William Calero-Cáceres

Animal production systems are simultaneously essential to global food security; however, the extensive use of antimicrobials in these systems has significantly contributed to the emergence and spread of antimicrobial resistance (AMR) (Clifford Astbury et al., 2025;Stehling et al., 2023). Food-producing animals account for most of the global antimicrobial consumption, sustaining selective pressure across food-producing animal sectors (Schar et al., 2020;Van Boeckel et al., 2015). This pressure has selected multidrugresistant Escherichia coli, Salmonella enterica, Campylobacter spp., Streptococcus spp., and other pathogens in animal, human, and environmental reservoirs (Izydorcyzk & Church, 2026;Tiseo et al., 2020). As the antibiotic pipeline fails to keep pace, bacteriophages have re-emerged as one of the most credible alternatives for veterinary use (Calero-Cáceres & Balcázar, 2025;Topa-Pila et al., 2026).Yet the path from an isolated phage to a validated veterinary product is long. It requires well-characterized phages and lysins, rational cocktail design, an understanding of prophage ecology and horizontal gene transfer, evolution-informed strategies to manage resistance, curated phage biobanks that enable precision selection, and regulatory frameworks (Bhale et al., 2026;Calero-Cáceres et al., 2026;Kaneko et al., 2026;Melaku et al., 2025;Mora-Domínguez & Calero-Cáceres, 2025). This Research Topic assembles twelve contributions that span this full arc: novel phage and endolysin discovery for animal-associated pathogens, prophage genomic surveillance, evolution-informed phage engineering, alternative in vivo models, speciesfocused translational syntheses, and integrative One Health roadmaps.Four original studies expand the phage and lysin repertoire against economically and zoonotically important pathogens of animal production. Wen et al. isolated phage WEN7 from hospital wastewater using a multidrug-resistant Pseudomonas aeruginosa PAO1-SZ1 host; the phage lysed 19 of 29 clinical P. aeruginosa isolates (65.5%), showed a burst size of ~165 PFU/cell and a 66,379 bp genome free of resistance and virulence determinants, remained stable across temperature, pH, UV, and ethanol stress, and displayed bactericidal activity in both milk and hospital wastewater -matrices that bridge clinical, food-safety, and environmental deployment. Zhao et al. characterized phage vB_RanS_GDF21 targeting Riemerella anatipestifer, the etiological agent of duck infectious serositis; the phage carries a 46,925 bp genome free of resistance and virulence genes, and its recombinant endolysin LysGDF21 both inhibited biofilm formation and disrupted mature biofilms, showing broad antibacterial activity across the R. anatipestifer panel. Shang et al. identified Ply691, a prophage-encoded lysin from Streptococcus suis SC267 with an unusual four-domain architecture (N-terminal Amidase-5, two central CW-7 binding domains, C-terminal Glucosaminidase), temperature adaptability from 4 to 37 °C and alkaline tolerance across pH 7-10, lytic activity against 11 S. suis serotypes, and, in a murine bacteremia model, 100% survival at 2 mg per mouse administered intraperitoneally one hour post-infection, with substantial reduction of bacterial burden across major organs and no detectable resistance emergence. Lu et al. isolated and genomically characterized Myoviridae phage LQ5 (171,908 bp, carrying endolysin and holin lysis-mediator genes) against avian pathogenic E. coli (APEC) serotype O₇₈; in a chick challenge model, phage administration reduced hepatic and splenic bacterial loads more effectively than amoxicillin.Two contributions turn to the bacterial genome itself as both a source of information and a substrate for engineering. Quelal-Madrid et al. performed the first systematic characterization of prophage diversity in 142 S. enterica genomes from poultry and clinical sources, using Phigaro, PHASTEST, and a terminase-based custom BLAST database. They found Peduovirus pro483 prevalent in S. Infantis, distinct cytosine-specific methyltransferase-bearing prophages in related S. Enteritidis, and Enterobacteria phage ST104 in S. Typhimurium encoding SieA and SieB superinfection-exclusion proteins that may confer competitive advantage within microbial communities. The study positions prophages as active drivers of serovar adaptation, virulence potential, and phage-resistance in a Latin American poultry system, and highlights that any deployed lytic-phage cocktail must be designed with awareness of the resident prophage landscape.Luo et al. take an explicitly evolution-informed approach by developing a sequential positive-negative selection strategy -omitting conventional liquid enrichment to preserve viral diversity -to isolate two TolC-dependent phages, PTolC-28 and PTolC-69, directly from poultry-farm environments. Both exploit the outer-membrane exit duct of the AcrAB-TolC multidrug efflux pump as their receptor, enabling two distinct therapeutic modes. PTolC-28 drives "phage steering" in a strain-specific manner: in isolate GDW21C03, phage escape was mediated by a >60% reduction in tolC mRNA (rather than coding-sequence mutation), producing collateral re-sensitization to fluoroquinolones (~5.3fold MIC drop), tetracyclines, and aminoglycosides. PTolC-69, in contrast, exhibited robust phage-antibiotic synergy with doxycycline and florfenicol, reducing effective antibiotic dose 8-fold in vitro and, in a chick infection model, reducing lung and spleen bacterial loads by nearly two orders of magnitude relative to either agent alone.Translating phage efficacy from bench to production animal is limited by ethical, economic, and methodological constraints of vertebrate studies. Ruiz-Santamaría et al. address this bottleneck for Campylobacter, the leading cause of foodborne zoonosis in the EU, by validating Galleria mellonella as a bridging host for phage evaluation. Their fourphage...

Antimicrobial resistance (AMR) continues to erode the antibiotic arsenal that has underpinned modern medicine for eight decades (Ho et al., 2025;Patra et al., 2025;Abdallah et al., 2026). Multidrug-resistant pathogens are spreading across human medicine, veterinary practice, agriculture, and aquaculture faster than new drug classes can replace the ones they defeat (Stehling et al., 2023;Marino et al., 2025;Nazir et al., 2025;Steve et al., 2026). Meeting this challenge requires both a precise understanding of how resistance arises and evolves, and innovative strategies to circumvent or reverse it (Jacobowski et al., 2025;El-Sayed et al., 2026).High-throughput sequencing, systems biology, analytical chemistry, and computational biology have transformed how we interrogate microbial physiology and antimicrobial response (Taj et al., 2025;Pant et al., 2026). Genomics, transcriptomics, metabolomics, proteomics, and acetyl-proteomics now reveal AMR determinants, therapeutic targets, and antimicrobial mechanisms invisible to any single technology (Luo et al., 2024;Matsumura et al., 2025;Napit et al., 2025). Paired with ecological and epidemiological methods, these tools sharpen our view of AMR as a One Health problem, in which humans, animals, and shared environments circulate resistance genes and resistant organisms alike. This Research Topic brings together thirteen contributions (original research articles, comprehensive reviews, and a mini-review) spanning mechanistic dissection of natural compounds, genome mining for antimicrobial discovery, clinical outbreak reconstruction, environmental surveillance, and critical evaluation of alternative therapies.No single omics platform captures the full mechanistic picture of AMR. Three contributions show what emerges when these layers are combined. Xia Wang et al. combined transcriptomics and metabolomics to show that isocorydine, a natural alkaloid, disrupts the Mycobacterium bovis cell envelope, triggering efflux-pump activation and lipidmetabolism reprogramming, a mechanistic lead for a pathogen that bridges veterinary and human tuberculosis. Hu et al. paired quantitative proteomics (255 differentially expressed proteins) with acetyl-proteomics (38 acetylated sites) to show that berberine sulfate acts against methicillin-resistant Staphylococcus aureus (MRSA) by acetylating residue K82 of the virulence regulator SarA, weakening its binding to the agr promoter, positioning protein acetylation as an underexplored antimicrobial target. Ju Zhang et al. tracked Aeromonas hydrophila under stepwise oxytetracycline exposure mimicking aquaculture practice: resistance emerged within three days across three independent lineages, driven by recurrent mutations in aheB and rpsJ alongside coordinated upregulation of efflux pumps and ribosomal genes among more than 1,000 differentially expressed genes. This adaptation, the result of a coordinated genetic and transcriptional response rather than an isolated mutation, has direct implications for antibiotic dosing strategies in aquaculture, where sub-lethal drug exposure is common.Together, these three studies convert static catalogs of resistance genes into dynamic, mechanistically grounded models of bacterial adaptation.Genome mining uncovered new biocontrol potential in two contributions. Opoku Gyamfi et al. characterized Streptomyces melanosporofaciens STM-2 from Taiwan, identifying 52 biosynthetic gene clusters (polyketide synthases, NRPS, RiPPs, siderophores, terpenes) plus antifungal hydrolytic enzymes validated by AlphaFold2 structural modeling, positioning STM-2 as a promising biocontrol candidate. Reichardt et al. found that endophytic Streptomyces from honeybee hives carry biosynthetic potential active against both plant and honeybee pathogens, expanding the chemical space available for antimicrobial screening from an underexplored ecological niche.A third contribution turned to genomic epidemiology in the clinic. Wang Zhang et al. used whole-genome sequencing to trace a Serratia marcescens bloodstream-infection cluster across eight pediatric wards, uncovering roughly ten months of silent intra-hospital transmission that standard surveillance had missed; bundled infection-control measures halted new cases within a month.From mining new antibiotic gene clusters to tracing a hospital outbreak, these three studies show how far genomic tools now reach in antimicrobial research.One experimental study and three reviews assessed where non-traditional antimicrobial strategies stand. Yaoqin Wang et al. showed that hyper-branched poly-L-lysine (HBPL), a synthetic cationic polymer, inhibits MRSA (MIC 0.5 mg/mL) and biofilm formation, synergizes with levofloxacin, but is antagonistic with daptomycin, a clinically significant interaction given daptomycin's role in MRSA treatment. Resistance under HBPL pressure carried measurable fitness costs, suggesting a higher barrier than conventional antibiotics face.Two reviews mapped where combination and peptide strategies stand. Wu et al. evaluated fosfomycin combinations against multidrug-resistant pathogens (with β-lactams, aminoglycosides, fluoroquinolones, polymyxins, and daptomycin) and traced how plasmid-borne fos resistance genes co-localize with extended-spectrum β-lactamase and carbapenemase genes, underscoring that combination regimens must suppress resistance co-selection, not just kill bacteria. Yuanyuan Zhang et al. reviewed hybrid and conjugated antimicrobial peptides, showing how conjugation with antibiotics, fatty acids, photosensitizers, and nanoparticles restores activity and broadens spectrum, while flagging pharmacokinetics and protease stability as the remaining barriers to clinical translation.A third review, by Daraghmeh et al., surveyed alternatives to antibiotics for bacterial pneumonia (phage therapy, mesenchymal stem cells, metal nanoparticles, probiotics, CRISPR-Cas, and vaccination) identifying manufacturing standardization and...

The recognition of the urinary microbiome (urobiome) has challenged the long-standing paradigm of urinary tract sterility, revealing diverse microbial communities associated with both urinary health and disease. However, the characterization of the urobiome remains highly variable across studies, particularly due to the low-biomass nature of urine samples and the strong influence of methodological bias. In this Mini Review, we examine how factors such as sample collection methods, contamination dynamics, DNA extraction protocols, sequencing approaches, and bioinformatic pipelines shape microbial profiles and contribute to inconsistencies in microbiome–disease associations. We further discuss the limitations of traditional culture-based diagnostics, which fail to detect many microorganisms identified through sequencing-based approaches, and highlight the need for standardized, contamination-aware, and function-oriented frameworks. Finally, we explore the potential of integrated multi-omics strategies to improve the reliability, reproducibility, and clinical relevance of urobiome research in urinary tract health and disease.

Theobroma cacao L., particularly the National “fino de aroma” variety, represents a strategic genetic resource for Ecuador; however, its cultivation is constrained by diseases and climate variability. This study aimed to characterize the fatty acid profile in biotechnological systems exposed to stress inducers (methyl jasmonate, salicylic acid, glycerol, and elevated temperature). Biomass proliferation was optimized in MS, B5, and WPM media, with WPM supplemented with NAA and KIN proving the most effective. Lipid extraction and quantification were conducted through acid hydrolysis, hexane extraction, and FTIR spectroscopy, focusing on oleic, palmitic, and stearic acids. Results indicated that salicylic acid and methyl jasmonate markedly enhanced fatty acid accumulation, whereas glycerol and high temperature had comparatively limited effects. These findings highlight the potential of stress inducers as effective tools to modulate lipid biosynthesis in cacao, offering promising applications for sustainable plant biotechnology.

Aquatic environments are key reservoirs and dissemination pathways of antimicrobial resistance (AMR). However, current water-based surveillance remains fragmented and inefficient for the timely detection of emerging threats. Integrating artificial intelligence with embedded metadata provides a powerful pathway to identify novel antimicrobial resistance genes, characterize resistome profiles, and predict AMR dynamics in real-time by combining omics, environmental, and hydrological data into spatiotemporal predictive models. Successful implementation of this framework will require robust governance, ethical safeguards, and capacity building to support predictive AMR monitoring aligned with the One Health approach.

Minimize safety concerns by excluding undesirable genetic elements and improving standardization (Kakkar et al., 2024;Banerjee et al., 2025). Collectively, these developments are accelerating the translation of antibacterial interventions toward more personalized and targeted approaches. This Research Topic highlights advances in phage therapeutics across engineered approaches, clinical translation, microbiome-informed evaluation, and enabling infrastructure for therapeutic deployment. Wiese et al. describe that high-throughput screening in-vitro platform ("i-screen") that evaluates phage activity against Clostridium perfringens within a complex chicken cecal microbiota, moving beyond simplified monoculture assays. In this microbiome-informed setting, a C. perfringens-specific phage suppressed pathogen growth with performance comparable to antibiotic benchmarks, while preserving microbiota structure more effectively and showing clear host specificity. The study provides a practical framework to prioritize candidate phages under ecologically relevant conditions, with implications for animal health and food production systems.Finally, Topa-Pila et al. highlight phage biobanks as enabling infrastructure for precision phage deployment in the AMR era. They argue that scalable therapeutic implementation requires curated collections supported by standardized genomic and functional characterization, harmonized metadata, and interoperable workflows that accelerate phage-host matching. By positioning biobanks as decision-support systems within a One Health perspective, the paper emphasizes that governance, standardization, and data harmonization are essential requirements for coordinated clinical, veterinary, agricultural, and environmental applications.While the promise of phage therapy is undeniable, its practical adoption necessitates addressing several challenges, including the optimization of phage cocktails for consistent and durable efficacy, clearer regulatory pathways, and scalable manufacturing with robust quality control (Morales and Hyman, 2024). In parallel, important knowledge gaps remain regarding phage-host dynamics in complex communities, the determinants of treatment success across different infection sites, and the evolutionary trajectories that shape both bacterial and phage resistance. Because genomics is increasingly central to phage discovery, characterization, and monitoring, it is also essential to recognize that methodological choices can introduce biases in phage sequencing and downstream interpretation, potentially limiting reproducibility and comparability across studies (Mora-Domí nguez and Calero-Ca ceres, 2025). Addressing these limitations will require harmonized protocols, transparent reporting standards, and standardized bioinformatic pipelines that support interoperable datasets and translation-ready evidence.In conclusion, we hope this special issue reinforces phage therapeutics as a rapidly advancing and increasingly practical complement to antibiotics in the response to antimicrobial resistance. By bringing together studies spanning engineered phageantimicrobial strategies, clinical implementation in complex infections, microbiomeinformed evaluation platforms, and the development of phage biobanks as enabling infrastructure, this collection highlights key directions shaping the field. Moving these advances toward broader uptake will require coordinated efforts to strengthen evidence generation and comparability, including standardized characterization frameworks, interoperable biobanking systems, and shared analytical workflows that support reliable phage-host matching across settings. Ultimately, global collaboration, interdisciplinary research, and innovation-driven translation will be essential to realize the full potential of phage-based interventions and to position them as a robust component of future AMR mitigation strategies.

The renewed interest in bacteriophage therapy as a response to antimicrobial resistance (AMR) has exposed a critical barrier to its scalable implementation: the lack of structured infrastructure to support precision phage deployment. Due to their narrow host specificity, bacteriophages require timely access to well-matched candidates, making ad hoc isolation and informal exchange insufficient for routine therapeutic use. In this Perspective, we argue that phage biobanks must be redefined as enabling infrastructure rather than passive repositories. We propose that next-generation phage biobanks should integrate curated biological diversity, systematic genomic and functional qualification, and predictive capacity to support rapid phage–host matching. Together, these elements transform biobanks into decision-support systems capable of informing translational applications under time-sensitive conditions. This infrastructural model is particularly relevant within a One Health framework, where resistant pathogens circulate across human, veterinary, agricultural, and environmental domains. Rather than reviewing methodologies, we outline conceptual principles to guide the design of phage biobanks as integrated, predictive, and sustainable assets. We contend that the future impact of phage therapy will depend less on individual phage discovery than on the development of interoperable biobank infrastructures that enable precision antimicrobial interventions at scale.