Difficult-to-treat (DTR) Pseudomonas aeruginosa harboring Verona-Integron metallo-ß-lactamase (blaVIM): infection management and molecular analysis.

Pseudomonas aeruginosa harboring Verona Integron-encoded metallo-ß-lactamase enzymes (VIM-CRPA) have been associated with infection outbreaks in several parts of the world. In the US, however, VIM-CRPA remain rare. Starting in December 2018, we identified a cluster of cases in our institution. Herein, we present our epidemiological investigation and strategies to control/manage these challenging infections. This study was conducted in a large academic healthcare system in Miami, FL, between December 2018 and January 2022. Patients were prospectively identified via rapid molecular diagnostics when cultures revealed carbapenem-resistant P. aeruginosa. Alerts were received in real time by the antimicrobial stewardship program and infection prevention teams. Upon alert recognition, a series of interventions were performed as a coordinated effort. A retrospective chart review was conducted to collect patient demographics, antimicrobial therapy, and clinical outcomes. Thirty-nine VIM-CRPA isolates led to infection in 21 patients. The majority were male (76.2%); the median age was 52 years. The majority were mechanically ventilated (n = 15/21; 71.4%); 47.6% (n = 10/21) received renal replacement therapy at the time of index culture. Respiratory (n = 20/39; 51.3%) or bloodstream (n = 13/39; 33.3%) were the most common sources. Most infections (n = 23/37; 62.2%) were treated with an aztreonam-avibactam regimen. Six patients (28.6%) expired within 30 days of index VIM-CRPA infection. Fourteen isolates were selected for whole genome sequencing. Most of them belonged to ST111 (12/14), and they all carried blaVIM-2 chromosomally. This report describes the clinical experience treating serious VIM-CRPA infections with either aztreonam-ceftazidime/avibactam or cefiderocol in combination with other agents. The importance of implementing infection prevention strategies to curb VIM-CRPA outbreaks is also demonstrated.

Durlobactam, a Diazabicyclooctane ß-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum ß-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb ß-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KI app 9.2 ± 0.9 µM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KI app 3.3 ± 0.6 µM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KI app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 µg/mL, similar to the ranges for meropenem (1-32 µg/mL) and imipenem (0.5-64 µg/mL). In ß-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel ß-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

AMPLON: Amplifying DNA with Multi-Arm Priming and Looping Optimization of Nucleic Acid.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, we present a novel approach for Amplifying DNA with Multi-arm Priming and Looping Optimization of Nucleic Acid (AMPLON). AMPLON relied on a novel polymeric material that utilizes a unique set of multi-arm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries ssDNA complementing the sense or antisense sequence of the target DNA (n = 6; 50% sense to 50% antisense sequences). The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multi-arm large DNA amplicon synthesis through successive DNA looping and unlooping steps. The developed AMPLON enables highly specific and sensitive detection of the targeted nucleic acid sequence. Using HIV-1 as a model clinical target, AMPLON exhibited high sensitivity, detecting target concentrations as low as 100 copies/mL, and selectively amplifying HIV-1 in the presence of other DNA and RNA viruses, such as HBV and HCV. Compared to a quantitative real-time PCR (qRT-PCR) assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 minutes, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology. This article is protected by copyright. All rights reserved.

Hetero-antagonism of avibactam and sulbactam with cefiderocol in carbapenem-resistant Acinetobacter spp.

The emergence of Gram-negative bacteria resistant to multiple antibiotics, particularly carbapenem-resistant (CR) Acinetobacter strains, poses a significant threat globally. Despite efforts to develop new antimicrobial therapies, limited progress has been made, with only two drugs-cefiderocol and sulbactam-durlobactam-showing promise for CR-Acinetobacter infections. Cefiderocol, a siderophore cephalosporin, demonstrates promising efficacy in the treatment of Gram-negative infections. However, resistance to cefiderocol has been reported in A. baumannii. Combination therapies, such as cefiderocol with avibactam or sulbactam, show reduced MICs against cefiderocol-non-susceptible strains with in vivo efficacy, although the outcomes can be complex and species-specific. In the present work, the molecular characterization of spontaneous cefiderocol-resistant variants, a CRAB strain displaying antagonism with sulbactam and an A. lwoffii strain showing antagonism with avibactam, were studied. The results reveal intriguing insights into the underlying mechanisms, including mutations affecting efflux pumps, transcriptional regulators, and iron homeostasis genes. Moreover, gene expression analysis reveals significant alterations in outer membrane proteins, iron homeostasis, and ß-lactamases, suggesting adaptive responses to selective pressure. Understanding these mechanisms is crucial for optimizing treatment strategies and preventing adverse clinical outcomes. This study highlights the importance of preemptively assessing drug synergies to navigate the challenges posed by antimicrobial resistance in CR-Acinetobacter infections.

14-Year Epidemiologic study of Pseudomonas aeruginosa bloodstream infection incidence and resistance in the Veterans Health Administration system, 2009-2022.

Background: Multidrug resistant Pseudomonas aeruginosa (PA) represents a serious threat to hospitalized patients. Characterizing the incidence of PA infection and degree of resistance can inform empiric treatment and preventative measures. Objectives: We sought to describe trends in incidence and resistance characteristics of PA bloodstream infections (BSI) observed within the Veterans Health Administration (VHA) system and identify factors contributing to higher observed mortality within this population. Methods: We characterized demographic and clinical features of unique patients among the VHA population presenting with their first episode of PA-BSI between 2009 and 2022 and summarized trends related to mortality and resistance phenotype based on year and geographical location. We additionally used logistic regression analysis to identify predictors of 30-day mortality among this cohort. Results: We identified 8039 PA-BSIs during the study period, 32.7% of which were hospital onset. Annual PA-BSI cases decreased by 35.8%, and resistance among all antimicrobial classes decreased during the study period, while the proportion of patients receiving early active treatment based on susceptibility testing results increased. Average 30-day mortality rate was 23.3%. Higher Charlson Comorbidity Index, higher mAPACHE score, VHA facility complexity 1b and hospital-onset cases were associated with higher mortality, and early active treatment was associated with lower mortality. Conclusions: PA-BSI resistance decreased across the VHA system during the study period. Further investigation of antimicrobial stewardship measures possibly contributing to the observed decreased resistance in this cohort and identification of measures to improve on the high mortality associated with PA-BSI in the VHA population is warranted.

Ω-Loop mutations control the dynamics of the active site by modulating a network of hydrogen bonds in PDC-3 ß-lactamase.

The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. To explore the relationships between structural dynamics and altered substrate specificity as a result of amino acid substitutions in PDC-3, innovative computational methods like machine learning driven adaptive bandit molecular dynamics simulations and markov state modeling of the wild-type PDC-3 and nine clinically identified variants were conducted. Our analysis reveals that structural changes in the Ω loop controls the dynamics of the active site. The E219K and Y221A substitutions have the most pronounced effects. The modulation of three key hydrogen bonds K67(sc)-G220(bb), Y150(bb)-A292(bb) and N287(sc)-N314(sc) were found to result in an expansion of the active site, which could have implications for the binding and inactivation of cephalosporins. Overall, the findings highlight the importance of understanding the structural dynamics of PDC-3 in the development of new treatments for antibiotic-resistant infections.

Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

Rational Design of Benzobisheterocycle Metallo-ß-Lactamase Inhibitors: A Tricyclic Scaffold Enhances Potency against Target Enzymes.

Antimicrobial resistance is a global public health threat. Metallo-ß-lactamases (MBLs) inactivate ß-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1-B3). We report design, synthesis, and evaluation of BTZ analogues. Structure-activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.

In vitro activity of sulbactam-durlobactam against colistin-resistant and/or cefiderocol-non-susceptible, carbapenem-resistant Acinetobacter baumannii collected in U.S. hospitals.

The activity of a novel ß-lactamase inhibitor combination, sulbactam-durlobactam (SUL-DUR), was tested against 87 colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant Acinetobacter baumannii clinical isolates collected from U.S. hospitals between 2017 and 2019. Among them, 89% and 97% were susceptible to SUL-DUR and imipenem plus SUL-DUR, with MIC50/MIC90 values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. The presence of amino acid substitutions in penicillin-binding protein 3, including previously reported A515V or T526S, was associated with SUL-DUR non-susceptibility.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid ß-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-ß-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Population pharmacokinetics and humanized dosage regimens matching the peak, area, trough, and range of amikacin plasma concentrations in immune-competent murine bloodstream and lung infection models.

Amikacin is an FDA-approved aminoglycoside antibiotic that is commonly used. However, validated dosage regimens that achieve clinically relevant exposure profiles in mice are lacking. We aimed to design and validate humanized dosage regimens for amikacin in immune-competent murine bloodstream and lung infection models of Acinetobacter baumannii. Plasma and lung epithelial lining fluid (ELF) concentrations after single subcutaneous doses of 1.37, 13.7, and 137 mg/kg of body weight were simultaneously modeled via population pharmacokinetics. Then, humanized amikacin dosage regimens in mice were designed and prospectively validated to match the peak, area, trough, and range of plasma concentration profiles in critically ill patients (clinical dose: 25-30 mg/kg of body weight). The pharmacokinetics of amikacin were linear, with a clearance of 9.93 mL/h in both infection models after a single dose. However, the volume of distribution differed between models, resulting in an elimination half-life of 48 min for the bloodstream and 36 min for the lung model. The drug exposure in ELF was 72.7% compared to that in plasma. After multiple q6h dosing, clearance decreased by ~80% from the first (7.35 mL/h) to the last two dosing intervals (~1.50 mL/h) in the bloodstream model. Likewise, clearance decreased by 41% from 7.44 to 4.39 mL/h in the lung model. The humanized dosage regimens were 117 mg/kg of body weight/day in mice [administered in four fractions 6 h apart (q6h): 61.9%, 18.6%, 11.3%, and 8.21% of total dose] for the bloodstream and 96.7 mg/kg of body weight/day (given q6h as 65.1%, 16.9%, 10.5%, and 7.41%) for the lung model. These validated humanized dosage regimens and population pharmacokinetic models support translational studies with clinically relevant amikacin exposure profiles.

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.

Structural basis of metallo-ß-lactamase resistance to taniborbactam.

The design of inhibitors against metallo-ß-lactamases (MBLs), the largest family of carbapenemases, has been a strategic goal in designing novel antimicrobial therapies. In this regard, the development of bicyclic boronates, such as taniborbactam (TAN) and xeruborbactam, is a major achievement that may help in overcoming the threat of MBL-producing and carbapenem-resistant Gram-negative pathogens. Of concern, a recent report has shown that New Delhi MBL-9 (NDM-9) escapes the inhibitory action of TAN by a single amino acid substitution with respect to New Delhi MBL-1 (NDM-1), the most widely disseminated MBL. Here, we report a docking and computational analysis that identifies that "escape variants" against TAN can arise by disruption of the electrostatic interaction of negative charges in the active site loops of MBLs with the N-(2-aminoethyl)cyclohexylamine side chain of TAN. These changes result in non-productive binding modes of TAN that preclude reaction with the MBLs, a phenomenon that is not restricted to NDM-9. This analysis demonstrates that single amino acid substitutions in non-essential residues in MBL loops can unexpectedly elicit resistance to TAN.

Draft genome sequence of Lacticaseibacillus rhamnosus CRL 2244, a strain with strong killing effect on carbapenem-resistant Acinetobacter baumannii.

We report here a draft genome assembly of Lacticaseibacillus rhamnosus CRL 2244, recovered from wastewater in Argentina. The genome has a size of 2,898,100 bp, with G + C content of 46.73%. Comparative analysis reveals that its closest relative is L. rhamnosus 1.0320 (GCF_006151905.1), with an average nucleotide identity of 97.46%.

Clinical Outcomes and Bacterial Characteristics of Carbapenem-resistant Acinetobacter baumannii Among Patients From Different Global Regions.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAb) is 1 of the most problematic antimicrobial-resistant bacteria. We sought to elucidate the international epidemiology and clinical impact of CRAb. METHODS: In a prospective observational cohort study, 842 hospitalized patients with a clinical CRAb culture were enrolled at 46 hospitals in five global regions between 2017 and 2019. The primary outcome was all-cause mortality at 30 days from the index culture. The strains underwent whole-genome analysis. RESULTS: Of 842 cases, 536 (64%) represented infection. By 30 days, 128 (24%) of the infected patients died, ranging from 1 (6%) of 18 in Australia-Singapore to 54 (25%) of 216 in the United States and 24 (49%) of 49 in South-Central America, whereas 42 (14%) of non-infected patients died. Bacteremia was associated with a higher risk of death compared with other types of infection (40 [42%] of 96 vs 88 [20%] of 440). In a multivariable logistic regression analysis, bloodstream infection and higher age-adjusted Charlson comorbidity index were independently associated with 30-day mortality. Clonal group 2 (CG2) strains predominated except in South-Central America, ranging from 216 (59%) of 369 in the United States to 282 (97%) of 291 in China. Acquired carbapenemase genes were carried by 769 (91%) of the 842 isolates. CG2 strains were significantly associated with higher levels of meropenem resistance, yet non-CG2 cases were over-represented among the deaths compared with CG2 cases. CONCLUSIONS: CRAb infection types and clinical outcomes differed significantly across regions. Although CG2 strains remained predominant, non-CG2 strains were associated with higher mortality. Clinical Trials Registration. NCT03646227.

The evolution of diverse antimicrobial responses in vancomycin-intermediate Staphylococcus aureus and its therapeutic implications.

Staphylococcus aureus causes endocarditis, osteomyelitis, and bacteremia. Clinicians often prescribe vancomycin as an empiric therapy to account for methicillin-resistant S. aureus (MRSA) and narrow treatment based on culture susceptibility results. However, these results reflect a single time point before empiric treatment and represent a limited subset of the total bacterial population within the patient. Thus, while they may indicate that the infection is susceptible to a particular drug, this recommendation may no longer be accurate during therapy. Here, we addressed how antibiotic susceptibility changes over time by accounting for evolution. We evolved 18 methicillin-susceptible S. aureus (MSSA) populations under increasing vancomycin concentrations until they reached intermediate resistance levels. Sequencing revealed parallel mutations that affect cell membrane stress response and cell-wall biosynthesis. The populations exhibited repeated cross-resistance to daptomycin and varied responses to meropenem, gentamicin, and nafcillin. We accounted for this variability by deriving likelihood estimates that express a population's probability of exhibiting a drug response following vancomycin treatment. Our results suggest antistaphylococcal penicillins are preferable first-line treatments for MSSA infections but also highlight the inherent uncertainty that evolution poses to effective therapies. Infections may take varied evolutionary paths; therefore, considering evolution as a probabilistic process should inform our therapeutic choices.

Effectiveness of Sotrovimab in Preventing COVID-19-Related Hospitalizations or Deaths Among US Veterans During Omicron BA.1.

Background: The real-world clinical effectiveness of sotrovimab in preventing coronavirus disease 2019 (COVID-19)-related hospitalization or mortality among high-risk patients diagnosed with COVID-19, particularly after the emergence of the Omicron variant, needs further research. Method: Using data from the US Department of Veterans Affairs (VA) health care system, we adopted a target trial emulation design in our study. Veterans aged ≥18 years, diagnosed with COVID-19 between December 1, 2021, and April 4, 2022, were included. Patients treated with sotrovimab (n = 2816) as part of routine clinical care were compared with all eligible but untreated patients (n = 11,250). Cox proportional hazards modeling estimated the hazard ratios (HRs) and 95% CIs for the association between receipt of sotrovimab and outcomes. Results: Most (90%) sotrovimab recipients were ≥50 years old, and 64% had ≥2 mRNA vaccine doses or ≥1 dose of Ad26.COV2. During the period that BA.1 was dominant, compared with patients not treated, sotrovimab-treated patients had a 70% lower risk of hospitalization or mortality within 30 days (HR, 0.30; 95% CI, 0.23-0.40). During BA.2 dominance, sotrovimab-treated patients had a 71% (HR, 0.29; 95% CI, 0.08-0.98) lower risk of 30-day COVID-19-related hospitalization, emergency room visits, or urgent care visits (defined as severe COVID-19) compared with patients not treated. Conclusions: Using national real-world data from high-risk and predominantly vaccinated veterans, administration of sotrovimab, compared with contemporary standard treatment regimens, was associated with reduced risk of 30-day COVID-19-related hospitalization or all-cause mortality during the Omicron BA.1 period.