# A combined genetic and phenotypic marker approach enables precise detection of hypervirulent Klebsiella pneumoniae and reveals associated traits of capsule overproduction and tellurite resistance

## TL;DR

This study combines genetic and phenotypic markers to accurately detect hypervirulent K. pneumoniae and identifies capsule overproduction and tellurite resistance as key traits.

## Contribution

A novel combined genetic and phenotypic marker approach for precise detection of hypervirulent K. pneumoniae is introduced.

## Key findings

- Five genetic markers and the string test effectively identify hypervirulent K. pneumoniae isolates.
- 5 genes-string test negative isolates produce the highest capsule levels and show higher lethality in infection models.
- Carbapenem-resistant classical K. pneumoniae isolates have significantly lower capsule production than hypervirulent strains.

## Abstract

We integrated genetic and phenotypic analyses to characterize carbapenem-resistant hypervirulent K. pneumoniae (CRhvKp) and liver abscess-associated K. pneumoniae (LAKp) isolates, aiming to identify potential biomarkers for the accurate detection of hvKp. A total of 660 carbapenem-resistant K. pneumoniae (CRKp) and 69 LAKp isolates were screened for five hvKp-associated biomarkers (peg-344, iroB, iucA, prmpA, prmpA2) and the string test. Among LAKp isolates, 49.3% were positive for all five biomarkers and the string test (5 genes-string test positive), while 39.1% carried all markers but were string test-negative (5 genes-string test negative). The seven-day post-infection survival rates of larvae infected with 5 genes-string test positive, 5 genes-string test negative, and other LAKp isolates were 5.9%, 10.4%, and 4.3%, respectively. In contrast, larvae infected with CRKp isolates showed higher survival rates of 7.1%, 17.9%, and 37.5% for the other groups. NTUH-K2044 and the hypervirulent isolates CRKp3, CRKp117, and LAKp88 exhibited higher lethality in bacteremia mice model compared to CRKp isolates lacking five virulence genes and string test negative. Moreover, 5 genes-string test negative LAKp produced the highest levels of capsule (117.7 ng/10⁶ CFU), followed by 5 genes-string test positive LAKp (101.4 ng/10⁶ CFU). Notably, carbapenem-resistant classical K. pneumoniae (CRcKp) isolates lacking five virulence genes and string test negative, markedly lower capsule levels (7.4 ng/10⁶ CFU) compared to other groups. These results suggest that the combined use of the five genetic markers and the string test enables effective identification of hvKp, and the capsule production is correlated with the virulence potential of the strains.

This study tackles the growing threat of K. pneumoniae strains that combine high antibiotic resistance with hypervirulence. By integrating genetic and phenotypic markers, we examined carbapenem-resistant K. pneumoniae (CRKp) and liver abscess-associated K. pneumoniae (LAKp) isolates. We found strong associations between five key virulence genes (peg-344, iroB, iucA, prmpA, prmpA2), positive string test results, high capsule production, and tellurite resistance. A 23-year surveillance of 1,815 carbapenem-susceptible isolates revealed that the proportion of blood isolates carrying all five virulence biomarkers, regardless of string test positivity, was significantly higher than that of urine isolates. Our findings provide a practical framework to enhance detection and risk assessment of hypervirulent K. pneumoniae, while deepening understanding of the traits that drive their clinical impact.