Signature-Tagged Transposon Mutagenesis Studies Demonstrate the Dynamic Nature of Cecal Colonization of 2-Week-Old Chickens by Campylobacter jejuni

Abstract

We have constructed plasmids to be used for in vitro signature-tagged mutagenesis (STM) of Campylobacter jejuni and used these to generate STM libraries in three different strains. Statistical analysis of the transposon insertion sites in the C. jejuni NCTC 11168 chromosome and the plasmids of strain 81-176 indicated that their distribution was not uniform. Visual inspection of the distribution suggested that deviation from uniformity was not due to preferential integration of the transposon into a limited number of hot spots but rather that there was a bias towards insertions around the origin. We screened pools of mutants from the STM libraries for their ability to colonize the ceca of 2-week-old chickens harboring a standardized gut flora. We observed high-frequency random loss of colonization proficient mutants. When cohoused birds were individually inoculated with different tagged mutants, random loss of colonization-proficient mutants was similarly observed, as was extensive bird-to-bird transmission of mutants. This indicates that the nature of campylobacter colonization in chickens is complex and dynamic, and we hypothesize that bottlenecks in the colonization process and between-bird transmission account for these observations.

Discrimination of Major Capsular Types of Campylobacter jejuni by Multiplex PCR

“…Noncapsulated mutants of C. jejuni were demonstrated to have reduced abilities to invade INT407 cells in vitro (6), to colonize chickens (5,12), and to cause diarrheal disease in ferrets (6). CPS also contributes to resistance to normal human serum (6).…”

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

“…57, 58 Although the vast majority of cases are self-limiting, campylobacter can cause severe post-infection complications, such as bacteraemia and polyneuropathies such as GuillainBarré and Miller-Fisher syndrome. 59 C. jejuni usually infects the avian gastrointestinal (GI) tract particularly of chickens. 60 During the slaughtering process, the GI contents may contaminate the meat products and ingestion and handling of contaminated meats are a main cause of sporadic cases of C. jejuni disease.…”

“…70 The primary reason for this is thought to be population bottlenecks during transit through the GI tract prior to establishment of bacterial colonies in the ceca indicating that STM may not be suitable for studying long-term colonization in C. jejuni. 59 In an attempt to overcome this problem the authors developed another method of screening known as wild-type isogenic tagged strains (WITS). As with STM each strain only differs from each other by a 40-bp DNA tag inserted into a pseudogene.…”

“…Therefore another screen was performed in C. jejuni in a 2-week old chick model. 59 An STM library was generated in three strains of C. jejuni using a modified marnier transposon system. In each STM library there was a high-frequency of random loss of colonization-proficient mutants from pools and experimental variation during mixed infections.…”

Phase-Variable Changes in the Position of O -Methyl Phosphoramidate Modifications on the Polysaccharide Capsule of Campylobacter jejuni Modulate Serum Resistance

“…jejuni CPS have been shown to be important for pathogenesis. Nonencapsulated mutants were attenuated in a ferret model of diarrheal disease and were reduced in their ability to colonize chickens, mice, and piglets (21)(22)(23)(24)(25), although there are conflicting reports on the role of MeOPN in pathogenesis of a Galleria mellonella model of disease (26,27) and in the invasion of intestinal epithelial cells in vitro (24,28). Capsules are a major factor in resistance to complement-mediated killing and, in the case of C. jejuni 81-176 (HS23/36), decoration of the CPS with MeOPN is essential for complement resistance.…”