Monitoring bacterial communities in raw milk and cheese by culture-dependent and -independent 16S rRNA gene-based analyses

Abstract

The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the Firmicutes (58% of the total clones) affiliated mainly with the orders Clostridiales and the Lactobacillales, followed by the phyla Proteobacteria (21.6%), Actinobacteria (16.8%), and Bacteroidetes (4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the Gammaproteobacteria phylum and Staphylococcus genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the Lactobacillales and subdominant species affiliated with the Actinobacteria, Bacteroidetes (Chryseobacterium sp.), or Gammaproteobacteria (Stenotrophomonas sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant Proteobacteria and Bacteroidetes (Flavobacteria) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant Actinobacteria and Staphylococcus organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.

FIG. 1.

Principal-component analysis of peak ratios from SSCP patterns obtained after direct amplification of milk and cheese DNA. (A) Plot of peaks composing the patterns. Labels refer to SSCP peak numbers indicated in Table 1. (B) Distribution of milk and cheese patterns along the first two principal components. Axis 1 indicates the first principal component, and axis 2 the second principal component. ▵, F1 milk; ○, F2 milk; □, F3 milk; , F1 1-day cheese; , F2 1-day cheese; , F3 1-day cheese; ▴, F1 28-day cheese; •, F2 28-day cheese; ▪, F3 28-day cheese.

FIG. 2.

Principal-component analysis of peak ratios from SSCP patterns of milk and cheese subcommunities culturable on PCAI. (A) Plot of peaks composing the patterns. Labels refer to SSCP peak numbers indicated in Table 3. (B) Distribution of milk and cheese patterns along the first two principal components. Axis 1 indicates the first principal component, and axis 2 the second principal component. ▵, F1 milk; ○, F2 milk; □, F3 milk; , F1 1-day cheese; , F2 1-day cheese; , F3 1-day cheese; ▴, F1 28-day cheese; •, F2 28-day cheese; ▪, F3 28-day cheese.

FIG. 3.

Principal-component analysis of peak ratios from SSCP patterns of milk and cheese subcommunities culturable on CRBM. (A) Plot of peaks composing the patterns. Labels refer to SSCP peak numbers indicated in Table 3. (B) Distribution of milk and cheese patterns along the first two principal components. Axis 1 indicates the first principal component, and axis 2 the second principal component. ▵, F1 milk; ○, F2 milk; □, F3 milk; , F1 1-day cheese; , F2 1-day cheese; , F3 1-day cheese; ▴, F1 28-day cheese; •, F2 28-day cheese; ▪, F3 28-day cheese.

FIG. 4.

Distribution of OTUs recovered from a clone library obtained from milk DNA and individual isolates retrieved from milk on culture media, according to phylogenetic groups. OTUs were defined by 97% sequence similarity or greater. Black bars, OTUs found among culture isolates only; gray bars, OTUs found among clones only; white bars, OTUs shared by isolates and clones presumably belonging to the same species according to their ≥97% sequence similarity.