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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2023; 51(4): 555-558

https://doi.org/10.48022/mbl.2309.09004

Received: September 19, 2023; Revised: November 30, 2023; Accepted: December 4, 2023

Complete Genome Sequence of Staphylococcus aureus strain 21SAU_AGRO3 Isolated from Korean Agricultural Products

Sojin Ahn1,2, Eunbyeol Ahn3, So Yun Jhang1,2, Misun Jeong1, Sangryeol Ryu3, and Seoae Cho1*

1eGnome Inc., Seoul 05836, Republic of Korea
2Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul 08826, Republic of Korea
3Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea

Correspondence to :
Seoae Cho,       seoae@egnome.co.kr

Abstract

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Staphylococcus aureus is a prominent multidrug-resistant pathogen known for its resistance to a variety of antibiotics. To combat this, a wide range of antibiotics, including quinolones, is utilized. While the efficacy of quinolones against S. aureus has been established, the rise in quinolone-resistant strains, particularly in methicillin-resistant S. aureus (MRSA), has necessitated a shift in their usage patterns. Genomic sequencing plays a crucial role as it offers insights into the genetic mechanisms of resistance. Thus, we report the complete genome sequence of an oxolinic acid-resistant strain of S. aureus isolated from sweet potato leaves, a crop commonly cultivated in Korea.

Keywords: Staphylococcus aureus, antimicrobial resistance, fluoroquinolones, complete genome

Body

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Staphylococcus aureus is notable for its resistance to various antibiotics, including methicillin-resistant S. aureus (MRSA). Historically, quinolone antibiotics have been used against S. aureus infections, but the emergence of fluoroquinolone-resistant strains has compromised this efficacy [1]. The widespread use of quinolones in the agricultural sector, including agents like oxolinic acid that target DNA gyrases, has further intensified the challenge of antibiotic resistance [2]. Resistance to quinolones often involves mutations within the quinolone resistance-determining region of the gyr and par genes [3]. Additionally, multidrug resistance efflux pumps are believed to play a role in mediating fluoroquinolone resistance, although the exact mechanisms are not fully understood [4]. Given these issues, monitoring quinoloneresistant strains is necessary. In this context, we report the complete genome sequence of S. aureus strain 21SAU_AGRO3, which possesses antimicrobial resistance genes that includes those specific to fluoroquinolones.

The S. aureus strain 21SAU_AGRO3, obtained from the leaves of sweet potato grown in Busan, South Korea, was subjected to antimicrobial susceptibility testing using the disk diffusion assay. For oxolonic acid (2 μg), we observed a zone of inhibition measuring 12 mm. In the absence of established guidelines for oxolonic acid, we followed the CLSI standards for quinolone resistance in S. aureus to evaluate the antibiotic susceptibility of 21SAU_AGRO3 [5]. The PureHelix Genomic DNA Prep Kit (Solution Type)-Bacteria was used for DNA extraction in anticipation of next generation sequencing. The quantity and quality of the isolated genomic DNA were assessed using gel electrophoresis at 260/230 nm and 260/280 nm absorbance ratio, and the Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen). The sequencing library was constructed using the Oxford Nanopore Technology ligation sequencing kit (SQK_LSK112) and the native barcoding expansion kit (EX_NBD114) according to the manufacturer’s instructions. Subsequently, the library was loaded onto a MinION Flowcell (MIN112, R10.4) for sequencing, utilizing a MinION MK1b and MinKNOW software (22.03.6, 22.05.5). We then performed de novo assembly of long reads using Flye v2.9.1 and validated the genome completeness using BUSCO v5.2.2 [6, 7]. As a result, a high-quality complete genome consisting of a single circular contig was produced with a BUSCO v5.2.2 completeness score of 100%. The overall genome length is 2,869,088 bp with a GC content of 32.82%. The annotated genome composed of 2,755 coding sequences (CDS), 60 tRNA genes, 19 rRNA genes, and a single transfer-messenger RNA gene. Comparison of our data with previously published complete S. aureus genomes using Average Nucleotide Identity (ANI) via FastANI identified the S. aureus FDAARGOS_412 strain (GCF_002386245.1) as the most closely related strain to ours, exhibiting an ANI value of 99.0453% [8].

Functional category annotation was performed using the Clusters of Orthologous Groups (COG) approach, resulting in the annotation of 2,344 CDS [9]. With the exclusion of Class S, which consists of 633 CDS of unknown function, the most abundant categories were Class E (Amino acid transport and metabolism) with 230 CDS, Class P (Inorganic ion transport and metabolism) with 186 CDS, Class K (Transcription) with 172 CDS, and Class J (Translation, ribosomal structure and biogenesis) with 169 CDS. Subsequently, antibiotic resistance genes in the strict and perfect categories of the 21SAU_ARGO3 strain were annotated using the Comprehensive Antibiotic Resistance Database (CARD) and the Resistance Gene Identifier (RGI) v5.2.1[10]. This led to the annotation of 12 resistance genes, of which 7 were found to be associated with the fluoroquinolone antibiotic drug class (see Table 1). The complete genome sequence of S. aureus strain 21SAU_ARGO3 were deposited in GenBank under the accession CP134047.

Figure 1.Circular genome map of S. aureus strain 21SAU_AGRO3. Each circle indicates CDS in the leading strand, CDS in the lagging strand, COG distribution, RNA, antibiotic resistance genes, and the GC contents from outer to inner. Antibiotic resistance genes are labeled.

Table 1 . Putative antimicrobial resistance gene in genome of 21SAU_ARGO3 strain based on the CARD and RGI tools.

Locus_tagNucleotide Start positionNucleotide End positionARO term (CARD Accession)RGI (percentage identity)Drug ClassAMR gene family
GLJKLCAL_000818609487482norC (ARO: 3007010)Strict (99.57)fluoroquinolone antibiotic; disinfecting agents and antisepticsMFS antibiotic efflux pump
GLJKLCAL_00306351859352278mepR (ARO: 3000746)Perfect (100.0)glycylcycline; tetracycline antibioticMATE transporter
GLJKLCAL_00309355725354367glpT (ARO: 3003901)Strict (99.78)fosfomycinantibiotic-resistant GlpT
GLJKLCAL_00667726927726484mgrA (ARO: 3000815)Perfect (100.0)fluoroquinolone antibiotic; cephalosporin; penam; tetracycline antibiotic; peptide antibiotic; disinfecting agents and antisepticsMFS antibiotic efflux pump; ABC antibiotic efflux pump
GLJKLCAL_00676734578735744norA (ARO: 3004667)Perfect (100.0)fluoroquinolone antibioticMFS antibiotic efflux pump
GLJKLCAL_0140414789681477613arlS (ARO: 3000839)Perfect (100.0)fluoroquinolone antibiotic; disinfecting agents and antisepticsMFS antibiotic efflux pump
GLJKLCAL_0140514796241478965arlR (ARO: 3000838)Strict (99.54)fluoroquinolone antibiotic; disinfecting agents and antisepticsMFS antibiotic efflux pump
GLJKLCAL_0198520679502068795blaZ (ARO: 3000621)Strict (94.31)penamBlaZ beta-lactamase
GLJKLCAL_0223023041792302737lmrS (ARO: 3004572)Strict (99.17)macrolide antibiotic; aminoglycoside antibiotic; oxazolidinone antibiotic; diaminopyrimidine antibiotic; phenicol antibioticMFS antibiotic efflux pump
GLJKLCAL_0223123049752304502sepA (ARO: 3007012)Strict (96.82)disinfecting agents and antisepticsSMR antibiotic efflux pump
GLJKLCAL_0223223064172305074sdrM (ARO: 3007013)Strict (99.78)fluoroquinolone antibiotic; disinfecting agents and antisepticsMFS antibiotic efflux pump

* ARO: Antibiotic Resistance Ontology; MFS: major facilitator superfamily; MATE: multidrug and toxic compound extrusion, ATP-binding cassette; ABC: ATP-binding cassette; SMR: small multidrug resistance

Acknowledgments

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This study was carried out with the support of “Cooperative Research Program for Agricultural Science and Technology Development (Project No. PJ01612001)”, Rural Development Administration, Republic of Korea

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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