ABSTRACT
Potential biological roles of the Campylobacter jejuni genes cj0641, cj0774c and cj1663 were investigated. The proteins encoded by these genes showed sequence similarities to the phosphonate utilisation PhnH, K and L gene products of Escherichia coli. The genes cj0641, cj0774c and cj1663 were amplified from the pathogenic C. jejuni strain 81116, sequenced, and cloned into pGEM-T Easy vectors. Recombinant plasmids were used to disrupt each one of the genes by inserting a kanamycin resistance (KmR) cassette employing site-directed mutagenesis or inverse PCR. Campylobacter jejuni 81116 isogenic mutants were generated by integration of the mutated genes into the genome of the wild-type strain. The C. jejuni mutants grew on primary isolation plates, but they could not be purified by subsequent passages owing to cell death. The mutant C. jejuni strains survived and proliferated in co-cultures with wild-type bacteria or in media in which wild-type C. jejuni had been previously grown. PCR analyses of mixed wild-type/mutant cultures served to verify the presence of the mutated gene in the genome of a fraction of the total bacterial population. The data suggested that each mutation inactivated a gene essential for survival. Rates of phosphonate catabolism in lysates of E. coli strain DH5 alpha were determined using proton nuclear magnetic resonance spectroscopy. Whole-cell lysates of the wild-type degraded phosphonoacetate, phenylphosphonate and aminomethylphosphonate. Significant differences in the rates of phosphonate degradation were observed between lysates of wild-type E. coli, and of bacteria transformed with each one of the vectors carrying one of the C. jejuni genes, suggesting that these genes were involved in phosphonate catabolism.
ABSTRACT
Sixty-two persistently colonising Campylobacter jejuni strains were tested for their ability to dominate colonisation of the chicken gastrointestinal tract in competition with each other leading to selection of dominant or "hyper-colonising"Campylobacter strains, which are able to displace others in the chicken intestinal tract. One such strain was shown to be a hyper-efficient coloniser of chickens, as it was able to displace other colonising strains, as well as maintain itself in the chicken intestinal tract for the duration of the 56-day broiler production cycle. Once colonisation was established, this hyper-colonising C. jejuni strain, 331, could not be displaced by other colonising or hyper-colonising strains. We proposed that a defined, hyper-colonising strain, or a cocktail of defined strains with a similar phenotype, could form the basis for biological control of unknown/uncharacterised Campylobacter strains from the environment that continuously colonise chicken flocks. To validate this approach, three different chicken infection trials were carried out. These trials demonstrated that the dominant strain of C. jejuni was able to colonise broiler chickens consistently and for the entire life of the birds irrespective of the day of inoculation and antimicrobial agents used in the feed to control other pathogenic micro-organisms. In addition, we have shown that the bio-control strain was able to replace other colonising strains at various points of a 56-day broiler production cycle irrespective of time and type of inoculation. This strain was also capable of re-establishing itself following the challenge with other strains, with and without re-challenge. This work represents a "proof of principle" that a defined C. jejuni strain could be used to biologically control circulation of uncharacterised environmental strains in commercial poultry flocks.
