Background: Masske is a traditional Iranian butter made from yoghurt. The first aim of this study was to isolate and identify the nonlactic pathogenic microflora by culture and molecular methods of identification, and the second purpose was to identify genetic similarity of the isolated bacteria in Masske. Materials and Methods: In order to detect pathogenic dominant indicator microorganisms, a number of 150 bacterial isolates from three Masske samples, which may comprise the repetitive isolates and could grow on appropriate media for Staphylococci and E.coli, were classified into 8 groups according to their phenotypic characterization followed by chemical tests. Then 2approximately similar isolates from each group were chosen (total 18 isolates; we selected 3 isolates from 2 groups of eight), and the sequencing of 16S rRNA gene was done for subsequent analysis. Results: Among 18 bacterial isolates, Staphylococcus hominis was the most frequently isolated species during the manufacture of Masske as the presence of this bacterium was confirmed in 14 out of 18 samples. Also, the presence of Staphylococcus epidermidis and Escherichia coli was identical across the samples (for each one, 2 out of 18). Conclusion: Our results based on cultural and molecular methods suggest making some improvements to the hygiene of Masske manufacture due to the high population of minor pathogens.

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