Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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).

Fig. 1

Temporal incidence and relative abundance of L. monocytogenes and co-enriching bacterial genera in BLEB, HFB-FB, and UVM-FB media every 4 h for 48 h. Incidence and abundance of L. monocytogenes (maroon) and co-enriching bacterial genera are shown at four hour intervals from hour 0 through 48 h of enrichment (a) for three enrichment protocols (B = BLEB, H = HFB-FB, and U = UVM-FB) (b). Four independent replicates for each enrichment at each time-point were pooled and sequenced to constitute each bar. The y-axis shows percent abundance of each taxonomic group within the total library

Fig. 2

Taxonomic profiles of co-occurring bacteria and L. monocytogenes strains derived from the shotgun metagenomic data employing Cosmos ID algorithms. Taxonomy and relative abundance of L. monocytogenes and co-occurring bacterial taxa, identified using the k-mer based approached developed by CosmosID

Fig. 3

Incidence and abundance of L. monocytogenes 16S rRNA gene sequence variants. Relative abundance of three Listeria monocytogenes 16S rRNA gene sequence variant types: AC, GT, and GC. Variants occurred at positions 159 and 174 (E. coli positions NCBI accession?) within the 16S rRNA gene. The y-axis shows the percent abundance of each L. monocytogenes16S rRNA gene sequence variant within the total microbial community at each time point

Fig. 4

Incidence of L. monocytogenes strains detected by Cosmos ID. Relative abundance and incidence of three putative strains of L. monocytogenes are shown for hours 0 to 48