Abstract
Here, the role of the dairy-processing chain as a reservoir of antimicrobial resistance (AR) determinants and a source of novel biocontrol quorum-sensing inhibitors is assessed through a functional metagenomics approach. A metagenomic library comprising ∼22,000 recombinant clones was built from DNA isolated from raw milk, raw milk cheeses, and cheese-processing environment swab samples. The high-throughput sequencing of 9,216 recombinant clones showed that lactic acid bacteria (LAB) dominated the microbial communities of raw milk cheese, while Gram-negative microorganisms of animal or soil origin dominated the microbiota of raw milk and cheese-processing environments. Although functional screening of the metagenomic library did not recover potential quorum-sensing inhibitors, in silico analysis using an in-house database built specifically for this study identified homologues to several genes encoding proteins with predicted quorum-quenching activity, among which, the QsdH hydrolase was the most abundant. In silico screening of the library identified LAB, and especially Lactococcus lactis, as a relevant reservoir of AR determinants in cheese. Functional screening of the library allowed the isolation of 13 recombinant clones showing an increased resistance toward ampicillin, which in all cases was accompanied by a reduced susceptibility to a wide range of β-lactam antibiotics. This study shows that the dairy-processing environment is a rich reservoir of AR determinants, which vary by sample source, and suggests that combining next-generation sequencing with functional metagenomics can be of use in overcoming the limitations of both approaches.IMPORTANCE The study shows the potential of functional metagenomics analyses to uncover the diversity of functions in microbial communities prevailing in dairy products and their processing environments, evidencing that lactic acid bacteria (LAB) dominate the cheese microbiota, whereas Gram-negative microorganisms of animal or soil origin dominate the microbiota of milk and cheese-processing environments. The functional and in silico screening of the library allowed the identification of LAB, and especially Lactococcus lactis, as a relevant reservoir of antimicrobial resistance (AR) determinants in cheese. Quorum-quenching (QQ) determinants were not recovered through the execution of wet-lab function-based screenings but were detected through in silico sequencing-based analyses.
Highlights
IMPORTANCE The study shows the potential of functional metagenomics analyses to uncover the diversity of functions in microbial communities prevailing in dairy products and their processing environments, evidencing that lactic acid bacteria (LAB) dominate the cheese microbiota, whereas Gram-negative microorganisms of animal or soil origin dominate the microbiota of milk and cheese-processing environments
To maximize the representation of the entire cheese microbiota and obtain genomic DNA representing a large proportion of the microbial taxa potentially entering the human gastrointestinal tract through cheese consumption, pools of samples coming from different compartments of the cheese-processing chain were used to build a library of ϳ22,000 recombinant clones (ϳ850 Mb of DNA), comprising ϳ185 Mb of DNA from raw milk, ϳ480 Mb of DNA from raw milk cheeses, and ϳ185 Mb of DNA from food-processing environments
These analyses showed that (i) raw milk-associated clones may contain a significant amount of eukaryotic DNA, likely coming from milk somatic cells, (ii) cheese-associated clones mainly contained DNA from Lactococcus lactis strains, likely used as the starter cultures during manufacturing, and (iii) clones associated with processing environments are more heterogeneous and contained DNA from a diverse set of microorganisms, such as Lactococcus spp., Psychrobacter spp., Stenotrophomonas spp., Klebsiella spp., or Moritella spp
Summary
IMPORTANCE The study shows the potential of functional metagenomics analyses to uncover the diversity of functions in microbial communities prevailing in dairy products and their processing environments, evidencing that lactic acid bacteria (LAB) dominate the cheese microbiota, whereas Gram-negative microorganisms of animal or soil origin dominate the microbiota of milk and cheese-processing environments. DeVirgiliis et al [12] showed that various fosmid-borne LAB-derived genes produce an AR phenotype in an E. coli host by screening a metagenomic library constructed from samples of mozzarella di Bufala Campana Italian cheese type. They described a low recovery of resistant recombinant clones, corresponding to a low occurrence of antibiotic-resistant bacteria, this study showed that functional metagenomics methodologies are sensitive and efficient for identifying AR determinants and can overcome the limitations of culture-dependent methods [3]
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