Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability

Abstract

The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens.

Figure 1. Morphology of the K. pneumoniae strains.

(a) Scanning electron micrographs of the K. pneumoniae strains. (b) Colonies of the strains on LB agar plates. Scale bar, 5 μm.

Figure 2. Oxidative stress tolerance and biofilm formation ability of K. pneumoniae strains.

(a) Survival of K. pneumoniae strains in oxidative stressed environment. The ATCC BAA-2146, D, M and T strains were incubated with different concentrations of H₂O₂ in sodium phosphate buffer, and then tested for their survival on LB plates. Growth is expressed as relative survival after 2 h incubation with H₂O₂. (b) Biofilm assays of K. pneumoniae strains. OD₅₉₅ readings were measured for each strain to determine the amount of biofilm formed. Data represent one of three independent experiments.

Figure 3. Growth curves of the K. pneumoniae strains in LB medium (a, c and d) or CDM (b).

Overnight-grown bacteria were diluted 100 folds in fresh LB medium or CDM and were cultivated at designated temperature with shaking (180 rpm). OD₆₀₀ readings were measured at designated time points for each strain. The data represent one of two independent experiments performed in triplicate, with s.d. indicated by error bars.

Figure 4. Circular representation of the genome features.

Genome sequences (ring 1), COG Annotated coding sequences (rings 2 + 3), KEGG enzyme (ring 4), RNA genes (ring 5: red, rRNA; blue, tRNA), GC content (rings 6), GC skew (ring 7), strain-specific SNPs (ring 8) are shown. All strain-specific SNPs from D (yellow), M (red), and T (blue) are shown on ring 8. Very short features were enlarged to enhance visibility. Clustered genes and SNPs, such as several rRNA genes, may appear as one line due to space limitations. The image was created by using the software Circos.

Figure 5. Expression of total genes (a) and the differentially expressed genes (b) identified among D, M and T strains (Fold changes >2).

The heatmap was generated from hierarchical cluster analysis of genes. The RPKM values were mean-centered and normalized across samples, with each row representing a different gene.

Figure 6. Distribution of differentially expressed genes in COG functional categories.

The y-axis represents the number of genes in each COG category.