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Draft genome sequences and antibiotic resistance genes of Enterococcus hirae YS00198 (KCCM 43413) isolated from piglet feces
Korean J. Microbiol. 2021;57(4):297-299
Published online December 31, 2021
© 2021 The Microbiological Society of Korea.

Eun Bae Kim1,2*, Jeong Ho Yoo1, Seojin Choi1, and Jongbin Park1

1Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
2Institute of Animal Life Science, Kangwon National University, Chuncheon 24341, Republic of Korea
Correspondence to: E-mail: itanimal@kangwon.ac.kr;
Tel.: +82-33-250-8642; Fax: +82-33-259-5574
Received November 3, 2021; Revised December 16, 2021; Accepted December 21, 2021.
Abstract
Enterococcus hirae is frequently found in the animal gut and regarded as a commensal, pathogen, or sometimes probiotic. We isolated E. hirae YS00198 strain from feces of 9-week-old piglet in a local farm in South Korea. Its genome was sequenced by using MGIseq system and assembled. The draft genome size was 2,948,865 bp in 77 contigs (≥ 500 bp in length, 132,295 bp in N50, and 36.67% in G + C content).
Keywords : Enterococcus hirae, antibiotic resistance, genome sequencing, piglet feces
Body

Enterococcus hirae is Gram-positive and catalase-negative bacterium that is frequently found in the gastrointestinal tract of animals. Certain E. hirae strains are associated with piglet diarrhea (Jang et al., 2019), while others are regarded as commensals or probiotics (Masduki et al., 2020). Commensal Enterococcus are very good indicators that reflect environmental changes such as antibiotic uses in the farm as shown with commensal Lactobacillus strains (Lee et al., 2017). Here, we sequenced a genome of an E. hirae strain, and compared it with other strains from animal or human origins for antibiotic-resistant (AR) genes and bacteriocins.

Enterococcus hirae YS00198 was isolated from feces of a 9-week-old piglet housed in a local farm in Gangwon-Do, South Korea. To extract its genomic DNA, a single colony of the strain grown on an Enterococcosel agar (MB cell) plate was selected and inoculated into the Brain-Heart Infusion broth (BHI, MB cell). The cells were incubated at 37°C for 24 h and harvested by centrifuging at 13,000 × g for 1 min. Its genomic DNA was extracted by using G-spin Total DNA Extraction kit (iNtRON Biotechnology, Cat. No. 17121) according to the manufacturer’s protocol. For a genomic DNA library, the extracted genomic DNA (1 mg) was sheared by S220 Ultra sonicator (Covaris), and the fragmented gDNA was prepared by using by AMPure XP magnetic beads for size selection (~300 bp). Size-selected DNA fragments were further processed for a genomic DNA sequencing library with MGIEasy DNA library prep kit according to the manufacturer’s instructions. The fragments were end-repaired for 30 min at 37°C and A-tailed for 15 min at 65°C. Indexing adapter was ligated to the ends of the DNA fragments at 23°C for 60 min. After cleanup of adapter-ligated DNA, we tried to PCR for enrich those DNA fragments that have adapter molecules. The double stranded library was quantified through the QauntiFluor ONE dsDNA System (Promega). The library is circularized at 37°C for 30 min, and then digested at 37°C for 30 min, followed by cleanup of circularization product. To make a DNA nanoball (DNB), the library was incubated with DNB enzyme for 25 min at 30°C. Finally, the library was quantified by QauntiFluor ssDNA System (Promega). Sequencing of the prepared DNB was conducted on the MGIseq system (MGI) for 150-bp paired-end reads. After sequencing, raw sequences were filtered to remove adapter regions and low-quality reads by using in-house Perl scripts. Briefly, we selected good-quality reads, in which at least 95% of bases have quality score 36 or higher. To avoid wrong assembly and improve the assembly quality, only ~119X-depth reads were randomly selected for the assembly. Genome Assembly using SPAdes (Bankevich et al., 2012) version 3.13.1 finally resulted in 2,948,865-bp draft genome (Table 1, contig length, ≥ 500 bp; 36.67% in G + C content). Annotation was carried out by using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) Version 5.2 (Haft et al., 2018). A total of 2,642 protein-coding sequences (CDS), 37 tRNAs, and 3 rRNAs were predicted. As we were interested in AR gene profiles, AR gene detection was conducted by using the Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., 2020). Three AR genes were identified in our draft genome: tetM (resistant to tetracycline), ermB (resistant to macrolide including erythromycin), and AAC(6')-Iid (resistant to aminoglycoside including kanamycin and gentamycin). These results are associated with its antibiotic resistance to erythromycin (5 µg/ml), kanamycin (MIC > 4,096 µg/ml), and gentamycin (MIC > 1,024 µg/ml). Such AR gene profile was compared with other E. hirae genomes from the pig’s gut (Strain names: CQP3-9, HDC14-2, and NCTC12368), mouse’s gut (FDAA RGOS_1125) and spleen (13144), cat’s gut (ATCC 9790), and human’s blood (13344 and FDAARGOS_1124), abscess (FDAARGOS_234) and gut (708). Interestingly, two Chinese E. hirae genomes from pig guts have 6 (CQP3-9) and 11 (HDC14-2) AR genes, respectively, while others have only 1 or less AR genes. Unlike pigs, we could not think that antibiotics were usually administered into mice, dogs, and humans on a daily basis unless they are not severely infected. That supports that, unlike Chinese strains, our Korean strain might have been influenced by fewer types and less quantity of antibiotics as guided by the governmental regulations on antibiotic uses since July, 2011. However, the 3 AR genes are still more than E. hirae strains from a pig (NCTC12368 probably from UK before 1985) (Farrow and Collins, 1985) with 1 AR gene, two mice, a dog, and 4 humans. Genes that are predicted as bacteriocins such as class II lanthipeptide (J8N11_03180), enterolysin A (J8N11_11245), and sactipeptide (J8N11_02380) were detected by BAGEL4, a bacteriocin gene prediction SW (van Heel et al., 2018). Antimicrobial activities mediated by such bacteriocin-like genes were not examined. Genes that are like class II Lanthipeptide and enterolysin A were found in all compared genomes, while no sactipeptide-like gene was found in other genomes of pig-origin strains. It is not clear whether such bacteriocin profile was influenced by the antibiotic regulations. Our results extended our understanding of animal commensal E. hirae to antibiotic responses by its genome when it is exposed to environmental changes, although more E. hirae genomes should be compared in sophisticated ways in the future.

Overview for E. hirae YS00198 (KCCM 43413)

Features Scaffolds
Genome Size (bp) 2,948,865
No. of contigs (≥ 500 bp) 77
N50 (bp) 132,295
GC content (%) 36.67
No. of tRNAs 37
No. of rRNAs 3
No. of protein-coding genes 2,642


Nucleotide sequence accession numbers

The draft genome sequences of E. hirae YS00198 (KCCM 43413) have been deposited in GenBank under accession number JAHENG000000000. The E. hirae YS00198 (KCCM 43413) strain has been deposited in the Korea Culture Center of Microorganisms, Seoul, Korea under accession number KCCM 43413.

적 요

본 연구에서는 9주령 이유자돈의 분변에서 분리된 Enteorococcus hirae YS00198 균주의 유전체 염기서열을 분석하였다. Draft 유전체는 77개의 contig들(500 bp 이상)로 구성되었으며, 크기는 2,948,865 bp이고, G + C content는 36.7%, N50는 132,295 bp이다. 3가지 박테리오신 유사 유전자들과 3가지의 항생제 저항성 유전자들(돼지 유래 중국 균주들보다 적고, 인체 유래 균주들보다 많음)이 발견되었다.

Acknowledgments

This study was supported by a grant from National Research Foundation of Korea (NRF-2019R1A2C1009406). This study has been worked with the support of a research grant of Kangwon National University in 2020.

Conflict of Interest

The authors have no conflict of interest to report.

References
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