A Functional Metagenomic Analysis of Tetracycline Resistance in Cheese Bacteria.

Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening. For this, the same cheese batch was sampled and analyzed on days 3 and 60 of manufacture. Samples were diluted and grown in the presence of tetracycline on plate count milk agar (PCMA) (non-selective) and de Man Rogosa and Sharpe (MRS) agar (selective for lactic acid bacteria, LAB). DNA from the cultured bacteria was then isolated and used to construct four fosmid libraries, named after the medium and sampling time: PCMA-3D, PCMA-60D, MRS-3D, and MRS-60D. Clones in the libraries were subjected to restriction enzyme analysis, PCR amplification, and sequencing. Among the 300 fosmid clones analyzed, 268 different EcoRI restriction profiles were encountered. Sequence homology of their extremes clustered the clones into 47 groups. Representative clones of all groups were then screened for the presence of tetracycline resistance genes by PCR, targeting well-recognized genes coding for ribosomal protection proteins and efflux pumps. A single tetracycline resistance gene was detected in each of the clones, with four such resistance genes identified in total: