High-Throughput 16S rRNA Gene Sequencing of Butter Microbiota Reveals a Variety of Opportunistic Pathogens

Abstract

Microbial contamination of dairy products with a high fat content (e.g., butter) has been studied insufficiently. No studies using modern molecular methods to investigate microbial communities in butter have been conducted so far. In this work, we used high-throughput sequencing and Sanger sequencing of individual bacterial colonies to analyze microbial content of commercially available butter brands. A total of 21 samples of commercially available butter brands were analyzed. We identified a total of 94 amplicon sequence variants corresponding to different microbial taxa. The most abundant lactic acid bacteria in butter were Lactobacillus kefiri, Lactobacillus parakefiri, Lactococcus taiwanensis and Lactococcus raffinolactis. A large amount of Streptococcus spp. bacteria (87.9% of all identified bacteria) was found in one of the butter samples. Opportunistic pathogens such as Bacillus cereus group, Pseudomonas aeruginosa, Cronobacter spp., Escherichia coli, Listeria innocua, Citrobacter spp., Enterococcus spp., Klebsiella pneumonia were detected. The analyzed butter samples were most strongly contaminated with bacteria from the Bacillus cereus group, and to a lesser extent - with Cronobacter spp. and Enterococcus spp. The plating and Sanger sequencing of individual colonies revealed the presence of Enterobacter cloacae and Staphylococcus epidermidis. The Sanger sequencing also showed the presence of Cronobacter sakazakii in butter which can be dangerous for children under the age of 1 year. We demonstrated that butter is a good growth medium for opportunistic pathogenic bacteria. Our data indicate that despite the fact that butter is a dairy product with a long shelf life, it should be subjected to quality control for the presence of opportunistic bacteria.