Abstract
BackgroundMicrobiota 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.ResultsDuring 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.ConclusionsAll 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0894-1) contains supplementary material, which is available to authorized users.
Highlights
Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery
Dynamics of L. monocytogenes and co-occurring bacteria 16S rRNA amplicon sequencing revealed that proportional abundances of L. monocytogenes remained low (0–10%) until 24 h of enrichment, even though each enrichment method employed selective antimicrobials (Fig. 1)
After 24 h of selective enrichment, relative abundances of L. monocytogenes increased at each successive time point until 40 h, at which time relative abundances ranged from 90%
Summary
Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. 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). We use culture independent generation sequencing (NGS) to characterize the microbiome at four hour intervals using three different enrichment methods used for recovery of Listeria monocytogenes from naturally contaminated ice cream. Analysis of L. monocytogenes in ice cream samples manufactured in the implicated production line provided information about the prevalence and level of L. monocytogenes [12, 13] and activity of L. monocytogenes in milkshakes prepared from the ice cream [14] These analyses did not identify Listeria species other than L. monocytogenes in the ice cream samples [12]
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