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


RESULTS
Time 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.


CONCLUSIONS
Our 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.
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, OARSA, Laurel, MD, USA. karen.jarvis@fda.hhs.gov.
Karen G
KG
Jarvis
Oak Ridge Institute for Science and Technology, Oak Ridge, TN, USA. james.dna.white@gmail.com.
James R
JR
White
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, OARSA, Laurel, MD, USA. Christopher.Grim@fda.hhs.gov.|Oak Ridge Institute for Science and Technology, Oak Ridge, TN, USA. Christopher.Grim@fda.hhs.gov.
Christopher J
CJ
Grim
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, OARSA, Laurel, MD, USA. Laura.Ewing-Peeples@fda.hhs.gov.
Laura
L
Ewing
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, ORS, College Park, MD, USA. Andrea.Ottesen@fda.hhs.gov.
Andrea R
AR
Ottesen
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, OARSA, Laurel, MD, USA. Junia.Jean-GillesBeaubrun@fda.hhs.gov.
Junia Jean-Gilles
JJ
Beaubrun
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, ORS, College Park, MD, USA. James.Pettengill@fda.hhs.gov.
James B
JB
Pettengill
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, ORS, College Park, MD, USA. Eric.Brown@fda.hhs.gov.
Eric
E
Brown
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, OARSA, Laurel, MD, USA. Darcy.Hanes@fda.hhs.gov.
Darcy E
DE
Hanes
D016000:Cluster Analysis; D028526:Coriandrum / Q000382:microbiology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D054892:Metagenome; D008828:Microbiological Techniques; D064307:Microbiota; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D014481:United States
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S