Antimicrobial Agents and Chemotherapy

Azoles remain the most common antifungal therapy worldwide. However, Nakaseomyces glabratus (previously named Candida glabrata) has a high intrinsic tolerance against azole drugs. The organism can also accrue additional chromosomal mutations to elevate its resistant level during treatment. These genetic alterations often result in overexpression of the ABC transmembrane transporter Cdr1, which has been shown to directly transport drugs out of the fungal cells. Another resistant mechanism is the upregulation of the ergosterol biosynthesis pathway, which is the direct target of azoles. Although the mechanisms of azole resistance in N. glabratus are well defined, knowledge of their regulation remains limited. Here, we show that the protein kinase Fpk1 is required for optimal azole response in vitro and in an in vivo mouse infection model. Loss of Fpk1 gene or its kinase function significantly enhances azole sensitivity in both azole-susceptible and -resistant clinical isolates. Fpk1 function is required for optimal expression of Cdr1 upon azole challenge. It also influences the intracellular trafficking of ergosterol, without affecting its biosynthesis. Together, our data demonstrates the important role of Fpk1 function in the N. glabratus azole response and characterizes it as a new regulator of the efflux pump and ergosterol biosynthesis pathways. IMPORTANCEAntifungal treatment against life-threatening bloodstream Candida infection remains limited to azoles, echinocandins, and polyenes. Among them, azoles are the most prescribed therapy worldwide. However, the pathogenic yeast Nakaseomyces glabrataus has a high level of resistance against azoles (> 10%) (1). This often complicates treatment and increases mortality and morbidity rates. Therefore, understanding the mechanism of azole resistance would reinforce the treatment strategy and bolster future therapy development. Here, we identify the protein kinase Fpk1 as an important regulator of the drug efflux plump and ergosterol biosynthesis pathways. Disruption of the Fpk1 function significantly enhances the azole efficacy in vitro and in a mouse model of Candida systemic infection. Protein kinases are druggable targets, and our data presents Fpk1 as a viable candidate for future antifungal development.

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.

Quinolone antibiotics such as ciprofloxacin inhibit DNA gyrase, leading to DNA double-strand breaks that result in rapid bacterial killing and induction of the mutagenic SOS DNA repair response. By contrast, the ciprofloxacin analogue IMP-1700 inhibits ciprofloxacin-induced SOS, suggesting a novel mechanism of action. Here, we provide evidence that IMP-1700 targets the quinolone binding domain of DNA gyrase but triggers a significantly higher frequency of division septa in S. aureus compared with other DNA gyrase targeting antibiotics, including ciprofloxacin. In keeping with this finding, the lipopeptide antibiotic daptomycin, which targets the division septum, bound more strongly to IMP-1700-treated cells relative to S. aureus exposed to other DNA gyrase inhibitors, leading to increased bacterial killing. This finding extended to a panel of paired daptomycin susceptible and resistant clinical isolates. We conclude that the ciprofloxacin analogue IMP-1700 has distinct effects on the cell envelope of S. aureus, despite appearing to share the same target as the parent drug, which result in the resensitisation of daptomycin resistant bacteria to the lipopeptide antibiotic.

Klebsiella aerogenes, a member of the Enterobacteriaceae, is a causative agent of healthcare-associated infections, and outbreaks caused by drug-resistant K. aerogenes have been reported worldwide. The range of antimicrobial agents available for treating infections caused by carbapenem-resistant K. aerogenes is limited. While in vivo animal experiments using clinical K. aerogenes isolates to evaluate antimicrobial therapy could facilitate selection of the most effective treatment, conducting infection experiments involving large numbers of mammals such as mice is challenging due to ethical concerns related to animal welfare. Silkworms are invertebrates increasingly used as experimental models for infectious disease research to evaluate antimicrobial efficacy. In this study, we aimed to establish a silkworm infection model using a clinical K. aerogenes isolate to evaluate its utility for determining effective antimicrobial doses. K. aerogenes strains were isolated from a patient at a Japanese hospital, and a silkworm infection model was established using the clinical isolate. The non-metallo-beta-lactamase-producing strain K. aerogenes TUM25562, isolated from a patient with a complicated urinary tract infection, was susceptible to meropenem (MEPM) and gentamicin (GM) in vitro. During treatment, additional isolates with increased resistance to MEPM and subsequently to both MEPM and GM emerged. K. aerogenes TUM25562 caused dose-dependent mortality in silkworms. Treatment with clinically equivalent weight-based doses of MEPM or GM did not cure the infected silkworms. The median effective (ED50) doses of MEPM and GM were therefore investigated using the silkworm infection model. Administration of higher doses corresponding to four times the ED50 significantly prolonged the survival of infected silkworms. These results suggest that a silkworm infection model using clinical K. aerogenes isolates may provide a practical approach for evaluating antimicrobial efficacy and determining effective antimicrobial doses.

Pharmacodynamic curves describe how changes in drug concentration affect pathogen growth. They are essential for designing treatments that promote pathogen eradication and minimize the evolution of antibiotic resistance. The classical function for modelling pharmacodynamics is a phenomenological, S-shaped curve with stable growth and death rates separated by a single drop. In this study, we characterized the pharmacodynamic curve of the {beta}-lactam antibiotic cefotaxime (CTX) acting against Escherichia coli. We found that the relationship between CTX concentration and net growth rate diverged from classical model predictions, instead yielding a two-step curve defined by distinct phases of growth, population maintenance, and killing. We hypothesized that the intermediate phase arose from antibiotic tolerance conferred by bacterial filaments. Microscopic assessment of treated cells indeed showed a difference in degree of filamentation with concentration. We further sought to explain this with a semi-mechanistic pharmacodynamic function, modelling the binding of CTX to its cellular targets, penicillin binding proteins (PBP) 1 and 3. By incorporating the preferential concentration-dependent binding of CTX to PBP3 and then PBP1, yielding filaments or lysed cells respectively, we replicated the two-step curve in silico. We also assessed the pharmacodynamics of CTX against mutants conferring resistance; these displayed further altered curves, in line with their fitness costs. Altogether, our results show that CTX has a two-step pharmacodynamic curve against E. coli arising from multiple targets separated in their affinity for the antibiotic. We present a model offering a mechanistically grounded framework for capturing such dynamics. These pharmacodynamic curves deserve careful consideration when defining optimal dosing.

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

BackgroundThe epidemiology of Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa is rapidly evolving in China. While blaKPC-2 remains the predominant KPC variant in P. aeruginosa, blaKPC-3 has rarely been documented in this pathogen. This study investigated the molecular epidemiology, resistance and virulence characteristics, and plasmid features of blaKPC-3-producing CRPA isolates collected from a tertiary hospital in eastern China. MethodsA total of 65 non-duplicate CRPA isolates collected in 2023 were subjected to whole-genome sequencing. Antimicrobial susceptibility testing, phylogenetic analysis, plasmid characterization, conjugation experiments, and virulence assays were performed. ResultsAmong the 65 CRPA isolates, 37 (56.9%) carried blaKPC-3. These blaKPC-3-positive isolates belonged to four sequence types (STs), including ST1076 (62.2%), ST463 (21.6%), ST646 (10.8%), and ST3393 (5.4%). To our knowledge, this is the first report of blaKPC-3 in P. aeruginosa ST463, ST646 and ST3393. All isolates exhibited extensive drug resistance, and 51.8% were resistant to ceftazidime-avibactam. Phylogenetic analysis indicated that blaKPC-3 dissemination was driven by both clonal expansion and horizontal transmission. Comparative genomic analysis identified three kinds of blaKPC-3 -carrying plasmid. A transferable IncP-2 megaplasmid was widely distributed among ST1076, ST646, and ST3393 isolates, whereas non-transferable IncP-10 plasmids were primarily restricted to ST463. The genetic environments and plasmid backbones of blaKPC-3 were highly conserved and closely related to those of blaKPC-2 and its variants, suggesting evolution from pre-existing blaKPC-2-associated plasmids. Virulence analysis demonstrated marked heterogeneity across lineages. ST463 isolates co-harbored exoU and exoS, exhibited enhanced biofilm formation and pyocyanin production, and caused significantly higher mortality in the G. mellonella infection model, indicating a hypervirulent phenotype. ConclusionsThe blaKPC-3 is becoming an increasingly important determinant of carbapenem resistance in P. aeruginosa in China. The IncP-2 megaplasmid and IncP-10 plasmid derived blaKPC-3 spread across multiple lineages. Continuous genomic surveillance and enhanced infection control measures are urgently needed to prevent its further prevalence in clinical settings.

The emergence of antibiotic tolerance in Staphylococcus aureus reduces antibiotic efficacy by allowing bacterial survival despite prolonged antibiotic exposure, the molecular basis of which remains poorly understood. Moreover, the phenotypic indistinguishability of tolerant isolates in antimicrobial susceptibility testing impedes effective diagnosis and therapy. Increased concentration of the second-messenger, cyclic-di-AMP (CDA), has recently been implicated in tolerance to {beta}-lactams as well as other cell-wall-reactive antibiotics. Using the ScanLag assay, Tolerance-Disk test, and traditional methodologies and employing isogenic mutagenized strains, we demonstrate that loss of GdpP function, a phosphodiesterase that hydrolyzes CDA, confers tolerance specifically to {beta}-lactam antibiotics independent of their class. The extent of {beta}-lactam tolerance correlated directly with the intracellular CDA concentration and inversely with the inhibition of bacterial cell-wall synthesis. {Delta}gdpP mutants caused higher mortality than wild-type strains in the Galleria mellonella infection model upon {beta}-lactam treatment, suggesting GdpP-mediated tolerance could lead to {beta}-lactam treatment failure. Large-scale within-host evolution analysis demonstrated that MRSA and MSSA strains isolated from patients acquire GdpP loss-of-function mutations during invasive infections but not during nasal carriage. Overall, this study highlights the clinical relevance of gdpP mutations, frequently selected in persistent S. aureus infections, as key mediators that could promote treatment failure due to {beta}-lactam tolerance.

Objectives: To investigate whether ethambutol resistance in Mycobacterium avium complex is associated with the emergence of macrolide resistance. Methods: Patients who developed macrolide resistance during guideline-based treatment were included, and longitudinal analyses of minimum inhibitory concentrations and mutations in embB or the upstream region of embA were performed. Clinical, microbiological, and radiological characteristics were compared according to the mutation status of embB or embA upstream region, prior to the emergence of macrolide resistance. We further evaluated the impact of embB mutation on the development of macrolide resistance using in vitro time-kill assays. Results: Sixteen patients developed macrolide resistance during guideline-based treatment. None of these patients had an ethambutol minimum inhibitory concentration >=16 ug/mL or embB or embA upstream mutations at treatment initiation; however, 8/16 patients (50.0%) had an ethambutol minimum inhibitory concentration >=16 ug/mL at the time of macrolide resistance detection, and 7/16 (43.8%) had developed embB or embA upstream mutations prior to the emergence of macrolide resistance. Cavitary lesions were present in 1/7 (14.3%) patients with embB or embA upstream mutations. In strains with embB mutations, the minimum inhibitory concentration of ethambutol increased by 1-2 dilutions relative to that of pretreatment isolates, with a corresponding increase in the concentration required to suppress macrolide resistance. Conclusions: Ethambutol resistance may contribute to the development of macrolide resistance in patients with M. avium complex pulmonary disease, particularly in those without cavitary lesions.

Pseudomonas aeruginosa remains one of the most important clinical pathogens for which new drugs are needed, due to its resistance machinery. Consequently, there is an increasing effort to develop new and effective treatments against this pathogen. We recently showed that tetrasodium ethylenediaminetetraacetic acid (tEDTA) exhibits promising antibacterial and antibiofilm activity against P. aeruginosa in advanced biofilm models. tEDTA is known to chelate divalent cations, with predicted effects on the outer membrane; however, a full understanding of how this kills P. aeruginosa is lacking. Also, it is currently not clear how slowly or rapidly P. aeruginosa will evolve resistance to this treatment. Using membrane disruption assays and RNA-seq, we showed that tEDTA disrupts bacterial membrane potential and permeabilises P. aeruginosa membranes. RNA-seq revealed the significant upregulation of genes involved in the transport of iron, phosphate, potassium, and magnesium ion. The arnABCD operon which is involved in lipid A biosynthesis was also upregulated. Using a 7-day evolutionary ramp approach, we showed that P. aeruginosa could not evolve resistance to tEDTA under strong selection. Lastly, we carried out a cytotoxicity assay with Human Epithelial type 2 (HEp-2) cells and showed that there was reduced cytotoxicity of tEDTA compared to meropenem. This study provides good insight into the mechanism of action of tEDTA and further evidence of its potential as an alternative to antibiotics for P. aeruginosa infections.

Reduced cephalosporin resistance in Enterococcus faecium has traditionally been reported in laboratory mutants and, more recently, in a single clinical ampicillin-susceptible (AmpS) isolate. Herein, we investigated whether this phenotype is widespread by analysing 95 clinical enterococcal isolates (78 AmpS and 17 ampicillin resistant [AmpR]) collected from three hospitals in Spain and Portugal (2009-2025). Low ceftriaxone MICs ([&le;]4 mg/L) were detected in 19/51 (37.3%) AmpS E. faecium and 7/27 (25.9%) E. lactis but in none of the AmpR isolates. Low ceftriaxone MICs were associated with older patient age in both species and with prior ampicillin therapy in E. faecium, but not with other clinical or epidemiological variables. Ceftaroline MICs were consistently low among AmpS isolates, while ceftriaxone and cefotaxime showed greater variability. Low-MIC isolates were distributed across multiple clonal lineages and hospitals and did not share a distinctive resistance or virulence gene profile. PBP5 phylogeny and variation at the psr-pbp5 region separated AmpS from AmpR E. faecium but did not explain variability in ceftriaxone MICs. Five AmpS isolates with reduced ceftriaxone MICs carried chromosomal deletions that included the psr-pbp5 region and genes with diverse cellular functions. Variation in other candidate resistance genes (pbpA, ponA, pbpF, croRS, stpA/stk and murAA) did not consistently explain the MIC differences. These results reveal unexpected heterogeneity in intrinsic cephalosporin resistance in clinical E. faecium and E. lactis and suggest that additional genetic or regulatory mechanisms underlie reduced susceptibility.

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.

Rhodotorula mucilaginosa is an emerging opportunistic fungal pathogen increasingly associated with catheter-related bloodstream infections. Although biofilm formation is considered a major virulence trait for R. mucilaginosa, factors contributing to biofilm persistence on medical devices remain poorly understood. Here, we characterized the thermotolerance, biofilm formation, UV resistance, and cell surface hydrophobicity profiles of eight R. mucilaginosa strains representing clinical and non-clinical (laboratory, environmental, and marine mammal) isolates. All strains grew optimally at 30C and exhibited restricted growth at 35C and 37C, although one environmental isolate maintained robust growth at 37C. All strains exhibited moderate to high cell surface hydrophobicity. We then assessed biofilm formation for each strain, including adherence to two different plastic substrates, development of biofilm biomass, comparison of biofilm metabolic activity, and the effects of temperature on biofilm formation. Under static conditions, biofilm biomass of most isolates on 96-well polystyrene plates was greatest at 24C. Clinical isolates generally maintained higher biofilm metabolic activity at 37C than nonclinical isolates, while at lower temperatures, clinical and non-clinical isolates did not differ significantly in metabolic activity. All strains readily formed biofilms on polyurethane intravenous catheters under dynamic conditions, as confirmed by scanning electron microscopy and metabolic activity. While planktonic cells already displayed substantial UV-C tolerance, biofilm-associated cells remained viable following exposure to UV-C doses up to eightfold higher than those that impaired planktonic growth. These findings document differences in thermotolerance and biofilm formation by isolate origin and identify biofilm formation as a major factor promoting persistence of R. mucilaginosa on clinically relevant materials and reduced susceptibility to UV-C sterilization.

We previously identified a series of heterobenzamides (HBAs) with potent growth inhibitory activity against Mycobacterium tuberculosis in axenic culture. We also provided evidence that these target QcrB, a component of the terminal cytochrome oxidase in the electron transport chain. We expanded our studies to look at the full microbiological profile: key molecules from the series were tested for activity under different conditions and against additional strains. HBA analogs were active against intracellular bacteria where they exhibited bacteriostatic activity. A strain of M. tuberculosis with a mutation in QcrB (T313I) was resistant to HBAs in both axenic culture and inside macrophages. HBAs retained potency against lineages and mono-resistant strains of M. tuberculosis. HBAs had a narrow spectrum of activity, since they were not active against the ESKAPEE pathogens. Combination of the key HBA with bedaquiline was synergistic, as expected for a QcrB inhibitor, but there was no strong synergy with other drugs. Exposure of M. tuberculosis to the key HBA led to ATP depletion and boosted the oxygen consumption rate. This effect was specific to M. tuberculosis, since human THP-1 macrophage-like cells were unaffected by exposure to the HBA. HBA did not induce the production of reactive oxygen species or affect membrane potential but did affect pH homeostasis. Taken together, these data provide further evidence to support the identification of QcrB as the target and indicate that they are suitable for further drug development.

Human-targeted non-antibiotic drugs are increasingly recognized for their intrinsic antibacterial activity, yet Gram-negative pathogens such as Acinetobacter baumannii exhibit substantial tolerance to these compounds. This tolerance is largely attributed to restricted outer membrane permeability and the activity of multidrug efflux systems. While Resistance Nodulation Division (RND) transporters have been extensively studied, the contribution of individual Major Facilitator Superfamily (MFS) transporters to non-antibiotic drug tolerance remains poorly understood. Here, we investigated H0N29_04330, designated Drug Resistance Transporter A (DrtA), a Bcr/CflA subfamily MFS transporter, to define its substrate specificity and contribution to antibiotic and non-antibiotic drug tolerance. DrtA was highly conserved across the A. calcoaceticus-baumannii complex and exhibited broad substrate specificity when heterologously expressed in an efflux-deficient Escherichia coli background, conferring resistance to benzalkonium, ethidium bromide, phenicols, and the antineoplastic agent mitomycin C. Intriguingly, drtA expression increased E. coli susceptibility to the antifolate compounds methotrexate and aminopterin, suggesting that DrtA may recognize folate-related metabolites rather than function as a dedicated antifolate transporter. In contrast, loss of drtA in its native A. baumannii host primarily impaired tolerance to mitomycin C, highlighting a context-dependent physiological role influenced by the extensive functional redundancy among A. baumannii efflux systems. Site-directed mutagenesis further identified M18 and the membrane-embedded protonatable residue D26 as critical determinants of DrtA transport activity and substrate recognition. Together with previous characterization of CraA, our findings demonstrate that Bcr/CflA subfamily MFS transporters contribute to protection against structurally diverse human-targeted compounds and expand the functional landscape of efflux-mediated intrinsic tolerance beyond conventional antibiotic resistance.

BackgroundOutbreaks of fluconazole-resistant Candida parapsilosis have recently emerged worldwide. In Spain, this phenomenon has been reported since 2020, mainly involving isolates from different clones harbouring the Y132F mutation at Erg11. MethodsWe analysed the expansion of fluconazole resistant C. parapsilosis strains within the national antifungal resistance surveillance program. Genetic clustering and relationships were assessed using microsatellite typing and whole genome sequencing. FindingsWe identified the expansion of three distinct clones carrying the Y132F mutation. Additionally, there was an increase in strains harbouring the G458S mutation, most of which belonged to a clonal complex, although other less prevalent clones were also detected. G458S isolates showed higher resistance to azoles than Y132F strains, particularly to voriconazole and isavuconazole. This increased resistance was associated with mutations in the Tac1 transcriptional regulator and duplication of a chromosomal region containing Tac1 and Erg11. One G458S isolate without mutation at Tac1 exhibited lower MIC values. Furthermore, two isolates carried the K143R mutation, and a distinct group of resistant strains without detectable ERG11 mutations was also identified. Resistant cases were detected across 31 hospitals in 12 autonomous regions. InterpretationOur findings indicate a concerning nationwide expansion of antifungal-resistant C. parapsilosis in Spain, involving multiple resistance mechanisms and clonal lineages, with implications for antifungal treatment and infection control strategies.

L-Serine is an important metabolic and immunomodulatory biomolecule with promising role in managing infections, and autoimmune diseases. L-Serine provides the energy requirements and triggers the toll-like receptor signalling collaterally. However, the role of L-Serine in host antimicrobial response against Mycobacterium tuberculosis (Mtb) remains unexplored. In this study, we investigated whether this metabolite could modulate the antibiotics efficacy against Mtb. Although L-Serine exhibits limited intrinsic anti-mycobacterial activity, but L-Serine demonstrates a synergistic effect when combined with rifampicin and moxifloxacin against both drug-sensitive and multidrug-resistant Mtb. Moreover, L-Serine particularly in combination with palmitic acid showed the enhanced intracellular bacterial clearance in a dose- and time-dependent manner in murine and human macrophages. This synergistic effect was accompanied by increased nitric oxide production and modulation of the host immune response. We identified elevated levels of pro-inflammatory cytokines and reduced IL-10 expression. Furthermore, the metabolic supplementation demonstrated enhanced antimicrobial activity in isolated primary CD14+ monocytes from TB patients. Similarly, the metabolic supplementation of L-Serine in combination with isoniazid and rifampicin significantly reduced bacterial burdens in the lungs and spleen, while improving tissue architecture in murine infection model. Our observations suggest that L-Serine contributes to the observed therapeutic effects. Collectively, this study concludes that L-Serine acts as a promising host-directed therapeutic adjunct, which enhances antimicrobial immunity and potentiating antibiotic efficacy, providing a potential strategy for improving tuberculosis treatment outcomes.

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.

Acinetobacter baumannii is a high priority Gram negative opportunistic pathogen known for its high rates of multidrug resistance (MDR). Minocycline (MIN), a tetracycline class antibiotic, is one of the most effective antibiotics for treating A. baumannii infections in patients. Unfortunately, MIN resistance is spreading internationally and has begun to emerge in the United States. While efflux pumps are correlated with MIN resistant A. baumannii, clinical data suggests alternative mechanisms of MIN resistance. To explore the genetic basis for MIN resistance in A. baumannii we employed a machine learning model to predict genetic resistance correlates from clinical isolates. Mutations in ruvB, a DNA repair protein, were strongly correlated with MIN resistant clinical strains of A. baumannii .Consistent with the prediction, tn26 insertion in ruvB in A. baumannii strain AB5075, and deletion of ruvB in strain ATCC 19606, increased MIN minimum inhibitory concentrations to a level that exceeds the MIN resistance breakpoint. RuvB complexes with RuvA and RuvC to resolve Holliday junctions during recombination. However, only ruvB mutants showed the resistance phenotype; neither ruvA nor ruvC mutants were MIN resistant, suggesting loss of the activity of the complex was not the basis for resistance. We observed ruvB mutants produced increased biomass during planktonic growth relative to the other two ruv mutants. Upon examination, the ruvB::tn26 mutant had a 451% increase in biomass and 360% thicker biofilms relative to wildtype. We determined the disruption of ruvB lead to thicker biofilms and enriched in extracellular DNA (eDNA), and DNase I treatment collapsed the enhanced biofilm phenotype and markedly reduced tetracycline class MICs. FLAG-RuvA accumulated within the biofilm matrix in the absence of RuvB, supporting a model in which RuvA contributes to stabilization of eDNA-rich structures. In a murine pneumonia model, ruvB disruption did not significantly alter survival or pulmonary burden in untreated infection but reduced bacterial dissemination and increased minocycline resistance. Together, these findings reveal an unexpected connection between Holliday junction processing, eDNA-rich biofilm architecture, and antibiotic resistance in A. baumannii.

Background & AimsCurrent nucleos(t)ide analogs efficiently suppress hepatitis B virus (HBV) replication but have limited effects on viral transcription from covalently closed circular DNA (cccDNA) and integrated HBV DNA. We aimed to identify clinically applicable compounds that directly inhibit HBV transcription by screening FDA-approved drugs. Approach & ResultsScreening of 1,470 FDA-approved compounds using an HBV enhancer I/X promoter reporter system identified vorapaxar and aripiprazole as potent inhibitors of viral promoter activity. Both compounds suppressed HBV replication in HBV-producing cells, HBV-infected HepG2-hNTCP cells, and primary human hepatocytes. Aripiprazole reduced hepatocyte nuclear factor 4 (HNF4) protein levels through an ERK/JNK-dependent pathway and inhibited HBV core promoter activity, whereas vorapaxar acted independently of HNF4. Both compounds suppressed enhancer I/X promoter activity through inhibition of STAT3 signaling. Vorapaxar inhibited PAR-1-mediated SRC, EGFR, and STAT3 activation, while aripiprazole suppressed SRC-STAT3 signaling independently of EGFR. PAR-1 activation enhanced HBV transcription, whereas PAR-1 knockdown reduced promoter activity and viral RNA expression. Both compounds also reduced HBV replication in human liver chimeric mice at clinically relevant exposure levels without apparent severe toxicity. ConclusionsVorapaxar and aripiprazole suppress HBV transcription and replication through distinct host signaling pathways. These findings identify PAR-1-STAT3 signaling as a previously unrecognized regulator of HBV transcription and suggest that host-targeting approaches may complement current therapies by suppressing viral gene expression from both cccDNA and integrated HBV DNA. Impact and implicationsCurrent nucleos(t)ide analogues effectively suppress HBV reverse transcription but have limited effects on viral transcription from cccDNA and integrated HBV DNA, highlighting the need for therapies targeting viral gene expression. We identify PAR-1- STAT3 signaling as a previously unrecognized regulator of HBV transcription and demonstrate that two clinically approved drugs, vorapaxar and aripiprazole, suppress HBV replication through distinct host signaling pathways. These findings are relevant to researchers developing host-targeting antivirals and to clinicians seeking complementary therapeutic strategies beyond current nucleos(t)ide analogue therapy. Although further clinical validation and combination studies are required, our results provide a rationale for repurposing approved drugs and for developing transcription-targeting therapies that may complement existing treatments for chronic hepatitis B. HighlightsO_LIVorapaxar and aripiprazole suppress HBV through distinct host pathways. C_LIO_LIBoth drugs inhibit HBV replication in vitro and in humanized liver mice. C_LIO_LIPAR-1 inhibition reduces HBV transcription by blocking SRC/EGFR/STAT3 signaling. C_LIO_LIPAR-1-STAT3 signaling is a novel regulator of HBV transcription. C_LIO_LIHost-targeting antiviral therapy complements current HBV treatment. C_LI