Complete genome sequences and genomic characterization of five plasmids harbored by environmentally persistent Cronobacter sakazakii strains ST83 H322 and ST64 GK1025B obtained from powdered infant formula manufacturing facilities

Flavia J Negrete; Katie Ko; Hyein Jang; Maria Hoffmann; Angelika Lehner; Roger Stephan; Séamus Fanning; Ben D Tall; Gopal R Gopinath

doi:10.1186/s13099-022-00500-5

Complete genome sequences and genomic characterization of five plasmids harbored by environmentally persistent Cronobacter sakazakii strains ST83 H322 and ST64 GK1025B obtained from powdered infant formula manufacturing facilities

Gut Pathog. 2022 Jun 6;14(1):23. doi: 10.1186/s13099-022-00500-5.

Authors

Flavia J Negrete^{1

2}, Katie Ko^{1

2}, Hyein Jang¹, Maria Hoffmann³, Angelika Lehner⁴, Roger Stephan⁴, Séamus Fanning^{5

6}, Ben D Tall¹, Gopal R Gopinath⁷

Affiliations

¹ Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, U. S. Food and Drug Administration, Laurel, MD, USA.
² Joint Institute for Food Safety and Applied Nutrition, University of Maryland College Park, College Park, MD, USA.
³ Center for Food Safety and Applied Nutrition, Office of Regulatory Science, Food and Drug Administration, College Park, MD, USA.
⁴ Institute for Food Safety and Hygiene, University of Zürich, Zurich, Switzerland.
⁵ WHO Collaborating Centre for Cronobacter, University College Dublin, Dublin, Ireland.
⁶ School of Public Health, Physiotherapy and Population Science, UCD Centre for Food Safety, University College Dublin, Dublin, Ireland.
⁷ Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, U. S. Food and Drug Administration, Laurel, MD, USA. [email protected].

Abstract

Background: Cronobacter sakazakii is a foodborne pathogen that causes septicemia, meningitis, and necrotizing enterocolitis in neonates and infants. The current research details the full genome sequences of two extremely persistent C. sakazakii strains (H322 and GK1025B) isolated from powdered infant formula (PIF) manufacturing settings. In addition, the genetic attributes associated with five plasmids, pH322_1, pH322_2, pGK1025B_1, pGK1025B_2, and pGK1025B_3 are described.

Materials and methods: Using PacBio single-molecule real-time (SMRT^®) sequencing technology, whole genome sequence (WGS) assemblies of C. sakazakii H322 [Sequence type (ST)83, clonal complex [CC] 83) and GK1025B (ST64, CC64) were generated. Plasmids, also sequenced, were aligned with phylogenetically related episomes to determine, and identify conserved and missing genomic regions.

Results: A truncated ~ 13 Kbp type 6 secretion system (T6SS) gene cluster harbored on virulence plasmids pH322_2 and pGK1025B_2, and a second large deletion (~ 6 Kbp) on pH322_2, which included genes for a tyrosine-type recombinase/integrase, a hypothetical protein, and a phospholipase D was identified. Within the T6SS of pH322_2 and pGK1025B_2, an arsenic resistance operon was identified which is in common with that of plasmids pSP291_1 and pESA3. In addition, PHASTER analysis identified an intact 96.9 Kbp Salmonella SSU5 prophage gene cluster in pH322_1 and pGK1025B_1 and showed that these two plasmids were phylogenetically related to C. sakazakii plasmids: pCS1, pCsa767a, pCsaC757b, pCsaC105731a. Plasmid pGK1025B_3 was identified as a novel conjugative Cronobacter plasmid. Furthermore, WGS analysis identified a ~ 16.4 Kbp type 4 secretion system gene cluster harbored on pGK1025B_3, which contained a phospholipase D gene, a key virulence factor in several host-pathogen diseases.

Conclusion: These data provide high resolution information on C. sakazakii genomes and emphasizes the need for furthering surveillance studies to link genotype to phenotype of strains from previous investigations. These results provide baseline data necessary for future in-depth investigations of C. sakazakii that colonize PIF manufacturing facility settings and genomic analyses of these two C. sakazakii strains and five associated plasmids will contribute to a better understanding of this pathogen's survival and persistence within various "built environments" like PIF manufacturing facilities.

Keywords: Built environment; Complete genomes; Cronobacter sakazakii; PHASTER; Phage-plasmids; Plasmids; Whole genome sequencing.