RECOVERY OF GENOTYPIC INFORMATION FRO NON-CULTIVABLE CAMPYLOBACTER JEJUNI ISOLATES

Authors: Callicott, Kenneth; Stern, Norman; Hiett, Kelli

Submitted to: Trade Journal Publication
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2005
Publication Date: 11/30/2005
Citation: Callicott, K., Stern, N.J., Hiett, K.L. 2005. Recovery of genotypic information fro non-cultivable campylobacter jejuni isolates. Poultry Science, 84:1530-1532.

Interpretive Summary: The most frequent cause of bacterial food poisoning in developed countries is Campylobacter jejuni. Because Campylobacter is found not only in farm-raised poultry but also in wild birds, studies on the dynamics of this bacterium must rely on shipments from one lab to another. Unfortunately, these shipped isolates often arrive in a non-cultivable state; in our current study of Campylobacter in the Icelandic poultry industry, almost half of all isolates shipped from our Icelandic collaborators arrived non-cultivable. Traditional practice within the field has been to discard these samples. In this paper, we describe a simple method of extracting DNA from the non-cultivable bacteria for use in molecular typing. This technique may allow other researchers to gather as much data as possible on this bacterium, and the increased knowledge of the biology of Campylobacter will allow the poultry industry to implement the best possible interventions to reduce the exposure to the consumer.

Technical Abstract: Isolates of Campylobacter jejuni shipped internationally often arrive in a non-cultivable state. We describe a polymerase chain reaction-based methodology whereby phylogenetic information can be recovered from non-cultivable C. jejuni stored in Wang's transport medium. The robustness of this methodology was initially tested using five strains of C. jejuni isolated from various sources associated with poultry production. These strains were stored in Wang's transport medium before being subjected to one of five treatments designed to render the stored cells non-cultivable: prolonged storage at room temperature, prolonged incubation at 42C, multiple rounds of freezing and thawing, boiling, or contamination with Pseudomonas aeruginosa (ATCC 27853). This method resulted in high molecular weight DNA appropriate for PCR. An approximately 400 nucleotide amplicon from the flaA gene and an approximately 800 nt amplicon from 16S rDNA were readily obtained, and a 1.5 kb section of the flaA locus was amplified from about half of the samples. These results indicate that this method may be useful for strain typing schemes based on PCR amplification of Campylobacter DNA, including flaA short variable region (flaA SVR) sequencing, multi-locus sequence typing (MLST), and flaA PCR-restriction fragment length polymorphisms (flaA PCR-RFLP). By using this method, isolates unrecoverable from 'Wang's' can still be used to provide phylogenetic information for epidemiological studies.