Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.
ABSTRACT
Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.
ANNOUNCEMENT
Shiga toxin-producing Escherichia coli (STEC) strains are responsible for serious outbreaks and sporadic cases of foodborne illness worldwide. Diseases associated with STEC infection include gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome, a life-threatening complication that occurs primarily in children, the elderly, and immunocompromised individuals (1). Serotype O157:H7 and serogroups O26, O103, O111, and O145 are the most epidemiologically relevant forms of STEC implicated in causing severe human illness (2) due to their low infectious dose and high virulence. The genes encoding the Shiga toxins (stx1 and stx2), as well as the eae gene encoding the intimin protein, are important STEC virulence factors.
To ensure consumer safety, methods have been reported for the detection of STEC in food (3–7). They rely on use of an enrichment step, using a selective broth to allow growth and improved recovery of the target STEC. The ISO13136:2012 method includes the use of novobiocin as a supplement in the enrichment broth due to its antimicrobial effect against Gram-positive bacteria (8). However, the growth of some serogroups, including O111, could be inhibited due to their sensitivity to novobiocin (9–11), which would result in false-negative detection results. Therefore, the genomes of seven STEC O111 strains isolated from humans and cattle (Table 1), previously shown to have variation in resistance to novobiocin (11), were sequenced. This information is important for understanding the characteristics of this pathogen at the molecular level, as well as for development of enrichment media and selective agars that will allow improved detection of STEC O111.
TABLE 1.
Characteristics and accession numbers of seven E. coli serogroup O111 strains
| Characteristic | Data for strain: |
||||||
|---|---|---|---|---|---|---|---|
| 00-4748 | 98-8338 | TB226A | 95-0586 | 3007-85 | CL-37 | 7-14 10A | |
| GenBank accession no. | QMHY00000000 | QMHZ00000000 | QMIA00000000 | QMIB00000000 | QMIC00000000 | QMHX00000000 | QMHW00000000 |
| SRA no.a | SRP155839 | SRP155841 | SRP155842 | SRP155848 | SRP155849 | SRP155838 | SRP155833 |
| Genome coverage (×)b | 76 | 100 | 88 | 94 | 82 | 81 | 100 |
| Genome size (bp)b | 4,966,944 | 5,288,007 | 5,429,255 | 5,242,733 | 5,179,065 | 5,281,527 | 5,212,079 |
| GC content (%)c | 50.50 | 50.40 | 50.40 | 50.40 | 50.40 | 50.40 | 50.40 |
| In silico serotyped | O111:H8 | O111:H8 | O111:H8 | O111:H8 | O111:H8 | O111:H8 | O111:H8 |
| Previously reported serotype | O111:NM | O111:NM | O111:H- | O111:H- | O111:NM | O111:H8 | O111:H8 |
| Shiga toxin variant(s)e | stx2a; stx2b | stx1a; stx1b | stx1a; stx1b; stx2a; stx2b | stx1a, stx1b | stx1a; stx1b; stx2a; stx2b | stx1a; stx1b | stx1a; stx1b |
| eae genee | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
| No. of contigsb | 254 | 295 | 333 | 316 | 300 | 305 | 279 |
| Total no. of genesb | 5,410 | 5,882 | 6,099 | 5,788 | 5,730 | 5,853 | 5,797 |
| No. of pseudogenesb | 316 | 343 | 353 | 318 | 339 | 356 | 331 |
| N50f | 105,049 | 99,577 | 99,577 | 99,880 | 104,026 | 104,100 | 104,239 |
| No. of CDSsb | 5,295 | 5,769 | 5,979 | 5,668 | 5,612 | 5,736 | 5,673 |
| No. of rRNAsb | 10 | 12 | 11 | 11 | 11 | 10 | 10 |
| No. of tRNAsb | 93 | 89 | 97 | 97 | 95 | 95 | 102 |
| No. of ncRNAsb | 12 | 12 | 12 | 12 | 12 | 12 | 12 |
| MLSTg | ST16; ST480 | ST16; ST480 | ST16; ST480 | ST16; ST480 | ST16; ST480 | ST16; ST480 | ST16; ST480 |
| Resistance gene(s)h | None found |
aac(3)-IId; aadA5; sul1; dfrA17; blaTEM-1B; catA1 |
aph(3)-Ia; aadA1; sul1; tet(A) |
None found | None found |
aph(3)-Ia; strA; strB; aph(6)-Ic |
None found |
| Origin | Human | Human | Human | Human | Human | Human | Cattle |
| Source or reference | 17 | 17 | 18 | Laboratory Centre for Disease Control, Canada |
CDC, STEC, Nebraska |
STEC Center, Canada |
Nebraska |
Sequence Read Archive (SRA) accession number.
Genome annotation was performed via the NCBI Prokaryotic Genome Annotation Pipeline (12). CDSs, coding sequences; ncRNAs, noncoding RNAs.
The GC content (%) was obtained using the Genome Assembler and Annotation Report.
The in silico serotype was obtained using E. coli Serotyping version 1.0.0 (13).
Virulence genes and Shiga toxin subtype were determined using VirulenceFinder (14).
The N50 value is the size of the shortest contig in the set of longest contigs that together cover at least 50% of the total genome size.
Multilocus sequence typing (MLST) information was obtained using the Bacterial Analysis Pipeline (15).
Antimicrobial resistance genes were identified using the Bacterial Analysis Pipeline (16).
STEC O111 strains (Table 1) were grown in tryptic soy broth (TSB; Becton, Dickinson and Company, Sparks, MD, USA) at 37°C for 18 h, and genomic DNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA) and a QIAcube instrument (Qiagen). The Nextera DNA Flex library prep kit (Illumina, San Diego, CA, USA) was used for library preparation, and the library concentration was assessed using a Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA). The libraries were denatured and loaded onto a flow cell (for cluster generation), and sequencing was performed using the Illumina MiniSeq platform with a 2 × 150-bp paired-end read protocol with approximately 100× coverage. BaseSpace Software (version 2.0.2018) found on the Illumina webpage was used to access the application for analysis of the raw data. This included FastQC (version 1.0.0) to improve the quality of the raw data, the SPAdes genome assembler (version 3.9.0), and the Bacterial Analysis Pipeline (version 1.0.4). Genome annotation was performed via the NCBI Prokaryotic Genome Annotation Pipeline (12). Strains used, genome size, GC content, coverage of the sequenced strains, serotype identification, virulence genes, antimicrobial resistance genes, multilocus sequencing typing (sequence types), and other characteristics are listed in Table 1.
Data availability.
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 1. The versions described in this paper are the first versions.
ACKNOWLEDGMENTS
This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under contract number DE-SC0014664.
All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of the USDA, ARS, DOE, or ORAU/ORISE. Mention of brand names does not constitute an endorsement.
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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
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 1. The versions described in this paper are the first versions.