Deciphering the relative importance of genetic elements in hypervirulent Klebsiella pneumoniae to guide countermeasure development

Background: Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed. Methods: Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log₁₀ LD₅₀ (range 1.5–2.9)) were created to measure the effects on LD₅₀ in a murine model of the hypervirulence-associated plasmid (pVir), iucA, _prmpA, _prmpA2 (truncated), irp2, and clbBC. Findings: Curing pVir had the greatest increase in survival (mean LD₅₀ to 7.6 (range 7.0–9.0, p ≤ 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD₅₀s for Δ_prmpA (5.9, p ≤ 0.0001), ΔiucA (3.6, p ≤ 0.0001), Δirp2 (3.4), ΔrmpAΔiucA (6.3, p ≤ 0.0001), and ΔpVirΔirp2 (8.7, p ≤ 0.0001). Notably ΔpVir had an additional mean LD₅₀ increase of 1.3 compared to the pVir-encoded Δ_prmpAΔiucA (p ≤ 0.01), suggesting presence of additional pVir-virulence genes. Truncated _pRmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, _pRmpA/aerobactin, _pRmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively. Interpretation: These data can guide countermeasure development. Funding: This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance.