Antimicrobial Agents and Chemotherapy最新文献

Carbapenemase OXA-48 and its variants pose a serious threat to the development of effective treatments for bacterial infections. OXA-48-producing Enterobacterales are the most prevalent carbapenemase-producing bacteria in large parts of the world. Although these bacteria exhibit low-level carbapenem resistance in vitro, the infections they cause are challenging to treat with conventional therapies, owing to their spread and complex detection in clinical settings. However, numerous β-lactamase inhibitors (BLIs) are currently in the pipeline or late clinical stages. To assess the potential of these compounds, this study compared the efficacy against OXA-48 of novel β-lactamase inhibitors, specifically the 1,6-diazabicyclo[3,2,1]octanes (DBOs) avibactam, relebactam, zidebactam, nacubactam, and durlobactam, along with the cyclic and bicyclic boronates vaborbactam, taniborbactam, and xeruborbactam. The extensive kinetics assays identified xeruborbactam, taniborbactam, and durlobactam, together with the already established avibactam, as BLIs with superior biochemical performance. Susceptibility testing further validated these findings but also demonstrated significantly improved bacterial killing by the DBOs zidebactam, nacubactam, and durlobactam. On the other hand, binding studies demonstrated the superior inhibitory capacity of the BLIs durlobactam and xeruborbactam. Combinations, such as cefepime/zidebactam, meropenem/nacubactam, and sulbactam/durlobactam, show promising activity against OXA-48-producing Enterobacterales, while ceftazidime/avibactam, cefepime/taniborbactam, and meropenem/xeruborbactam combinations also appear highly active, largely due to the excellent kinetics of these new inhibitors. Overall, this comprehensive analysis provides important insights into the effectiveness of new BLIs against OXA-48-producing Enterobacterales, highlighting xeruborbactam, durlobactam, and avibactam as leading candidates. Additionally, BLIs like zidebactam, nacubactam, and taniborbactam also showed potential in addressing the clinical challenges posed by OXA-48-mediated antimicrobial resistance.

Novel and repurposed antiviral drugs are available for the treatment of coronavirus disease 2019 (COVID-19). However, antiviral combinations may be more potent and lead to faster viral clearance, but the methods for screening antiviral combinations against respiratory viruses are not well established and labor-intensive. Here, we describe a time-efficient (72-96 h) and simple in vitro drug-sensitivity assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using standard 96-well plates. We employ different synergy models (zero interaction potency, highest single agent, Loewe, Bliss) to determine the efficacy of antiviral therapies and synergistic combinations against ancestral and emerging clinical SARS-CoV-2 strains. We found that monotherapy of remdesivir, nirmatrelvir, and active metabolite of molnupiravir (EIDD-1931) demonstrated baseline EC50s within clinically achievable levels of 4.34 mg/L (CI: 3.74-4.94 mg/L), 1.25 mg/L (CI: 1.10-1.45 mg/L), and 0.25 mg/L (CI: 0.20-0.30 mg/L), respectively, against the ancestral SARS-CoV-2 strain. However, their efficacy varied against newer Omicron variants BA.1.1.15 and BA.2, particularly with the protease inhibitor nirmatrelvir. We also found that remdesivir and nirmatrelvir have a consistent, strong synergistic effect (Bliss synergy score >10) at clinically relevant drug concentrations (nirmatrelvir 0.25-1 mg/L with remdesivir 1-4 mg/L) across all SARS-CoV-2 strains tested. This method offers a practical tool that streamlines the identification of effective combination therapies and the detection of antiviral resistance. Our findings support the use of antiviral drug combinations targeting multiple viral components to enhance COVID-19 treatment efficacy, particularly in the context of emerging viral strains.

Hand, foot, and mouth disease (HFMD) is a serious pediatric infectious disease that causes immeasurable physical and mental health burdens. Currently, there is a lack of information on the mechanisms of HFMD severity and early diagnosis. We performed metabolomic profiling of sera from 84 Enterovirus A71 (EV-A71) infections and 45 control individuals. Targeted metabolomics assays were employed to further validate some of the differential metabolic molecules. We identified significant molecular changes in the sera of HFMD patients compared to healthy controls (HCs). A total of 54, 60, 35, and 62 differential metabolites were screened between mild cases and HCs, severe cases and HCs, severe cases and mild cases, and among the three groups, respectively. These differential metabolites implicated dysregulation of the tricarboxylic acid cycle, alanine, aspartate, and glutamate metabolism, and valine, leucine, and isoleucine biosynthesis. The diagnostic panel based on some overlapped differential metabolites could effectively discriminate severe cases from mild cases with an AUC of 0.912 (95% CI: 0.85-0.97) using the logistic regression model. Next, we found the elevation of serum sphingosine 1-phosphate (S1P) level in EV-A71 infection mice, which was similar to clinical observation. Importantly, after blocking the release of S1P by MK571, the clinical symptoms and survival of mice were significantly improved, involving the reduction of leukocyte infiltration in infected brain tissues. Collectively, our data provided a landscape view of metabolic alterations in EV-A71 infected children and revealed regulating S1P metabolism was an exploitable therapeutic target against EV-A71 infection.

Vancomycin causes kidney injury by accumulating in the proximal tubule, likely mediated by megalin uptake. Protamine is a putative megalin inhibitor that shares binding sites with heparin and is approved for the treatment of heparin overdose. We employed a well-characterized Sprague-Dawley rat model to assess kidney injury and function in animals that received vancomycin, protamine alone, or vancomycin plus protamine over 5 days. Urinary KIM-1 was used as the primary measure for kidney injury, while plasma iohexol clearance was calculated to assess kidney function. Animals had samples drawn pre-treatment in order to serve as their own controls. Additionally, since protamine is not a known nephrotoxin, the protamine group also served as a control. Cellular inhibition studies were performed to assess the ability of protamine to inhibit organic anion transporter (OAT1 and OAT3) and organic cation transporter-2 (OCT2). Rats that received vancomycin alone had significantly increased urinary KIM-1 on day 2 (24.9 ng/24 h, 95% CI 1.87-48.0) compared to the protamine alone group. By day 4, animals that received protamine with their vancomycin had KIM-1 amounts that were elevated compared to protamine alone as a base comparison (KIM-1 29.0 ng/24 h, 95% CI 5.0-53.0). No statistically observed differences were identified for iohexol clearance changes between drug groups or when comparing clearance change from baseline (P > 0.05). No substantial inhibition of OAT1, OAT3, or OCT2 was observed with protamine. IC₅₀ values for protamine were 0.1 mM for OAT1 and OAT3 and 0.043 mM for OCT2. Protamine, when added to vancomycin therapy, delays vancomycin-induced kidney injury as defined by urinary KIM-1 in the rat model by 1-3 days. Protamine putatively acts through the blockade of megalin and does not appear to have significant inhibition on OAT1, OAT3, or OCT2. Since protamine is an approved FDA medication, it has clinical potential as a therapeutic to reduce vancomycin-related kidney injury; however, greater utility may be found by pursuing compounds with fewer adverse event liabilities.

Artemisinin-based combination therapies (ACTs) are vital for malaria treatment, but these are threatened by blood-stage persisters-dormant forms of Plasmodium parasites that can survive drug exposure and cause recrudescent infections. Here, we present improved protocols for efficient preparation of pure Plasmodium falciparum persister populations without the need for magnetically activated columns, sorbitol exposure, or prolonged manipulations. Our protocols transformed actively replicating parasites into persister populations by exposing mixed blood-stage parasites to three or four consecutive daily 6 h pulses of 700 nM or 200 nM dihydroartemisinin (DHA). In micrographs of Giemsa-stained cells, we observed different persister morphologies: Type I persisters containing a rounded magenta-stained nucleus accompanied by a local region of blue-stained cytoplasm; and the more-prevalent Type II persisters characterized by a dark round or irregular-appearing nucleus and faded or no detectable cytoplasm. We also observed cells with disorganized nuclear and cytoplasmic structure, suggesting possible autophagic processes of destruction and remodeling. Recrudescence of actively replicating parasites to starting parasitemia or higher occurred around 17-22 days after initial DHA exposure. Differential expression patterns of the acetyl CoA carboxylase (acc) and skeleton binding protein 1 (sbp1) genes during DHA treatment, dormancy, and recrudescence highlighted the evolution of physiologic states and metabolic changes underlying persister formation and recovery. Our findings suggest hypotheses and questions for further research to understand the cellular pathways of dormancy and uncover strategies to thwart parasite survival after drug exposure.

Pseudomonas aeruginosa is a clinically important Gram-negative pathogen responsible for a wide variety of serious nosocomial and community-acquired infections. Antibiotic resistance is a major concern, as this organism has a wide variety of resistance mechanisms, including chromosomal class C (bla_PDC) and D (bla_OXA-50 family) β-lactamases, efflux pumps, porin channels, and the ability to readily acquire additional β-lactamases. Surveillance studies can reveal the diversity and distribution of β-lactamase alleles but are difficult and expensive to conduct. Herein, we apply a novel approach, using publicly available data derived from whole genome sequences, to explore the diversity and distribution of β-lactamase alleles across 30,452 P. aeruginosa isolates. The most common alleles were bla_PDC-3, bla_PDC-5, bla_PDC-8, bla_OXA-488, bla_OXA-50, and bla_OXA-486. Interestingly, only 43.6% of assigned bla_PDC alleles were encountered, and the 10 most common bla_PDC and intrinsic bla_OXA alleles represent approximately 75% of their respective total alleles, while many other assigned alleles were extremely uncommon. As anticipated, differences were observed over time and geography. Surprisingly, more distinct unassigned alleles were encountered than distinct assigned alleles. Understanding the diversity and distribution of β-lactamase alleles helps to prioritize variants for further research, select targets for drug development, and may aid in selecting therapies for a given infection.

Klebsiella pneumoniae carbapenemases (KPCs) are widespread β-lactamases that are a major cause of clinical non-susceptibility of Gram-negative bacteria to carbapenems and other β-lactam antibiotics. Ceftazidime combined with the β-lactamase inhibitor avibactam (CAZ-AVI) has been effective in treating infections by KPC-producing bacteria, but emerging KPC variants confer resistance to the combination. Taniborbactam and ledaborbactam are bicyclic boronate β-lactamase inhibitors currently under development with cefepime and ceftibuten, respectively, to treat carbapenem-resistant bacterial infections. Here, we assessed the effects of clinically important KPC-2 and KPC-3 variants (V240G, D179Y, and D179Y/T243M) on the antibacterial activity of cefepime-taniborbactam (FEP-TAN) and ceftibuten-ledaborbactam (CTB-LED) and examined catalytic activity and inhibition of these variants. Unlike CAZ-AVI, FEP-TAN and CTB-LED were highly active against Escherichia coli strains expressing these KPC variants. Experiments with purified enzymes showed that FEP and CTB were poorly hydrolyzed by the KPC variants and had weak affinity for variants containing D179Y. In addition, the D179Y substitution in KPC-2 reduced inhibition by TAN and LED, but inactivation efficiencies (k₂/K) for these inhibitors were significantly higher than those for AVI. K₂/K was less affected for D179Y-containing KPC-3 variants, and robust inhibition was observed by TAN, LED, and AVI. Together, the findings illustrate a biochemical basis for FEP-TAN and CTB-LED efficacy in KPC variant-mediated CAZ-AVI resistance backgrounds, whereby the boronate inhibitors have sufficient inhibitory activity, while FEP and CTB are poor substrates and bind to the variant enzymes with reduced affinity.

Acinetobacter baumannii is a clinically important, Gram-negative pathogen responsible for a wide variety of nosocomial and community-acquired infections. Antibiotic resistance is a serious concern, as the organism has a wide variety of intrinsic resistance mechanisms, including chromosomal class C (bla_ADC) and D (bla_OXA-51 family) β-lactamases, and the ability to readily acquire additional β-lactamases. Surveillance studies can reveal the diversity and distribution of β-lactamase alleles, but are difficult and expensive to conduct. Herein, we describe an approach using publicly available data derived from whole genome sequences, to explore the diversity and distribution of β-lactamase alleles across 28,330 isolates. The most common intrinsic alleles at the time of writing were bla_ADC-73, bla_ADC-30, bla_ADC-222, bla_ADC-33, and bla_OXA-66, and the most common acquired allele was bla_OXA-23. Interestingly, only 63.0% of assigned bla_ADC alleles were encountered and the 10 most common bla_ADC and intrinsic bla_OXA alleles represented approximately 75% of their respective gene totals while dozens were extremely infrequent. Differences were observed over time and geography. Surprisingly, more distinct unassigned (i.e., lacking a bla_ADC or bla_OXA number) alleles were encountered than distinct, assigned alleles. Understanding the diversity and distribution of β-lactamase alleles helps to prioritize variants for further research, selects targets for drug development, and may aid in selecting therapies for a given infection.

Xpert MTB/RIF Ultra (Ultra)-detected rifampicin-resistant tuberculosis (TB) is often programmatically confirmed using MTBDRplus. There are limited data on discordant results, including when re-tested using newer methods, like FluoroType MTBDR (FT-MTBDR) and targeted deep sequencing. MTBDRplus rifampicin-susceptible isolates from people with Ultra rifampicin-resistant sputum were identified from a South African programmatic laboratory. FT-MTBDR and single molecule-overlapping reads (SMOR; rpoB, inhA, katG) on isolate DNA were done (SMOR was used as a reference standard). Between 1 April 2021 and 30 September 2022, 8% (109/1347) of Ultra rifampicin-resistant specimens were MTBDRplus-susceptible. Of 89% (97/109) isolates with a sequenceable rpoB, SMOR resolved most in favor of Ultra (79% [77/97]). Sputum with lower mycobacterial load was associated with Ultra false-positive resistance (46% [11/24] of "very low" Ultra had false resistance vs 12% [9/73; P = 0.0004] of ≥"low"), as were Ultra heteroresistance calls (all wild-type probes, ≥1 mutant probe) (62% [23/37 vs 25% 15/60] for Ultra without heteroresistance calls; P = 0.0003). Of the 91% (88/97) of isolates successfully tested by FT-MTBDR, 55% (48/88) were FT-MTBDR rifampicin-resistant and 45% (40/88) susceptible, translating to 69% (47/68) sensitivity and 95% (19/20) specificity. In the 91% (99/109) of isolates with inhA and katG sequenced, 62% (61/99) were SMOR isoniazid-susceptible. When Ultra and MTBDRplus rifampicin results are discordant, Ultra is more likely to be correct, and FT-MTBDR agrees more with Ultra than MTBDRplus; however, lower load and the Ultra heteroresistance probe pattern were risk factors for Ultra false rifampicin-resistant results. Most people with Ultra-MTBDRplus discordant resistance results were isoniazid-susceptible. These data have implications for drug-resistant TB diagnosis.