Rapid Confirmation of Listeria monocytogenes from Artificially Inoculated Food Matrices Using Partial 16S rDNA Sequence Analysis

Introduction: The confirmation of Listeria to the species level can add up to nine days to the total analysis time when performing a conventional culturing-based isolation starting from a contaminated food product. Nucleic acid sequencing of specific gene targets has the potential to identify L. monocytogenes directly from selective/differential media within 8-12 hours, significantly reducing analysis time.

Purpose: A 16S rDNA fragment sequence library was constructed and subsequently used to rapidly confirm L. monocytogenes from three types of artificially inoculated food matrices.

Methods: A sequence library was created using multiple foodborne isolates of L. monocytogenes serotype 1/2a, 1/2b, 1/2c, and 4b and foodborne isolates of L. innocua, L. welshimeri and L. seeligeri. A 527 bp region between nucleotide positions 5 and 531 from the 5´end of the 16S rDNA gene was amplified and sequenced using the MicroSeq 500ä Bacterial Identification System and an Applied Biosystems 3500xL Genetic Analyzer. Conventional and sequence-based species confirmation was compared using artificially inoculated food matrices.

Results: No intra-species variation was observed for any members of Listeria tested. The majority of the inter-species variation within the sequenced region occurred between positions 175 and 200 starting from the 5´ end of the intact 16S rDNA gene of L. monocytogenes. L. monocytogenes could be distinguished from L. innocua by base pair substitutions at positions 180, 197,and 201. L. monocytogenes could be distinguished by substitutions at positions 181, 183, 187, 194, 197, 198, 199 and 268 and at positions 176, 181, 182, 183, 196 and 198 from L. seeligeri and L. welshimeri, respectively. In each case, sequence-based species determination matched traditional biochemical confirmation for artificially inoculated food matrices.

Significance: The use of 16S rDNA sequencing reduced the confirmation and total sample turnaround time for Listeria species identification. The ability to confirm the presence of Listeria in food products in a shorter time allows regulatory agencies and manufacturers to react sooner to potential human health issues.