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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(4): 509-512

https://doi.org/10.48022/mbl.2409.09009

Received: September 17, 2024; Revised: November 26, 2024; Accepted: December 2, 2024

Complete Genome of Multidrug-Resistant Enterococcus faecalis JF4A2234 Belonging to Sequence Type 620

Jae Hong Jeong, Jae Young Oh, Su Min Kwak, and Jong-Chan Chae*

Division of Biotechnology, Jeonbuk National University, Iksan 54596, Republic of Korea

Correspondence to :
Jong-Chan Chae,        chae@jbnu.ac.kr

Abstract

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Enterococcus faecalis strain JF4A2234 was isolated from the feces of a pig in the Republic of Korea. The complete genome of E. faecalis JF4A2234 was obtained through a hybrid assembly of long- and short-read sequences. It consisted of one chromosome (2,796,215 bp with 37.7% GC content) and one circular plasmid (92,100 bp with 34.6% GC content). The annotation process identified 2,803 protein-coding sequences, 12 rRNA genes, and 62 tRNA genes. The genome harbored multiple antibiotic resistance genes, such as aac(6”)-le-aph(2”)-Ia, aad(6), ant(6)-Ia, ant(9)-Ia, aph(3’)-IIIa, tetM, tetL, ermB, dfrG, cat, sat-4, IsaE, and lnuB, which confer resistance to aminoglycoside, tetracycline, chloramphenicol, trimethoprim, erythromycin, lincosamide, and streptothricin.

Keywords: Enterococcus faecalis, pig, plasmid, multiple resistance genes

Body

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Enterococci are Gram-positive, facultative anaerobic bacteria that inhabit the gastrointestinal tract of various organisms, including humans [1]. To date, more than 50 species are known with Enterococcus faecalis and Enterococcus faecium accounting for more than 80% of clinical isolates and being most common in the intestines of humans and animals [2, 3]. Differences in antibiotic resistance among enterococci from various farm animals are known to be one of the important factors in determining the level of resistance based on the use of antibacterial agents [4]. E. faecalis strain JF4A2234 was isolated from a fecal swab sample from a 15-week-old pig in South Korea. The rectal swab collected from the pig was suspended in brain heart infusion broth (BD Bioscience, USA). The inoculum was streaked on enterococcosel agar (BD Bioscience, USA) and incubated at 37℃ for 48 h. Suspected enterococci colonies, which were translucent with brownish-black to black zones, were cultured on blood agar plates (Synergy Innovation, South Korea). Subsequently. E. faecalis was identified by PCR and sequencing analysis using the housekeeping gene gdh (5’-GGCGCACTAAAAGATATGGT-3’ and 5’-CCAAGATTGGGCAACTTCGTCCCA-3’), and the nucleotide sequences of the 16S rRNA gene were determined from a species-specific PCR-positive colony. The strain E. faecalis JF4A2234 exhibited 100% similarity to E. faecalis strain ATCC 19433 (GenBank accession No. ASDA01000001.1) when compared using the 16S rRNA sequence in EzBioCloud database [5]. The multilocus sequence type (ST) of strain E. faecalis JF4A2234 was identified as ST620 using the E. faecalis scheme in PubMLST (https://pubmlst.org/organisms/enterococcus-faecalis). Disk diffusion and broth microdilution methods were employed for an antibiotic susceptibility test following the Clinical and Laboratory Standards Institute guidelines. Strain JF4A2234 was resistant to various antibiotics, such as fluoroquinolone (ciprofloxacin), aminoglycoside (gentamicin, streptomycin), tetracycline (tetracycline), streptogramin (quinupristin-dalfopristin), and phenicol (chloramphenicol) but was sensitive to β-lactams (ampicillin, penicillin), nitrofuran (nitrofurantoin), glycopeptide (vancomycin, teicoplanin), oxazolidinone (linezolid), and glycylcycline (tigecycline).

Genomic DNA was extracted using the LaboPassTM Bacteria Mini DNA purification kit (Cosmogentech, Republic of Korea) according to the manufacturer’s instructions. Genome sequencing of E. faecalis strain JF4A2234 was conducted using two sequencing platforms, Illumina iSeq100 (Illumina, USA) and MinION sequencer (Oxford Nanopore Technologies, UK). While the library for the iSeq platform was prepared using the Illumina DNA Prep Kit (Illumina) and Nextera DNA CD indexes (Illumina), that for the MinION platform was prepared using a Flow Cell R10 Version (Oxford Nanopore Technologies, UK) according to the manufacturer's instructions. Illumina short-read sequencing and Nanopore long-read sequencing produced 1,157,269 and 515,568 reads, respectively. Adapter/primer sequences and low-quality reads from short-read raw sequence file were trimmed using Trimmomatic (v.0.39) [6]. Low-quality reads and adapter sequences from the long-read sequence files were trimmed using Filtlong (v.0.2.0)(https://github.com/rrwick/Filtlong) and Porechop (v.0.2.4)(https://github.com/rrwick/Porechop). The trimmed short-and long-read sequence files were used for hybrid de novo assembly using Unicycler (v.0.4.9b) [7]. Functional categorization of all predicted protein-coding genes was performed using the COGclassifier (v.1.0.5) (https://github.com/moshi4/COGclassifier). Potential antimicrobial resistance genes in E. faecalis strain JF4A2234 were predicted using the Comprehensive Antibiotic Resistance Database (CARD) and the Resistance Gene Identifier (RGI) v.6.0.3 [8]. Potential mobile genetic elements were predicted using the Prokka v.1.14.6 [9]. The complete genome of E. faecalis strain JF4A2234 contains one circular chromosome (2,796,215 bp with 37.7%GC content) and one circular plasmid designated as pJF4A2234 (92,100 bp with 34.6% GC content) (Table 1). Furthermore, the complete genome comprises 2,803 protein-coding sequences (CDSs) and 74 non-coding genes (12 rRNA and 62 tRNA genes). The average nucleotide identity (ANI) analysis of our strain with E. faecalis strain ATCC 19433, the closest strain based on 16S rRNA gene sequence, was calculated using the OrthoANI algorithm exhibiting an ANI value of 98.75%. A total of 2,203 proteins were classified based on functional categorization by the Clusters of Orthologous Groups (COG) database. The top five most abundant COG categories were ‘carbohydrate transport and metabolism (G)’ (248 genes, 11.3%), ‘translation, ribosomal structure and biogenesis (J)’ (235 genes, 10.7%), ‘transcription (K)’ (191 genes, 8.7%), ‘amino acid transport and metabolism (E)’ (169 genes, 7.7%), and ‘function unknown (S)’ (146 genes, 6.6%) (Fig. 1). The chromosome contained erythromycin and trimethoprim resistance genes (lsaA and dfrE). In addition, ciprofloxacin resistance in strain JF4A2234 resulted from a mutation in gyrA on the chromosome. The plasmid (contig 2), pJF4A2234, contained multiple antibiotic resistance genes, including gentamicin (aac(6’)-le-aph(2”)-la), streptomycin (aad(6), ant(6)-Ia, and ant(9)-Ia), streptothricin (sat-4), kanamycin (aph(3’)-IIIa), quinupristin-dalfopristin (IsaE), tetracyclines (tetL and tetM), erythromycin (lnuB), trimethoprim (dfrG), and chloramphenicol (catA8). Although no domestic studies on E. faecalis ST620 have been reported, the ST620 clone from live-stock in China was found to carry the optrA gene, which is associated with linezolid resistance in 2015 [10]. In contrast, the JF4A2234 strain possessed multiple antibiotic resistance genes, excluding optrA. Additionally, various mobile genetic elements were found in the plasmid, such as transposases (ISSsu9, ISBce13, IS1216E, IS1297, IS1476) and transposon (Tn552). Surveillance of multidrug-resistant E. faecalis clones in farm animals could be helpful for controlling the spread of antibiotic-resistant bacteria in the food production chain.

Table 1 . Genome features of Enterococcus faecalis strain JF4A2234.

Genome featuresChromosomePlasmid
Genome size (bp)2,796,21592,100
GC content (%)37.734.6
No. of CDSs2,686117
No. of rRNA12-
No. of tRNA62-

CDS, protein coding sequence

Figure 1.Cluster of Orthologous Group (COG) functional categories of the predicted protein coding sequence in the genome of Enterococcus faecalis strain JF4A2234.

Nucleotide Sequence Accession Number

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The genome sequence of Enterococcus faecalis strain JF4A2234 has been deposited in GenBank/EMBL/DDBJ under accession numbers CP133450.1 (chromosome) and CP133451.1 (plasmid).

The GenBank/EMBL/DDBJ accession numbers for the complete genome sequence of strain JF4A2234 are CP133450.1 (chromosome) and CP133451.1 (plasmid).

The strain was deposited in KCTC under deposition number BP1918526.

Acknowledgments

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This work was supported by grants of the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT)(2022R1A2C1011800) and Ministry of Food and Drug Safety (23194MFDS012).

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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