Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola

Abstract

BackgroundKlebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.ResultThis work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.ConclusionsThis multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0396-6) contains supplementary material, which is available to authorized users.

Klebsiella variicola and Klebsiella quasipneumoniae with capacity to adapt to clinical and plant settings

“…8 Excluding transposons and tra-genes that are involved in horizontal transfer and selecting those that are involved in metabolism and cellular structure, 13 structural proteins were found to be unique for K. variicola, from which a fimbrial protein was chosen for further analysis (fimbria have been shown to mediate host interaction). Plant-associated proteins in the K. variicola, K. quasipneumoniae and K. pneumoniae genomes Plant-associated proteins were identified in K. variicola and K. quasipneumoniae isolates.…”

“…K. variicola has been isolated from plant tissues, 2,3 fungal gardens of leaf-cutter ant colonies, 4 cotton disease vectors (specifically of the insect, Nezara viridula), 5 human [6][7][8][9][10] and animal infections 11 including bovine mastitis. 12 Unlike K. variicola, K. quasipneumoniae has been described exclusively in hospital settings.…”

Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans

“…1,3 With this criterium K. pneumoniae 342 from maize, 14 and K. pneumoniae KP5-1 from insect 15 were found to be misclassified as K. pneumoniae.…”

“…genomes that corresponded to K. variicola and K. quasipneumoniae Both K. variicola as K. pneumoniae and K. quasipneumoniae isolates exhibit very similar biochemical features, 1 and to date there are no reported biochemical tests for their appropriate differentiation. 3,4,10,11 The phylogenetic analysis of the rpoB gene is more adequate than the 16S rRNA for Klebsiella spp. differentiation.…”

“…genomes was first identified by whole-genome and rpoB phylogenies. 3,4 Subsequently by K. quasipneumoniae and other K. variicola genomes the misclassification was evidenced by Average Nucleotide Identity (ANI). 5 The aim of this work was detect misclassified K. variicola and K. quasipneumoniae genomes.…”