ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) bacteria are zoonotic pathogens. We report here the high-quality complete genome sequences of three STEC O177:H- (fliCH25) strains, SMN152SH1, SMN013SH2, and SMN197SH3. The assembled genomes consisted of one optical map-verified circular chromosome for each strain, plus two plasmids for SMN013SH2 and three plasmids for SMN152SH1 and SMN197SH3, respectively.
GENOME ANNOUNCEMENT
Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause hemorrhagic colitis and hemolytic uremic syndrome (1–3). Cattle are major reservoirs of STEC and are the most common source for foodborne infections (4–6). During a longitudinal survey of STEC in healthy cattle, we isolated three non-O157 STEC strains that share phenotypic similarity with the O157 serogroup (7). Because these strains were nonmotile when isolated, DNA microarray for STEC serotyping (8) confirmed that these strains (SMN152SH1, SMN013SH2, and SMN197SH3, formerly named SH1, SH2 and SH3 respectively.) belonged to serogroup O177:H25. Based on these data we reassigned the serotype of these strains to O177:H- (fliCH25: fliC genotype H25). Among the surveyed cattle in Washington and Idaho, US, and Alberta, Canada, the O177:H- serotype was more prevalent than the O157:H7 serotype (7). We report here the availability of three high-quality complete genomes of STEC O177:H- assembled using a hybrid approach that combines PacBio and Illumina reads.
Genomic DNA was prepared using a GenElute bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO) per the manufacturer’s instructions. Whole-genome sequencing was carried out on a PacBio RS II (Pacific Biosciences, Menlo Park, CA) platform with 6-kb and 20-kb insert libraries by using P6 polymerase-binding and C4-sequencing kits for 240-min acquisition. Short-read sequencing was done at the IBEST Genomics Resources Core at the University of Idaho, using an Illumina MiSeq and v2 500-cyles kit (PE250) following construction of barcoded Illumina libraries using the IntegenX Apollo 324 PrepX ILM DNA library kit and custom Illumina TruSeq barcoded adapters. The Illumina reads were cleaned using HTStream (https://github.com/ibest/HTStream) to trim adapter sequences and low-quality ends and to overlap paired-end reads. Long reads were assembled with Canu v1.6 using default parameters (9). Gaps between contigs were sealed with GapBlaster (10). The small plasmids of SMN152SH1 and SMN197SH3 were assembled with Unicycler (11). The assembled genomes were oriented and trimmed using Circlator v1.5.2 (12), then corrected with Pilon v1.22 using cleaned Illumina reads (13). The resulting assemblies were verified using restriction enzyme NcoI whole-genome maps generated according to the OpGen protocol (OpGen, Inc., Gaithersburg, MD).
The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Features of the three O177:H- isolates include an stx2c variant gene which is 100% identical to the stx2c carried by a clinical isolate E. coli O177 (14); nearly identical pO157-like 88-kb and 48-kb plasmids, a 6.7-kb colicinogenic plasmid in SMN152SH1 and SMN197SH3; and chromosomally encoded AmpC type β-lactamase.
A detailed report on additional analysis of these genomes will be included in a future publication.
Accession number(s).
Accession numbers and assembly metrics for each complete genome sequence are listed in Table 1.
TABLE 1.
Accession numbers and assembly metrics of the three annotated STEC O177:H- genomes
| Strain | Plasmid | Accession no. | Genome size (bp) | GC content (%) | Plasmid size (bp) |
|---|---|---|---|---|---|
| SMN152SH1 | CP024618 | 5,302,502 | 50.7 | ||
| pO177A1 | CP024617 | 88,896 | |||
| pO177B1 | CP024616 | 48,983 | |||
| pO177C1 | CP024615 | 6,675 | |||
| SMN013SH2 | CP023673 | 5,143,533 | 50.5 | ||
| pO177A2 | CP023674 | 87,524 | |||
| pO177B2 | CP023675 | 46,664 | |||
| SMN197SH3 | CP024056 | 5,267,207 | 50.7 | ||
| pO177A3 | CP024055 | 88,839 | |||
| pO177B3 | CP024054 | 48,982 | |||
| pO177C3 | CP024053 | 6,675 |
ACKNOWLEDGMENTS
This work was funded by the National Institute of General Medical Sciences of the National Institutes of Health under award number P30GM103324 through the IBEST Genomics and Computational Resources Cores and P20GM103408 (Idaho INBRE Program) and the USDA National Institute for Food and Agriculture, Hatch Project numbers IDA01467 (C.J.H.) and IDA01406 (S.A.M.), with additional support from the University of Idaho Agriculture Experiment Station and New Mexico INBRE NM-INBRE P20 GM103451.
Footnotes
Citation Sheng H, Duan M, Hunter SS, Minnich SA, Settles ML, New DD, Chase JR, Fagnan MW, Hovde CJ. 2018. High-quality complete genome sequences of three bovine Shiga toxin-producing Escherichia coli O177:H- (fliCH25) isolates harboring virulent stx2 and multiple plasmids. Genome Announc 6:e01592-17. https://doi.org/10.1128/genomeA.01592-17.
REFERENCES
- 1.Edelman R, Karmali MA, Fleming PA. 1988. Summary of the International Symposium and Workshop on Infections Due to Verocytotoxin (Shiga-like toxin)-producing Escherichia coli. J Infect Dis 157:1102–1104. doi: 10.1093/infdis/157.5.1102. [DOI] [PubMed] [Google Scholar]
- 2.Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis 151:775–782. doi: 10.1093/infdis/151.5.775. [DOI] [PubMed] [Google Scholar]
- 3.Smith HR, Scotland SM. 1988. Vero cytotoxin-producing strains of Escherichia coli. J Med Microbiol 26:77–85. doi: 10.1099/00222615-26-2-77. [DOI] [PubMed] [Google Scholar]
- 4.Blanco M, Blanco JE, Mora A, Dahbi G, Alonso MP, Gonzalez EA, Bernardez MI, Blanco J. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-ξ). J Clin Microbiol 42:645–651. doi: 10.1128/JCM.42.2.645-651.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Boerlin P, McEwen SA, Boerlin-Petzold F, Wilson JB, Johnson RP, Gyles CL. 1999. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J Clin Microbiol 37:497–503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Karmali MA. 1989. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev 2:15–38. doi: 10.1128/CMR.2.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Sheng H, Davis MA, Knecht HJ, Hancock DD, Van Donkersgoed J, Hovde CJ. 2005. Characterization of a Shiga toxin-, intimin-, and enterotoxin hemolysin-producing Escherichia coli ONT:H25 strain commonly isolated from healthy cattle. J Clin Microbiol 43:3213–3220. doi: 10.1128/JCM.43.7.3213-3220.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Lacher DW, Gangiredla J, Jackson SA, Elkins CA, Feng PC. 2014. Novel microarray design for molecular serotyping of Shiga toxin- producing Escherichia coli strains isolated from fresh produce. Appl Environ Microbiol 80:4677–4682. doi: 10.1128/AEM.01049-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. doi: 10.1101/gr.215087.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.de Sá PHCG, Miranda F, Veras A, de Melo DM, Soares S, Pinheiro K, Guimarães L, Azevedo V, Silva A, Ramos RTJ. 2016. GapBlaster, a graphical gap filler for prokaryote genomes. PLoS One 11:e0155327. doi: 10.1371/journal.pone.0155327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi: 10.1371/journal.pcbi.1005595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Hunt M, Silva ND, Otto TD, Parkhill J, Keane JA, Harris SR. 2015. Circlator: automated circularization of genome assemblies using long sequencing reads. Genome Biol 16:294. doi: 10.1186/s13059-015-0849-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. doi: 10.1371/journal.pone.0112963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Gilmour MW, Tabor H, Wang G, Clark CG, Tracz DM, Olson AB, Mascarenhas M, Karmali MA, Mailman T, Ng LK. 2007. Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli. J Clin Microbiol 45:3771–3773. doi: 10.1128/JCM.01125-07. [DOI] [PMC free article] [PubMed] [Google Scholar]