Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.

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Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4-98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2 T (=DSM 18061 T =LMG 22410 T ) is the type strain of Agrococcus casei sp. nov.The complex consortium of micro-organisms found on the surfaces of smear-ripened cheeses includes major populations of coryneform bacteria (catalase-positive irregular rods and cocci), staphylococci (catalase-positive cocci) and yeasts (Valdés-Stauber et al., 1997;Carnio et al., 1999;Bockelmann & Hoppe-Seyler, 2001). Until recently, cheese coryneform bacteria were assigned to groups based on a few subjectively weighted morphological and staining properties (Piton-Malleret & Gorrieri, 1992;Eliskases-Lechner & Ginzinger, 1995). Such studies have been replaced by polyphasic taxonomic investigations, which show that the coryneform component of smear-ripened cheeses contains members of novel taxa, as exemplified by the isolation and description of Corynebacterium casei and Microbacterium gubbeenense from the surface of Gubbeen cheese (Brennan et al., 2001a(Brennan et al., , b, 2002 and Arthrobacter arilaitensis and Arthrobacter bergerei from diverse French smear-ripened cheeses (Irlinger et al., 2005). The present study was designed to determine the taxonomic status of a homogeneous group of coryneform bacteria isolated from smear-ripened cheeses and presumptively assigned to the genus Agrococcus.The genus Agrococcus was proposed by Groth et al. (1996) for two Gram-positive, coryneform bacteria that could be distinguished from members of other genera classified in the family Microbacteriaceae using genotypic and phenotypic criteria. The genus currently contains four species with validly described names: Agrococcus baldri Zlamala et al. The phylogenetic positions of the four representative cheese isolates were determined by 16S rRNA gene sequence analysis. Biomass from growth in brain-heart infusion broth (BHI; Difco) for 5 days at 30 uC was checked for purity, harvested by centrifugation, washed in NaCl/EDTA buffer (0.1 M EDTA, 0.1 M NaCl, pH 8.0) and stored at 220 u C until required. Genomic DNA was extracted as described by Sambrook & Russell (2001) and used as a template for PCR amplification and sequencing following the procedure of Kim et al. (1998). The resultant almost complete 16S rRNA gene sequences (1466-1470 nt) were manually aligned ...

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