Patterns of Klebsiella pneumoniae bacteremic dissemination from the lung

Abstract

Bacteremia, a leading cause of death, generally arises after bacteria establish infection in a particular tissue and transit to secondary sites. Studying dissemination from primary sites by solely measuring bacterial burdens does not capture the movement of individual clones. Here, by barcoding Klebsiella pneumoniae, a leading cause of bacteremia, we tracked pathogen dissemination following pneumonia. Variability in organ bacterial burdens was attributable to two distinct dissemination patterns distinguished by the extent of clonal expansion in the lungs. In metastatic dissemination, bacterial clones underwent heterogeneous clonal expansion within the lung and the dominant clones spread to secondary organs. In direct dissemination, bacterial clones exited the lungs without clonal expansion, leading to lower burdens in systemic sites. We uncover bacterial and host factors that govern these two modes of dissemination. Our data reveal unexpected heterogeneity in the dynamics of Klebsiella bacteremia and define a new paradigm for understanding within-host bacterial dissemination.

Quantification ofSalmonella entericaserovar Typhimurium Population Dynamics in Murine Infection Using a Highly Diverse Barcoded Library

“…Typhimurium isolated from the liver to those in the intestine. The similarity between the initial site of infection (in our case the intestine) and secondary sites differentiates whether the secondary population arose soon after inoculation, before the population experienced the profound intestinal bottleneck (‘early’ in Figure 3B), or at a later point, after the initial bottlenecks and subsequent replication (‘late’ in Figure 3B) (18).…”

“…To gain further insight into when S. Typhimurium disseminates to extraintestinal sites, we compared the similarity of barcodes found in S. Typhimurium isolated from the liver to those in the intestine. The similarity between the initial site of infection (in our case the intestine) and secondary sites differentiates whether the secondary population arose soon after inoculation, before the population experienced the profound intestinal bottleneck ('early' in Figure 3B), or at a later point, after the initial bottlenecks and subsequent replication ('late' in Figure 3B) (18). We expect that early spread would yield dissimilar barcode frequencies between intestinal and extraintestinal sites, whereas, with late spread, there would be greater barcode similarity in the two populations.…”

“…The barcoded library was created using the pSM1 plasmid donor library, which contains >70,000 barcodes, and helper pJMP1339, which contains the Tn7 transposon and conjugation/replication machinery, as described (16)(17)(18)(19)(20)(21)(22). S. Typhimurium was heat shocked for 4 h at 42⁰C.…”

“…Samples were prepared for sequencing as described (16)(17)(18)(19)(20)(21)(22). Briefly, bacteria from large (150 mm x 15 mm) plates were resuspended in PBS + 20% glycerol and stored at -80⁰C.…”

“…In addition, the STAMPR analysis pipeline can determine the relative similarity of bacterial populations at different sites within the same animal by comparing the frequency and identity of barcodes. The quantification of both founding populations and similarity between populations can reveal new insights into host bottlenecks to infection and unexpected patterns of bacterial spread within the host (16)(17)(18)(19)(20)(21)(22).…”