Detection of Klebsiella pneumoniae in Veterinary and Food Matrices Using Loop-Mediated Isothermal Amplification.

Abstract

Klebsiella pneumoniae is an opportunistic human pathogen of high relevance due to its ability to acquire antibiotic resistance. This pathogen is included, along with Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp., in the ESKAPE group, which consists of the most important bacterial pathogens resistant to antibiotics in clinical setups. Due to the importance of the rapid identification of infection-causative agents, a novel method for the rapid identification of K. pneumoniae was developed in the present work. This novel method was based on loop-mediated isothermal amplification (LAMP) and evaluated in real-time LAMP, as well as in end-point colorimetric LAMP. Additionally, the method was evaluated in two different clinical samples, namely, blood and urine, along with a food sample, namely, milk; four DNA purification protocols were also evaluated (thermal lysis, chelex, magnetic beads, and glass milk). The results revealed differences in the performance of the LAMP assays depending on the specific combination of the matrix-DNA purification protocol. Overall, the protocol reporting the best results in all the matrices was the one based on chelex, with which it was possible to reach an LOD50 below 10 CFU/mL after a short pre-enrichment step of 6 h in TSB. The method demonstrated reliability, sensitivity, and simplicity and could be performed by non-trained personnel thanks to the colorimetric format.