Brevundimonas brasiliensis sp. nov.: a New Multidrug-Resistant Species Isolated from a Patient in Brazil

ABSTRACT To increase knowledge on Brevundimonas pathogens, we conducted in-depth genomic and phenotypic characterization of a Brevundimonas strain isolated from the cerebrospinal fluid of a patient admitted in a neonatal intensive care unit. The strain was identified as a member of the genus Brevundimonas based on Vitek 2 system results and 16S rRNA gene sequencing and presented a multidrug resistance profile (MDR). Several molecular and biochemical tests were used to characterize and identify the species for in-depth results. The draft genome assembly of the isolate has a total length of 3,261,074 bp and a G+C of 66.86%, similar to other species of the genus. Multilocus sequence analysis, Type (Strain) Genome Server, digital DNA-DNA hybridization, and average nucleotide identity confirmed that the Brevundimonas sp. studied represents a distinct species, for which we propose the name Brevundimonas brasiliensis sp. nov. In silico analysis detected antimicrobial resistance genes (AMRGs) mediating resistance to β-lactams (penP, blaTEM-16, and blaBKC-1) and aminoglycosides [strA, strB, aac(6′)-Ib, and aac(6′)-Il]. We also found AMRGs encoding the AcrAB efflux pump that confers resistance to a broad spectrum of antibiotics. Colistin and quinolone resistance can be attributed to mutation in qseC and/or phoP and GyrA/GyrB, respectively. The Brevundimonas brasiliensis sp. nov. genome contained copies of type IV secretion system (T4SS)-type integrative and conjugative elements (ICEs); integrative mobilizable elements (IME); and Tn3-type and IS3, IS6, IS5, and IS1380 families, suggesting an important role in the development and dissemination of antibiotic resistance. The isolate presented a range of virulence-associated genes related to biofilm formation, adhesion, and invasion that can be relevant for its pathogenicity. Our findings provide a wealth of data to hinder the transmission of MDR Brevundimonas and highlight the need for monitoring and identifying new bacterial species in hospital environments. IMPORTANCE Brevundimonas species is considered an opportunistic human pathogen that can cause multiple types of invasive and severe infections in patients with underlying pathologies. Treatment of these pathogens has become a major challenge because many isolates are resistant to most antibiotics used in clinical practice. Furthermore, there are no consistent therapeutic results demonstrating the efficacy of antibacterial agents. Although considered a rare pathogen, recent studies have provided evidence of the emergence of Brevundimonas in clinical settings. Hence, we identified a novel pathogenic bacterium, Brevundimonas brasiliensis sp. nov., that presented a multidrug resistance (MDR) profile and carried diverse genes related to drug resistance, virulence, and mobile genetic elements. Such data can serve as a baseline for understanding the genomic diversity, adaptation, evolution, and pathogenicity of MDR Brevundimonas.

Multidrug-Resistant Brevundimonas brasiliensis sp. nov. Microbiology Spectrum associated with resistance to antibiotics and toxic compounds (16 copper homeostasis, 2 resistance to fluoroquinolones, 1 beta-lactamase, 1 multidrug resistance efflux pump, 1 copper tolerance), while 14 were related to invasion and intracellular resistance (Fig. 1C). The data of whole-genome sequencing, circular representations, and subsystem category distributions are shown in Fig. 1. The distribution of protein-coding genes into the cluster of orthologous groups (COG) functional category showed a total of 2,743 genes (Fig. 1D). The majority of known protein-coding genes were associated with "metabolism" (n = 1,013; 36.93%), followed by those related to "cellular processes and signaling" (n = 666; 24.28%), and "information storage and processing" (n = 508; 18.51%). The number of genes associated with "unknown functions" was 556 (20.26%) and with defense was 34 (1.23%) (Fig. 1D).
Phylogenetic tree and biochemical analysis. The genomic sequence of Brevundimonas sp. presented only one 16S rRNA gene sequence, indicating that the genome assembly was not contaminated by other organisms. Therefore, a phylogenetic tree was constructed based on the 16S rRNA gene sequence (1,459 bp) of our strain and all 16S rRNA gene sequences (n = 44) of known Brevundimonas species deposited in GenBank. The 16S rRNA reference sequence of Henriciella pelagia strain LA220 was used as an outgroup. The results confirmed that the Brevundimonas sp. represents a member of the genus Brevundimonas. In this initial taxonomic classification, Brevundimonas sp. was most related to Brevundimonas olei with a sequence identity of 99.71% (with 68.5% bootstrap support), followed by Brevundimonas naejangsanensis (BIO TAS2-2) ( Fig. 2A). To define the characteristics of Brevundimonas sp., biochemical tests were performed and compared with Brevundimonas olei, Brevundimonas naejangsanensis, Brevundimonas diminuta, and Brevundimonas vesicularis (Fig. 2B). Unlike B. olei, our strain was oxidase positive and motile. The results also showed that Brevundimonas sp. had a yellow color, it was catalase positive, and it only assimilated Multidrug-Resistant Brevundimonas brasiliensis sp. nov.
Microbiology Spectrum L-arabinose. The nonutilization of D-mannitol is unique to our strain when compared with other Brevundimonas species (Fig. 2B). Genetic relatedness. To further determine the taxonomic affiliation of Brevundimonas sp., a multilocus sequence analysis (MLSA) was performed with five housekeeping genes found in complete genomic and reference sequences of Brevundimonas (see Table S3 in the supplemental material). The phylogenetic trees (Fig. 3A) were generated based on the concatenated sequences in the following order: atpD (1,536 bp), recA (1,080 bp), ileS (2,922 bp), rpoD (1,923 bp), and trpB (1,224 bp), which yielded an alignment of 8,684 bp. The MLSA tree exhibited the close association between our Brevundimonas sp. and Brevundimonas naejangsanensis FS1091 (Fig. 3A), followed by Brevundimonas naejangsanensis DSM 23858.
A phylogenetic tree based on 19 reference genome sequences and the Brevundimonas sp. was constructed using Type (Strain) Genome Server (TYGS). The TYGS-based results showed that Brevundimonas sp. are most closely related to Brevundimonas naejangsanensis DSM 23858 (Fig. 3D), with dDDH values (formula d4) of 50.8%, also positioning Brevundimonas sp. as a novel species.
Although Brevundimonas olei presented .99% 16S rRNA sequence identity with the Brevundimonas sp., it had no housekeeping genes or genome sequence available in genetic sequence database for comparison. Therefore, Brevundimonas olei was not included in MLSA, ANI, dDDH, or TYGS analysis.
Genome properties and comparative functional analysis. To investigate general evolutionary patterns of genomes, we constructed two phylogenetic trees based on the set of core and accessory genomes of our strain with 49 reference and complete genomes of Brevundimonas deposited in GenBank. The trees were divided into seven showing the relationship between Brevundimonas sp. strain with Brevundimonas reference sequence strains deposited at NCBI. Tree inferred with FastME 2.1.6.1 (147) from GBDP (BLAST genome distance phylogeny method) distances calculated from genomic sequences. Branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above the branches are GBDP pseudobootstrap support values . 60% of 100 replications, with an average branch support of 75.1%. The tree was rooted at the midpoint (148). main clusters, according to topological structure and evolutionary distance. The relative positions of Brevundimonas brasiliensis sp. nov., Brevundimonas naejangsanensis DSM 23858, Brevundimonas naejangsanensis FS1091, and Brevundimonas naejangsanensis B1 species (clade 5) varied between the two trees. Brevundimonas brasiliensis sp. nov. and Brevundimonas naejangsanensis DSM 23858 were segregated under a common node in the core genome tree, although the strains segregated together under distinct nodes in the accessory genome tree ( Fig. 4A and B).
Additionally, we found amino acid alterations in PhoP (Arg81Cis) and qseC (Ile283Leu) that mediate resistance to colistin antibiotics, as well as double amino acid substitution in GyrA (S83L and D87H) and single amino acid substitution in GyrB (Leu-466), which are associated with quinolone resistance (see Fig. S1 in the supplemental material).

DISCUSSION
The prevalence of certain MDR Gram-negative bacteria is increasing dramatically in patient care settings (25). Here, we reported a phenotypic and a systematic genomic characterization of a Brevundimonas clinical strain isolated from the cerebrospinal fluid of an infant admitted in the neonatal intensive care unit (NICU). Although it is considered a rare human pathogen, there has been an increase of infections caused by Brevundimonas spp. in recent years (26,27), including in hospitalized children. Explanations for this include the following. Babies are more vulnerable to colonization and infection with pathogens due to an immature immune system. Novel molecular and phenotypic methods are providing more accurate and robust identification of these pathogens (28)(29)(30).
As Brevundimonas spp. are becoming known for their resistance properties to many different antibiotics (10,31,32), we analyzed the resistance profile to the antibiotics most commonly used to treat infections caused by Gram-negative bacteria. The studied Brevundimonas sp. was classified as MDR, presenting resistance to b-lactams, polymyxin, aminoglycosides, and fluoroquinolones. In contrast, we only observed susceptibility to tigecycline. Although the resistance mechanisms in the Brevundimonas genus remain poorly understood (10), it is known that the resistance profile can be highly varied. For instance, Brevundimonas vesicularis and Brevundimonas diminuta are the main species isolated from human infections (26). Studies have reported that both species may be resistant (17,31,32) or susceptible (9,26,33) to most antibiotics tested in this study.
Since species identification of the Brevundimonas isolate was not possible with the Vitek 2 system, WGS was performed on the Brevundimonas sp. for a more accurate identification and characterization of the isolate. The genome size and GC content were similar to most of the Brevundimonas spp. deposited in NCBI. The RAST and eggNOG analysis showed that most genes were related to cellular processes, which are essential to the bacteria (34). Notably, the genes related to the defense mechanisms present in eggNOG and disease in RAST analysis were associated with a multidrug resistance profile.
Preliminary phylogenetic analysis based on 16S rRNA gene sequences confirmed that our strain belongs to the genus Brevundimonas. It shared the highest similarity to Brevundimonas olei MJ15 (99.71%) followed by Brevundimonas naejangsanensis BIO TAS2-2 (99.37%). Although the 16S rRNA gene is widely used to differentiate strains at the genus level (35)(36)(37), it has poor discriminatory power at the species level since 16S rRNA genes are identical or highly homologous among different species (38). To aid bacterial identification, biochemical tests were performed, revealing that our strain is an oxidase positive and motile bacillus, unlike Brevundimonas olei. Multilocus sequence analysis (MLSA) based on several housekeeping genes has become a high-resolution technique to elucidate taxonomic relationship and phylogenetic analysis of closely related strains and subspecies (39,40). The MLSA scheme based on five housekeeping genes (atpD, recA, ileS, rpoD, and trpB) showed that the Brevundimonas sp. isolate was clearly separated from Brevundimonas naejangsanensis FS1091 and Brevundimonas naejangsanensis DSM 23858, indicating a novel species within the Brevundimonas genus.
ANI or dDDH analysis has been most widely used as a gold standard for species delineation (24). Studies have reported that dDDH is considered necessary when strains share more than 97% 16S rRNA gene sequence similarity (41,42), as it was observed for Brevundimonas sp., Brevundimonas olei, and Brevundimonas naejangsanensis BIO TAS2-2.
To provide more accurate evidence to support that the Brevundimonas sp. strain is a novel species, ANI and dDDH analyses were performed with the Brevundimonas sp. and complete genomic and reference sequences from the genus Brevundimonas available in GenBank. Our data revealed that values for ANI (,95%) and dDDH (,70%) were lower than those generally accepted for species-level, showing that the isolate Brevundimonas sp. represents a novel species. To further validate our results, a phylogenetic tree inferred with genome BLAST distance phylogeny (GBDP) was constructed with the Type (Strain) Genome Server (TYGS), using the strain Brevundimonas sp. and reference sequences deposited in GenBank. The TYGS results also indicate that the strain Brevundimonas sp. is a novel species. Based on these findings, the name Brevundimonas brasiliensis sp. nov. was proposed.
To gain insights into similarity and distance within the genus Brevundimonas, we constructed two phylogenetic trees based on the set of core and accessory genomes (19). Brevundimonas brasiliensis sp. nov., B. naejangsanensis DSM 23858, B. naejangsanensis strain FS1091, and B. naejangsanensis B1 were grouped into the same clade in both trees. However, the phylogenetic trees presented a different topology. Brevundimonas brasiliensis sp. nov. showed evolutionary relatedness to the B. naejangsanensis DSM 23858 on the core gene tree, but they were no longer sisters on the accessory genome tree, suggesting that noncore genes were likely to make them diverged. KEGG analysis showed that most important pathways in core, accessory, and unique genes among four Brevundimonas strains are associated with "metabolism." Among these genes, most were related to "amino acid metabolism," "carbohydrate metabolism," and "overview," suggesting important roles in the maintenance of cellular function and survival. Important drug resistance genes were identified in unique gene clusters for human disease. Brevundimonas brasiliensis sp. nov. also harbored the highest number of singletons among the four strains, presenting specific genes associated with resistance and virulence genes. Singleton genes such as species-specific or strain-specific genes are those present in only one genome, which are usually acquired by horizontal gene transfer (43). All of these genomic features suggest a high versatility of Brevundimonas species in adapting to a wide range of environments, including health care environments.
We checked if the presence of AMRGs corresponded to phenotypic profiles and observed that the b-lactams in Brevundimonas brasiliensis sp. nov can be associated with penP, bla TEM-16 , and bla BKC-1 genes. The penP gene encodes a narrow-spectrum b-lactamase that displays a more effective hydrolysis only of first-and second-generation penicillins and cephalosporins (44)(45)(46). The bla TEM-116 gene has been reported in a variety of clinical isolates (28,47). Studies have related that TEM-116 b-lactamase can confer resistance to ceftazidime, cefotaxime, and aztreonam (48,49). The bla BKC-1 gene encodes a Brazilian Klebsiella carbapenemase (BKC-1) that can confer resistance to penicillins, broad-spectrum cephalosporins, and aztreonam and decreased susceptibility to carbapenems (50). Interestingly, BKC-1 was described for the first time in Brazil in three Klebsiella pneumoniae strains (51) and more recently in a Citrobacter freundii strain (52), further showing that the bla BKC-1 gene is spreading to other pathogens.
Colistin resistance in Gram-negative bacteria can be attributed to mutation in PhoPQ, PmrAB, qseC, and plasmid-borne genes, such as mcr and its variants (59)(60)(61). Our strain displayed amino acid alterations at position 283 in qseC (Ile283Leu) and position 81 in phoP (Arg81Cis). Similar mutations have been reported by Pitt et al. (62) as conferring colistin resistance in K. pneumoniae.
Resistance to quinolones is frequently acquired by mutations in the quinolone resistance-determining regions (QRDRs) of the target genes, such as gyrA, gyrB, parC, and parE (28, 63). Our strain displayed a double amino acid substitution in GyrA, serine to leucine at codon 83, and aspartic acid to histidine at 87 (GyrA-S83L-D87H). Studies have reported that Ser83-Leu substitution in GyrA is usual, but an additional mutation in codon 87 is associated with higher levels of quinolone resistance than mutations at other codons within the QRDR (63,64). Although the GyrB subunit is less commonly associated with quinolone resistance (65), B. brasiliensis sp. nov presented amino acid substitutions at position 466 in GyrB (Glu466-Leu). Similar findings have been reported in quinolone-resistant B. diminuta (32).
Antimicrobial resistance can also be acquired by altered expression of porins leading to decreased penetration of antibiotic within bacteria or increased efflux of antibiotics from the bacterial cell due to overexpression of efflux pump acting synergistically with the outer membrane mutation (66). The oqxBgb gene present in Brevundimonas brasiliensis sp. nov. can encode proteins that are part of multidrug efflux pumps responsible for fluoroquinolone resistance (67,68) The acrA-like, acrB-like, and tolC-like genes found in B. brasiliensis sp. nov. encode a well-studied RND-based tripartite efflux pump (AcrAB-TolC) in Escherichia coli, which is able to export chloramphenicol, fluoroquinolone, tetracycline, rifampin, novobiocin, fusidic acid, nalidixic acid, and b-lactam antibiotics (69)(70)(71). Brevundimonas brasiliensis sp. nov. also carried oprM and mexL genes. OprM is the outer membrane component present in Burkholderia vietnamiensis and Pseudomonas aeruginosa (72,73). This outer membrane protein is a component of MexAB-OprM, MexXY-OprM, MexJK-OprM, and MexVW-OprM efflux systems, and it mediates multidrug resistance in P. aeruginosa (74,75). Although mexAB, mexXY, mexJK, and mexVW genes were not found in our strain, the mexL encoded a TetR family repressor (MexL) that is a negative regulator of MexJK expression that can be associated to tetracycline and erythromycin resistance (76)(77)(78).
Mobile genetic elements (MGEs) play an important role in the dissemination of antibiotic resistance and emergence of MDR pathogens worldwide (96). Still, the distribution of mobile genetic elements in the Brevundimonas genus remains scarce. In our study, whole-genome assemblies of B. brasiliensis sp. nov. presented several MGEs that can be associated with antibiotic resistance and/or virulence, including transposons, insertions, putative ICE with T4SS, and putative IME. Antibiotic gene cassettes [strA, strB, aac(6')-Ib, aac (6')-Il, sul1, dfrA21], IS6100, and Tn6001 were located closely at scaffold 38. IS6100 plays a role in strA and strB expression in Xanthomonas campestris pv. vesicatoria (97) and have been identified in many bacteria (98). Although the bla VIM-3 gene was not found in our isolate, studies have shown that Tn6001 can contain a bla VIM-3 -harboring integron In450 and is associated to the dissemination of carbapenem-nonsusceptible Pseudomonas aeruginosa and extensively drug-resistant P. aeruginosa (99,100). Bouallègue-Godet et al. (101) showed that dfrA21, which encodes resistance to trimethoprim, may be located in plasmids and inserted as a single resistance cassette in a class I integron of Salmonella enterica. The aac(6')-Ib gene, responsible for most amikacin-resistant strains, is usually found in integrons, transposons, plasmids, and chromosomes of different bacterial species (102-104). Brevundimonas brasiliensis sp. nov. also presented ISKpn23 and putative ICE with T4SS harboring resistance genes (bla BKC-1 and floR) and virulence genes (tufA and/or virB11). Studies have shown that ISKpn23 plays an important role in expression of bla BKC-1 of K. pneumoniae (51,105). The floR gene has been described for the small plasmid p1807 (106) of Glaesserella parasuis and on the multidrug resistance region of an incomplete Tn4371-like integrative and conjugative element (ICE) in the P. aeruginosa chromosome (107). The virB11 virulence gene in our strain was associated with IME and putative ICE with T4SS. Campylobacter jejuni carries the virB11 gene localized on the pVir plasmid that encodes various genes that are homologous to a type IV secretion system (91,108). In our study, plasmid sequences were not detected using WGS, so it is uncertain whether many virulence and resistance genes are localized on plasmids. Furthermore, we could not correlate all the detected antibiotic resistance or virulence genes to MGEs due to the lack of literature.
In conclusion, we characterized a novel species of Brevundimonas, which is capable of infecting patients admitted to neonatal intensive care units. Since cases of Brevundimonas infection are being reported with increasing frequency, our report provides valuable information on this novel species that may be useful for surveillance, particularly in health care settings.

MATERIALS AND METHODS
Bacterial isolate. The Brevundimonas sp. was recovered from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit (NICU) of the Hospital Geral de Palmas, Palmas, Tocantins, Brazil. This isolate was sent to the Central Laboratory of Public Health of Tocantins-Brazil (LACEN/TO/BR), a health care facility from the Brazilian Ministry of Health that receives samples of antimicrobial resistance for surveillance. The sample was sent for identification and antimicrobial susceptibility testing using the Vitek 2 system (bioMérieux, Marcy l'Etoile, France). However, species identification of Brevundimonas sp. was not possible using the Vitek system. Identification of the bacterial isolate at the genus and species level was further analyzed using whole-genome sequencing (WGS) by our research group. We also used 49 representative and complete sequences of Brevundimonas type strains in this study. Data is available in GenBank as of June 2022 (https://www.ncbi.nlm.nih.gov/genbank/) (see Table S1 in the supplemental material).
Antimicrobial susceptibility. The drug susceptibility of the Brevundimonas sp. was performed using the Vitek 2 system (bioMérieux, Inc., Hazelwood, MO, United States) following the Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute) (109). Phenotypic detection for the production of carbapenemases was carried out by modified Hodge test, synergy test, and the EDTA test under the CLSI guidelines (109) as described elsewhere (110)(111)(112)(113). The MIC values of colistin and tigecycline were determined by the broth microdilution method, and results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2021; https://www.eucast.org/) criteria. The Brevundimonas sp. isolate was tested for susceptibility against 16 antibiotics as follows: amikacin, ampicillin, ampicillin/sulbactam, cefepime, cefoxitin, ceftazidime, ceftriaxone, cefuroxime axetil, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, piperacillin-tazobactam, and tigecycline. Multidrug-resistant (MDR) Brevundimonas sp. isolate was defined by nonsusceptibility to at least one agent in three or more antibiotic categories (114).
DNA isolation and library preparation for sequencing. Total DNA extraction was performed using the Wizard Genomic DNA purification kit (Promega, Madison, WI, United States). The quantification of DNA was made using NanoVue Plus (GE Healthcare Life Sciences, Marlborough, MA, United States). The integrity of DNA was verified by electrophoresis analysis. Bacterial DNA concentration was also measured fluorometrically (Qubit 3.0, kit Qubit dsDNA broad-range assay kit; Life Technologies, Carlsbad, CA, United States). Samples were submitted to sequencing reaction using 1 ng of total DNA. Nextera XT DNA library prep kit (Illumina, San Diego, CA, United States) was used for library production. The libraries were amplified using a short cycle PCR program. In the first PCR step, the index 1 (i7) adapters and index 2 (i5) adapters were added for sequencing cluster generation. The purification of the library was performed using 0.6Â Agencourt AMPure XP beads (Beckman Coulter). For checking the library quality and DNA fragment size, samples were analyzed by electrophoresis on 1.5% agarose gel. The libraries were quantified with a fluorometric method Qubit 3.0 using Qubit dsDNA broad-range assay kit (Life Technologies, Carlsbad, CA, United States) and normalized to 4 nM by standard dilution method. Libraries were pooled, denatured by addition of 0.2 N NaOH, and diluted to the final concentration of 1.8 pM. A PhiX control reaction was made in the final concentration of 1.5 pM. The run-length was a paired-end run of 75 cycles for each read (2 Â 75), plus up to eight cycles each for two index reads.
16S rRNA phylogeny and biochemical identification. We identified a 16s rRNA gene sequence from our genome annotation. All curated 16S rRNA gene sequences from genus Brevundimonas were searched for in the GenBank database (see Table S2 in the supplemental material). The nucleotide sequences of 16s rRNA were aligned using multiple sequence alignment software (MAFFT) (124) (https://www.ebi.ac.uk/Tools/msa/ mafft/). The construction of the maximum likelihood (ML) phylogenetic tree and the selection of the best assembly model were performed using the PhyML v3.0 program (125) and JModelTest (126), respectively.
The Brevundimonas sp. was subjected to biochemical tests using the Bactray I, II, III Systems according to the manufacturer's instructions (LaborClin, Paraná, Brazil). The results were compared with other Brevundimonas species reported in the literature.
Multilocus sequence analysis. Multilocus sequence analysis (MLSA) was conducted with five housekeeping genes, atpD (beta subunit of ATP synthase), ileS (isoleucina-tRNA ligase), recA (RecA protein), rpoC (DNA-directed RNA polymerase beta subunit), and trpB (beta chain of tryptophan synthase), which were retrieved from Brevundimonas reference species and the complete genome from the NCBI (National Center for Biotechnology Information) (https://www.ncbi.nlm.nih.gov/) (see Table S3 in the supplemental material). The genes were aligned and concatenated in the following order: atpD, recA, ileS, rpoC, and trpB. The phylogenetic tree was built with the PhyML v3.0 program (125) based on the best model chosen by JModelTest (126).
Core and accessory genome comparison. The complete and reference genomes of the genus Brevundimonas (see Table S6 in the supplemental material) were analyzed together with Brevundimonas brasiliensis sp. nov. using the Roary pipeline to infer the core and accessory genome trees (130).
Genome analysis with OrthoVenn and KEGG. For these analyses, we used the species closest to our strain according to the genomic core tree. Whole-genome comparison analysis of Brevundimonas brasiliensis sp. nov. against the selected genomes of Brevundimonas was performed using the OrthoVenn2 web server (https://orthovenn2.bioinfotoolkits.net) (131). Annotation of high-level functions and other high-throughput metabolism data was performed by Bacterial Pangenome Analysis Pipeline (BPGA) (132) against the Kyoto Encyclopedia Genomics and Genes Database (KEGG) (133). Thus, detailed identification of core genes, accessory genes, and unique genes was possible.
Characterization of resistance and virulence factors. The draft genome was screened for the presence of antimicrobial resistance (AMR) genes with the Rapid Annotation using Subsystem Technology server (RAST) (120) (https://rast.nmpdr.org/). BLAST was performed using two databases as follows: the comprehensive antibiotic resistance database (CARD; https://card.mcmaster.ca/) (134) and the antibiotic resistance gene ANNotation (ARG-ANNOT database) (135).
Ethics statement. In this work, we did not access the medical records of the patient. The Brevundimonas sp. and the anonymous archival data related to sample type were obtained from the Central Laboratory of Public Health of Tocantins (LACEN/TO, data's owner). The studies involving human participants were reviewed and approved by the Committee of Ethics in Human Research of the Federal University of São Carlos (CEHRFUSC), and the need for informed consent for conducting this study was waived by the committee (no. 1.595.268). 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 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. Data availability. The raw reads are available in the Sequence Read Archive under BioProject accession number PRJNA882454. This strain was deposited at the Bacteria Collection from Environment and Health (CBAS) of the Oswaldo Cruz Foundation (FIOCRUZ) (http://cbas.fiocruz.br/), under the (accession number CBAS 910).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.4 MB.