The bacterial populations in Danish raw milk cheeses were identified using denaturating gradient gel
electrophoresis (DGGE) of PCR amplicons of the V3 region of the 16S rRNA gene and pyrosequencing of
tagged amplicons of the V3 and V4 regions of the 16S rRNA gene. Both DNA and RNA extracted from
cheeses were studied in order to determine the metabolically active bacteria. The main bacteria, which
included Lactococcus, Lactobacillus and Streptococcus, were detected by pyrosequencing and DGGE in
both 16S rDNA and cDNA obtained from cheeses indicating their viability and contribution to cheese
ripening. Other bacteria like Corynebacterium, Halomonas, Pediococcus, Micrococcus and Staphylococcus,
which were encountered in some cheese samples at low percentages compared with the total bacterial
populations, were only detected by pyrosequencing. 16S rRNA gene pyrosequencing is an efficient
method for deep sequencing of microbial communities and it expands our knowledge of the bacterial
diversity in raw milk cheese.