Antimicrobial Agents and Chemotherapy

Linezolid is a key component of the bedaquiline, pretomanid, linezolid (BPaL) regimen for rifampin-resistant tuberculosis, but dose- and duration-dependent mitochondrial toxicity frequently limits its use. TBD09 (formerly MK-7762) is a novel oxazolidinone developed to maintain antitubercular potency comparable to linezolid while reducing related adverse events. We evaluated the contribution of TBD09 to the bactericidal and sterilizing activities of BPa-based regimens in a murine tuberculosis model. BALB/c mice were aerosol-infected with Mycobacterium tuberculosis H37Rv and treated starting 2 weeks post-infection. In Experiment 1, dose-ranging bactericidal activity of TBD09 (50, 100, 200 mg/kg) was assessed alone and in combination with BPa, with comparison to linezolid and sutezolid. In Experiment 2, the sterilizing activity of TBD09 (200 mg/kg) in combination with BPa and BPa plus moxifloxacin (M) was assessed by evaluating relapse after treatment durations of 4-22 weeks. TBD09 monotherapy was bacteriostatic, similar to linezolid over the first 4 weeks of treatment. The addition of TBD09 to BPa significantly enhanced bactericidal activity comparable to the addition of linezolid. In relapse assessments, BPa+TBD09 resulted in significantly fewer relapses compared to BPa alone after 14 weeks of treatment, and BPaM+TBD09 resulted in significantly fewer relapses compared to BPaM after 10 weeks. Model-based analysis estimated the treatment duration to 95% cure for BPa+TBD09 at 2.7 weeks, compared to 3.2 weeks for BPaL, although the time to 50% cure was similar. These results demonstrate that TBD09 provides bactericidal and sterilizing activity similar to linezolid in combination with BPa(M), supporting further clinical development of TBD09 for tuberculosis.

Rationale: Tuberculosis preventive therapy (TPT) is essential for global tuberculosis control, yet estimated coverage remains well below global targets. Current World Health Organization (WHO)-recommended regimens require 1-9 months of treatment and are associated with suboptimal completion rates. Long-acting injectable (LAI) formulations offer the potential to improve TPT uptake and completion, with ideal products providing "one-and-done" options via a single administration. Sorfequiline, a second-generation diarylquinoline with superior potency over bedaquiline and lower QT prolongation risk, is a promising candidate for LAI TPT.

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.

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.

The emergence of antimicrobial resistance restricts the clinical efficacy of therapeutic antibiotic drugs. Elucidating the molecular mechanisms by which bacteria evade antibiotic activities will lead to novel therapeutic approaches. Therefore, it is critical to understand the mechanisms that govern susceptibility to antibiotics. Fluoroquinolones are a class of antibiotics widely used to treat both Gram-positive and Gram-negative bacterial infections. Resistance to fluoroquinolones is widespread in multiple pathogenic bacteria, limiting their therapeutic usefulness. Resistance to fluoroquinolones is well-known to be mediated by Point mutations in the genes encoding the cellular targets of fluoroquinolones, type-II topoisomerases, leads to clinically relevant resistance. However, several recent studies have revealed that dysregulation of translation influences fitness during fluoroquinolone exposure. For example, several screens have identified insertions in gene that encodes elongation factor P (EF-P) increases survival to fluoroquinolones. Here, we sought to understand the impact of EF-P on fluoroquinolone survival using the Gram-positive model organism Bacillus subtilis. We found that loss of EF-P increases the survival of B. subtilis exposed to fluoroquinolones by several orders of magnitude. Furthermore, we demonstrate that fluoroquinolone sensitivity is dependent on post-translational modification of EF-P, indicating that it is dependent on EF-P activity. Remarkably, loss of EF-P increases survival of cells lacking RecA, a key DNA repair protein, which is crucial for the survival of fluoroquinolone drugs. Using transcriptomics, we identify key cellular response pathways differentially regulated in cells lacking EF-P during fluoroquinolone treatment. We conclude that EF-P regulates diverse mechanisms that drive the susceptibility to fluoroquinolones.

Screening of the open-access CRESTdb small-molecule library identified the imidazothiazole carboxamide sCQG200 (1) as an initial hit, with activity against H37Rv and Erdman Mycobacterium tuberculosis strains in cholesterol-containing medium (MIC90= 6.88 uM and 4.48 uM, respectively) and against intracellular Mtb (EC50= 3.80 uM). sCQG200 retained activity against drug-resistant Mtb strains, including clinical MDR/XDR isolates. SAR optimization led to analogs 21 and 24 with substantially improved antimycobacterial potency. Mouse pharmacokinetic studies following oral and intravenous administration showed 62.1% and 29.5% oral bioavailability for 21 and 24, respectively. This structurally differentiated chemotype provides a promising starting point for further optimization as antitubercular agent.

Successful treatment of primary Clostridioides difficile infection (CDI) while preventing its recurrence remains a major clinical challenge, with recurrent CDI (R-CDI) affecting 20-30% of treated patients. Persistence of C. difficile spores within the intestinal mucosa is thought to drive R-CDI following antibiotic treatment. Here, we show that intracellular C. difficile spores remain protected from taurocholate-triggered germination, and that nystatin, an FDA-approved antifungal that blocks spore entry into host cells, reduces this protected intracellular population and redirects spores toward an extracellular, germinating population accessible to antibiotics. In a murine model of R-CDI, oral co-administration of taurocholate with nystatin and antibiotics during primary CDI reduced R-CDI incidence from 100% to 44%. When administered during an active R-CDI episode, the same formulation reduced a second R-CDI from 100% to 62%. To improve bioavailability, we encapsulated nystatin and vancomycin in chitosan nanoparticles. Oral administration of nanoparticle-encapsulated vancomycin, alone or combined with nanoparticle-encapsulated nystatin, reduced R-CDI colonization to 33% and 44%, respectively, compared with 100% in mice receiving free vancomycin. Together, these findings support a therapeutic strategy that repurposes nystatin and taurocholate, in combination with antibiotics and nanoparticle-based delivery, to target intracellular spore persistence and reduce the incidence of C. difficile recurrence..

Co-administration of phages and antibiotics has been proposed as a therapeutic approach against antibiotic-resistant bacteria. The relationship, however, between antibiotic resistance and phage resistance in clinical isolates is unclear. Here, we examine associations between phage and antibiotic resistance profiles across a panel of Pseudomonas aeruginosa clinical isolates from the Centers for Disease Control (CDC) and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate (ARI) Bank comprising 55 clinical strains with full genome sequences and antibiotic susceptibility testing (AST) data for 11 clinically relevant antibiotics. As phages in this study, we use three well-characterized, morphologically distinct phages, OMKO1, Luz19, and PAML31-1. We screen for phage resistance using a growth suppression assay, then conduct statistical analysis against antibiotic MIC (Minimum Inhibitory Concentration) data provided by the CDC to define association patterns across this dataset. We find multiple significant susceptibility correlations between pairs of antibiotics and phages, and a positive overall association between average phage resistance and antibiotic resistance across the 55 strains, even controlling for phylogenetic associations (=0.358, p<0.005). We conclude that phage and antibiotic resistance are positively associated across this clinical isolate collection, suggesting that the two resistance phenotypes are not independent in P. aeruginosa. These findings have implications for the development of phage-antibiotic cocktails.

Fosfomycin is increasingly used to treat multidrug-resistant Gram-negative infections, yet resistance surveillance largely relies on homology-based detection of known fos genes. Here, we identify Gfa, a previously unrecognized integron-borne fosfomycin resistance determinant uncovered through functional screening of 129 integron gene cassettes. Gfa confers high-level fosfomycin resistance across Enterobacterales and Pseudomonas aeruginosa and is resistant to phosphonoformate inhibition. Structural modelling and mutagenesis reveal a conserved cysteine-rich, zinc-organized architecture essential for activity and distinct from canonical Fos enzymes. Crude-extract assays confirm glutathione-dependent fosfomycin inactivation under metal-chelating conditions. Distant Gfa homologs also conferred resistance, although activity was restricted to a subset of proteins tested. Together, these findings establish Gfa as a previously unrecognized fosfomycin resistance family and highlight the limitations of homology-based mining for discovering novel resistance determinants and predicting which family members are functional.

In a previous screen for compounds that inhibit Plasmodium falciparum merozoite invasion of red blood cells, we identified the Medicines for Malaria Venture compound MMV006833. This compound inhibits PfSTART1, a protein implicated in the expansion of the nascent parasitophorous vacuole membrane following invasion, to accommodate the developing ring-stage parasite. Live-cell lattice light-sheet microscopy of invading merozoites revealed that mNeonGreen-tagged PfSTART1 is released from structures within the merozoite into the nascent parasitophorous vacuole approximately 109 seconds after invasion. Expansion microscopy of PfSTART1-HA merozoites further showed that these punctate PfSTART1-containing structures do not colocalise with known secretory organelles (rhoptries, micronemes and dense granules). Although analogues of MMV006833 are highly potent against P. falciparum, they were previously found to be ineffective against P. berghei parasites in the mouse malaria model. Here, we demonstrate that PbSTART1 is highly resistant to MMV006833 and its analogues when expressed in P. falciparum, indicating that structural differences between the orthologous proteins reduce inhibitor potency. The crystal structure of PfSTART1 in complex with WEHI-991 revealed the molecular basis for inhibition and provided a structural explanation for the reduced potency of this family of compounds against P. berghei. To sensitise P. berghei parasites to MMV006833 analogues, the parasites were engineered to express PfSTART1; however, these chimeric parasites remained insensitive to the compounds. This suggests that factors beyond target engagement, such as compound half-life or bioavailability, contribute to the lack of efficacy observed in the mouse malaria model.

Powassan virus (POWV) is an emerging tick-borne orthoflavivirus that causes severe encephalitis, long-term neurologic sequelae, and death, yet no approved antiviral therapies exist. Here, we evaluated several previously characterized nucleoside analogs as candidate countermeasures for POWV encephalitis. Favipiravir, molnupiravir, 4'-fluorouridine (4'-FlU), and GS-441524 inhibited POWV replication in vitro and demonstrated comparable activity against West Nile virus (WNV). We established C3H/HeNCrl mice as a stringent POWV disease model, showing increased susceptibility, higher day 7 brain titers, and more pronounced neurologic signs resembling severe human disease, compared with C57BL/6 mice. In prophylactic and therapeutic studies, 4'-fluorouridine was the most efficacious compound tested, significantly reducing brain viral burden even when treatment was delayed. Direct comparison with molnupiravir, favipiravir, and GS-441524 in mice confirmed superior survival benefit and antiviral activity for 4'-FlU against both POWV and WNV. These data identify 4'-fluorouridine as a promising therapeutic candidate for neurotropic flavivirus disease.

Objective Heme-dependent metronidazole (MTZ) resistance has emerged in Clostridioides difficile and is primarily mediated by a T-to-G single-nucleotide polymorphism (SNP) in the promoter of nimB (PnimBG). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the PnimBG mutation associated with MTZ resistance. Methods A series of primers and CRISPR RNAs (crRNAs) were designed and optimized to identify the most effective combination for discriminating between PnimBG (MTZ-resistant) and PnimBT (MTZ-susceptible) alleles. Detection was evaluated by visual fluorescence using blue-light and UV imaging systems, as well as by quantitative fluorescence measurements. Results The CRISPR-Cas12a assay accurately differentiated PnimBG and PnimBT alleles in C. difficile. In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the PnimBT allele and all 10 MTZ-resistant isolates carrying the PnimBG allele. Fluorescence signals were readily detected both visually and quantitatively. Furthermore, colony PCR using crude colony lysates yielded results comparable to those obtained with purified genomic DNA, enabling rapid colony-based detection without conventional DNA extraction. Conclusions The CRISPR-Cas12a assay enables rapid molecular detection of the PnimBG mutation associated with heme-dependent MTZ resistance in C. difficile. This approach could complement conventional susceptibility testing for MTZ resistance detection.

BackgroundFebrile neutropenia requires rapid empirical antibiotic therapy, with regimen selection shaped by local prescribing practice and pathogen patterns. We described starting treatment, later drug changes, microbiological findings, and inpatient outcomes. MethodsThis ambispective cohort comprised 96 episodes from 90 patients admitted to a tertiary-level teaching hospital. Treatment and outcome analyses used each episode as the unit of observation. Recorded variables included the starting antibiotics, later regimen modification, microbiology, hospitalization length, invasive fungal infection (IFI) as captured in study records, and disposition. Comparisons based on escalation status were exploratory. ResultCefoperazone-sulbactam appeared in 62/96 initial regimens (64.6%), amikacin in 42 (43.8%), teicoplanin in 28 (29.2%), and meropenem in 14 (14.6%). Eighteen episodes (18.8%) required a change that broadened or substituted antibacterial therapy. Among 31 microbiologically documented infections, 22 (71.0%) yielded Gram-negative organisms and 9 (29.0%) were polymicrobial. Median length of admission was 18.5 days in the escalation group and 9 days in the remainder (p<0.001). Mortality was 27.8% (5/18) with escalation and 5.1% (4/78) without it (p=0.011), with nine inhospital deaths overall (9.4%). ConclusionAntibacterial management differed within this cohort, with treatment escalation required in 18 of 96 episodes. Gram-negative organisms comprised most microbiologically confirmed infections. Escalation marked a clinically complex course with prolonged hospitalization and greater observed mortality; the observational design does not establish comparative efficacy or causation.

BackgroundSevere community-acquired pneumonia (CAP) remains a leading cause of mortality in children under five. The PediCAP trial evaluated oral step-down therapy using dispersible amoxicillin or amoxicillin/clavulanate tablets in African children hospitalised with severe CAP. Dispersible tablet formulations are preferred in children; however, pharmacokinetic data for oral amoxicillin/clavulanate in paediatric populations remain limited. We characterised the pharmacokinetics of 4:1, 7:1, and 14:1 dispersible amoxicillin/clavulanate tablets in children enrolled in the PediCAP trial and predicted target attainment under different dosing schemes. MethodsChildren were randomised within 24 hours of starting intravenous antibiotics to oral step-down therapy with weight-banded dosing of twice-daily amoxicillin/clavulanate (4:1, 7:1, or 14:1). Plasma concentrations were quantified following intensive sampling and analysed using nonlinear mixed-effects modelling with allometric scaling and postmenstrual age-dependent clearance maturation. Pharmacodynamic indices included %fT>MIC for amoxicillin and %fT>Ct for clavulanate. ResultsA total of 157 children (median [range] age, 12 [2-70] months; weight, 8.4 [4.0-22.0] kg) contributed 707 paired concentrations. Amoxicillin was described by a two-compartment model and clavulanate by a one-compartment model, both with sequential zero-and first-order absorption. Typical clearance for an 8-kg individual at full maturation was 11.8L/h for amoxicillin and 6.1L/h for clavulanate. Pharmacokinetics were similar between the formulation ratios. Weight-banded dosing achieved balanced exposures across weight groups. Amoxicillin achieved 40%fT>MIC for MICs[&le;]2mg/L. A proposed clavulanate target of 20%fT>0.5mg/L was achieved in most children even at the lowest amoxicillin/clavulanate ratio. ConclusionWeight-banded dosing using dispersible amoxicillin/clavulanate tablets provide consistent exposure profiles in African children with severe CAP. SUMMARYPopulation pharmacokinetic modelling of dispersible amoxicillin/clavulanate tablets (4:1, 7:1, 14:1) in 157 African children with severe pneumonia showed weight-banded dosing achieved consistent and adequate amoxicillin and clavulanate exposures across formulations.

Mosquito-transmitted alphaviruses, including chikungunya (CHIKV), Mayaro, Eastern equine encephalitis, and Venezuelan equine encephalitis viruses, cause severe arthritic or neurologic disease in humans. There are currently no approved antiviral treatments against alphavirus infection. Here, we report that the purine ribonucleoside EIDD-3525 is a potent inhibitor of alphavirus replication in vitro with broad spectrum antiviral activity against the Alphavirus genus. Using a trans-replicase assay, we find that EIDD-3525 blocks alphavirus RNA replication. Mutational profiling suggests that EIDD-3525 targets the alphavirus nsP4 RNA-dependent RNA polymerase that is conserved across medically important alphaviruses. In a pre-clinical mouse model of CHIKV infection and disease, oral treatment with EIDD-3525 reduced viral tissue burdens and the severity of virus-induced disease. Collectively, our findings support the continued development of the purine ribonucleoside EIDD-3525 as an orally available antiviral therapeutic for use against a broad group of alphaviruses.

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new drug development.

Methicillin-resistant Staphylococcus aureus (MRSA) is non-susceptible to beta-lactams. Blockade of cell wall biosynthesis is a potential target for beta-lactam enhancement but requires further investigation. A butyrolactone derivative enhanced beta-lactams against MRSA strains by reducing the availability of D-Ala-D-Ala. Unlike D-cycloserine, it did not inhibit D-Ala-D-Ala ligase (Ddl). Nor did it show an additive or synergistic effect when combined with cycloserine, indicating a unique mechanism for blocking cell wall precursor production that does not involve the traditional Lipid II pathway. Notably, beta-lactam potentiation by our chemical or D-cycloserine was highly dependent on the intrinsic autolytic ability of the tested MRSA strains. Strains that resisted lysis upon Triton X-100 exposure showed a minimal increase in beta-lactam susceptibility, whereas highly autolytic strains showed significant changes in their beta-lactam MICs. We have thus identified autolytic ability as the Achilles Heel in the strategy of targeting cell wall biosynthesis for beta-lactam potentiation.

Mycobacterium tuberculosis has a flavin-intensive metabolism, with more than 180 proteins that require flavin or deazaflavin cofactors. M. tuberculosis requires riboflavin biosynthesis for growth in standard culture conditions in vitro, which can be overcome by providing exogenous riboflavin. It is unknown whether M. tuberculosis also requires riboflavin synthesis to grow or persist in infected host cells or tissues. Here we used a M. tuberculosis strain that conditionally expresses ribA2, which encodes a bifunctional enzyme that catalyzes the first steps in both branches of riboflavin biosynthesis, to determine whether M. tuberculosis requires riboflavin biosynthesis during infection. Transcriptional repression of ribA2 caused death of M. tuberculosis in vitro without riboflavin supplementation. RNAseq revealed that riboflavin starved M. tuberculosis induce a transcriptional program indicating decreased electron transport chain function and broad changes to central metabolism. Transcriptional repression of ribA2 prevented M. tuberculosis growth in cultured macrophages and caused clearance of M. tuberculosis from infected tissues at all stages of infection in both C57BL/6 mice that form cellular lung lesions and C3HeB/FeJ mice that develop necrotic lung granulomas. Our results demonstrate that M. tuberculosis requires RibA2 for viability at all stages of infection and support further characterization of the riboflavin biosynthesis pathway as a potential target for development of new anti-tubercular drugs.

Heme metabolism regulates the availability of heme, an iron-containing molecule essential for oxygen transport, mitochondrial respiration, electron transfer, and enzymatic reactions. Increasing evidence implicates heme metabolism in regulating viral infection, potentially representing an important innate antiviral mechanism. Components of both heme biosynthesis and degradation pathways exhibit antiviral activity against different viruses; however, their contributions and conservation across human coronaviruses remain incompletely defined. We investigated the impact of modulating heme metabolism on infection by seasonal human coronaviruses HCoV-OC43 and HCoV-229E. Treatment with 5-aminolevulinic acid (5-ALA) produced cell line-dependent antiviral effects, with robust inhibition in H1299 cells and selective HCoV sensitivity in MRC-5 cells. These differences correlated with intracellular accumulation of protoporphyrin IX (PPIX), suggesting that cellular metabolic capacity influences antiviral efficacy. In contrast, components of the heme degradation pathway, including heme oxygenase-1 (HO-1), carbon monoxide, and biliverdin, did not exhibit antiviral effects against HCoV-OC43 or HCoV-229E. PPIX and synthetic porphyrin CoPPIX inhibited early infection through direct viral inactivation. Collectively, these findings indicate that antiviral effects of heme metabolism against seasonal human coronaviruses are primarily driven by direct inactivation by PPIX rather than cellular antiviral responses induced by the heme degradation pathway

Background: Bovine respiratory disease (BRD) is a major cause of antimicrobial use in cattle, raising concerns about antimicrobial resistance (AMR). This systematic review estimated pooled AMR proportions in BRD pathogens: Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, and assessed temporal and geographic trends in resistance. Methods: Five antimicrobials commonly used for BRD treatment were evaluated: penicillin G (PEN), tetracycline (TET), florfenicol (FLO), tulathromycin (TUL), and enrofloxacin (ENR). Following PRISMA guidelines, 1,097 records were screened and 24 studies met inclusion criteria. Study quality and risk of bias were assessed, and random-effects meta-analyses estimated pooled proportions of resistant isolates by antimicrobial, continent and period. Results: A total of 13,949 bacterial isolates were analysed including P. multocida (5,615), M. haemolytica (5,585), and H. somni (2,749). TET resistance was highest across all pathogens (12-30%), followed by TUL (6-11%), PEN (4-12%), FLO (0.5-1.6%) and ENR (0.2-1.1%). TET resistance in M. haemolytica and P. multocida, and FLO resistance in P. multocida were significantly more frequent in North America than in Europe (p[&le;]0.05). TUL resistance increased significantly in P. multocida after 2013 (p=0.038), with similar non significant trends in M. haemolytica and H. somni. FLO resistance decreased significantly in H. somni after 2013 (p=0.013). Egger's test identified significant funnel plot asymmetry for ENR resistance in H. somni and PEN resistance in M. haemolytica, suggesting possible small study effects. Conclusion: These results offer insights into distribution of phenotypic resistance across pathogens and regions, informing BRD antimicrobial treatment guidelines and One-Health antimicrobial stewardship aimed at preserving the effectiveness of medically important antimicrobials.