Assessment of Bacterial Communities in Raw Milk Cheeses from Central Poland Using Culture-Based Methods and 16S rRNA Amplicon Sequencing.

Traditional raw milk cheeses are valued for their distinctive sensory properties and microbial richness but may pose microbiological safety risks. The objective of this research was to investigate the bacterial communities present in cheeses sourced from dairies from different regions of one voivodeship using culture-based methods and 16S rRNA amplicon sequencing. Artisanal Polish "twarog"-type cheeses produced from raw cow's milk were obtained from four small local dairies in the Łódź Voivodeship. Microbial diversity and safety were assessed by culture-based enumeration following ISO standards and 16S rRNA amplicon sequencing targeting the V3-V4 region. Microbial counts included total viable microorganisms, lactic acid bacteria (LAB), Enterobacteriaceae, coagulase-positive staphylococci, yeasts, molds, and pathogens (Salmonella spp., Listeria monocytogenes). Significant differences (p < 0.05) were observed among cheeses, with samples ZJ-473 and ZJ-505 showing the highest LAB and total counts, while Salmonella spp. and L. monocytogenes were undetected. 16S rRNA amplicon sequencing revealed dominance of Firmicutes and Proteobacteria, mainly Lactococcus lactis, Leuconostoc pseudomesenteroides, and Lactobacillus delbrueckii. Alpha diversity and co-occurrence analyses indicated higher microbial complexity in samples ZJ-473 and ZJ-505. The integration of culture-based and sequencing data provided a comprehensive view of cheese microbiota and safety, confirming the protective role of LAB and emphasizing the need for strict hygiene in artisanal cheese production.