Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses

Hyein Jang, Hannah R. Chase, Jayanthi Gangiredla, Christopher J. Grim, Isha R. Patel, Mahendra H. Kothary, Scott A. Jackson, Mark K. Mammel, Laurenda Carter, Flavia Negrete, Samantha Finkelstein, Leah Weinstein, Qiong Qiong Yan, Carol Iversen, Franco Pagotto, Roger Stephan, Angelika Lehner, Athmanya K. Eshwar, Seamus Fanning, Jeffery FarberGopal R. Gopinath, Ben D. Tall*, Monica Pava-Ripoll

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)
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Abstract

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010–2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this “pattern of circulation” has continued over decades.

Original languageEnglish
Article number561204
JournalFrontiers in Microbiology
Volume11
DOIs
Publication statusPublished - 25 Sept 2020
Externally publishedYes

Bibliographical note

Funding Information:
We thank the University of Maryland, Joint Institute for Food Safety and Applied Nutrition (JIFSAN) for supporting JIFSAN interns SF, FN, and LW through a cooperative agreement with the FDA, #FDU001418. We thank the Oak Ridge Institute for Science and Education of Oak Ridge, Tennessee for sponsoring research fellow HC and HJ. Funding. Funds supporting this work were obtained internally through U.S. FDA appropriations.

Publisher Copyright:
© Copyright © 2020 Jang, Chase, Gangiredla, Grim, Patel, Kothary, Jackson, Mammel, Carter, Negrete, Finkelstein, Weinstein, Yan, Iversen, Pagotto, Stephan, Lehner, Eshwar, Fanning, Farber, Gopinath, Tall and Pava-Ripoll.

Keywords

  • Cronobacter
  • flies as insects pests
  • microarray
  • phylogenetic analysis
  • whole-genome sequencing

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

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