Genetic diversity of the species Debaryomyces hansenii and the use of chromosome polymorphism for typing of strains isolated from surface-ripened cheeses.

AIMS: To investigate the genetic diversity among strains of Debaryomyces hansenii and further to evaluate chromosome polymorphism determined by pulse-field gel electrophoresis (PFGE) as a tool for strain typing. METHODS AND RESULTS: In total 56 isolates of D. hansenii were analysed by PFGE. The isolates included type strains and other strains obtained from culture collections as well as strains collected during production of Danish surface-ripened cheeses. By use of the PFGE technique the number and size of the chromosomal bands were calculated and the total genome size estimated. The number of chromosomal bands observed was found to vary from five to 10. The most common chromosome number was found to be six and for strains with six chromosomes the total genome size was found to vary from 9.4 to 12.6 Mb. The chromosome numbers for the type strain of each variety of D. hansenii (D. hansenii var. hansenii and D. hansenii var. fabryi) appeared to be six and seven respectively. By use of the PFGE technique it was possible to differentiate between all the investigated CBS strains and the vast majority of the dairy isolates. The dairy isolates that were found to have identical profiles (three of 56 isolates) were all isolated during production of one batch of surface-ripened cheeses and are likely to be the same strain isolated several times during cheese production. Further it was shown that the PFGE analysis did not result in a division of the two D. hansenii varieties, i.e. D. hansenii var. fabryi and D. hansenii var. hansenii into separate groups. CONCLUSION: The present study shows that the chromosomal arrangement of D. hansenii strains is heterogenic and does have a distinct chromosome polymorphism. Further the PFGE technique was proved to have a high discriminative power for strain typing of D. hansenii. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained add to the first knowledge on the genetic diversity of the species D. hansenii. Further the distinct chromosome polymorphism of D. hansenii strains as shown in this study makes the PFGE technique a useful tool for strain typing of D. hansenii, e.g. during cheese production.

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses.

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses.

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.