Identification of Biomarkers for Differentiation of Hypervirulent Klebsiella pneumoniae from Classical K. pneumoniae

Abstract

A hypervirulent Klebsiella pneumoniae (hvKp) pathotype is undergoing global dissemination. In contrast to the usual health care-associated epidemiology of classical K. pneumoniae (cKp) infections, hvKp causes tissue-invasive infections in otherwise healthy individuals from the community, often involving multiple sites. An accurate test to identify hvKp strains is needed for improved patient care and epidemiologic studies. To fill this knowledge gap, clinical criteria or random blood isolates from North American and United Kingdom strain collections were used to assemble hvKp-rich (n = 85) and cKp-rich (n = 90) strain cohorts, respectively. The isolates were then assessed for multiple candidate biomarkers hypothesized to accurately differentiate the two cohorts. The genes peg-344, iroB, iucA, plasmid-borne rmpA gene ( _prmpA), and _prmpA2 all demonstrated >0.95 diagnostic accuracy for identifying strains in the hvKp-rich cohort. Next, to validate this epidemiological analysis, all strains were assessed experimentally in a murine sepsis model. peg-344, iroB, iucA, _prmpA, and _prmpA2 were all associated with a hazard ratio of >25 for severe illness or death, additionally supporting their utility for identifying hvKp strains. Quantitative siderophore production of ≥30 μg/ml also strongly predicted strains as members of the hvKp-rich cohort (accuracy, 0.96) and exhibited a hazard ratio of 31.7 for severe illness or death. The string test, a widely used marker for hvKp strains, performed less well, achieving an accuracy of only 0.90. Last, using the most accurate biomarkers to define hvKp, prevalence studies were performed on two Western strain collections. These data strongly support the utility of several laboratory markers for identifying hvKp strains with a high degree of accuracy.

Keywords: biomarkers; classical Klebsiella pneumoniae; diagnosis; diagnostic test; hypervirulent Klebsiella pneumoniae.

FIG 1

The survival of CD1 mice as a function of dose after subcutaneous challenge. Outbred CD1 mice underwent subcutaneous challenge with all strains from the hvKp-rich cohort (n = 85) and the cKp-rich cohort (n = 90). Animals were monitored for up to 14 days for the development of severe illness (in extremis state) or death. (A) Mice challenged with strains that possessed peg-344 had a 64-fold increase in the hazard ratio of severe illness or death compared to the levels in strains that did not. (B) Mice challenged with strains that produced >30 μg/ml of siderophores had a 31.7-fold increase in the hazard ratio of severe illness or death compared to strains that produced <30 μg/ml.

FIG 2

A quantitative siderophore concentration of >30 μg/ml is strongly predictive of strains that belong to the hvKp-rich versus the cKp-rich cohort. A quantitative measurement of total siderophore production was performed for all strains from the hvKp-rich cohort (n = 85) and the cKp-rich cohort (n = 90). (A) Data presented as a receiver operating characteristic curve. The point on the curve that corresponds to a concentration of 30 μg/ml is marked. (B) Data are presented as a box plot (minimum, first quartile, median, third quartile, and maximum). The horizontal line indicates a concentration of 30 μg/ml.