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Applied and Environmental Microbiology, April 2008, p. 2321-2331, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02269-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of a Strain-Specific Molecular Method for Quantitating Individual Campylobacter Strains in Mixed Populations{triangledown}

Karen T. Elvers,1* Christopher R. Helps,1 Trudy M. Wassenaar,3 Vivien M. Allen,1 and Diane G. Newell2

University of Bristol, Langford, Bristol BS40 5DU, United Kingdom,1 Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, United Kingdom,2 Molecular Microbiology and Genomics Consultants, Tannenstrasse 7, 55576 Zotzenheim, Germany3

Received 5 October 2007/ Accepted 8 February 2008

The identification of sites resulting in cross-contamination of poultry flocks in the abattoir and determination of the survival and persistence of campylobacters at these sites are essential for the development of intervention strategies aimed at reducing the microbial burden on poultry at retail. A novel molecule-based method, using strain- and genus-specific oligonucleotide probes, was developed to detect and enumerate specific campylobacter strains in mixed populations. Strain-specific oligonucleotide probes were designed for the short variable regions (SVR) of the flaA gene in individual Campylobacter jejuni strains. A 16S rRNA Campylobacter genus-specific probe was also used. Both types of probes were used to investigate populations of campylobacters by colony lift hybridization. The specificity and proof of principle of the method were tested using strains with closely related SVR sequences and mixtures of these strains. Colony lifts of campylobacters were hybridized sequentially with up to two labeled strain-specific probes, followed by the generic 16S rRNA probe. SVR probes were highly specific, differentiating down to 1 nucleotide in the target sequence, and were sufficiently sensitive to detect colonies of a single strain in a mixed population. The 16S rRNA probe detected all Campylobacter spp. tested but not closely related species, such as Arcobacter skirrowi and Helicobacter pullorum. Preliminary field studies demonstrated the application of this technique to target strains isolated from poultry transport crate wash tank water. This method is quantitative, sensitive, and highly specific and allows the identification and enumeration of selected strains among all of the campylobacters in environmental samples.

* Corresponding author. Mailing address: Division of Farm Animal Science, School of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, United Kingdom. Phone: 44 (0)1173319131. Fax: 44 (0)1179289324. E-mail: Karen.Elvers{at}bristol.ac.uk

{triangledown} Published ahead of print on 15 February 2008.

Applied and Environmental Microbiology, April 2008, p. 2321-2331, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02269-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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