Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Use of Rep PCR to identify Staphylococci

Approximately 160 coccus-shaped 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 60 reference strains was included for identification by rep-PCR. The set of reference strains included the genera Staphylococcus, Micrococcus, Macrococcus, Kocuria and Rothia. Each species was represented by several strains. The reference set was chosen accordingly to the most common species present on red smear cheeses. DNA of cocci was prepared using a lysostaphin and a phenol-chloroform step. PCR was performed according to Versalovic et al. (1994, Molecular and Cellular Biology. 5: 25-40) with the primer (GTG)5. Band patterns were analyzed 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 Staphylococcus cohnii/equorum (between which rep-PCR is not able to distinguish), and Staphylococcus saprophyticus. Two representatives of each strain were deposited in the BCCM culture collection for research purposes, for a total of 51 staphylococci and other coccus-shaped bacteria.

The project enabled the construction of a comprehensive database on various coccus-shaped 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 cocci isolated from foods.

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