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Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil

Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (blaIND–13, blaCIA–3, blaTEM–116, blaOXA–209, blaVEB–15), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.


Introduction
Chryseobacterium indologenes is a non-motile, catalase-positive, oxidase-positive, indole-positive, nonglucose-fermenting Gram-negative bacilli (1) widely distributed in nature, but it is not normally present in the human microflora (2,3). In humans, C. indologenes was isolated for the first time from a tracheal aspirate of a patient with ventilator-associated pneumonia (4). Although considered a relatively uncommon human pathogen, the number of hospitalacquired infections caused by C. indologenes has been increasing (5)(6)(7)(8). Since C. indologenes can survive in inanimate objects, it may be isolated from hospital environments and cultured from specimens of sinks, indwelling vascular catheters, vials, feeding tubes, and other equipment that are in contact with fluids and water (9). Therefore, C. indologenes is considered a potential reservoir for various types of serious infections (8,10), including pneumonia, meningitis, wound infection, intraabdominal infection, primary bacteremia, intravascular catheter-related bacteremia, and cellulitis (7,(11)(12)(13)(14). Generally, major risk factors for C. indologene infection are patients with predisposing diseases, immunocompromised status, long-term broad-spectrum antibiotics treatment, and long-term hospitalization with indwelling devices (1,15,16).
Whole-genome sequencing (WGS) has become a wellestablished technique for high-resolution characterization of the genetic repertoire of bacterial pathogens, including antibiotic resistance, molecular epidemiology, and virulence (25). It is a promising technique for surveillance and monitoring infection control and outbreak cases of many microbial pathogens of interest in public health (26). Hence, we conducted the complete genome sequencing of MDR C. indologenes to understand the genomic diversity and genes responsible for antibiotic resistance and virulence. To the best of our knowledge, there is no published data on C. indologenes whole-genome sequencing from the cerebrospinal fluid sample of a hospitalized infant in Tocantins, Brazil.

Patient and bacterial isolate
Chryseobacterium indologenes was isolated from the cerebrospinal fluid (CSF) of an infant hospitalized at the Neonatal Intensive Care Unit (NICU) of Hospital Geral de Palmas, Palmas, Tocantins, Brazil. This isolate was initially identified as C. indologenes by a clinical microbiology laboratory using conventional methods. It was sent to the Central Laboratory of Public Health of Tocantins (LACEN/TO) for Frontiers in Medicine 02 frontiersin.org species confirmation and drug susceptibility testing. LACEN is a healthcare facility in the Brazilian Ministry of Health that receives samples for the surveillance of antimicrobial resistance.

Bacteria information and antimicrobial susceptibility
Once the sample was received at LACEN, bacterial identification and drug susceptibility assays were performed by the Vitek 2 system (bioMérieux, Marcy-l'Etoile, France) and interpreted according to Clinical and Laboratory Standards Institute guidelines (27). Minimum inhibitory concentration (MICs) of colistin and tigecycline was determined by the broth microdilution (BMD) method according to the EUCAST (28) recommendations. The results obtained with the VITEK 2 system were compared to those obtained by the BMD method.
Phenotypic detection for the production of carbapenemases was carried out by modified Hodge test, synergy test, and the ethylenediaminetetraacetic acid (EDTA) test under the CLSI guidelines (27) as described elsewhere (29-32). Multidrugresistant (MDR) C. indologenes isolate was defined by nonsusceptibility to at least one agent in three or more antibiotic categories (33).

DNA isolation and genome sequencing
Genomic DNA extraction was done in an overnight culture using the Wizard R Genomic DNA Purification Kit (Promega, Madison, WI, United States), according to the manufacturer's instructions. The DNA extract concentration and purity were determined by measuring absorbance at wavelengths of 260 and 280 nm (NanoVue Plus; GE Healthcare Life Sciences, Marlborough, MA, United States). The integrity of genomic DNA was tested by way of electrophoresis. Bacterial DNA concentration was also measured fluorometrically (Qubit R 3.0, kit Qubit R dsDNA Broad Range Assay Kit, Life Technologies, Carlsbad, CA, United States). The sample from the isolate was prepared for sequencing using 1 ng of input genomic DNA. Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, United States) was used for library preparation. The libraries were amplified using a limited cycle PCR program. The PCR step adds Index 1(i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation. The purification of the amplified library was performed using 0.6x Agencourt AMPure XP beads (Beckman Coulter). For checking the library quality and size of fragmented DNA, the samples were evaluated on 1.5% electrophoresis agarose gel. The libraries were quantified with a fluorometric method Qubit R 3.0 using kit Qubit R dsDNA Broad Range Assay Kit, Life Technologies, Carlsbad, CA, United States) and normalized to 4 nM using a Standard Dilution Method. The libraries were pooled, denatured with 0.2 N NaOH, and diluted to the final concentration of 1.8 pM. A PhiX control was added to a final concentration of 1.5 pM. The run-length was a paired-end run of 75 cycles for each read (2 × 75), plus up to eight cycles each for two index reads.

Genome assembly and annotation
Raw reads were assessed for quality using FastQC v.0.11.9, a quality control tool for high throughput sequence data, 1 and filtered for quality, length, and adapter regions using TrimGalore! v.0.6.5, a wrapper tool specific for FastQC. 2 The de novo genome assembly was made with SPAdes v.3.15.3 ("careful" and "cov-cutoff auto" options selected) (34) and SSPACE software (35). Plasmid detection and assembly attempts were made using PlasmidFinder 2.1 3 (36) and PlasmidSPAdes (37), respectively. Scaffolds of less than 200 bp were discarded. We assessed the general statistics of the assembled genome using QUAST v5.0.2 (38). The graphical map of the circular genome was generated using CGView Server (39).
Phylogenetic inferences using 16S rRNA gene, average nucleotide identity, and DNA-DNA hybridization We identified the C. indologenes 16S rRNA gene sequence from our genome annotation. We considered reference sequences of the 16S rRNA gene from other 14 Chryseobacterium species available at the GenBank database, including another C. indologenes strain. Elizabethkingia miricola was used as the outgroup. The accession numbers of these sequences are shown in the phylogeny. Nucleotide sequences were aligned using MAFFT v.7 (45). With the software MEGA-X (46), we estimated the best-fitting nucleotide model of substitution. This information was used to construct the phylogenetic tree by the Maximum Likelihood (ML) method. The robustness of branches was assessed by bootstrap analysis of 1,000 replicates (47). The average nucleotide identity (ANI) values among our C. indologenes genome and other twelve Chryseobacterium genomes (Supplementary Table 1) were calculated using OrthoANI 7 (48). From these same genomes, we calculated the in silico DNA-DNA hybridization (DDH)-analogous values using the GGDC v.3.0 tool 8 (49). Heat maps of ANI and DDH were generated using CIMminer. 9 Comparative pan-genome analysis of Chryseobacterium indologenes strains We constructed a whole-genome sequence-based phylogenetic tree considering our C. indologenes genome and other 15 Chryseobacterium indologenes genomes (Supplementary Table 2) using the online pipeline REALPHY v.1.13 (50) with default settings. The four closest strains of our C. indologenes were then analyzed with OrthoVenn2 10 (51), a webserver used for genome-wide annotation and comparison of orthologous gene clusters. Bacterial Pangenome Analysis Pipeline (BPGA) (52) was used for the identification of the core, accessory, and unique genes, and their functional distribution in KEGG categories.

Identifications of mutations associated with quinolone and colistin resistance
Genes responsible for colistin and quinolone resistance were identified through the rapid prokaryotic genome annotation (PROKKA). The amino acid sequences obtained from lpxA, lpxC, lpxD, and pmrC were manually analyzed for known mutations conferring resistance to colistin. Acinetobacter multispecies (WP_196075311.1) and Acinetobacter baumanii (SUU42982.1) lpxA sequences were manually aligned with our C. indologenes sequence (58). Other genes associated with polymyxin resistance, such as pmrB, pmrA, phoP, and phoQ were not found in the annotation executed by PROKKA.
Genomic islands, insertion sequences, and other mobile genetic elements detection IslandViewer 4 webserver 14 (59) was used for the identification of the genomic island using FDAARGOS_379 as a reference strain. Insertion sequences, transposons, and integrons were predicted using ISFinder 15

Sequences accession number
The raw reads were submitted to Sequence Reads Archives, 21 and the Bioproject accession id is PRJNA830910. Moreover, all the sequence data that we analyzed are related to this Bioproject id.

General features of the Chryseobacterium indologenes genome
The total length of the draft genome assembled was 4,836,765 bp, comprising one circular chromosome, with 37.32% G + C content. We detected a COIRNAI plasmid replicon but could not assemble any plasmid. The statistics  Figure 1A. The assembly comprised 58 scaffolds and 4,409 genes that covered 88.20% of the genome. Of these genes, 4,341 were predicted to be coding sequences (CDSs). Of the 68 RNA genes predicted, three were rRNAs, 64 transfer RNAs (tRNAs), and one transfer-messenger RNA (tmRNA) (Figures 1A,B). The RAST analysis showed that the genome of C. indologenes comprises 366 subsystems that could be classified into 27 categories. The five most significant categories in this genome were "amino acids and derivatives" that accounted for 384 genes, followed by "carbohydrates" (232 genes), "cofactors, vitamins, prosthetic groups, pigments" (224 genes), "protein metabolism" (222 genes), and "virulence, disease, and defense" (120). In the category of "virulence, disease, and defense, " 94 genes were found to be related to "resistance to antibiotics and toxic compounds, " including beta-lactamase (13 genes), multidrug resistance efflux pumps (13 genes), multidrug resistance, tripartite systems found in gramnegative bacteria (12 genes), resistance to fluoroquinolones (5 genes), and aminoglycoside adenylyltransferases (1 gene) ( Figure 1C).

Orthologous genes and gene ontology of Chryseobacterium indologenes
The distribution of protein-coding genes into the Cluster of Orthologous Groups (COG) functional category using EggNog resulted in a total of 3,759 genes. The majority of known protein-coding genes were related to "amino acid metabolism and transport" (n = 310; 8.25%), followed by those associated with "cell wall/membrane/envelope biogenesis" (n = 304; 8.09%), and "transcription" (n = 295; 7.85%). The number of genes associated with defense mechanisms was 70 (1.86%), and with "unknown functions" it was 973 (25.88%) (Figure 2A).

Phylogenetic position and similarity of whole genomes
A phylogenetic tree based on the 16S rRNA sequence was constructed with fourteen 16S rRNA reference sequences related to Chryseobacterium and one 16S rRNA reference sequence of Elizabethkingia miricola as an outgroup. This aimed to define the evolutionary position of our C. indologenes strain. Analyses revealed that our C. indologenes was most closely related to Chryseobacterium indologenes (AM232813). Nevertheless, it also showed a similarity between different species, including Chryseobacterium cucumeris (KX146463), Chryseobacterium arthrosphaerae (FN398101), Chryseobacterium gleum (AM232812), and Chryseobacterium flavum (EF154516) (Figure 3A).
The genomic similarity between our sample and the twelve Chryseobacterium species that had complete genome sequences in the NCBI (Figure 3A) was evaluated using in silico ANI (Supplementary Table 3 Table 4) analyses (Figures 3B,C) to confirm the species relatedness inferred from the phylogenetic tree and ensure an accurate assignment at the species level. The ANI analysis found that our C. indologenes and C. indologenes (GCF_900460995.1) had a high similarity of 99.06% (Figure 3B). The DDH value between C. indologenes and C. indologenes (GCF_900460995.1) was 91.7% ( Figure 3C). Therefore, both ANI and DDH analysis indicated that our C. indologenes strain belongs to the C. indologenes species.

PhyloGenetic tree and genetic relatedness
We performed a phylogenetic analysis based on C. indologenes genomes downloaded from the NCBI database. The resulting tree topology was assessed to identify genetic relatedness between our C. indologenes isolate and 15 C. indologenes strains ( Figure 4A). Our analysis showed that our C. indologenes strains is more closely related to Chryseobacterium indologenes 742 (SAMN22445227), Chryseobacterium indologenes FDAARGOS_648 (SAMN11056363) (Figure 4A). The characteristics of the 16 C. indologenes strains are shown in Figure 4B.

Chryseobacterium indologenes genomes
Using OrthoVenn web server, comparison and annotation of orthologous gene clusters were performed between our C. indologenes and its four most closely related neighbors. Data indicates that there are 4,060 core-conserved genes shared by all five strains and a total of 6 strain-specific gene clusters in our C. indologenes strain. These unique genes were related to metal ion transport, response to cadmium ion, and copper ion transport ( Figure 5A). The meanings of these unique gene clusters are not clear, and further studies are needed to better understand the functions of these singular genes. Our Chryseobacterium indologenes strain contained the highest number of singletons (n = 182), followed by C. indologenes 742 (n = 109), C. indologenes FDAARGOS_379 (n = 29), C. indologenes FDAARGOS_537 (n = 18), and C. indologenes FDAARGOS_648 (n = 11) (Figure 5B). We also found sul2, bla TEM−116 , aadS, bla VEB−15 , bla OXA−209 , catB e ermF resistance genes in singletons of our C. indologenes strain.

Resistome of Chryseobacterium indologenes
The whole-genome sequence analysis of C. indologenes corroborated with the phenotypic analyses, which revealed several antibiotic resistance-related genes (Table 1 and    genes that encode an efflux pump which confers resistance to aminoglycosides, 1 outer-membrane protein (TolC) and 1 gene of membrane fusion protein (HlyD) genes that mediate resistance to colistin antibiotic (Table 1). Additionally, our in silico analysis found genes that can confer resistance to tetracycline [tet(A)] (68), streptomycin (aadS) (69), phenicol (cat3, catB3) (70), sulfonamide (sul2) (71), and macrolides (ermF) (72); however, our strains were not tested phenotypically to these antibiotics ( Table 1). Gene functions annotated using the RAST/PROKKA tool identified gyrA, gyrB, and lpxA gene, in which amino acid substitution can also confer antibiotic resistance for fluoroquinolone and colistin, respectively. Amino acid substitutions related to quinolone resistance were also found at positions 83 of DNA gyrase subunit A (gyrA: Ser83Tyr) and at positions 425 and 473 of DNA gyrase subunit B (gyrB: Leu425Ile and Lys473Arg). Mutations that may play a role in the development of colistin resistance were found at lpxA (G68D) of C. indologenes (Figures 6A,B).

Genomic islands and other mobile genetic elements genes in Chryseobacterium indologenes
Our analyses indicate that the C. indologenes genome contained 13 potential Genomic Islands (GI) (Figure 7A). These had some elements associated with one integrative and conjugative element (ICE), in the 8 and 12 GI regions, such as a copy of a TrbC, and a relaxase (xerC). The ICE, named ICECind1, contained further open reading frames (ORFs) encoding putative TrbL, TrbC (1 copy), TrwB, integrase, and it was bordered by a 16-bp direct repeat (attL; 5 -TTGTGGGTCCTGAGG-3 , and attR: 5 -TTGTGGGTCCTGAGGG-3 ) at both ends. The attR was included in the 3' end of the tRNA-Val(TAC). In GI, we also found a sul2 gene encoding for resistance to sulfonamide, flanked by IS91-like element (ISVsa3 family transposase); an IS91 family transposase ISTha3; 2 copies of xerC; and other "accessory" sequences ( Figures 7B,C).

Discussion
Multidrug-resistant Gram-negative bacteria are usually associated with nosocomial infection and pose an important health problem for neonates admitted to neonatal intensive care units worldwide (9,73). Chryseobacterium indologenes is an emerging nosocomial pathogen that has acquired clinical significance due to ubiquitous and intrinsical resistance to several antibiotics, life-threatening infection potential, and ability to persist in hospital settings (9,74). The number of studies about C. indologenes is very limited, mainly in terms of their genomes. To build on current information, we performed a systematic genotypic characterization of a C. indologenes strain isolated from the cerebrospinal fluid of an infant.
Although there has been an increasing trend of C. indologenes bacteremia related to infants over the last few decades (5,75,76), most cases of infections caused by C. indologenes are still reported in adults (8,10). The precise reason for fewer reports of C. indologenes infections in children remains unclear. Studies have suggested it may relate to the  lower frequency of comorbidities in the pediatric population (77). Considering the pediatric population can present significant morbidity and immune dysfunction, we postulate that the relevance of C. indologenes isolated from clinical samples of infants can be challenging since novel molecular and phenotypic tests are providing rapid and accurate identification of many bacterial species, including glucose non-fermenting gram-negative bacilli (78,79) such as C. indologenes. Chryseobacterium indologenes often exhibit resistance to a wide variety of broad-spectrum antibiotics, including aminoglycosides, tetracyclines, chloramphenicol, macrolides, clindamycin, and teicoplanin; extended-spectrum penicillins; and first-, second-and third-generation cephalosporin, aztreonam, ticarcillin-clavulanate and the carbapenems (19,23,80). According to our phenotypic results, our C. indologenes strain was resistant to most of these antibiotics. It revealed a multidrug resistance profile, presenting in vitro resistance to a wide range of antibiotic classes including β-lactams, aminoglycosides, quinolone, glycylcycline, and polymyxin. Although some studies have reported that C. indologenes remains susceptible to trimethoprim/sulfamethoxazole, quinolones, minocycline, cefepime, ceftazidime, piperacillin-tazobactam, and rifampin (10,18,81,82), our strain was resistant not only to piperacillintazobactam, ciprofloxacin, cefepime, and ceftazidime but also to colistin and tigecycline. Our findings are in accordance with other studies (3,9,24) that show an increasing trend in resistance of C. indologenes against most commonly used antimicrobial agents.
In recent years, whole-genome sequencing (WGS) has become an efficient method not only for understanding the evolution of a wide range of infectious pathogens, such as emerging bacteria, but also for outbreak surveillance and implementation of rapid infection control protocols (83). Thus, we decided to investigate the C. indologenes genome using WGS. Our draft genome revealed one circular chromosome that had a similar length (4,836 kb) to most of the sequenced genomes from C. indologenes deposited in NCBI. The genomic features of chromosomes annotated using RAST, eggNOG, and GO showed similar characteristics, presenting cellular processes that are essential to the bacteria (84). The genes related to the disease found in RAST and the defense mechanisms present in eggNOG analysis indicate our C. indologenes strain is associated with the multidrug resistance profile. Liang et al. (84) obtained similar results when they analyzed the genomic features and antimicrobial susceptibility patterns of the Chryseobacterium arthrosphaerae strain ED882-96 isolated from a patient in Taiwan.
To evaluate the taxonomic position and to confirm the species identification of our strain, we conducted a 16S rRNA analysis and found that our strain is affiliated with the species C. indologenes (GenBank: AM232813). Although 16S rRNA gene sequences are highly conserved among strains of the same bacterial species and are frequently used to identify and classify microorganisms, taxonomic classifications based only on the analysis of the 16S rRNA gene can lead to misclassifications in some cases (85). This happens because this analysis lacks sufficient discriminatory power to differentiate species in many genera, such as Aeromonas, Bacillus, Pseudomonas, Streptococcus, etc. (86,87). Therefore, we used average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) to validate species identity, which confirmed that the most closely related species of our strain was C. indologenes (GCF_900460995.1).
The phylogenetic and corresponding taxonomic analysis is fundamental not only to establish the genetic novelty and the genotype-phenotype relationships of the isolates but also to identify the closest relatives of microorganisms within assembled genomes (88,89). When we assessed the genetic relatedness between C. indologenes isolate and fifteen C. indologenes strains (90-92), we found that the five closest relatives of C. indologenes strains were isolated from clinical human samples. We then analyzed the distribution of shared gene families (sequence clusters) among the proteomes of our sample and four C. indologenes strains. Interestingly, our C. indologenes strain harbored the highest number of singleton genes compared to its closest four relatives. Some of the singleton genes were related to antibiotic resistance. The genomic variability and diversity among the isolates are highlighted by the analysis of singletons and non-core genes that may be acquired from distal lineages through horizontal gene transfer and represent the genetic source for the emergence of novel functions (89).
The biological functions of the KEGG pathway genes were analyzed to further characterize the genomic differences among five strains. KEGG pathway annotation revealed that most of the genes of the core genome are related to metabolism. Similar results were obtained in previous studies on Chryseobacterium genomes (93). Moreover, drug resistance genes that may contribute to the wide resistance of strain C. indologenes to antimicrobials were found in a unique genome. All these distinctive features of our C. indologenes isolate provide evidence of its genomic plasticity that may contribute to antibiotic resistance and environmental adaptation.
When investigating the genetic basis of multidrug resistance (MDR) profile in C. indologenes, we observed a high level of concordance between the phenotypic and the genotypic results. Our analysis showed that the resistance gene profile to βlactams in C. indologenes isolate may be due to the carriage The bla CIA and bla IND genes have previously been identified in C. indologenes (94), and they usually provide intrinsic resistance to carbapenems and cephalosporins due to their production of class A β-lactamase (CIA) and class B carbapenem-hydrolyzing β-lactamase (IND) (23,95,96). The blaTEM−116 gene was described for the first time in Korea in a clinical isolate of E. coli (97). In Brazil, it was previously found in Klebsiella pneumoniae (98), Vibrio parahaemolyticus (99), Aeromonas hydrophila, and Aeromonas jandaei (100). Studies have related that TEM-116 β-lactamase can confer resistance to ceftazidime, cefotaxime, and aztreonam (97,101). The bla VEB−15 gene was first identified from genomic DNA of E. coli isolate that had reduced susceptibility to ceftazidime-avibactam (102). However, the bla VEB group has been identified in a variety of species of Enterobacteriaceae and non-fermenting species such as P. aeruginosa and Acinetobacter baumannii (102). The VEB enzymes confer a high level of resistance to an expanded-spectrum cephalosporin (103,104). The bla OXA−209 is a β-lactamase gene that was first described in Riemerella anatipestifer strain isolated from ducks and geese (105). It was also described more recently in pan-drug-resistant Myroides odoratimimus PR63039 strain isolated from a patient presenting with post-injury urinary tract infection (106). Although there were a few reports on the substrate profile of beta-lactamase class D OXA-209, we suggest this enzyme can contribute to the resistance of C. indologenes to β-lactam antibiotics. Our analysis revealed the presence of genes that can mediate resistance to tigecycline (tet(X)), quinolones (mcbG and mutation in the gyrA and gyrB genes), polymyxin (HlyD, TolC, and mutation in the lpxA gene), and aminoglycosides (RanA and RanB). Tet(X) is an enzyme capable of modifying the antibiotic tetracycline and its derivates, including tigecycline and glycylcycline (68,107). Since the identification of the tet(X) gene from the obligately anaerobic Bacteroides spp. (108,109), it has been reported among strains of Enterobacteriaceae, Comamonadaceae, Flavobacteriaceae, and Moraxellaceae (23,110,111). Resistance to quinolones can be conferred by mutations in quinolone-resistance-determining region (QRDRs) (gyrA, gyrB, parC, and parE subunits), efflux pumps (QepA and OqxAB), DNA topoisomerase protection protein Qnr, and quinolone acetyltransferase Aac (6 )-Ib-cr (23,112,113). Our strain displayed amino acid alternations at position 83 in GyrA (Ser83Tyr) (23,114) and positions 425 and 473 in gyrB (Leu425 Ile and Lys473Arg) (23). These findings are in line with studies showing these mutations in C. indologenes (23). Furthermore, the mcbG gene, found in our C. indologenes strain, encodes a pentapeptide-repeat protein with 19.6% amino acid identity with QnrA (115). The mcbG protein protects DNA gyrase from the action of microcin B17 (MccB17) and some quinolones antibiotics (115,116).
We also examined whether mutations in pmrC, lpxA, lpxC, and lpxD genes were associated with polymyxin resistance found in our strain. Our analysis revealed only a substitution in lpxA (Gly68Asp). Amino acid substitutions, frameshifts, or truncation of lpxD lpxA and lpxC have been demonstrated to lead to a complete loss of LPS (117). These changes may play a role in colistin resistance, as shown in clinical A. baumannii and Chryseobacterium oranimense isolates (117,118).
Efflux systems have been described in several bacteria isolated from clinical specimens and can be related to multidrug resistance phenotypes (119). We identify genes associated with multidrug effux pumps, such as HlyD, TolC, RanA, and RanB. HlyD belongs to the membrane fusion protein family (120) and forms a continuous channel by docking to the TolC. HlyD has been shown to contribute to polymyxin resistance in A. baumannii (121,122). TolC, an outer-membrane channel protein, is often the final portal in the pathways of protein toxin transport or export of unwanted molecules, such as antibiotics (123)(124)(125). RanARanB genes encode an efflux pump of the ABC efflux pump system. RanA alongside RanB mediates resistance to aminoglycosides in Riemerella anatipestifer (126), a member of the Flavobacteriaceae family.
Genomic Islands (GIs) are cluster genes in prokaryotic genomes of probable horizontal origin, which harbor components of mobile genetic elements (MGEs) that may be associated with mobilizing DNA (151,152). GI regions often carry genes related to pathogenicity, symbiosis, metabolic, fitness, or resistance islands (153) that confer a selective advantage to the host bacterium (152). We searched the C. indologenes genome for the MGEs able to transfer genes between DNA molecules (insertion sequences, gene cassettes, integrons, and transposons) and for those able to transfer genes between cells (conjugative and mobilizable plasmids, and integrative and conjugative element (ICEs) (154). Our analysis showed that the most prevalent ORFs are hypothetical proteins, which are frequently found in GIs and ICEs (152). We also found a GI region with ICE features (ICECind1) that contained copies of a relaxase-encoding gene; genes related to a type IV secretion system such as three conjugative transfer protein-encoding genes (1 copy trbL and 2 copies of trbC) (155,156); a gene related to essential integral membrane protein (TrwB), important for the conjugation process (157); and a gene encoding a site-specific integrase (xerC), which ensures the sitespecific chromosomal integration of the ICE as well as effective excision of the element, where it may be aided by an excisionase or recombination directionality factor (158)(159)(160). In addition, we found a transposon Tn5393 that usually carries the strA and strB genes, responsible for the resistance to streptomycin (161,162). However, these resistance genes were not present in this region. The sul2 gene, encoding for resistance to sulfonamide, was flanked by ISVsa3 family transposase (IS91-like element). This has been previously described in a plasmid (pEPMS-18199) from Edwardsiella piscicida (163). The cat variant genes usually participate in the composition of gene cassette or integron, and confer the ability of antibiotic resistance (164). Chryseobacterium indologenes contained a catB3, a chloramphenicol acetyltransferase gene, inserted in integron In31. Interestingly, Laraki et al. (165) showed that catB6, a chloramphenicol acetyltransferase−encoding allele of the catB family, may be inserted in this integron (In31). The authors also observed it decreased the in vitro antibiotic susceptibilities of Pseudomonas aeruginosa strains.
Although WGS allows the analysis of large datasets using in silico plasmid typing methods, short reads from popular high-throughput sequencers can be difficult to assemble. Therefore, complete plasmid sequences may not be accurately reconstructed (166). When antimicrobial resistance genes are localized on incomplete contigs, it is uncertain whether they are localized on plasmid or chromosome (167). In our C. indologenes strain, most resistance genes (bla TEM−116 , bla VEB−15 , bla OXA−209 , aadS, catB3, ermF) detected in singletons and the replicon (ColRNAI_DQ298019) were located on unaligned scaffolds. These findings partially corroborate the results of Evans et al. (168), who found a ColRNAI plasmid harboring genes associated with β-lactams (bla OXA−9 , bla TEM−1A ), chloramphenicol (catA1) antibiotic resistance. Furthermore, studies have shown that catB3 is one member of the gene cassette aacA7-catB3-aadB-oxa2-orf D. It can be mobilized by the integron-encoded DNA integrase and plays a role in chloramphenicol resistance of plasmid pBWH301 (169,170). However, this complete gene cassette was not found in our analysis.
The scarcity of data on the properties of clinical isolates of C. indologenes makes it challenging to characterize the transmission and evolution of this pathogen. Therefore, we believe that our detailed data of the WGS will contribute to the understanding of the genomic diversity, pathogenic potential, and multidrug resistance profile presented by C. indologenes. In addition, our data may guide future public health policy and MDR C. indologenes infection control.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the link: https: //www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA830910. Bioproject accession id is PRJNA830910.

Ethics statement
The studies involving human participants were reviewed and approved by the Committee of Ethics in Human Research of the Federal University of São Carlos (no. 1.595.268). In this work, C. indologenes and the anonymous archival data related patient age, gender, and sample type were obtained from the Central Laboratory of Public Health of Tocantins (LACEN/TO, data's owner). Patient consent was not required since the data presented in this study do not relate to any specific person or persons. Written informed consent from the participants or their legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Permission to conduct the present study was obtained from the Health Department of the State of Tocantins (Secretaria da Saúde do Estado do Tocantins -SESAU) and LACEN/TO.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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