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Complete genome sequence of a commensal bacterium, Hafnia alvei CBA7124, isolated from human feces



Members of the genus Hafnia have been isolated from the feces of mammals, birds, reptiles, and fish, as well as from soil, water, sewage, and foods. Hafnia alvei is an opportunistic pathogen that has been implicated in intestinal and extraintestinal infections in humans. However, its pathogenicity is still unclear. In this study, we isolated H. alvei from human feces and performed sequencing as well as comparative genomic analysis to better understand its pathogenicity.


The genome of H. alvei CBA7124 comprised a single circular chromosome with 4,585,298 bp and a GC content of 48.8%. The genome contained 25 rRNA genes (9 5S rRNA genes, 8 16S rRNA genes, and 8 23S rRNA genes), 88 tRNA genes, and 4043 protein-coding genes. Using comparative genomic analysis, the genome of this strain was found to have 72 strain-specific singletons. The genome also contained genes for antibiotic and antimicrobial resistance, as well as toxin–antitoxin systems.


We revealed the complete genome sequence of the opportunistic gut pathogen, H. alvei CBA7124. We also performed comparative genomic analysis of the sequences in the genome of H. alvei CBA7124, and found that it contained strain-specific singletons, antibiotic resistance genes, and toxin–antitoxin systems. These results could improve our understanding of the pathogenicity and the mechanism behind the antibiotic resistance of H. alvei strains.


Hafnia alvei was first identified by Moller in 1954. It belongs to the family Enterobacteriaceae, and was isolated from the feces of mammals, birds, reptiles, and fish, as well as from soil, water, sewage, and foods [2]. H. alvei is a Gram-negative, rod-shaped, and facultative anaerobic bacterium. It is an opportunistic pathogen, and has been implicated in intestinal and extraintestinal infections in humans [2]. In addition, several strains of H. alvei have been known to produce acyl lactones and form biofilms [3]. Biofilm formation is considered an important virulence factor involved in bacterial attachment and settlement [4]. However, the pathogenesis and mechanisms of action of H. alvei are still not clear [5]. So far, 11 strains of H. alvei have been sequenced, and only three genomes of them were completed.

In this study, we isolated the strain, Hafnia alvei CBA7124, from human feces, and performed sequencing and comparative genomic analysis with other H. alvei strains in order to understand its pathogenicity. The complete genome sequence of H. alvei CBA7124 would improve our understanding of different strains of opportunistic infectious pathogens.


Bacterial strain and DNA preparation

The strain H. alvei CBA7124 was isolated from a fecal sample of 66-year old Korean female from Geochang, Republic of Korea. The fecal sample was cultured in a brain heart infusion agar (BD) in anaerobic conditions at 37 °C for 24 h. The isolate was transferred at least thrice in the same conditions. The cell morphology of the strain was examined using a scanning electron microscope (SEM). The strain was then preserved in 20% (v/v) glycerol at −80 °C. The genomic DNA of the isolated strain was extracted using the QuickGene DNA tissue kit S (Kurabo, Japan) and purified using the MG Genomic DNA purification kit (Doctor Protein, Korea). The quality and concentration of the extracted DNA were determined using 1%-agarose gel electrophoresis and a NanoDrop spectrophotometer (Nanodrop Technologies, UK).

Genome sequencing, assembly, and gene annotation

Whole genome sequencing was performed using Pacific Biosciences RS II (Pacific Biosciences, Menlo Park, USA) (Additional file 1: Table S1). A 20-kb sequencing library was constructed using SMRTbell™ Template Prep Kit and sequenced with P6 polymerase and C4 chemistry. The genome was assembled according to the protocol in the Hierarchical Genome Assembly Process version 2 with PacBio SMRT analysis version 2.3, and polishing was performed with Quiver. Identification of rRNA and tRNA genes was performed with the RNAmmer 1.21 [6] server and the tRNA scan-SE 1.21 [7], respectively. Functional genes were predicted and annotated using the SEED subsystems in the RAST server (rapid annotation using subsystem technology) [8, 9] and the COG (clusters of orthologous groups of proteins) databases [10]. The presence of CRISPRs was detected using the CRISPRfinder server [11]. PathogenFinder was used for predicting pathogenicity towards humans [12]. The ResFinder program was used to screen for antimicrobial resistance genes [13].

Comparative genomic analysis

Comparative genomic analysis was performed on 11 Hafnia alvei strains, ATCC 29926, ATCC 13337T, DSM 30099, FB1, HUMV-5920, DSM 30098, LE8, GB001, FDAARGOS_158, bta3-1, and ATCC 51873. The orthologous average nucleotide identity (orthoANI) algorithm was used to measure the phylogenetic distances between these strains [14]. Pan-genome orthologous groups (POGs) were identified using the EzBioCloud Comparative Genomics Database ( The heat map was clustered according to the presence or absence of genes [15].

Quality assurance

Before the genome sequencing, the identity of the H. alvei CBA7124 strain was verified through 16S rRNA gene sequencing and cell morphology analysis (Additional file 1: Figure S1). In addition, the identity of the strain CBA7124 was confirmed through analysis of the 16S rRNA gene obtained after genome sequencing. In addition, we used the orthoANI values with the genome sequence of H. alvei.

Results and discussion

Genome characteristics

The analysis of the whole genome sequence of H. alvei CBA7124 revealed a single circular chromosome with 4,585,298 bp, after quality control of 150,292 raw reads with an average read length of 5885 bp (Table 1). The genome coverage was found to be 168.69-fold and the GC content was 48.8%. The genome contained 25 rRNA genes (9 5S rRNA genes, 8 16S rRNA genes, and 8 23S rRNA genes) and 88 tRNA genes. Four confirmed CRISPRs (with at least three motifs and at least two exactly identical direct repeats) and four questionable CRISPRs (small CRISPRs or structures where the repeated motifs are not 100% identical) were found. The CRISPR-associated (Cas) proteins belong to the types I (Cas3), II (Cas1), IF (Csy1, Csy2, and Csy3 family), and IIB (Csy4 family), as confirmed from the SEED database. The strain had a 0.65% chance of being pathogenic, and was found to match with 28 pathogenic families. The genome contained 4043 protein-coding genes (CDSs) and 3838 genes were allotted to 18 COG functional categories. In the COG distribution, amino acid transport and metabolism (E; 341 ORFs), carbohydrate transport and metabolism (G; 314 ORFs), transcription (K; 299 ORFs), general function prediction only (R; 282 ORFs), and function unknown (S; 728 ORFs) were the major functional categories (Fig. 1). In the SEED subsystem distribution, carbohydrates (552 ORFs), amino acids and derivatives (430 ORFs), cofactors, vitamins, prosthetic groups, pigments (319 ORFs), and RNA metabolism (215 ORFs) were the abundant categories.

Table 1 Complete genome features of Hafnia alvei CBA7124
Fig. 1
figure 1

Circular genome map of Hafnia alvei CBA7124. From outer to inner rings, the individual circles indicate rRNAs and tRNAs, reverse CDSs, forward CDSs, GC skew, and GC ratio

Comparative genomic analysis

The genome of the H. alvei CBA7124 strain was compared with those of 11 other H. alvei strains. The orthoANI values of strain CBA7124 with ATCC 13337T, ATCC 29926, DSM 30099, FB1, HUMV-5920, DSM 30098, LE8, GB001, FDAARGOS_158, bta3-1, and ATCC 51873 were 99.1, 99.0, 97.8, 97.7, 95.8, 95.7, 94.3, 92.9, 82.6, 82.5, and 82.5%, respectively, indicating that the strain CBA7124 was closely related to the H. alvei strains ATCC 13337T and ATCC 29926 (Additional file 1: Figure S2). According to the heat map generated based on core pan-genome orthologous groups (POGs), the strain CBA7124 was clustered with H. alvei genomes of strains HUMV-5920 and DSM 30098, based on the presence or absence of genes (Fig. 2). Based on the POG comparison analysis of the 12 genomes, 72 strain-specific singletons, including “transposase for insertion sequence element IS200”, “protein SamB”, “protein RhsA”, and others, were identified in strain CBA7124 (Additional file 1: Table S2). The number of strain-specific POGs in the H. alvei genomes ranged from 72 to 354 (Additional file 1: Table S3). These results indicated that the genome of strain CBA7124 was separate from, but highly homologous to, that of other H. alvei genomes.

Fig. 2
figure 2

Heat map of strain CBA7124 with the related Hafnia alvei strains, constructed based on the presence or absence of POGs. The presence and absence of POGs are indicated by blue and red, respectively

Antibiotic and antimicrobial resistance genes

In the genome of strain CBA7124, 12 kinds of subsystems were found to be associated with the subcategory “resistance to antibiotics and toxic compounds” on the SEED database. These subsystems included 6 mdtABCD multidrug resistance clusters, 2 lysozyme inhibitors, 1 multiple antibiotic resistance (MAR) locus, 6 copper homeostasis, 2 bile hydrolysis, 6 cobalt-zinc-cadmium resistance, 3 multidrug resistance tripartite systems found in gram negative bacteria, 4 resistance to fluoroquinolones, 3 arsenic resistance, 7 copper homeostasis: copper tolerance, 3 beta-lactamase, and 10 multidrug resistance efflux pumps (Additional file 1: Table S4). In addition, the antimicrobial resistance gene, blaACC-3, was also found to be associated with the beta-lactam resistance AmpC-type gene from the ResFinder server.

Toxin–antitoxin systems

Several toxin–antitoxin (TA) systems were annotated in the genome of Hafnia alvei for stabilization, based on the SEED database. We detected the TA systems of yefM/yoeB, ccdAB, parDE, and ygiUT in H. alvei CBA7124, which have been reported to inhibit replication by inhibiting DNA gyrase and translation. Among them, the antitoxin of yefM is involved in the formation of biofilms [16] and the ability of the biofilm-forming bacteria to withstand antibiotics; therefore, it has a significant impact on therapy and patient care [17]. In addition, the overproduction of the toxin of yoeB is known to inhibit the growth of E. coli [18, 19].

Future directions

We described a genome sequence of H. alvei, a known opportunistic pathogen isolated from a Korean fecal sample. This genome was found to have strain-specific singletons through comparative genomic analysis with the other H. alvei strains. In addition, this genome contained antibiotic and antimicrobial resistance genes, toxin–antitoxin systems, and several Cas proteins against pathogen defence systems. This information provides new insights into the multidrug resistance, biofilm formation, and antibacterial activity of H. alvei for surviving in the intestinal environment. Furthermore, it can help us comprehend the pathogenesis and mechanisms of action of H. alvei. The data presented in this report provide important genetic information and a framework for further research. However, further in vivo studies are needed to characterize the pathogenicity of H. alvei.



protein coding gene


clusters of orthologous groups


pan-genome orthologous group


orthologous average nucleotide identity


rapid annotation using subsystem technology




  1. McBee ME, Schauer DB. The genus Hafnia. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes. New York: Springer; 2006. p. 215–8.

    Chapter  Google Scholar 

  2. Janda JM, Abbott SL. The genus Hafnia: from soup to nuts. Clin Microbiol Rev. 2006;19:12–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Viana ES, Campos ME, Ponce AR, Mantovani HC, Vanetti MC. Biofilm formation and acyl homoserine lactone production in Hafnia alvei isolated from raw milk. Biol Res. 2009;42:427–36.

    CAS  PubMed  Google Scholar 

  4. Vivas J, Padilla D, Real F, Bravo J, Grasso V, Acosta F. Influence of environmental conditions on biofilm formation by Hafnia alvei strains. Vet Microbiol. 2008;129:150–5.

    Article  CAS  PubMed  Google Scholar 

  5. Tan JY, Yin WF, Chan KG. Gene clusters of Hafnia alvei strain FB1 important in survival and pathogenesis: a draft genome perspective. Gut Pathog. 2014;6:29.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005;33:W686–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genom. 2008;9:75.

    Article  Google Scholar 

  9. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014;42:D206–14.

    Article  CAS  PubMed  Google Scholar 

  10. Galperin MY, Makarova KS, Wolf YI, Koonin EV. Expanded microbial genome coverage and improved protein family annotation in the COG database. Nucleic Acids Res. 2015;43:D261–9.

    Article  CAS  PubMed  Google Scholar 

  11. Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35:W52–7.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Cosentino S, Voldby Larsen M, Moller Aarestrup F, Lund O. PathogenFinder—distinguishing friend from foe using bacterial whole genome sequence data. PLoS ONE. 2013;8:e77302.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee I, Kim YO, Park SC, Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 2016;66:1100–1103.

    Article  CAS  Google Scholar 

  15. Wilkinson L, Friendly M. The history of the cluster heat map. Am Stat. 2009;63:179–84.

    Article  Google Scholar 

  16. Kim Y, Wang X, Ma Q, Zhang XS, Wood TK. Toxin-antitoxin systems in Escherichia coli influence biofilm formation through YjgK (TabA) and fimbriae. J Bacteriol. 2009;191:1258–67.

    Article  CAS  PubMed  Google Scholar 

  17. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284:1318–22.

    Article  CAS  PubMed  Google Scholar 

  18. Zheng C, Xu J, Ren S, Li J, Xia M, Chen H, Bei W. Identification and characterization of the chromosomal yefM-yoeB toxin-antitoxin system of Streptococcus suis. Sci Rep. 2015;5:13125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nieto C, Cherny I, Khoo SK, de Lacoba MG, Chan WT, Yeo CC, et al. The yefM-yoeB toxin-antitoxin systems of Escherichia coli and Streptococcus pneumoniae: functional and structural correlation. J Bacteriol. 2007;189:1266–78.

    Article  CAS  PubMed  Google Scholar 

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Authors’ contributions

YDN and SWR designed and coordinated all the experiments. HSS and SWR performed gene annotation and prepared the manuscript. JYK and YBK performed the sequence assembly and gene prediction. MSJ, JK, and JKR discussed and analyzed the data. JSK and JSC performed the comparative genomic analysis. JK and HJC checked the manuscript. All authors read and approved the final manuscript.


Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The complete genome data of Hafnia alvei CBA7124 has been deposited in DDBJ/EMBL/GenBank, with Accession Number AP017469.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The study protocol was approved by the institutional review board of the Theragen ETEX Bio Institute (700062-20160804-JR-005-02).


This research was supported by the Korean Food Research Institute (E0131600-04); the World Institute of Kimchi funded by the Ministry of Science, ICT and Future Planning, Republic of Korea (KE1702-2); the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2015R1D1A1A09061039); and the Ewha Womans University scholarship of 2016.

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Correspondence to Seong Woon Roh.

Additional file


Additional file 1: Table S1. Genome sequencing information for Hafnia alvei CBA7124. Table S2. Strain-specific singletons of Hafnia alvei CBA7124 based on the comparison of POGs (without 3 uncharacterized proteins and 54 hypothetical proteins). Table S3. Comparison of genome characteristics and strain-specific singletons in Hafnia alvei strains. Table S4. Antibiotic and antimicrobial resistance genes of Hafnia alvei CBA7124. Figure S1. Cell Morphology of Hafnia alvei CBA7124 viewed using SEM. Bar size = 1 μm. Figure S2. OrthoANI dendrogram of Hafnia alvei CBA7124 with other H. alvei genomes.

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Song, H.S., Kim, J.Y., Kim, Y.B. et al. Complete genome sequence of a commensal bacterium, Hafnia alvei CBA7124, isolated from human feces. Gut Pathog 9, 41 (2017).

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