Abstract
The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively.
Highlights
The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy
The Gram-negative members of the ESKAPE pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are especially associated with nosocomial infections, and are frequently resistant to antimicrobial chemotherapy[3] and identification can trigger a change in treatment
Each DNA sample generated a peak for the 16S rRNA control, including DNA from an isolate of E. cloacae, a Gram-negative not included in the panel
Summary
The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. The Gram-negative members of the ESKAPE pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are especially associated with nosocomial infections, and are frequently resistant to antimicrobial chemotherapy[3] and identification can trigger a change in treatment. A. baumannii is frequently resistant to first line cephalosporins, such as ceftriaxone, and aminoglycosides[8]
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