Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307

Spatial relationships of ESBL-producing K. pneumoniae strains from the multiple hospitals in the Houston Methodist system with clonal groups and associated patient deaths. This circular cladogram displays the first isolate from each patient, color coded by the five hospitals of origin (teal, yellow, red, purple, and gray). The circles surrounding the cladogram, from innermost to outermost, indicate the clonal group of the strain, yersiniabactin locus, colibactin locus, and patient death. In the innermost circle, the CG258 strains are red and the CG307 strains are blue. Other clonal types are indicated with a gray bar. The yersiniabactin circle is color coded to indicate which yersiniabactin locus is present, if any, with the two most common loci, ybt17 and ybt10, in yellow and teal, respectively; all other ybt loci are gray. The colibactin circle is purple to indicate the presence of clb3, the only colibactin synthesis locus detected in our collection. In the outermost circle, in-hospital death is indicated by a black line. For patients with multiple strains, only the first isolate from the patient is represented on the tree.

ESBL-producing K. pneumoniae strains recovered during the course of this study. The first strain recovered from each patient is graphed (circles, green linear regression). The number of strains classified as CG307 (triangles, blue linear regression) and CG258 (inverted triangles, red linear regression) are also shown. This figure demonstrates that CG258 and CG307 strains were abundant throughout the study period.

Gene content differences between the reference genomes of CG258 and CG307. CG258 is the globally abundant strain of K. pneumoniae, and CG307 is the locally abundant strain in our study. Bidirectional BLAST was performed by using the PATRIC resource to illustrate the differences in gene content between these two reference genomes. The color indicates the percent identity of the BLAST hit for each gene, with darker shading indicating a bidirectional hit and lighter shading indicating a unidirectional hit (top). A progressive Mauve alignment of the CG258 and CG307 reference genomes is shown. Local colinear blocks are organized by color (bottom). NJST258_2 is the CG258 reference genome, and KPN11 is the CG307 reference assembly.

Genetic variation in ESBL-producing K. pneumoniae clinical strains in the same patient over time. (A) Clonal group of each strain recovered from 196 patients with multiple strains. (B) Clonal group of each isolate recovered from 10 patients (numbered 1 to 10) with multiple strains spanning the longest period. Each circle represents one isolate. The vertically overlapping circles shown for patient 5 indicate that two different strains were recovered from different specimens on the same day or consecutive days.

RNA-seq analysis of 10 genomically diverse ESBL-producing K. pneumoniae strains. Strains of CG258 (n = 4, red), CG307 (n = 3, blue), and other STs (n = 3, gray) were analyzed. (A) Growth curves of strains. RNA-seq analysis of duplicate cultures was done (the mean absorbance of each strain pair is shown); the growth curves of all of the strains are similar. Cells were harvested at the mid-exponential phase of growth (OD₆₀₀ of 0.6; horizontal dashed green line). (B) RNA-seq transcriptome data for chromosomally located genes of each strain were compared to those of CG258 reference strain BK30684. Plotted are expression relationships among the strains based on principal component 1 (PC1) and principal component 2 (PC2), which account for the two largest unrelated variances in the data. The strains are distributed across both axes, showing transcriptome diversity within and between CGs. (C) Number of genes with significantly altered expression relative to reference strain BK30684. Plotted are the numbers of genes with significantly increased (positive y axis) and decreased (negative y axis) expression for each strain. (D) Box plot summarizing data from the 10 strains combined or within CGs.

Host immune response to ESBL-producing K. pneumoniae in a mouse model of pneumonia. Flow cytometry analysis of leukocytes recovered from infected lungs, including neutrophils (A), monocytes (B), T cells (C), B cells (D), alveolar macrophages (E), CD4 T cells (F), γδ T cells (G), IL-17-producing CD4 T cells (H), and IL-17-producing γδ T cells (I). (J) IL-17-producing γδ T cells were also recovered from the spleen.