Polyphasic approach for differentiating P. nordicum from P. verrucosum.

Surface moulding of meat products by environmental contaminating species, mainly belonging to the genera Penicillium and Aspergillus, should be always avoided, as some of such species proved to be toxigenic. In particular, on such products the presence of moulds such as Penicillium verrucosum, Penicillium nordicum and Aspergillus ochraceus must be kept under control, since they can produce ochratoxin A (OTA), a secondary metabolite that proved to be highly toxic and has been classified by the IARC as potentially carcinogenic to humans (Group 2B). With regard to this, although differentiation between Penicillium and Aspergillus is reliably attainable by the morphological method, such method cannot be used to differentiate P. verrucosum from P. nordicum, as they are morphologically undistinguishable: only further cultural tests on selective media and the latest PCR techniques can at present do this. Moreover, as only little experimentations have been carried out on these strains by using additional techniques such as the measurement of consumption of carbon-based sources i automated systems (i.e. Biolog MicrostationTM) and the measurement of OTA produced by RP-HPLC, we applied a polyphasic approach to differentiate and to characterize some Penicillium verrucosum and Penicillium nordicum strains. In particular, we use: (i) a cultural technique with two substrates (DG18 and YES) selective for these species; (ii) a molecular diagnostic PCR recently set up; (iii) an automated system based on fungal carbon source utilisation (Biolog MicrostationTM); (iv) and an RP-HPLC analysis to quantify OTA production. Thirty strains from the SSICA collection, isolated from meat products and formerly identified as Penicillium verrucosum by the morphological method, were re-examined by both cultural tests and a PCR test: they all resulted to belong to the species Penicillium nordicum. Their biochemical and chemical characterization supported the results obtained by cultural and molecular techniques applied and showed a different ability in P. verrucosum and P. nordicum to metabolize carbon-based sources and to produce OTA at different concentrations, respectively. In particular, by means of Biolog MicrostationTM we succeed to carry out a metabolite profiling of P. nordicum, in order to create a reliable “P. nordicum User Database” suitable for future identifications of isolates belonging to this species. The metabolic profiles of all the strains were then compared by using the RetroSpectTM Trending & Tracking Software and we found out that ß-Hydroxy-butyric acid and D-Cellobiose proved to be diagnostic carbon-based metabolized sources for P. verrucosum and P. nordicum, respectively.