Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.