Phenotypic screening of hypervirulent Klebsiella pneumoniae using tellurite resistance and carbohydrate fermentation testing

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ABSTRACT Phenotypic identification of hypervirulent Klebsiella pneumoniae in routine laboratories remains challenging. We evaluated tellurite resistance and selected carbohydrate fermentation tests as phenotypic tools for screening hypervirulent strains and improving lineage identification. A total of 193 K. pneumoniae species complex strains with available whole-genome sequencing data were analyzed, including K. pneumoniae ( n = 152), K. variicola ( n = 29), and K. quasipneumoniae ( n = 12). Hypervirulent strains were defined as those carrying ≥4 of 5 virulence-associated genes ( iucA , iroB , peg-344 , rmpA , and rmpA2 ). A total of 68 strains (35.2%) were classified as hypervirulent, and 74 (38.3%) carried ter genes. Hypervirulent strains were strongly associated with ter gene carriage, and the tellurite-resistant phenotype on MacConkey agar containing 4 μg/mL potassium tellurite closely matched ter gene carriage. Among tellurite-resistant strains, adonitol non-fermentation was useful for excluding non-hypervirulent K. variicola . Using combined tellurite resistance and adonitol fermentation, the assay identified hypervirulent strains with a sensitivity of 86.8%, specificity of 89.6%, and accuracy of 88.6%, all higher than the string test. All 27 ST23 strains showed dulcitol fermentation and l -sorbose non-fermentation. Adonitol fermentation improved screening performance, whereas dulcitol and l -sorbose fermentation patterns supported presumptive identification of prototypical hypervirulent lineages. These findings support the potential utility of this culture-based approach for phenotypic screening of hypervirulent K. pneumoniae in clinical microbiology laboratories. IMPORTANCE Hypervirulent Klebsiella pneumoniae is associated with severe invasive infections, but practical methods for early recognition in routine clinical laboratories remain limited. Leveraging the strong association between hypervirulence genotype and ter gene carriage, we developed a simple culture-based screening approach for the early presumptive identification of hypervirulent K. pneumoniae . This approach, based on combined tellurite resistance and adonitol fermentation, shows higher sensitivity and specificity than the string test for identifying hypervirulent strains. Additionally, dulcitol and l -sorbose fermentation patterns support presumptive identification of prototypical hypervirulent lineages, including ST23. These phenotypic assays may expedite the identification of hypervirulent K. pneumoniae and selected hypervirulent lineages in clinical microbiology laboratories.

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