PCR system for the correct differentiation of the main bacterial species of the Klebsiella pneumoniae complex

Accurate recognition of the closely related species Klebsiella pneumoniae , Klebsiella quasipneumoniae and Klebsiella variicola by phenotypic, biochemical and automated tests is notoriously unreliable in hospitals’ diagnostic laboratories. A comparative genomics approach was conducted for the correct differentiation of the main bacterial species in the K. pneumoniae complex. Analysis of the deduced proteomes of 87 unique genomes of the Klebsiella in public databases, was used for the identification of unique protein family members. This allowed the design of a multiplex-PCR assay for the correct differentiation of these three species from different origins. This system allowed us to determine the prevalence of K. pneumoniae , K. quasipneumoniae and K . variicola among a collection of 552 clinical isolates. Of these, 87.3% (482/552) isolates corresponded to K. pneumoniae , 6.7% (33/552) to K. quasipneumoniae and 5.9% (33/552) to K. variicola . The multiplex-PCR results showed a 100% accuracy for the correct identification of the three species evaluated, which was validated with rpoB phylogenetic sequence analysis.