title: Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak.
abstract: Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively.
  
  
  
  During enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes.
  
  
  
  All three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes. Both shotgun and 16S rRNA data supported the presence of three slightly variable genomes of L. monocytogenes. Moreover, the draft assembly of a consensus genome of L. monocytogenes from shotgun metagenomic data demonstrated the potential utility of this approach to expedite trace-back of outbreak-associated strains, although further validation will be needed to confirm this utility.
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA. Andrea.Ottesen@fda.hhs.gov.
authors.forename: Andrea
authors.identifier: 0000-0002-6425-7943
authors.initials: A
authors.lastname: Ottesen
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA.
authors.forename: Padmini
authors.initials: P
authors.lastname: Ramachandran
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Reed
authors.affiliation: Resphera Biosciences, 1529 Lancaster Street, Baltimore, MD, 21231, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: White
authors.affiliation: CosmosID, 155 Gibbs Street, Rockville, MD, 20850, USA.
authors.forename: Nur
authors.initials: N
authors.lastname: Hasan
authors.affiliation: CosmosID, 155 Gibbs Street, Rockville, MD, 20850, USA.
authors.forename: Poorani
authors.initials: P
authors.lastname: Subramanian
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA.
authors.forename: Gina
authors.initials: G
authors.lastname: Ryan
authors.affiliation: Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD, 20708, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Jarvis
authors.affiliation: Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD, 20708, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Grim
authors.affiliation: Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD, 20708, USA.
authors.forename: Ninalynn
authors.initials: N
authors.lastname: Daquiqan
authors.affiliation: Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD, 20708, USA.
authors.forename: Darcy
authors.initials: D
authors.lastname: Hanes
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Allard
authors.affiliation: CosmosID, 155 Gibbs Street, Rockville, MD, 20850, USA.
authors.forename: Rita
authors.initials: R
authors.lastname: Colwell
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Brown
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Drive, College Park, MD, 20740, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004196:Disease Outbreaks; D005516:Food Microbiology / Q000379:methods* / Q000592:standards; D005517:Foodborne Diseases / Q000382:microbiology; D006801:Humans; D007054:Ice Cream / Q000382:microbiology*; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008088:Listeriosis / Q000382:microbiology*; D064307:Microbiota; D014481:United States; D016885:United States Department of Agriculture; D014486:United States Food and Drug Administration
chemical_list: D004269:DNA, Bacterial
keywords: 16S rRNA; Buffered Listeria enrichment broth (BLEB); Co-enriching bacteria; Enrichment; FDA; Fraser broth (FB); Half-Fraser broth (HFB); ISO; Ice cream; Listeria monocytogenes; Microbiota; NGS; Next-generation sequencing; Shotgun metagenomics; USDA; University of Vermont modified broth (UVM)