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. 2023 Mar 15;12(4):e01177-22. doi: 10.1128/mra.01177-22

Draft Genome Sequences of 110 Shiga Toxin-Producing Escherichia coli Isolates Collected from Bovine Manure Samples in the Southern United States

Christopher A Baker a,*, Alan Gutierrez b, Rebecca Bell c, Keith R Schneider a,✉
Editor: Irene L G Newtond
PMCID: PMC10112231  PMID: 36920193

ABSTRACT

Shiga toxin-producing Escherichia coli (STEC) bacteria continue to impact the food industry. Environmental sampling of potential sources of contamination is important to aid epidemiologic efforts in tracking foodborne illnesses throughout the United States. Here, the draft genome sequences of 110 STEC isolates from bovine manure collected in Florida and Texas are reported.

ANNOUNCEMENT

Shiga toxin-producing Escherichia coli (STEC) bacteria naturally reside in the intestinal tract of cattle (1, 2), which can be transmitted to foods, such as fresh produce, and result in illness following consumption (3–10).

In efforts to track foodborne pathogens, the FDA collects whole-genome sequence (WGS) data on food and environmental-derived enteric pathogens and makes the data public in real time at NCBI (11). As part of a multi-institute study to determine the prevalence and concentration of STEC in bovine manure based on geographical regions, bovine manure was sampled (n = 514) on 12 farms (dairy, feedlot, and livestock) from August 2017 to May 2018 (12). The draft genome sequences of 110 STEC isolates were generated from isolates obtained from surface and subsurface samples. All of these isolates were collected in Florida, except for one collected in Texas, which is denoted in Table 1. Samples were collected from manure piles, lagoons, barns, and/or screened solids at each farm and were sampled for stx+ E. coli by adding 30 g to 270 mL of tryptic soy broth (TSB), which was massaged by hand (1 min) and incubated for 2 h at 25°C and 100 rpm and then incubated for 8 h at 42°C and 100 rpm. A total of 1 mL of the enriched slurry was added to 9 mL of modified enterohemorrhagic E. coli (mEHEC) broth and incubated for 12 h at 42°C. Enrichments were streaked for isolation onto CHROMagar STEC and incubated for 24 h at 37°C, after which up to 12 presumptive positive colonies were restreaked for isolation. Isolated colonies were tested for the presence of stx1 and stx2 via quantitative PCR (qPCR), after which stx+ slurry sample bags were tested via a most probable number method to quantify stx+ E. coli in manure samples. Positive sample wells were streaked for isolation onto CHROMagar STEC. A separate analysis was performed to detect E. coli O157 presence via immunomagnetic separation, selective plating, and qPCR targeting of the rfbE gene (12). Isolates that were stx+ and/or rfbE+ (O157) as determined by qPCR were stored at −80°C until shipped to FDA Center for Food Safety and Applied Nutrition for sequencing. Genomic DNA was extracted after overnight incubation at 35°C in TSB using the DNeasy blood and tissue kit (Qiagen Inc, Valencia, CA). DNA concentrations were measured using a Qubit 3.0 fluorometer (Life Technologies, MD). Sequencing libraries were prepared according to Nextera XT or Illumina DNA prep protocols using 0.2 ng/μL of DNA and sequenced on the MiSeq desktop sequencer (Illumina, San Diego, CA) using a MiSeq reagent V2 or V3 kits (500 cycles) following the manufacturer’s guidelines. Raw sequence data were assembled using SKESA 2.3.0 (13). Taxonomy assignments were verified using Kraken (14). Sequencing records with quality metrics above minimum thresholds accessed by the “MicroRunQC v1” workflow (15) and correctly identified as E. coli were submitted to NCBI. MicroRunQC is a galaxy quality-control (QC) workflow that includes FastQC (15), assembly QC, and multilocus sequence typing (MLST). Genomes were annotated automatically upon upload using NCBI Prokaryotic Genome Annotation Pipeline (16). Default parameters were used for all software unless otherwise specified.

TABLE 1.

Genome statistics for 110 STEC isolates recovered from bovine manure samples between 2017 and 2018

CFSAN no. No. of contigs Total length (bp) N50 value (bp) Genome coverage (×) No. of reads GC content (%) No. of genes SRA accession no. GenBank accession no.
CFSAN086474a 92 4,929,277 134,673 85 2,068,962 51.4 4,930 SRR9997957 AARKQE000000000
CFSAN086475a 86 4,939,900 134,671 71 1,685,216 51.6 4,932 SRR9998125 AARKQD000000000
CFSAN086476a 89 4,937,030 142,502 75 1,697,742 51.5 4,935 SRR9998224 AARKQB000000000
CFSAN086477a 80 4,939,244 142,502 85 1,937,874 51.3 4,935 SRR9997746 AARKQJ000000000
CFSAN086478a 91 4,941,853 151,729 94 2,216,136 51.6 4,940 SRR9997745 AARKQG000000000
CFSAN086479a 106 4,988,585 131,411 83 1,968,518 51.4 4,993 SRR9997722 AARKQI000000000
CFSAN086480a 92 5,021,692 134,673 116 2,685,170 51.3 5,030 SRR9997611 AARKQK000000000
CFSAN086481a 99 5,022,375 134,673 104 2,430,606 51.1 5,034 SRR9997734 AARKQH000000000
CFSAN086482a 121 4,997,932 101,607 45 2,322,998 51.7 5,028 SRR10156122 AASXNZ000000000
CFSAN086483a 214 5,191,833 124,373 81 1,800,666 51.5 5,216 SRR18610094 ABFQTA000000000
CFSAN086489a 159 5,354,420 118,620 155 3,906,100 50.7 5,411 SRR9997960 AARKQF000000000
CFSAN086490a 106 4,763,617 98,184 47 1,197,768 49.2 4,697 SRR10013029 AAQWGC000000000
CFSAN086491a 238 5,379,495 108,435 47 1,318,536 50.7 5,448 SRR10012698 AARBWV000000000
CFSAN086492a 372 5,075,194 66,193 86 1,874,670 50.9 5,119 SRR18663670 ABFQSX000000000
CFSAN086493a 369 4,792,599 44,064 51 1,285,004 50.9 4,879 SRR10012674 AARKOU000000000
CFSAN086494a 149 4,687,902 94,333 51 1,233,896 50.9 4,630 SRR10012677 AARKOQ000000000
CFSAN086497a 93 4,903,382 193,199 79 2,109,382 49.6 4,851 SRR10013021 AAQWGB000000000
CFSAN086498a 91 4,944,872 160,866 75 2,090,478 50.5 4,900 SRR10013127 AAQWGP000000000
CFSAN086499a 91 4,894,969 121,865 47 976,510 51.7 4,888 SRR10156282 AASNDW000000000
CFSAN086500a 98 4,898,424 130,727 50 1,269,644 51.6 4,896 SRR10013114 AARKOT000000000
CFSAN086502a 91 4,894,701 124,861 54 1,339,974 51.5 4,883 SRR10012704 AAQWGH000000000
CFSAN086503a 89 4,901,233 121,488 75 1,870,852 51.8 4,890 SRR10013003 AARKOR000000000
CFSAN086504a 95 4,900,239 124,861 53 1,312,274 51.7 4,893 SRR10012673 AARKOW000000000
CFSAN086505a 93 5,193,660 176,150 55 1,432,358 50.9 5,071 SRR10012676 AARKOV000000000
CFSAN086506a 94 4,896,106 133,339 56 1,441,796 51.9 4,888 SRR10013348 AARKOO000000000
CFSAN086507a 93 4,897,708 124,373 52 1,316,304 51.4 4,890 SRR10012913 AAQWGN000000000
CFSAN086508a 235 5,548,683 89,576 64 4,278,596 50.7 5,699 SRR19619724 AAQWGE000000000
CFSAN086509a 195 5,378,022 144,775 73 2,044,106 50.9 5,416 SRR10013157 AARBXF000000000
CFSAN086511a 159 5,377,663 160,449 45 1,045,134 50.5 5,388 SRR10049372 AARBFX000000000
CFSAN086513a 162 5,380,271 149,646 50 1,195,770 50.6 5,393 SRR10049365 AARBEU000000000
CFSAN086514a 100 5,073,438 158,513 73 1,730,124 50.1 5,064 SRR10049353 AASNJS000000000
CFSAN086516a 89 5,190,325 142,934 57 1,380,666 49.8 5,124 SRR10049915 AASKHF000000000
CFSAN086517a 209 5,363,107 124,147 37 906,572 50.2 5,358 SRR10049768 AARBEO000000000
CFSAN086518a 195 5,368,952 124,148 57 1,486,602 50.3 5,376 SRR10049865 AARBEA000000000
CFSAN086519a 391 5,080,779 48,614 83 1,830,094 50.5 5,160 SRR18662672 ABFQSY000000000
CFSAN086520a 198 5,400,027 127,101 78 1,795,548 51.0 5,404 SRR10141123 AARAGE000000000
CFSAN086521a 185 5,368,544 118,175 86 2,505,386 50.7 5,405 SRR10051028 AASKHD000000000
CFSAN086522b 95 5,310,334 168,230 61 1,357,182 50.9 5,344 SRR10050136 AASOEX000000000
CFSAN086523b 190 5,407,417 183,844 83 1,939,826 50.4 5,468 SRR18610082 ABFPZD000000000
CFSAN086524b 183 5,347,686 185,629 45 1,005,100 50.8 5,405 SRR10049456 AARBEI000000000
CFSAN086525b 145 4,708,302 81,649 63 1,241,962 51.0 4,694 SRR10049408 AASKHK000000000
CFSAN086526b 173 5,331,457 185,772 49 1,098,784 50.9 5,378 SRR10049658 AARBFS000000000
CFSAN086527b 47 5,223,533 349,671 78 1,714,334 50.9 5,114 SRR10049661 AASOFD000000000
CFSAN086528b 148 5,425,778 150,959 126 2,872,330 51.0 5,435 SRR10049352 AARBEM000000000
CFSAN086529b 56 5,238,479 328,083 124 2,761,940 51.2 5,103 SRR10051010 AASOEW000000000
CFSAN086531b 164 4,955,606 110,646 79 1,663,362 50.9 4,930 SRR10051009 AASKIU000000000
CFSAN086533b 88 4,840,208 112,816 87 1,774,266 50.9 4,739 SRR10050106 AASLBP000000000
CFSAN086534b 101 4,890,378 130,915 107 2,218,120 50.9 4,810 SRR10051131 AASKGW000000000
CFSAN086537b 80 4,917,914 181,415 133 2,775,564 51.4 4,897 SRR10051132 AASOET000000000
CFSAN086538b 153 4,949,050 119,247 77 1,603,794 51.2 4,965 SRR10049553 AASOFE000000000
CFSAN086539b 87 4,899,843 181,415 73 1,496,546 51.2 4,883 SRR10049520 AASLNE000000000
CFSAN086541b 89 4,935,011 181,696 61 1,258,366 51.2 4,924 SRR10050983 AASNMU000000000
CFSAN086542b 97 4,962,098 181,415 83 1,738,650 51.1 4,971 SRR10050135 AASKHB000000000
CFSAN086543b 73 4,970,574 260,303 72 1,515,064 50.7 4,842 SRR19919805 ABGXCD000000000
CFSAN086544b 90 4,943,851 181,415 73 1,511,694 51.1 4,942 SRR10051040 AASLNC000000000
CFSAN086545b 87 4,936,889 181,415 79 1,635,664 51.2 4,930 SRR10049370 AASKHL000000000
CFSAN086546b 80 4,913,588 181,457 87 1,831,044 50.9 4,833 SRR18610002 ABFQTE000000000
CFSAN086547b 82 4,919,616 181,415 97 2,036,186 50.9 4,844 SRR18610087 ABFQTB000000000
CFSAN086548b 116 4,770,104 124,126 93 1,907,446 50.7 4,643 SRR18610096 ABFQSZ000000000
CFSAN086549b 83 4,897,745 151,719 59 1,378,958 50.9 4,879 SRR10051123 AASKHA000000000
CFSAN086550b 84 4,906,966 181,415 42 975,008 51.0 4,896 SRR10050986 AASOEY000000000
CFSAN086551b 40 4,817,120 268,732 130 2,868,048 50.9 4,684 SRR10140638 AASOBL000000000
CFSAN086552b 89 4,907,656 130,727 63 1,470,850 51.2 4,898 SRR10049457 AASLCH000000000
CFSAN086553b 47 4,797,923 268,732 48 985,612 50.7 4,676 SRR10140640 AASKOS000000000
CFSAN086554b 77 4,765,697 175,155 55 1,231,694 50.6 4,648 SRR10049413 AASLBS000000000
CFSAN086555b 69 4,917,593 151,729 72 1,497,874 51.1 4,894 SRR10051052 AASKGZ000000000
CFSAN086556b 120 4,981,814 135,485 87 1,876,566 50.5 4,898 SRR18610005 ABFPZI000000000
CFSAN086557b 78 4,953,769 181,450 77 1,629,178 51.1 4,944 SRR10049412 AASLBT000000000
CFSAN086559b 75 4,917,531 181,415 87 1,823,706 51.0 4,898 SRR10050998 AASLBM000000000
CFSAN086560b 87 4,902,336 181,415 90 1,887,420 50.9 4,828 SRR18610077 ABFPZF000000000
CFSAN086561b 80 4,913,910 142,502 66 1,415,364 51.3 4,903 SRR10050114 AASLND000000000
CFSAN086562b 93 5,039,470 166,520 89 1,924,180 50.6 4,899 SRR18610081 ABFQTD000000000
CFSAN086563b 99 4,935,994 133,339 89 1,910,340 51.3 4,925 SRR10050056 AAVPXB000000000
CFSAN086564b 98 4,950,852 150,999 41 862,556 51.1 4,906 SRR10049881 AASKIN000000000
CFSAN086566b 83 4,921,082 181,415 88 1,851,800 50.9 4,842 SRR18610078 ABFPZE000000000
CFSAN086567b 129 5,376,965 177,440 80 1,827,092 50.3 5,369 SRR18610000 ABFQTO000000000
CFSAN086569b 124 5,141,577 148,635 55 1,461,942 50.3 5,145 SRR9998331 AARKQA000000000
CFSAN086570b 65 4,812,490 177,907 75 1,806,930 50.5 4,691 SRR9998290 AARKQC000000000
CFSAN086571b 145 5,336,455 111,649 77 1,750,800 49.6 5,346 SRR18610093 ABFPYZ000000000
CFSAN086572b 137 5,200,706 154,569 82 1,825,898 50.5 5,135 SRR18610086 ABFQTC000000000
CFSAN086574b 236 5,529,286 137,284 60 1,548,764 50.2 5,443 SRR9998286 AARBVT000000000
CFSAN086576b 441 5,426,999 40,841 65 1,676,440 50.2 5,704 SRR9995150 AARKQN000000000
CFSAN086579b 76 4,997,366 145,428 65 1,392,996 50.7 4,954 SRR10059538 AASKGY000000000
CFSAN086583b 283 5,105,534 67,955 80 1,762,766 51.5 5,172 SRR10051053 AASKHE000000000
CFSAN086584b 103 5,200,119 142,431 82 1,849,524 50.4 5,121 SRR18743873 ABFUHB000000000
CFSAN086588b 157 5,182,699 196,165 73 1,613,054 48.9 5,197 SRR18743868 ABFUHF000000000
CFSAN086591b 188 5,378,042 187,934 75 1,726,938 50.4 5,419 SRR18743679 ABFUGS000000000
CFSAN086592b 188 5,293,530 188,037 48 1,087,124 51.2 5,322 SRR10049375 AARBEB000000000
CFSAN086593b 203 5,311,811 145,813 45 1,017,372 51.1 5,356 SRR10049366 AARBEQ000000000
CFSAN086594b 348 5,309,259 64,059 38 851,272 49.9 5,418 SRR10049324 AASKHO000000000
CFSAN086596b 274 5,207,318 134,558 39 869,236 50.9 5,293 SRR10051039 AASOFC000000000
CFSAN086599b 133 5,387,493 169,843 73 1,684,608 50.3 5,336 SRR18743964 ABFUSR000000000
CFSAN086600b 232 5,344,814 121,594 71 2,074,638 50.3 5,362 SRR9883201 AARCWG000000000
CFSAN086601b 202 5,543,285 136,965 66 1,557,912 50.8 5,545 SRR9883212 AARCUY000000000
CFSAN086604b 218 5,379,512 121,127 63 1,453,604 50.9 5,404 SRR9883194 AARCWF000000000
CFSAN086605b 75 5,027,903 135,750 81 1,776,382 50.9 4,976 SRR9883175 AASHCD000000000
CFSAN086607b 220 5,386,924 138,961 89 2,276,162 50.7 5,411 SRR9998315 AARBYI000000000
CFSAN086608b 217 5,197,148 90,472 83 1,886,862 50.8 5,287 SRR9883173 AAYORT000000000
CFSAN086609b 188 4,781,392 62,450 38 1,364,702 50.2 4,737 SRR10059537 AASLQD000000000
CFSAN086614b 378 5,443,939 91,892 85 2,336,406 50.4 5,679 SRR9943104 AARCJS000000000
CFSAN086615b,c 255 5,398,012 89,303 63 1,662,176 50.5 5,277 SRR9943086 AARCON000000000
CFSAN086619b 106 4,826,822 102,696 42 865,212 50.7 4,817 SRR9883180 AASHCC000000000
CFSAN086620b 101 4,845,164 152,672 96 2,091,298 50.8 4,840 SRR9943059 AASKIW000000000
CFSAN086621b 93 4,836,552 152,516 53 1,081,244 50.7 4,825 SRR9883174 AASHCE000000000
CFSAN086622b 86 4,834,271 121,636 53 1,073,210 50.7 4,817 SRR9883193 AASHFC000000000
CFSAN086623b 98 4,861,701 141,668 142 3,501,034 50.4 4,845 SRR9943083 AASKIS000000000
CFSAN086625b 124 5,359,034 219,640 42 949,422 50.4 5,272 SRR9883221 AASHCB000000000
CFSAN086627b 60 5,057,053 304,057 77 1,829,580 50.5 4,975 SRR9883203 AASHCA000000000
CFSAN086628b 281 5,078,853 40,403 49 1,057,850 51.2 5,192 SRR9883186 ABCYGI000000000
CFSAN086629b 272 5,088,583 40,444 48 1,040,026 51.1 5,214 SRR9883209 AARCXN000000000
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a

Collected in 2017.

b

Collected in 2018.

c

Collected in Texas.

Data availability.

The draft genome sequences reported in this article were deposited in GenBank, and accession numbers for each SRA and whole-genome shotgun sequencing project are available in Table 1.

ACKNOWLEDGMENTS

We thank Ploy Kurdmongkoltham, Chris Pabst, and Karla Sanchez (University of Florida) and Peiman Aminabadi (Western Center for Food Safety) for technical assistance and Colleen Larson and Mary Sowerby (University of Florida) for their assistance with farm identification and sample collection. We also thank Yuhuan Chen, David Oryang, David Ingram, and Jane Van Doren from FDA Center for Food Safety and Applied Nutrition for assistance with the study design.

The contents of the manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the FDA.

This research was conducted as part of a larger study performed in cooperation with the University of California, Davis, University of Arizona, University of Delaware, and the University of Florida.

This project was funded by Western Center for Food Safety contract U19-FD004995 from the United States Food and Drug Administration (FDA).

Contributor Information

Keith R. Schneider, Email: keiths29@ufl.edu.

Irene L. G. Newton, Indiana University, Bloomington

REFERENCES

Associated Data

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

Data Availability Statement

The draft genome sequences reported in this article were deposited in GenBank, and accession numbers for each SRA and whole-genome shotgun sequencing project are available in Table 1.

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