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

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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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13099-022-00500-5.

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Most cited references 42

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Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads

Ryan Wick, Louise Judd, Claire Gorrie … (2017)

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

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PHASTER: a better, faster version of the PHAST phage search tool

David L. Arndt, Jason Grant, Ana Marcu … (2016)

PHASTER (PHAge Search Tool – Enhanced Release) is a significant upgrade to the popular PHAST web server for the rapid identification and annotation of prophage sequences within bacterial genomes and plasmids. Although the steps in the phage identification pipeline in PHASTER remain largely the same as in the original PHAST, numerous software improvements and significant hardware enhancements have now made PHASTER faster, more efficient, more visually appealing and much more user friendly. In particular, PHASTER is now 4.3× faster than PHAST when analyzing a typical bacterial genome. More specifically, software optimizations have made the backend of PHASTER 2.7X faster than PHAST, while the addition of 80 CPUs to the PHASTER compute cluster are responsible for the remaining speed-up. PHASTER can now process a typical bacterial genome in 3 min from the raw sequence alone, or in 1.5 min when given a pre-annotated GenBank file. A number of other optimizations have also been implemented, including automated algorithms to reduce the size and redundancy of PHASTER's databases, improvements in handling multiple (metagenomic) queries and higher user traffic, along with the ability to perform automated look-ups against 14 000 previously PHAST/PHASTER annotated bacterial genomes (which can lead to complete phage annotations in seconds as opposed to minutes). PHASTER's web interface has also been entirely rewritten. A new graphical genome browser has been added, gene/genome visualization tools have been improved, and the graphical interface is now more modern, robust and user-friendly. PHASTER is available online at www.phaster.ca.

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Mauve: multiple alignment of conserved genomic sequence with rearrangements.

Aaron C E Darling, Bob Mau, Frederick R Blattner … (2004)

As genomes evolve, they undergo large-scale evolutionary processes that present a challenge to sequence comparison not posed by short sequences. Recombination causes frequent genome rearrangements, horizontal transfer introduces new sequences into bacterial chromosomes, and deletions remove segments of the genome. Consequently, each genome is a mosaic of unique lineage-specific segments, regions shared with a subset of other genomes and segments conserved among all the genomes under consideration. Furthermore, the linear order of these segments may be shuffled among genomes. We present methods for identification and alignment of conserved genomic DNA in the presence of rearrangements and horizontal transfer. Our methods have been implemented in a software package called Mauve. Mauve has been applied to align nine enterobacterial genomes and to determine global rearrangement structure in three mammalian genomes. We have evaluated the quality of Mauve alignments and drawn comparison to other methods through extensive simulations of genome evolution. Copyright 2004 Cold Spring Harbor Laboratory Press ISSN

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Author and article information

Contributors

Flavia J. Negrete: flavianegrete@yahoo.com

Katie Ko: Katie.Ko@fda.hhs.gov

Hyein Jang: Hyein.Jang@fda.hhs.gov

Maria Hoffmann: Maria.Hoffmann@fda.hhs.gov

Angelika Lehner: lehnera@fsafety.uzh.ch

Roger Stephan: stephanr@fsafety.uzh.ch

Séamus Fanning: sfanning@ucd.ie

Ben D. Tall: shakee2326@aol.com

Gopal R. Gopinath: Gopal.Gopinathrao@fda.hhs.gov

Journal

Journal ID (nlm-ta): Gut Pathog

Journal ID (iso-abbrev): Gut Pathog

Title: Gut Pathogens

Publisher: BioMed Central (London )

ISSN (Electronic): 1757-4749

Publication date (Electronic): 6 June 2022

Publication date PMC-release: 6 June 2022

Publication date Collection: 2022

Volume: 14

Electronic Location Identifier: 23

Affiliations

[1 ]GRID grid.417587.8, ISNI 0000 0001 2243 3366, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, , U. S. Food and Drug Administration, ; Laurel, MD USA

[2 ]GRID grid.164295.d, ISNI 0000 0001 0941 7177, Joint Institute for Food Safety and Applied Nutrition, , University of Maryland College Park, ; College Park, MD USA

[3 ]GRID grid.483501.b, ISNI 0000 0001 2106 4511, Center for Food Safety and Applied Nutrition, Office of Regulatory Science, Food and Drug Administration, ; College Park, MD USA

[4 ]GRID grid.7400.3, ISNI 0000 0004 1937 0650, Institute for Food Safety and Hygiene, , University of Zürich, ; Zurich, Switzerland

[5 ]GRID grid.7886.1, ISNI 0000 0001 0768 2743, WHO Collaborating Centre for Cronobacter, , University College Dublin, ; Dublin, Ireland

[6 ]GRID grid.7886.1, ISNI 0000 0001 0768 2743, School of Public Health, Physiotherapy and Population Science, , UCD Centre for Food Safety, University College Dublin, ; Dublin, Ireland

Article

Publisher ID: 500

DOI: 10.1186/s13099-022-00500-5

PMC ID: 9169379

SO-VID: f3405d33-f968-455b-ba5a-0919775a5cb0

License:

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