Comprehensive Microbiome Profiling of Cheese Production Environments Using Long-Read Sequencing

Environmental microbiota in cheese-production facilities can influence both product quality and hygienic conditions. This study characterised airborne and surface-associated microbial communities in production and ripening rooms of eight Slovak bryndza cheese facilities using culture-dependent methods and long-amplicon Nanopore sequencing. Air samples were collected with PBS-filled impingers and surface-swab suspensions were processed for DNA extraction. Bacterial 16S rRNA and fungal 28S rRNA gene amplicons were sequenced and taxonomically classified using Kraken2 and EMU. Community analyses included alpha-diversity estimates, Bray-Curtis ordination, PERMANOVA and supplementary ALDEx2 comparisons. Microbial loads varied among facilities and rooms, with elevated bacterial or fungal counts detected in several ripening environments. Sequencing revealed diverse bacterial and fungal communities comprising ripening-associated microorganisms, environmental contaminants, potential spoilage organisms and low-abundance taxa of hygienic relevance. Surface material had limited effects on overall bacterial and fungal community composition, although selected differences in fungal richness were observed among materials and individual facilities showed distinct microbial profiles. To our knowledge, this is among the first studies applying long-amplicon Nanopore sequencing to jointly characterise airborne and surface-associated bacterial and fungal communities in dairy-production environments. These findings support integrated environmental monitoring that considers microbial load, community diversity and facility-specific production conditions.