Antimicrobial resistance is a significant issue, and it threatens the prevention and effective treatment of a range of bacterial infections. Here, we report the whole-genome sequence of the multidrug-resistant isolate Serratia sp. strain HRI. A hybrid assembly was created using sequences from a first (MiSeq) and second (PacBio) sequencing run. This work is imperative for understanding antimicrobial resistance and adds to the knowledge base for combating multidrug-resistant bacteria.
ABSTRACT
Antimicrobial resistance is a significant issue, and it threatens the prevention and effective treatment of a range of bacterial infections. Here, we report the whole-genome sequence of the multidrug-resistant isolate Serratia sp. strain HRI. A hybrid assembly was created using sequences from a first (MiSeq) and second (PacBio) sequencing run. This work is imperative for understanding antimicrobial resistance and adds to the knowledge base for combating multidrug-resistant bacteria.
ANNOUNCEMENT
The emergence of antibiotic resistance and the threat of a postantibiotic era has promoted research into antimicrobial resistance. Serratia sp. strain HRI is of interest, as the MICs of this bacterium suggest that it has high disinfectant resistance capabilities.
Serratia sp. HRI was first isolated from a bottle of didecyldimethylammonium chloride (DDAC)-based disinfectant (1%) at room temperature at the University of the Free State (UFS) in South Africa.
For both isolation and sequencing procedures, Serratia sp. HRI was cultivated on tryptic soy broth agar at 37°C. Total genomic DNA was isolated by manual extraction, as done by Labuschagne and Albertyn (1). The isolate was identified by sequencing the 16S rRNA gene and comparing it to other isolates on the EzBioCloud database (2–5); the closest related type strain was Serratia marcescens subsp. marcescens ATCC 13880. The type strain was subsequently obtained from the American Type Culture Collection, and MICs were determined using methods described by Wiegand and coworkers (6) (Table 1).
TABLE 1.
MICs for Serratia sp. strain HRI and the closest related type strain for a variety of quaternary ammonium compound disinfectants
| Disinfectant | MIC (μg/ml) of: |
|
|---|---|---|
| Serratia marcescens subsp. marcescens ATCC 13880 | Serratia sp. strain HRI | |
| Virukill | 2.8671 | 43.12494 |
| Benzalkonium chloride | 17.227 | 1,837.5 |
| DDAC | 12.79 | 2,873 |
Genomic DNA was sent to the UFS Next Generation Sequencing Unit for library preparation and whole-genome sequencing on the Illumina MiSeq platform. An index PCR was done to attach dual indices and Illumina sequencing adapters using the Nextera XT index kit, as per the manufacturer’s instructions. Library concentrations were equalized, pooled, denatured, and transferred to a V3 sequencing cartridge. The MiSeq run proceeded for 600 cycles (2 × 150-bp paired-end reads) and generated 376,021 reads.
A second round of sequencing was conducted at Inqaba Biotechnical Industries (Pretoria, South Africa) to improve sequence quality. Sequencing was done using single-molecule real-time (SMRT) technology using the Sequel PacBio equipment in CCS mode with 50× coverage. Genomic DNA was sheared to ∼10-kb fragments using a Covaris g-TUBE. Thereafter, the sample was end repaired and SMRTbell adaptor ligated. Finally, primer annealing and polymerase binding were completed before sequencing on the PacBio Sequel system, as per the manufacturer’s instructions, generating 180,789 reads.
A hybrid assembly was created using sequences from the MiSeq and PacBio sequencing runs. The rpoB gene of Serratia sp. HRI was annotated using a previous sequencing run by Rapid Annotations using Subsystems Technology (RAST), and a BLASTn search of this sequence revealed that Serratia marcescens CAV1492 (GenBank accession number CP011642.1) was the closest relative (maximum score, 7,424; total score, 7,424; query cover, 100%; E value, 0.0; percent identity, 99.93%) (7–10). Thus, the genome of CAV1492 was utilized as a reference within the hybrid assembly. Raw reads were quality assessed using FastQC v0.11.8; default parameters were used for all software unless otherwise specified (11), followed by quality filtering using PRINSEQ v0.20.3 (12). Reads obtained were assembled using SPAdes via the hybrid assembly pipeline, and contigs were evaluated using QUAST (13, 14). The assembled genome is 5,885,723 bp long, with a G+C content of 58.4% originating from 105 contigs. This assembly has an N50 value of 611,273 bp, an L50 value of 4, and 5,935 coding sequences with 123 RNAs.
Data availability.
The assembled genome has been deposited into NCBI as BioProject PRJNA580358 and BioSample SAMN13155787, in the Sequence Read Archive under accession numbers SRX7885200 and SRX7109663 for the MiSeq and PacBio data, respectively, and in the DDBJ/ENA/GenBank database under the accession number WIXF00000000. The version described in this paper is the second version, WIXF02000000.
ACKNOWLEDGMENTS
This work was partially funded by the National Research Foundation of South Africa and the Department of Microbial, Biochemical and Food Biotechnology at the University of the Free State.
REFERENCES
- 1.Labuschagne M, Albertyn J. 2007. Cloning of an epoxide hydrolaseencoding gene from Rhodotorula mucilaginosa and functional expression in Yarrowia lipolytica. Yeast 24:69–78. doi: 10.1002/yea.1437. [DOI] [PubMed] [Google Scholar]
- 2.Wilson K, Blitchington R, Greene RC. 1990. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol 28:1942–1946. doi: 10.1128/JCM.28.9.1942-1946.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Rainey FA, Ward-Rainey N, Kroppenstedt RM, Stackebrandt E. 1996. The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46:1088–1092. doi: 10.1099/00207713-46-4-1088. [DOI] [PubMed] [Google Scholar]
- 4.Turner S, Pryer K, Miao V, Palmer J. 1999. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot Microbiol 46:327–338. doi: 10.1111/j.1550-7408.1999.tb04612.x. [DOI] [PubMed] [Google Scholar]
- 5.Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67:1613–1617. doi: 10.1099/ijsem.0.001755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wiegand I, Hilpert K, Hancock RE. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163–175. doi: 10.1038/nprot.2007.521. [DOI] [PubMed] [Google Scholar]
- 7.Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. doi: 10.1186/1471-2164-9-75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. 2014. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res 42:D206–D214. doi: 10.1093/nar/gkt1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA, Stevens R, Vonstein V, Wattam AR, Xia F. 2015. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5:8365. doi: 10.1038/srep08365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. doi: 10.1089/10665270050081478. [DOI] [PubMed] [Google Scholar]
- 11.Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc.
- 12.Schmieder R, Edwards R. 2011. Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864. doi: 10.1093/bioinformatics/btr026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.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]
- 14.Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086. [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
The assembled genome has been deposited into NCBI as BioProject PRJNA580358 and BioSample SAMN13155787, in the Sequence Read Archive under accession numbers SRX7885200 and SRX7109663 for the MiSeq and PacBio data, respectively, and in the DDBJ/ENA/GenBank database under the accession number WIXF00000000. The version described in this paper is the second version, WIXF02000000.