Citrobacter braakii and Citrobacter freundii are Gram-negative opportunistic pathogens associated with many infectious diseases, including septicemia, in humans and animals. Here, we report the draft genome sequences of seven C. braakii strains and one C. freundii strain isolated from Canadian wastewater treatment facilities.
ABSTRACT
Citrobacter braakii and Citrobacter freundii are Gram-negative opportunistic pathogens associated with many infectious diseases, including septicemia, in humans and animals. Here, we report the draft genome sequences of seven C. braakii strains and one C. freundii strain isolated from Canadian wastewater treatment facilities.
ANNOUNCEMENT
Citrobacter braakii and Citrobacter freundii, two of the 15 species in the genus Citrobacter, are Gram-negative coliform bacteria in the Enterobacteriaceae family (http://www.bacterio.net/citrobacter.html). They can be found in soil, water, sewage, and the intestinal tracts of animals and humans. They are opportunistic pathogens, causing infections of the urinary, gastrointestinal, and pulmonary systems, as well as blood infections, often nosocomial in nature; they are a major cause of neonatal meningitis (1, 2). Citrobacter freundii generally exhibits high antimicrobial resistance to penicillin and third-generation cephalosporins (3), which harbor a β-lactam ring.
This article reports the draft genome sequences of seven C. braakii strains and one C. freundii strain isolated from pre- or posttreated samples collected at three Canadian wastewater treatment facilities. Strains HH1, HH5, HH6, HH8, HH10, and HH11 were isolated using a Compendium of analytical methods of Health Canada method (MFHPB-19) (4) for the isolation of Escherichia coli. Strains HH7 and HH9 were isolated using a Compendium of analytical methods of Health Canada method (MFLP-75) (5) for the isolation of Salmonella species. All isolates were confirmed to be Citrobacter at the genus level by a combination of the API 20E biochemical test (bioMérieux, Canada, Inc.), Biolog (carbohydrate analysis), and Sherlock (fatty acid analysis) (Midi, Inc., Newark, DE, USA) microbial identification systems (Table 1). The isolates were further identified as C. braakii or C. freundii by the API 20E test. Species identifications were confirmed by comparing the draft genome sequences reported here (Table 1) with the reference genome of each species.
TABLE 1.
Sequencing and assembly metrics for the strains used in this study
| Strain | Species | Assembly size (bp) | No. of contigs | N50 value (bp) | GC content (%) | No. of CDS | Coverage (×) | No. of reads | Assembly accession no. | SRA accession no. |
|---|---|---|---|---|---|---|---|---|---|---|
| HH1 | C. braakii | 4,824,576 | 18 | 803,039 | 52.1 | 4,638 | 99 | 1,643,354 | GCA_002923765 | SRR6664068 |
| HH5 | C. braakii | 4,873,670 | 21 | 757,685 | 52.0 | 4,664 | 63 | 673,281 | GCA_004331575 | SRR8643105 |
| HH6 | C. braakii | 5,128,900 | 54 | 254,837 | 51.9 | 4,958 | 91 | 1,064,342 | GCA_004331585 | SRR8643104 |
| HH7 | C. braakii | 4,825,329 | 15 | 803,041 | 52.1 | 4,577 | 90 | 848,821 | GCA_004331545 | SRR8643107 |
| HH8 | C. braakii | 5,401,766 | 77 | 235,111 | 51.9 | 5,236 | 71 | 819,903 | GCA_004331535 | SRR8643106 |
| HH9 | C. braakii | 5,001,420 | 26 | 435,834 | 52.1 | 4,811 | 67 | 790,355 | GCA_004331635 | SRR8643109 |
| HH10 | C. braakii | 5,326,488 | 84 | 210,999 | 51.8 | 5,222 | 70 | 852,877 | GCA_004331445 | SRR8643108 |
| HH11 | C. freundii | 5,449,209 | 97 | 183,552 | 51.7 | 5,396 | 79 | 1,031,234 | GCA_004331565 | SRR8643110 |
Isolates were kept at −80°C in tryptic soy broth with 25% glycerol before sequencing. Genomic DNA was extracted from overnight cultures grown at 37°C on tryptic soy agar using the DNeasy blood and tissue DNA purification kit (catalog number 69504; Qiagen, Inc., Toronto, Ontario, Canada) and quantified using a Qubit 3.0 fluorometer (Life Technologies). Sequencing libraries were constructed using Nextera XT DNA sample preparation kits (Illumina, Inc., San Diego, CA), and the quality of libraries was analyzed using a Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced with a MiSeq platform (Illumina, Inc.) using 300-bp v3 reagent kits.
Sequencing adapter sequences and low-quality bases were trimmed (BBduk), and overlapping pairs were merged (BBMerge) using the BBTools v37.68 software suite (https://jgi.doe.gov/data-and-tools/bbtools/). The remaining reads were then assembled de novo with Unicycler v0.4.4 (6) running SPAdes v3.11.1 (7) and then polished with Pilon v1.22 (8). Other programs used by Unicycler were BLAST v2.7.1+ (9), Bowtie 2 v2.3.4 (10), and SAMtools v1.6 (11). The parameters used for each step of the assembly pipeline are detailed at https://github.com/duceppemo/bacteria_genome_assembly. Gene predictions and annotations were performed using the NCBI Prokaryotic Genome Annotation Pipeline (12). Sequencing and assembly metrics are summarized in Table 1. Antimicrobial resistance genes were detected using ResFinder v3.1.0 (13), with default parameters.
The median total length, protein count (number of coding sequences [CDS]), and GC content of the C. braakii genome assemblies in the NCBI are 5.1 Mb, 4,822, and 52.1%, respectively. Similarly, the median values of the seven C. braakii strains presented here are 5.1 Mb, 4,672, and 52.0%, respectively. The median total length, protein count (number of CDS), and GC content of the C. freundii genome assemblies on NCBI are 5.3 Mb, 4,991, and 51.7%, respectively, which are similar to those (5.499 Mb, 5,396, and 51.7%, respectively) of the C. freundii strain analyzed in this study (Table 1). All isolates were predicted to be resistant to β-lactam antibiotics.
Data availability.
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers PQWA00000000, SJSJ00000000, SJSI00000000, SJSH00000000, SJSG00000000, SJSF00000000, SJSE00000000, and SJSD00000000 for strains HH1, HH5, HH6, HH7, HH8, HH9, HH10, and HH11, respectively. The versions described in this paper are versions PQWA01000000, SJSJ01000000, SJSI01000000, SJSH01000000, SJSG01000000, SJSF01000000, SJSE01000000, and SJSD01000000, respectively. The Illumina raw sequencing reads are available in the NCBI Sequence Read Archive through the accession numbers found in Table 1.
ACKNOWLEDGMENTS
This work was funded by the Canadian Food Inspection Agency.
We thank Stephan Brière at the Ottawa Laboratory – Fallowfield, Canadian Food Inspection Agency, for conducting tests using the Biolog and Sherlock microbial identification systems.
REFERENCES
- 1.Doran TI. 1999. The role of Citrobacter in clinical disease of children: review. Clin Infect Dis 28:384–394. doi: 10.1086/515106. [DOI] [PubMed] [Google Scholar]
- 2.Badger JL, Stins MF, Kim KS. 1999. Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infect Immun 67:4208–4215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Barlow M, Hall BG. 2002. Origin and evolution of the AmpC β-lactamases of Citrobacter freundii. Antimicrob Agents Chemother 46:1190–1198. doi: 10.1128/AAC.46.5.1190-1198.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Health Canada. 2002. Enumeration of coliforms, faecal coliforms and E. coli in foods using the MPN method. Compendium of analytical methods, vol 2. Method MFHPB-19. Health Canada, Ottawa, Ontario, Canada. [Google Scholar]
- 5.Health Canada. 2016. Procedure for the isolation of Salmonella species by the modified semi-solid Rappaport-Vassiliadis (MSRV) method. Compendium of analytical methods, vol 3. Method MFLP-75. Health Canada, Ottawa, Ontario, Canada. [Google Scholar]
- 6.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]
- 7.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. doi: 10.1371/journal.pone.0112963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- 10.Langmead B, Salzberg S. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.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 (SAM) format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
- 13.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]
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 PQWA00000000, SJSJ00000000, SJSI00000000, SJSH00000000, SJSG00000000, SJSF00000000, SJSE00000000, and SJSD00000000 for strains HH1, HH5, HH6, HH7, HH8, HH9, HH10, and HH11, respectively. The versions described in this paper are versions PQWA01000000, SJSJ01000000, SJSI01000000, SJSH01000000, SJSG01000000, SJSF01000000, SJSE01000000, and SJSD01000000, respectively. The Illumina raw sequencing reads are available in the NCBI Sequence Read Archive through the accession numbers found in Table 1.