Efficacies of three anti-Salmonella additives - bacteriophage, formaldehyde, and formic acid - within the animal food microbiota

Abstract Bacteriophage, formaldehyde, and formic acid are anti- Salmonella additives used in animal food but their effects on its microbiota are largely unknown. We applied 16S rRNA gene amplicon sequencing and shotgun metagenomics to measure animal food microbiota shifts following treatment with three commercial products: SalmoLyse (bacteriophage) in dry dog food, and Termin-8 (formaldehyde) and Amasil NA (formic acid) in poultry feed. Both Termin-8 and Amasil NA were efficacious at reducing Salmonella in poultry feed (100% and 98.4% reductions in MPN, respectively) but not SalmoLyse in dry dog food (no reduction by MPN or APC). Shotgun metagenomics showed predominant genera in inoculated & unenriched metagenomes being Salmonella in dry dog food (due to very low background microbiota), and Pseudomonas and Pantoea in poultry feed, which remained the same after anti- Salmonella additive applications. In inoculated & enriched metagenomes, there were relative 99.9%, 97.7%, and 33.1% decreases in Salmonella relative abundance for Termin-8, Amasil NA, and SalmoLyse, respectively. Significant shifts in microbiota composition included Salmonella in SalmoLyse-treated dry dog food and Clostridium in Termin-8-treated poultry feed ( P < 0.01). Compared to shotgun metagenomics, 16S rRNA gene amplicon sequencing showed similar trends in respective animal food microbial composition shifts. Molecular screening with loop-mediated isothermal amplification agreed with both sequencing datasets for Salmonella detection. These findings reveal how Salmonella inoculation, antimicrobial treatment, and culture enrichment dynamically impact animal food microbiota. This knowledge will strengthen future microbial risk assessment efforts in animal food and the control of various microbial hazards in these products.