ABSTRACT Biofilm formation is a key determinant of persistence and pathogenicity in fungal infections, yet the genetic basis underlying its variability among clinical isolates remains poorly understood. In this study, a collection of 95 N. glabratus clinical isolates was analyzed in terms of biofilm formation capacity, revealing large phenotypic diversity. Interestingly, urinary tract associated isolates were found to produce, on average, twice as much biofilm as the remaining isolates. A more in‐depth analysis was carried out focusing on a selection of three of the lowest biofilm producing and four of the highest biofilm producing isolates. Higher total biofilm mass was found to consistently correlate with higher metabolic activity and carbohydrate extracellular matrix content, while no correlation with virulence against the Galleria mellonella infection model could be detected. Whole‐genome sequencing of these high and low biofilm producing strains led to a list of 184 genes whose mutation may underlie biofilm formation variability. Deletion of 24 of these genes, identified BST1 , EST3 , LSM7 , AMS1 and ZCF21 as previously uncharacterized contributors to biofilm formation, whose disruption results in a measurable reduction in biofilm biomass. Altogether, the obtained knowledge suggests within‐host positive selection in the urinary tract, for N. glabratus specialization towards biofilm growth. The identification of new effectors of biofilm formation is consistent with the existence of a multi‐layered network that connects transcriptional regulation and cell surface architecture in biofilm development.
ABSTRACT The escalating crisis of antibiotic resistance has driven the global livestock and poultry industry to seek effective alternatives. Bacteriophages (phages) are promising candidates because their high host specificity allows targeted elimination of key intestinal pathogens, including Salmonella , Campylobacter , pathogenic Escherichia coli , and Clostridium perfringens , while sparing commensal microbiota. This review summarizes the mechanisms by which phages promote intestinal health in livestock and poultry, covering pathogen lysis, modulation of gut microbiota, reinforcement of the intestinal mucosal barrier, and regulation of host immune responses. Evidence from broiler chickens, weaned piglets, and ruminants shows that phage therapy can improve growth performance and help control diseases such as necrotic enteritis and edema disease. We further discuss how administration route, dosage, timing, and formulation affect intestinal delivery, and how encapsulation systems such as microcapsules and liposomes improve phage survival under gastrointestinal conditions. Strategies to delay resistance evolution through phage cocktails, and combinations of phages with probiotics or plant extracts, are also evaluated, with a staged screening framework proposed for assessing compatibility prior to field deployment. Because positive results dominate the controlled‐trial literature, we critically appraise failed, limited, and non‐durable in vivo trials, attributing these outcomes to rapid clearance, anti‐phage immunity, inappropriate dosing, and spatial exclusion coupled with ecological mismatch. Commercial translation additionally depends on scalable production and downstream removal of endotoxins and host cell proteins at acceptable cost. Overcoming these challenges would allow phage‐based interventions to serve as precision antibiotic alternatives that support sustainable animal farming and global food security.
ABSTRACT Diet composition shapes gut microbiota, influencing nutrient utilization, health, and growth. The shift toward sustainable ingredients such as soybean meal (SB) and seaweed (SW) requires an understanding of their effects on the gut microbiota. This study investigated how SB and SW diets modulate gut microbiota and predicted functional profile in Nile tilapia. Fish were assigned to four dietary treatments for 62 days: fishmeal (FM), SB, SW, and a natural meal (NM, non‐formulated diet). Gut microbiota were characterized using 16S rRNA gene sequencing, and functional potential was inferred using PICRUSt2. Diet significantly influenced microbial beta diversity (PERMANOVA, R 2 = 0.60, F = 4.05, p = 0.001). Alpha diversity was highest in the SW and lowest in the NM, with a significant difference detected only between these groups. Fusobacteriota and Pseudomonadota predominated across treatments, while Cetobacterium was consistently identified as a core genus. SB and SW diets were associated with distinct taxonomic and predicted functional profiles. SB diet showed higher representation of pathways related to nucleotide turnover, central carbon metabolism, and stress response, and was associated with growth performance. SW showed higher predicted abundances of amino acid biosynthesis and carbohydrate metabolism pathways, and achieved the highest protein digestibility. In contrast, NM showed lower predicted functional potential and lower growth indices. Overall, SB and SW were associated with changes in microbial composition and predicted metabolic functions while maintaining relatively stable core microbiota. These findings support the relevance of gut microbiota as a biological indicator when evaluating sustainable feed ingredients for Nile tilapia aquaculture.
ABSTRACT Klebsiella pneumoniae is an increasingly important opportunistic pathogen worldwide, particularly due to the emergence and spread of hypervirulent K. pneumoniae (hvKP), a clinically significant pathotype first identified in Taiwan. This study aimed to molecularly identify hvKP isolates screened for coexisting virulence determinants and evaluate the association between specific virulence genes and hypervirulence. Clinical specimens were collected from hospitalized patients, and K. pneumoniae isolates were identified phenotypically using microscopic culture characteristics and biochemical assays and genotypically confirmed by PCR targeting the khe gene. Hypermucoviscosity regulators p rm pA and p rmp A2, siderophore‐encoding genes iuc A and iro N, and other virulence‐associated genes including wzi , mag A, wzy K2, wab G, and peg‐ 344 were investigated. Eighty‐two capsulated non‐motile K. pneumoniae isolates were confirmed with p rm pA and p rmp A2 detected in 42 and 29 isolates, respectively, whereas iuc A was identified in 31 isolates and iro N in 6 isolates only. Based on the molecular criteria, 34 isolates were classified as hvKP and 48 as classical K. pneumoniae (cKP). A strong association was observed between the peg‐ 344 gene and hypervirulence ( p = 0.0001), whereas hypermucoviscosity showed a weaker correlation. However, low‐speed sedimentation assay (LSSA) analysis demonstrated a significant relationship between the hypermucoviscous phenotype and genotypic hypervirulence ( p = 0.041). These findings highlight the molecular determinants of hvKP and identify peg ‐344 as the gene most strongly associated with hypervirulence; however, its moderate sensitivity (58.8%) and absence in over 40% of hvKP isolates indicate that it is better suited as a supportive confirmatory marker than a standalone diagnostic biomarker for hvKP
ABSTRACT Early‐life gut microbiota plays a critical role in livestock health and productivity. Probiotic interventions, particularly those employing bacteriocin‐producing lactic acid bacteria (LAB), may contribute to modulation of microbial development during this critical period. This study evaluated Lactiplantibacillus pentosus AA86 (formerly Lactobacillus pentosus ) and Pediococcus pentosaceus AA94 for genomic safety, bacteriocin potential, and their effects on calf gut microbiota. Whole‐genome sequencing assessed antimicrobial resistance genes, virulence determinants, plasmid‐associated elements, and bacteriocin‐encoding genes. Both strains lacked detectable antimicrobial resistance genes, virulence determinants, and plasmid‐related elements, while harboring pediocin‐encoding genes consistent with potential antimicrobial functionality. In a 30‐day feeding trial, eight calves (50 ± 10 kg) were assigned to a control group (sterile skimmed milk, n = 2) or a probiotic group (milk supplemented with both strains at ~8.6 × 10 10 CFU/day, n = 6). Gut microbiota was evaluated at baseline, mid‐, and post‐intervention using 16S rRNA gene (V3/V4) sequencing and QIIME2. Alpha diversity and overall community structure did not differ significantly between groups. However, supplementation was associated with selective temporal compositional shifts, particularly in Firmicutes and Bacteroidetes. At the genus level, Lactobacillus showed significant temporal changes within treatment group, although the treatment‐by‐time interaction was not significant. L. pentosus AA86 and P. pentosaceus AA94 exhibited favorable genomic safety profiles and bacteriocin‐associated antimicrobial potential. Their supplementation was associated with selective temporal modulation of the developing calf gut microbiota without substantial alteration of overall community structure. These findings provide a foundation for larger and longer‐term studies incorporating strain‐resolved, functional, metabolomic, and host health analyses.
