Identification of biofilm-associated genes in Arcobacter butzleri

<i>Arcobacter butzleri</i> is an emerging zoonotic and foodborne pathogen isolated from human diarrhoeal samples and environmental sources. Despite its increasing clinical relevance, functional studies in this species have been limited by the lack of effective genetic tools. In this study, we report the first successful application of random transposon mutagenesis using the EZ-Tn5 transposome system in three <i>A. butzleri</i> strains. Transformation efficiency was found to be strain-dependent, with only one strain (P8) yielding enough mutants for phenotypic screening. A total of 56 mutants were characterised with 29 different disrupted genes, all exhibiting significantly reduced biofilm formation, and ten strains showing reduced or abolished motility. Furthermore, the phenotype of some mutants was also associated with lactate metabolism, methionine auxotrophy, cold sensibility and resistance to several antimicrobials. The identification of multiple independent insertions at identical nucleotide positions suggests possible insertional hotspots, although no conserved sequence motifs were identified. Overall, our findings provide novel insights into the gene functions associated with biofilm formation and other phenotypes in <i>A. butzleri</i>. This work not only represents a significant technical advancement for the genetic manipulation of this bacterium species but also establishes a foundation for future functional genomics studies aimed at elucidating the pathogenicity and environmental adaptability of <i>A. butzleri</i>.