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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): 496-499

https://doi.org/10.48022/mbl.2404.04008

Received: April 9, 2024; Revised: June 10, 2024; Accepted: June 14, 2024

대한민국 농업 환경에서 분리된 다제내성 Pseudomonas asiatica CJ23의 전장 유전체 서열

Complete Genome Sequence of Multidrug-Resistant Pseudomonas asiatica CJ23, Isolated from the Agricultural Environment, South Korea

Yong-Seok Kim† and Chang-Jun Cha*

Department of Systems Biotechnology and Center for Antibiotic Resistome, Chung-Ang University, Anseong 17546, Republic of Korea
†Present address: DNA Link, Inc., Seoul 07793, Republic of Korea

Correspondence to :
Chang-Jun Cha,        cjcha@cau.ac.kr

Abstract

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Multidrug-resistant Pseudomonas asiatica CJ23 was isolated from the soil in a lettuce cultivation field, South Korea. The genome of strain CJ23 comprising 5,715,627 bp was assembled to a single contig with a G + C content of 62.7%. The complete genome sequence contained 5,092 protein-coding genes, 22 rRNA genes, and 74 tRNA genes. The genome of strain CJ23 possessed resistance-nodulation-division (RND)-type multidrug efflux pump, ampC β-lactamase and two metal resistance genes.

Keywords: Pseudomonas asiatica, complete genome, multidrug-resistant bacteria

Body

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The genus Pseudomonas, belonging to the family Pseudomonadaceae in the phylum Pseudomonadota, is one of the most diverse and complex bacterial genera. It is widely distributed in a variety of environmental habitats, including soil, water, animal gut and plant.

Members of the genus are generally aerobic, Gram-staining-negative, rod-shaped, and motile with one or more polar flagella. The genome sequences of Pseudomonas spp. show an average genome size of 6.37 Mbp and a G+C content of 63.7% [1]. Pseudomonas asiatica was first isolated from stool samples of hospitalized patients in Japan and Myanmar [2]. P. asiatica is considered a potential human pathogen belonging to the Pseudomonas putida group. The species has been reported to harbor New Delhi metallo-beta-lactamases (NMD) and Verone integron-encoded metallo-beta-lactamase (VIM), conferring resistance to carbapenem [3].

In this study, a multidrug-resistant Pseudomonas asiatica strain CJ23 was isolated from the soil in an agricultural field, South Korea (37°09'29.3"N 127°17'19.9"E) and the complete genome sequence of the strain is reported.

The genomic DNA was extracted using FastDNA™ Spin Kit for Soil (MP Biomedicals) following the manufacturer’s protocols. Whole genome sequencing of strain CJ23 was carried out at DNA Link, Inc. (Republic of Korea) using the Oxford Nanopore MinION flow cell (R9.4.1 FLO-MIN106, Oxford Nanopore) and the Illumina NovaSeq 6000 sequencing platforms. The genomic DNA of strain CJ23 for the nanopore sequencing was prepared using the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore, UK) following the manufacturer’s protocol. The genomic DNA library for Illumina sequencing was prepared according to TruSeq DNA PCR-Free kit manufacturer’s protocol. Genome assembly was performed using Trycycler v0.5.4 [4] following the recommended workflow [5], and error correlation and polishing were performed using medaka v1.11.3 (https://github.com/nanoporetech/medaka) based on long-read sequencing data. To obtain a more refined genome sequence, additional error correlation and polishing steps were performed using polypolish v0.5.0 [6] and polca v3.4.2 [7] based on short-read sequencing data. Genome completeness was evaluated using BUSCO v5.6.1 [8] and CheckM v1.2.2 [9]. Average nucleotide identity (ANI) value was calculated by the OrthoANI algorithm [10]. The assembled genome was visualized using Genovi v0.2.16 [11] and genome annotation was performed using bakta v 1.9.1 [12]. Antibiotic resistance genes and metal resistance genes were predicted using NCBI Antimicrobial Resistance Gene Finder v3.11.2 [13]. The antibiotic susceptibility of strain CJ23 was tested by the disk diffusion assay using antibiotic impregnated disks (Lioflchem, Italy) including amoxicillin (10 μg), cephalexin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), meropenem (10 μg), streptomycin (10 μg), sulfamethoxazole (50 μg), tetracycline (30 μg), and trimethoprim (5 μg). Susceptibility results were recorded as susceptible (S), intermediate (I) or resistant (R) based on breakpoints according to the CLSI M100 clinical breakpoint table.

The sequencing depth was 281.14 × coverage and the completeness of genome assembly was determined to be 99.8% and 99.9% based on BUSCO and CheckM, respectively. The genome size of strain CJ23 was 5,715,627 bp comprising a single contig with a G + C content of 62.7%(Fig. 1). The genome sequence contained 5,092 protein-coding genes (CDSs), 22 rRNA genes (seven rRNA operons and one 5S ribosomal RNA gene) and 74 tRNA genes (Table 1). The 16S rRNA sequence similarity between strain CJ23 and Pseudomonas asiatica RYU5T was 100%, and the ANI value was 99.3%, indicating that strain CJ23 can be classified as P. asiatica. The strain CJ23 displayed multidrug resistance to amoxicillin, cephalexin, chloramphenicol, ciprofloxacin, gentamicin, meropenem, streptomycin, and trimethoprim. Based on the analysis of antibiotic resistance genes from the complete genome sequence of strain CJ23, an ampC β-lactamase gene and a resistance-nodulation-division (RND)-type multidrug efflux pump, ttgAB, were identified. Two metal resistance genes, chrR (chromate resistance gene) and cadR (cadmium resistance gene), were also present in the genome.

Table 1 . Genome feature of Pseudomonas asiatica CJ23.

FeaturesValue
No. of contigs1
Genome size (bp)5,715,627
G + C ratio (%)62.7
N505,715,627
No. of CDSs5,092
No. of rRNA (5S, 16S, 23S) genes22 (8, 7, 7)
No. of tRNA genes74
No. of ncRNA genes67
GenBank accession No.CP150846
Antibiotic resistance profile (R)*Amoxicillin, Cephalexin, Gentamicin, Meropenem, Streptomycin, Trimethoprim
Antibiotic resistance profile (I)*Chloramphenicol, Ciprofloxacin

*Susceptibility results were recorded as resistant (R) or intermediate (I) based on breakpoints according to the CLSI M100 clinical breakpoint table.
Figure 1.The complete genome map of Pseudomonas asiatica CJ23. From inner to outer tracks: The first track shows the GC skew values with dark green indicating lower than zero and green indicating higher than zero. The second track represents the GC content. The third, fourth and fifth tracks display the locations of COG, CDS, tRNA and rRNA on reverse strand of the genome, respectively. The sixth, seventh, and eighth tracks show the locations of tRNA, rRNA, CDS, and COG on forward strand of the genome, respectively.

적요

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다제내성 Pseudomonas asiatica CJ23 균주가 대한민국의상추 재배지 토양에서 분리되었다. 5,715,627 bp로 이루어진CJ23 균주의 유전체는 G + C 함량이 62.7%인 한 개의 contig로 조립되었다. 완전한 유전체 서열은 5,092개의 단백질 코딩유전자, 22개의 rRNA 유전자, 74개의 tRNA 유전자를 포함하였다. CJ23 균주의 유전체는 내성-결절-세포 분열 (RND)-유형의 다중 약물 배출 펌프, ampC β-락타마아제 및 두 개의 금속내성 유전자를 보유하였다.

Nucleotide sequence accession number(s)

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Pseudomonas asiatica CJ23 the complete genome sequence has been deposited to the DDBJ/EMBL/Gen-Bank under the accession number CP150846.

Acknowledgments

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This research was supported by the research program for Agricultural Science & Technology Development under the National Institute of Agricultural Science (NAS)-Rural Development Administration (RDA), South Korea, under grant numbers PJ015130, and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2023R1A2C1003654).

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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