Enterobacterales use capsules, transporters, mobile genetic elements, and other evolutionary adaptations to survive antibiotics exposure in the absence of resistance genes

Abstract

Methods: Whole-genome sequencing, transcriptomic profiling, and epigenomic analyses were performed. Phenotypic assays were used to evaluate the effects of various inhibitors on antibiotic susceptibility, while bioinformatic pipelines were used to characterize resistance determinants, virulence factors, and mobile genetic elements (MGEs).

Results: Phylogenetic analysis revealed widespread carriage of diverse resistance genes, particularly on plasmids of K. pneumoniae, while Enterobacter species possessed fewer known ARGs. Despite lacking known carbapenemase and mcr genes, several isolates demonstrated colistin or carbapenem resistance mediated by upregulation of efflux pumps, overproduction of capsular polysaccharides, mutations in outer membrane proteins, and potential lipopolysaccharide-modifying enzymes. Transcriptomic analysis revealed significant differential gene expression upon antibiotic exposure. Notably, genes encoding ABC transporter proteins were significantly downregulated (p-value <0.0001, fold change > 10), while genes encoding transposases were significantly upregulated (p-value <0.0001, fold change > 11). These changes underscore the critical role of transporters and MGEs in antibiotic resistance adaptation.

Conclusions: In the absence of canonical carbapenemase and mcr genes, K. pneumoniae and Enterobacter species can deploy a spectrum of adaptive mechanisms, including efflux pumps, mobile elements, and altered outer membrane/capsule structures, to overcome colistin and carbapenem treatments. These findings support the need for ongoing surveillance of novel or underrecognized resistance mechanisms to preserve the efficacy of last-line antibiotics.

Keywords: Multi-drug resistance; RNA-sequencing; epigenomics; genomics; transcriptomic profiling.

Graphical abstract

Figure 1.

Phylogenetic and resistome dynamics of K. pneumoniae isolates from South Africa collected from human samples. Each strain is represented by its strain identifier, MLST designation, and country of origin. Strains belonging to the same clade are highlighted with the same colour on the branches. The resistome is depicted through green and white blocks, representing the presence and absence of antibiotic resistance genes, respectively.

Figure 2.

Phylogenetic and resistome dynamics of K. pneumoniae isolates from Africa collected from human samples. Each strain is represented by its strain identifier, MLST designation, and country of origin. Strains belonging to the same clade are highlighted with the same colour on the branches. The resistome is depicted through green and white blocks, representing the presence and absence of antibiotic resistance genes, respectively.

Figure 3.

Global phylogenetic and resistome dynamics of E. asburiae isolates, collected from human samples. Each strain is represented by its strain identifier, MLST designation, and country of origin. Strains belonging to the same clade are highlighted with the same colour on the branches. The resistome is depicted through blue and white blocks, representing the presence and absence of antibiotic resistance genes, respectively.

Figure 4.

Global phylogenetic and resistome dynamics of E. bugandensis isolates, collected from human samples. Each strain is represented by its strain identifier, MLST designation, and country of origin. Strains belonging to the same clade are highlighted with the same colour on the branches. The resistome is depicted through blue and white blocks, representing the presence and absence of antibiotic resistance genes, respectively.

Figure 5.

Global phylogenetic and resistome dynamics of E. cloacae complex isolates, collected from human samples. Each strain is represented by its strain identifier, MLST designation, and country of origin. Strains belonging to the same clade are highlighted with the same colour on the branches. The resistome is depicted through blue and white blocks, representing the presence and absence of antibiotic resistance genes, respectively.

Figure 6.

A volcano plot was used to compare the Differentially expressed genes (DEGs) between the carbapenem-resistant K. pneumoniae KP_4 and the susceptible Kp_13 isolate as a reference genome. Each data point represents a gene, and its position was determined by the Fold change (log2FC) and the statistical significance (log p-value). The x-axis shows the log2 Fold change, indicating the magnitude and direction of expression changes (left: downregulated, right: upregulated). The y-axis shows the negative log10-transformed p-values, indicating the significance of the differential expression (higher values are more significant). Red points highlight genes considered significantly differentially expressed, while grey points represent those not meeting the significance threshold. The blue dashed line represents the threshold for a p-value of 0.05, above which genes are considered significantly differentially expressed. The green dashed line marks the threshold for no fold change.

Figure 7.

A volcano plot for strain Kp_14, visually representing the differential expression analysis results. in this plot, each point represents a gene. The x-axis shows the log2 Fold change, indicating the magnitude and direction of expression changes (left: downregulated, right: upregulated). The y-axis shows the negative log10-transformed p-values, indicating the significance of the differential expression (higher values are more significant). Red points highlight genes considered significantly differentially expressed, while grey points represent those not meeting the significance threshold. The blue dashed line represents the threshold for a p-value of 0.05, above which genes are considered significantly differentially expressed. The green dashed line marks the threshold for no fold change.

Figure 8.

A volcano plot for strain Kp_15, visually representing the differential expression analysis results. in this plot, each point represents a gene. The x-axis shows the log2 Fold change, indicating the magnitude and direction of expression changes (left: downregulated, right: upregulated). The y-axis shows the negative log10-transformed p-values, indicating the significance of the differential expression (higher values are more significant). Red points highlight genes considered significantly differentially expressed, while grey points represent those not meeting the significance threshold. The blue dashed line represents the threshold for a p-value of 0.05, above which genes are considered significantly differentially expressed. The green dashed line marks the threshold for no fold change.

Figure 9.

A volcano plot for strain Kp_24, visually representing the differential expression analysis results. in this plot, each point represents a gene. The x-axis shows the log2 Fold change, indicating the magnitude and direction of expression changes (left: downregulated, right: upregulated). The y-axis shows the negative log10-transformed p-values, indicating the significance of the differential expression (higher values are more significant). Red points highlight genes considered significantly differentially expressed, while grey points represent those not meeting the significance threshold. The blue dashed line represents the threshold for a p-value of 0.05, above which genes are considered significantly differentially expressed. The green dashed line marks the threshold for no fold change.

Figure 10.

Structural changes in OmpK36 (OmpC) porins in K. pneumoniae compared to K. pneumoniae wild type strain. AlphaFold was used to analyse the amino acid sequences of the wild-type and carbapenem-resistance K. pneumoniae strains’ OmpK36 porins. As shown in a-e, the mutations in the porins led to structural adjustments or conformations that likely affected the permeability of the porins to the carbapenems. Regions showing structural transformations are circled in red.

Figure 11.

Structural changes in OmpK36 (OmpC) and OmpK37 porins in K. pneumoniae compared to K. pneumoniae wild type strain. Evo 2 was used to analyse the amino acid sequences of the wild-type and carbapenem-resistance K. pneumoniae strains’ OmpK36 and OmpK37 porins. Regions showing structural transformations are circled in broken circles of red, orange, and yellow. The comparison shows how the mutations (shown as inserts in e, f, and g) affects the structure of the porins and evidently, their permeability.