Background/Objective: The rise of multidrug-resistant bacteria underscores the urgent need for alternative antimicrobial strategies. Metal-based compounds and bacteriophage (phage) therapy have emerged as promising candidates, but the evolutionary trade-offs associated with these selective pressures and their combination remain poorly understood. This study aimed to investigate how prior exposure to T4 phage influences Escherichia coli B’s subsequent adaptation to iron (III) stress and to assess the resulting phenotypic and genomic signatures of dual resistance. Method: In this study, we performed experimental evolution using Escherichia coli B to investigate adaptive responses under four conditions: control (LB broth), T4 phage-only, iron (III) sulfate-only, and sequential phage followed by iron (III) exposure. Each treatment consisted of ten independently evolved populations (biological replicates), all derived from a common ancestral strain and passaged daily for 35 days. Phage resistance evolved rapidly, with complete resistance observed within 24 h of exposure. Results: In contrast, iron-selected populations evolved tolerance to high iron concentrations (1000–1750 mg/L) over time at a cost to resistance in other metals (gallium and iron (II) and antibiotics (tetracycline). Notably, prior phage exposure altered these outcomes: phage/iron-selected populations retained phage resistance and iron tolerance but showed diminished resistance to iron (II) and distinct antibiotic sensitivity profiles. Whole-genome sequencing revealed stressor-specific adaptations: large deletions in phage receptor-related genes (waaA and waaG) under phage pressure, and selective sweeps in iron-adapted populations affecting regulatory and membrane-associated genes (qseB, basR, aroK, fieF, rseB, and cpxP). Conclusions: These results demonstrate that the sequence of environmental stressors significantly shapes phenotypic and genetic resistance outcomes. Our findings highlight the importance of fitness epistasis and historical contingency in microbial adaptation, with implications for the design of evolution-informed combination therapies.

The antimicrobial resistance crisis has led to the use of metals and bacteriophages as possible alternatives to antibiotics. Experimental studies have examined interactions between ionic/nano-silver and bacteriophages against multidrug-resistant bacteria. However, these approaches have often failed to examine whether silver affects the stability and infectivity of bacteriophages. Here, we utilized experimental evolution to evolve resistance to ionic silver in bacteriophage T7. High ionic silver concentrations that do not represent physiological exposure conditions were used to impose strong selective pressure. Evolution of ionic silver resistance in phage T7 was rapid, as evidenced by recovery of bacteriophage growth in E. coli following repeated exposures to ionic silver, enhanced infectivity of silver-selected populations relative to parallel control and ancestral populations under increasing ionic silver concentrations, and greater suppression of E. coli growth in standard medium. Furthermore, silver resistance evolved without loss of thermal or pH stability under the conditions tested. The genomic foundation of silver resistance was relatively simple, with positive and negative natural selection differentiating the silver-selected populations from the controls and ancestral populations across serial passages in silver. Support for replication-associated adaptation under ionic silver selection may be reflected in recurrent mutations identified in genes involved in transcription, DNA replication, and genome maintenance, including T7p07 (RNA polymerase), T7p10 (DNA ligase), and T7p29 (DNA polymerase I). These findings highlight the importance of evaluating phage –silver combination strategies within an evolutionary framework that accounts for the adaptive capacity of bacteriophages under silver selection.

Antimicrobial resistance (AMR) disproportionately affects low- and middle-income countries, yet perspectives and innovations from these regions remain underrepresented. During World Antimicrobial Awareness Week 2025, the AMR Knowledge Hub convened a four-day webinar series on AMR challenges and One Health solutions across three regions, with a country spotlight on Nigeria. Discussions highlight critical gaps in political commitment, sustainable financing, and locally relevant evidence, particularly on antibiotic stewardship. Regional and country experiences demonstrate the importance of context-specific research, locally driven innovation, and stronger governance. Integrating these voices into global AMR governance is essential for a more equitable, effective, and enduring response.

Background/Objectives: Antimicrobial resistance (AMR) and virulence represent co-evolving dimensions of microbial pathogenic potential whose ecological organization in building-scale wastewater systems remains poorly understood. Methods: Using shotgun metagenomic sequencing, we characterized the temporal dynamics and ecological associations of antimicrobial resistance genes (ARGs) and virulence factors (VFs) in 12 wastewater grab samples (2 per semester) collected from a university residence hall designated for COVID-19 quarantine between 2021 and 2023. Results: The wastewater microbiome was anchored by a stable core of gut-associated anaerobic bacteria, with community composition exhibiting significant Spring-versus-Fall structuring and a year × semester interaction that explained 60% of the community variation. A marked shift toward opportunistic taxa, particularly Acinetobacter, during Fall 2023 represented the most pronounced temporal perturbation. Total ARG abundance remained stable across semesters, while resistome composition shifted significantly, indicating that temporal dynamics were driven by compositional turnover rather than changes in overall resistance burden. VF functional categories were broadly conserved across sampling periods, consistent with their structural embedding within the persistent fecal core microbiome. Correlation and network analyses revealed modular ecological coupling between resistance and virulence functional categories, with metal/co-resistance and fosfomycin classes showing the strongest associations with virulence functions. At the community level, a Benjamini–Hochberg–corrected co-occurrence network resolved into taxa-anchored resistance modules and separate virulence-function clusters, with Acinetobacter and fluoroquinolone resistance as the principal connectors. Conclusions: These findings indicate that building-scale wastewater metagenomics can capture ecologically structured functional gene dynamics, highlighting its potential as a surveillance tool for monitoring AMR and virulence in built environments.

Background/Objective: Antimicrobial resistance poses a critical public health crisis, highlighting the urgent requirement to investigate bacterial evolutionary adaptations and pioneer alternative therapeutics. Consequently, bacteriophages and metal-based compounds are emerging as viable options to combat drug-resistant infections. Building on our finding that T4 phage resistance in E. coli B also confers adaptation to high iron(III), we used RNA-sequencing (RNA-seq) to explore bacterial gene expression in resistant and control populations. We analyzed samples from our five experimental groups—Ancestor (ANC), control (CON), phage-selected (Phage), iron(III)-selected (FE), and phage/iron(III)-selected (PF), to understand how these regimes drive transcriptional changes. Method: Total RNA was extracted using the TRIzol protocol, and sequencing libraries were prepared with the Illumina RNA Total Library Prep Kit. Sequencing was performed on the Illumina NextSeq 1000/2000 platform. Reads were aligned to the E. coli B ATCC 11303 reference genome, and pairwise comparisons between the five experimental groups were conducted to determine differential gene expression profiles. Results: Principal component analysis (PCA) showed that iron-adapted populations (FE and PF) separated distinctly from the Ancestor and control populations along PC1 (capturing 40% of the variance), while the phage-selected replicates were split, with one (Phage5) clustering with CON3 and two (Phage2, Phage4) falling closer to, but clearly separated from, the Ancestor. Differential expression analysis (P_adj < 0.05 and |log₂FC| ≥ 1) revealed extensive transcriptional rewiring, with 482 and 381 differentially expressed genes (DEGs) in the FE and PF populations, respectively, compared to the Ancestor, and 177 DEGs in the phage-selected population compared to the Ancestor. The direct pairwise comparison between the iron-selected and phage/iron-selected populations yielded zero DEGs, demonstrating that both iron-adapted populations converged on a near-identical gene expression profile regardless of their distinct genetic and evolutionary backgrounds. Conclusions: This study suggests that pmrB and arn pathway genes may serve as primary markers for resistance to iron stress. These results are significant because they demonstrate a coordinated, multi-gene defense mechanism in E. coli B against high iron(III) stress, in which the arn operon and eptA remodel lipid A and the outer membrane, while glycerol-3-phosphate metabolism and phage-shock/chaperone pathways are repressed.

Background: Built environments function as hubs of microbial exchange, and kitchens serve as key sites for interaction between occupants and physical surfaces. Understanding how spatial connectivity and regional variation influence bacterial and fungal colonization on kitchen surfaces is relevant to the protection of human health and food safety. Here, we applied long-read 16S rRNA and ITS amplicon sequencing to samples collected from countertops, drain holes, and faucet spouts (n = 123) in 41 residential kitchens across two counties in North Carolina, USA to quantify variation in microbial community structure across multiple spatial scales. Results: We found lower taxonomic resolution for bacteria than for fungi, with species-level assignments representing ~ 30% of bacterial reads and ~ 88% of fungal reads. Community structure was driven primarily by kitchen identity (45% of bacterial and 37% of fungal variation), with ZIP code, surface type and county each explaining smaller, yet significant, proportions of the observed variation. Countertops exhibited the highest bacterial and fungal diversity, whereas drain holes and faucet spouts were dominated by taxa including Pseudomonas , Acinetobacter , Penicillium , and Candida . Targeted taxonomic screening identified Stenotrophomonas maltophilia , Acinetobacter junii , Aspergillus fumigatus , and Penicillium brevicompactum , indicating that sink surfaces can harbor opportunistic taxa associated with infections in immunocompromised individuals. Conclusions: Our findings demonstrate that kitchen identity signatures dominate microbial community structure, with moisture-retaining surfaces acting as key niches. This multi-scale structuring supports the framework that indoor microbiomes are seeded by outdoor inputs but structured by human occupancy.