we find imposing symmetry across the orbital plane does not play a significant role in the final outcome of the mergers. Our results suggest that, as in the black hole-neutron star merger scenario, an incipient jet emerges only when the seed magnetic field has a sufficiently large-scale poloidal component aligned to the initial orbital angular momentum. The lifetime [Δt ≳ 140(M NS/1.625 M ⊙) ms] and Poynting luminosities [L EM ≃ 1052 erg/s] of the jet, when it forms, are consistent with typical short gamma-ray bursts, as well as with the Blandford-Znajek mechanism for launching jets.

Black hole-neutron star coalescence: Effects of the neutron star spin on jet launching and dynamical ejecta mass.

Black hole-neutron star (BHNS) mergers are thought to be sources of gravitational waves (GWs) with coincident electromagnetic (EM) counterparts. To further probe whether these systems are viable progenitors of short gamma-ray bursts (SGRBs) and kilonovas, and how one may use (the lack of) EM counterparts associated with LIGO/Virgo candidate BHNS GW events to sharpen parameter estimation, we study the impact of neutron star spin in BHNS mergers. Using dynamical spacetime magnetohydrodynamic simulations of BHNSs initially on a quasicircular orbit, we survey configurations that differ in the BH spin (a BH/M BH = 0 and 0.75), the NS spin (a NS/M NS = -0.17, 0, 0.23, and 0.33), and the binary mass ratio (q = M BH:M NS = 3:1 and 5:1). The general trend we find is that increasing the NS prograde spin increases both the rest mass of the accretion disk onto the remnant black hole, and the rest mass of dynamically ejected matter. By a time Δt ~ 3500-5500M ~ 88-138(M NS/1.4 M ⊙) ms after the peak gravitational-wave amplitude, a magnetically driven jet is launched only for q = 3:1 regardless of the initial NS spin. The lifetime of the jets [Δt ~ 0.5-0.8(M NS/1.4 M ⊙) s] and their outgoing Poynting luminosity [L Poyn ~ 1051.5±0.5 erg/s] are consistent with typical SGRBs' luminosities and expectations from the Blandford-Znajek mechanism. By the time we terminate our simulations, we do not observe either an outflow or a large-scale magnetic-field collimation for the other systems we consider. The mass range of dynamically ejected matter is 10-4.5-10-2(M NS/1.4 M ⊙) M ⊙, which can power kilonovas with peak bolometric luminosities L knova ~ 1040-1041.4 erg/s with rise times ≲6.5 h and potentially detectable by the LSST.

Dynamically Stable Ergostars Exist: General Relativistic Models and Simulations.

We construct the first dynamically stable ergostars (equilibrium neutron stars that contain an ergoregion) for a compressible, causal equation of state. We demonstrate their stability by evolving both strict and perturbed equilibrium configurations in full general relativity for over a hundred dynamical timescales (≳30 rotational periods) and observing their stationary behavior. This stability is in contrast to earlier models which prove radially unstable to collapse. Our solutions are highly differentially rotating hypermassive neutron stars with a corresponding spherical compaction of C=0.3. Such ergostars can provide new insights into the geometry of spacetimes around highly compact, rotating objects and on the equation of state at supranuclear densities. Ergostars may form as remnants of extreme binary neutron star mergers and possibly provide another mechanism for powering short gamma-ray bursts.

Development of Broth Microdilution MIC and Disk Diffusion Antimicrobial Susceptibility Test Quality Control Ranges for the Combination of Cefepime and the Novel ß-Lactamase Inhibitor Enmetazobactam.

Third-generation cephalosporin resistance among Enterobacteriaceae, mediated by the spread of extended-spectrum ß-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone ß-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections. Using the Clinical and Laboratory Standards Institute (CLSI) M23 tier 2 study design, broth microdilution MIC and disk diffusion quality control (QC) ranges were determined for cefepime-enmetazobactam. Enmetazobactam was tested at a fixed concentration of 8 µg/ml in the MIC assay, and a cefepime-enmetazobactam disk mass of 30/20 µg was used in the disk diffusion assay. Escherichia coli ATCC 25922, E. coli ATCC 35218, E. coli NCTC 13353, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853 were chosen as reference strains. The CTX-M-15-producing E. coli NCTC 13353 isolate is recommended for routine testing to control for inhibition of ESBL activity by enmetazobactam. Broth microdilution MIC QC ranges spanned 3 to 4 doubling dilutions and contained 99.6% to 100.0% of obtained MIC values for the five reference strains. Disk diffusion yielded inhibition zone diameter QC ranges that spanned 7 mm and encompassed 97.1% to 100.0% of the obtained values. Quality control ranges were approved by the CLSI in 2017 (broth microdilution MIC) and 2019 (disk diffusion). The established QC ranges will ensure that appropriate assay performance criteria are attained using CLSI reference methodology when determining the susceptibility of clinical isolates to cefepime-enmetazobactam.

In Vitro Activity of Cefepime-Enmetazobactam against Gram-Negative Isolates Collected from U.S. and European Hospitals during 2014-2015.

Enmetazobactam, formerly AAI101, is a novel penicillanic acid sulfone extended-spectrum ß-lactamase (ESBL) inhibitor. The combination of enmetazobactam with cefepime has entered clinical trials to assess safety and efficacy in patients with complicated urinary tract infections. Here, the in vitro activity of cefepime-enmetazobactam was determined for 1,993 clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa collected in the United States and Europe during 2014 and 2015. Enmetazobactam at a fixed concentration of 8 µg/ml lowered the cefepime MIC90 from 16 to 0.12 µg/ml for Escherichia coli, from >64 to 0.5 µg/ml for Klebsiella pneumoniae, from 16 to 1 µg/ml for Enterobacter cloacae, and from 0.5 to 0.25 µg/ml for Enterobacter aerogenes Enmetazobactam did not enhance the potency of cefepime against P. aeruginosa Applying the Clinical and Laboratory Standards Institute susceptible-dose-dependent (SDD) breakpoint of 8 µg/ml to cefepime-enmetazobactam for comparative purposes resulted in cumulative inhibitions of 99.9% for E. coli, 96.4% for K. pneumoniae, 97.0% for E. cloacae, 100% for E. aerogenes, 98.1% for all Enterobacteriaceae assessed, and 82.8% for P. aeruginosa Comparator susceptibilities for all Enterobacteriaceae were 99.7% for ceftazidime-avibactam, 96.2% for meropenem, 90.7% for ceftolozane-tazobactam, 87% for cefepime (SDD breakpoint), 85.7% for piperacillin-tazobactam, and 81.2% for ceftazidime. For the subset of ESBL-producing K. pneumoniae isolates, the addition of 8 µg/ml enmetazobactam to cefepime lowered the MIC90 from >64 to 1 µg/ml, whereas the shift for 8 µg/ml tazobactam was from >64 to 8 µg/ml. Cefepime-enmetazobactam may represent a novel carbapenem-sparing option for empirical treatment of serious Gram-negative infections in settings where ESBL-producing Enterobacteriaceae are expected.

Lessons for general vaccinology research from attempts to develop an HIV vaccine.

In the past when large investments have been made in tackling narrow scientific challenges, the enormous expansion in our knowledge in one small area has had a spill-over effect on research and treatment of other diseases. The large investment in HIV vaccine development in recent years has the potential for such an effect on vaccine development for other diseases. HIV vaccine developers have experienced repeated failure using the standard approaches to vaccine development. This has forced them to consider immune responses in greater depth and detail. It has led to a recognition of the importance of epitopic specificity in both antibody and T cell responses. Also, it has led to an understanding of the importance of affinity maturation in antibody responses and the quality of T cell responses in T cell-mediated immunity. It has advanced the development of many novel vaccine vectors and vehicles that are now available for use in other vaccines. Further, it has focused attention on the impact of research funding mechanisms and community engagement on vaccine development. These developments and considerations have implications for vaccinology more generally. Some suggestions are made for investigators working on other "hard-to-develop" vaccines.

Beyond Piperacillin-Tazobactam: Cefepime and AAI101 as a Potent ß-Lactam-ß-Lactamase Inhibitor Combination.

Impeding, as well as reducing, the burden of antimicrobial resistance in Gram-negative pathogens is an urgent public health endeavor. Our current antibiotic armamentarium is dwindling, while major resistance determinants (e.g., extended-spectrum ß-lactamases [ESBLs]) continue to evolve and disseminate around the world. One approach to attack this problem is to develop novel therapies that will protect our current agents. AAI101 is a novel penicillanic acid sulfone ß-lactamase inhibitor similar in structure to tazobactam, with one important difference. AAI101 possesses a strategically placed methyl group that gives the inhibitor a net neutral charge, enhancing bacterial cell penetration. AAI101 paired with cefepime, also a zwitterion, is in phase III of clinical development for the treatment of serious Gram-negative infections. Here, AAI101 was found to restore the activity of cefepime against class A ESBLs (e.g., CTX-M-15) and demonstrated increased potency compared to that of piperacillin-tazobactam when tested against an established isogenic panel. The enzymological properties of AAI101 further revealed that AAI101 possessed a unique mechanism of ß-lactamase inhibition compared to that of tazobactam. Additionally, upon reaction with AAI101, CTX-M-15 was modified to an inactive state. Notably, the in vivo efficacy of cefepime-AAI101 was demonstrated using a mouse septicemia model, indicating the ability of AAI101 to bolster significantly the therapeutic efficacy of cefepime in vivo The combination of AAI101 with cefepime represents a potential carbapenem-sparing treatment regimen for infections suspected to be caused by Enterobacteriaceae expressing ESBLs.