Comprehensive phenotyping combined with multi-omics of Salmonella Infantis and its H2S negative variant - Resolving adaption mechanisms to environmental changes.

The zoonotic pathogen S. Infantis is of emerging importance, making detection in poultry critical. Phenotypic changes, which are significant for standardized control programs via EN/ISO 6579-1:2017, could lead to pathogens remaining undetected, increasing the risk of food-borne outbreaks. This study investigates an S. Infantis strain with both normal growth (NCP) and atypical H₂S-negative colony variant (ACV) from an Austrian broiler farm. NCP and ACV underwent comprehensive analyses, including stability tests, electron microscopy, whole-genome sequencing, transcriptomics, and proteomics. Our findings demonstrate a stable atypical colony variant exhibiting acquired resistance against cefoxitin in ACV. Genomic analysis identified 9 single nucleotide polymorphisms (SNPs) and two deletions, affecting genes involved in porphyrin and sulfur metabolism. Key factors were a mutation disrupting cysG, which is essential for siroheme biosynthesis and a vital cofactor in sulfur metabolism, and a stop codon in menD (2-oxoglutarate decarboxylase), crucial for small colony variant appearance. Consequently, we hypothesize that these mutations lead to a deficiency in siroheme, as well as anaerobic sulfur respiration altogether resulting in the H₂S-negative phenotype. Functional network analysis highlighted compensatory upregulation of alternative metabolic pathways, including nitrate metabolism, propanoate metabolism and mixed-acid fermentation, which may aid ACV's persistence and adaptation under anaerobic conditions. Reduced flagellin expression suggests a mechanism for immune evasion. These genetic and metabolic adaptations likely respond to environmental stressors, such as oxidative stress from disinfectants or antimicrobial pressure, leading to the emergence of the H₂S-negative phenotype. Consequently, this study provides insights into the genetic and biochemical adaptations of an atypical S. Infantis variant.