This report presents the draft genome sequences of two Campylobacter novaezeelandiae and four unclassified Campylobacter isolates from Canadian agricultural surface water. Phylogenomic analysis revealed that the six isolates formed unique clades, closely related to the disease-causing species C. jejuni, C. coli, and C. hepaticus.
ABSTRACT
This report presents the draft genome sequences of two Campylobacter novaezeelandiae and four unclassified Campylobacter isolates from Canadian agricultural surface water. Phylogenomic analysis revealed that the six isolates formed unique clades, closely related to the disease-causing species C. jejuni, C. coli, and C. hepaticus.
ANNOUNCEMENT
Contaminated surface water can spread pathogens and pose a threat to human health through a variety of exposure routes. Unclassified thermophilic Campylobacter spp. have been frequently recovered from various surface water types and sources in Canada (1–4). Here, six Campylobacter sp. isolates recovered between 2014 and 2015 from the surface water of two agricultural watersheds located at the South Nation River Basin east of Ottawa, Ontario (5), were subjected to whole-genome sequencing (WGS). Water samples were filtered through 0.47-μm membranes and the filters incubated in modified Preston broth (Oxoid, Lenexa, KS, USA) containing polymyxin B, rifampin, trimethoprim, and amphotericin B at 42°C under microaerophilic conditions (5% O2, 85% N2, and 10% CO2) for 48 h. The enriched samples were streaked onto modified Karmali agar (Oxoid) containing the same antibiotic supplement and incubated under the above-mentioned conditions (6). For WGS, the isolates were grown on modified Campylobacter blood-free selective agar (Thermo Fisher Scientific) or BD BBL Trypticase soy agar with 5% sheep blood (Thermo Fisher Scientific) under the same conditions described above. Biomass from the isolated colonies was subjected to genomic DNA extraction using the GenElute bacterial genomic DNA kit (Sigma-Aldrich) or the MagNA Pure LC total nucleic acid isolation kit (Roche). Nextera XT v2 libraries were prepared and 2 × 250-bp MiSeq Illumina reads generated. The paired-end reads were trimmed using Trimmomatic v0.38 (7) (-phred33). The quality of the sequence data before and after trimming was assessed using FastQC v0.11.8 (8). The trimmed reads were assembled using metaSPAdes v3.13.0 (k-mers 65, 77, 99, and 115) (9) and annotated using the Prokaryotic Genome Annotation Pipeline v4.2 (10). The quality of the assemblies was evaluated by Quast v5.0.2 (11) (Table 1). The average nucleotide identity (ANIb) was calculated using pyani v0.2.9 (12), and in silico DNA-DNA hybridization (isDDH) estimates were determined using the Genome-to-Genome Distance Calculator v2.1, formula 2 (http://ggdc.dsmz.de/ggdc.php). GFF files produced by Prokka v1.13.3 (13) were used as input for Roary v3.13.0 (14) to generate a core gene alignment at 55% protein identity cutoff (parameter -i 55). Gblocks v0.91b (15) was used to remove ambiguous alignments and phylogenetically uninformative positions (parameters: minimum length of a block was set to minimum, and positions where at least 50% of the sequences had a gap were treated as gap positions). The maximum-likelihood method in RAxML-NG v0.9.0 (16) was used to infer a phylogenetic tree from the alignment under the GTR+G substitution model with 100 bootstrap replicates. Default parameters were used for all software except where otherwise stated.
TABLE 1.
Characteristics and accession numbers of the six thermophilic Campylobacter genome sequences
| Parameter | Data for strain: |
|||||
|---|---|---|---|---|---|---|
| Campylobacter sp. CW4087 (2018MI34) | Campylobacter sp. CW4409 (TTU-622) | Campylobacter sp. CW4516 (TTU_617) | C. novaezeelandiae CW4519 (TTU_619) | C. novaezeelandiae CW4600 (TTU_621) | Campylobacter sp. CW4073 (2018MI35) | |
| No. of reads | 1,476,472 | 205,168 | 916,870 | 1,195,800 | 164,400 | 1,003,376 |
| No. of bases (Mbp) | 333.4 | 47.5 | 220.8 | 266.6 | 34.4 | 233 |
| No. of contigs (≥500 bp) | 80 | 215 | 49 | 123 | 369 | 51 |
| k-mer coverage (×) | 108 | 14 | 69 | 84 | 11 | 77 |
| Nucleotide coverage (×)a | 199 | 26 | 127 | 154 | 20 | 142 |
| Genome assembly size (bp) (contigs ≥ 500 bp) | 1,487,666 | 1,540,934 | 1,483,241 | 1,494,070 | 1,419,279 | 1,504,203 |
| N50 (bp) | 52,357 | 15,455 | 115,198 | 44,342 | 6,180 | 53,282 |
| No. of CDSs (total)b | 1,711 | 1,700 | 1,495 | 1,559 | 1,701 | 1,589 |
| GC content (%) | 26.81 | 26.75 | 26.73 | 27.44 | 27.65 | 28.32 |
| No. of 16S rRNA genes | 1 | 1 | 1 | 1 | 1 | 2 |
| No. of 23S rRNA genes | 3 | 1 | 1 | 1 | 1 | 1 |
| No. of tRNA genes | 40 | 39 | 39 | 39 | 39 | 40 |
| No. of ncRNA genesc | 3 | 3 | 3 | 3 | 3 | 3 |
| No. of CRISPR/Cas genes | 1 | |||||
| SRA accession no. | SRR13349625 | SRR13349624 | SRR10695724 | SRR10084915 | SRR9894734 | SRR13349623 |
| GenBank accession no. | JAENKS000000000.1 | JAENKR000000000.1 | JAENKV000000000.1 | JAENKU000000000.1 | JAENKT000000000.1 | JAENKQ000000000.1 |
| BioProject accession no. | PRJNA686218 | PRJNA686218 | PRJNA296704 | PRJNA296704 | PRJNA296704 | PRJNA686218 |
| BioSample accession no. | SAMN17119060 | SAMN17119061 | SAMN13569984 | SAMN12715343 | SAMN12476126 | SAMN17119062 |
Nucleotide coverage = (Kmer coverage × read length)/(read length – Kmer length + 1), where Kmer length is the length of the longest k-mer used for the assembly (115 bp).
CDSs, coding DNA sequences.
ncRNA, noncoding RNA.
The Campylobacter sp. isolates clustered into three phylogenomic groups (Fig. 1). Strains CW4519 and CW4600 were identified as C. novaezeelandiae, having 98% ANIb and 84.1% isDDH to C. novaezeelandiae isolated in New Zealand (17). Campylobacter sp. strain CW4073 had C. taeniopygiae MIT10-5678 as its closest relative (91% ANIb, 42.3% isDDH), while Campylobacter sp. strains CW4087, CW4409, and CW4516 formed a unique cluster closest to C. novaezeelandiae (91% ANIb, 40.8% isDDH). The latter strains had the lowest GC content (26.8% ± 0.04% standard deviation [SD]) and genome size (1.50 Mbp ± 0.03 Mbp SD) among the Campylobacter spp. In all genomes, blaOXA gene variants commonly found in Campylobacter were identified. The lack of other antimicrobial resistance genes would suggest that these isolates are commensally prevalent in the environment, with a low health risk to humans and animals.
FIG 1.
Maximum-likelihood phylogenetic tree showing the placement of the two Canadian C. novaezeelandiae (in red bold font) and the four unclassified Campylobacter (in blue and green bold font) isolates described in this study within the 38 currently known Campylobacter species. The tree was based on the alignment of 135 core genes (110 kb) and generated using the RAxML GTR+G substitution model with 100 bootstrap replicates. Geneious Prime v2020.0.4 (Biomatters, Ltd.) was used to visualize the tree. Arcobacter butzleri ATCC 49616 was used as an outgroup and to root the tree.
Data availability.
Isolates CW4409, CW4516, CW4519, and CW4600 were sequenced as part of the GenomeTrakr Project at the Texas Department of State Health Services (Austin, TX), and isolates CW4087 and CW4073 were sequenced at the Center for Biotechnology & Genomics, Texas Tech University (Lubbock, TX). Alignment and newick files were deposited into the Dryad Digital Repository (https://doi.org/10.5061/dryad.qrfj6q5f2).
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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
Isolates CW4409, CW4516, CW4519, and CW4600 were sequenced as part of the GenomeTrakr Project at the Texas Department of State Health Services (Austin, TX), and isolates CW4087 and CW4073 were sequenced at the Center for Biotechnology & Genomics, Texas Tech University (Lubbock, TX). Alignment and newick files were deposited into the Dryad Digital Repository (https://doi.org/10.5061/dryad.qrfj6q5f2).