Skip to main content
NCBI home page
Microbiology Resource Announcements logoLink to Microbiology Resource Announcements
. 2020 Apr 9;9(15):e00168-20. doi: 10.1128/MRA.00168-20

Whole-Genome Draft Assemblies of Difficult-to-Classify Escherichia coli O157 and Non-O157 Isolates from Feces of Canadian Feedlot Cattle

Vinicius Silva Castro a, Eduardo Eustáquio de Souza Figueiredo b, Tim McAllister c, Robin King d, Tim Reuter e, Rodrigo Ortega Polo c, Carlos Adam Conte Jr a, Kim Stanford e,✉
Editor: David Raskof
PMCID: PMC7380519  PMID: 32273360

Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx1a, stx1b, stx2a, and/or stx2b. Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N50 values between 79,648 and 294,166 bp and G+C contents between 50.

ABSTRACT

Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx1a, stx1b, stx2a, and/or stx2b. Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N50 values between 79,648 and 294,166 bp and G+C contents between 50.3% and 51.4%.

ANNOUNCEMENT

Escherichia coli bacteria are ubiquitous microorganisms that are most often commensals, but some groups, such as Shiga toxin-producing E. coli (STEC), possess genes that confer pathogenicity in humans, leading to vomiting, bloody diarrhea, and hemolytic uremic syndrome (1, 2). Ruminants are the main reservoir of STEC (3, 4), but STEC may also be present in other vectors, including birds, rodents, flies, and water (5–8). Shedding of STEC in cattle appears to be influenced by season, housing density, and the host (9, 10). In this project, 48 E. coli strains were selected based on consistent or inconsistent detection of stx and/or serogroup by PCR. (9). Within our collection of approximately 15,000 E. coli strains, relatively few, other than those selected for whole-genome sequencing (WGS), have had inconsistent PCR results. Thirty-one strains were consistent for serogroup detection by PCR, and they were confirmed by WGS (Table 1) using the E. coli O and H group (EcOH) database in ABRicate version 0.8.10 (https://github.com/tseemann/ABRICATE) (11). All 48 strains were classified as STEC by PCR based on carriage of stx1 and/or stx2. Fourteen strains were consistent for PCR detection of stx1 and/or stx2, but only 11 of these were STEC based on WGS. These strains were positive for antimicrobial resistance genes for beta-lactams (100%), tetracycline (81.2%), and sulfonamides (8.3%), as assessed by using the Comprehensive Antibiotic Resistance database (12) and ABRicate. In addition, the virulence profile was analyzed using the ABRicate E. coli_VF database, and strains possessed stx1a, stx1b, stx2a, stx2b, and adhesion genes (i.e., toxB, fdeC, csg, and variants) related to biofilm formation and several types of secretion systems, including the type III secretion system encoded in the locus of enterocyte effacement (LEE) or non-LEE-encoded type III effector. For all ABRicate analyses, default parameters were used except for minimum DNA percent coverage, which was set to 60%.

TABLE 1.

Overview of the E. coli draft genome assemblies

Strain Serotype BioSample accession no. SRA accession no. Assembly accession no. Total no. of reads Sequencing depth (×) G+C content (%) N50 (bp) No. of contigs Genome size (bp) No. of CDSsj Resistance gene(s); stx subtype(s)
CAP01g O103:H2 SAMN13870004 SRR10947171 ASM1102935v1 2,436,574 85 51.2 127,894 211 5,114,061 4,883 arsB(mob)k , blaEC-18; csg
CAP02a,h O103:H11 SAMN13870005 SRR10947170 ASM1102932v1 2,235,508 75 51.0 93,966 316 5,434,829 5,263 arsB(mob), blaEC-18, toxB; csg, stx1a, stx1b
CAP03a ,h O157:H7 SAMN13870006 SRR10947160 ASM1102933v1 2,375,528 82 51.0 142,962 216 5,380,304 5,104 arsB(mob), blaEC-15, toxB; csg, stx1a, stx1b, stx2a, stx2b
CAP04a ,h O26:H11 SAMN13870007 SRR10947159 ASM1102928v1 2,473,316 86 51.1 99,468 292 5,556,121 5,401 arsB(mob), blaEC-18, toxB; csg, stx1a, stx1b
CAP05b ,i O9:H30 SAMN13870008 SRR10947158 ASM1102924v1 2,257,402 75 51.1 141,506 100 4,802,339 4,457 aph(3'')-Ib, aph(6)-Id, arsB(mob), blaEC-15, blaTEM-1, dfrA5, sul2; csg
CAP06c ,i O110:H30 SAMN13870009 SRR10947157 ASM1103269v1 2,333,812 80 50.9 151,454 123 5,369,514 5,158 arsB(mob), blaEC-13, tet(A); csg
CAP08h O103:H2 SAMN13870010 SRR10947156 ASM1103268v1 2,612,588 86 51.1 200,865 179 5,230,549 5,032 arsB(mob), blaEC-18; csg
CAP09d H34 SAMN13870011 SRR10947155 ASM1103265v1 2,478,842 88 51.2 104,339 117 4,782,368 4,456 arsB(mob), blaEC; csg
CAP10a ,h O157:H7 SAMN13870012 SRR10947154 ASM1103264v1 2,161,150 71 50.9 157,234 188 5,285,310 4,978 arsB(mob), blaEC-15, toxB; csg, stx1a, stx1b
CAP11g O121:H7 SAMN13870013 SRR10947153 ASM1103262v1 2,173,434 75 51.1 241,028 90 5,016,814 4,700 arsB(mob), blaEC-18; csg
CAP12c ,i O9:H4 SAMN13870014 SRR10947169 ASM1103254v1 2,220,446 76 51.1 83,564 137 4,775,627 4,469 arsB(mob), blaEC-18; csg
CAP13b ,i H:28 SAMN13870015 SRR10947168 ASM1103259v1 2,193,184 73 50.9 216,462 91 5,273,949 4,971 blaEC; csg
CAP14g O103:H2 SAMN13870016 SRR10947167 ASM1103258v1 2,238,086 77 51.1 92,301 214 5,156,047 4,965 arsB(mob), blaEC-18; csg
CAP15i O45:H51 SAMN13870017 SRR10947166 ASM1103252v1 2,443,526 82 51.1 135,724 173 5,184,896 4,990 arsB(mob), blaEC-18, tet(C); csg
CAP16a ,h O26:H11 SAMN13870018 SRR10947165 ASM1103253v1 2,447,720 85 51.2 98,248 312 5,547,588 5,401 arsB(mob), blaEC-18, toxB; csg, stx1a, stx1b
CAP17b ,i O17:H18 SAMN13870019 SRR10947164 ASM1103244v1 2,220,086 75 50.5 142,741 274 5,463,072 5,174 aph(3'')-Ib, aph(6)-Id, arsB(mob), blaEC-8, tet(B); csg
CAP18a ,h O145:H28 SAMN13870020 SRR10947163 ASM1103247v1 2,274,708 74 50.3 139,371 220 5,318,832 5,028 blaEC, toxB; csg, stx1a, stx1b, stx2a, stx2b
CAP19a ,g O121:H7 SAMN13870021 SRR10947162 ASM1103248v1 2,020,850 67 50.6 187,631 114 5,105,071 4,856 arsB(mob), blaEC-18; csg, stx1a, stx1b
CAP20d ,i O17:H18 SAMN13870022 SRR10947161 ASM1103242v1 2,506,734 84 50.8 211,387 126 5,137,726 4,774 arsB(mob), blaEC-8; csg
CAP21d ,i O153:H8 SAMN13870023 SRR10958942 ASM1103243v1 1,767,498 65 50.9 252,910 90 5,140,948 4,863 arsB(mob), blaEC-18, qacG2, tet(A), tet(M); csg
CAP22e ,i O8:H2 SAMN13870024 SRR10958941 ASM1103235v1 2,055,856 66 50.9 101,879 169 5,377,305 5,056 arsB(mob), blaEC-18; csg
CAP23a ,h O145:H28 SAMN13870025 SRR10958930 ASM1103234v1 1,975,732 68 50.9 213,717 222 5,315,836 5,033 blaEC, toxB; csg, stx1a, stx1b
CAP24e ,i O76:H34 SAMN13870026 SRR10958920 ASM1103236v1 2,383,008 82 50.9 119,506 108 4,796,204 4,483 aadA2, blaEC, dfrA12, qacEdelta1, sul1, tet(A); csg
CAP25g O121:H7 SAMN13870027 SRR10958919 ASM1103232v1 2,381,330 86 51.2 185,921 115 5,149,544 4,862 arsB(mob), blaEC-18; csg
CAP26g O45:H11 SAMN13870028 SRR10958918 ASM1103233v1 2,021,072 71 51.1 109,224 124 4,989,320 4,688 arsB(mob), blaEC-13; csg
CAP27g O103:H8 SAMN13870029 SRR10958917 ASM1103228v1 1,971,998 70 51.0 128,935 187 5,300,501 4,991 arsB(mob), blaEC-18; csg
CAP28d ,i O5:H32 SAMN13870030 SRR10958916 ASM1103226v1 1,985,702 72 51.3 79,648 172 5,386,841 5,141 arsB(mob), blaEC-15, tet(B); csg
CAP29d ,i O5:H19 SAMN13870031 SRR10958915 ASM1103227v1 2,525,640 82 51.1 189,310 98 5,252,991 5,053 arsB(mob), blaEC-18, qacG2, tet(A), tet(M); csg
CAP30f ,i H34 SAMN13870032 SRR10958914 ASM1103223v1 1,561,088 56 51.1 205,590 57 4,718,264 4,362 arsB(mob), blaEC-15; csg
CAP31g O157:H29 SAMN13870033 SRR10958940 ASM1103222v1 1,736,606 60 50.7 142,303 140 4,948,575 4,579 arsB(mob), blaEC-15, tet(C); csg
CAP32a ,g O145:H28 SAMN13870034 SRR10958939 ASM1103218v1 2,050,004 74 51.3 140,944 251 5,265,290 4,997 blaEC, toxB; csg, stx1a, stx1b
CAP33a ,h O103:H25 SAMN13870035 SRR10958938 ASM1103219v1 2,546,878 82 51.2 103,076 250 5,325,616 5,150 arsB(mob), blaEC-18; csg, stx1a, stx1b
CAP34b ,i O8:H10 SAMN13870036 SRR10958937 ASM1103216v1 2,462,412 82 50.7 116,765 125 4,963,326 4,722 aadA1, aph(3'')-Ib, aph(6)-Id, arsB(mob) blaEC, blaTEM-1, floR, sul2; csg
CAP35h O45:H45 SAMN13870037 SRR10958936 ASM1103213v1 1,886,694 61 50.9 85,497 183 5,124,512 4,916 arsB(mob), blaEC-15, tet(C); csg
CAP36g O26:H9 SAMN13870038 SRR10958935 ASM1103212v1 2,219,820 73 51.4 150,844 99 4,745,341 4,414 arsB(mob), blaEC; csg
CAP37d ,i O187:H52 SAMN13870039 SRR10958934 ASM1103205v1 1,980,034 64 51.2 215,461 100 4,933,181 4,634 arsB(mob), blaEC-18; csg
CAP38g O157:H29 SAMN13870040 SRR10958933 ASM1103204v1 1,834,588 59 50.4 186,647 113 5,066,397 4,714 arsB(mob), blaEC-15, tet(A); csg
CAP39g O45:H4 SAMN13870041 SRR10958932 ASM1103209v1 1,705,628 54 51.1 161,396 126 4,971,004 4,633 aph(3'')-Ib, aph(6)-Id, arsB(mob), blaEC-18, floR, sul2, tet(A); csg
CAP40f,i O53:H32 SAMN13870042 SRR10958931 ASM1103207v1 2,552,634 81 51.2 147,707 80 4,667,418 4,308 arsB(mob), blaEC-15; csg
CAP41g O103:H19 SAMN13870043 SRR10958929 ASM1103202v1 2,058,560 65 50.9 203,947 56 4,774,483 4,398 arsB(mob), blaEC-18; csg
CAP42g O26:H32 SAMN13870044 SRR10958928 ASM1102813v1 2,590,136 84 51.0 294,166 59 4,774,449 4,441 arsB(mob), blaEC; csg
CAP43f ,i O51:H14 SAMN13870045 SRR10958927 ASM1102814v1 2,378,144 74 50.8 132,565 104 5,133,685 4,729 arsB(mob), blaEC; csg
CAP44g O45:H38 SAMN13870046 SRR10958926 ASM1102812v1 2,140,178 69 51.0 252,725 89 4,953,772 4,583 arsB(mob), blaEC-18; csg
CAP45g O157:H12 SAMN13870047 SRR10958925 ASM1102810v1 1,790,792 57 51.2 213,375 81 4,764,211 4,517 arsB(mob), blaEC; csg
CAP46g O103:H21 SAMN13870048 SRR10958924 ASM1102808v1 2,181,136 68 51.2 165,437 110 5,031,905 4,705 arsB(mob), blaEC-18; csg
CAP47a ,g O145:H28 SAMN13870049 SRR10958923 ASM1102805v1 2,181,386 73 51.0 147,767 222 5,280,428 5,009 blaEC; csg, stx1a, stx1b
CAP48g O157:H38 SAMN13870050 SRR10958922 ASM1102802v1 3,040,268 102 50.8 120,842 151 5,340,491 5,101 arsB(mob), blaEC-18; csg
CAP49g O103:H14 SAMN13870051 SRR10958921 ASM1102804v1 1,353,030 47 50.7 122,791 243 5,458,338 5,263 arsB(mob), blaEC-18; csg
Open in a new tab
a

Strains confirmed as STEC by WGS.

b

Formerly identified as O26 by PCR.

c

Formerly identified as O45 by PCR.

d

Formerly identified as O103 by PCR.

e

Formerly identified as O145 by PCR.

f

Formerly identified as O157 by PCR.

g

Consistent serogroup by PCR and inconsistent virulence factors.

h

Consistent serogroup and virulence factors by PCR.

i

Inconsistent serogroup and virulence factors by PCR.

j

CDS, coding DNA sequences.

k

arsB(mob), mobile version of arsB.

The isolation was performed as described by Stanford et al. (9). Briefly, fecal aliquots were enriched in E. coli broth (EMD Millipore, Darmstadt, Germany) (6 h at 37°C) and then subjected to immunomagnetic separation using RapidChek Confirm STEC kits (Romer Labs Technology, Inc., Newark, DE, USA) and magnetic-bead separation using Pickpen (BioControl Systems, Bellevue, WA, USA). The bead-bacteria mixture was then plated on MacConkey agar (Dalynn Biologicals, Calgary, Canada) and incubated (18 to 24 h at 37°C). Three to nine sorbitol-negative colonies/plate were subjected to PCR screening for E. coli target genes (13).

For genomic DNA analysis, the methodology was as described by Bumunang et al. (14), where the DNA of isolates was extracted from overnight bacterial cultures grown in 9 ml of Luria-Bertani broth (Merck, Kirkland, Canada) using the ZR fungal/bacterial DNA miniprep kit (Epigenetics Company, Irvine, CA, USA) according to the manufacturer’s instructions. WGS was performed at the Agri-Food Laboratories (Alberta Agriculture and Forestry, Edmonton, Canada). DNA was quality checked and quantified using a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA) and Tapestation 4200 system (Agilent, Santa Clara, CA, USA).

Sequencing was performed on an Illumina MiSeq platform using the MiSeq reagent kit V2 (Illumina, San Diego, CA, USA) to produce 251-bp paired-end reads. Sequencing reads were de novo assembled into contigs using the Shovill pipeline v1.0.4 (https://github.com/tseemann/shovill). Shovill included trimming, which was performed with Trimmomatic v0.39, and de novo assembly was performed with SPAdes v3.13.1 (15). The quality report for the assembly was measured using QUAST v5.0.0, and draft genome assemblies were annotated with Prokka (16). The assignment of the strain to species was verified using Centrifuge 1.04 (17) and the p-compressed index of 5,202 taxa from the RefSeq database of NCBI. Default parameters were used for these software. Across strains, the assembled genome sizes varied between 4,667,418 and 5,556,121 bp and 56 and 316 contigs, with sequence coverages between 47× and 102×. The N50 values varied between 79,648 and 294,166 bp, and the G+C contents varied between 50.3% and 51.4%.

Data availability.

The raw Illumina data (sequence read archive [SRA]) and genome contigs with respective annotations were deposited in NCBI and are described in Table 1. All SRA and genome annotation data were included in the BioProject number PRJNA601484.

ACKNOWLEDGMENTS

We thank Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (sandwich doctorate process E-26/201.859/2019) and Coordenação de Aperfeicoamento de Pessoal de Nível Superior (CAPES/Brazil-visiting professor; process, PVEX-88881.169965/2018-01) for funding. Additional funding came from the Canada-Alberta Project (CAP) Accelerating the Advancement of Agriculture of Alberta Agriculture and Forestry.

Many thanks go to Ashwin Deo, Yidong Graham, Susanne Trapp, and Homayoun Zahiroddini for technical assistance.

REFERENCES

  • 1.Clements A, Young JC, Constantinou N, Frankel G. 2012. Infection strategies of enteric pathogenic Escherichia coli. Gut Microbes 3:71–87. doi: 10.4161/gmic.19182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Griffin PM, Karmali MA. 2017. Emerging public health challenges of Shiga toxin–producing Escherichia coli related to changes in the pathogen, the population, and the environment. Clin Infect Dis 64:371–376. doi: 10.1093/cid/ciw708. [DOI] [PubMed] [Google Scholar]
  • 3.Zschöck M, Hamann HP, Kloppert B, Wolter W. 2000. Shiga-toxin-producing Escherichia coli in faeces of healthy dairy cows, sheep and goats: prevalence and virulence properties. Lett Appl Microbiol 31:203–208. doi: 10.1046/j.1365-2672.2000.00789.x. [DOI] [PubMed] [Google Scholar]
  • 4.Oporto B, Esteban JI, Aduriz G, Juste RA, Hurtado A. 2008. Escherichia coli O157:H7 and non‐O157 Shiga toxin‐producing E. coli in healthy cattle, sheep and swine herds in northern Spain. Zoonoses Public Health 55:73–81. doi: 10.1111/j.1863-2378.2007.01080.x. [DOI] [PubMed] [Google Scholar]
  • 5.Conrad CC, Stanford K, Narvaez-Bravo C, Neumann NF, Munns K, Tymensen L, Jokinen C, McAllister TA. 2018. Zoonotic fecal pathogens and antimicrobial resistance in Canadian petting zoos. Microorganisms 6:70. doi: 10.3390/microorganisms6030070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Nielsen EM, Skov MN, Madsen JJ, Lodal J, Jespersen JB, Baggesen DL. 2004. Verocytotoxin-producing Escherichia coli in wild birds and rodents in close proximity to farms. Appl Environ Microbiol 70:6944–6947. doi: 10.1128/AEM.70.11.6944-6947.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Puri-Giri R, Ghosh A, Thomson JL, Zurek L. 2017. House flies in the confined cattle environment carry non-O157 Shiga toxin-producing Escherichia coli. J Med Entomol 54:726–732. doi: 10.1093/jme/tjw240. [DOI] [PubMed] [Google Scholar]
  • 8.Olsen SJ, Miller G, Breuer T, Kennedy M, Higgins C, Walford J, McKee G, Fox K, Bibb W, Mead P. 2002. A waterborne outbreak of Escherichia coli O157:H7 infections and hemolytic uremic syndrome: implications for rural water systems. Emerg Infect Dis 8:370–375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Stanford K, Johnson RP, Alexander TW, McAllister TA, Reuter T. 2016. Influence of season and feedlot location on prevalence and virulence factors of seven serogroups of Escherichia coli in feces of Western-Canadian slaughter cattle. PLoS One 11:e0159866. doi: 10.1371/journal.pone.0159866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Wang O, Liang G, McAllister TA, Plastow G, Stanford K, Sellinger B, Guan LL. 2016. Comparative transcriptomic analysis of rectal tissue from beef steers revealed reduced host immunity in Escherichia coli O157:H7 super-shedders. PLoS One 11:e0151284. doi: 10.1371/journal.pone.0151284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ingle DJ. 2016. EcOH database: GitHub. https://github.com/katholt/srst2.
  • 12.Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, Huynh W, Nguyen AV, Cheng AA, Liu S, Min SY, Miroshnichenko A, Tran HK, Werfalli RE, Nasir JA, Oloni M, Speicher DJ, Florescu A, Singh B, Faltyn M, Hernandez-Koutoucheva A, Sharma AN, Bordeleau E, Pawlowski AC, Zubyk HL, Dooley D, Griffiths E, Maguire F, Winsor GL, Beiko RG, Brinkman FSL, Hsiao WWL, Domselaar GV, McArthur AG. 2020. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res 48:D517–D525. doi: 10.1093/nar/gkz935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Conrad C, Stanford K, McAllister TA, Thomas J, Reuter T. 2014. Further development of sample preparation and detection methods for O157 and the top 6 non-O157 serogroups in cattle feces. J Microbiol Methods 105:22–30. doi: 10.1016/j.mimet.2014.06.020. [DOI] [PubMed] [Google Scholar]
  • 14.Bumunang EW, McAllister TA, Zaheer R, Ortega Polo R, Stanford K, King R, Niu YD, Ateba CN. 2019. Characterization of non-O157 Escherichia coli from cattle faecal samples in the north-west province of. South Africa Microorganisms 7:272. doi: 10.3390/microorganisms7080272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
  • 17.Kim D, Song L, Breitwieser FP, Salzberg SL. 2016. Rapid and sensitive classification of metagenomic sequences. Genome Res 26:1721–1729. doi: 10.1101/gr.210641.116. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The raw Illumina data (sequence read archive [SRA]) and genome contigs with respective annotations were deposited in NCBI and are described in Table 1. All SRA and genome annotation data were included in the BioProject number PRJNA601484.

Articles from Microbiology Resource Announcements are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES