Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

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.

Glucose-6-phosphate dehydrogenase mutations in malaria endemic area of Thailand by multiplexed high‐resolution melting curve analysis

“…HRM assays have been widely used to detect gene mutations [ 43 – 45 ]. However, for G6PD genotyping, most of the developed HRM assays are singleplex or duplex, which can only detect one or two mutations simultaneously [ 38 , 46 , 47 ].…”

“…However, after PCR amplification, an additional gel electrophoresis step is required to check the size of the amplified PCR products, which is impractical for large population screening. The HRM assay is a powerful and reliable tool that has been widely used in the detection of gene mutations [ 43 – 45 ]. Previously, HRM assays were applied to detect G6PD mutations in different population groups [ 38 , 46 – 48 ].…”

Resistance Patterns and Phenotypic Detection of β-lactamase Enzymes among Enterobacteriaceae Isolates from Referral Hospitals in Khartoum State, Sudan

“…Likewise, a study in Uganda reported that K. pneumoniae (52%) and E. coli (44%) were the most frequent Gram-negative organisms from patients' clinical specimens [17]. As well, global studies proved that infections caused by Gram-negative bacteria are predominant compared to other pathogens [18,19].…”

ChloS-HRM, a novel assay to identify chloramphenicol-susceptible Escherichia coli and Klebsiella pneumoniae in Malawi

“…We report here the design of an RT-PCR assay to detect chloramphenicol susceptibility in E. coli and Klebsiella spp. employing high-resolution melt (HRM) analysis, which enables a high degree of multiplexing, without expensive fluorescent probes 22 …”