Construction of a Mutant Library of Avibacterium paragallinarum Transposons and Screening and Preliminary Study of Genes Related to Biofilm Formation

Avibacterium paragallinarum (Av. paragallinarum), the causative agent of infectious coryza, imposes substantial economic burdens on the poultry industry by inducing growth retardation in broilers and reducing egg production in laying hens by up to 40%. Disease control is hindered by the limited efficacy of available vaccines and the increasing prevalence of antibiotic resistance—challenges that are exacerbated by the pathogen’s capacity to form biofilms, which facilitate bacterial persistence and enhance drug tolerance. To systematically elucidate the genetic determinants underlying biofilm formation in Av. Paragallinarum, we constructed a high-density random mutant library using mini-Tn5 transposon mutagenesis, comprising 3106 individual mutants. Phenotypic screening via crystal violet staining identified 188 mutants displaying altered biofilm-forming capacity relative to the wild-type strain, including 172 with enhanced and 16 with reduced biofilm formation. Sequencing of transposon insertion sites in these mutants revealed 105 disrupted genes involved in diverse biological pathways, including amino acid metabolism, quorum sensing, and transmembrane transport. A representative subset of eight mutants was selected for detailed phenotypic characterization. Their biofilm phenotypes were consistent with the initial screening results; certain mutants exhibited markedly enhanced biofilm formation (e.g., Tn-2206), whereas others, including Tn-1504, Tn-2428, and Tn-2859, showed significant reductions in biofilm production. Notably, these three biofilm-deficient mutants—harboring disruptions in a TonB-dependent receptor (Tn-1504), a GntP family permease (Tn-2428), and a hypothetical protein (Tn-2859)—displayed drastically attenuated virulence in vitro. Compared with the wild-type strain, these mutants exhibited reductions in cytotoxicity (up to 66.38%), cell adhesion (up to 50.68%), and invasive capacity, while maintaining normal growth kinetics. These findings indicate that the identified genes may play crucial roles in biofilm-associated virulence and highlight Tn-1504, Tn-2428, and Tn-2859 as promising candidates for the development of live attenuated vaccines. Collectively, this study provides a comprehensive genetic foundation for the rational design of novel anti-biofilm strategies against Av. paragallinarum.