International Journal of Antimicrobial Agents

The minimum inhibitory concentration (MIC) assay is the gold standard for evaluating antimicrobial activity1. However, conventional agar-based MIC methods often underestimate the potency of physicochemically complex compounds[1, 2]. Hydrophobic and adhesive molecules, such as lipoglycopeptide antibiotics, exhibit poor diffusion and non-specific binding to agar, leading to artificially elevated MIC values compared to broth-based methods[3]. This issue complicates accurate potency assessment and is particularly an issue when attempting resistance frequency (FOR) studies, which must be conducted on solid media. Here, we developed a modified miniaturised agar MIC assay using 1% agarose, 0.002% Tween 80-supplemented tryptic soy broth (TSB), and a 24-well plate format[4]. These modifications improved compound dispersion, reduced matrix interactions, and lowered compound requirements. The optimised assay was validated with vancomycin, oritavancin, and dalbavancin against Staphylococcus aureus ATCC 43300 (MRSA) and Streptococcus pneumoniae ATCC 700677. This efficient, cost-effective, high-throughput platform overcomes the limitations of traditional agar methods, enhancing reliability in evaluating challenging antimicrobials and supporting next-generation antibiotic development.

Sepsis is a global health problem. Patients in the Intensive Care Unit (ICU) are at risk of having sepsis because of their vulnerability and use of invasive medical devices. Emerging antimicrobial-resistance complicates treatment. Understanding bacterial profiles and antibiotic sensitivity patterns in the ICU plays an important role in managing sepsis, optimizing antimicrobial use, and improving patient outcomes. The aim of this study was to show bacterial profiles and antibiotic sensitivity patterns among patients who were admitted to the ICU at Mbeya Zonal Referral Hospital for 12 months. This was a prospective cross-sectional study among 187 sepsis-suspected patients. Data analysis was performed using SPSS Version 27. Categorical data were summarized by frequency and percentage. Fishers exact test, chi-square test, and modified Poisson regression were used to show the association between patients clinical variables and culture positivity at a 95% CI. Statistical significance was set at a p-value < 0.05. Out of 187 samples, 41.7% were culture positive. Gram-negative bacteria were commonly isolated (65.4%, with Klebsiella pneumoniae and Escherichia coli being commonly isolated. Commonly isolated Gram-positive bacteria were Coagulase-negative Staphylococcus species (19%). 100% of gram-positive bacteria and 96% of gram-negative bacteria were Multi-Drug Resistant (MDR), respectively. In multivariable analysis, patients who were intubated and mechanically ventilated (aPR = 2.208, 95% CI: 1.133-4.305, p = 0.020) and post-laparotomy patients (aPR = 3.241,95% CI: 1.320-7.958, p = 0.010) independently had a higher proportion of Gram-negative bacteria. The study reveals a high level of resistance to commonly used antimicrobials in our setting, necessitating strengthening of the antimicrobial stewardship program and routine surveillance of antimicrobial resistance patterns.

Purpose: To compare the efficacy and safety of intravenous cefazolin versus antistaphylococcal penicillins for the treatment of MSSA bacteremia in adults using evidence exclusively from randomized controlled trials. Methods: We searched 5 databases for RCTs comparing definitive intravenous cefazolin with ASPs (oxacillin, cloxacillin, flucloxacillin, nafcillin) in adults with MSSA bacteremia. Primary outcomes were 90-day all-cause mortality and acute kidney injury (AKI). Secondary outcomes included adverse event-related treatment discontinuation and persistent bacteremia (>72h). Dichotomous outcomes were synthesized as risk ratios (RRs) with 95% confidence intervals (CIs) using random-effects models with Paule-Mandel estimation and Hartung-Knapp-Sidik-Jonkman inference. Fixed-effect Mantel-Haenszel models were performed as sensitivity analyses. Results: Two RCTs randomized 1,656 participants (SNAP, n = 1,341; CloCeBa, n = 315), of whom 1,579 contributed to the 90-day mortality analysis. There was no statistically significant difference in 90-day mortality (RR 0.90, 95% CI 0.57-1.41; I2 = 0%). Both trials favoured cefazolin for acute kidney injury, but the random-effects estimate was highly imprecise (RR 0.26, 95% CI 0.00-939,970.59; I2 = 81.7%). Treatment discontinuation also favoured cefazolin directionally, although the random-effects estimate was imprecise (RR 0.24, 95% CI 0.00-24.41; I2 = 36.2%). Microbiological outcomes were synthesized narratively because definitions and assessment time points differed between trials. Conclusion: Randomized evidence supports cefazolin as a potential definitive treatment option when adequate source control, appropriate antimicrobial dosing, and clinical monitoring can be achieved. However, the limited number of trials and imprecision in pooled estimates warrant cautious interpretation.

Nontuberculous mycobacterial (NTM) infections are a scourge that affect growing patient populations due to the rise of risk factors associated with these opportunistic pathogens. Cure rates are unacceptably low despite intensive multidrug and multi-year therapy. The issue of poor drug exposure relative to inferior potency of repurposed antibiotics against NTM pathogens is broadly recognized as a major factor underlying dismal success rates. Yet, a quantitative and systematic evaluation of probabilities of pharmacokinetic-pharmacodynamic (PK-PD) target attainment (PTA) is lacking. Here, we leveraged published population PK models and minimum inhibitory concentration distributions across large patient populations to generate PTAs for standard of care antibiotics used in the treatment of NTM infections. PTAs of tuberculosis drugs were included in our analysis as a baseline since tuberculosis cure rates are excellent compared to NTM diseases. We found that PK-PD target attainment for NTM was low overall and that PTAs were generally consistent with reported clinical outcomes. Specifically, drugs for which genetic resistance is associated with worse outcome showed favorable PTAs, consistent with their contribution to cure. These include macrolides for M. abscessus and the M. avium complex (MAC), and rifampicin for M. kansasii. Bedaquiline emerged as a promising option against M. abscessus and MAC, and clofazimine against the latter, consistent with recent clinical reports. Omadacycline PTAs predict improved efficacy compared to tigecycline for patients with M. abscessus and suggest clinical utility for MAC. As is common practice for antibacterials, PTA considerations could inform NTM treatment guidelines and help select drug combinations in future trials.

Introduction Miltefosine is the only approved oral antileishmanial agent, but its use in women of childbearing potential (WOCBP) is restricted due to preclinical teratogenicity. Current labeling recommends contraception during treatment and for at least five months thereafter, based solely on its long terminal elimination half-life. This study re-evaluated the required contraceptive duration using an exposure margin-based approach. Methods Virtual populations were generated from anthropometric data of 382 Indian, 4,462 Eastern African, and 4,019 Brazilian WOCBP with leishmaniasis. Published population pharmacokinetic models were used to simulate miltefosine exposure following 14-42-day regimens for visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL). A developmental safety exposure threshold was derived from the rat no-observed-adverse-effect level (0.6 mg/kg/day for 10 days) and adjusted using a 10-fold safety margin. Contraceptive durations resulting in median residual post-contraception exposure (AUCEOC-{infty}) below this threshold were considered supportive of contraceptive discontinuation. Results The developmental safety exposure threshold was estimated at 2.5 mg{middle dot}day/L. Despite pharmacokinetic differences across geographical regions and disease manifestations, required minimum contraceptive durations were consistent: three months from treatment initiation for the 14-day regimen, four months for 21- and 28-day regimens, and five months for the 42-day regimen. For the 14-day regimen, a single dose of a long-acting injectable contraceptive administered at treatment initiation would provide sufficient coverage. Conclusion An exposure margin-based approach supports shorter contraceptive durations than current recommendations. For the 14-day VL regimen, three months of contraception may provide a practical alternative to current labeling.

Objective: Carbapenem-resistant Acinetobacter baumannii (CRAB) infections present a significant public health challenge in the United States due to multidrug resistance, substantial added inpatient costs, and subsequently high mortality rates. Method: A decision-analytic model was developed to evaluate the cost-effectiveness of sulbactam-durlobactam compared to colistin for treating CRAB infections, using data from the Acinetobacter Treatment Trial Against Colistin (ATTACK) trial, in which patients received either sulbactam-durlobactam or colistin alongside imipenem-cilastatin. The model incorporated survival rates, clinical outcomes (complete or incomplete cure), and the incidence and costs of acute kidney injury, which is more frequent in colistin-treated patients. Deterministic sensitivity analysis and two scenario analyses were performed. Results: The base case analysis estimated an expected per-patient cost of $57,887 for sulbactam-durlobactam compared to $55,157 for colistin. Patients treated with sulbactam-durlobactam had higher survival than those treated with colistin (0.66 versus 0.46), corresponding to 0.19 additional life saved per patient. The incremental cost-effectiveness ratio (ICER) for sulbactam-durlobactam compared with colistin was $14,097 per additional life saved. The ICER was most sensitive to the proportion of survivors with incomplete clinical cure for both treatments, the costs of sulbactam-durlobactam and cefiderocol, and survival outcomes in patients treated with sulbactam-durlobactam. Scenario analyses showed that the cost-effectiveness of sulbactam-durlobactam was sensitive to clinical cure outcomes, with ICERs ranging from $69,758 per additional life saved to dominance favoring sulbactam-durlobactam under different assumptions. Conclusions: Although treatment costs were higher, sulbactam-durlobactam has a favorable cost-effectiveness profile as compared with colistin when accounting for the costs of managing acute kidney injury.

Nitrofurantoin is commonly prescribed as a first-line treatment for urinary tract infections (UTIs) and is effective against most uropathogens; however, resistance among Klebsiella spp. remains high. In this study, we investigated synergistic drug combinations--defined as pairs of drugs whose combined effect exceeds that of each agent alone--to enhance treatment efficacy against drug-resistant Klebsiella pneumoniae. A high-throughput drug screen identified six and two small molecules that exhibited >50% synergistic activity in tested strains in combination with nitrofurantoin and ciprofloxacin, respectively. We further validated the top three candidates using nitrofurantoin-susceptible and resistant clinical isolates. Notably, the combination of nitrofurantoin and dequalinium demonstrated strong synergistic activity, observed in 68% of susceptible Escherichia coli strains and 94% of resistant Klebsiella pneumoniae strains tested. Combined inhibition of the citric acid cycle by nitrofurantoin and F1-ATPase by dequalinium resulted in a significant reduction in ATP levels compared to either treatment alone. Importantly, decreased ATP levels did not increase persister cell formation, and dequalinium alone reduced persister cell populations more effectively than nitrofurantoin. However, nitrofurantoin is processed into a poorly understood reactive intermediate whose specific metabolic targets have not been fully elucidated. Using metabolomic analyses, we identified aconitase and isocitrate dehydrogenase--key enzymes in the citric acid cycle--as altered in response to nitrofurantoin. Together, these findings demonstrate that dual targeting of bacterial metabolism by nitrofurantoin and dequalinium represents a promising therapeutic strategy for treating drug-resistant Klebsiella spp. in UTIs.

Background: Polymorphisms in the N-acetyltransferase 2 (NAT2) gene explain much of the interindividual variation in isoniazid (INH) metabolism and determine risk of toxicities. However, there is limited evidence to guide INH dose adjustment according to the NAT2 acetylator profile in weekly rifapentine-INH tuberculosis preventive therapy (TPT). Methods: In a prospective, multicenter, within-subject PK trial (NCT05413551), adults initiating 3HP in Brazil were assigned genotype-guided INH doses (slow: 5 mg/kg <=300 mg; intermediate: 15 mg/kg <=900 mg; rapid: 25 mg/kg <=1,500 mg) alongside a standard 900 mg flat dose on an alternate occasion. AUC0-24 and C24 were estimated from serial blood samples; a two-compartment Michaelis-Menten population PK model characterized NAT2 effects on clearance. Results: Among 228 participants, 47.4% (108/228) were intermediate, 43.4% (99/228) slow, and 9.2% (21/228) rapid acetylators. Genotype-guided dosing reduced AUC0-24 variability approximately two-fold versus standard dosing (CV 58.8% vs 76.8%) and increased exposure uniformity (median AUC0-24 27.2 [IQR 18.8-41.3] vs 43.2 [27.3-71.0] mg h/L). Among slow acetylators, C24 >0.15 ug/mL decreased from 27/42 (64%) with standard dosing to 1/42 (2%) with genotype-guided dosing (P<0.0001). In 104 participants with intensive PK sampling, rapid acetylators receiving guided doses had AUC0-24 similar to standard-dose intermediate acetylators (42.8 vs 39.5 mg h/L; P=.63). Monte Carlo simulations supported doses of 600, 900, and 1,200 mg for slow, intermediate, and rapid acetylators, respectively. Conclusions: NAT2-guided isoniazid dosing reduced variation in drug levels, averting very low and high AUC and C24. These findings inform genotype-stratified dosing of INH for TPT, which might reduce toxicities and improve outcomes.

The human opportunistic pathogen Pseudomonas aeruginosa produces copious quantities of the secondary metabolite pyocyanin (PYO). PYO has recently been shown to interact with the aryl hydrocarbon receptor (AhR), a transcription factor controlling expression of a number of genes, including the cytochrome P450 CYP1A family involved in fluoroquinolone antibiotic clearance. In this study, we investigated whether Pseudomonas aeruginosa could influence the metabolism of ciprofloxacin through human CYP1A2 induction. Primary human hepatocytes were exposed to 1-100 M PYO, with high concentrations resulting in apparent cytotoxicity. Treatment with 5 {micro}M PYO led to a 6.2-fold change in CYP1A2 mRNA and a 3.6-fold increase in oxociprofloxacin metabolite formation. Our results suggest that sub-toxic concentrations of PYO induce CYP1A2 expression and increase ciprofloxacin metabolism, which could lead to sub-efficacious antibiotic concentrations and further drive resistance. This finding directs us to a novel mechanism by which Pseudomonas aeruginosa may escape antimicrobial therapy by hijacking host xenobiotic metabolism pathways. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/740721v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1b350forg.highwire.dtl.DTLVardef@388eb7org.highwire.dtl.DTLVardef@121bc9aorg.highwire.dtl.DTLVardef@1e53060_HPS_FORMAT_FIGEXP M_FIG C_FIG

Introduction Rifampicin-resistant tuberculosis poses a significant threat worldwide. Clofazimine is considered important for the construction of effective individualized multidrug regimens to treat rifampicin-resistant tuberculosis. Our goal was to characterize clofazimine pharmacokinetics in novel combination treatment regimens and to identify participant characteristics associated with variations in drug exposure. Methods Clofazimine pharmacokinetic data were obtained from adults and adolescents enrolled in the PandrTB pharmacokinetic sub-study of the endTB and endTB-Q Phase 3 randomized controlled therapeutic trials for rifampicin-resistant tuberculosis. Plasma concentrations were analyzed using nonlinear mixed-effects modeling to quantify clofazimine exposure and explore the impact of relevant covariates. Results 100 participants from six countries with high burdens of rifampicin-resistant tuberculosis contributed pharmacokinetic data. Their median age was 34 years, 32% were female, 18% were living with diabetes mellitus, and 18% were living with HIV. Clofazimine plasma concentration values were best described by a 2-compartment pharmacokinetic model with first-order absorption. Co-administration with delamanid and HIV co-infection resulted in a 37% increase and 20% decrease in clofazimine bioavailability, respectively. Diabetes was associated with a 43% decrease in clofazimine clearance. Clofazimine remained in the body for a median of 2.5 years (95th percentile: 0.5 to 8) following 9 months of treatment. Conclusion Co-administration with delamanid, diabetes mellitus, and HIV were associated with variation in clofazimine exposure. We estimated that clofazimine remained present in the body for longer than two years in over half of participants following 9 months of treatment. Follow-up studies are recommended to confirm these associations before adjusting clofazimine dose or clinical care decisions.

The rapid rise of multidrug-resistant (MDR) bacterial infections has severely limited treatment options, particularly for Gram-negative pathogens such as Klebsiella pneumoniae, a leading contributor to pneumonia, bloodstream, urinary tract, and surgical-site infections. One strategy to restore antibiotic efficacy is the use of resistance-mitigating agents (RMAs), compounds that re-sensitize bacteria to existing antibiotics without displaying independent antibacterial activity. Herein, we report the results of a high-throughput screen of a 3,200-compound fragment-based library against an MDR K. pneumoniae isolate in the presence of subinhibitory ciprofloxacin. This screen identified a tetrahydrocarbazole-containing compound, 1, as a ciprofloxacin potentiator. Subsequent structure-activity relationship studies yielded a difluorinated analog, compound 5, which potentiated multiple antibiotic classes in MDR K. pneumoniae, reducing MICs up to [&ge;]16 fold. Further testing demonstrated synergistic interactions between compound 5 and ciprofloxacin, ceftriaxone, cefoxitin, and tetracycline across four genetically diverse MDR K. pneumoniae strains. These findings suggest that tetrahydrocarbazole-containing compounds constitute a promising new class of RMAs with potential for future development as therapies against MDR K. pneumoniae infections.

Introduction Praziquantel (PZQ) is the only widely available chemotherapy that is effective against all species of schistosomes. Environmental enteric dysfunction (EED) is an acquired intestinal disorder of altered gut function whose effect on drug pharmacokinetics has not been directly explored. Methods Preschool-age children infected with S. mansoni were randomized to receive 40mg/kg or 80mg/kg of crushed PZQ tablets. Plasma PZQ concentrations were quantified using ultra-high performance liquid chromatography mass spectrometry. Maximum concentration (Cmax), time to Cmax (Tmax) and area under the curve (AUC) of PZQ were calculated. Biomarkers of intestinal inflammation (stool calprotectin), epithelial damage (plasma Intestinal Fatty Acid Binding Protein (IFABP)), permeability (urine lactulose:mannitol (LM) ratio and alpha-1 antitrypsin (AAT)), microbial translocation (plasma Endotoxin core antibodies (EndoCAb), systemic inflammation (plasma C-reactive protein (CRP)), and presence of faecal occult blood (FOB) were measured. Using linear regression, we assessed association of AUC, Cmax, Tmax and R- to S-PZQ exposure with each biomarker, adjusting for dose, age, and sex. Results Of the 184 participants included in the final analysis, 91 received 40mg/kg and 93 received 80mg/kg of PZQ. The Tmax was associated with LM ratio ({beta}=0.06, 95% CI 0.02 - 0.11, p=0.003) and calprotectin ({beta}=0.001, 0.0002 - 0.002, p=0.013). CRP was associated with AUC ({beta}=0.13, 95% CI 0.06 - 0.21, p=0.001) and Cmax ({beta}=0.12, 95% CI 0.05 - 0.21, p=0.002). Calprotectin ({beta}=0.12, 95% CI 0.04 - 0.20, p=0.003) and AAT ({beta}=0.09, 95% CI 0.02 - 0.15, p=0.008) were associated with a higher R-PZQ/S-PZQ AUC ratio, while CRP was not (p=0.38). Conclusion Elevated intestinal inflammatory markers were associated with increased Tmax, indicating reduced rate of absorption and relative increase in exposure to the active R-enantiomer. Systemic inflammation was associated with higher Cmax and AUC, implying increased exposure to PZQ. This is the first report linking alterations in praziquantel pharmacokinetics to EED markers and systemic inflammation.

Purpose: Vancomycin-resistant Gram-positive organisms represent a potential therapeutic challenge in endophthalmitis. To evaluate alternative treatment options, the FDA-approved oxazolidinones linezolid (LZD) and tedizolid (TZD) were tested against a collection of Gram-positive bacterial endophthalmitis isolates. Methods: Minimum inhibitory concentrations (MICs) were determined using epsilometers (Etest strips) for coagulase negative staphylococci (CoNS, n=20), Staphylococcus aureus (n=18), Bacillus species (n=10), Enterococcus species (n=5), and Streptococcus pneumoniae (n=1). Available CLSI and EUCAST breakpoints were applied to determine susceptibility. Results: TZD exhibited a >5-fold reduction in median MIC compared with LZD across all organisms tested (p<0.0001). MIC90 values for CoNS were 2.0 /ml for LZD and 0.25 /ml for TZD. For S. aureus, MIC90 values were 1.5 /ml (LZD) and 0.19 /ml (TZD). For Bacillus spp., MIC90 values were 1.0 /ml (LZD) and 0.25 /ml (TZD). Among Enterococcus species, the MIC range was 0.5-4.0 /ml (LZD) and 0.064-0.75 /ml (TDZ). The single S. pneumoniae strain had a MIC of 0.5 for LZD and 0.064 for TZD. All isolates were susceptible to TZD. One Enterococcus isolate was non-susceptible to LZD; the remaining isolates were susceptible. Conclusions: LZD and TZD demonstrated in vitro activity against Gram-positive bacterial endophthalmitis isolates, with TZD displaying consistently lower MICs than LZD. These findings suggest that oxazolidinones may represent potential alternatives when vancomycin cannot be used, including infections caused by vancomycin-resistant Gram-positive organisms. Given their excellent oral bioavailability, favorable safety profile, and reported anti-virulence effects in certain pathogens, further evaluation of oxazolidinones for the treatment of intraocular infections is warranted.

Drug repositioning has emerged as an attractive strategy to accelerate the development of new antimalarial therapies, particularly by evaluating compounds already used against pathologies co-endemic with malaria. This approach offers the advantage of leveraging existing pharmacokinetic, toxicological, and safety data, thereby potentially shortening the drug development pipeline. However, transposing a compound from its original therapeutic indication to an antimalarial use is far from straightforward: differences in target biology, parasite stage specificity, pharmacodynamic requirements, and host-parasite interactions can result in a loss of efficacy despite promising in vitro or structural rationale. Rigorous in vivo validation therefore remains indispensable before any repositioning hypothesis can be considered translationally relevant. In this context, we evaluated the blood-stage antimalarial activity of triclabendazole, an antihelminthic drug used against co-endemic fascioliasis, together with its metabolite triclabendazole sulfoxide, and sutezolide, an oxazolidinone antibiotic, in a murine model of Plasmodium berghei ANKA infection following oral administration. None of the three compounds demonstrated significant antimalarial activity under these experimental conditions, contradicting a previously published repositioning hypothesis. Beyond these specific findings, our study is deliberately framed within the 3Rs principles (Replacement, Reduction, Refinement) governing animal experimentation. We argue that publishing negative in vivo results is not only scientifically legitimate but ethically necessary: sharing such data allows research teams working on similar preclinical models to build on existing knowledge, avoid unnecessary experimental duplication, and ultimately reduce the number of animal procedures performed across the field. We advocate for wider dissemination of negative outcomes in antimalarial drug repositioning research as a concrete contribution to more responsible and efficient use of animal models in preclinical pharmacology. Graphical Abstract

Background: Individuals living with HIV have been under-represented in operational research and trials of all-oral regimens for multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB) treatment. We report outcomes from a prospective observational cohort study of a nine-month regimen comprised of bedaquiline, linezolid, a fluoroquinolone, delamanid, and clofazimine, in Lesotho, a setting with a high prevalence of HIV infection. Methods: We analyzed patients who initiated the regimen between 2020 and 2023. Overall, and by HIV status, we characterized treatment safety by summarizing the frequency and timing of clinically relevant adverse events. We described treatment effectiveness at the end of treatment and sustained treatment success eighteen months after treatment initiation. Results: Two hundred and fifty-five patients who initiated the shortened all-oral regimen were included in the analysis, of whom 161 (63.1%) were living with HIV. Regardless of HIV status, the most frequent clinically relevant adverse event was peripheral neuropathy, followed by myelosuppression. The estimated unadjusted rates of sustained treatment success at 18 months post-treatment initiation were 86.4% (95% confidence interval (CI): 81.4%, 90.5%) overall, 85.0% (95% CI: 78.2%, 90.4%) among patients living with HIV, and 88.8% (95% CI: 80.3%, 94.5%) among people without HIV. Conclusions: The effectiveness of the nine-month all-oral regimen was excellent. Although adverse events were common, they were manageable and did not appear to substantially impact treatment success. Results were similar regardless of HIV status, supporting the use of this regimen in this population.

Genomic prediction of antimicrobial susceptibility aims to identify resistance determinants and thereby to report those antibiotics for which therapeutic failure is to be expected. We investigated what this approach can achieve in Pseudomonas aeruginosa, a species in which resistance often arises through changes in gene expression. For this purpose, 5,749 consecutive clinical isolates from the routine laboratories of two tertiary care centers were sequenced, each with paired phenotypic susceptibility test results for ciprofloxacin, ceftazidime, meropenem, and tobramycin. Genomic variation could be interpreted only after three adaptations. Variants were called against the population-wide major allele rather than against PAO1. This removes markers that are artefacts of the reference. Loss of gene function was captured systematically, as many genetic determinants of resistance act through loss-of-function mechanisms. Furthermore, we introduced a specificity ratio - enabled by testing all four antibiotics on the same isolates - which separated markers associated with resistance mechanisms from those that merely tracked clonal lineage; without this distinction, expanding the catalogue reduced rather than improved predictive performance. In addition to classical resistance determinants, markers of susceptibility were taken into account, such as the functional loss of an efflux pump that exports the antibiotic out of the cell. While resistance could only be assigned with corresponding confidence for a few percent of isolates, the absence of ciprofloxacin and meropenem resistance could be reported for substantially more isolates, in a conservative deduplicated analysis even for 43% and 87%, respectively. The remaining isolates were left blank rather than forced into one of the two categories. The blank is not an absence of information: resistance rates within it lay between those of the two reported groups, and its isolates clustered around the clinical breakpoint. It is the genomic counterpart of a population the phenotype already recognizes and reports as susceptible, increased exposure or as an area of technical uncertainty. For meropenem, the residual risk of a susceptibility finding already meets the accepted threshold of 3%, while for ciprofloxacin it approaches this threshold. For tobramycin, validation in a larger cohort, possibly specifically enriched for resistant isolates, is required before our results can be considered reliable. For ceftazidime, by contrast, it appears that additional markers need to be identified, in particular those associated with increased AmpC expression. Taken together, the present data show that, for two of the four antibiotics investigated here, clinically relevant findings can already be generated today: the genome allows the identification of an isolate group with no indication of resistance, marks a smaller group as resistant, and assigns the remaining isolates to an intermediate range close to the clinical breakpoint.

Background. Optimal antibiotic dosing regimens depend on the pharmacokinetic/pharmacodynamic (PK/PD) index that best predicts antibacterial efficacy. PK/PD targets are traditionally determined using murine infection models based on a limited number of bacterial isolates. Objective. This study aimed to investigate whether animal experiments could be replaced by in vitro time-kill experiments performed on a large collection of clinical isolates and analyzed using a modelling approach accounting for inter-strain variability. The proposed framework was evaluated using meropenem against Pseudomonas aeruginosa. Materials and Methods. In vitro time-kill experiments were performed on 66 clinical isolates of P. aeruginosa. A population pharmacodynamic model was developed from experimental data. A murine pharmacokinetic model was reproduced from literature and combined with the pharmacodynamic model to simulate in vivo bacterial burden over time. The relationships between simulated bacterial counts at 24 h and the three main PK/PD indices (fCmax/MIC, fAUC/MIC and %fT>MIC) were characterized using nonlinear mixed-effects Imax models. Results. The PK/PD index showing the strongest correlation with meropenem efficacy at 24 h was %fT>MIC (R2 = 0.989), compared with fAUC/MIC (R2 = 0.373) and fCmax/MIC (R2 = 0.284). These findings are consistent with previous studies using murine thigh infection models. The %fT>MIC target required to achieve a 2-log CFU reduction was estimated at 44%, with substantial inter-strain variability (10th and 90th percentiles: 27% and 71%, respectively). Conclusions. Using meropenem against P. aeruginosa as a proof of concept, we demonstrate that in vitro time-kill experiments combined with pharmacometric modelling can identify the same PK/PD efficacy targets as animal infection models. Moreover, performing experiments on a large panel of clinical isolates enables the quantification of inter-strain variability in PK/PD targets, providing information that may improve their translation to clinical dosing optimization.

Background: Most patients with melioidosis receive prolonged intravenous ceftazidime during the intensive phase of their antibiotic therapy. Contemporary guidelines use weight and renal function to guide dosing, but therapeutic drug monitoring (TDM) might enable further individualisation of therapy. Objective: To examine the potential utility of ceftazidime TDM in the management of melioidosis. Methods: We reviewed consecutive serum free ceftazidime concentrations in patients with culture-confirmed melioidosis at an Australian referral hospital. We documented the minimum inhibitory concentration (MIC) for ceftazidime Burkholderia pseudomallei isolates of the patients. We then recorded the ceftazidime dosing regimen for each patient, their serum free ceftazidime concentration and if any adverse drug reactions occurred during their treatment. Results: Trough concentrations were measured in 31 patients receiving intermittent ceftazidime dosing, while random concentrations were measured in 91 patients receiving a continuous infusion. The median (range) trough concentration:MIC ratio was 37.7 (2.7-156.6) in those receiving intermittent dosing and 47.5 (8.1-181.5) in those receiving a continuous infusion. Serum ceftazidime concentrations correlated with neurotoxicity, which was documented in 5/31 (16%) receiving intermittent dosing and in 4/91 (4%) receiving a continuous infusion. Serum ceftazidime concentrations were also higher in individuals who died from their infection than in those who survived. There was no association between ceftazidime concentrations and subsequent disease recurrence. Conclusion: Current dosing recommendations for the treatment of melioidosis achieve serum ceftazidime concentrations that greatly exceed the MIC of B. pseudomallei in this region of Australia. TDM-guided reductions in the ceftazidime dose and/or dosing frequency may mitigate the risk of ceftazidime toxicity.

BACKGROUND: BK polyomavirus (BKPyV) reactivation is a major complication in kidney and hematopoietic stem cell transplant recipients, yet no specific antiviral therapy is currently available. Antiviral discovery is complicated by the restricted tropism and slow replication kinetics of BKPyV and its extensive dependence on cellular processes. RESULTS: We established a phenotypic high-throughput screening and validation pipeline to identify small molecule inhibitors of BKPyV infection. Using an SV40-infected CV1 reporter system, approximately 28,000 small molecules were screened, yielding 98 primary candidates. Confirmatory testing identified 33 compounds with reproducible activity, of which 16 subsequently inhibited BKPyV in human renal proximal tubular epithelial cells. Concentration response and cytotoxicity analyses revealed distinct antiviral potency and selectivity profiles, and integration of these data with predicted toxicity, physicochemical properties, and synthetic accessibility enabled further compound prioritization. Time of addition experiments revealed distinct temporal windows of antiviral activity, and MOI dependent concentration response analyses demonstrated that the potency of selected inhibitors varied with viral inoculum. Further characterization of prioritized compounds identified differential effects on BKPyV attachment and viral gene expression. Transcriptomic profiling of three selected compounds C5, C8, and C9 revealed distinct compound-associated cellular responses, supporting interference with different host-dependent processes during BKPyV infection. CONCLUSIONS: We identified a pharmacologically diverse panel of small-molecule inhibitors active against BKPyV in human renal epithelial cells. Their distinct potency, selectivity, temporal activity, and cellular response profiles indicate multiple modes of antiviral interference and establish C5, C8, and C9 as candidates for further target identification and optimization. More broadly, our findings demonstrate the utility of surrogate phenotypic screening for discovering inhibitors of BKPyV and provide new chemical tools to investigate host dependencies of the BKPyV life cycle.

Malaria remains a critical public health threat in sub-Saharan Africa and Southeast Asia owing to the emergence of resistance to gold-standard artemisinin-based combination therapies. This epidemiological shift drives an urgent need to explore novel bioactive compounds for malaria treatment. Microorganisms produce diverse secondary metabolites with potent biological activities. To develop microbe-derived antimalarial drugs, rigorous safety evaluations are crucial, particularly regarding hepatotoxicity. Therefore, we evaluated the hepatotoxic potential of three compounds with antimalarial attributes: thiolutin (NATPP0650), cochliodinol (NATPP0437), and 4'-hydroxy-mycophenolic acid (NATPP0604). Acute hepatotoxic responses were determined by biochemical assays for cell viability and damage in HepG2 cells following 24 h exposure, comparing conventional two-dimensional cultures with an organ-on-a-chip (OOC) system featuring in vivo-like functionality and long-term culture capabilities. In both culture platforms, NATPP0650 and NATPP0437 caused significant toxicity at concentrations of 10 and 50 {micro}M, whereas NATPP0604 was relatively non-toxic. Based on the OOC data, NATPP0650 and NATPP0437 exhibited CC50 values of 3.04 and 2.21 {micro}M, respectively, whereas NATPP0604 had a CC50 value > 50 {micro}M, offering a superior safety profile with a selectivity index > 23.7. Functional assessment of HepG2 OOCs showed that NATPP0650 and NATPP0437 markedly suppressed albumin production at 10 and 50 {micro}M, whereas NATPP0604 showed minimal inhibition. However, long-term (7 days) toxicity testing on the OOC model revealed a critical finding: NATPP0604 exerted delayed hepatotoxic effects that were undetectable in the 24-h study, as evidenced by partial reductions in cell viability at 10 and 50 {micro}M and decreased albumin production across all tested concentrations. These findings demonstrate the therapeutic safety potential of NATPP0604 for further development as an antimalarial drug and underscore the importance of the long-term OOC data in establishing accurate safety margins for future clinical trials.