Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing

Abstract

BackgroundSalmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq.ResultsTime zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples.ConclusionsOur cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve detection sensitivity for foodborne enteric pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0497-2) contains supplementary material, which is available to authorized users.

Mungo bean sprout microbiome and changes associated with culture based enrichment protocols used in detection of Gram-negative foodborne pathogens

“…Similar observations were also previously described during enrichment of Salmonella in cilantro and tomato [13, 14]. In addition, among Firmicutes , the genus Paenibacillus was shown to inhibit and kill the Proteobacterium Salmonella in the enrichment of the tomato phyllosphere.…”

“…In other studies, the impact of enrichment procedures on the phyllosphere microflora associated with tomatoes and cilantro has been investigated [13, 14]. However, for sprouted seeds, the identity of bacterial taxa co-enriched during enrichment procedures and the microbial dynamics influencing the detection of target pathogens are still poorly understood.…”

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

“…Frequently, antagonistic microorganisms are co-enriched along with target pathogens [21]. For example, Paenibacillus spp ., which are capable of inhibiting and killing Salmonella [22, 23] , were also enriched using protocols outlined in the FDA BAM to recover Salmonella from tomatoes [21, 24] and cilantro [25]. Thus, continued optimization of reference enrichment protocols is still needed.…”

Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing

“…16S rRNA and/or shotgun metagenomic sequencing has been used to determine the abundances of bacterial species on pre-enriched samples as well as to determine shifts in the microbial community during enrichment for foodborne pathogens such as Salmonella enterica on tomatoes [30] and cilantro [31] and STEC on spinach [16]. It is clear from these studies that the pathogen is growing amidst a dynamically changing microbial community structure as the enrichment proceeds.…”