Skip to main content
Log in

Genomic and Phenotypic Characterization of a Lytic Bacteriophage CF1 Infecting the Multi-drug Resistant Bacterium Citrobacter freundii

  • Research Paper
  • Systems Biology
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

Citrobacter freundii is a facultative anaerobic and Gram-negative bacterium belonging to the Enterobacteriaceae family. Citrobacter spp. are opportunistic pathogens that are commonly found in water, soil, food, animals, or intestines in humans and warm-blooded animals and are a major concern for antibiotic resistance, especially C. freundii that exists in the farms, medical environment, and is difficult to eradicate. In this study, genomic and phenotypic characterization of a newly isolated bacteriophage CF1 capable of specifically infecting C. freundii is presented. The bacteriophage CF1 was isolated from sewage water sample near a livestock farm. The bacteriophage was morphologically and microbiologically characterized including one stage growth curve, and host range, on top of which the genome sequence of bacteriophage CF1 was determined. Microbiological characterization demonstrated that bacteriophage CF1 is stable in a range of pH4-9, and at the temperature below 70°C for 60 min. Genomic analysis revealed that bacteriophage CF1 is a new one with a double-stranded DNA of 50,339 bp, GC content of 42.65% that encodes 89 predicted open reading frames. Comparative analysis showed that bacteriophage CF1 is similar to C. freundii bacteriophage Stevie. Thus, this study provides genomic and phenotypic characteristics of a newly isolated bacteriophage, which can serve as a knowledge-base to use bacteriophage CF1 in the treatment of pathogenic C. freundii particularly with antimicrobial resistance in livestock industry.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

Data Availability

Nucleotide sequence accession number

The complete genome sequence and annotation of bacteriophage CF1 has been deposited to GenBank under the accession number KY694971.

Reference

  1. Shanks, R. M. Q., A. Dashiff, J. S. Alster, and D. E. Kadouri (2012) Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii. Arch. Microbiol. 194: 575–587.

    Article  CAS  Google Scholar 

  2. Janda, J. M., S. L. Abbott, W. K. Cheung, and D. F. Hanson (1994) Biochemical identification of citrobacteria in the clinical laboratory. J. Clin. Microbiol. 32: 1850–1854.

    Article  CAS  Google Scholar 

  3. Ocholi, R. A., J. C. Chima, E. M. Uche, and I. L. Oyetunde (1988) An epizootic infection of Citrobacter freundii in a guineapig colony: short communication. Lab. Anim. 22: 335–336.

    Article  CAS  Google Scholar 

  4. Kilonzo-Nthenge, A., S. N. Nahashon, F. Chen, and N. Adefope (2008) Prevalence and antimicrobial resistance of pathogenic bacteria in chicken and guinea fowl. Poult. Sci. 87: 1841–1848.

    Article  CAS  Google Scholar 

  5. Anyadoh-Nwadike, S. O., J. N. Okereke, R. Odah, O. Obijekwu, S. I. Okorondu, and K. O. Obasi (2015) Bacteriological quality of water contaminated with faecal wastes from livestock. Sci. J. Public Health. 3: 29–33.

    Google Scholar 

  6. Antonelli, A., M. M. D’Andrea, G. Vaggelli, J. D. Docquier, and G. M. Rossolini (2015) OXA-372, a novel carbapenem-hydrolysing class D beta-lactamase from a Citrobacter freundii isolated from a hospital wastewater plant. J. Antimicrob. Chemother. 70: 2749–2756.

    Article  CAS  Google Scholar 

  7. Liu, X., Y. Huang, X. Xu, Y. Zhao, Q. Sun, Z. Zhang, X. Zhang, Y. Wu, J. Wang, D. Zhou, X. An, G. Pei, Y. Wang, Z. Mi, Z. Yin, and Y. Tong (2016) Complete genome sequence of multidrug-resistant Citrobacter freundii strain P10159, isolated from urine samples from a patient with esophageal carcinoma. Genome Announc. 4: e01754–15.

    PubMed  PubMed Central  Google Scholar 

  8. Khadke, S. K., J. H. Lee, J. T. Woo, and J. Lee (2019) Inhibitory effects of honokiol and magnolol on biofilm formation by Acinetobacter baumannii.Biotechnol. Bioprocess Eng. 24: 359–365.

    Article  CAS  Google Scholar 

  9. Chen, Y., Z. Zhou, Y. Jiang, and Y. Yu (2011) Emergence of NDM-1-producing Acinetobacter baumannii in China. J. Antimicrob. Chemother. 66: 1255–1259.

    Article  CAS  Google Scholar 

  10. Cho, S. W., J. Yang, S. Park, B. Kim, and S. W. Seo (2019) Complete genome sequence of lactic acid bacterium Pediococcus acidilactici strain ATCC 8042, an Autolytic anti-bacterial peptidoglycan hydrolase producer. Biotechnol. Bioprocess Eng. 24: 483–487.

    Article  CAS  Google Scholar 

  11. Joo, H. S. (2018) Immobilized lipid affinity capture for antimicrobial peptides screening. Biotechnol. Bioprocess Eng. 23: 598–604.

    Article  CAS  Google Scholar 

  12. Lee, D., Y. Seo, M. S. Khan, J. Hwang, Y. Jo, J. Son, K. Lee, C. Park, S. Chavan, A. A. Gilad, and J. Choi (2018) Use of nanoscale materials for the effective prevention and extermination of bacterial biofilms. Biotechnol. Bioprocess Eng. 23: 1–10.

    Article  Google Scholar 

  13. Liao, X., L. Fang, L. Li, J. Sun, X. Li, M. Chen, H. Deng, Q. Yang, X. Li, and Y. Liu (2015) Characterization of chromosomal qnrB and ampC alleles in Citrobacter freundii isolates from different origins. Infect. Genet. Evol. 35: 214–220.

    Article  CAS  Google Scholar 

  14. Kim, M. K. (2019) Staphylococcus aureus toxins: from their pathogenic roles to anti-virulence therapy using natural products. Biotechnol. Bioprocess Eng. 24: 424–435.

    Article  CAS  Google Scholar 

  15. Guentzel, M. N. (1996) Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter, and Proteus. In: S. Baron (ed.). Medical Microbiology. The University of Texas Medical Branch at Galveston, Galveston, TX, USA.

    Google Scholar 

  16. Nilsson, A. S. (2014) Phage therapy—constraints and possibilities. Ups J. Med. Sci. 119: 192–198.

    Article  Google Scholar 

  17. Park, J., G. M. Lee, D. Kim, D. H. Park, and C. S. Oh (2018) Characterization of the lytic bacteriophage phiEaP-8 effective against both Erwinia amylovora and Erwinia pyrifoliae causing severe diseases in apple and pear. Plant Pathol. J. 34: 445–450.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Millman, J. M., K. Waits, H. Grande, A. R. Marks, J. C. Marks, L. B. Price, and B. A. Hungate (2013) Prevalence of antibiotic-resistant E. coli in retail chicken: comparing conventional, organic, kosher, and raised without antibiotics. F1000Res. 2: 155.

    Article  Google Scholar 

  19. Chaudhry, W. N., I. U. Haq, S. Andleeb, and I. Qadri (2014) Characterization of a virulent bacteriophage LK1 specific for Citrobacter freundii isolated from sewage water. J. Basic Microbiol. 54: 531–541.

    Article  CAS  Google Scholar 

  20. Hockett, K. L. and D. A. Baltrus (2017) Use of the soft-agar overlay technique to screen for bacterially produced inhibitory compounds. J. Vis Exp. 119: e55064.

    Google Scholar 

  21. Pajunen, M., S. Kiljunen, and M. Skurnik (2000) Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7. J. Bacteriol. 182: 5114–5120.

    Article  CAS  Google Scholar 

  22. Ellis, E. L. and M. Delbrück (1939) The growth of bacteriophage. J. Gen. Physiol. 22: 365–384.

    Article  CAS  Google Scholar 

  23. Boulanger, P. (2009) Purification of bacteriophages and SDS-PAGE analysis of phage structural proteins from ghost particles. Methods Mol. Biol. 502: 227–238.

    Article  CAS  Google Scholar 

  24. Deveau, H., M. R. Van Calsteren, and S. Moineau (2002) Effect of exopolysaccharides on phage-host interactions in Lactococcus lactis.Appl. Environ. Microbiol. 68: 4364–4369.

    Article  CAS  Google Scholar 

  25. Brettin, T., J. J. Davis, T. Disz, R. A. Edwards, S. Gerdes, G. J. Olsen, R. Olson, R. Overbeek, B. Parrello, G. D. Pusch, M. Shukla, J. A. Thomason 3rd, R. Stevens, V. Vonstein, A. R. Wattam, and F. Xia (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.

    Article  Google Scholar 

  26. Edgar, R. C. (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 26: 2460–2461.

    Article  CAS  Google Scholar 

  27. Sonnhammer, E. L. and G. Ostlund (2015) InParanoid 8: orthology analysis between 273 proteomes, mostly eukaryoticn Nucleic Acids Res. 43: D234–D239.

    Article  CAS  Google Scholar 

  28. Buchfink, B., C. Xie, and D. H. Huson (2015) Fast and sensitive protein alignment using DIAMOND. Nat. Methods. 12: 59–60.

    Article  CAS  Google Scholar 

  29. Craigie, J. and C. H. Yen (1938) The demonstration of types of B. Typhosus by means of preparations of type II Vi phage: I. principles and technique. Can. Public Health J. 29: 448–46

    Google Scholar 

  30. Brussow, H. and F. Desiere (2001) Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages. Mol. Microbiol. 39: 213–222.

    Article  CAS  Google Scholar 

  31. Young, R. (2014) Phage lysis: three steps, three choices, one outcome. J. Microbiol. 52: 243–258.

    Article  CAS  Google Scholar 

  32. Oliveira, H., G. Pinto, A. Oliveira, C. Oliveira, M. A. Faustino, Y. Briers, L. Domingues, and J. Azeredo (2016) Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates. Appl. Microbiol. Biotechnol. 100: 10543–10553.

    Article  CAS  Google Scholar 

  33. Li, E., Z. Yin, Y. Ma, H. Li, W. Lin, X. Wei, R. Zhao, A. Jiang, J. Yuan, and X. Zhao (2016) Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans.Sci. Rep. 6: 34300.

    Article  CAS  Google Scholar 

  34. Yang, Y., S. Lu, W. Shen, X. Zhao, M. Shen, Y. Tan, G. Li, M. Li, J. Wang, F. Hu, and S. Le (2016) Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa.Sci. Rep. 6: 38795.

    Article  CAS  Google Scholar 

  35. Hyman, P. and S. T. Abedon (2010) Bacteriophage host range and bacterial resistance. Adv. Appl. Microbiol. 70: 217–248.

    Article  CAS  Google Scholar 

  36. Hamdi, S., G. M. Rousseau, S. J. Labrie, R. S. Kourda, D. M. Tremblay, S. Moineau, and K. B. Slama (2016) Characterization of five Podoviridae phages infecting Citrobacter freundii.Front. Microbiol. 7: 1023.

    Article  Google Scholar 

  37. Mizuno, C. M., M. Krupovic, L. Debarbieux, and D. R. Roach (2018) Comparative genomics and phylogeny unveil lineage diversification of Citrobacter rodentium polyvalent bacteriophages. bioRxiv. 248153.

    Google Scholar 

  38. Kim, Y., S. Ko, Y. E. Yeon, J. Lim, B. K. Han, H. Kim, J. K. Ahn, and D. Kim (2018) Draft genome sequence of lytic bacteriophage CF1 infecting Citrobacter freundii isolates. Korean J. Microbiol. 54: 79–80.

    Google Scholar 

  39. Chaudhari, N. M., V. K. Gupta, and C. Dutta (2016) BPGA- an ultra-fast pan-genome analysis pipeline. Sci. Rep. 6: 24373.

    Article  CAS  Google Scholar 

  40. Shaw, J. P., C. A. Aviles Medina, Y. Chen, A. J. Luna, A. C. Hernandez, and G. F. Kuty Everett (2015) Complete genome of Citrobacter freundii siphophage Stevie. Genome Announc. 3: e01434–14.

    Article  Google Scholar 

Download references

Acknowledgements

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

Author information

Authors and Affiliations

Authors

Contributions

Y. K. and D. K. designed the study. Y. K. and Y. E. Y. performed experiments. S. K. performed computational analysis, and data analysis. Y. K., S. K., Y. E. Y., B. K. H., H. K., C. S. O., Y. R., and D. K. wrote the manuscript with contributions from all other authors.

Corresponding authors

Correspondence to Jeong Keun Ahn or Donghyuk Kim.

Additional information

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, Y., Ko, S., Yeon, Y.E. et al. Genomic and Phenotypic Characterization of a Lytic Bacteriophage CF1 Infecting the Multi-drug Resistant Bacterium Citrobacter freundii. Biotechnol Bioproc E 25, 384–393 (2020). https://doi.org/10.1007/s12257-019-0505-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-019-0505-8

Keywords

Navigation