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Detection of Klebsiella pneumoniae in Veterinary and Food Matrices Using Loop-Mediated Isothermal Amplification
Icía Bermúdez-Fornos1, Alberto Cepeda1, Alejandro Garrido-Maestu2
1Food Hygiene, Inspection and Control Laboratory (LHICA), Department of Analytical Chemistry, Nutrition and Bromatology, Veterinary School, Campus Terra, Universidade de Santiago de Compostela, 27002 Lugo, Spain.
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.
Insights
A new loop-mediated isothermal amplification (LAMP) method rapidly identifies Klebsiella pneumoniae, a key antibiotic-resistant pathogen. The chelex DNA purification protocol combined with LAMP achieved high sensitivity in clinical samples.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Klebsiella pneumoniae is a critical opportunistic pathogen, part of the ESKAPE group of antibiotic-resistant bacteria.
- Rapid identification of K. pneumoniae is crucial for effective clinical management and infection control.
Purpose of the Study:
- To develop and evaluate a novel, rapid method for identifying Klebsiella pneumoniae.
- To assess the performance of loop-mediated isothermal amplification (LAMP) for K. pneumoniae detection.
Main Methods:
- Loop-mediated isothermal amplification (LAMP) was employed for K. pneumoniae detection.
- The method was tested using real-time and colorimetric LAMP formats.
- Evaluations included clinical samples (blood, urine) and a food sample (milk) with four DNA purification protocols (thermal lysis, chelex, magnetic beads, glass milk).
Main Results:
- Performance varied based on the matrix-DNA purification protocol combination.
- The chelex DNA purification protocol demonstrated superior results across all tested matrices.
- An LOD50 below 10 CFU/mL was achieved with chelex and a 6-hour pre-enrichment step.
Conclusions:
- The developed LAMP method is reliable, sensitive, and simple for K. pneumoniae identification.
- The colorimetric format allows for use by non-trained personnel.
- This method offers a valuable tool for rapid pathogen detection in clinical and food settings.
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