Multi-drug resistant Enterobacter bugandensis species isolated from the International Space Station and comparative genomic analyses with human pathogenic strains

Abstract

Background: The antimicrobial resistance (AMR) phenotypic properties, multiple drug resistance (MDR) gene profiles, and genes related to potential virulence and pathogenic properties of five Enterobacter bugandensis strains isolated from the International Space Station (ISS) were carried out and compared with genomes of three clinical strains. Whole genome sequences of ISS strains were characterized using the hybrid de novo assembly of Nanopore and Illumina reads. In addition to traditional microbial taxonomic approaches, multilocus sequence typing (MLST) analysis was performed to classify the phylogenetic lineage. Agar diffusion discs assay was performed to test antibiotics susceptibility. The draft genomes after assembly and scaffolding were annotated with the Rapid Annotations using Subsystems Technology and RNAmmer servers for downstream analysis.

Results: Molecular phylogeny and whole genome analysis of the ISS strains with all publicly available Enterobacter genomes revealed that ISS strains were E. bugandensis and similar to the type strain EB-247^T and two clinical isolates (153_ECLO and MBRL 1077). Comparative genomic analyses of all eight E. bungandensis strains showed, a total of 4733 genes were associated with carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes). In addition, 112 genes identified in the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the MDR tripartite system were also identified in the ISS strains. A multiple antibiotic resistance (MAR) locus or MAR operon encoding MarA, MarB, MarC, and MarR, which regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12, was also observed in the ISS strains.

Conclusion: Given the MDR results for these ISS Enterobacter genomes and increased chance of pathogenicity (PathogenFinder algorithm with > 79% probability), these species pose important health considerations for future missions. Thorough genomic characterization of the strains isolated from ISS can help to understand the pathogenic potential, and inform future missions, but analyzing them in in-vivo systems is required to discern the influence of microgravity on their pathogenicity.

Keywords: Enterobacter; International Space Station; Phylogenomic analyses.

Fig. 1

Multiple-locus sequence types (MLST) analysis of ISS strains and related species of the Enterobacter. The obtained genomic contigs of the ISS isolates (in bold) were searched for gene sequences of dnaA, fusA, gyrB, leuS, pyrG, rplB, and rpoB, which are standardized for the use in MLST analysis and reported for E. cloacae species [29]. The retrieved sequences were compared with the sequence types deposited at the Enterobacter MLST database, concatenated according to the MLST scheme. The reconstruction was based on the RAxML algorithm [4], and the bootstrap values were calculated using 1000 replicates. The bar indicates 2% sequence divergence

Fig. 2

Single nucleotide polymorphism (SNP) based phylogenetic tree, showing the relationship between the ISS isolates (in bold) and members of the Enterobacter genus. The tree was generated using CSI Phylogeny [28] version 1.4

Fig. 3

Metabolic functional profiles and subsystem categories distribution of strain IF3SW-P2. 4733 genes were identified that dominated by carbohydrate metabolism followed by amino acid and derivatives

Fig. 4

Global comparison of ISS E. bugandensis with other Enterobacter WGS from NCBI Microbial Genomes Resource was done using BRIG. Genome sequence assemblies were aligned using BLASTN and the diagrammatic view was created using BRIG software. The innermost ring indicates the genomic position of the reference genome (E. bugandensis 247^T), next ring indicates GC content, and the third ring indicates GC skewness. The remaining 21 rings indicate the presence or absence of BLASTN hits at that position. Each ring represents WGS of single Enterobacter species, each shown in different color. Positions covered by BLASTN alignments are indicated in solid colors and gaps (white spaces) represent genomic regions not covered by BLASTN alignments. Order of genome from inner ring to outer is as follow: E. aerogenes KCTC 2190, E. asburiae ATCC 35953 T, E. bugandensis EB-247^T, E. cancerogenus ATCC 35316, E. bugandensis 153_ECLO, E. cloacae ATCC 13047^T, E. bugandensis MBRL1077, E. hormaechei ATCC 49162^T, E. kobei DSM 13645^T, E. lignolyticus SCF1, E. ludwigii EN-119^T, E. massiliensis JC163^T, E. mori LMG 25706^T, E. muelleri JM-458^T, Enterobacter soli ATCC BAA-2102^T, Enterobacter xiangfangensis LMG 27195^T, E. bugandensis IF2SW-B1, E. bugandensis IF2SW-B5, E. bugandensis IF2SW-P2, E. bugandensis IF2SW-P3, E. bugandensis IF3SW-P2, Xenorhabdus nematophila ATCC 19061^T