Vancomycin-resistant bacteria of various of the antibiotics, one of the main characteristics of the strain in this study, vancomycin-resistant enterococci, namely Enterococcus faecium and E. faecalis were isolated in Great Britain in 1988 for the first time (Courvalin, 2006). Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from a Japanese patient in 1996 for the first time (Hiramatsu, 2001) and some strains in the genus Sphingobacterium isolated from various environments such as milk, decaying fern and aquifers also had vancomycin resistance (Schmidt et al., 2012; Cheng et al., 2019; He et al., 2019). These vancomycin-resistant bacteria have shown potential applications in biomediation as an important indicator of the decline of the clinical effectiveness of vancomycin.

The genus Sphingobacterium belonging to the family Sphingobacteriaceae of the phylum Bacteroidetes, was proposed in 1983 by Yabuuchi et al. (1983) with Sphingobacterium spiritivorum as the type species. Members of this genus are Gram-stain-negative, aerobic, rod-shaped bacteria that have menaquinone-7 as the major isoprenoid quinone and phosphatidylethanolamine as a major polar lipid and represent the G + C contents ranging from 35 to 44 mol% (Zhou et al., 2020; Kakumanu et al., 2021) and have the ability to degrade various pollutants and the antibiotics resistance (Liu et al., 2020). Interestingly, according to previous studies, vancomycin-resistant strains of the genus Sphingobacterium had vancomycin B-type resistant gene vanW in common (Schmidt et al., 2012; Cheng et al., 2019; He et al., 2019). The present genomic analysis results thus supported that vanW gene influences the determination of vancomycin resistance.

For whole genome sequencing, genomic DNA was extracted using a G-spin^TM Genomic DNA Extraction Kit (iNtRON). Whole genome sequencing of the strain SRCM116780 was performed by Macrogen, using a combination of PacBio RSII and Illumina HiSeq platform. After the raw reads assembled using Hierarchical Genome Assembly Process version 4 (HGAP4) (Chin et al., 2013), Illumina reads were applied for sequence compensation to construct contigs more accurately using the Pilon (version 1.21) software tool. Then, genes were identified and annotated using the Prokka pipeline version 1.14.5. (Seemann, 2014) and gene functions were annotated using the EggNOG database (Huerta-Cepas et al., 2016). The overall genome related index (OGRI) was compared to Sphingobacterium genomes available in NCBI. Digital DNA-DNA hybridization (dDDH) (http://ggdc.dsmz.de/ggdc.php/) values between SRCM116780 and other Sphingobacterium species were calculated as described by Chun et al. (2018). Average nucleotide identity (ANI) between SRCM116780 and Sphingobacterium species were calculated using the Orthologous Average Nucleotide Identity Tool (OAT) (Lee et al., 2015).

The genome of strain SRCM116780 was 4.3 Mb in length with a genome coverage of 137.0x, and the N50 value was 4.3 Mb, indicating a circular chromosome. (GenBank accession no. NZ_CP090446) (Fig. 1

Fig. 1. Graphical circular map of Sphingobacterium sp. SRCM116780. From bottom to top: genes on the forward strand (colored by COG categories), genes on the reverse strand (colored by COG categories), RNA genes (tRNA-green, rRNA-red, other RNAs-black), GC content, and GC skew (green/lavender).

). The DNA G + C content of the SRCM116780 genome was 35.5 mol%, complying with the range of 35–44 mol% reported for the genus Sphingobacterium. Of the 3,687 predicted genes, 3,578 were protein-coding genes, 102 were RNA genes (22 complete rRNAs, 77 tRNAs, and 3 ncRNAs) and 7 were pseudo genes. The general features of the genome of strain of SRCM116780, which was smaller than those of closely related reference strains but, including DNA G + C contents, and the numbers of rRNA and tRNA genes, were similar with those of closely related reference strains (Table 1). As the genome of SRCM116780 was compared with currently known genomes of Sphingobacterium, the ANI and dDDH values were 69.8–78.9% and 19.3–22.7%, respectively (Table 1). The values were below the 95% (for ANI) and 70% (for dDDH) cut-offs proposed for bacterial species delineation (Chun et al., 2018). Based on the EggNOG database functional annotation, of the 3,578 protein-coding genes, 3,404 genes were assigned and strain SRCM116780 had 67 genes (2.0%) associated with defense mechanisms, including vancomycin B-type resistant genes vanW, a multidrug resistant gene mdtK, a colistin resistance gene emrA, and so on (Table 2)

Table 1

Strains (genome accession): 1, strain SRCM116780 (CP090446); 2, S. faecium DSM11690^T (QBKH01); 3, S. multivorum IAM1431^T (CP068086); 4, S. siyangense SY1^T (MCAQ01); 5, S. detergens CECT 7938^T (RAPY01); 6, Sphingobacterium athyrii M46^T (QCXX01); 7, S. puteale M05W1-28^T (RBWS01); 8, S. thalpophilum DSM 11723^T (LR590484); 9, S. spiritivorum ATCC 33861^T (ACHA02); 10, S. nematocida M-SX103^T (FUZF01).

Characteristic	1	2	3	4	5	6	7	8	9	10
General genomic features
Genome size (Mb)	4.3	5.3	6.0	6.8	6.8	6.9	6.7	6.0	5.1	5.1
No. of contigs	1	22	3	31	15	23	49	1	15	50
G + C contents (mol%)	35.5	36.3	40.1	39.7	39.8	40.6	40.7	43.6	39.8	39.9
No. of total genes	3687	4473	5175	5584	5541	5710	5571	5047	4332	4322
No. of protein coding genes	3578	4383	5059	5496	5458	5616	5471	4936	4269	4241
No. of pseudogenes	7	18	31	17	17	18	30	30	13	26
No. of rRNA genes (5S/16S/23S)	22 (8/7/7)	9 (7/1/1)	21 (7/7/7)	13 (6/3/4)	3 (1/1/1)	23 (7/7/9)	6 (1/3/2)	21 (7/7/7)	3 (1/1/1)	7 (4/1/2)
No. of tRNA genes	77	72	85	71	66	76	70	81	50	55
Vancomycin resistant genes	vanW	-	vanW	vanW	vanW	vanW	vanW	vanW	-	-
OGRI relative to strain SRCM116780
16S rRNA gene (%)	100	96.2	93.3	93.3	92.8	93.4	93.8	93.5	92.4	92.8
ANI (%)	100	78.9	71.6	71.6	71.4	71.2	71.1	70.5	70.3	69.8
dDDH (%)	100	19.3	21.9	22.6	22.2	22.3	22.4	22.7	22.2	20.2

Table 2

Based on the EggNOG database genes functional annotation description of strain SRCM116780

Code	Description	Count	Ratio (%)
J	Translation, ribosomal structure and biogenesis	141	4.14
A	RNA processing and modification	0	0.00
K	Transcription	165	4.85
L	Replication, recombination and repair	147	4.32
B	Chromatin structure and dynamics	1	0.03
D	Cell cycle control, cell division, chromosome partitioning	18	0.53
Y	Nuclear structure	0	0.00
V	Defense mechanisms	67	1.97
T	Signal transduction mechanisms	116	3.41
M	Cell wall/membrane/envelope biogenesis	194	5.70
N	Cell motility	1	0.03
Z	Cytoskeleton	1	0.03
W	Extracellular structures	0	0.00
U	Intracellular trafficking, secretion, and vesicular transport	27	0.79
O	Posttranslational modification, protein turnover, chaperones	115	3.38
C	Energy production and conversion	130	3.82
G	Carbohydrate transport and metabolism	155	4.55
E	Amino acid transport and metabolism	187	5.49
F	Nucleotide transport and metabolism	63	1.85
H	Coenzyme transport and metabolism	82	2.41
I	Lipid transport and metabolism	75	2.20
P	Inorganic ion transport and metabolism	173	5.08
Q	Secondary metabolites biosynthesis, transport and catabolism	23	0.68
R	General function prediction only	300	8.81
S	Function unknown	1223	35.93

. In particular, the molecular function of the vanW gene and its role in vancomycin resistance remain unknown. However, a previous study, reported that vanW-like domains are found in proteins such as penicillin-insensitive L, D-transpeptidase from E. faecium, and the gene encoding this enzyme is proximal to a gene coding for L-lactate dehydrogenase; with these two activities reminiscent of vanY and vanH, respectively (Stogios and Savchenko, 2020). Interestingly, according to previous studies, vancomycin-resistant strains of the genus Sphingobacterium had vancomycin B-type resistant gene vanW in common (Schmidt et al., 2012; Cheng et al., 2019; He et al., 2019). The present genomic analysis results thus supported that vanW gene influences the determination of vancomycin resistance.

According to the whole genome analysis and antibiotic resistance results, strain SRCM116780 presented notable resistance to vancomycin. Based on these results, we tried to determine the survival rate by concentration for vancomycin. The survival curves of strain SRCM116780 at various concentrations for vancomycin showed more than 94.5% cell survival up to a concentration of 40 mg/L and the survival rate rapidly decreased at the concentration above that (Fig. 2). In comparison, the closely related species, S. kitahiroshimense KACC 13399^T, S. anhuiense KACC 14208^T were affected by cell growth from 10 mg/L of vancomycin, whereas S. faecium KACC 12160^T showed more than 91.7% cell survival until 60 mg/L of vancomycin concentration (Fig. 2). Based on these results, the MIC value of strain SRCM116780 was determined by 40 mg/L, despite that it showed cell a survival rate of more than 50% at higher concentrations. Strain SRCM116780 showed a higher survival rate compared to closely related species except for S. faecium KACC 12160^T. These results indicate remarkable vancomycin resistance, but further research is required to define vancomycin resistance determinants of SRCM116780, such as a genome comparison between species.

Fig. 2. Representative survival curves of strain SRCM116780 and three related species by various concentration for vancomycin. ●, strain SRCM116780; ◇, S. kitahiroshimense; ▼, S. anhuiense; ○, S. faecium.

Nucleotide sequence accession number

The GenBank accession numbers for 16S rRNA sequence and genome sequence of strain SRCM116780 (= KCCM 43460 = JCM 35369) are MZ753665 and CP090446, respectively.

Sphingobacterium sp. SRCM116780은 순창군 논 토양에서 분리하였으며, vancomycin에 대하여 높은 저항성을 나타내었다. SRCM116780 균주의 전장유전체 염기서열은 PacBio RS II와 Illumina HiSeq platform으로 분석하였고, 137.0x의 genome coverage를 통해 4.3 Mb의 길이를 나타내며, 35.5 mol%의 GC 함량을 가지는 환형 구조를 나타내었다. 총 3,687개의 유전자 중 3,578개의 protein coding gene과 102개의 RNA genes (22 rRNA, 77 tRNA, 3 non-coding RNA), 7개의 pseudo genes의 유전자 구성을 가지는 것을 확인하였다. 3,578개의 protein coding gene 중 3,404개의 유전자가 기능이 알려져 있으며, vancomycin 저항성 유전자(vanW) 및 다제내성 유전자(mdtK), colistin 저항성 유전자(emrA) 등과 같은 방어 메커니즘과 관련된 67개의 유전자를 가지고 있음을 확인하였다.

This research was supported by a grant from the Establishment of Integrated Biobank for Agriculture, Food and Livestock Microbiome Project funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea.

The authors have no conflict of interest to report.