A Klebsiella pneumoniae strain isolated from houseflies in a trash disposal truck in the United States was resistant to colistin, a last-resort drug for treating infections caused by multidrug-resistant Gram-negative bacteria. Complete genome sequencing resulted in a total genome size of 5,337,408 bp for this isolate with a plasmid of 224,442 bp.
ABSTRACT
A Klebsiella pneumoniae strain isolated from houseflies in a trash disposal truck in the United States was resistant to colistin, a last-resort drug for treating infections caused by multidrug-resistant Gram-negative bacteria. Complete genome sequencing resulted in a total genome size of 5,337,408 bp for this isolate with a plasmid of 224,442 bp.
ANNOUNCEMENT
Klebsiella pneumoniae is an opportunistic pathogen and a leading cause of nosocomial infections (1). The emergence of K. pneumoniae resistance to colistin, a last-resort antimicrobial used to treat carbapenem-resistant Enterobacteriaceae, has led to fear of pan-drug resistance.
Houseflies have been linked to various bacterial infections as a mechanical vector, but their role in the dissemination of antimicrobial resistance, particularly colistin resistance, remains poorly understood (2–5). Here, we report the complete genome sequence of a colistin-resistant K. pneumoniae strain isolated from houseflies trapped in a trash disposal truck in Auburn, Alabama.
Colistin selective medium CHROMagar COL-APSE (CHROMagar, Paris, France) was used for the isolation of colistin-resistant Enterobacteriaceae from fly homogenates at 37°C for 18 h. A mucoid, metallic blue colony suggestive of coliform from Fly ID AU-87 was selected and grown in lysogeny broth supplemented with 3.5 μg/ml colistin sulfate (Sigma-Aldrich, St. Louis, MO) at 37°C for 18 h. A volume of 400 μl from the cultured isolate was used for extraction of genomic DNA with the High Pure PCR template preparation kit (Roche Diagnostic, Indianapolis, IN) according to the manufacturer’s instructions. The isolate was characterized as K. pneumoniae (isolate Kp8701) based on PCR amplification of the full-length 16S rRNA gene using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) as previously described, (6) followed by Sanger sequencing (Elim Biopharmaceuticals, Inc., San Francisco, CA). A BLAST search of the obtained sequence was conducted using the NCBI Nucleotide Basic Local Alignment Tool (BLASTn) database.
The single-molecule real-time (SMRT) sequencing library was constructed with the SMRTbell prep kit 1.0 (Pacific Biosciences, Menlo Park, CA) and performed on the PacBio Sequel platform (Oebiotech, Shanghai, China). Reads with low quality were removed with SMRT Analysis 2.3.0 (Pacific Biosciences), and the filtered reads were assembled to generate one contig without gaps using FALCON 1.1 (7). The corrected subreads were used as an input for the single-pass read accuracy improver software SPRAI 0.9.9.23 (8) to maximize the continuity of the assembled contig. Genes were predicted and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (9, 10). Default parameters were used for all software.
SMRT sequencing of Kp8701 resulted in three data sets with roughly 3.65 Gbp total bases available for de novo assembly. There were 397,836 total reads with a minimum contig length of 51 bp and a maximum length of 70,234 bp (average length, 9,162 bp). Assuming an average genome size of 5 Mbp for K. pneumoniae, the total bases obtained from SMRT sequencing theoretically had 730-fold coverage of the genome.
The de novo assembly of the genome is composed of a single circular chromosome of 5,337,408 bp with a GC content of 57.42% and a circular plasmid (pKp8701) of 224,442 bp with a GC content of 52.37%. There was a total of 5,569 genes predicted in the genome of Kp8701, consisting of 5,319 protein-coding genes, 131 RNA-coding genes, and 119 pseudogenes. The RNA-coding genes included 90 sets of tRNA genes encoding all 21 amino acids, 25 rRNA genes (9 sets for 5S rRNA, 8 sets for 16S rRNA, and 8 sets for 23S rRNA), and 16 noncoding RNA (ncRNA) genes.
Data availability.
The raw sequencing reads were deposited in the Sequence Read Archive (SRA) under accession number SRR11212804 (BioProject number PRJNA609348). The de novo genome assemblies of the K. pneumoniae Kp8701 chromosome and plasmid were deposited in GenBank under accession numbers CP049604 and CP049605, respectively.
ACKNOWLEDGMENTS
This research was supported by a USDA-ARS program (58-6040-9-017) and by the Alabama Agricultural Experimental Station and the USDA National Institute of Food and Agriculture, Hatch project (ALA052-1-17026).
REFERENCES
- 1.Nordmann P, Cuzon G, Naas T. 2009. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis 9:228–236. doi: 10.1016/S1473-3099(09)70054-4. [DOI] [PubMed] [Google Scholar]
- 2.Onwugamba FC, Fitzgerald JR, Rochon K, Guardabassi L, Alabi A, Kühne S, Grobusch MP, Schaumburg F. 2018. The role of “filth flies” in the spread of antimicrobial resistance. Travel Med Infect Dis 22:8–17. doi: 10.1016/j.tmaid.2018.02.007. [DOI] [PubMed] [Google Scholar]
- 3.Iwasa M, Makino S-I, Asakura H, Kobori H, Morimoto Y. 1999. Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan. J Med Entomol 36:108–112. doi: 10.1093/jmedent/36.1.108. [DOI] [PubMed] [Google Scholar]
- 4.Davari B, Kalantar E, Zahirnia A, Moosa-Kazemi S. 2010. Frequency of resistance and susceptible bacteria isolated from houseflies. Iran J Arthropod Borne Dis 4:50–55. [PMC free article] [PubMed] [Google Scholar]
- 5.Bahrndorff S, de Jonge N, Skovgård H, Nielsen JL. 2017. Bacterial communities associated with houseflies (Musca domestica L.) sampled within and between farms. PLoS One 12:e0169753. doi: 10.1371/journal.pone.0169753. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703. doi: 10.1128/jb.173.2.697-703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Chin C-S, Peluso P, Sedlazeck FJ, Nattestad M, Concepcion GT, Clum A, Dunn C, O’Malley R, Figueroa-Balderas R, Morales-Cruz A, Cramer GR, Delledonne M, Luo C, Ecker JR, Cantu D, Rank DR, Schatz MC. 2016. Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods 13:1050–1054. doi: 10.1038/nmeth.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Miyamoto M, Motooka D, Gotoh K, Imai T, Yoshitake K, Goto N, Iida T, Yasunaga T, Horii T, Arakawa K, Kasahara M, Nakamura S. 2014. Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes. BMC Genomics 15:699. doi: 10.1186/1471-2164-15-699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O’Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res 46:D851–D860. doi: 10.1093/nar/gkx1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
The raw sequencing reads were deposited in the Sequence Read Archive (SRA) under accession number SRR11212804 (BioProject number PRJNA609348). The de novo genome assemblies of the K. pneumoniae Kp8701 chromosome and plasmid were deposited in GenBank under accession numbers CP049604 and CP049605, respectively.