ABSTRACT
Here, we report the complete genome of Bordetella parapertussis strain 400431-b isolated from a nasopharyngeal swab from a 4-year-old patient presenting with a protracted course of whooping cough, vaccinated with three doses of diphtheria-tetanus-acellular pertussis-hepatitis B-poliomyelitis-Haemophilus influenzae type b vaccine.
KEYWORDS: whooping cough, WGS, antimicrobial resistance, Bordetella, genomics
ANNOUNCEMENT
Bordetella parapertussis (Bpp) 400431-b was isolated from a 4-year-old patient and vaccinated with three doses of diphtheria-tetanus-acellular pertussis-hepatitis B-poliomyelitis-Haemophilus influenzae type b vaccine, showing a protracted clinical course of whooping cough. The nasopharyngeal swab was plated onto Bordetella Selective Medium (Oxoid, Waltham, MA, USA) and incubated 48 hours at 37°C. Presumptive Bordetella spp. colonies were subcultivated on blood agar (COS, Biomérieux, Marcy-l'Étoile, France) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Microflex LT/SH, MBT Compass IVD4.2, Flex Analysis v3.4, Bruker, Billerica, MA, USA).
Antimicrobial susceptibility testing was performed as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) with minimum inhibitory concentration (MIC) gradient strips containing erythromycin, azithromycin, clarithromycin, and trimethoprim-sulfamethoxazole (ETEST, Biomérieux, Marcy-l'Étoile, France) on Mueller-Hinton agar + 5% blood + 20 mg/L β-NAD (Biomérieux, Marcy-l'Étoile, France). Due to the lack of EUCAST breakpoints, susceptibility results were compared with strain FR6242 (1).
Genomic DNA was isolated from cells grown on COS, for 48 h at 37°C. The MagAttract HMW (Qiagen, Hilden, Germany) and the Wizard HMW (Promega, Madison, WI, USA) DNA Extraction Kits were used for Illumina and Oxford Nanopore sequencing, respectively. Sequencing libraries were prepared using the DNA preparation (M) kit (Illumina, San Diego, CA, USA) and the rapid barcoding kit SQK-RBK004 [Oxford Nanopore Technologies (ONT), Oxford, UK]. Libraries were 2 × 150 bp sequenced on NextSeq 2000 (Illumina, San Diego, CA, USA) as described (2) and with FLO-MIN114 R10.4.1 SpotON flow cell on a GridION (ONT, Oxford, UK). Default parameters were used for all software unless otherwise specified. A total of 3,146,612 Illumina reads with average length of 142 bp were quality controlled using FastQC v0.11.9.12 (3). A total of 293,539 Nanopore reads (N50: 15,211 bp) were obtained using Guppy v6.3.8 (4) in super-accurate basecalling mode and filtered using Filtlong v0.2.1 (parameters: -t 400000000 min_length 1000 --keep_percent 95). Assembly was performed using Flye v2.9.2-b1786 (5). Draft assembly was polished using Medaka_consensus v.1.8.0 with model r1041_e82_260bps_sup_g632 followed by Polypolish v.0.5.0 (6), which resulted in one contig with 92-fold average coverage, a genome size of 4.8 Mb, and a GC content of 68.1% (Table 1). Burrows-Wheeler Aligner Maximal-Exact-Matches algorithm (BWA-MEM) (7) and SAMtools v.1.17 (8) were used to manually inspect reads at the contig ends to check circularity of the final assembly.
TABLE 1.
Characteristics of B. parapertussis 400431-b
| Parameter | Finding/strain/accession | |
|---|---|---|
| Total genome size (bp) | 4,773,379 (circular) | |
| No. of contigs | 1 | |
| Hybrid assembly genome coverage (×) | 92 | |
| No. of raw short reads | 3,146,612 | |
| No. of raw long reads | 293,539 | |
| Long-read fragment N50 (bp) | 15,211 | |
| GC content (%) | 68.1 | |
| Total no. of genes | 4,521 | |
| Total no. of coding sequences | 4,223 | |
| No. of RNA genes | 67 | |
| No. of complete rRNAs (5S, 16S, 23S) | 3, 3, 3 | |
| No. of tRNAs | 54 | |
| No. of noncoding RNAs | 4 | |
| Total no. of pseudogenes | 231 | |
| No. of CRISPR/Cas elements | 0 | |
| No. of insertion sequences IS1001 (identity >99.0%) | 20 (with 4 to 7 substitutions) | |
| No. of insertion sequences IS1002 (identity >98.6%) | 9 (with 11 to 13 substitutions) | |
| No. of composite transposons_IS1001 | 3 (with 5 to 7 substitutions) | |
| No. of plasmids | 0 | |
| No. of phages | 0 | |
| Sequence type | 19 | |
| Virulence genes | Present: - Fimbrial precursors (fim2, fim3) - Pertussis toxin promoter (ptxP) - Pertussis toxin (ptxA, ptxB, ptxC, ptxD, ptxE) - Filamentous hemagglutinin (fhaB) - Dermonecrotic toxin (dnt) - Adenylate cyclase (cyaA) -Pertactin (prn) a |
Absent: - Tracheal colonization factor (tcfA) |
| Efflux pumps |
adeF (two sequences) qacG |
|
| MIC trimethoprim-sulfamethoxazole (concentration 1/19) (mg/L) | 6 | |
| MIC erythromycin (mg/L) | 1.5 | |
| MIC clarithromycin (mg/L) | 1.5 | |
| MIC azithromycin (mg/L) | 0.75 | |
| cgMLST reference genome | Strain 12822: NC_002928.3 | |
| cgMLST query genomes | Bpp5: NC_018828.1, NC018830.1; H904: NZ_CP016342.1; H889: NZ_CP018897.1; A747: NZ_CP020655.1; B337: NZ_CP020654.1; D577: NZ_CP020652.1; E738: NZ_CP020648; I440: NZ_LS483424.1; NCTC10520: NZ_LS483424.1; NCTC10524: NZ_LR134329.1; Bpp01: NZ_AP019378.2; A005: NZ_CP025070.1; B114: NZ_CP025068.1; B149: NZ_CP025071.1; B160: NZ_CP025072.1; B271: NZ_CP025067.1; F585: NZ_CP024174.1; E762: NZ_CP019932.1; E843: NZ_CP019931.1; NCTC10522: NZ_LR590480.1; H100: NZ_CP043165.1; H101: NZ_CP043164.1; H102: NZ_CP043163.1; H104: NZ_CP043162.1; H105: NZ_CP043161.1; H242: NZ_CP043159.1; H314: NZ_CP043155.1; H554: NZ_CP043145.1; H555: NZ_CP043144.1; H580: NZ_CP043140.1; H581: NZ_CP043139.1; I133: NZ_CP043121.1; I139: NZ_CP043120.1; FDAARGOS_1540: NZ_CP085972.1; FDAARGOS_1541: NZ_CP085971.1. | |
prn promoter is absent; prn is non-functional due to deletion in position 1893.
AMRFinderPlus v3.11.2 (9), MOB-suite v3.1.4 (10), MobileElementFinder v1.0.5 (11), and VFDB v2020-Feb-28 (12) were used for the detection of antimicrobial resistance genes, virulence genes, and mobile genetic elements.
The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.6 (13) identified 4,521 genes, 4,223 coding sequences, and 67 RNA genes (Table 1). An ad hoc core genome multilocus sequence typing scheme comprising 3,479 core targets was created using the Target-Definer v1.5 from SeqSphere + v8.5.1 (Ridom, Münster, Germany), using strain 12822 (14) as reference and 35 Bpp query genomes from GenBank (Table 1 ). Comparison of Bpp 400431-b with all available genomes from GenBank showed 37, 16, and 10 allelic differences to strains 12822, FR6242 (1), and J456 (15), respectively.
Bpp 400431-b carried blaBOR-1 , three efflux pumps, and had 50-, 12-, and 8-fold higher MICs for trimethoprim-sulfamethoxazole, erythromycin, and clarithromycin than strain FR6242, respectively.
ACKNOWLEDGMENTS
We thank Loredana Ingrosso for her feedback throughout the project and on the manuscript.
Maria João Cardoso was supported by a grant from the European Public Health Microbiology Training Program (EUPHEM), European Centre for Disease Prevention and Control (specific grant agreement no. 1/ECD.13493 implementing ECDC/GRANT/2020/010).
AFTER EPUB
[This article was published on 29 November 2023 with an error in Table 1. The error was corrected in the current version, posted on 12 December 2023.]
Contributor Information
Daniela Schmid, Email: daniela.schmid@meduniwien.ac.at.
Werner Ruppitsch, Email: werner.ruppitsch@ages.at.
David Rasko, University of Maryland School of Medicine, Baltimore, Maryland, USA .
DATA AVAILABILITY
This whole genome project was deposited in NCBI GenBank database under the accession number CP134883, BioProject accession number PRJNA1013794, and BioSample accession number SAMN37311000. This is the first version of this genome. Illumina raw sequence reads have been deposited in the Sequence Read Archive (SRA) under the accession number SRR26175772 and Oxford Nanopore reads under SRA accession number SRR25937767.
ETHICS APPROVAL
The study received a waiver from the ethics committee of Vienna (number MA15-EK/23-177-VK_NZ).
REFERENCES
- 1. Toubiana J, Azarnoush S, Bouchez V, Landier A, Guillot S, Matczak S, Bonacorsi S, Brisse S. 2019. Bordetella parapertussis bacteremia: clinical expression and bacterial genomics. Open Forum Infect Dis 6:fz122. doi: 10.1093/ofid/ofz122 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Martinovic A, Cabal A, Nisic A, Sucher J, Stöger A, Allerberger F, Ruppitsch W. 2021. Genome sequences of Lactococcus garvieae and Lactococcus petauri strains isolated from traditional montenegrin brine cheeses. Microbiol Resour Announc 10:e0054621. doi: 10.1128/MRA.00546-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc
- 4. Wick RR, Judd LM, Holt KE. 2019. Performance of neural network basecalling tools for oxford nanopore sequencing. Genome Biol 20:129. doi: 10.1186/s13059-019-1727-y [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37:540–546. doi: 10.1038/s41587-019-0072-8 [DOI] [PubMed] [Google Scholar]
- 6. Wick RR, Holt KE. 2022. Polypolish: short-read polishing of long-read bacterial genome assemblies. PLoS Comput Biol 18:e1009802. doi: 10.1371/journal.pcbi.1009802 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Li H. 2013. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. q.bioGN - arXiv. doi: 10.1099/mgen.0.000122 [DOI]
- 8. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H. 2021. Twelve years of SAMtools and BCFtools. Gigascience 10:giab008. doi: 10.1093/gigascience/giab008 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Feldgarden M, Brover V, Haft DH, Prasad AB, Slotta DJ, Tolstoy I, Tyson GH, Zhao S, Hsu C-H, McDermott PF, Tadesse DA, Morales C, Simmons M, Tillman G, Wasilenko J, Folster JP, Klimke W. 2019. Validating the AMRFinder tool and resistance gene database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates. Antimicrob Agents Chemother 63:e00483-19. doi: 10.1128/AAC.00483-19 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. 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]
- 11. Johansson MHK, Bortolaia V, Tansirichaiya S, Aarestrup FM, Roberts AP, Petersen TN. 2021. Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: mobileelementfinder . J Antimicrob Chemother 76:101–109. doi: 10.1093/jac/dkaa390 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Chen L, Zheng D, Liu B, Yang J, Jin Q. 2016. VFDB 2016: hierarchical and refined dataset for big data analysis—10 years on. Nucleic Acids Res 44:D694–7. doi: 10.1093/nar/gkv1239 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Li W, O’Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ, Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res 49:D1020–D1028. doi: 10.1093/nar/gkaa1105 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Parkhill J, Sebaihia M, Preston A, Murphy LD, Thomson N, Harris DE, Holden MTG, Churcher CM, Bentley SD, Mungall KL, et al. 2003. Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet 35:32–40. doi: 10.1038/ng1227 [DOI] [PubMed] [Google Scholar]
- 15. Weigand MR, Peng Y, Loparev V, Batra D, Bowden KE, Cassiday PK, Davis JK, Johnson T, Juieng P, Miner CE, Rowe L, Sheth M, Tondella ML, Williams MM. 2016. Complete genome sequences of four different Bordetella sp. isolates causing human respiratory infections. Genome Announc 4:e01080-16. doi: 10.1128/genomeA.01080-16 [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
This whole genome project was deposited in NCBI GenBank database under the accession number CP134883, BioProject accession number PRJNA1013794, and BioSample accession number SAMN37311000. This is the first version of this genome. Illumina raw sequence reads have been deposited in the Sequence Read Archive (SRA) under the accession number SRR26175772 and Oxford Nanopore reads under SRA accession number SRR25937767.