Antimicrobial Agents and Chemotherapy最新文献

Conventional in vitro susceptibility testing methods may underestimate the bactericidal activity of antibiotics that are chemically unstable in aqueous media, thereby limiting their clinical translatability. Tigecycline is considered a representative example of such compounds, exhibiting notable therapeutic efficacy against a broad range of pathogens despite poor in vitro susceptibility profiles, as reflected by elevated MIC values. This discrepancy is likely attributable, at least in part, to the chemical instability of TGC under standard MIC assay conditions. In this manuscript, we propose a mechanism-based PK/PD modeling approach as a framework to overcome the limitations of traditional MIC assessments and to address potential discrepancies between intrinsic and experimentally measured apparent antibacterial activity. Dynamic time-kill curves for single and multiple dose scenarios of TGC against Mycobacterium abscessus (Mab) were experimentally simulated in 24-well plates, leveraging the chemical instability of TGC. Based on the resulting in vitro data, a mechanism-based model was developed to perform simulations for characterizing intrinsic efficacy and potency of TGC. While the in vitro MIC of TGC determined under standard conditions was determined as 3.125 mg/L, an intrinsic MIC simulated based on model predicted bacterial time-time kill curves was 0.5 mg/L. Model-based analysis also revealed that MIC under standard conditions was stemming from drug instability and bacterial growth rate in the utilized media. In conclusion, the PK/PD modeling and simulation-based MIC determination indicated that clinically achievable exposure levels of TGC are sufficient to kill Mab, underlining the therapeutic potential of TGC against Mab infections.

The persistence of multidrug-resistant Acinetobacter baumannii remains a clinical challenge. Cefepime/enmetazobactam is a novel combination with demonstrated activity against extended-spectrum β-lactamase-producing Enterobacterales, but its activity against Acinetobacter has not yet been thoroughly explored. We aimed to assess its activity against Acinetobacter spp., including multidrug-resistant strains producing carbapenem-hydrolyzing class D β-lactamases (CHDLs). We analyzed 208 clinical isolates of Acinetobacter spp., including 67 carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic susceptibility testing was conducted with cefepime, sulbactam, and imipenem, alone and in combination with enmetazobactam; the latter was also tested individually. Additionally, MICs of enmetazobactam/durlobactam and sulbactam/durlobactam were determined for CRAB and CHDL-producing A. baumannii ATCC 17978 transformants. PBP binding assays (IC₅₀), molecular docking, simulation studies with the enmetazobactam/OXA-23 adduct, hydrolysis kinetics (k_cat, K_m), and OXA-23 inhibition assays (IC₅₀, k_off, t_₁/₂) were performed to elucidate the mechanism of enmetazobactam and detect reduced susceptibility. Enmetazobactam showed high intrinsic activity against Acinetobacter spp., displaying reduced MICs against carbapenem-susceptible isolates. MIC_50/90 of the enmetazobactam/durlobactam combination was 2/2 mg/L for CHDL-producing A. baumannii. Enmetazobactam exhibited bactericidal activity comparable to sulbactam. Binding assays revealed that the antimicrobial activity is driven by selective affinity for PBP2 (IC₅₀ 3.6 mg/L) and PBP3 (IC₅₀ 4.2 mg/L). OXA-23 readily inactivated enmetazobactam, confirming the major role of CHDLs in resistance to enmetazobactam, via substrate-assisted de-acylation. This study evidences the potent antimicrobial activity of enmetazobactam against A. baumannii via inhibition of PBP2 and PBP3. Its combination with new OXA-type inhibitors (e.g., durlobactam) represents a potential therapeutic alternative for multidrug-resistant A. baumannii.

Increased resistance to β-lactams/β-lactamase inhibitors by mutations in β-lactamase genes, porins, and efflux pumps complicates the management of carbapenem-resistant Klebsiella pneumoniae (CRKP). Polymyxin B (PMB)-based combination therapy is the best alternative treatment for middle and low-income countries that cannot access the latest medicines. It is crucial to know both phenotypic and genotypic characteristics of a pathogen to understand the killing effect of each drug and its combinations. Hence, our objective was to incorporate mechanistic insights gained from resistance mechanisms of each isolate to develop a mechanism-based pharmacokinetic/pharmacodynamic model. Six clinical CRKP isolates with diverse genotypic resistance expressing bla_KPC, bla_NDM, porin, and mgrB mutations were used for static concentration time kill (SCTK) assays to evaluate the rate and extent of killing by monotherapy, double and triple combinations using PMB (0.5-64 mg/L), meropenem (10-120 mg/L), and fosfomycin (75-500 mg/L). Isolate BRKP28 expressed non-functional MgrB (a regulatory protein) and high-level phenotypic resistance (PMB MIC: >128 mg/L). In line with the observed resistance, the model estimated that BRKP28 had a reduced maximum killing rate constant for PMB (3.61 h⁻¹) relative to other isolates. The mechanistic synergy of PMB, due to outer membrane disruption, was incorporated into three isolates with porin mutations. PMB demonstrated 83%-88% mechanistic synergy with meropenem and 81%-98% with fosfomycin. The model further estimated that a very low concentration of PMB (0.49-0.64 mg/L) was sufficient to achieve 50% of the maximum synergy. Simulations using population pharmacokinetic models showed that combination therapy of PMB (1 mg/kg q12h) and fosfomycin (8 g q8h) achieved >73% reduction in area under the bacterial load-versus-time curve across four isolates. The triple combination therapy achieved a 67.7% reduction in non-carbapenamase producing isolate. These findings demonstrates that a low PMB dosing regimen (1 mg/kg q12h) can produce synergistic effects in combination therapy and may be effective in managing infections caused by CRKP, including PMB resistant isolates.

Rifampin, isoniazid, and ethambutol are the backbone of the regimen used to treat Mycobacterium kansasii-complex (MKC) lung disease. However, ethambutol pharmacokinetics/pharmacodynamics (PK/PD) studies to inform on optimal exposure target and clinical dose for MKC are lacking. We performed studies to determine ethambutol minimum inhibitory concentration (MIC), mutation frequency (3× MIC), a PK/PD study using the hollow fiber system model of MKC (HFS-MKC) using the reference ATCC#12478 strain, and Monte Carlo simulation experiments for clinical dose selection and susceptibility breakpoint. We also performed a literature search to generate ethambutol MIC distribution for MKC. First, nine studies were identified with MIC of 587 isolates, and MIC₅₀ and MIC₉₀ identified as 4 and 16 mg/L, respectively. Second, the ethambutol MIC of the ATCC strain was 8 mg/L, and the mutation frequency was 4.23 × 10^-2 CFU/mL. Third, in the HFS-MKC, ethambutol failed to kill M. kansasii below stasis (B₀), and resistance emerged rapidly. The target exposure was an AUC_0-24/MIC of 5.47 (95% confidence interval: 1.17-9.77). Fourth, Monte Carlo experiments of 10,000 virtual subjects identified doses of 1,200 and 3,000 mg to achieve or exceed target exposure in 18.21% and 58.57% of patients; and PK/PD MIC susceptibility breakpoints were determined as 2 and 4 mg/L, respectively. Doses >1,200 mg/day may have a higher likelihood of ocular toxicity. The risk of toxicity versus no microbial kill benefit in HFS-MKC suggests the need for better drugs compared to ethambutol in the treatment of MKC lung disease.

Francisella tularensis is a gram-negative, intracellular bacterium that causes the disease tularemia. Tularemia is prevalent in North America, Europe, and Asia and is typically treated with injected and orally administered antibiotics, including streptomycin, gentamicin, doxycycline, and ciprofloxacin, administered for 10 to 21 days. New therapeutic options are required to reduce the potential of a relapse of disease. Inhaled liposomal-encapsulated ciprofloxacin has demonstrated protection in a murine model of tularemia. The efficacy was further assessed in a nonhuman primate model of tularemia. Mixed-sex common marmosets were challenged with F. tularensis by the inhalational route, and the efficacy of ciprofloxacin delivered by either the inhalational (Apulmiq liposomal formulation) or oral route was compared. Antibiotics were initiated either at 24 h post-challenge (post-exposure prophylaxis) or at the onset of fever (treatment) and continued for 7 days. All control (untreated) animals succumbed to infection by 8 days post-challenge. All animals that received antibiotics, by either route, survived the duration of the study, with bacterial clearance in all but one animal that received inhalational ciprofloxacin. Antibiotic treatment also reduced the physiological and immunological responses observed when compared to animals that received no antibiotics. Histological changes in the lungs were less frequent, although mild, resolving lesions were present in animals treated with ciprofloxacin delivered at the onset of fever by either route.

The Mycobacterium avium complex (MAC) is the primary cause of pulmonary disease (PD) among nontuberculous mycobacteria, presenting a significant treatment challenge on a global scale. A long-term (≥12 months) three-drug regimen, typically including a macrolide, such as clarithromycin (CLR) or azithromycin, along with rifampicin and ethambutol, is recommended. However, many patients fail to respond adequately to therapy, and some eventually develop macrolide resistance, making the disease even more difficult to treat. This highlights the urgent need for improved therapeutic strategies. Here, we investigated the efficacy of clofazimine (CFZ) and bedaquiline (BDQ), both repurposed from multidrug-resistant tuberculosis therapy, against macrolide-resistant MAC. In macrophage infection assays, both CFZ and BDQ showed significant intracellular inhibitory activity against macrolide-resistant clinical isolates, with CFZ generally exhibiting stronger effects. In a chronic murine model of MAC-caused progressive PD, substitution of CLR with CFZ and BDQ in the treatment regimen led to marked reductions in bacterial loads in both lung and spleen compared with the standard regimen, achieving up to 0.86 log₁₀ CFU reduction in lung and 2.17 log₁₀ CFU in spleen tissues. These findings demonstrate that CFZ and BDQ retain potent activity against macrolide-resistant MAC and highlight their potential as promising components of alternative treatment regimens.

Retained hardware/prosthetic infections frequently require antimicrobial suppression therapy. Treatment options are often limited by resistance, allergies, and dosing frequencies. Dalbavancin (DAL) is a potentially attractive option for suppression of gram-positive infections given its potential for infrequent dosing. However, optimal DAL suppression dosing is unknown. An in silico pharmacokinetic/pharmacodynamic simulation was performed to assess the predicted dalbavancin concentration resulting from suppressive regimens. Serum levels were deemed adequate if the fAUC₂₄/MIC was above the PK target of 27.1. Patients at a U.S. medical center receiving DAL as suppressive therapy were reviewed. PK simulation of DAL dosed 1,500 mg monthly resulted in free serum concentrations above the PK target. Because many clinicians opt to initiate these regimens with two doses given 1 week apart, the next modeled regimen included this load, before initiating 1,500 mg monthly. This initial load did not significantly alter total drug exposure. The final simulated regimen was 1,000 mg monthly. With this simulation, the lower 95% CI fAUC₂₄/MIC fell just below the PK target for an isolate at the breakpoint. Nine patients who received dalbavancin 1,500 mg monthly as suppressive therapy were reviewed. All had retained hardware and received DAL for a median 591 days, with 7 patients still receiving treatment and no reported suppressive therapy failure. Monthly 1500 mg dalbavancin dosing for suppressive therapy is supported by this case series and PK simulation data. An initial weekly loading dose appears unnecessary. Reducing the monthly dose to 1000 mg may also be appropriate for certain patients, though clinical data is needed to support this practice.

Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of acute respiratory infections resulting in significant morbidity and mortality. Although approved influenza antivirals (e.g., oseltamivir and baloxavir) exist, concerns persist about the potential for emergence of drug-resistant variants, highlighting the continuing need for new antiviral therapies. Here, we describe the development of an orally bioavailable, direct-acting antiviral (VNT-101) with a novel mechanism of action: disrupting homo-oligomerization of the influenza nucleoprotein (NP) and thereby inhibiting viral RNA synthesis. Selection of drug-resistant mutants revealed amino acid substitutions mapping to the oligomerization domain of NP, and X-ray crystallography co-structure determination of VNT-101 complexed with recombinant NP confirmed VNT-101 binding in the oligomerization pocket. Biochemical experiments using size exclusion chromatography confirmed disruption of oligomerization when this chemotype is added to preparations of recombinant NP in vitro. VNT-101 has potent and specific activity against the currently circulating IAV subtypes H1N1 and H3N2, with mean EC₅₀ values ranging from 2 to 18 nM, and displays strong efficacy in a murine model of lethal influenza infection when administered either prophylactically or therapeutically. Importantly, VNT-101 remains active against influenza variants that are resistant to either oseltamivir or baloxavir and also has potent activity against highly pathogenic avian H5N1 and H7N9 isolates that have transmitted to humans and represent strains of potential pandemic concern. These studies support the continued development of VNT-101 to augment our therapeutic arsenal against both seasonal and pandemic influenza.

Candida albicans (C. albicans) is a commensal, drug-resistant opportunistic pathogen, and the eradication of invasive candidiasis represents a significant clinical challenge. This study investigated the inhibitory effect of isobavachalcone (IBC) on C. albicans growth and elucidated its mechanism. The antifungal activity of IBC was evaluated using minimum inhibitory concentration 90% (MIC₉₀) and minimum fungicidal concentration (MFC), combined with murine vaginitis and oral thrush models to assess in vivo efficacy. An MTT assay was used to assess drug safety. Mechanistic investigations included cell membrane/wall damage assessments, virulence factor inhibition, oxidative stress evaluation, ATP metabolism analysis, protein expression profiling, and target identification (including RT-qPCR, inhibitor intervention experiments, and related methodologies). The antifungal potency of IBC against C. albicans was demonstrated with a MIC₉₀ of 2 µg/mL and an MFC of 8 μg/mL. Against strain S393, IBC exhibited potent efficacy with median effective and effective concentrations of 1.301 µg/mL and 1.449 µg/mL, respectively. In vivo, IBC significantly alleviated vulvovaginal candidiasis and oropharyngeal thrush, outperforming fluconazole in therapeutic efficacy and mucosal protection. Mechanistic studies revealed that IBC prevented fungal invasion by inhibiting C. albicans adhesion and colonization on mucosal surfaces, mitigated inflammation through suppression of proinflammatory cytokine release, and downregulated the expression of ADE13, TPI1, and ADK1 genes, with ADK1 demonstrating the most significant suppression. Furthermore, IBC specifically bound to ADK1, inhibiting ATP synthesis and disrupting cellular energy metabolism in C. albicans. IBC represents a promising antifungal drug that acts by downregulating the ADE13, TPI1, and ADK1 genes. Its downregulation of ADK1 leads to impaired ATP synthesis, induced DNA damage, and suppressed fungal proliferation.

Antibacterial drug mechanisms have traditionally been examined through a drug-pathogen lens, with limited attention to host influences on drug activity. However, growing evidence suggests that the host environment is crucial for antibacterial efficacy. Pyrazinamide (PZA), a key component of modern tuberculosis therapy, exemplifies this complexity, exhibiting potent in vivo activity despite its inability to reduce Mycobacterium tuberculosis viability in standard in vitro culture. Here, using macrophage and murine infection models, we identify a critical role for CD4⁺ T cell-dependent cell-mediated immunity in PZA's antitubercular action. Using MHC class II knockout mice, we demonstrate that CD4 T-cell help is essential for PZA efficacy. While interferon gamma (IFN-γ) is required for PZA-mediated clearance of M. tuberculosis at extrapulmonary sites, bacterial reduction in the lungs occurs, independent of IFN-γ signaling. We show that PZA leverages cell-mediated immunity in part through activation of the oxidative burst. Our findings underscore the need to incorporate host factors into antibacterial drug evaluation and highlight potential avenues for host-directed therapies and adjunctive antibiotics in first- and second-line tuberculosis treatment.