Identification of Campylobacter jejuni genes implicated in pig infection by Tn-seq.

To identify the genes in Campylobacter jejuni implicated in infection of piglets, a newly developed gnotobiotic piglet infection model was used to screen a mariner Tn mutant library in C. jejuni M1cam. Two groups of piglets (isolator 1: n = 11 and isolator 2: n = 10) were infected with 10^9 CFU of the Tn mutant library (~9,951 unique Tn insertion mutants). Tn mutants that populated the gastrointestinal tract were recovered at five days post-infection. To identify genes required for infection of piglets, the composition of the Tn mutant library in the inoculum (input) and the library recovered from infected piglets (output) was determined by Tn insertion site sequencing, referred to as Tn-seq. Tn-seq sequencing libraries were sequenced (50 bp single-end) on the Illumina HiSeq 2500 platform at Cancer Research UK (Cambridge). Tn-seq data analysis was performed using the ESSENTIALS pipeline. To identify genes of which Tn mutants showed attenuated or enriched infection of piglets, read counts were collected per gene and compared between output (piglets group 1; n = 4 and piglets group 2; n = 5) and the input (inoculum; n = 3). Selection criteria were as follows: genes represented > 100 sequence reads in the input, a log2 fold-change (FC) of <-2 (attenuated survival) or >3 (enhanced survival), Benjamini & Hochberg false discovery rate < 0.05, and 2 or more Tn mutants showing a log2 fold-change of <-2 or >3 (analysed using a custom Python script). In addition, genes that were required for in vitro growth (“fitness” genes) were eliminated from our analysis.