Genomic Characterization of Enterococcus gallinarum and Enterococcus avium Infecting Broiler Embryos; A Shared Integrative and Conjugative Element Associated with Avian Isolates

Enterococcus species are common opportunistic pathogens infecting a wide range of hosts, and often presenting multidrug resistance. Our research has identified several species of Enterococcus infecting broiler embryos or newly hatched chicks. Genome assemblies for Enterococcus avium and Enterococcus gallinarum isolated from broilers were compared with genomes from poultry and other hosts for phylogenomic relationships and pangenome associations. Three different methods demonstrated that host preference in E. gallinarum is associated with genome evolution, with three prominent clusters. In E. avium there is no such evidence for clustering of isolates with respect to host range. The pange-nome analysis identified a 50- to 51-gene cluster spanning approximately 36 kbp enriched in avian and poultry isolates, relative to human isolates for both species. ChatGPT+ analysis of gene annota-tions from different pipelines for this cluster predicted the cluster is likely a new Integrative and Conjugative Element (ICE). Representative ICE inserts were identified: ICE3 in E. gallinarum EGM181, with ICE1 and ICE2 from our hybrid assembly for E. avium 1788. Boundaries of these ICEs were identified by alignment to closely related genomes that lacked the ICE. ICE1 and ICE3 are 99% identical, while ICE2 is 94% identical to ICE1/ICE3. Two pipelines were constructed using ChatGPT+ to screen all available genomes from both species, for ICE1-like and ICE2-like, and subsequently extract intact ICEs, including identification of flanking genes. E. gallinarum genomes contained 13 ICE1-like and 1 ICE2-like, while E. avium genomes contained 15 ICE1-like and 4 ICE2-like. Only 1788 contained two ICEs. For 31 genomes the ICE insert is downstream of UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase (murF) and upstream of DEAD-box ATP-dependent RNA helicase (cshA), with all inserts in the same orientation relative to the flanking genes. In 1788 the upstream gene for ICE2 is murF, and downstream gene is glucose uptake protein, while for ICE1 the upstream gene is sapS and downstream gene is cshA. The pipelines identified an additional 6 E. gallinarum and 5 E. avium genomes with an ICE1 or ICE2 split across two contigs. While 20 of the gene annotations are for hypothetical or poorly defined polypeptides ChatGPT+ ascribes the remainder to Replication Control/Regulatory/Accessory, Conjugative Transfer, Stability/Maintenance, Recombination/Mobil-ity, or Adhesion/Biofilm. Therefore, the role of the ICE in associating bacterial isolates with avian/poultry cannot be determined at this time but likely resides in the 20 hypothetical/poorly defined genes in the adaptation region of these new ICE.