Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Abstract

Klebsiella spp. are opportunistic pathogens with clinical, veterinary and plant-associated isolates. A previous study showed that bacterial ooze from wetwood of severely declined ironwood trees in Guam contained Ralstonia solanacearum, Klebsiella variicola and K. oxytoca. In this study, Loop-Mediated Isothermal Amplification (LAMP) detected K. variicola and K. oxytoca specifically, using unique primer sets designed individually for each organism. Each LAMP detected its target specifically, while showing negative results for non-target bacteria and negative controls. LAMP detected Klebsiella in inoculated-ironwood stem tissues and bacterial ooze. Due to the presence of plant inhibitors, different sampling protocols were tested. Soaking plant tissue samples to allow diffusion of bacteria into solution, followed by boiling, provided optimum detection of Klebsiella directly from plant samples. False negatives obtained when using crushed plant samples were eliminated by including an enrichment step, involving plating and 12-h incubation. DGGE (denaturing gradient gel electrophoresis) and a colony-blot immunoassay using a Klebsiella-specific antibody also detected Klebsiella in inoculated ironwood. DGGE bands and antibody crossreactions from closely related enterobacters showed the potential for false positive results. The nature of LAMP makes it ideal for point-of-care testing, and when combined with the specificity of the LAMP primers developed in this study, demonstrates its potential as a routine field test for Klebsiella in ironwood in Guam, as well as clinical and veterinary diagnosis of Klebsiella infection. Additionally, the regions targeted for detection in this study have application across all forms of molecular-based diagnostics.

Klebsiella variicola : an emerging pathogen in humans

“…This assay was accomplished by whole-genome comparisons to obtain specific proteins for both bacterial species. Two proteins were selected: i) hemolysin-coregulated protein 1 (Hcp1) unique to K. variicola and ii) UGH, a glucuronyl hydrolase unique to K. oxytoca [32]. Practically simultaneously, Garza-Ramos et al (2015) [7] and Berry et al (2015) [6] proposed the first efficient molecular methods for proper differentiation of K. variicola from K. pneumoniae.…”