The Integrative and Conjugative Element ICECspPOL2 Contributes to the Outbreak of Multi-Antibiotic-Resistant Bacteria for Chryseobacterium Spp. and Elizabethkingia Spp.

Antibiotic resistance genes (ARGs) and horizontal transfer of ARGs among bacterial species in the environment can have serious clinical implications as such transfers can lead to disease outbreaks from multidrug-resistant (MDR) bacteria. Infections due to antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the multi-antibiotic-resistant strain Chryseobacterium sp. POL2 was isolated from the wastewater of a livestock farm. Whole-genome sequencing and annotation revealed that the POL2 genome encodes dozens of ARGs. The integrative and conjugative element (ICE) ICECspPOL2, which encodes ARGs associated with four types of antibiotics, including carbapenem, was identified in the POL2 genome, and phylogenetic affiliation analysis suggested that ICECspPOL2 evolved from related ICEEas of Elizabethkingia spp. Conjugation assays verified that ICECspPOL2 can horizontally transfer to Elizabethkingia species, suggesting that ICECspPOL2 contributes to the dissemination of multiple ARGs among Chryseobacterium spp. and Elizabethkingia spp. Because Elizabethkingia spp. is associated with clinically significant infections and high mortality, there would be challenges to clinical treatment if these bacteria acquire ICECspPOL2 with its multiple ARGs, especially the carbapenem resistance gene. Therefore, the results of this study support the need for monitoring the dissemination of this type of ICE in Chryseobacterium and Elizabethkingia strains to prevent further outbreaks of MDR bacteria. IMPORTANCE Infections with multiple antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the mobile integrative and conjugative element ICECspPOL2, which was associated with the transmission of a carbapenem resistance gene, was identified in the genome of the multi-antibiotic-resistant strain Chryseobacterium sp. POL2. ICECspPOL2 is closely related to the ICEEas from Elizabethkingia species, and ICECspPOL2 can horizontally transfer to Elizabethkingia species with the tRNA-Glu-TTC gene as the insertion site. Because Elizabethkingia species are associated with clinically significant infections and high mortality, the ability of ICECspPOL2 to transfer carbapenem resistance from environmental strains of Chryseobacterium to Elizabethkingia is of clinical concern.

INTRODUCTION

Antibiotic resistance has become a serious threat to human health (1, 2), and antibiotic resistance genes (ARGs) are considered a type of genetic pollution in the environment (3). As such, the effects of antibiotics on the environment in terms of ARGs, antibiotic-resistant bacteria, and horizontal transfer of ARGs have become an area of interest in the environmental sciences. There are four ways for bacteria to obtain ARGs: transformation (4), transduction (5), conjugation (6), and potentially through the fusion of two cells or the fusion of cells with DNA-containing vesicles (7). Of these four ways, conjugation is the more common method of horizontal gene transfer among bacteria in the environment. A study of 1,124 complete prokaryotic genomes revealed 180 putative conjugative plasmids and 335 putative integrative and conjugative elements (ICEs), suggesting that ICEs are likely more common than conjugative plasmids in prokaryotes (8).

ICEs are typically mosaic and modular genome-integrated mobile genetic elements, ranging from ∼20 kb to 500 kb in size, and are passively proliferated during genome replication, segregation, and cell division. Two typical features characterize ICEs: ICEs are integrated into a host genome, and they encode a type IV secretion/conjugation system, which enables them to transfer to other bacteria via conjugation (9–11). Many ICEs encode ARGs that confer antibiotic resistance on the recipient strain, and when the recipient strain is a human pathogen, this resistance can lead to difficulty in eradicating the pathogen and therefore potentially lead to outbreaks of infection. Elizabethkingia anophelis is an emerging opportunistic human pathogen that is associated with clinically significant infections and high mortality and has caused outbreaks in Singapore, Taiwan, Hong Kong, and the US state of Wisconsin (12, 13). An ICE named ICEEa1 was identified in the Wisconsin outbreak strains and Singapore outbreak strains (12). Recently, three types of ICEs, ICEEaI, ICEEaII, and ICEEaIII, have been identified in pathogenic E. anophelis strains (13).

Livestock and poultry farms are commonly considered to be the major sources of ARGs. In this study, we isolated the multi-antibiotic-resistant strain Chryseobacterium sp. POL2 from a wastewater sample from a livestock farm in Shandong, China. In the chromosome of POL2, we identified a typical ICE, which we named ICECspPOL2, that carries a carbapenem resistance gene. ICECspPOL2 was closely related with ICEEas found in E. anopheles, and ICECspPOL2 was found to horizontally transfer to Elizabethkingia sp., raising a warning regarding the need to track this kind of ICE in the environment to prevent further outbreaks of infection.

RESULTS

Chryseobacterium sp. POL2 is a multi-antibiotic-resistant strain.

After preliminary 16S rRNA gene analysis and then alignment of the whole-genome sequence, strain POL2 was identified as Chryseobacterium sp. MIC assays revealed that strain POL2 had some resistance to all tested antibiotics (Table 1), including ampicillin (MIC ≥32 mg/liter), cefixime (MIC ≥16 mg/liter), meropenem (MIC ≥64 mg/liter), amikacin (MIC ≥64 mg/liter), ciprofloxacin (MIC ≥128 mg/liter), tetracycline (MIC ≥128 mg/liter), florfenicol (MIC ≥128 mg/liter), sulfamethoxazole (MIC ≥128 mg/liter), vancomycin (MIC ≥32 mg/liter), and polymyxin E (MIC ≥16 mg/liter). Surprisingly, environmental isolate POL2 had striking resistance to meropenem, which is used in clinical disease treatment.

TABLE 1

Antibiotic susceptibility test of strain Chryseobacterium sp. POL2, Elizabethkingia sp. M6, and transconjugant Elizabethkingia sp. M6-P2

Antimicrobial agents	MIC (mg/liter)/antibiotic susceptibilitya
	POL2	M6	M6-P2
Ampicillin	32/R	72	96
Cefixime	16/R	<2/S	<2/S
Meropenem	64/R	8/R	36/R
Amikacin	64/R	16/R	48/R
Ciprofloxacin	>128/Rb	>128/R	>128/R
Tetracycline	>128/R	32/R	72/R
Florfenicol	>128/R	16/R	96/R
Sulfamethoxazole	>128/R	32/R	32/R
Vancomycin	32/R	<2/S	<2/S
Polymyxin E	16/R	<2/S	<2/S

Bacterial antibiotic susceptibility was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

R, resistant; S, susceptible.

Genomic features and phylogenetic relationship of strain POL2.

To understand the basis for its antibiotic resistance profile, the whole-genome sequence of strain POL2 was further analyzed (Fig. 1). POL2 contains only one circular chromosome, which is 3,243,462 bp in size, with an average GC content of 35.2%. The genome annotation revealed 3,003 genes in the POL2 genome, including 2,877 protein-coding genes, 15 rRNA genes, 49 tRNA genes, and three other RNA genes. Consistent with its antibiotic resistance profile (Table 1), genome annotation revealed that POL2 harbored dozens of antibiotic resistance-associated genes (Dataset S2), including genes coding for class D beta-lactamase, tetracycline-inactivating enzyme, and aminoglycoside 6-nucleotidyltransferase. In addition, a search for putative virulence genes led to the identification of 115 virulence factor-encoding genes (Dataset S3) based on the PHI database, indicating that POL2 is potentially pathogenic for humans and/or other organisms.

FIG 1

Comprehensive genomic analysis of Chryseobacterium sp. POL2. (A) Circular graphical display of genomic features. From inner to outer rings: GC skew; GC content; coding sequences (CDS) with homology to known antimicrobial resistance (AMR) genes; non-CDS features; CDS on the reverse strand; CDS on the forward strand; contigs/chromosome; and the position label (Mbp). (B) An overview of the subsystems/genes found in the Chryseobacterium sp. POL2 genome.

A phylogenetic tree analysis was also performed to determine the evolutionary relationship between POL2 and other Chryseobacterium/Elizabethkingia species (Fig. 2). The results revealed that strain POL2 is most closely related to strain Chryseobacterium bovis DSM 19482, although it is also related to Elizabethkingia spp., which like Chryseobacterium, belong to the Flavobacteriaceae family.

FIG 2

Molecular phylogenetic analysis of Chryseobacterium sp. POL2 based on the genome sequence. The whole-genome phylogenetic tree was constructed using the PATRIC server. The position of POL2 in the phylogenetic tree is indicated by the black arrow.

Identification of the integrative and conjugative element ICECspPOL2.

Preliminary analysis with the IslandViewer 4 program indicated that nucleotide positions 1,765,804 to 1,883,110 of the POL2 genome contained a potential antibiotic genomic island. Further analysis by ICEberg 2.0 verified a novel ICE in this region, which was named ICECspPOL2 (Fig. 3). ICECspPOL2 extends from position 1,767,025 to 1,883,295 in the POL2 genome and contains 116,271 bp. ICECspPOL2 is bordered by an 18 bp direct repeat (DR) (5′-ATTCCCCTACGGGCTACT-3′) at both ends and is inserted into the 3′ end of the tRNA-Glu-TTC gene (G6R40_RS08190). Gene annotation revealed that ICECspPOL2 contains 124 open reading frames (ORFs) (Table S2), including genes encoding conjugative elements, such as conjugative relaxases, ATPases of type IV secretion systems and the type IV coupling proteins, T4CP, and genes encoding DNA replication or partitioning-associated proteins and site-specific integrases. ICECspPOL2 also contains ARGs, including two genes (floR and catB) associated with chloramphenicol/florfenicol resistance; two genes (mphG and mefC) associated with macrolide resistance; one gene (tet(X), encoding tetracycline-inactivating enzyme) associated with tetracycline resistance; one gene (ant(6)-I) associated with aminoglycoside resistance, and one gene encoding a Class D beta-lactamase OXA-10 like protein, which might be partially responsible for the meropenem resistance of strain POL2. Hence, ICECspPOL2 has two typical characteristics of ICEs: it was integrated into the POL2 genome at the 3′ end of the tRNA-Glu-TTC gene and it encodes a type IV secretion/conjugation system.

FIG 3

Schematic view of the integrative and conjugative element ICECspPOL2. Top image: the predicted position of ICECspPOL2 in the Chryseobacterium sp. POL2 genome using IslandViewer 4. Bottom image: gene arrangement and characteristics of ICECspPOL2 identified using ICEberg 2.0. ICECspPOL2 is bordered by an 18 bp DR (5′-ATTCCCCTACGGGCTACT-3′), indicated by black bars, in the chromosome of POL2. Violet arrows, ARGs; blue arrows, conjugative transfer genes; green arrows, DNA replication or partitioning genes; gray arrows, genes with other functions.

Phylogenetic relationship of ICECspPOL2.

The whole nucleotide sequence of ICECspPOL2 was analyzed by BLAST, and pairwise alignment results revealed that ICECspPOL2 has strong homology with the ICEs ICEEaI and ICEEaIII in Elizabethkingia anophelis. Further BLASTn analysis revealed that genes in ICECspPOL2 relevant to horizontal conjugative transfer were highly similar to genes in ICEEaIII(10) of the E. anophelis NUH6 strain; ICEEaIII (5) of the E. anophelis NUHP1 strain; and ICEEaI(1) of the E. anophelis CSID_3015183678 strain (Fig. 4), indicating that ICECspPOL2 likely evolved from related ICEEas of E. anophelis strains.

FIG 4

Schematic showing potential sources of genes in ICECspPOL2. Gene sequences in ICECspPOL2 of Chryseobacterium sp. POL2 were compared with gene sequences from ICEEaIII(10) of E. anophelis NUH6, ICEEaIII (5) of E. anophelis NUHP1, and ICEEaI(1) of E. anophelis CSID_3015183678. Violet arrows, ARGs; blue arrows, conjugative transfer genes; green arrows, DNA replication or partitioning genes; gray arrows, genes with other functions. The 18 bp DR sequences are indicated by black bars.

To further track the evolutionary history of ICECspPOL2, eight related ICES of the ICEEas group were selected based on the BLASTn alignment results, and their nucleotide sequences were compared (Fig. 5). ICECspPOL2 was most closely related to ICEEaIII(16) in E. anopheles 12012-2PRCM; ICEEaIII(10) in E. anopheles NUH6; and ICEEaIII(5) in E. anopheles NUHP1, with these ICEs forming a clade separate from ICEEaI(1) in E. anopheles CSID_3015183678. Consistent with ICECspPOL2, both ICEEaIII(10) and ICEEaIII(5) are also inserted into a tRNA-Glu-TTC gene (14), suggesting that these three ICEs are evolutionarily related to each other. Notably, ICECspPOL2 harbors one Class D beta-lactamase gene, associated with carbapenem resistance, whereas the other reported ICEEas do not carry carbapenem resistance genes.

FIG 5

Phylogenetic relationships of ICECspPOL2. Based on the ICECspPOL2 whole nucleotide sequence alignment results, eight ICE sequences were selected, and the phylogenetic tree was constructed using MEGA7 software. The position of ICECspPOL2 in the phylogenetic tree is indicated by the black arrow.

Transfer of ICECspPOL2 to another bacterial strain.

ICECspPOL2 is a typical ICE, and the phylogenetic tree and BLASTn analysis revealed that it may have evolved from ICEEaIII of E. anophelis. To determine whether ICECspPOL2 could be horizontally transferred among bacteria, conjugation assays were conducted using POL2 as the donor strain with different recipient strains. When Elizabethkingia sp. M6 was used as the recipient strain with meropenem (16 mg/liter) and sodium azide (220 mg/liter) as the selective pressure, the transconjugation frequency was about 1.02 × 10−7 colony forming units (CFU)/donor. One of the transconjugants was selected and named M6-P2. To test whether ICECspPOL2 was inserted into the M6 genome, a PCR test and DNA sequencing were performed. The results revealed that the four chosen gene fragments of ICECspPOL2 were present in the transconjugant M6-P2 but not in the recipient strain M6 (Fig. 6). Additionally, MIC analysis showed that the transconjugant Elizabethkingia sp. M6-P2 acquired resistance to antibiotics associated with ICECspPOL2 resistance genes, especially meropenem (Table 1). The above data indicated that ICECspPOL2 was horizontally transferred from the donor strain POL2 to the recipient strain Elizabethkingia sp. M6. However, when using the sodium azide-resistant Escherichia coli 25DN and E. coli DL21 as the recipient strains, no transconjugant was obtained.

FIG 6

Verification of the presence of ICECspPOL2. (A) Primer positions in ICECspPOL2 are indicated using bent arrows. (B) Four ICECspPOL2 fragments were amplified by PCR using total DNA of strain M6 (lane 1), transconjugant M6-P2 (lane 2), strain POL2 (lane 3) as the templates.

DISCUSSION

Vigilance against the spread of ARGs and antibiotic-resistant bacterial infections has become increasingly important. Chryseobacterium spp. and Elizabethkingia spp. are opportunistic pathogens belonging to the family Flavobacteriaceae (15, 16). Chryseobacterium spp. and Elizabethkingia spp. are reported to be resistant to multiple different antibiotics such as lactam antibiotics (imipenem, piperacillin, meropenem, ceftazidime, cefepime, cefpirome), aminoglycoside antibiotics (gentamicin, tobramycin, amikacin), and quinolone antibiotics (ciprofloxacin, levofloxacin) in many isolates (17–20). This resistance is of clinical concern as the acquisition of Chryseobacterium and Elizabethkingia infections in intensive care units has been increasing in recent years (12, 13). Chryseobacterium species are ubiquitous inhabitants of the water, soil, and hospital environments (21–23), whereas Elizabethkingia species have aroused much concern in recent years (12, 13) because they are associated with disease-carrying mosquitoes, including the dengue fever vector Aedes and the malaria vector Anopheles (24–28), which are associated with clinically significant infections and high mortality.

In this study, the multi-antibiotic-resistant Chryseobacterium sp. POL2 was isolated from livestock wastewater. POL2 had notable resistance to carbapenem antibiotics. An ICE named ICECspPOL2 was identified in the Chryseobacterium sp. POL2 strain, and our bioinformatics analyses suggested that ICECspPOL2 evolved from related ICEEas of E. anophelis strains. Further analysis indicated that ICECspPOL2 could be horizontally transferred to Elizabethkingia sp. M6, a strain that was isolated from hospital wastewater. Because ICECspPOL2 contains ARGs that could confer resistance to meropenem, acquisition of ICECspPOL2 by environmental strains of Elizabethkingia could result in infections that would be difficult to treat. Reports have shown that ICEEas spread in Elizabethkingia spp., and that these ICEs mediate horizontal transfer of genes associated with antibiotic resistance, virulence, and stress response (12, 14, 27, 29–31). Therefore, these ICEEas can enable their bacterial hosts to quickly adapt to changing ecological niches and enable them to establish infections in humans. In this report, ICECspPOL2 was found to disseminate multiple ARGs among Chryseobacterium and Elizabethkingia species (Fig. 7), highlighting the risks associated with the potential transmission of such ICEs in the environment, particularly with regard to their potential to cause outbreaks of multi-antibiotic resistant Chryseobacterium and Elizabethkingia species.

FIG 7

A simple model showing that ICECspPOL2 mediated the dissemination of ARGs in the environment.

Bioinformatics analysis suggested that ICEs, rather than conjugative plasmids, are likely more important in the transmission of antimicrobial resistance in prokaryotes (8). The current NCBI genome database documents only five plasmids among the 261 genomes of Chryseobacterium species and two plasmids among the 202 genomes of Elizabethkingia species. In contrast, ICEs are commonly identified in the genomes of these two genera, such as the three types of ICEs (ICEEaI, ICEEaII, and ICEEaIII) that have been identified in E. anophelis strains isolated from around the world (14), suggesting that ICEs play an important role in horizontal gene transfer in these genera. Chryseobacterium sp. POL2, which was isolated in this study, contains only one chromosome, and as noted, ICECspPOL2 in the POL2 genome is closely related to the ICEEas from Elizabethkingia species, suggesting that ICEs might contribute to the dissemination of antimicrobial resistance among Chryseobacterium spp. and Elizabethkingia spp.

Phylogenetic analysis showed that ICECspPOL2 was most closely related to several members of the ICEEaIII group of E. anopheles ICEs. Some evidence suggests that ICEs of different groups favor different integration sites. ICEEaI can integrate into a gene or intragenic regions, such as the mutY gene of the Wisconsin outbreak strain of E. anophelis (12, 14). ICEEaII and ICEEaIII were found to insert next to tRNA genes, with ICEEaII mainly inserted into tRNA-Leu-CAA, whereas ICEEaIII can insert into different tRNA genes, such as tRNA-Glu-TTC, tRNA-Arg-ACG, and tRNA-Asp-GTC (14). Analysis by ICEberg 2.0 verified that ICECspPOL2 was inserted into the 3′ end of the tRNA-Glu-TTC gene of the Chryseobacterium sp. POL2 genome, and further analysis indicated that ICECspPOL2 was also inserted into the 3′ end of the tRNA-Glu-TTC gene of the transconjugant Elizabethkingia sp. M6-P2 strain. Together with the phylogenetic analysis, this insertion pattern further suggests that ICECspPOL2 belongs to the ICEEaIII group of ICEs.

Because ICECspPOL2 encodes a type IV secretion/conjugation system, we tested whether this ICE could be horizontally transferred to other bacteria, such as Escherichia coli strains 25DN and DL21. However, we did not obtain transconjugants using these recipient strains, and further analysis found that both the att site (5′-ATTCCCCTACGGGCTACT-3′) and insertion site (tRNA-Glu-TTC gene) of ICECspPOL2 do not exist in the 25DN genome (GenBank accession no. CP049298) or DL21 genome (GenBank accession no. CP079747). Further bioinformatics analysis with the tRNA-Glu-TTC gene sequence using BLASTn revealed that tRNA-Glu-TTC mainly exists in Chryseobacterium and Elizabethkingia species, indicating that ICECspPOL2 may not have a broad host range and that Chryseobacterium and Elizabethkingia species may be the major hosts of ICECspPOL2.

In conclusion, the integrative and conjugative element ICECspPOL2, which was associated with the transmission of a carbapenem resistance gene, was identified in the genome of the multi-antibiotic-resistant strain Chryseobacterium sp. POL2. ICECspPOL2 is closely related to the ICEEas from Elizabethkingia species, and conjugation assays found that ICECspPOL2 can horizontally transfer to Elizabethkingia species, with the tRNA-Glu-TTC gene as the insertion site. Because Elizabethkingia species are associated with clinically significant infections and high mortality, the ability of ICECspPOL2 to transfer carbapenem resistance from environmental strains of Chryseobacterium to Elizabethkingia is of clinical concern. Indeed, as ICEEas, including ICECspPOL2, could contribute to the dissemination of multiple types of ARGs among Chryseobacterium and Elizabethkingia spp., it will be important to track the spread of ICEEas in hospital and environmental strains of Chryseobacterium and Elizabethkingia to prevent further outbreaks of MDR bacteria.

MATERIALS AND METHODS

Strains.

Strain POL2 was isolated from a wastewater sample from a livestock farm in Shandong, China. 10-fold serial dilutions of the wastewater sample were prepared with sterilized water, plated onto LB agar supplemented with 16 mg/liter tetracycline, and incubated overnight at 28°C to obtain single colonies. Next, a single colony was picked and streaked three consecutive times onto LB agar containing tetracycline to obtain a pure culture. A single colony was then selected and grown as a pure culture, which was named strain POL2.

The sodium azide-resistant strains Escherichia coli 25DN and DL21 were obtained from our laboratory stocks (32) and grown at 37°C in LB medium. Elizabethkingia sp. M6 strain, which was previously isolated from hospital wastewater, was stored in our laboratory before growing at 28°C in LB medium.

Antibiotic susceptibility testing.

To determine the MICs of different antibiotics for strain POL2, antibiotic susceptibility testing was performed using the broth microdilution method (33). The recipient strain M6 and the transconjugant M6-P2 used in the conjugation assays below were also tested for MICs. All the antibiotic susceptibility tests in this study were carried out in triplicate.

Whole-genome sequencing and genomic analysis.

The POL2 genome was sequenced using the Nanopore and BGISEQ-500 platform (BGI, Wuhan, China) and assembled using Unicycler software (34). Genome annotation was performed using the Prokaryotic Genome Annotation Pipeline (PGAP) on the NCBI website (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Additional POL2 genome annotation was performed using the RASTtk server (35, 36) and the Pathosystems Resource Integration Center (PATRIC) server (37). The virulence factors in the POL2 genome were predicted using the Pathogen Host Interactions (PHI) database (38). Sequence alignment was performed using the BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and UniProt server (https://www.uniprot.org/blast/).

Phylogenetic affiliation analysis of strain POL2.

To determine the taxonomic status of POL2, molecular phylogenetic analysis of Chryseobacterium sp. POL2 was analyzed based on its genome sequences. Twenty-seven genome sequences belonging to Chryseobacterium and Elizabethkingia species were selected, and the whole-genome phylogenetic tree was constructed using the PATRIC server (37).

Identification of the integrative and conjugative element.

The ICE in the chromosome of strain POL2 was first predicted by IslandViewer 4 (39) and then further analyzed using ICEberg 2.0 software (40). The novel ICE was named ICECspPOL2, and genes in ICECspPOL2 were annotated using NCBI and the RASTtk server (35, 36). Insertion sequences in ICECspPOL2 were predicted using IS-Finder (41).

Evolutionary analysis of ICECspPOL2.

Pairwise alignment of ICECspPOL2 and other genetic elements was conducted using the BLAST search tool, and further alignment was conducted using BioXM 2.6 software. To analyze the phylogenetic affiliation, the whole nucleotide sequence of ICECspPOL2 was first compared with other genetic elements using the BLAST website, and then nine nucleotide sequences belonging to the relevant ICEs were selected from the NCBI database. Evolutionary analyses were conducted in MEGA7 (42).

Conjugation assays.

To verify the horizontal transferability of the ICECspPOL2 among different bacteria, conjugation assays were performed as previously described with some modifications (32). E. coli strains 25DN and DL21 and the Elizabethkingia sp. strain M6 were used as the recipient strains, and Chryseobacterium sp. POL2 was used as the donor strain. After mixing POL2 and a recipient strain, the mixture was cultured on LB agar plates with meropenem (16 mg/liter), sodium azide, and X-Gluc (5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid) to screen the transconjugants. The presence of ICECspPOL2 in the transconjugants was demonstrated by PCR, using primers listed in Table S1, and DNA sequencing.

Data availability.

The complete sequence of the chromosome of Chryseobacterium sp. strain POL2 was deposited in GenBank under accession no. CP049298.