Antimicrobial Agents and Chemotherapy

Ethambutol (EMB) is a vital drug for treating Mycobacterium avium-intracellulare (MAI) infections; however, the genomic mutations underlying EMB resistance in MAI remain unclear. Herein, we evaluated eight sets of MAI clinical isolates, each containing at least two serial isolates collected from the same patient who received EMB in Japan. In four sets, the isolates independently increased EMB MIC by 4-fold, coinciding with mutations in the upstream region of embA or those corresponding to Mycobacterium tuberculosis (Mtb) embB Met306Val and Gln497Arg. Based on the increased EMB MIC values, we defined normal and elevated EMB MICs as [≤]8 {micro}g/mL and [≥]16 {micro}g/mL, respectively. In the other four sets, all of the isolates had elevated EMB MICs. In silico promoter prediction and expression analysis indicated that the upstream region of embA corresponds to the embA-embB promoter region, and mutations in this region increased the transcription of embA and embB, increasing EMB MICs. Furthermore, the analysis of 60 epidemiologically unrelated strains revealed that isolates with mutations in the embA-embB promoter and at embB codons 306/497 exhibited significantly higher EMB MICs compared with those without mutations. Publicly available genomic data demonstrate the worldwide occurrence of these mutations in clinical isolates. These results establish an association between elevated EMB MICs and mutations at embB codons 306/497 and the embA-embB promoter and are expected to predict EMB resistance.

Background. The intrapulmonary pharmacokinetics of antimicrobial agents used to treat nontuberculous mycobacterial (NTM) pulmonary disease remain poorly characterized, limiting the optimization of dosing regimens. This study characterized the plasma and intrapulmonary pharmacokinetics of azithromycin, ethambutol, rifampicin, clofazimine, and amikacin, as well as their penetration into pulmonary lesion sites. Methods. We prospectively enrolled patients undergoing guideline-based treatment for NTM pulmonary disease who were indicated for surgical resection at a single center in Japan. Drug concentrations were measured in the plasma and lung samples, and analyzed using a population pharmacokinetic model. The lung lesion site, cavity, or nodule/bronchiectatic were evaluated as covariates of the plasma-to-lung partition ratios. Results. Twenty-four patients were enrolled in the study. Antimicrobial agents other than rifampicin and amikacin accumulate in the lungs at concentrations > 40-fold higher than those in the plasma. Notably, the intrapulmonary half-life of ethambutol, which has not been well-characterized to date, is estimated to be approximately 2 months, indicating prolonged retention within the lungs. Evaluation of drug penetration into cavities and nodular/bronchiectatic lesions showed no clearly reduced concentration compared to that of normal lung tissue. However, in the single case where the caseum was obtained, azithromycin, ethambutol, and rifampicin levels exhibited clearly lower concentrations. Conclusions. Ethambutol shows a prolonged intrapulmonary half-life, suggesting sustained lung exposure even with intermittent dosing. The absence of clearly reduced drug penetration into lesion sites suggests that lesion phenotype alone may have limited value in guiding drug selection.

IntroductionErgosterol-targeting azoles are widely used in the treatment of Candida albicans infection. In addition to direct antifungal activity, azoles are known to enhance neutrophil-mediated killing of C. albicans, but the underlying mechanisms remain unclear, particularly whether ergosterol depletion directly modulates host immune responses. Gap StatementIt remains unknown whether reduced ergosterol levels alone, independent of broader disruption to sterol biosynthesis and fungal morphogenesis, influence neutrophil antifungal activity. AimThis study aimed to determine how genetic disruption of late-stage ergosterol biosynthesis affects neutrophil-mediated responses to C. albicans. MethodologyDoxycycline-repressible GRACE mutants targeting late-stage ergosterol biosynthesis genes (ERG4, ERG5, ERG3 and ERG28) were co-incubated with primary human neutrophils. Fungal survival, oxidative burst, phagocytosis, neutrophil extracellular trap (NET) formation and cell wall composition were assessed. ResultsAll ergosterol-deficient strains induced elevated neutrophil reactive oxygen species (ROS) production; however, only ERG4 depletion was associated with enhanced fungal clearance. This phenotype correlated with increased phagocytosis and reduced NET formation. Cell wall analysis revealed no changes in total chitin or mannan content but demonstrated significantly increased surface exposure of {beta}-1,3-glucan in ERG4-depleted cells. ConclusionThese findings indicate that disruption of late-stage ergosterol biosynthesis, particularly via ERG4, enhances neutrophil antifungal responses and is associated with increased {beta}-glucan exposure. This study highlights a potential role for ergosterol in immune evasion and suggests that targeting terminal steps of the pathway may improve host-mediated clearance of C. albicans.

Adjunctive therapies that enhance the efficacy of existing antitubercular drugs are needed for drug-resistant tuberculosis. We evaluated the efficacy of intranasally administered recombinant D29 LysB, a mycobacteriophage-derived mycolylarabinogalactan esterase, in murine and guinea pig models of pulmonary tuberculosis. BALB/c mice and guinea pigs were aerosol-infected with Mycobacterium tuberculosis H37Rv and treated for 4 weeks with LysB alone or with standard antitubercular therapy (ATT: rifampicin, isoniazid, pyrazinamide). Outcomes included pulmonary and extrapulmonary bacterial burden (CFU), lung and spleen histopathology, cytokine profiling, and humoral immune responses. LysB monotherapy produced modest pulmonary CFU reductions. When given adjunctively with ATT, LysB produced an additional 0.6-0.7 log10 reduction in lung CFU compared with ATT alone and decreased splenic dissemination in both species. Combination therapy improved tissue pathology, reducing granulomatous involvement and preserving pulmonary architecture. LysB treatment increased TNF- with a moderate rise in IL-10, a profile consistent with enhanced antibacterial immunity without excessive inflammatory damage. Repeated intranasal administration was well tolerated; no IgE-mediated hypersensitivity was detected. LysB-specific IgG developed but did not diminish therapeutic efficacy. These results show that intranasal D29 LysB augments the bactericidal and histopathological effects of standard ATT in vivo and support further development of inhaled phage-derived lysins as adjunctive therapies for drug-resistant tuberculosis. ImportanceTuberculosis remains a major cause of infectious mortality worldwide, and the increasing burden of multidrug-resistant and extensively drug-resistant disease continues to challenge effective treatment. New therapeutic approaches that complement conventional antibiotics are urgently needed. In this study, intranasally delivered recombinant mycobacteriophage-derived LysB was well tolerated and enhanced treatment efficacy in experimental pulmonary tuberculosis. Adjunctive LysB improved bacterial clearance, reduced tissue pathology, and modulated host immune responses in both murine and guinea pig models. These findings highlight phage-derived endolysins as promising inhalable adjunctive therapeutics for drug-resistant tuberculosis.

Biofilms pose a significant challenge to antimicrobial therapy. Bacteria in biofilms differ from planktonic counterpart in their altered metabolism, collective behavior, protective role of extracellular matrix and diversified microbial subpopulations. These attributions significantly influence bioavailability and activity of antibiotics. The presence of bacterial aggregates during acute infections expands the problem to many other conditions previously not discussed in the biofilm context. Klebsiella pneumoniae is a leading cause of life-threatening hospital-acquired infections and is included in the WHO Bacterial Priority Pathogens List due to increasing antimicrobial resistance. The combination of antimicrobial resistance and the ability to form biofilms severely limits the efficacy of antibiotic treatments. In this study, we investigated the in vitro susceptibility of mature biofilms to 13 antimicrobials of K. pneumoniae clinical isolates from a single hospital. The resistance profiles of the local clinical isolates were consistent with the global epidemiology of K. pneumoniae. Minimal biofilm eradication concentrations (MBEC) for mature biofilms were defined with two assays (biomass and metabolic activity measurements) and brought into relation with susceptibility breakpoints and plasma (Cmax). Colistin sulfate, tigecycline, cephalosporins and combination of imipenem with cilastatin were the most potent biomass eradicators, while suppression of metabolic activity was barely reachable. Moreover, we observed a notable increase in metabolic activity upon exposure to sub-MBEC concentrations of antibiotics. Finally, our data broach a subject of antibiotic prioritization with respect to biofilm tolerance. IMPORTANCEThis study addresses the critical gap between standard antibiotic susceptibility testing and the tolerance of biofilm and microbial aggregates during infections caused by K. pneumoniae. By systematically evaluating mature biofilms from a significant number of clinical isolates, we demonstrate that colistin and tigecycline show potent activity against both biofilm biomass and metabolic activity, whereas cephalosporins primarily reduce biomass without effectively suppressing bacterial metabolism, and other drugs have only weak effects on biofilms at clinically achievable concentrations. Furthermore, the alarming observation that sub-inhibitory biofilm eradication concentration (sub-MBEC) of antibiotic can paradoxically increase the metabolic activity of biofilms highlights a potential risk factor for therapy failure and resistance development. Our findings contribute to the necessary evidence base for prioritizing existing antibiotics in the limited armamentarium against biofilm-forming K. pneumoniae.

Klebsiella pneumoniae (Kp) is a common antibiotic-resistant pathogen that colonizes the gastrointestinal tract and can disseminate to peripheral sites, causing a range of infections including bacteremia, urinary tract infections, and pneumonia. Intestinal colonization with Kp is a risk factor for subsequent infection, as the colonizing strain frequently corresponds to the infecting isolate. Accordingly, targeting Kp prior to dissemination at the site of colonization through decolonization strategies offers a promising approach to mitigate infection risk. In this study, we evaluated the repurposing of existing drugs with previously uncharacterized antibacterial activity as candidates for Kp decolonization. To this end, we screened an antiviral compound library for their activity against Kp. We identified and validated six compounds with previously uncharacterized activity against Kp. Then, we screened a library of clinical Kp strains against a subset of these compounds and found that their activity was strain-specific to degrees that differed based on the compound. Finally, we tested the activity of these compounds in conditions relevant to the human gut. We determined the activity of these candidates was dependent on biological context. Collectively, these findings support further investigation of antiviral drugs as potential gut decolonization therapies for Kp.

Rationale: Efficacious dose selection for anti-tuberculosis drugs has traditionally relied on achieving plasma exposures above the minimum inhibitory concentration, but this approach has not consistently aligned with clinical outcomes. Objectives: We sought to identify early pharmacokinetic-pharmacodynamic targets most predictive of clinical efficacious dose. Methods: We conducted a back-translational, pharmacokinetic-pharmacodynamic simulation-based analysis of 15 anti-tuberculosis drugs. Using pharmacokinetic data from multiple biological matrices and a range of pharmacodynamic metrics, we established candidate exposure-response targets for attainment. We systematically evaluated the predictive accuracy of each target pair against established clinical doses to formulate a decision-making framework linking key drug properties to the most predictive targets. Measurements and Main Results: Depending on the target used, projected clinical doses varied widely - both within and across compounds - highlighting the importance of target selection for dose projection and go/no-go decisions. In general, targeting cellular lesion-level drug exposures relative to in vivo preclinical potency provided an effective approach for early dose selection. However, for highly penetrating drugs, targeting site-of-action therapeutic exposures in the caseum was more predictive of clinical dose. Based on these findings, we developed a preliminary dose prediction tool that enables drug developers to estimate clinically relevant dose ranges of compounds using in vitro and early in vivo data. Conclusions: This work establishes and validates a simple, evidence-based framework to standardize early translational decision-making on dose selection of anti-tuberculosis candidates in development.

We report the isolation and identification of a Paenibacillus polymyxa strain from the citizen science project; Swab and Send. Through whole genome sequencing we are able to describe the biosynthetic gene cluster of polymyxin A produced by P. polymyxa 1G (NCBI accession no. JBVPZV000000000), compare the pmxA, pmxB and pmxE genes to five other polymyxin genes encoding known polymyxin variants, and provide mass spectrometry data that supports the production of polymyxin A1 (1157 m/z) and A2 (1143 m/z). Polymyxins are ranked in the highest priority critically important antimicrobials classification by the WHO and are of particular importance for treating gram-negative multidrug resistant pathogens. Due to the discovery of polymyxins occurring in the 1940s, there is little genetic research around polymyxins, and the literature focusses primarily on clinically used polymyxin E (colistin) and polymyxin B. Previous literature suggests that polymyxin A1 has similar/lower toxicity to clinically used polymyxins E and B. To test if polymyxin A was able to overcome current resistance mechanisms to clinically used polymyxins, the cell free supernatant from P. polymyxa 1G was tested against a panel of clinical isolates with various resistance genes. We found that resistance genes mcr-1 and mcr-4 confer resistance to polymyxin A produced by our isolate meaning that, while polymyxin A has good antimicrobial activity, clinical resistance mechanisms already confer resistance to this variant of polymyxin.

Ganciclovir (GCV), and its orally available pro-drug valganciclovir (VGCV), are preferred therapies for treating congenital cytomegalovirus (cCMV), however, their use carries a significant risk of neutropenia for the child. This risk limits dosing and effectiveness of VGCV, particularly in the treatment of infants with cCMV infection, who are at increased risk for sensorineural hearing loss (SNHL). We hypothesized that an improved understanding of the pharmacokinetics (PK) and pharmacodynamics (PD) of VGCV in cCMV-infected infants at risk for SNHL would inform strategies for optimizing safe and effective VGCV dosing. Participants were enrolled in one of two clinical studies interrogating the PK, safety, and efficacy of VGCV treatment in cCMV-infected infants at risk for SNHL. GCV exhibited a short median half-life of 2.02 h and the median (range) area under the 24 h concentration-time curve (AUC24) was 60.8 (26.8, 99.4) g*h/mL. An AUC24 > 70 g*h/mL was associated with an elevated risk of neutropenia (Fisher's Exact p = 0.029). No associations between GCV PK and hearing outcomes were observed. Taken together, these results indicate vast inter-individual variability in GCV PK that is associated with dose-related toxicity, supporting the need for individualized dosing in the cCMV-infected population.

Maintaining the efficacy of {beta}-lactam antibiotics against Staphylococcus aureus is a clinical priority given the prevalence of methicillin-resistant S. aureus (MRSA). We previously showed that the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd) synergize with {beta}-lactams. Here, we extended this by evaluating additional nucleotide metabolism-targeting agents. Gemcitabine (Gem) and mitomycin C (Mito), like 5-FU and 5-FUrd, exhibited intrinsic anti-MRSA activity and potentiated {beta}-lactams, whereas the purine analogue 6-thioguanine (6-TG) showed distinct, often antagonistic effects. Transcriptomic analysis revealed that pyrimidine-targeting agents repress lysine and glutamate biosynthesis, while 6-TG induced these pathways, implicating amino acid metabolism in {beta}-lactam potentiation. Consistent with this, pyrimidine analogues also suppressed GlmS expression, potentially limiting UDP-GlcNAc production required for cell wall synthesis, and synergized with fosfomycin. Fluorescence microscopy confirmed that the potentiation of oxacillin activity by pyrimidine-targeting agents, but not 6-TG, was accompanied by impaired peptidoglycan synthesis. Additionally, glutathione-mediated attenuation of killing implicated reactive oxygen species in the bactericidal activity of cloxacillin combinations. Finally, these agents displayed strong anti-biofilm activity, further enhanced in combination with daptomycin and rifampicin. Together, these findings highlight the potential of pyrimidine analogues to potentiate cell wall-targeting antibiotics and identify an important role for modulation of cell wall precursor pathways in this anti-MRSA activity. ImportanceDrug interactions can complicate the treatment of antimicrobial resistant infections in patients undergoing treatment for cancer highlighting the importance of understanding the effects of anti-cancer drugs on pathogens like MRSA. Here, we investigated several drugs that target nucleotide metabolism and are used to treat cancer, fungal, and viral infections, both alone and in combination with commonly used penicillin-type antibiotics. We found that pyrimidine analogue drugs enhanced the activity of these antibiotics against MRSA, whereas the purine analogue 6-thioguanine reduced antibiotic effectiveness. These drugs altered the bacterial cell wall and other metabolic pathways linked to antibiotic susceptibility. Our findings reveal the potential to repurpose certain anticancer drugs to improve treatment of MRSA infections, while also cautioning that some drug combinations may interfere with antibiotic therapy.

BackgroundPhage therapy is increasingly considered a promising alternative for treating multidrug-resistant (MDR) infections. However, its clinical application remains limited by challenges in isolating effective phages against resistant clinical strains and by the limited ability of in vitro assays to predict performance in real biological environments. While biological matrices are known to influence phage activity, these effects are not well characterised. MethodsA phage-resistant Pseudomonas aeruginosa isolate from a patient with recurrent MDR urinary tract infection was used as the model organism. Conventional isolation methods failed to recover effective phages, leading to the development of TEASER-i (Transient EDTA- and Ion-Assisted Sequential Enrichment & Recovery). Recovered phages were characterised using adsorption assays, one-step growth kinetics, and time-kill experiments. Their antibacterial activity was evaluated both in vitro and in ex vivo human matrices (whole blood, serum, plasma, and urine). Phage efficacy was quantified using maximum log reduction (Emax), area under the curve (AUC), and phage-to-bacteria ratio (PBR). ResultsA novel TEASER-i method optimised for difficult-to-treat Gram-negative infections, enabled recovery of a functionally effective Osewage-derived P. aeruginosa phage, which outperformed a Ourine-derived P. aeruginosa phage that showed slower replication and lower burst size. Phage activity varied significantly in blood, serum, and plasma. Urine supported the most sustained antibacterial effect. In many cases, early bacterial reduction was followed by regrowth. Sustained activity was associated with maintenance of favourable PBR values, while negative PBR corresponded to treatment failure. At 96 h, only two conditions maintained favourable phage load (log 10 PBR > 0): the S. aureus phage in urine (+1.66) and the sewage-derived P. aeruginosa phage in serum (+1.32). ConclusionsPhage efficacy depends not only on intrinsic lytic capacity but also on the ability to persist and amplify within specific biological environments. Conventional isolation and in vitro screening may therefore overestimate therapeutic potential. Combining optimised isolation strategies with ex vivo evaluation provides a more realistic framework for phage selection and clinical translation.

The development of novel therapeutics for infectious diseases remains a global health priority. To accelerate the treatment development, innovative strategies through new approach methodology (NAM) are needed to bridge speed of in vitro with predictive power of in vivo studies, while reducing mammalian experiments. The zebrafish (Danio rerio), particularly the embryo/larva, has been established as a valuable non-mammalian in vivo model in biomedical research. We developed a standardized and streamlined workflow for the zebrafish as NAM, which consisted of 3 steps: drug selection and efficacy evaluation, internal exposure assessment, and PKPD modelling. Compounds with higher tolerated doses than minimum inhibitory concentration were selected. Drug efficacy was quantified through longitudinal individual fluorescence microscopy at baseline and 24 and 48h on treatment. Drug exposure was quantified in larval homogenates and exposure medium from 0-48h on treatment. The PKPD relationship was quantified by non-linear mixed effects modelling. For case study bedaquiline, PKPD was quantified using a one-compartment model with age-depending elimination, and an Emax concentration-response relationship on the delayed logistic bacterial growth function, with an EC50 of 26.6 {micro}g/mL and an Emax of 1.07-1.37. In the case of clarithromycin, in contrast, negligible internal exposure after waterborne treatment were observed, illustrating the risk of false negatives without internal exposure assessments. Bactericidal efficacy was confirmed by intravenous drug injections, showing a clear dose dependent antimycobacterial effect. The standardized zebrafish NAM workflow presented here facilitates the translation of drug efficacy to higher vertebrates, reducing rodent studies to confirmatory or replacing them completely, thus accelerating drug development.

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

Tuberculosis (TB) remains a leading cause of infectious disease mortality, and the continued emergence of drug-resistant Mycobacterium tuberculosis (MTB) strains threatens the effectiveness of standard treatment regimens. Culture-based antibiotic susceptibility testing (AST) remains the clinical reference standard for resistance determination but typically requires six to eight weeks, delaying initiation of optimized therapy for patients with drug-resistant disease. Whole-genome sequencing (WGS)-based approaches provide a rapid alternative for predicting antimicrobial resistance directly from genomic data and are increasingly being incorporated into diagnostic workflows. This survey reviews computational approaches for genomic resistance prediction in MTB, focusing on two major classes of methods: catalog-based tools that identify established resistance-conferring variants, and de novo machine learning approaches that infer resistance from genome-wide sequence features. We examine the strengths and limitations of these approaches with respect to interpretability, scalability, computational requirements, and concordance with phenotypic testing. We further discuss emerging directions in quantitative minimum inhibitory concentration (MIC) prediction, challenges in pyrazinamide susceptibility testing, and the limited availability of resistant isolates for newer and repurposed drugs used in multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) treatment regimens. Continued expansion of paired phenotypic and genomic datasets, standardized MIC testing protocols, and rigorous lineage-aware evaluation frameworks will be essential for improving the clinical reliability and global deployment of genomic resistance prediction for tuberculosis diagnostics.

OXA-232, an OXA-48 like carbapenemase stands amongst newly identified beta-lactamases that causes of the extensive of beta-lactam resistance. While active-site residues are well characterised, the contributions of conserved non-active-site residues in exerting enzymatic activity remain unexplored, limiting our understanding about the roles of these residues in the overall OXA-232 function. To address these gaps, the conserved residues S118, V120, L158, and D159 of OXA-232 positioned adjacent to the active-site motifs and within the omega-like loop were substituted with alanine. Substitutions of S118A and D159A rendered the expressing cells susceptible to penicillins, cephalosporins, and carbapenems, whereas the cells harbouring OXA-232V120A and OXA-232L158A proteins exhibited substrate-selective susceptibility changes. Kinetic analysis with purified proteins revealed the reduction in catalytic efficiency of all the mutants compared to wild-type protein. Though the L158A and D159A mutated proteins become deacylation-deficient, the mutations S118A and V120A exhibited selective acylation defects without trapping intermediates. It is evident from circular dichroism spectroscopy and molecular dynamics simulations that OXA-232S118A, OXA-232V120A, and OXA-232L158A nearly retained their secondary structures and compactness, except for OXA-232D159A, which presumably triggered a misfolding leading to destabilisation of the omega-loop. Interestingly, bicarbonate supplementation partially rescued the lost activities in soluble mutants, underscoring the carbamylation dependence. Taken together, these findings establish S118 and D159 as essential for core catalysis and structural integrity, with V120 and L158 modulating substrate-specific turnover and orientation. The current study reappraised the mechanistic insights of OXA-48-like carbapenemases, providing significant resources in rationally designing future therapeutics to combat carbapenem resistance.

The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 g/ml for CAZ, 8 g/ml for LVX, 16 g/ml for MEM, and 8 g/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 g/ml, 8 g/ml, and 16 g/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document. IMPORTANCEThe Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for four antimicrobials, we encountered significant challenges in our analyses, including limitations with data for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide clinicians on treatment and supported the eventual removal of ECVs from the CLSI M100 standards document.

BackgroundThe gut-kidney axis plays a direct role in gastrointestinal and kidney health. Gut-derived metabolites like uremic toxins are associated with the pathophysiology of feline chronic kidney disease (CKD). The aim of the study was to identify novel fecal biomarkers and investigate the roles of gastrointestinal metabolites in feline CKD. ResultsFecal samples from 41 healthy non-CKD (control) and 67 CKD cats, including 5 IRIS stage 1 (CKD1), 37 stage 2a (CKD2a), 18 stage 2b (CKD2b), and 7 stage 3 (CKD3), were subject to fecal untargeted metabolomics and targeted short-chain fatty acid (SCFA) analyses. Multiple linear regression, adjusted for sex, age, body weight and study site, identified 64 differential metabolites between control and across CKD groups (P<0.0001 and FDR<0.10). Approximately 65% of the metabolites were lipids, including polyunsaturated long-chain fatty acids, acylcarnitines, and ceramides. Random Forest algorithm selected N1-methyl-2-pyridone-5-carboxamide (2PY), a uremic toxin from nicotinamide catabolism, as the top fecal marker for classifying feline CKD. Fecal 2PY was increased in CKD1 (P = 0.03), CKD2a, CKD2b, and CKD3 (all P<0.0001) compared to the controls. Data mining revealed serum concentration of 2PY was significantly increased with severity of CKD in cats, possibly due to impaired renal excretion. Cholesterol and arachidonic acid, markers for enterocyte shedding and inflammation, were increased in CKD3 versus control (both P<0.05). In healthy non-CKD cats, evident suggested fecal lipids increased with age (P<0.0001), and were higher in females versus males (P<0.0001). While fecal indole and p-cresol were increased in CKD3 versus control (both P<0.05), no change was observed in indoxyl sulfate (IS) or p-cresol sulfate (PCS). Fecal indole-3-acetic acid (IAA) was decreased in several CKD groups compared to the controls (all P<0.05). Finally, two branched SCFAs, isobutyrate and isovalerate, were increased in CKD3 versus control (both P<0.05). ConclusionsThe study revealed 2PY as a novel marker and unveiled profound alterations in intestinal lipid compositions with a potential link to gut barrier integrity and inflammation in CKD.

BackgroundTuberculous meningitis (TBM) is the most sinister form of extrapulmonary tuberculosis (EPTB), associated with high mortality due to delayed diagnosis and limited sensitivity of conventional and molecular tests. Current study evaluated the diagnostic utility of Lipoarabinomannan antigen (LAM) detection in CSF and urine and explored host inflammatory biomarkers for diagnosis and prognosis of TBM. MethodsThis prospective observational study enrolled 80 patients with presumptive TBM at a tertiary care centre. CSF samples were subjected to AFB microscopy, liquid culture(MGIT-960), GeneXpert MTB/RIF (GX), and LAM lateral flow assay. Urine LAM was performed at baseline. Serum and CSF levels of IL-1{beta}, IL-6, TNF-, IFN-{gamma}, IL-17A, and IP-10 were measured at baseline and after 1 month treatment. ResultsAmong 80 participants, 23 (28.7%) had definite TBM and 46 (57.5%) had probable TBM. CSF LAM sensitivity and specificity against microbiological reference standards was 43.5% and 80.7%, while urine LAM sensitivity (60.9%) and specificity 82.5% was higher. Against composite reference standards, both CSF and urine LAM showed reduced sensitivity but achieved 100% specificity. Serum IL-1{beta} showed the best diagnostic performance (AUC 0.943; sensitivity 88.9%, specificity 90.9%). Elevated serum and CSF IP-10 levels were associated with poor outcomes, whereas declining IL-6 and TNF- levels correlated with treatment response. ConclusionLAM detection in CSF and urine may serve as a highly specific, rapid rule-in test for TBM. Host inflammatory biomarkers, especially IL-1{beta} and IP-10, show additional diagnostic and prognostic value. Combining LAM testing with cytokine biomarkers may improve early diagnosis and efficient clinical management of TBM.

Antimicrobial resistance is an impactful One Health issue. One of its drivers is the extensive use of antibiotics in both human and animal production systems, and despite regulatory restrictions on antibiotic use in poultry farming, antimicrobial resistance remains a major challenge. Consequently, animals are at higher risk of harder-to-treat diseases and play a role as resistance reservoirs, highlighting the need for alternative antimicrobial strategies. Towards this end, antimicrobial peptides (AMPs) have emerged as promising candidates due to their broad-spectrum activity and lower propensity to induce resistance. However, the effectiveness of AMPs against poultry pathogens, and in particular multi drug-resistant strains, is largely unclear. To tackle this question, we evaluated the synthetic AMP SET-M33 against four species of clinically relevant pathogens in poultry, namely Escherichia coli, Salmonella enterica, Enterococcus faecalis and Enterococcus cecorum. Using a panel of 141 field isolates, we found that SET-M33 broadly inhibited bacterial growth at low micromolar concentrations (median MICs of 2.5 M and 5 M for Gram-negative and Gram-positive strains, respectively), including in multi drug-resistant isolates. To examine the potential impact of SET-M33 on the host, we established a new in vitro co-cultivation system using chicken intestinal organoids. We found that SET-M33 retains its antimicrobial activity in organoid-microbe co-cultures at concentrations that preserved host viability. These findings demonstrate the potential of SET-M33 as a new antimicrobial agent against pathogens in poultry.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation or ciprofloxacin resistance have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotic challenge remains incompletely understood. In this study we employed a whole genome screen to assay the impact of gene disruptions or altered gene expression on survival of P. aeruginosa biofilms exposed to different concentrations of ciprofloxacin. Genes impacting fitness in the biofilm context were identified by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Genes associated with c-di-GMP regulation and Gac/Rsm signalling were identified as primary regulators for biofilm formation in the presence and absence of ciprofloxacin. In addition, a group of genes involved in respiration, metabolism (especially polyamine metabolism), and various transporter and efflux systems were identified as important for biofilm fitness. Ciprofloxacin specifically imposed a selective pressure on flagellar function and Psl production which were essential for survival in early biofilms. Moreover, transposon insertions within the CPA gene clusters (PA5448-PA5451 and PA5455-PA5456) and the salvage peptidoglycan recycling pathway showed reduced fitness in late biofilms at high concentration of ciprofloxacin, indicating that cell envelope integrity is beneficial for mature biofilms. This study identifies important determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin and implicates potential therapeutic targets for antibiofilm drug development.