Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Use of Rep PCR to identify corynebacteria

Over 400 coryneform isolates from three different batches of cheese (or cheesemaking facilities) and three different stages of ripening (early middle and late) of 5 European cheeses are deposited in our R-collection (Research collection, Laboratory of Microbiology, University of Ghent). Gram staining, motility test, cell and colony morphology, catalase test and oxidase test were performed on all isolates.

A set of over 120 reference strains was included for identification by rep-PCR. The set of reference strains includes the genera Arthrobacter, Brevibacterium, Brachybacterium, Corynebacterium, Clavibacter, Curtobacterium, Leucobacter, Mycetocola, Microbacterium and Rhodococcus. Each species was present with several strains. The reference set was chosen accordingly to the most common species present on red smear cheeses. DNA of coryneforms was prepared with a mutanolysin and a phenol-chloroform step. PCR was performed according to Versalovic et al. (1994, Molecular and Cellular Biology. 5: 25-40) with the primer BOXA1R. Band patterns were analysed using the Pearson coefficient and the UPGMA dendrogram type with the Bionumerics software (Applied Maths). If a cheese isolate did not cluster with any of the reference strains, a partial 16S sequence analysis was performed in order to assign the isolate to a species group. All isolates were identified either to species or species-group level confirming rep-PCR as a rapid tool for identification.

The most common species present on the cheeses were Arthrobacter arilaitensis and Brevibacterium aurantiacum, two newly described species, Corynebacterium casei, Corynebacterium variabile and Microbacterium gubbeenense. Two representatives of each strain were deposited in the BCCM culture collection for research purposes, for a total of 122 coryneforms.

The project enabled the construction of a comprehensive database on various coryneform species, improving the process of identification of such isolates. Hence, the BCCM/LMG Bacteria Collection can offer a service for identification and molecular typing of coryneforms.

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GENT UNIVERSITY
35 K.L. Ledeganckstraat 35
9000 GENT
Belgium
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