Antimicrobial Agents and Chemotherapy最新文献

Candida glabrata is the second most common cause of invasive candidiasis and is widely known to have reduced susceptibility to fluconazole relative to many other Candida spp. Upc2A is a transcription factor that regulates ergosterol biosynthesis gene expression under conditions of sterol stress such as azole drug treatment or hypoxia. Through an in vitro microevolution experiment, we found that loss-of-function mutants of the ATF/CREB transcription factor CST6 suppresses the fluconazole hyper-susceptibility of the upc2A∆ mutant. Here, we confirm that the cst6∆ upc2A∆ mutants are resistant to fluconazole but not to hypoxia relative to the upc2A∆ mutant. Sterol analysis of these mutants indicates that this suppression phenotype is not due to restoration of ergosterol levels in the cst6∆ upc2A∆ mutant. Furthermore, increased expression of CDR1, the efflux pump implicated in the vast majority of azole-resistant C. glabrata strains, does not account for the suppression phenotype. Instead, our data suggest that this effect is due in part to increased expression of the adhesin EPA3, which has been shown by others to reduce fluconazole susceptibility in C. glabrata. In addition, we find that loss of both UPC2A and CST6 reduces the expression of mitochondrial and respiratory genes and that this also contributes to the suppression phenotype as well as to the resistance of cst6∆ to fluconazole. These latter data further emphasize the connection between mitochondrial function and azole susceptibility.

Mycobacterium abscessus exhibits intrinsic resistance to most antibiotics, hence leading to infections that are difficult to treat. To address this issue, the identification of new molecular targets is essential for the development or repositioning of therapeutic agents. This study demonstrated that the MAB_2362-knockout strain, Mab^Δ2362, became significantly susceptible to a range of antibiotics, not only in vitro but also exhibited susceptibility to rifabutin, bedaquiline, and linezolid in vivo. While the bacterial burden of the wild-type M. abscessus (Mab^Wt) increased by over 1 log₁₀ CFU/lung in a murine infection model 16 days post-infection, that of Mab^Δ2362 strain decreased by more than 1 log₁₀ CFU/lung, which suggests that the disruption leads to attenuation. Bioinformatics analysis revealed that MAB_2362 shares the highest similarity (41.35%) with SteA, a protein known to influence cell division in Corynebacterium glutamicum, suggesting that MAB_2362 might be involved in cell division. Mab^Δ2362 cells exhibited a median length of 2.62 µm, which was substantially longer than the 1.44 µm recorded for Mab^Wt cells. Additionally, multiple cell division septa were observed in 42% of Mab^Δ2362 cells, whereas none were seen in Mab^Wt cells. An ethidium bromide uptake assay further suggested a higher cell envelope permeability in Mab^Δ2362 compared to Mab^Wt. Collectively, these findings underscore the role of MAB_2362 in intrinsic resistance and virulence of M. abscessus possibly through the regulation of cell division. Thus, MAB_2362 emerges as a promising candidate for targeted interventions in the pursuit of novel antimicrobials against M. abscessus.

Carbapenemase-producing Klebsiella pneumoniae (KPC) are globally emerging pathogens that cause life-threatening infections. Novel treatment alternatives are urgently needed. We therefore investigated the effectiveness of three novel bacteriophages (Spivey, Pharr, and Soft) in a neutropenic murine model of KPC gastrointestinal colonization, translocation, and disseminated infection. Bacteriophage efficacy was determined by residual bacterial burden of KPC (CFU/g) in kidneys. Parallel studies were conducted of bacteriophage pharmacokinetics and resistance. Treatment of mice with 5 × 10⁹ PFU of phage cocktail via intraperitoneal injection was effective in significantly reducing renal KPC CFU by 100-fold (P < 0.01) when administered every 24 h and 1000-fold (P < 0.01) every 12 h. Moreover, a combination of bacteriophage and ceftazidime-avibactam produced a synergistic effect, resulting in a 10⁵-fold reduction in bacterial burden in cecum and kidney (P < 0.001 in both tissues). Prophylactic administration of bacteriophages via oral gavage did not prevent KPC translocation to the kidneys. Bacteriophage decay determined by linear regression of the ln of mean concentrations demonstrated R² values in plasma of 0.941, kidney 0.976, and cecum 0.918, with half-lives of t_1/2 = 2.5 h. Furthermore, a phage-resistant mutant displayed increased sensitivity to serum killing in vitro, but did not show significant defects in renal infection in vivo. A combination of bacteriophages demonstrated significant efficacy alone and synergy with ceftazidime/avibactam in the treatment of experimental disseminated KPC infection in neutropenic mice.

The pipeline for new drugs against multidrug-resistant Pseudomonas aeruginosa remains limited, highlighting the urgent need for innovative treatments. New strategies, such as membrane-targeting molecules acting as adjuvants, aim to enhance antibiotic effectiveness and combat resistance. RW01, a cyclic peptide with low antimicrobial activity, was selected as an adjuvant to enhance drug efficacy through membrane permeabilization. RW01's activity was evaluated via antimicrobial susceptibility testing in combination with existing antibiotics on 10 P. aeruginosa strains and analog synthesis. Synergy was assessed using checkerboard assays, and one-step mutants were generated to identify altered pathways through whole-genome sequencing and variant analysis. Permeabilizing activity was studied using flow cytometry and real-time fluorescence measurement. In vivo toxicity was assessed in female C57BL/6J mice, and possible interaction with mouse serum was also evaluated. Susceptibility testing revealed specific synergy with tetracyclines, with up to a 16-fold reduction in minimum inhibitory concentrations. Sequencing revealed that resistance to the RW01-minocycline combination involved mutations in the pmrB gene, affecting outer membrane lipopolysaccharide composition. This was further confirmed by the identification of cross-resistance to colistin in these mutants. RW01 reduced the mutant prevention concentration of minocycline from 64 to 8 mg/L. RW01 was demonstrated to enhance membrane permeabilization and therefore minocycline uptake with statistical significance. Synthetic derivatives of RW01 showed a complete loss of activity, highlighting the importance of RW01's D-proline(NH2) residue. No acute or cumulative in vivo toxicity was observed in mice. These findings suggest that RW01 could revitalize obsolete antimicrobials and potentially expand therapeutic options against multidrug-resistant P. aeruginosa.

Macrolides are the first-line compounds used for the treatment of campylobacteriosis. Macrolide resistance remains low in France, with mutations in 23S rDNA being the main associated resistance mechanism. However, two erythromycin methyltransferases have also been identified: erm(B), which is mainly described in animal reservoirs, and erm(N), which is strictly described in humans. In France, between 2018 and 2023, erythromycin-resistant Campylobacter species strains were systematically sequenced and analyzed via an in-house bioinformatics pipeline, leading to the identification of the resistomes, MLST and cgMLST, as well as the characterization of the source of contamination. In this study, the genomes of 280 erythromycin-resistant strains were sequenced over a 6-year period. The identification of erythromycin-associated resistance markers revealed a predominance of 23S rDNA mutations, in 90% of cases, but also erm-type methyltransferases in 10% of cases: 75% for erm(N) and 25% for erm(B). Over this period, an important increase in the rate of erm-positive isolates was observed: 2% in 2018 compared with 13% in 2023, with 10% for erm(N) and 3% for erm(B). erm(N) has been found exclusively within a CRISPR-Cas9 operon, whereas erm(B) has been found within diverse types of resistance genomic islands. Each erm(N)- or erm(B)-positive isolate had at least two other resistance markers (mostly ciprofloxacin, tetracycline, or ampicillin) and often carried aminoglycoside-associated resistance genes. The majority of the erm-positive isolates were obtained from chicken. The increasing rates of erm-positive and multiresistant isolates make the monitoring of erythromycin-resistant Campylobacter strains, specifically within the chicken meat production, a topic of serious importance.

This Phase 1 trial described the intrapulmonary pharmacokinetics and safety profile of IV fosfomycin in healthy participants. Fosfomycin, a broad-spectrum antibiotic mainly used to treat urinary tract infections, is being considered for treatment of more complex conditions, including lung infections, due to the emergence of multidrug-resistant (MDR) organisms. Despite its potential, the pharmacokinetics and safety profile of intravenous (IV) fosfomycin, particularly its penetration into the lower respiratory tract, are unknown. To address this gap, we conducted a Phase 1, open-label trial to assess the safety and pulmonary pharmacokinetics of IV fosfomycin in healthy participants. Thirty-seven healthy volunteers aged 18-45 years received three doses of 6 g IV fosfomycin every 8 hours. Bronchoscopy with bronchoalveolar lavage (BAL) was performed at randomly assigned time points after the third dose. BAL fluid, BAL cell pellets, and blood plasma samples for fosfomycin were analyzed using validated assays of liquid chromatography with tandem mass spectrometry (LC-MS/MS). Adverse events (AEs) were assessed. Fosfomycin exhibited penetration into alveolar macrophages (AM) at a rate of 16.8% and into the extracellular lining fluid (ELF) at 30.8%. Mean AM fosfomycin concentration ranged from 14.8 to 32 μg/mL, while the mean ELF concentration ranged from 15.7 to 82.5 μg/mL. All participants experienced at least one treatment-emergent adverse event (TEAE), mostly mild/grade 1, with no serious adverse events (SAEs) reported. Intravenous fosfomycin effectively penetrates both the extracellular (ELF) and intracellular (AM) compartments of the lower respiratory tract in healthy participants. The overall tolerability of IV fosfomycin was favorable, suggesting its potential as an effective antibacterial treatment for lower respiratory tract infections.

Clinical trials: This study is registered with ClinicalTrials.gov as NCT03910673.

The combination of aztreonam (ATM) and avibactam (AVI) is an attractive option to treat infections caused by extended spectrum β-lactamase plus NDM-1-producing Enterobacteriaceae. Since ATM activity was shown to be severely impacted by an increase in the inoculum size in vitro, we wondered whether ATM-AVI activity could be impaired in high-inoculum infections. We analyzed the impact of the inoculum size on ATM-AVI activity in vitro and in a murine model of peritonitis due to susceptible Escherichia coli CFT073-pTOPO and its isogenic derivatives producing NDM-1 (E. coli CFT073-NDM1) and CTX-M-15 plus NDM-1 (E. coli CFT073-CTXM15-NDM1). The impact of the inoculum size on bacterial morphology was studied by microscopic examination. In vitro, at standard (10⁵) inoculum, E. coli CFT073-CTXM15-NDM1 was resistant to ATM but susceptible to the ATM-AVI combination. At high (10⁷) inoculum, MICs of ATM alone and of the ATM-AVI combination reached >512 and 64 mg/L, respectively, against all tested strains. ATM led to bacterial filamentation when active against the bacteria, i.e., in monotherapy or in combination with AVI against susceptible E. coli CFT073-pTOPO and only in combination with AVI against E. coli CFT073-CTXM15-NDM1. In vivo, increase in the inoculum led to a drastic decrease in the activity of ATM alone against E. coli CFT073-pTOPO and ATM-AVI against E. coli CFT073-CTXM15-NDM1. Our results suggest a high in vivo impact of the inoculum increase on the activity of ATM alone against ATM-susceptible E. coli and of ATM-AVI against CTX-M-15 plus NDM-1 producing E. coli. Clinicians must be aware of the risk of failures when using ATM-AVI in high-inoculum infections.

Ventriculitis in neurocritical care patients leads to significant morbidity and mortality. Antibiotic dose optimization targeting pharmacokinetic/pharmacodynamic (PK/PD) exposures associated with improved bacterial killing may improve therapeutic outcomes. We sought to develop and apply a population PK model in infected critically ill patients to determine optimal piperacillin-tazobactam (PTZ) dosing regimens to achieve target cerebrospinal fluid (CSF) exposures. Neurosurgical patients with external ventricular drains and receiving PTZ treatment were recruited and had plasma and CSF samples collected and assayed. A population PK model was developed using plasma and CSF piperacillin and tazobactam concentrations. Eight patients were recruited. Median age was 59 years, median weight was 70 kg, and five patients were female. The median creatinine clearance was 84 mL/min/1.73 m² (range 52-163). Substantial inter-individual PK variability was apparent, particularly in CSF. Piperacillin penetration into CSF had a median of 3.73% (range 0.73%-7.66%), and tazobactam CSF penetration was not predictable. Dosing recommendations to optimize CSF exposures for the treatment of ventriculitis were not possible due to substantial PK variability and very low drug penetration. High plasma PTZ exposures may not translate to effective exposures in CSF.

This study aimed to describe the population pharmacokinetics of caspofungin in critically ill patients receiving extracorporeal membrane oxygenation (ECMO) and to identify dosing regimens with a high likelihood of achieving effective exposures. Serial blood samples were collected over a single-dosing interval during ECMO. Total plasma concentrations were measured by a validated chromatographic assay. A population pharmacokinetic model was built and Monte Carlo dosing simulations were performed using Monolix. The probability of target attainment (PTA) and fractional target attainment (FTA) rates were simulated for various caspofungin dosing regimens against Candida albicans, Candida glabrata, and Candida parapsilosis. In all, 64 plasma concentration-time points were obtained from 8 critically ill patients receiving ECMO. Plasma concentration-time data for caspofungin were best described by a one-compartment model with first-order elimination. Lean body weight was identified as a significant covariate of volume of distribution. The typical volume of distribution and clearance of caspofungin in this cohort were 8.13 L and 0.55 L/h, respectively. The licensed caspofungin dosing regimen (a loading dose of 70 mg on day 1 followed by a maintenance dose of either 50 mg/day or 70 mg/day) demonstrated optimal PTA rates (≥90%) against C. albicans with an MIC of ≤0.064 mg/L, C. glabrata with an MIC of ≤0.125 mg/L, and C. parapsilosis with an MIC of ≤0.064 mg/L. The FTA analysis suggested that the licensed dosing regimen is only optimal (≥95%) against Candida glabrata, regardless of lean body weight. A higher-than-standard empirical dosing regimen (e.g., a loading dose of 100 mg on day 1, followed by a maintenance dose of 100 mg daily) is likely advantageous for critically ill patients receiving ECMO.

The impact of heteroresistance on tuberculosis (TB) treatment outcomes is unclear, as is the role of different rifampin and isoniazid exposures on developing resistance mutations. Hollow fiber system model of TB (HFS-TB) units were inoculated with drug-susceptible Mycobacterium tuberculosis (Mtb) and treated with isoniazid and rifampin exposure identified in a clinical trial as leading to treatment failure and acquired drug resistance. Systems were sampled for drug concentration measurements, estimation of total and drug-resistant Mtb, and small molecule overlapping reads (SMOR) analysis for the detection of heteroresistance. In the second HFS-TB study, systems were inoculated with an isoniazid-resistant clinical strain and treated with various combinations of isoniazid, rifampin, moxifloxacin, and levofloxacin for 28 days. Linear regression and exponential decline models were used for data analysis. Suboptimal isoniazid and rifampin exposures failed to kill drug-susceptible Mtb in the HFS-TB. Standard susceptibility methods failed to detect drug resistance, but SMOR detected isoniazid and rifampin heteroresistance, as well as fluoroquinolone, to which bacilli were not exposed. rpoB mutations arising from low rifampin exposures were Q513K and H526N, whereas those from regimen adequate rifampin but low isoniazid concentrations were S531L. Moxifloxacin-rifampin combination sterilized the HFS-TB units inoculated with the isoniazid-resistant Mtb in 14 days compared with 21 days of treatment with levofloxacin-rifampin, with no further emergence of drug resistance. Early detection of isoniazid and rifampin heteroresistance could provide an opportunity to individualize the therapy and protect fluoroquinolones when added to the MDR-TB treatment regimen.