Enrichment Dynamics of Listeria monocytogenes and the Associated Microbiome from Naturally Contaminated Ice Cream Linked to a Listeriosis Outbreak

Introduction: Microbiota that co-enrich during efforts to recover pathogens interfere with efficient detection and recovery. Here we describe co-enriching microbiota from ice cream linked to an outbreak of listeriosis using protocols from: 1) the Food and Drug Administration (FDA), 2) the International Organization of Standardization (ISO), and 3) the United States Department of Agriculture (USDA).

Purpose:   This study was conducted to optimize recovery of pathogens from enrichments for expedited trace-back of outbreak-associated strains.

Methods: Enrichment methods were followed according to the published protocols for each method. Aliquots were taken every 4 h throughout 48 h of incubation. DNA was extracted using the Qiagen Dneasy Kit according to the manufacturer’s specifications. Shotgun libraries were prepared using Truseq, and 16S rRNA gene amplification targeted the V1/V2 region. Sequencing was performed on an Illumina Miseq. Taxonomic profiling was performed using Resphera Insight and the Genius bioinformatics software package from CosmosID.

Results: Dominant co-enriching bacterial taxa were identified as Anoxybacillus spp., Geobacillus spp., Serratia spp., Pseudomonas spp., Erwinia spp., and Streptococcus spp. Anoxybacillus spp. and Geobacillus spp. appear to competitively exclude Listeria monocytogenes (Lm) during early enrichment hours. Lm became dominant after 24 h in all three methods. Shotgun reads of Lm from hour 48 produced a draft genome of Lm with a similar tracking utility to pure culture isolates of Lm.

Significance: All three methods performed equally well for enrichment of Lm. The observation that Anoxybacillus spp. and Geobacillus spp. competitively excluded Lm provided novel information that can be used to further optimize enrichment formulations. Draft assembly of a genome of Lm from shotgun metagenomic data demonstrated the utility of this approach to expedite trace-back of outbreak-associated strains directly from enrichments.