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

Abstract

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 (bla _IND-13, bla _CIA-3, bla _TEM-116, bla _OXA-209, bla _VEB-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.

Keywords: Chryseobacterium indologenes; metabolic features; mobile genetic elements; neonatal intensive care unit; virulence and resistance genes; whole-genome sequencing.

FIGURE 1

Basic data of Whole-Genome Sequencing, circular representations, and subsystem category distributions of Chryseobacterium indologenes. (A) Assembly and annotation statistics. (B) Circles are numbered from 1 (outer) to 4 (inner). The outer two circles represent the coding sequence (CDS), transfer ribonucleic acid (tRNA), ribosomal ribonucleic acid (rRNA), and transfer-messenger RNA (tmRNA). The third circle shows the GC content (black). The fourth circle demonstrates the GC skew curve (positive GC skew, green; negative GC skew, violet). (C) The genome of C. indologenes annotated by the Rapid Annotation System Technology (RAST) server was classified into subsystems and categories. The green part in the bar chart at the leftmost position corresponds to the percentage of proteins included. The pie chart and count of the subsystem features in the right panel show the percentage distribution and category of the subsystems.

FIGURE 2

Functional annotations of Chryseobacterium indologenes strain. (A) Clusters of orthologous groups (COGs) based on functional annotation using eggNOG-mapper. (B) Distribution of gene ontology (GO).

FIGURE 3

(A) Phylogenetic tree based on 16S rRNA showing the relationship among our C. indologenes strain and other reference sequences of 16S rRNA obtained from public databases. The number next to the node is the statistical bootstrap value. In brackets are GenBank accession numbers of the 16S rRNA genes. The scale bar indicates 0.020 substitutions per nucleotide position. The heat maps of average nucleotide identity (ANI) (B) and in silico DNA–DNA hybridization (DDH) (C) between our C. indologenes genome and twelve Chryseobacterium genomes. The yellow box represents our strain.

FIGURE 4

(A) Phylogenetic tree showing the evolutionary similarity between C. indologenes and other 15 selected strains of Chryseobacterium indologenes. (B) Genome Assembly and Annotation report (https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/14653/) of 16 strains of C. indologenes. The yellow box represents our strain.

FIGURE 5

Comparative genomic analysis. (A) Venn diagram and bar chart showing the numbers of unique and shared orthologous genes in the five most closely related C. indologenes. (B) Number of proteins, clusters, and singletons. (C) KEGG pathway classification in core, accessory, and unique genomes. (D) Distribution of KEGG pathway classification.

FIGURE 6

(A) Amino acid sequence alignment for the GyrA QRDR (Ser83Tyr) of Chryseobacterium ureilyticum, Chryseobacterium (Multispecies), Chryseobacterium indologenes*; GyrB QRDR (Leu425Ile) of Chryseobacterium aurantiacum, Chryseobacterium joostei, Chryseobacterium indologenes* and GyrB QRDR (Lys473Arg) of Chryseobacterium arachidis, Chryseobacterium taiwanense, Chryseobacterium indologenes*; and mutations in lpxA (Gly68Asp) of Chryseobacterium (Multispecies), Acinetobacter baumanii, Chryseobacterium indologenes*. (B) Known mutations in genes conferring resistance to quinolones (GyrA and GyrB QDDR) and colistin (lpxA). *Strain isolated in this study.

FIGURE 7

Schematic representation of the Genomic Islands (GIs) and Mobile Genetic Elements (MGEs) in C. indologenes strain. (A) Circular genomic representation of GIs. (B) Genetic composition of the GIs. (C) MGEs found in C. indologenes. Arrow: Light blue (resistance genes), dark blue (transposase genes), golden [elements associated with Integrative and Conjugative Element (ICE)], green (virulence genes), purple (tRNAs). Red/green triangles represent the attL and attR sequences, respectively. Orange squares represent Insertion Sequences. The yellow ellipse represents transposons. The gray circle represents integron.