Background: Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the developed world. To improve our understanding of this important human pathogen, the C. jejuni NCTC11168 genome was sequenced and published in 2000. The original annotation was a milestone in Campylobacter research, but is outdated. We now describe the complete re-annotation and re-analysis of the C. jejuni NCTC11168 genome using current database information, novel tools and annotation techniques not used during the original annotation. Results: Re-annotation was carried out using sequence database searches such as FASTA, along with programs such as TMHMM for additional support. The re-annotation also utilises sequence data from additional Campylobacter strains and species not available during the original annotation. Re-annotation was accompanied by a full literature search that was incorporated into the updated EMBL file [EMBL: AL111168]. The C. jejuni NCTC11168 re-annotation reduced the total number of coding sequences from 1654 to 1643, of which 90.0% have additional information regarding the identification of new motifs and/or relevant literature. Re-annotation has led to 18.2% of coding sequence product functions being revised. Conclusions: Major updates were made to genes involved in the biosynthesis of important surface structures such as lipooligosaccharide, capsule and both O- and N-linked glycosylation. This re-annotation will be a key resource for Campylobacter research and will also provide a prototype for the re-annotation and re-interpretation of other bacterial genomes.

Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N -linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

Campylobacter jejuni is the predominant cause of bacterial gastroenteritis worldwide, but traditional typing methods are unable to discriminate strains from different sources that cause disease in humans. We report the use of genomotyping (whole-genome comparisons of microbes using DNA microarrays) combined with Bayesian-based algorithms to model the phylogeny of this major food-borne pathogen. In this study 111 C. jejuni strains were examined by genomotyping isolates from humans with a spectrum of C. jejuni -associated disease (70 strains), chickens (17 strains), bovines (13 strains), ovines (5 strains), and the environment (6 strains). From these data, the Bayesian phylogeny of the isolates revealed two distinct clades unequivocally supported by Bayesian probabilities ( P = 1); a livestock clade comprising 31/35 (88.6%) of the livestock isolates and a “nonlivestock” clade comprising further clades of environmental isolates. Several genes were identified as characteristic of strains in the livestock clade. The most prominent was a cluster of six genes ( cj1321 to cj1326 ) within the flagellin glycosylation locus, which were confirmed by PCR analysis as genetic markers in six additional chicken-associated strains. Surprisingly these studies show that the majority (39/70, 55.7%) of C. jejuni human isolates were found in the nonlivestock clade, suggesting that most C. jejuni infections may be from nonlivestock (and possibly nonagricultural) sources. This study has provided insight into a previously unidentified reservoir of C. jejuni infection that may have implications in disease-control strategies. The comparative phylogenomics approach described provides a robust methodological prototype that should be applicable to other microbes.

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram‐negative bacteria. Campylobacter jejuni produces OMVs that trigger IL‐8, IL‐6, hBD‐3 and TNF‐α responses from T84 intestinal epithelial cells and are cytotoxic to Caco‐2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E‐cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co‐incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E‐cadherin and occludin. The addition of 11168H OMVs to the co‐culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time‐dependent and dose‐dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells.

Poultry is frequently associated with campylobacteriosis in humans, with Campylobacter jejuni being the most usual Campylobacter associated with disease in humans. Far-reaching research on Campylobacter was undertaken over the past two decades. This has resulted in interventions being put in place on farms and in processing plants. Despite these interventions, coupled with increased media coverage to educate the consumer on Campylobacter prevalence and campylobacteriosis, human health incidents are still high. Recent research is now shifting toward further understanding of the microorganisms to challenge interventions in place and to look at further and more relevant interventions for the reduction in human incidents. Farm practices play a key role in the control of colonization within poultry houses and among flocks. Prevalence at the farm level can be up to 100% and time of colonization may vary widely between flocks. Considerable research has been performed to understand how farm management and animal health practices can affect colonization on farms. This review will focus on farm practices to date as a baseline for future interventions as the microorganism becomes better understood. Further research is required to understand the chicken microbiome and factors influencing vertical transmission. The persistence of Campylobacter in animal and environmental reservoirs within and around farms requires further investigation to tailor farm practices toward preventing such reservoirs. IMPLICATIONS This review gives an overview of farm practices and their effect on Campylobacter prevalence in poultry. Various elements of farm practices have been captured in this review.

Chickens are a key food source for humans yet their microbiome contains bacteria that can be pathogenic to humans, and indeed potentially to chickens themselves. Campylobacter is present within the chicken gut and is the leading cause of bacterial foodborne gastroenteritis within humans worldwide. Infection can lead to secondary sequelae such as Guillain-Barré syndrome and stunted growth in children from low-resource areas. Despite the global health impact and economic burden of Campylobacter, how and when Campylobacter appears within chickens remains unclear. The lack of day to day microbiome data with replicates, relevant metadata, and a lack of natural infection studies have delayed our understanding of the chicken gut microbiome and Campylobacter. Here, we performed a comprehensive day to day microbiome analysis of the chicken cecum from day 3 to 35 (12 replicates each day; final n = 379). We combined metadata such as chicken weight and feed conversion rates to investigate what the driving forces are for the microbial changes within the chicken gut over time, and how this relates to Campylobacter appearance within a natural habitat setting. We found a rapidly increasing microbial diversity up to day 12 with variation observed both in terms of genera and abundance, before a stabilization of the microbial diversity after day 20. In particular, we identified a shift from competitive to environmental drivers of microbial community from days 12 to 20 creating a window of opportunity whereby Campylobacter can appear. Campylobacter was identified at day 16 which was 1 day after the most substantial changes in metabolic profiles observed. In addition, microbial variation over time is most likely influenced by the diet of the chickens whereby significant shifts in OTU abundances and beta dispersion of samples often corresponded with changes in feed. This study is unique in comparison to the most recent studies as neither sampling was sporadic nor Campylobacter was artificially introduced, thus the experiments were performed in a natural setting. We believe that our findings can be useful for future intervention strategies and help reduce the burden of Campylobacter within the food chain.