In total, 12 quinolone-resistant Escherichia coli (QREC) strains containing qnrS1 were submitted to long-read sequencing using a FLO-MIN106 flow cell on a MinION device. The long reads were assembled with short reads (Illumina) and analyzed using the MOB-suite pipeline. Six of these QREC genome sequences were closed after hybrid assembly.
ABSTRACT
In total, 12 quinolone-resistant Escherichia coli (QREC) strains containing qnrS1 were submitted to long-read sequencing using a FLO-MIN106 flow cell on a MinION device. The long reads were assembled with short reads (Illumina) and analyzed using the MOB-suite pipeline. Six of these QREC genome sequences were closed after hybrid assembly.
ANNOUNCEMENT
The presence of quinolone-resistant Escherichia coli (QREC) in the animal reservoir is a potential public health concern, especially related to plasmid-mediated quinolone resistance genes, as they might spread to more pathogenic bacteria. The qnrS1 gene is known to be situated on plasmids with different incompatibility (Inc) groups (1, 2). Here, we aimed to select QREC strains encoding qnrS1 on plasmids with different Inc groups to complete circular plasmid contigs.
We previously sequenced 280 QREC isolates from broilers, pigs, red foxes, and wild birds, collected through the NORM-VET program from 2006 to 2017, using short-read sequencing (Illumina, San Diego, CA) (3). The samples were either selectively isolated on MacConkey agar containing 0.06 mg/liter ciprofloxacin or randomly collected from E. coli isolated on MacConkey agar. In total, 12 QREC isolates encoding qnrS1 from these four animal species were selected for long-read sequencing. Here, we report the hybrid assembly of these isolates, including six closed genome sequences. The hybrid assemblies were further analyzed using MOB-suite (4).
Extraction of genomic DNA was performed using the Genomic-tip 100/G kit (Qiagen, Hilden, Germany). Bacteria were enriched overnight at 37°C in 2 to 3 ml heart infusion broth (Difco, Omagh, UK). The DNA concentration was determined using the Qubit double-stranded DNA (dsDNA) broad-range (BR) assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and the DNA was quality assessed using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). Approximately 400 ng of high-quality DNA was subjected to library preparation using a rapid barcoding kit (SQK-RBK004; Oxford Nanopore Sequencing [ONT], Oxford, UK). Four samples were run with smaller amounts (104, 154, 324, and 369 ng), as only a maximum volume of 7.5 μl of template was allowed into the library preparation reaction. The constructed libraries were indexed using barcodes RB1 to RB12, loaded onto a FLO-MIN106 flow cell on a MinION device (Oxford Nanopore Sequencing), and run for 40 h. The raw sequence data were base called separately after the run using Guppy v.3.4.5 (5) and demultiplexed using qcat v.1.1.0 (ONT, https://github.com/nanoporetech/qcat). The sequence quality of the demultiplexed data sets was checked with NanoPlot v.1.30.0 (6). Default parameters were used for all software unless otherwise specified.
Canu v.1.9 (7) was used to improve the accuracy of the long reads, followed by Filtlong v.0.2.0 (https://github.com/rrwick/Filtlong) to remove reads of <1,000 bp from the corrected long reads. Hybrid assemblies were generated using Unicycler v.0.4.8 (8), followed by Prokka v.1.14.5 (9) to annotate the hybrid assemblies. The GC content of each assembly was calculated using the EMBOSS v.6.6.0 (10) commands “union” and “infoseq.” MOB-suite v.1.4.9 (4) was used to predict plasmid sequences from the hybrid assemblies and identify their respective replicon types. Each plasmid FASTA file generated by MOB-suite was subjected to ResFinder v.4.0 (11), VirulenceFinder v.2.0 (12), and PlasmidFinder v.2.1 (13). Plasmids containing qnrS1 were confirmed by genome annotation with Prokka. The Illumina reads were mapped back to the assembly using BWA v.0.7.17 (14), and the depth of coverage was calculated using SAMtools v.1.10 (15) using the depth (genome-wide) and coverage (replicon) options.
The characteristics and accession numbers are presented in Table 1. The plasmid assemblies with Inc groups that allowed further typing were run on pMLST v.2.0 (13) on the Center for Epidemiology Genomics website to further determine the respective replicon types.
TABLE 1.
Characteristics and accession numbers of the quinolone-resistant Escherichia coli qnrS1 strains
| Strain | Plasmid Inc type (pMLST) | STa | No. of Illumina reads for: |
Data for Nanopore reads: |
No. of contigs | Total size (Mbp) | Replicon size (bp) | GC content (%) | No. of genes | Coverage (×) | ENA accession no. for: |
|||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Read 1 | Read 2 | No. of reads | Avg length (bp) | Raw reads | Assembly | |||||||||
| 2015-01-2097 | 1421 | 818,798 | 865,881 | 331,312 | 5,292.7 | 2c | 4.68 | 50.8 | 4,507 | 275.1 | ERR4592247 | LR881940.1 | ||
| IncX1b | 21,374 | 44.5 | 28 | 437.9 | LR881941.1 | |||||||||
| 2015-01-466 | 10 | 761,941 | 819,989 | 211,523 | 5,227.7 | 5c | 4.87 | 50.6 | 4,695 | 247.4 | ERR4592248 | LR882052.1 | ||
| IncF (F-:A1:B1) | 113,096 | 52.6 | 137 | 256.3 | LR882053.1 | |||||||||
| IncH | 87,822 | 47.9 | 96 | 164.3 | LR882054.1 | |||||||||
| IncFb (F2:A-:B-) | 50,909 | 53.0 | 63 | 316.1 | LR882055.1 | |||||||||
| IncX1 | 46,065 | 40.7 | 55 | 294.5 | LR882056.1 | |||||||||
| 2016-02-324 | 7036 | 654,152 | 713,188 | 258,316 | 4,232.1 | 2c | 4.90 | 51.0 | 4,656 | 213.1 | ERR4592249 | LR882050.1 | ||
| IncFb (F-:A-:B53) | 94,955 | 52.8 | 108 | 225.2 | LR882051.1 | |||||||||
| 2016-02-418 | 58 | 596,773 | 650,657 | 174,481 | 2,309.8 | 29 | 4.96 | 50.8 | 4,786 | 191.0 | ERR4592250 | CAJGEF01 | ||
| IncX1b | 46,447d | 42.9 | 55 | 310.5 | ||||||||||
| 2016-02-522 | 1011 | 795,118 | 867,426 | 166,584 | 4,176.7 | 4 | 4.94 | 50.6 | 4,596 | 255.6 | ERR4592251 | CAJGEG01 | ||
| IncYb | 78,634 | 50.3 | 103 | 244.5 | ||||||||||
| 2016-02-620 | 694 | 676,465 | 740,782 | 438,687 | 3,794.8 | 5 | 4.71 | 50.8 | 4,494 | 227.8 | ERR4592252 | CAJGEH01 | ||
| IncX3b | 44,425 | 46.3 | 59 | 251.6 | ||||||||||
| 2016-17-164 | 7593 | 654,299 | 713,350 | 588,805 | 2,983.2 | 8 | 4.93 | 50.8 | 4,672 | 211.0 | ERR4592253 | CAJGEI01 | ||
| IncFb (F89:A-:B53) | 118,361 | 50.1 | 133 | 106.0 | ||||||||||
| 2016-17-292 | 23 | 695,093 | 720,319 | 310,224 | 5,196.4 | 3c | 4.99 | 50.4 | 4,849 | 217.5 | ERR4592254 | LR882493.1 | ||
| IncF (F24:A-:B1) | 97,083 | 48.7 | 99 | 121.3 | LR882494.1 | |||||||||
| IncI2 | 59,944 | 42.1 | 83 | 136.4 | LR882495.1 | |||||||||
| 2016-17-363 | 48 | 761,196 | 825,502 | 404,780 | 2,644.6 | 5 | 4.67 | 50.7 | 4,478 | 258.2 | ERR4592255 | CAJGWN01 | ||
| IncHb (unknown) | 86,214 | 48.5 | 100 | 221.7 | ||||||||||
| 2016-17-550 | 2165 | 988,537 | 1,058,892 | 218,828 | 4,398.6 | 2c | 4.82 | 50.8 | 4,559 | 326.5 | ERR4592256 | LR883965 | ||
| IncYb | 104,732 | 48.0 | 118 | 128.2 | LR883966 | |||||||||
| 2015-01-2838 | 117 | 388,306 | 418,338 | 129,950 | 3,457.6 | 15 | 5.14 | 50.7 | 4,899 | 98.0 | ERR4592257 | CAJGWP01 | ||
| IncX2b | 39,630 | 46.0 | 50 | 337.3 | ||||||||||
| 2014-01-7375 | 453 | 472,494 | 482,585 | 209,994 | 4,667.7 | 5c | 5.27 | 50.6 | 5,119 | 34.1 | ERR4592258 | LR882057.1 | ||
| IncI1 | 98,997 | 49.4 | 110 | 62.5 | LR882058.1 | |||||||||
| IncF (F-:A-:B56) | 82,142 | 47.8 | 89 | 46.4 | LR882059.1 | |||||||||
| IncX1b | 47,686 | 43.1 | 56 | 64.0 | LR882060.1 | |||||||||
| IncF (F-:A-:B114) | 42,660 | 52.5 | 54 | 88.3 | LR882061.1 | |||||||||
ST, sequence type.
Plasmid with qnrS.
Genome closed.
Plasmid not circularized.
Data availability.
All data sets are deposited in ENA under accession number PRJEB40547 (Table 1).
ACKNOWLEDGMENTS
Illumina sequencing was provided by the Norwegian Sequencing Centre (www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and Oslo University Hospital, supported by the Functional Genomics and Infrastructure programs of the Research Council of Norway and the South-Eastern Norway Regional Health Authority. The work was performed on resources provided by UNINETT Sigma2, the National Infrastructure for High Performance Computing and Data Storage in Norway.
This work was part of the One Health EJP project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 773830 (2018 to 2022).
REFERENCES
- 1.Dobiasova H, Dolejska M. 2016. Prevalence and diversity of IncX plasmids carrying fluoroquinolone and beta-lactam resistance genes in Escherichia coli originating from diverse sources and geographical areas. J Antimicrob Chemother 71:2118–2124. doi: 10.1093/jac/dkw144. [DOI] [PubMed] [Google Scholar]
- 2.Dolejska M, Villa L, Minoia M, Guardabassi L, Carattoli A. 2014. Complete sequences of IncHI1 plasmids carrying blaCTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution. J Antimicrob Chemother 69:2388–2393. doi: 10.1093/jac/dku172. [DOI] [PubMed] [Google Scholar]
- 3.Kaspersen H, Sekse C, Zeyl Fiskebeck E, Slettemeas JS, Simm R, Norstrom M, Urdahl AM, Lagesen K. 2020. Dissemination of quinolone-resistant Escherichia coli in the Norwegian broiler and pig production chain and possible persistence in the broiler production environment. Appl Environ Microbiol 86:e02769-19. doi: 10.1128/AEM.02769-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Robertson J, Nash JHE. 2018. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb Genom 4:e000206. doi: 10.1099/mgen.0.000206. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Ueno Y, Arita M, Kumagai T, Asai K. 2003. Processing sequence annotation data using the Lua programming language. Genome Inform 14:154–163. [PubMed] [Google Scholar]
- 6.De Coster W, D'Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34:2666–2669. doi: 10.1093/bioinformatics/bty149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.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]
- 8.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]
- 9.Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
- 10.Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16:276–277. doi: 10.1016/s0168-9525(00)02024-2. [DOI] [PubMed] [Google Scholar]
- 11.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640–2644. doi: 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Joensen KG, Scheutz F, Lund O, Hasman H, Kaas RS, Nielsen EM, Aarestrup FM. 2014. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol 52:1501–1510. doi: 10.1128/JCM.03617-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Carattoli A, Zankari E, Garcia-Fernandez A, Voldby Larsen M, Lund O, Villa L, Moller Aarestrup F, Hasman H. 2014. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58:3895–3903. doi: 10.1128/AAC.02412-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup . 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352. [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
All data sets are deposited in ENA under accession number PRJEB40547 (Table 1).