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. 2019 Oct 17;8(42):e00974-19. doi: 10.1128/MRA.00974-19

Draft Genome Sequences of 43 Enterococcus faecalis and Enterococcus faecium Isolates from a Commercial Beef Processing Plant and Retail Ground Beef

Devin B Holman a,, Katherine E Gzyl a, Rahat Zaheer b, Tineke H Jones a, Tim A McAllister b
Editor: Catherine Putontic
PMCID: PMC6797533  PMID: 31624168

Here, we report the draft genome sequences of 36 Enterococcus faecalis and 7 Enterococcus faecium isolates recovered from a beef processing facility and retail ground beef. The beef processing facility samples were collected from beef carcasses, conveyor belts, and ground product.

ABSTRACT

Here, we report the draft genome sequences of 36 Enterococcus faecalis and 7 Enterococcus faecium isolates recovered from a beef processing facility and retail ground beef. The beef processing facility samples were collected from beef carcasses, conveyor belts, and ground product.

ANNOUNCEMENT

Enterococcus faecalis and Enterococcus faecium are commensal microorganisms present in the gastrointestinal tract of both humans and cattle; therefore, enterococci are often used as an indicator of fecal contamination. We collected samples from four different locations in a commercial beef processing facility in Alberta, Canada, over an 18-month period. Enterococcus spp. were collected from a randomly selected 10- by 10-cm area on carcasses after hide removal (H) and final washing (W), as well as from conveyer belts (C) using a 2- by 2-cm sterile gauze swab (n = 150 each). Ground product (n = 150) and retail ground beef (n = 60) samples (25 g each) were also collected during the same time period. The swabs and ground samples were homogenized and preenriched in a Stomacher 400 circulator (Seward, Norfolk, UK) at 260 rpm for 2 min in 10 ml or 225 ml of buffered peptone water, respectively, and incubated overnight at 37°C. A 1-ml aliquot of this mixture was then added to 9 ml of Enterococcosel broth (BD, Mississauga, Ontario, Canada) and incubated overnight at 37°C to enrich for Enterococcus spp. Enterococcosel broth tubes displaying evidence of esculin hydrolysis (black) were streaked onto Enterococcosel agar and incubated at 37°C. After 48 h, the plates were examined for colonies with black zones, which is indicative of esculin hydrolysis. The groES-EL spacer region of presumptive enterococcal colonies was amplified using the EntES-211-233-F and Ent-EL-74-95-R primers (1), and the product was sequenced using an ABI Prism 3130xl genetic analyzer (Thermo Fisher Scientific, Inc., Mississauga, Ontario, Canada) for confirmation and species identification. From the confirmed Enterococcus spp., 36 E. faecalis and 7 E. faecium isolates were selected for whole-genome sequencing.

Briefly, genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Mississauga, Ontario, Canada) with the modification that cells were incubated with agitation (150 rpm) for 45 min at 37°C in 280 μl of lysis buffer (20 mM Tris-HCl [pH 8.0], 2 mM sodium EDTA, 1.2% Triton X-100, and 20 mg/ml lysozyme; Sigma-Aldrich Canada, Toronto, Ontario, Canada). The Nextera XT DNA library preparation kit (Illumina, Inc., San Diego, CA, USA) was used to prepare sequencing libraries that were sequenced on a MiSeq instrument (Illumina, Inc.) with the MiSeq reagent kit v3 (Illumina, Inc.; 600 cycles) as per the manufacturer’s instructions.

FastQC v0.11.5 (2) was used to assess read quality before (678,235 ± 23,253 [standard error of the mean {SEM}] reads per isolate) and after (596,916 ± 21,456 reads per isolate) quality filtering. Sequencing adapters, reads with a quality score of less than 15 over a 4-bp sliding window, and reads that were less than 50 bp in length were removed with Trimmomatic v0.38 (3). The paired-end reads were assembled with SPAdes v3.11.1 (4), with the default parameters in the “careful” mode, and the quality of the assemblies was determined using QUAST v5.0.1 (5). Contigs less than 500 bp in length were removed prior to confirming the taxonomy of each assembly with Kraken 2 v2.0.7-beta and the minikraken2 database v2 (6). The assemblies were then annotated with Prokka v1.13.3 (7) using the default parameters. Multilocus sequence typing (MLST) was done using the Enterococcus faecalis MLST website (https://pubmlst.org/efaecalis/) (8) and the Enterococcus faecium MLST website (https://pubmlst.org/efaecium/) (9). The assembly statistics, GenBank and SRA accession numbers, and MLST results for each isolate are presented in Table 1.

TABLE 1.

Assembly statistics for Enterococcus faecalis and Enterococcus faecium isolates from a beef processing facility and retail ground beefa

Isolate name Species GenBank accession no. SRA accession no. No. of contigs No. of reads Genome size (bp) N50 value (bp) Coverage (×) No. of coding sequences G+C content (%) MLST
C112 E. faecalis GCA_006541215 SRR9321129 26 722,884 2,988,147 605,310 73 2,886 37.43 Unknown
C138 E. faecalis GCA_006541395 SRR9321128 30 899,074 2,695,188 213,629 100 2,557 37.59 228
C144 E. faecalis GCA_006541075 SRR9321127 34 689,845 2,697,493 222,684 77 2,519 37.58 228
C146 E. faecalis GCA_006541305 SRR9321126 28 502,816 2,697,692 351,123 56 2,567 37.59 228
G109 E. faecalis GCA_006541225 SRR9321133 65 463,324 2,853,332 140,707 49 2,739 37.57 Unknown
G127E E. faecalis GCA_006541295 SRR9321132 105 633,257 3,053,018 80,851 62 2,997 37.48 Unknown
G138E E. faecalis GCA_006541335 SRR9321131 119 564,994 3,016,964 86,924 56 2,917 37.45 21
G149 E. faecalis GCA_006541345 SRR9321130 125 446,360 2,994,467 59,749 45 2,854 37.42 40
G42 E. faecalis GCA_006541405 SRR9321125 97 630,933 2,872,254 73,537 66 2,712 37.56 21
G69E E. faecalis GCA_006541355 SRR9321124 17 529,454 2,959,381 645,373 54 2,784 37.51 202
G81 E. faecalis GCA_006541905 SRR9321137 59 859,935 2,835,934 144,986 91 2,697 37.55 Unknown
G85 E. faecalis GCA_006541465 SRR9321136 64 640,416 2,876,123 124,461 67 2,732 37.52 76
H102 E. faecalis GCA_006541755 SRR9321139 106 416,566 2,830,742 73,730 44 2,747 37.6 147
H112E E. faecium GCA_006541175 SRR9321138 42 512,378 2,655,194 141,400 58 2,519 38.05 212
H134E E. faecium GCA_006541535 SRR9321141 66 535,680 2,507,908 101,754 64 2,365 38.07 29
H136 E. faecalis GCA_006541265 SRR9321140 22 883,588 3,052,803 378,930 87 3,001 37.34 Unknown
H22 E. faecalis GCA_006541095 SRR9321143 35 657,288 2,786,536 250,863 71 2,564 37.49 16
H4 E. faecalis GCA_006541805 SRR9321142 59 641,369 2,996,880 146,743 64 2,860 37.42 40
H44 E. faecalis GCA_006541185 SRR9321135 46 649,040 2,816,920 228,593 69 2,629 37.57 76
H74 E. faecalis GCA_006541115 SRR9321134 38 769,279 2,733,857 166,527 84 2,575 37.57 Unknown
H96E E. faecalis GCA_006541255 SRR9321160 32 664,950 2,847,191 191,075 70 2,644 37.53 708
R2 E. faecium GCA_006541615 SRR9321161 145 784,155 2,743,995 46,108 86 2,682 38.24 76
R20 E. faecalis GCA_006541155 SRR9321162 163 623,226 2,985,199 78,020 63 2,859 37.52 260
R26E E. faecium GCA_006541645 SRR9321163 237 261,875 2,688,512 23,732 29 2,594 38.38 76
R29 E. faecalis GCA_006541545 SRR9321156 45 468,536 2,911,452 206,037 48 2,785 37.47 260
R30 E. faecalis GCA_006541705 SRR9321157 49 505,869 2,647,103 84,312 57 2,488 37.7 228
R37 E. faecalis GCA_006541775 SRR9321158 70 482,350 2,929,864 135,712 49 2,769 37.39 260
R43E E. faecalis GCA_006541085 SRR9321159 73 591,636 2,833,916 87,778 63 2,734 37.49 Unknown
R49 E. faecalis GCA_006541715 SRR9321154 36 390,483 3,029,112 153,103 39 2,921 37.37 192
R4E E. faecium GCA_006541685 SRR9321155 100 667,395 2,738,432 75,527 73 2,683 38.24 Unknown
R50 E. faecalis GCA_006541785 SRR9321147 113 519,617 2,976,798 77,751 52 2,863 37.46 21
R51 E. faecalis GCA_006541855 SRR9321146 62 463,047 3,005,276 121,849 46 2,957 37.45 84
R52 E. faecalis GCA_006541795 SRR9321145 66 449,634 3,046,695 119,574 44 2,931 37.37 260
R53 E. faecalis GCA_006541655 SRR9321144 79 596,560 3,030,982 119,130 59 2,966 37.42 260
R5E E. faecalis GCA_006541525 SRR9321151 76 611,462 2,934,492 97,444 63 2,785 37.39 Unknown
R7 E. faecalis GCA_006541595 SRR9321150 51 507,624 2,911,924 184,949 52 2,821 37.47 260
W100 E. faecalis GCA_006541515 SRR9321149 55 452,646 2,961,448 130,717 46 2,832 37.42 260
W133 E. faecalis GCA_006541445 SRR9321148 364 678,090 2,944,917 14,728 69 2,799 37.19 Unknown
W141 E. faecium GCA_006541625 SRR9321153 82 626,699 2,761,265 123,573 68 2,695 38.22 76
W148E E. faecium GCA_006541485 SRR9321152 96 501,112 2,758,663 89,896 54 2,687 38.23 76
W19 E. faecalis GCA_006541875 SRR9321122 76 865,657 2,956,998 112,037 88 2,846 37.5 Unknown
W84 E. faecalis GCA_006541885 SRR9321123 99 564,979 3,018,223 89,324 56 2,896 37.45 Unknown
W97 E. faecalis GCA_006541435 SRR9321121 61 741,306 3,050,168 155,000 73 2,961 37.37 40
a

The number of coding sequences is based on Prokka annotations. Annotations in GenBank are based on the Prokaryotic Genome Annotation Pipeline (PGAP).

The genome assemblies were also screened for the presence of antimicrobial resistance (AMR) genes using BLASTn 2.6.0+ and the Comprehensive Antibiotic Resistance Database (CARD; v3.0.2) (10), with a minimum identity of 90%. The most prevalent AMR genes in the E. faecalis genomes were tet(M) (25%) and erm(B) (8%); in the E. faecium assemblies, msrC (100%), aac(6′)-II (100%), and tet(M) (29%) were most frequently detected.

Data availability.

All sequences and draft genome assemblies have been deposited in the Sequence Read Archive and GenBank, respectively, under the accession numbers listed in Table 1.

ACKNOWLEDGMENTS

This project was financially supported by the Beef Cattle Research Council and Agriculture and Agri-Food Canada.

We thank Cara Service, Scott Hrycauk, and Nicole Lassel for their valuable technical assistance.

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Associated Data

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

Data Availability Statement

All sequences and draft genome assemblies have been deposited in the Sequence Read Archive and GenBank, respectively, under the accession numbers listed in Table 1.


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